In silico evidence that substitution of glycine for valine (p.G8V) in a common variant of TMPRSS2 isoform 1 increases accessibility to an endocytic signal: Implication for SARS-cov-2 entry into host cells and susceptibility to COVID-19.

The TMPRSS2 protease plays a key role in the entry of the SARS-CoV-2 into cells. The TMPRSS2 gene is highly polymorphic in humans, and some polymorphisms may affect the susceptibility to COVID-19 or disease severity. rs75603675 (c.23G > T) is a missense variant that causes the replacement of glycine with valine at position 8 (p.G8V) in the TMPRSS2 isoform 1. According to GnomAD v4.0.0 database, the allele frequency of the rs75603675 on a global scale is 38.10 %, and range from 0.92 % in East Asian to 40.77 % in non-Finnish European (NFE) population. We analyzed the occurrence of the rs75603675 in two cohorts of patients, the first with severe/critical COVID-19 enrolled in a French hospital (42 patients), and the second with predominantly asymptomatic/pauci-symptomatic/mild COVID-19 enrolled in an Italian hospital (69 patients). We found that the TMPRSS2-c.23T minor allele frequency was similar in the two cohorts, 46.43 % and 46.38 %, respectively, and higher than the frequency in the NFE population (40.77 %). Chi-square test provided significant results (p < 0.05) when the genotype data (TMPRSS2-c.23T/c.23T homozygotes + TMPRSS2-c.23G/c.23T heterozygotes vs. TMPRSS2-c.23G/c.23G homozygotes) of the two patient groups were pooled and compared to the expected data for the NFE population, suggesting a possible pathogenetic mechanism of the p.G8V substitution. We explored the possible effects of the p.G8V substitution and found that the N-terminal region of the TMPRSS2 isoform 1 contains a signal for clathrin/AP-2-dependent endocytosis. In silico analysis predicted that the p.G8V substitution may increase the accessibility to the endocytic signal, which could help SARS-CoV-2 enter cells.

Expression of Transposable Elements in the Brain of the Drosophila melanogaster Model for Fragile X Syndrome.

Fragile X syndrome is a neuro-developmental disease affecting intellectual abilities and social interactions. Drosophila melanogaster represents a consolidated model to study neuronal pathways underlying this syndrome, especially because the model recapitulates complex behavioural phenotypes. Drosophila Fragile X protein, or FMRP, is required for a normal neuronal structure and for correct synaptic differentiation in both the peripheral and central nervous systems, as well as for synaptic connectivity during development of the neuronal circuits. At the molecular level, FMRP has a crucial role in RNA homeostasis, including a role in transposon RNA regulation in the gonads of D. m. Transposons are repetitive sequences regulated at both the transcriptional and post-transcriptional levels to avoid genomic instability. De-regulation of transposons in the brain in response to chromatin relaxation has previously been related to neurodegenerative events in Drosophila models. Here, we demonstrate for the first time that FMRP is required for transposon silencing in larval and adult brains of Drosophila "loss of function" dFmr1 mutants. This study highlights that flies kept in isolation, defined as asocial conditions, experience activation of transposable elements. In all, these results suggest a role for transposons in the pathogenesis of certain neurological alterations in Fragile X as well as in abnormal social behaviors.

The Drosophila simulans Genome Lacks the crystal-Stellate System.

The cry-Ste system is a genetic interaction system between heterochromatin and euchromatin in Drosophila melanogaster, regulated via the piRNA pathway. Deregulation of this system leads to meiotic defects and male sterility. Although the cry-Ste system is peculiar to D. melanogaster, ancestors of Ste and Su(Ste) elements are present in the three closely related species, D. simulans, D. sechellia, and D. mauritiana. The birth, evolution, and maintenance of this genetic system in Drosophila melanogaster are of interest. We investigate the presence of sequences homologous to cry and Ste elements in the simulans complex and describe their chromosomal distribution. The organization and expression of cry- and Ste-like sequences were further characterized in the D. simulans genome. Our results allow us to conclude that the cry-Ste genetic interaction system is absent in the D. simulans genome.

