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As presented in this report numerous fossils like forms resembling a variety of marine arthropodsincluding crustaceans, sea spiders, scorpions, arachnids, nematodes, annelids, tube worms, sea snakes,Kimberlla, Namacalathus, Lophotrochozoa, armored trilobites and millipedes have been found in GaleCrater (on Sols 302, 553, 753, 781, 809, 869, 880, 905, 1032), and (annelids, tube worms, crustaceans) inMeridiani Planum both of which have hosted rivers, lakes, and inland seas. Similar specimens are mixedwithin a variety of divergent fossil-like forms and are also found on distant sediment and mud stone. Allspecimens are distinct from underlying substrate and there are no obvious patterns or repetitions typicallyproduced by erosion or weathering. Although without extraction and direct examination it is impossibleto precisely determine the identity of all these specimens, the same problems bedevil identification ofBurgess Shale fossils some of which are presented in this report for comparative analysis. The discoveriespresented here and in other reports supports the theory that metazoans and other marine organismsevolved in the lakes, oceans and inland seas of Mars

An array of formations resembling the fossilized remains of Ediacaran and Cambrian fauna and other marine organisms have been observed embedded atopsediments in the dried lake beds of Gale Crater, Mars. Specimens similar and diverse in morphology have been found together and upon adjacent and nearbyrocks and mudstone. These include forms morphologically similar to polychaete and segmented annelids, tube worms, "Kimberella,” crustaceans, lobopods,chelicerates, Haplophrentis carinatus, and the “ice-cream-cone-shaped” “Namacalathus” and “Lophophorates” and other biomineralized metazoans. Allspecimens may have dwelled in a large body of water and fossilized/mineralized following the rapid receding of these waters. Statistical quantitative micro- andmacro- morphological comparisons with analog organisms from Earth support the fossil-hypothesis. It is not likely so many similar and diverse specimens, side byside, oriented differently, some on top of each other, were fashioned via abiogenic forces such as wind, mineralization, crystallization, dried mud, or water-erosionscenarios as there are no terrestrial abiogenic analogs. Interplanetary transfer of life may explain the parallels with Earth. Collectively these putative fossils mayrepresent the equivalent of a “Cambrian Explosion” and the remnants of Martian organisms that long ago flourished in the lakes and inland seas of Gale Crater (PDF) Fossils on Mars: A "Cambrian Explosion" and "Burgess Shale" in the Lake Beds of Gale Crater?. Available from: https://www.researchgate.net/publication/365926923_Fossils_on_Mars_A_Cambrian_Explosion_and_Burgess_Shale_in_the_Lake_Beds_of_Gale_Crater

What is life and how could it originate? This question lies at the heart of understanding the cell as the smallest living unit. Although we are witnessing a golden age of life sciences, we are ironically still far from giving a convincing answer to this question. With the aim to examines potential source of energy available to protocells on early Earth and/or elsewhere and mechanisms by which the energy could be used to drive polymer synthesis, experiments aimed at revealing the ability of meteorites and some terrestrial rocks to perform catalytic reactions operative in present-day life have been performed. 1. Indroduction The aim of this work is to present and discuss results of recent and ongoing wet-lab experiments supporting Multiple Root Genesis Hypothesis (MuGeRo) already proposed elsewhere [1] seeking approaches surrounding the mysterious primeval steps of life emergence on Earth or on planets around distant stars beyond our Galaxy. This is an additional hypothesis to that microbial or early forms of life were already present in our solar system at the time of our Earth’s formation so that we can reconsider that panspermia and abiogenesis are not rival theories but complementary theories [2]. Life on Earth is carbon-based, uses water as solvent, and photosynthesis and chemosynthesis as way to obtain energy. Following a bottom-up approach, I utilized as a model for the emergence of earliest life on Earth, the self-organizing M4 material that I’m producing from L6 condrites and some terrestrial rocks and minerals (olivine and magnetite ) [3]. 2. Figures 3. Summary and Conclusions Searching for the very first instants of life on Earth, with several hypotheses in play [4], the challenge has been to replicate the conditions that could have allowed the emergence of early life to emerge. I developed a new approach to stimulate physicochemical processes that may have led to the emergence of the first life forms from inanimate matter on Earth or Earth-like planets via photo-metabolic pathways. My studies does not starts from ground zero, but provide evidence of non-enzymatic catalysis that modifies sugars, aminoacids, urea and other molecules produced in the prebiotic environments on the planets or satellites of our solar system by investigating the effect of physico-chemical stress on the formation of the metal-organic material M4 [3]. References [1] del Gaudio, R.: Understanding the key requirement and the conditions that sparked life on Earth and beyond:clues and new knowledges supporting MuGeRo hypothesis., Europlanet Science Congress 2020, online, https://doi.org/10.5194/epsc2020-167, 2020. [2] del Gaudio, R.: Transition from Non-living to living Matter: can integration of MuGeRo hypothesis and synthetic prebiotic biology laboratory approach shed light on the origin of Life? , Europlanet Science Congress 2021, online, 13–24 Sep 2021, EPSC2021-347, https://doi.org/10.5194/epsc2021-347, 2021. [3] Geraci, G., D’Argenio, B., del Gaudio R. Patent US9328337 B2, granted, 2016. [4] Bartlett, S. and Wong, L., Defining Lyfe in the Universe: From Three Privileged Functions to Four Pillars Life 2020, 10(4), 42; https://doi.org/10.3390/life10040042

