Who Is the Intermediate Host of RNA Viruses? A Study Focusing on SARS-CoV-2 and Poliovirus.

The COVID-19 pandemic has sparked a surge in research on microbiology and virology, shedding light on overlooked aspects such as the infection of bacteria by RNA virions in the animal microbiome. Studies reveal a decrease in beneficial gut bacteria during COVID-19, indicating a significant interaction between SARS-CoV-2 and the human microbiome. However, determining the origins of the virus remains complex, with observed phenomena such as species jumps adding layers to the narrative. Prokaryotic cells play a crucial role in the disease's pathogenesis and transmission. Analyzing previous studies highlights intricate interactions from clinical manifestations to the use of the nitrogen isotope test. Drawing parallels with the history of the Poliovirus underscores the need to prioritize investigations into prokaryotic cells hosting RNA viruses.

Neuroprotective Effects of gH625-lipoPACAP in an In Vitro Fluid Dynamic Model of Parkinson's Disease.

Parkinson's disease (PD) is an aggressive and devastating age-related disorder. Although the causes are still unclear, several factors, including genetic and environmental, are involved. Except for symptomatic drugs, there are not, to date, any real cures for PD. For this purpose, it is necessary develop a model to better study this disease. Neuroblastoma cell line, SH-SY5Y, differentiated with retinoic acid represents a good in vitro model to explore PD, since it maintains growth cells to differentiated neurons. In the present study, SH-SY5Y cells were treated with 1-methyl-4-phenylpyridinium (MPP+), a neurotoxin that induces Parkinsonism, and the neuroprotective effects of pituitary adenylate cyclase-activating polypeptide (PACAP), delivered by functionalized liposomes in a blood-brain barrier fluid dynamic model, were evaluated. We demonstrated PACAP neuroprotective effects when delivered by gH625-liposome on MPP+-damaged SH-SY5Y spheroids.

gH625-liposomes deliver PACAP through a dynamic in vitro model of the blood-brain barrier.

The blood-brain barrier (BBB) selectively protects the central nervous system (CNS) from external insults, but its function can represent a limit for the passage of therapeutic molecules. Numerous in vitro models of the BBB have been realized in order to study the passage of drugs for neurodegenerative diseases, but these in vitro models are not very representative of the physiological conditions because of a limited supply of oxygen and nutrients due to static conditions. To avoid this phenomenon, we used a millifluidic bioreactor model that ensures a circulation of the medium and, therefore, of the nutrients, thanks to the continuous laminar flow. This dynamic model consists of a double-culture chamber separated by a membrane on which brain endothelial cells are cultured in order to evaluate the passage of the drug. Furthermore, in the lower chamber, SH-SY5Y were seeded as 3D spheroids to evaluate the drug passage through these cells. As nanodelivery system, we used liposomes functionalized with viral fusion peptide to evaluate the passage of a neuroprotective agent, pituitary adenylate cyclase-activating polypeptide (PACAP), through the dynamic in vitro model of the BBB. We showed that our nanodelivery system, made of functionalized liposomes and loaded with specific molecules, efficiently crosses the in vitro fluid-dynamic model of the BBB. Our findings represent an important step for further experimental investigations on PACAP administration as a therapeutic agent by an enhanced drug delivery system. Our results can improve the diffusion of good practice in neuroscience laboratories, helping to spread the 3R rules.

Could SARS-CoV-2 Have Bacteriophage Behavior or Induce the Activity of Other Bacteriophages?

SARS-CoV-2 has become one of the most studied viruses of the last century. It was assumed that the only possible host for these types of viruses was mammalian eukaryotic cells. Our recent studies show that microorganisms in the human gastrointestinal tract affect the severity of COVID-19 and for the first time provide indications that the virus might replicate in gut bacteria. In order to further support these findings, in the present work, cultures of bacteria from the human microbiome and SARS-CoV-2 were analyzed by electron and fluorescence microscopy. The images presented in this article, in association with the nitrogen (15N) isotope-labeled culture medium experiment, suggest that SARS-CoV-2 could also infect bacteria in the gut microbiota, indicating that SARS-CoV-2 could act as a bacteriophage. Our results add new knowledge to the understanding of the mechanisms of SARS-CoV-2 infection and fill gaps in the study of the interactions between SARS-CoV-2 and non-mammalian cells. These findings could be useful in suggesting specific new pharmacological solutions to support the vaccination campaign.