The Role of HSP90 in Preserving the Integrity of Genomes Against Transposons Is Evolutionarily Conserved.

The HSP90 protein is a molecular chaperone intensively studied for its role in numerous cellular processes both under physiological and stress conditions. This protein acts on a wide range of substrates with a well-established role in cancer and neurological disorders. In this review, we focused on the involvement of HSP90 in the silencing of transposable elements and in the genomic integrity maintenance. The common feature of transposable elements is the potential jumping in new genomic positions, causing chromosome structure rearrangements, gene mutations, and influencing gene expression levels. The role of HSP90 in the control of these elements is evolutionarily conserved and opens new perspectives in the HSP90-related mechanisms underlying human disorders. Here, we discuss the hypothesis that its role in the piRNA pathway regulating transposons may be implicated in the onset of neurological diseases.

IgM and IgG Profiles Reveal Peculiar Features of Humoral Immunity Response to SARS-CoV-2 Infection.

The emergence of coronavirus disease 2019 (COVID-19) is globally a major healthcare threat. There is little information regarding the mechanisms and roles of the humoral response in SARS-CoV-2 infection. The aim of this study was to analyze the antibody levels (IgM and IgG) by chemiluminescence immunoassay in 54 subjects positive to SARS-CoV-2 swab test in relation to their clinical status (whether asymptomatic, pauci-symptomatic or with mild, sever or critical symptoms), the time from the symptom onset, sex, age, and comorbidities. Overall, the presence of comorbidities and the age of subjects were associated with their clinical status. The IgG concentrations were significantly higher in patients who developed critical and severe symptoms and seemed to be independent from age, sex and comorbidities. IgG titers peaked around day 60, and then began gradually to drop, decreasing by approximately 50% on the 180th day, while the IgM titers progressively decreased as early as the tenth day, but they could be detected even at later time points. Despite the small number of individuals, some peculiar characteristics of the humoral response in COVID-19 emerged. We observed a high inter-individual variability, an ephemeral IgG half-life in several patients, and a persistence of IgM in others.

Assessment of CYP2C9, CYP2C19, and CYP2D6 Polymorphisms in Allergic Patients with Chemical Sensitivity.

BACKGROUND: Self-reported chemical sensitivity (SCS) is characterized by adverse effects due to exposure to low levels of chemical substances. The clinical manifestations of SCS are similar to the allergy, and a high percentage of individuals with both diseases have been found. Various genes, especially genes of importance to the metabolism of xenobiotic compounds, have been associated with SCS. OBJECTIVES: The purpose of this study was to investigate whether allergic individuals with chemical sensitivity differed from allergic patients without chemical sensitivity with regard to the distribution of genotype and phenotype of CYP2C9, CYP2C19, and CYP2D6 polymorphisms. METHODS: A total of 180 patients were enrolled for this study. A questionnaire was employed to collect information on individual chemical sensitivity, while the Skin prick test and the PATCH test were used to verify the presence of an allergic condition against inhalants or contact allergens, respectively. For the evaluation of the CYP2C9, CYP2C19, and CYP2D6 polymorphisms, we used a strategy based on the amplification of the entire gene coupled to direct genomic DNA sequencing analysis. RESULTS: Overall, a total of 15 different CYP2C9, CYP2C19, and CYP2D6 haplotypes were identified in our population. If the 5 CYP2C9 and the 2 CYP2C19 identified alleles correspond to the previously described ones, 4 of the 8 CYP2D6 haplotypes, detected in the study group, present new SNPs combinations. These new suballeles were categorized as CYP2D6*2M Sa-lento Variant 1, CYP2D6*35B Salento Variant 2, CYP2D6*41 Salento Variant 3, and CYP2D6*4P Salento Variant 4 due to the presence of the key SNPs 2,850 C>T, 31G>A, 2,988 G>A, and 1,846 G>A, respectively. When the allergic individuals are divided into 2 groups according to their SCS score, we observed that the distribution of the CYP2D6 phenotypes was significantly different between the 2 groups. CONCLUSIONS: Our idea is that the application of the questionnaire that we have adopted has enabled us to diagnose a degree of chemical sensitivity, which results as comorbid of the allergic disease and in which a condition of poor or intermediate metabolizes for the detrimental CYP2D6 alleles, could represent a discriminant between the chemical sensitivity and the health state.