Mars has been subject to repeated waxing and waning episodes of extreme chaotic obliquity (axial tilting)for at least four billion years. Obliquity is currently at 25.19 degrees and has exceeded 80 ◦ . Each timeobliquity exceeds 40 ◦ Martian atmospheric pressures and global temperatures increase causing themelting of glaciers and permafrost and subsurface ice, and resulting in oceans, lakes and rivers of waterflooding across the surface then stabilizing and enduring for hundreds of thousands of years or longer.There is evidence that within these seas evolved stromatolite constructing cyanobacteria, green algae,acritarchs,foraminifera,seaweed, and marine metazoan invertebrates including sponges, tube worms,crustaceans, reef-building corals, and those resembling Kimberella , Namacalathus and Lophophorates; almost all of which (with the exception of algae, fungi and lichens) may have become extinct. The lastepisode of extreme obliquity may have begun over a million years in the past and endured until 110,000years ago. Subsequently, as axial tilting declined, the waters of Mars seeped back beneath the surfaceforming vast aquifers and glacial deposits of water-ice and the remainder froze at the poles and atop dustylayers of icy-sediment: the remnants of previous obliquity-driven freeze-thaw cycles that may havecaused life to evolve and oceans and lakes to repeatedly form, stabilize, endure then freeze.

Mars has been subject to repeated waxing and waning episodes of extreme chaotic obliquity (axial tilting) for at least four billion years. Obliquity is currently at 25.19 degrees and has exceeded 80◦. Each time obliquity exceeds 40◦ Martian atmospheric pressures and global temperatures increase causing the melting of glaciers and permafrost and subsurface ice, and resulting in oceans, lakes and rivers of water flooding across the surface then stabilizing and enduring for hundreds of thousands of years or longer. There is evidence that within these seas evolved stromatolite constructing cyanobacteria, green algae, acritarchs, foraminifera, seaweed, and marine metazoan invertebrates including sponges, tube worms, crustaceans, reef-building corals, bivalves, and those resembling Kimberella, Namacalathus and Lophophorates; almost all of which (with the exception of algae, fungi and lichens) may have become extinct. The last episode of extreme obliquity may have begun over a million years in the past and endured until 110,000 years ago. Subsequently, as axial tilting declined, the waters of Mars seeped back beneath the surface forming vast aquifers and glacial deposits of water-ice and the remainder froze at the poles and atop dusty layers of icy-sediment: the remnants of previous obliquity-driven freeze-thaw cycles that may have caused life to evolve and oceans and lakes to repeatedly form, stabilize, endure then freeze.

The Scientific Degree Plan (PLS) is Italian intervention plan focused at orientation, teaching and training actions that are directed at high school students, universities, and teachers. The PLS is organized into National Disciplinary Projects: Biology and Biotechnology, Chemistry, Physics, Geology, Computer Science, Mathematics, Natural and Environmental Sciences, Materials Science, Statistics. At the Federico II University of Naples, 8 of the 9 national projects are active (PLS-UNINA) and every year, as part of the activities of each of them, many activities are launched for students and teachers from Campania and beyond, among them the second edition of PLS Virtual Summer School for Students (PVS3). The PVS3 will be directed at students attending the last two years of secondary schools who wish to deepen their knowledge of the subjects of the scientific area, going beyond the aspects usually dealt with in the course of school activities. The activities, including highly interdisciplinary virtual lessons and laboratories, will take place from 6th to 10th September 2021 on the Microsoft Teams. A positive side of this pandemic is that it has exposed people to new technologies developed over the past decade. Since March 2020, university professors teaching science subjects requiring lab work, such as biology, had to become particularly creative and had to adapt their courses and structures to the online synchronous format. Due to the beginning of the COVID-19 pandemic- which incidentally coincided with the beginning of my molecular biology academic course -it was impossible to carry out any activity in person, because all the teachers and all the students could not access the classrooms and the educational laboratories. To give a sense of continuity and normality to my students, I started documenting and learning myself, choosing and using some new technologies, in order to provide to my students a suitable alternative to face-to-face lab activities. The first solutions adopted were Science Education Resources at “Jove.com” (ICERI, 2020) and later “Labster.com”, an application that offers virtual laboratory simulations whose use license was offered free of charge for over six months. With Labster.com, in a risk-free simulated learning environment, the students performed experiments interacting with the laboratory equipment, and repeated the actvities as many times as they wanted to improve their learning. Based on this experience, from now on I will divide PLS summer school students into smaller groups since working groups help build friendships which is a component of the traditional lab experience. Moreover, the English language of the virtual laboratory simulations will be an additional value and a different learning context useful to potentiate the learning also of foreign language and an inclusive experience in learning key science concepts. In conclusion, although it is difficult to replace the "hands-on" experience of a real laboratory environment, the very positive in silico experience with my students will be adapted in the near future to PVS3 2021 edition (www.pls.unina.it), by overturning the theoretical lessons into synchronous virtual simulations that shall be the basis for new creative activities that students will be able to carry out in the future with greater mastery and safety in a real laboratory with teachers of the high school science laboratories. Protocols for new activities will be one of the expected results.