Morphological, Gene, and Hormonal Changes in Gonads and In-Creased Micrococcal Nuclease Accessibility of Sperm Chromatin Induced by Mercury.

Mercury is one of the most dangerous environmental pollutants. In this work, we analysed the effects of exposure of Mytilus galloprovincialis to 1, 10 and 100 pM HgCl2 for 24 h on the gonadal morphology and on the expression level of three stress genes: mt10, hsp70 and πgst. In this tissue we also evaluated the level of steroidogenic enzymes 3ß-HSD and 17ß-HSD and the expression of PL protein genes. Finally, we determined difference in sperm chromatin accessibility to micrococcal nuclease. We found alterations in gonadal morphology especially after exposure to 10 and 100 pM HgCl2 and hypo-expression of the three stress genes, particularly for hsp70. Furthermore, decreased labelling with both 3ß-HSD and 17ß-HSD antibodies was observed following exposure to 1 and 10 pM HgCl2 and complete absence at 100 pM HgCl2 exposure. Gonads of mussels exposed to all HgCl2 doses showed decreased expression of PL protein genes especially for PLIII. Finally, micrococcal nuclease digestions showed that all doses of HgCl2 exposure resulted in increased sperm chromatin accessibility to this enzyme, indicative of improper sperm chromatin structure. All of these changes provide preliminary data of the potential toxicity of mercury on the reproductive health of this mussel.

Molecular Alterations in Spermatozoa of a Family Case Living in the Land of Fires. A First Look at Possible Transgenerational Effects of Pollutants.

In our previous work, we reported alterations in protamines/histones ratio, in DNA binding of these proteins and their involvement in DNA oxidative damage in 84% of the young men living in the Land of Fires. In the present work, we extended our findings, evaluating any alterations in spermatozoa of a family case, a father and son, living in this area, to also give a first look at the possibility of transgenerational inherited effects of environmental contaminants on the molecular alterations of sperm nuclear basic proteins (SNBP), DNA and semen parameters. In the father and son, we found a diverse excess of copper and chromium in the semen, different alterations in SNBP content and low DNA binding affinity of these proteins. In addition, DNA damage, in the presence of CuCl2 and H2O2, increased by adding both the father and son SNBP. Interestingly, son SNBP, unlike his father, showed an unstable DNA binding and were able to produce DNA damage even without external addition of CuCl2, in line with a lower seminal antioxidant activity than the father. The peculiarity of some characteristics of son semen could be a basis for possible future studies on transgenerational effects of pollutants on fertility.

Spermatogenesis and regulatory factors in the wall lizard Podarcis sicula.

Spermatogenesis is an extraordinarily complex process, regulated by several factors, which leads to the differentiation of spermatogonia into spermatozoa. Among vertebrates, several reports have been focused on the lizard Podarcis sicula, a seasonal breeder and a good model for the study of reproductive processes. The goal of this review is to resume all the available data about systemic and above all local control factors involved in the control of P. sicula testicular activity. During the seasonal reproductive cycle, the variation of the expression levels of these factors determines significant variations that induce the activation or blocking of spermatogenesis. The data supplied in this review, in addition to analyze the current literature regarding the main actors of Podarcis sicula spermatogenesis, will hopefully provide a basic model that can be used for further studies on the intratesticular interaction between molecular factors that control spermatogenesis.

Immunolocalization of aromatase P450 in the epididymis of Podarcis sicula and Rattus rattus.