Drosophila melanogaster as a Model to Study the Multiple Phenotypes, Related to Genome Stability of the Fragile-X Syndrome.

Fragile-X syndrome is one of the most common forms of inherited mental retardation and autistic behaviors. The reduction/absence of the functional FMRP protein, coded by the X-linked Fmr1 gene in humans, is responsible for the syndrome. Patients exhibit a variety of symptoms predominantly linked to the function of FMRP protein in the nervous system like autistic behavior and mild-to-severe intellectual disability. Fragile-X (FraX) individuals also display cellular and morphological traits including branched dendritic spines, large ears, and macroorchidism. The dFmr1 gene is the Drosophila ortholog of the human Fmr1 gene. dFmr1 mutant flies exhibit synaptic abnormalities, behavioral defects as well as an altered germline development, resembling the phenotypes observed in FraX patients. Therefore, Drosophila melanogaster is considered a good model to study the physiopathological mechanisms underlying the Fragile-X syndrome. In this review, we explore how the multifaceted roles of the FMRP protein have been addressed in the Drosophila model and how the gained knowledge may open novel perspectives for understanding the molecular defects causing the disease and for identifying novel therapeutical targets.

The RNA Helicase BELLE Is Involved in Circadian Rhythmicity and in Transposons Regulation in Drosophila melanogaster.

Circadian clocks control and synchronize biological rhythms of several behavioral and physiological phenomena in most, if not all, organisms. Rhythm generation relies on molecular auto-regulatory oscillations of interlocked transcriptional-translational feedback loops. Rhythmic clock-gene expression is at the base of rhythmic protein accumulation, though post-transcriptional and post-translational mechanisms have evolved to adjust and consolidate the proper pace of the clock. In Drosophila, BELLE, a conserved DEAD-box RNA helicase playing important roles in reproductive capacity, is involved in the small RNA-mediated regulation associated to the piRNA pathway. Here, we report that BELLE is implicated in the circadian rhythmicity and in the regulation of endogenous transposable elements (TEs) in both nervous system and gonads. We suggest that BELLE acts as important element in the piRNA-mediated regulation of the TEs and raise the hypothesis that this specific regulation could represent another level of post-transcriptional control adopted by the clock to ensure the proper rhythmicity.

dFmr1 Plays Roles in Small RNA Pathways of Drosophila melanogaster.

Fragile-X syndrome is the most common form of inherited mental retardation accompanied by other phenotypes, including macroorchidism. The disorder originates with mutations in the Fmr1 gene coding for the FMRP protein, which, with its paralogs FXR1 and FXR2, constitute a well-conserved family of RNA-binding proteins. Drosophila melanogaster is a good model for the syndrome because it has a unique fragile X-related gene: dFmr1. Recently, in addition to its confirmed role in the miRNA pathway, a function for dFmr1 in the piRNA pathway, operating in Drosophila gonads, has been established. In this review we report a summary of the piRNA pathways occurring in gonads with a special emphasis on the relationship between the piRNA genes and the crystal-Stellate system; we also analyze the roles of dFmr1 in the Drosophila gonads, exploring their genetic and biochemical interactions to reveal some unexpected connections.

Novel mutants of the aubergine gene.