Understanding the origin of life (OoL) remains one of the most challenging challenges today with many scientific questions still unanswered. The complexity of the problem of the origin of life has spawned a large number of possible evolutionary scenarios. Their number, however, can be dramatically reduced by the simultaneous consideration of various bioenergetic, physical, and geological constraints. It is a widely accepted hypothesis that life originated from inanimate matter, being somehow a synthetic product of molecular organic aggregates, and as such the result of a kind of synthetic prebiotic biology [1]. In a multiverse perspective, supported by experimental data, I have already proposed the MuGeRo hypothesis in which both cosmic biotic “infection” and abiotic geochemistry contributing to trigger the emergence of life on Earth and beyond [2]. I proposed that spontaneous reactions demonstrated to occur in the environment supposed to exist in the young Earth might have provided the starting organic molecules on which peculiar component of rock (also meteorites) might have act as catalysts of molecular complexity initiating early life processes that, as has been proposed from other Authors are spontaneously and inevitable. In recent decades, the new scientific discipline of synthetic biology has set ambitious goals by pursuing the full design and manufacture of complete genetic circuits, or entire genomes, or even the whole minimal life in the laboratory. I hold that synthetic biology could also throw new and interesting perspectives on the topic of the origins of life and that, moreover, it could help challenge the most commonly accepted definitions of life, thus changing the way we might rethink life and its origins. Infact, following the bottom-up approach I reported experimental evidence that both extraterrestrial and terrestrial minerals and rocks containing iron produces the self-organizing M4 material (Fig.1 a-h) endowed of amino sugar-driven prebiotic synthetic processes supporting abiogenesis [2,3]. This is in addition to the hypothesis that early forms of life were already present in our solar system at the time of Earth formation [4]. Following the bottom-up approach and using the self-organized M4 material [5] obtained from meteorites and terrestrial rocks and minerals as a model for the emergence and early evolution of life on Earth, this work proposes an evolutionary scenario that satisfies the known constraints by proposing that life on Earth emerged, fueled by solar radiation, from non-enzymatic, photochemical and self-sustaining reactions, surviving through some form of redox chemistry. The M4 material autopoietically self-assembled on hypersthenic chondrite or siderite and on fragments of same terrestrial rocks containing iron could have contributed to the chemical evolution of biomolecules by own catalytic activities that modify sugars and amino acids produced in the prebiotic environments. Here I’m proposing that these non-enzymatic, self-sustaining, photochemical reactions might be a primitive examples of reaction network supporting abiogenic development of early life on Earth and/or beyond. Indeed, these non-enzymatic reactions appear efficient enough to provide the variety and abundance of biologically favorable molecules from which Darwinian selection operating at molecular level may have seeded protometabolic reaction in the pre-biotic contexts. The aim of this work here is to present and discuss the new results of my ongoing experiments compared to those obtained in the past and more recently. Indeed, the results that I obtained so far do not prove or exclude the possibility that M4 materials could be a kind of non-conventional form of pre-terrestrial life or earthly early life but demonstrate that they are certainly the results of reactions depending on mineral or rock (meteorites too) that might be considered prebiotic catalysis probably responsible for the emergence of a large pre-biotic pool of biologically favorable molecules. Furthermore, I proposed that the photo-geochemical abiotic reactions could have given rise to a variety of independent biogenic events leading to a plurality of Darwinian Initial Life Forms (IDAs) from which the earliest forms of terrestrial life emerged giving rise to more than one LUCA (Last Universal Common Ancestor). References 1.Longo, A. and Damer, B: Factoring origin of Life Hypotheses into the Search for Life in the Solar System and Beyond., Life, 2020, 10, 5:52, https://doi.org/10.3390/life10050052 2.del Gaudio, R.: Understanding the key requirement and the conditions that sparked life on Earth and beyond:clues and new knowledges supporting MuGeRo hypothesis., Europlanet Science Congress 2020, online, https://doi.org/10.5194/epsc2020-167, 2020. 3.del Gaudio, R.,: Transition from Non-Living to Living Matter: filling the gap with a laboratory-based approach according to a Land-based birthplace and Iron catalysis hypotheses at the origin of life on the early Earth and beyond., Europlanet Science Congress-DPS Joint Meeting 2019, Vol. 13, EPSC-DPS2019-80-1 EPSC-DPS2019-80-1. 4. Geraci, G., del Gaudio, R., D'Argenio, B., Rendiconti Lincei, Vol. 12, pp. 51-68, 2001. 5. Geraci, G., D’Argenio, B., del Gaudio R. Patent US9328337 B2, granted, 2016.

More than twenty years ago the reverse vaccinology paradigm came to light trying to design new vaccines based on the analysis of genomic information in order to select those pathogen peptides able to trigger an immune response. In this context, focusing on the proteome of Trypanosoma cruzi, we investigated the link between the probabilities for pathogen peptides to be presented on a cell surface and their distance from human self. We found a reasonable but, as far as we know, undiscovered property: the farther the distance between a peptide and the human-self the higher the probability for that peptide to be presented on a cell surface. We also found that the most distant peptides from human self bind, on average, a broader collection of HLAs than expected, implying a potential immunological role in a large portion of individuals. Finally, introducing a novel quantitative indicator for a peptide to measure its potential immunological role, we proposed a pool of peptides that could be potential epitopes and that can be suitable for experimental testing. The software to compute peptide classes according to the distance from human self is free available at http://www.iasi.cnr.it/~dsantoni/nullomers.