The goal of this study was to evaluate P450 aromatase localization in the epididymis of two different vertebrates: the lizard Podarcis sicula, a seasonal breeder, and Rattus rattus, a continuous breeder. P450 aromatase is a key enzyme involved in the local control of spermatogenesis and steroidogenesis and we proved for the first time that this enzyme is represented in the epididymis of both P. sicula and R. rattus. In details, P450 aromatase was well represented in epithelial and myoid cells and in the connective tissue of P. sicula epididymis during the reproductive period; instead, during autumnal resumption this enzyme was absent in the connective tissue. During the non-reproductive period, P450 aromatase was localized only in myoid cells of P. sicula epididymis, whereas in R. rattus it was localized both in myoid cells and connective tissue. Our findings, the first on the epididymis aromatase localization in the vertebrates, suggest a possible role of P450 aromatase in the control of male genital tract function, particularly in sperm maturation.

Pituitary adenylate cyclase-activating peptide (PACAP) and its receptors in Mus musculus testis.

To enlighten the involvement of PACAP/receptors system in the control of mammal testis, we investigated the expression of PACAP and the localization of PACAP and its receptors PAC1, VPAC1, and VPAC2 in the testis of Mus musculus. By molecular and immunohistochemical investigations, we highlighted that PACAP and its receptors are widely represented in germ cells of Mus testis, particularly in spermatocytes I, spermatids, and spermatozoa, strongly suggesting their involvement in spermatogenesis process. Moreover, for the first time in the adult mouse testis we highlighted that PACAP is present within Leydig cells, as PACAP receptors, confirming its involvement in the control of steroidogenesis in mouse.

Seasonal expression and cellular distribution of star and steroidogenic enzymes in quail testis.

The quail Coturnix coturnix is a seasonal breeder with a physiological switch on/off of gonadic activity. Photoperiod and temperature are the major environmental factors regulating the spermatogenesis. To more thoroughly comprehend the steroidogenic pathways that govern the seasonal reproductive cycle, we have investigated the localization of StAR protein and steroidogenic enzymes (3ß-HSD, 17ß-HSD, P450 aromatase, and 5α-Red) as well as androgen and estrogen levels, in the testis of reproductive and nonreproductive quails. We demonstrated that StAR, 3ß-HSD, 17ß-HSD, P450 aromatase, and 5α-Red were always present in the somatic (Leydig and Sertoli cells) and germ cells (spermatogonia, spermatocytes I and II, spermatids, and spermatozoa). In addition, by western blot analysis, we demonstrated that 17ß-HSD, P450 aromatase, and 5α-Red showed the highest expression levels during the reproductive testis compared with nonreproductive one. Accordingly, we also found that during the reproductive phase the highest titres of testosterone, 17ß-estradiol, and 5α-dihydrotestosterone are recorded. In conclusion, our findings demonstrated that in C. coturnix: (a) both somatic and germ cells are involved in the local synthesis of sex hormones; (b) 17ß-HSD, P450 aromatase, and 5α-Red expressions, as well as testicular androgens and estrogens, increased in reproductive quail testis. This study strongly indicates that the steroidogenic process in quail testis exhibits seasonal changes with the promotion of both androgenic and estrogenic pathways in the reproductive period, suggesting their synergic mechanism in the spermatogenesis regulation.

The expression of estrogen receptors during the Mytilus galloprovincialis ovarian cycle.

The aim of this paper is to assess, by real-time polymerase chain reaction and in situ hybridization, the expression of estrogen receptors ER1 and ER2 during the ovarian cycle of Mytilus galloprovincialis. By considering four phases of the reproductive cycle, that is stasis and previtellogenic stage (Stage 0), early vitellogenesis (Stage I), vitellogenesis (Stage II), full-grown oocyte (Stage III), our investigation demonstrates that the two receptors are differently expressed during the phases investigated of the ovarian cycle: ER1 reaches the highest level at Stage III, whereas ER2 reaches the highest level at Stage II, with ER2 always present at higher levels than ER1. The stage-dependent receptor expression was recorded within oocytes, follicle cells, and adipogranular cells. No ER1 and ER2 messenger RNAs (mRNAs) were found within vesicular cells. It is to be noted that the ER1 and ER2 expression within the growing oocytes, the follicular, and adipogranular cells overlaps with that of the mRNA for vitellogenin in the same cells, strongly suggesting that in Mytilus, as in vertebrates studied so far, the vitellogenin expression is under the control of estrogens.