Aubergine is an RNA-binding protein of the Piwi clade, functioning in germline in the piRNA pathway that silences transposons and repetitive sequences. Several mutations of this gene exist, but they mostly result in truncated proteins or correspond to mutations that also affect neighboring genes. We have generated complete aubergine knock-out mutants that do not disrupt the neighboring genes. These novel mutants are characterized by PCR and sequencing. Their nature is confirmed by female sterility and by the presence of crystals in testes, common to the aubergine loss of function mutations. These mutants provide novel and more appropriate tools for the study of the piRNA pathway that controls genome stability.

Loss of Pol32 in Drosophila melanogaster causes chromosome instability and suppresses variegation.

Pol32 is an accessory subunit of the replicative DNA Polymerase δ and of the translesion Polymerase ζ. Pol32 is involved in DNA replication, recombination and repair. Pol32's participation in high- and low-fidelity processes, together with the phenotypes arising from its disruption, imply multiple roles for this subunit within eukaryotic cells, not all of which have been fully elucidated. Using pol32 null mutants and two partial loss-of-function alleles pol32rd1 and pol32rds in Drosophila melanogaster, we show that Pol32 plays an essential role in promoting genome stability. Pol32 is essential to ensure DNA replication in early embryogenesis and it participates in the repair of mitotic chromosome breakage. In addition we found that pol32 mutants suppress position effect variegation, suggesting a role for Pol32 in chromatin architecture.

The Drosophila fragile X mental retardation protein participates in the piRNA pathway.

RNA metabolism controls multiple biological processes, and a specific class of small RNAs, called piRNAs, act as genome guardians by silencing the expression of transposons and repetitive sequences in the gonads. Defects in the piRNA pathway affect genome integrity and fertility. The possible implications in physiopathological mechanisms of human diseases have made the piRNA pathway the object of intense investigation, and recent work suggests that there is a role for this pathway in somatic processes including synaptic plasticity. The RNA-binding fragile X mental retardation protein (FMRP, also known as FMR1) controls translation and its loss triggers the most frequent syndromic form of mental retardation as well as gonadal defects in humans. Here, we demonstrate for the first time that germline, as well as somatic expression, of Drosophila Fmr1 (denoted dFmr1), the Drosophila ortholog of FMRP, are necessary in a pathway mediated by piRNAs. Moreover, dFmr1 interacts genetically and biochemically with Aubergine, an Argonaute protein and a key player in this pathway. Our data provide novel perspectives for understanding the phenotypes observed in Fragile X patients and support the view that piRNAs might be at work in the nervous system.

Transposons, environmental changes, and heritable induced phenotypic variability.

The mechanisms of biological evolution have always been, and still are, the subject of intense debate and modeling. One of the main problems is how the genetic variability is produced and maintained in order to make the organisms adaptable to environmental changes and therefore capable of evolving. In recent years, it has been reported that, in flies and plants, mutations in Hsp90 gene are capable to induce, with a low frequency, many different developmental abnormalities depending on the genetic backgrounds. This has suggested that the reduction of Hsp90 amount makes different development pathways more sensitive to hidden genetic variability. This suggestion revitalized a classical debate around the original Waddington hypothesis of canalization and genetic assimilation making Hsp90 the prototype of morphological capacitor. Other data have also suggested a different mechanism that revitalizes another classic debate about the response of genome to physiological and environmental stress put forward by Barbara McClintock. That data demonstrated that Hsp90 is involved in repression of transposon activity by playing a significant role in piwi-interacting RNA (piRNAs)-dependent RNA interference (RNAi) silencing. The important implication is that the fixed phenotypic abnormalities observed in Hsp90 mutants are probably related to de novo induced mutations by transposon activation. In this case, Hsp90 could be considered as a mutator. In the present theoretical paper, we discuss several possible implications about environmental stress, transposon, and evolution offering also a support to the concept of evolvability.

Functional characterization of the Bari1 transposition system.