The aim of this work is to present and discuss the results of recent and ongoing wet and in silico laboratory experiments supporting Multiple Root Genesis (MuGeRo) hypothesis already proposed in search of approaches surrounding the mysterious primeval steps of life emergence on Earth and elsewhere in the Universe. There are and have been reported many theories on how the very first life began on Earth, and also on how life itself evolved, some even say that life might have arisen on Earth more than once, and since it is hard to prove or disprove them there is no fully accepted theory. Approaching the primordial step of life emerging and possibile evolutionary scenarios from nonliving-matter towards answering the fundamental question about when, where and how life was born on Earth I’ll discuss essential requirement for the first emergence of life on Earth and Earth-like planets. As examples of proto-metabolic reactions occurred in a pre-biotic hydrogel context and as a model for the emergence and early evolution of life on Earth, I'm proposing the self-organizing M4 materials, having a complex chemistry, that I’ve obtained from both some meteorites and terrestrial rocks and minerals. Moreover, they are certainly the result of several coordinated activities and only some of them can be attributed to the meteorite or terrestrial rock components. The results so far obtained could point a way towards understanding how Earth kick-started metabolism emerged on landmass that arose from Archean oceans rather than in the depths near a deep sea hydrothermal vent. This work puts forward also an evolutionary scenario that satisfies the known constraints by proposing that life on Earth emerged, powered by solar radiation because the M4 catalytic activities might be a primitive form of reaction network supporting abiogenic development of life on Earth or elsewhere in the Universe. This in addition the idea that microbial or virus or early forms of life were already present in our solar system at the time of Earth’s formation so that panspermia or abiogenesis results are not rival but two complementary theories. Concerning the role that minerals may have played in organizing organic matter rising towards life from supports to scaffolds or energy sources or molecular-level information. There are some hints concerning the role that some minerals may have played in organizing matter in its rise towards life, from simple supports to scaffolds, from energy sources to even maybe providers of molecular-level information. The work that remains to be done is huge is there an "interfacial" path from the disorganized complexity of prebiotic and "primitive-jam" to the functional systems that have been the precursors of life? To answer this question I hope soon to analyze with systems chemists the results already obtained and they will be obtained from new weighted experiments designed in scientific cooperation.

The story of life’s origins is one of the deep, intriguing, and unsolved mysteries of our Science. With several hypotheses in play, the challenge is to replicate the conditions that allowed life to emerge. I developed a new approach to stimulate the energetic processes that may have led to the emergence of proto-metabolic pathway on Earth or earth-like planet. Current status of my laboratory experiments in hydrogel environments, utilizing the self-organizing M4 material obtained from meteorites and terrestrial rocks and minerals as model for the emergence and early evolution of life on Earth, will be presented and discussed. My findings give support to MuGeRo hypothesis that I have already proposed are in line with recent paper showing that mineral containing Iron and Nickel that would have been common on the early Earth and those reported by other authors on a land-based birthplace supporting the theory that life could have emerged on Earth from inanimate matter via mineral/rocks-organic interfacial processes.

In mammals, the RXFP3 is the cognate receptor of the relaxin-3 peptide (RLN3). In teleosts, many different orthologue genes for RXFP3 are present. In particular, two paralogue genes, rxfp3-2a and rxfp3-2b, likely encode the receptors for the Rln3a peptide. The transcription of these two rxfp3 genes is differentially regulated early during zebrafish embryogenesis. Indeed, reverse transcription–polymerase chain reaction analyses show that the rxfp3-2b transcript is always present during embryo development, while the rxfp3-2a transcript is detectable only at larval stage. By in situ hybridization experiments on embryos and larvae, the rxfp3-2b transcript was revealed in the brain and in the retinal ganglion cell layer and thymus. Particularly in the brain, many territories are involved in the rxfp3-2b expression, among them the optic tectum, thalamus, preoptic area, different nerve nuclei, habenula and pineal gland. The RXFP3 spatiotemporal expression pattern appears to be conserved between Danio rerio and mammals, as also previously showed for the corresponding ligand, the RLN3. Interestingly, the brain areas expressing the rxfp3-2b receptor gene are involved in the visual system, emotional behaviors and circadian rhythm and could be functionally related to the neurotransmitter Rln3a-expressing territories.

RXFP2 is one of the 4 receptors for relaxin insulin-like peptides, in particular it binds with high affinity the INSL3 peptide. INSL3/RXFP2 pair is essential for testicular descent during placental mammalian development. The evolutionary history of this ligand/receptor pair has received much attention, since its function in vertebrate species lacking testicular descent, such as the fishes, remains elusive. Herein, we analyzed the expression pattern of three rxfp2 homologue genes in zebrafish embryonic development. For all the three rxfp2 genes (rxfp2a, rxfp2b, and rxfp2-like) we showed the presence of maternally derived transcripts. Later in the development, rxfp2a is only expressed at larval stage, whereas rxfp2b is expressed in all the analyzed stage with highest level in the larvae. The rxfp2-like gene is expressed in all the analyzed stage with a transcript level that increased starting at early pharyngula stage. The spatial localization analysis of rxfp2-like gene showed that it is expressed in many cell clusters in the developing brain. In addition, other rxfp2-like-expressing cells were identified in the retina and oral epithelium. This analysis provides new insights to elucidate the evolution of rxfp2 genes in vertebrate lineage and lays the foundations to study their role in vertebrate embryonic development.