Brain Metabolic DNA Is Reverse Transcribed in Cytoplasm: Evidence by Immunofluorescence Analysis.

In a previous study (Mol Neurobiol 55:7476-7486, 2017), newly synthesized brain metabolic DNA (BMD) from rat subcellular fractions has been shown to behave as a DNA-RNA hybrid when analyzed in cesium gradients at early [3H] thymidine incorporation times but to assume the double-stranded configuration at later times. Conversely, BMD from purified nuclei displayed the dsDNA configuration even at early incorporation times. The results were interpreted to support the BMD origin by reverse transcription in the cytoplasm and its later acquisition of the double-stranded configuration before the partial transfer to the nuclei. This interpretation has now been confirmed by immunofluorescence analyses of newly synthesized BrdU-labeled BMD from the mouse brain that demonstrates its cytoplasmic localization and colocalization with DNA-RNA hybrids. In addition, BrdU-labeled BMD has been shown to colocalize with astroglial anti-GFAP antibodies and with presynaptic anti-synaptophysin antibodies.

Pituitary adenylate cyclase-activating polypeptide in the testis of the quail Coturnix coturnix: Expression, localization, and phylogenetic analysis.

To evaluate the involvement of pituitary adenylate cyclase-activating polypeptide (PACAP)/receptors system in the control of testis activity, we have investigated the expression and localization of PACAP and the distribution of its receptors in the testis of mature samples of quail Coturnix coturnix, and we have performed a phylogenetic analysis of PACAP in birds. Using histological, molecular, and bioinformatics tools, we demonstrated that (a) PACAP messenger RNA shows a high sequence identity with that reported in other birds studied so far and in other vertebrates. Furthermore, we showed that purifying selection acts on PACAP; (b) the PACAP peptide is present only in Leydig cells, whereas its receptors are localized within both Leydig and germ cells; (c) the synthesis of PACAP does not take place in seminiferous tubules. The role of PACAP in the control of spermatogenesis and steroidogenesis in birds is discussed. Finally, we talk about the phylogenetic and evolutionary relationships between PACAP in birds and in other vertebrates.

The Mussel Mytilus galloprovincialis in the Bay of Naples: New Insights on Oogenic Cycle and Its Hormonal Control.

The aim of the present article was to investigate the oogenic cycle of Mytilus galloprovincialis sampled in the Bay of Naples, and to immunolocalize 3ß-hydroxysteroid dehydrogenase (3ß-HSD), 17ß-hydroxysteroid dehydrogenase (17ß-HSD), and P450 aromatase, enzymes involved in the synthesis of two sex hormones: testosterone and 17ß-estradiol. We demonstrate that the oogenic cycle starts in late summer-early fall and continues in early winter when the first event of spawning occurs; other spawning events take place until June, when the ovary is spent and contains a few empty ovarian follicles and numerous somatic cells, that is, adipogranular cells and vesicular connective tissue cells. During the oogenic cycle, apoptotic events occur at the level of oogonia, previtellogenic oocytes, as well as follicle cells; by contrast, necrosis events probably take place in vitellogenic oocytes, which, once degenerated, transfer their content to healthy oocytes. Finally, the present data demonstrate that 3ß-HSD, 17ß-HSD, and P450 aromatase are present in the ovary both during the reproductive and nonreproductive phases. The possible role of these enzymes during the Mytilus galloprovincialis reproductive cycle is discussed. Anat Rec, 302:1039-1049, 2019. © 2019 Wiley Periodicals, Inc.

Aromatase immunolocalization and activity in the lizard's brain: Dynamic changes during the reproductive cycle.