The transposons of the Bari family are mobile genetic elements widespread in the Drosophila genus. However, despite a broad diffusion, virtually no information is available on the mechanisms underlying their mobility. In this paper we report the functional characterization of the Bari elements transposition system. Using the Bari1 element as a model, we investigated the subcellular localization of the transposase, its physical interaction with the transposon, and its catalytic activity. The Bari1 transposase localized in the nucleus and interacted with the terminal sequences of the transposon both in vitro and in vivo, however, no transposition activity was detected in transposition assays. Profiling of mRNAs expressed by the transposase gene revealed the expression of abnormal, internally processed transposase transcripts encoding truncated, catalytically inactive transposase polypeptides. We hypothesize that a post-transcriptional control mechanism produces transposase-derived polypeptides that effectively repress transposition. Our findings suggest further clues towards understanding the mechanisms that control transposition of an important class of mobile elements, which are both an endogenous source of genomic variability and widely used as transformation vectors/biotechnological tools.

The "Special" crystal-Stellate System in Drosophila melanogaster Reveals Mechanisms Underlying piRNA Pathway-Mediated Canalization.

The Stellate-made crystals formation in spermatocytes is the phenotypic manifestation of a disrupted crystal-Stellate interaction in testes of Drosophila melanogaster. Stellate silencing is achieved by the piRNA pathway, but many features still remain unknown. Here we outline the important role of the crystal-Stellate modifiers. These have shed light on the piRNA pathways that defend genome integrity against transposons and other repetitive elements in the gonads. In particular, we illustrate the finding that HSP90 participates in the molecular pathways of piRNA production. This observation has relevance for the mechanisms underlying the evolutionary canalization process.

Different aubergine alleles confirm the specificity of different RNAi pathways in Drosophila melanogaster.

The crystal-Stellate system is one of the best-known examples of heterochromatin-euchromatin interaction. The components of this system are homologous repetitive sequences clustered in three regions: 12E1 and h27 on the X and h11 on the Y. The symptom of a disrupted crystal-Stellate interaction is the presence of crystals in the spermatocytes of males lacking the crystal region. Stellate silencing is based on the RNAi process. Many modifiers of this system have been isolated and many of these are involved in RNAi. One of these modifiers is aubergine(sting); this is a "gain of function" allele in somatic tissues. Here we report the different behavior of two aubergine alleles with respect to the RNAi pathway: aub(sting) and a "loss of function" heteroallelic combination aub(HN)/aub(QC42). An increased amount of Aub interferes with the correct functioning of the somatic yellow hairpin RNAi, whereas the Aub reduction does not. We also demonstrate the different behavior of these alleles on the I transposon silencing in ovaries. Intriguingly, neither of these aubergine alleles silence the Stellate locus. We can conclude that the crystal-Stellate system reveals different RNAi pathways even though much still remains to be done to completely explain the molecular bases of the crystal-Stellate interaction.

Porin isoform 2 has a different localization in Drosophila melanogaster ovaries than porin 1.

Eukaryotic porins or VDACs are a class of transmembrane proteins mainly localized in the outer mitochondrial membrane, whose function is to allow the diffusion of metabolites between the cytosol and the mitochondrion. In Drosophila melanogaster, as in other organisms, a small family of genes encoding porins has been discovered from the sequence of the genome. It is of general interest to understand whether these genes represent functional entities or not, and whether their product is associated to any particular tissue. In previous work we reported about the transcriptional and translational analysis of porin 1 and porin 2, and we proposed the specific presence of the latter in spermatozoa of the fly. In this paper we performed real time RT-PCR quantification of porin 1 and porin 2 transcripts in germ cells of the fly. It indicates that Porin 1 is abundantly expressed in both male and female tissues; Porin 2 instead, is very abundant in testis and it is present in ovaries as well, but in a small amount. The immuno-histological stain of ovaries shows that Porin isoform 1 is selectively targeted to follicular cells while Porin isoform 2 is present in mitochondria of the epithelial sheath cells of the ovariole. The implications of the subcellular distribution of these porin isoforms and the specific localization in germ tissues are discussed.

aubergine gene overexpression in somatic tissues of aubergine(sting) mutants interferes with the RNAi pathway of a yellow hairpin dsRNA in Drosophila melanogaster.