Relaxin peptides exert different functions in reproduction and neuroendocrine processes via interaction with two evolutionarily unrelated groups of receptors: RXFP1 and RXFP2 on one hand, RXFP3 and RXFP4 on the other hand. Evolution of receptor genes after splitting of tetrapods and teleost lineage led to a different retention rate between mammals and fish, with the latter having more gene copies compared to the former. In order to improve our knowledge on the evolution of the relaxin ligands/receptors system and have insights on their function in early stages of life, in the present paper we analyzed the expression pattern of five zebrafish RXFP3 homologue genes during embryonic development. In our analysis, we show that only two of the five genes are expressed during embryogenesis and that their transcripts are present in all the developmental stages. Spatial localization analysis of these transcripts revealed that the gene expression is restricted in specific territories starting from early pharyngula stage. Both genes are expressed in the brain but in different cell clusters and in extra-neural territories, one gene in the interrenal gland and the other in the pancreas. These two genes share expression territories with the homologue mammalian counterpart, highlighting a general conservation of gene expression regulatory processes and their putative function during evolution that are established early in vertebrate embryogenesis.

We report the gene characterization, the cDNA cloning and the temporal and spatial expression pattern of the relaxin receptor rxfp1 gene in the zebrafish Danio rerio. The zebrafish rxfp1 gene has the same syntenic genomic organization, and a similar exon-intron structure to the homologue human gene. Furthermore, the deduced Rxfp1 protein sequence shows a high degree of amino acid similarity when compared with the human protein and the conservation of all amino acid identity necessary for the binding with relaxin. Our results show that rxfp1 gene is active either during embryogenesis or in the adult organism, showing a wide expression pattern. Moreover, we provide the first description of rxfp1 spatial expression pattern during embryo development, showing that the transcript is already present at the early developmental stage and is distributed in all of the embryonic cells until somitogenesis. Starting at the pharyngula stage the gene expression becomes mainly restricted in the brain territories. In fact, at the larval stage, the transcript is detectable in the epiphysis, postoptic region, posterior tuberculum, hypothalamus, optic tectum, tegmentum/pons, medulla and also in the structure of a peripheral nervous system, the terminal nerve. The rxfp1 expression pattern in Danio rerio embryos is very similar to that reported in the adult mammalian brain, suggesting a pivotal role of this receptor in the neurophysiology processes already at very early developmental stages.

We show that ptma, a single copy gene found in all organisms investigated so far, is duplicated in zebrafish. The two genes, ptmaa and ptmab, are individually controlled as indicated by their different expression patterns during embryonic development. Only the ptmab transcript is observed at 4 and 8 hpf of development in all embryonic cells, whereas both genes are expressed at later stages as revealed by in situ hybridization studies. In most cases, the two genes are expressed in the same territories, but only the ptmaa transcript was found in the trigeminal ganglion and in endodermal pouches. In the eye, at 72 hpf, the ptmaa and ptmab transcripts were found in amacrine cells, whereas only the ptmab transcript appeared in horizontal cells. The existence of two prothymosin genes indicates that their function in cell proliferation and differentiation is more complex in fishes than in mammals. Developmental Dynamics 237:1112–1118, 2008. © 2008 Wiley-Liss, Inc.

RXFP2 is one of the 4 receptors for relaxin insulin-like peptides, in particular it binds with high affinity the INSL3 peptide. INSL3/RXFP2 pair is essential for testicular descent during placental mammalian development. The evolutionary history of this ligand/receptor pair has received much attention, since its function in vertebrate species lacking testicular descent, such as the fishes, remains elusive. Herein, we analyzed the expression pattern of three rxfp2 homologue genes in zebrafish embryonic development. For all the three rxfp2 genes (rxfp2a, rxfp2b, and rxfp2-like) we showed the presence of maternally derived transcripts. Later in the development, rxfp2a is only expressed at larval stage, whereas rxfp2b is expressed in all the analyzed stage with highest level in the larvae. The rxfp2-like gene is expressed in all the analyzed stage with a transcript level that increased starting at early pharyngula stage. The spatial localization analysis of rxfp2-like gene showed that it is expressed in many cell clusters in the developing brain. In addition, other rxfp2-like-expressing cells were identified in the retina and oral epithelium. This analysis provides new insights to elucidate the evolution of rxfp2 genes in vertebrate lineage and lays the foundations to study their role in vertebrate embryonic development.

Relaxin peptides exert different functions in reproduction and neuroendocrine processes via interaction with two evolutionarily unrelated groups of receptors: RXFP1 and RXFP2 on one hand, RXFP3 and RXFP4 on the other hand. Evolution of receptor genes after splitting of tetrapods and teleost lineage led to a different retention rate between mammals and fish, with the latter having more gene copies compared to the former. In order to improve our knowledge on the evolution of the relaxin ligands/receptors system and have insights on their function in early stages of life, in the present paper we analyzed the expression pattern of five zebrafish RXFP3 homologue genes during embryonic development. In our analysis, we show that only two of the five genes are expressed during embryogenesis and that their transcripts are present in all the developmental stages. Spatial localization analysis of these transcripts revealed that the gene expression is restricted in specific territories starting from early pharyngula stage. Both genes are expressed in the brain but in different cell clusters and in extra-neural territories, one gene in the interrenal gland and the other in the pancreas. These two genes share expression territories with the homologue mammalian counterpart, highlighting a general conservation of gene expression regulatory processes and their putative function during evolution that are established early in vertebrate embryogenesis.