The purpose of the present study is to highlight the role of aromatase in the neuroendocrine control of the reproductive cycle of the male lizard Podarcis sicula during the three significant phases, i.e. the pre-reproductive, reproductive, and post-reproductive stages. Using immunohistochemical, biochemical, and hormonal tools, we have determined the localization and the activity of P450 aromatase (P450 aro) in the lizard's brain together with the determination of hormonal profile of sex steroids, i.e. testosterone and 17ß-estradiol. The present data demonstrated that the localization of P450 is shown in brain regions involved in the regulation of the reproductive behavior (medial septum, preoptic area, and hypothalamus). Its activity, as well as the intensity of the signal, is modified according to the period of reproduction, resulting in functional dynamic changes. P450 aro activity and signal intensity decrease in the pre-reproductive period and progressively increase during the reproductive stage until it reaches the maximum peak level at the post-reproductive phase. P450 aro determines a local estrogen synthesis, balancing the testosterone and estradiol levels, and therefore its role is crucial, since it plays an important role in the neuroendocrine/behavioral regulation of the reproductive processes in the male lizard P. sicula.

DNA in Squid Synaptosomes.

The synthesis of brain metabolic DNA (BMD) is modulated by learning and circadian oscillations and is not involved in cell division or DNA repair. Data from rats have highlighted its prevalent association with the mitochondrial fraction and its lack of identity with mtDNA. These features suggested that BMD could be localized in synaptosomes that are the major contaminants of brain mitochondrial fractions. The hypothesis has been examined by immunochemical analyses of the large synaptosomes of squid optic lobes that are readily prepared and identified. Optic lobe slices were incubated with 5-bromo-2-deoxyuridine (BrdU) and the isolated synaptosomal fraction was exposed to the green fluorescent anti-BrdU antibody. This procedure revealed that newly synthesized BrdU-labeled BMD is present in a significant percent of the large synaptosomes derived from the nerve terminals of retinal photoreceptor neurons and in synaptosomal bodies of smaller size. Synaptosomal BMD synthesis was strongly inhibited by actinomycin D. In addition, treatment of the synaptosomal fraction with Hoechst 33258, a blue fluorescent dye specific for dsDNA, indicated that native DNA was present in all synaptosomes. The possible role of synaptic BMD is briefly discussed.

Human Milk and Donkey Milk, Compared to Cow Milk, Reduce Inflammatory Mediators and Modulate Glucose and Lipid Metabolism, Acting on Mitochondrial Function and Oleylethanolamide Levels in Rat Skeletal Muscle.

Scope: Milk from various species differs in nutrient composition. In particular, human milk (HM) and donkey milk (DM) are characterized by a relative high level of triacylglycerol enriched in palmitic acid in sn-2 position. These dietary fats seem to exert beneficial nutritional properties through N-acylethanolamine tissue modulation. The aim of this study is to compare the effects of cow milk (CM), DM, and HM on inflammation and glucose and lipid metabolism, focusing on mitochondrial function, efficiency, and dynamics in skeletal muscle, which is the major determinant of resting metabolic rate. Moreover, we also evaluated the levels of endocannabinoids and N-acylethanolamines in liver and skeletal muscle, since tissue fatty acid profiles can be modulated by nutrient intervention. Procedures: To this aim, rats were fed with CM, DM, or HM for 4 weeks. Then, glucose tolerance and insulin resistance were analyzed. Pro-inflammatory and anti-inflammatory cytokines were evaluated in serum and skeletal muscle. Skeletal muscle was also processed to estimate mitochondrial function, efficiency, and dynamics, oxidative stress, and antioxidant/detoxifying enzyme activities. Fatty acid profiles, endocannabinoids, and N-acylethanolamine congeners were determined in liver and skeletal muscle tissue. Results: We demonstrated that DM or HM administration reducing inflammation status, improves glucose disposal and insulin resistance and reduces lipid accumulation in skeletal muscle. Moreover, HM or DM administration increases redox status, and mitochondrial uncoupling, affecting mitochondrial dynamics in the skeletal muscle. Interestingly, HM and DM supplementation increase liver and muscle levels of the N-oleoylethanolamine (OEA), a key regulator of lipid metabolism and inflammation. Conclusions: HM and DM have a healthy nutritional effect, acting on inflammatory factors and glucose and lipid metabolism. This beneficial effect is associated to a modulation of mitochondrial function, efficiency, and dynamics and to an increase of OEA levels in skeletal muscle.