AUBERGINE (AUB) is a member of the PPD family of proteins. These proteins are implicated in RNA interference. In this article we demonstrate that the expression of the aub gene and protein increase in aub(sting) mutants. We used a genetic method to test whether aub(sting) overexpression could interfere with proper functioning of the process of RNA interference in somatic tissues of Drosophila melanogaster. This method is based on a transgenic line bearing a construct in which a fragment of the yellow (y) gene is cloned to form an inverted repeat (y-IR) under the control of the upstream activation sequence (UAS) of the yeast transcriptional activator GAL4. The UAS-y-IR transgene and the Act5C-GAL4 driver were brought together on chromosome 3 via recombination. In the resulting strain (Act5C-y-IR), transcriptional activation by GAL4 constitutively produces a dsRNA hairpin bearing cognate sequences to the yellow gene causing continuing degradation of y mRNA resulting in yellow(1) (y(1)) phenocopies. In this genetic background, the mutation of any factor involved in RNAi should repress degradation of y mRNA, restoring the wild-type phenotype. We employed this genetic approach to show that an increased amount of AUBERGINE interferes with the regular functioning of the somatic RNAi pathway.

The voltage dependent anion selective channel family in Drosophila melanogaster.

VDACs (voltage-dependent anion-selective channels) or mitochondrial PORINS are transmembrane proteins forming pores in the outer membrane. In eukaryotic genomes multiple genes coding for VDAC homologues have been discovered, but the functional meaning of this gene redundancy is unknown. In Drosophila melanogasterthree additional genes homologous to the gene porin (CG6647) have been found. As in other occurences, the presence of a gene revealed by genome analysis raises the questions: are these genes really expressed? What are the molecular features of the putative proteins, if they are expressed? Where and when in the organism are they expressed? Consequently have they any specific activity justifying the presence of more isoforms in the organism? To answer to these questions we have produced antibodies against the recombinant proteins corresponding to the whole (VDAC1 and 2) or to substantial portions (VDAC3 and 4) of the known or predicted proteins. Immunohistological and transcriptional analysis has been performed, showing that VDAC2 and 3 are expressed, while VDAC4 was not detected. Structural predictions of VDAC3 are consistent with the presence of additional alpha-helices at the N-terminus of the protein.

Expression and localization in spermatozoa of the mitochondrial porin isoform 2 in Drosophila melanogaster.

Mitochondrial porins or VDACs (voltage-dependent anion-selective channels) are transmembrane pore-forming proteins. In eukaryotic genomes multiple genes coding for VDAC homologues have been discovered, but their function remains unknown. In Drosophila melanogaster three additional genes homologous to the gene porin have been found. In a previous report we have expressed in vitro Porin 2 (gene GC17137) and we have found that the reconstituted protein shows pore-forming activity but it is cation-selective and poorly dependent from voltage. In this work we have characterized the expression pattern of Porin 2. Amplification upon germinal and somatic or stage specific mRNA showed that the highest transcription level of Porin 2 is in testis. Western blot analysis performed with antibodies raised against the recombinant Porin 2 confirmed a high level of expression in the fly spermatozoa. Immuno-histochemical studies indicate that Porin 2 is selectively present in spermatozoa tail, where the mitochondria are located, but not in spermatocytes. A lethal mutant of D. melanogaster carrying a P-element in the first intron of the porin (Porin 1) gene hinders the expression of both Porin 1 and 2. Our results suggest that Porin 2 is truly expressed and that it is required for functional germinal tissues.