The aim of this study is to assess, by RT-PCR, in situ hybridization, electron microscopy, and immunohistochemistry, the site/s of vitellogenin (VTG) synthesis in the mussel Mytilus galloprovincialis. Our investigations demonstrate that, among the analyzed tissues, the synthesis of VTG occurs only in the female gonad, that is, within the oocyte and follicle and connective cells. Such a synthesis is just evident in early vitellogenic oocytes, whose cytoplasm is characterized by numerous RER cisternae and an extended Golgi complex surrounded by nascent yolk platelets. The synthesis of VTG goes on in vitellogenic oocytes assuming a pear form, and progressively reduces once the oocyte shows the pear or polygonal form, typical of those oocytes that have concluded the growth. The expression of VTG occurs also within follicle (auxiliary) and connective cells. In particular, it is noteworthy that follicle cells are characterized by numerous RER cisternae and an active Golgi complex surrounded by numerous vesicles and vacuoles containing electron dense material. The same material is also present along their plasma membrane, within the intercellular space between oocyte and follicle cells, and finally within invaginations of the oocyte surface, thus suggesting a VTG transfer to the oocyte via endocytosis. Differently, no VTG synthesis was observed within digestive gland. All together the findings here reported strongly suggest that in M. galloprovincialis, inside the gonad, the VTG synthesis occurs in the oocyte (autosynthesis) and in the follicle and adipogranular cells (heterosynthesis). J. Cell. Physiol. 228: 547–555, 2013. © 2012 Wiley Periodicals, Inc.

We report the identification, the cDNA cloning, the temporal and spatial expression pattern analysis of the rln gene in the Zebrafish Danio rerio. The deduced Rln B and A domains show different evolutionary conservation. Rln B domain shows higher similarity when compared to zebrafish and human RLN3 B domain than human RLN1 and RLN2 B domain. Differently, the zebrafish Rln A domain shows relatively low amino acid sequence similarity when compared with the same sequences. The rln gene is transcribed both during embryogenesis and in adult organism, where higher transcript level has been particularly evidenced in the brain. Moreover, we provide the first description of rln spatial expression pattern during embryonic development. In particular, we show restricted transcript localization starting at the pharyngula stage in olfactory placode, branchial arch region, and in a cell cluster near to otic vesicle. In larval stage, new transcription territories have been detected in both neural and non-neural regions. In particular, in the brain, rln expression has been revealed in telencephalic region around anterior commissure, in the preoptic area, and in restricted rombencephalic cell clusters. Expression of rln gene in extra-neural territories has been detected in the pancreatic and thyroid gland regions. Danio rerio rln expression pattern analysis reveals shared features with the mammalian RLN gene, particularly in the brain, where it might have a role in the neurophysiological processes. In addition, expression in the thyroid and pancreas region suggests a function as a paracrine and endocrine hormone.

Protein-inspired biomaterials have gained great interest as an alternative to synthetic polymers, in particular, for their potential use as biomedical devices. The potential inspiring models are mainly proteins able to confer mechanical properties to tissues and organs, such as elasticity (elastin, resilin, spider silk) and strength (collagen, silk). The proper combination of repetitive sequences, each of them derived from different proteins, represents a useful tool for obtaining biomaterials with tailored mechanical properties and biological functions. In this report we describe the design, the production, and the preliminary characterization of a chimeric polypeptide, based on sequences derived from the highly resilient proteins resilin and elastin and from collagen-like sequences. The results show that the obtained chimeric recombinant material exhibits promising self-assembling properties. Young's modulus of the fibers was determined by AFM image analysis and lies in the range of 0.1-3 MPa in agreement with the expectations for elastin-like and resilin-like materials.

Innexins are a family of transmembrane proteins involved in the formation of gap junctions, specific intercellular channels, in invertebrates. Analyses of the entire innexin family during Drosophila melanogaster embryonic development shows the occurrence of complex and specific patterns of expression of the different genes. Innexins inx-2 and inx-7, in general, do not appear to exhibit extensive co-expression in different D. melanogaster cellular compartments. We propose here a new and robust mechanism, based on our analysis of the genomic organization of inx-2 and inx-7, that structurally justifies the reciprocal expression of genes. © 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

DNA methylation plays a central role in the control of gene expression during development and cell differentiation, thus DNA methylation and demethylation processes are expected to be strictly regulated during these events. We have explored the expression levels of the genes encoding DNA methylases, methyl-CpG binding proteins and demethylases during in vitro differentiation of human carcinoma colon cells (CaCO-2) used as a model system. The results show that the global DNA methylation pattern remains constant during CaCO-2 cells differentiation indicating that required genome methylation pattern in cell differentiation was already established in the seeded cells. On the contrary, the timing of expression of several of the explored genes is tightly regulated, suggesting they are involved in the regulation of the differentiation program. In particular, the timing of expression of DNMT3b and of MBD2b and 5-MCDG shows two peaks not observed in the time courses of the expression of other genes belonging to the same families. These events, not dependent on the cell cycle synchronization, have apparently no significant impact on the overall methylation status of the genome.

Innexins are a family of membrane proteins involved in the formation of gap junctions in invertebrates. They have been found to participate in several aspects of cell differentiation and in embryonic patterning through the formation of specific intercellular communication channels. We present here data showing that the recently identified innexin of the marine worm Chaetopterus variopedatus is expressed only in particular cells of the early stage, demonstrating cell specificity of innexin expression also in polychaete annelids. Phylogenetic analysis of all known innexins results in a phylogenetic tree clearly distinguishing insect, nematode, and other invertebrate innexins. Comparative analysis of proteins and known related genes shows that the apparent similarity of protein composition, overall structural organization, and specificity of cellular expression, typical of innexins of all studied organisms, correspond to highly heterogeneous gene structures even for genes that are in close contiguity on the same chromosome. A possible evolutionary motive producing this situation is discussed.