Testicular steroidogenic enzymes in the lizard Podarcis sicula during the spermatogenic cycle.

Steroidogenic Acute Regulatory protein (StAR), 3ß-hydroxysteroid dehydrogenase (3ß-HSD), 17ß-hydroxysteroid dehydrogenase (17ß-HSD), 5α-Reductase (5α-Red), P450 aromatase are key enzymes involved in steroidogenesis. Recently, we showed the expression and the localization of P450 aromatase in Podarcis sicula testis during the different phases of the reproductive cycle, showing its involvement in the control of steroidogenesis, particularly in 17ß-estradiol synthesis. Now, we have investigated the presence and distribution of the other enzymes involved in steroidogenesis, i.e. StAR, 3ß-HSD, 17ß-HSD and 5α-Red, during three significant periods of the reproductive cycle: summer stasis (July-August), autumnal resumption (November) and reproductive period (May-June). We demonstrated for the first time that all these enzymes are always present in somatic cells (Leydig and Sertoli) and germ cells (spermatogonia, spermatocytes I and II, spermatids and spermatozoa) of Podarcis testis, mainly in spermatids and spermatozoa. The present results strongly suggest that in Podarcis testis both somatic and germ cells could be involved in local sex hormone synthesis and that 5α-Red and P450 could carry out a pivot role.

Vasoactive intestinal peptide (VIP) localization in the epididymis of two vertebrate species.

VIP and its receptors (VPACRs) are largely investigated in vertebrate testis, as well as their functions in the control of spermatogenesis and steroidogenesis. By contrast, a few data are available about the presence and role of VIP in the epididymis. The aim of the present paper was to investigate the localization of VIP and its receptors in the epididymis of two vertebrates: Podarcis sicula, a seasonal reproducer, and Rattus rattus, a continuous reproducer. By immunohystochemical investigation, we demonstrated for the first time that VIP and its receptors are widely represented in the epididymis of Podarcis sand Rattus; in particular in Podarcis, we showed that during the reproductive period, as well as in Rattus, VIP and its receptors are well represented in all the epithelial cells and the connective tissue of the epididymis; by contrast, during the non-reproductive period, VIP and its receptors are represented only in the connective tissue. The possible role of the VIP/VPACR system in the control of reproduction is discussed.

Spermatogenic Cycle and Steroidogenic Control of Spermatogenesis in Mytilus galloprovincialis Collected in the Bay of Naples.

The aim of the present article was to study the spermatogenic cycle of Mytilus galloprovincialis collected in the Bay of Naples during a whole year and to acquire new insights into the mechanism of control. Knowledge of the Mytilus cycle in this geographic area is of particular interest as, to the best of our knowledge, the male gonad cycle has been hitherto unexplored. Testis organization was evaluated together with the localization of the enzymes 3ß-HSD, 17ß-HSD, and P450-aromatase, which are strictly connected to the synthesis of two key hormones involved in the testis activity: testosterone and 17ß-estradiol. It was demonstrated that: (1) the spermatogenic cycle starts in late Summer-early Fall and continues until early Winter, when the first spawning occurs; after rapid gonad restoration, several spawning events take place until June, when the testis becomes non-active again; (2) in the testis, true Leydig and Sertoli cells are present; (3) during the reproductive period, Sertoli, Leydig, germ, and adipogranular cells (ADGs) are positive to 3ß-HSD and 17ß-HSD, while only germ cells are positive to P450 aromatase; by contrast, during the resting period, only ADGs are positive to 3ß-HSD and 17ß-HSD, and P450-aromatase is no longer recognizable. The presence of a hermaphrodite sample is also described. Anat Rec, 2017. © 2017 Wiley Periodicals, Inc. Anat Rec, 300:1881-1894, 2017. © 2017 Wiley Periodicals, Inc.