A novel member of the innexin family (cv-inx) has been isolated from the annelid polychaete worm Chaetopterus variopedatus using a PCR approach on genomic DNA and sequence analysis on genomic DNA clones. The gene is present in a HindIII-HindIII segment of 2250 bp containing an uninterrupted open reading frame of 1196 bp encoding a protein of 399 amino acids. The predicted protein shows the typical structural features of innexins and consensus sites for phosphorylation. Analyses on genomic DNA demonstrate that cv-inx is a single copy gene with no introns in the coding region, exactly corresponding to the cDNA sequence. The gene expression is regulated during development as shown by Northern blots analyses of the RNA and by immunoreaction with antibodies against the protein at several embryonic stages. The finding of an innexin in the phylum Annelida, outside of the Ecdysozoa clade, and its peculiar gene structure suggest the necessity to reconsider the current hypothesis on the origin and evolution of gap junctional proteins.

The turnover and localization of the enzyme DNA (cytosine-5) methyltransferase (Dnmt1) were studied during Paracentrotus lividus sea urchin embryo development using antibody preparations against the NH2 and COOH-terminal regions of the molecule. The antibodies reveal, by Western blots and whole-mount analyses, that the enzyme is differently required during embryonic development. The changeover point is at blastula stage, where a proteolytic mechanism hydrolyses the enzyme present in all embryonic cells by removing a peptide of about 45 kDa from the amino terminal region of the 190 kDa enzyme initially synthesized on maternal transcripts. The resulting 145 kDa enzyme shows modified catalytic properties, different antibody reactivity and is rapidly destroyed in the few hours before gastrulation. At more advanced stages of development the enzyme is newly synthesized but only in particular cell types, among which neurons. The data show that Dnmt1 is removed from embryonic cells before gastrulation to be synthesized again at different levels in different cell types, indicating that the concentration of Dnmt1 is critical for the various differentiated cells of the developing sea urchin embryo. © 2001 Elsevier Science B.V. All rights reserved.

Crystals, rocks and mineral ores of different origins contain viable microbial life that appears actively swimming under the microscope when the sample is properly fragmented and suspended in a nutrient medium. This form of life in rocks is unaffected by time, since microbes have been found in samples of all geological ages, from about 2.8 Ga to recent rocks, and by pressure and temperature, since it is present in metamorphic and in igneous rocks. From the tests performed, among which those to secure from sample pollution, it emerges that this form of life is not destroyed, as indeed expected, when the rock is heated above 500 °C in a kiln. However, all cloned microbes are sensitive to growth inhibition by specific antibiotics. A similar search, for the presence of microbes in meteorites, shows that also these materials are rich in microorganisms, indicating that these already existed in early Earth formation stages. Some different microbial species, derived from different samples of rocks and meteorites, have been cultured, cloned and classified by 16S rDNA typing and found to be not essentially different from present day organisms. An interesting consequence of these findings, among others, is the support to the hypothesis that life came from outside Earth with the additional indication that it was already present in those materials that accreted to form the solar planetary system.

The enzyme S-adenosylmethionine-DNA (cytosine-5)-methyltransferase has been identified, first time for invertebrates, in embryos of the marine polychaete annelid worm Chaetopterus variopedatus. The molecule has been isolated from embryos at 15 h of development. It is a single peptide of about 200 kDa molecular weight, cross-reacting with antibodies against sea urchin DNA methyltransferase. The enzymatic properties of the molecule are similar to those of Dnmt1 methyltransferases isolated from other organisms, but with the peculiarity to be unable to make 'de novo' methylation on double stranded DNA. Copyright (C) 1999 Federation of European Biochemical Societies.

Histone genes were identified and their nucleotide sequences were determined in the polychaete marine worm Chaetopterus variopedatus. The genes are organized in about 390 clusters of 7.3 kbp. Each cluster contains one copy of the five histone genes. The H1 histone gene present in the clusters is the first ever isolated in the phylum Annelida. The cluster has the unique peculiarity that all genes contain both the replication-dependent and the replication-independent 3' mRNA termination signals. Despite the differences in cluster organization and transcription polarity of the individual histone genes between C. variopedatus and Platynereis dumerilii, the other annelid in which histone genes have been studied, phylogenetic analysis of the encoded amino acid sequences clearly groups together those two organisms in a tree in which the other studied worms find closely related positions on the same evolutionary branch.

Hydrolysis by methylation-dependent restriction enzymes shows that the genomic DNA of the polychaete annelid worm Chaetopterus variopedatus is methylated. Electrophoretic analyses of the digestion products indicate that the degree of methylation is lower in adult tissues than in sperm and embryonic DNA. 5-Methylcytosine was identified by HPLC, absorption spectroscopy and mass spectrometry analyses of free bases obtained by acid hydrolysis of the DNA. An average value of 1.6% methylated cytosines was determined in sperm DNA. Partial methylation was also found in an actively expressed H1 histone gene. This is the first time that genomic DNA methylation is demonstrated to occur in a worm.

Abstract: In HIV-1 the nucleocapsid protein (NC) is essential in the virion assembly process. The discovery of substances able to interfere with the HIV-1 NC functions could be a starting point for the design of new drugs that might inhibit the HIV-1 viral infectivity. A single peptide sequence binding to the NC was isolated using a conformationally homogeneous phage displayed-peptide library. The minimal requirement for the NCp7/peptide interaction was the NCp7 region encompassing the second zinc finger.

CO binding kinetics to the homodimeric myoglobin (Mb) from Nassa mutabilis has been investigated between pH 1.9 and 7.0. Protonation of the proximal imidazole at low pH (less than or equal to 3.0) and the consequent cleavage of the HisF8NE2-Fe proximal bond brings about a approximately 20-fold increase of the second-order rate constant for CO binding. This process displays a pKa = 4.0 +/- 0.2, significantly higher than that observed in all other deoxygenated hemoproteins investigated up to now. Such a feature underlies a decreased energy for the HisF8NE2-Fe proximal bond in the unliganded form and it also appears supported by resonance Raman spectroscopy in the low frequency region of the Fe(II) deoxygenated hemoprotein. Further, the pH-rate profile of N. mutabilis Mb, like that of the homodimeric hemoglobin (Hb) from Scapharca inaequivalvis (Coletta, M., Boffi, A., Ascenzi, P., Brunori, M. and Chiancone, E. (1990) J. Biol. Chem. 265, 4828-4830), can be described only by assuming a concerted proton-linked transition with n = 1.8 +/- 0.1. Such a characteristic suggests, also on the basis of the amino acid sequence homology between N. mutabilis Mb and S. inaequivalvis Hb in the region forming the subunit interface, that the interaction mechanism is similar for the two homodimeric proteins, and drastically different Hb in the region forming the subunit interface, that the interaction mechanism is similar for the two homodimeric proteins, and drastically different from that operative in other hemoproteins.

Here I present activities of a Dissemination Science Project that will be realized in the framework of MEETmeTONIGHT 2021 Event, the annual initiative included in the circuit of the European Researchers' Night events promoted by the European Commission in all European countries. This project aims to create a unique opportunity to meet and to speak with researchers in order to understand how scientific research has an impact in everyday life of all of us. The activities that I’ll be proposed also aim to consolidate a bridge between the scientific community of Federico II Biology Department and the local high school students. To this aim, I’ll involve a selection of undergraduate students of the second academic year of biology degree in practical activities that will be included in their Thesis. In collaboration with my colleagues and the selected university students, I have conceived interdisciplinary experimental activities that will revolve around the theme of diet and epigenetics that we will propose during the event. We all know that a diet rich in fruit and vegetables is healthy for our everyday life, but it is becoming increasingly clear that everything we eat will impact our DNA. During the activity here proposed students will learn about the connection between nutrients in their diet and the stability of their genome. Moreover they will be able also to: -Describe nutrient compositions of healthy and unhealthy diets -Explain how genomic instability may lead to the development of diseases such as cancer -Summarize the impact of diet on genomic stability. Among the products of the innovative teaching activities there will be that of verifying how many of the visitors know which are the genetic factors that allow to inherit that phenotype and/or modify it, enriching the biodiversity of our planet. In addition, students will prepare popular posters and models to illustrate how our diet can influence the epigenetic changes in our genome that underlie various diseases, including cancer and obesity. A 3-D cut-and-paste model depicting how histone, acetyl and methyl molecules control access to DNA and affect gene expression, DNA or protein extraction from greens, gel electrophoresis, extraction and dosage of pigments will be among of the proposed and participated activities. Memory-themed games that will be used to involve also the young student by correlating epigenetic and diet. In conclusion, this year's novelty will be also the event that I'm organizing with virtual laboratory activities aimed also to High Schools teachers that should draw inspiration for an innovative teaching springed out from research on a captivating theme that fits in the interest that the plants are taking in the field of the green economy and healthy eating having significant implications for our long-term health and our life expectancy. I got a free trial from Labster.com, an application that offers virtual lab simulations. Labster, recreates the hands-on lab experience because students can run experiments and interact with lab equipment so that students aren't just watching a video of someone else performing the experiments, but taking part in it. Students will carry out laboratory simulations alone or in groups. For these activities one of the labster simulations I have chosen is entitled Your Diet and Your DNA (https://www.labster.com/simulations/your-diet-and-your-dna/)

Here we present a new interdisciplinary teaching proposal to improve high school students involvement through digital learning resources for science education, through laboratory experiences of comparison and sharing that increase student participation and bring the class to a much higher communication level also in English language. During a pandemic period and/or in the modern teaching innovation proces, the activity of a scientific laboratory within home walls plays an important role. The topics and activity selected, here presented, aims therefore, to make pupils aware of their attitudes, highlights the possibility of discovering the laws of nature employing “easy-to-find” materials, developing "doing to learn", increases the critical sense, self-assessment and communication. The characteristics of this method provide multiple stimuli to learning through imitation and action, through observation of the behavior of others and comments received on one's own, through analysis of the entire process. In addition, other objectives of the teaching here presented, are to bring out the knowledge acquired by pupils in the various disciplines - physics and biology - interactively, but also the behavioral rules within a laboratory, the interactions between gases and biomolecules in one case concrete study, as during DNA electrophoresis and, last but not least, the pupil's creativity. The activity took place in several phases that saw the involvement of multiple teachers: chemistry, physics, biology, English. The initiative involved several institutions: the biology department of the "Federico II" University of Naples, the "Galileo Galilei" Scientific High School of Naples. at the 33rd edition of "Remote Future - TEACH and education 4.0 "on November 23, 2019 in Naples. Some of teaching and learning results,here presented, were also showed last november during the 33rd Edition of Futuro Remoto in Naples as Teach and education 4.0. New lessons and amazing classroom activities, also added, were designed in collaboration and will realized integrating CLIL, ISBE methodologies and ICT tool (from Science Education section of Journal of Visualized Experiments, styled JoVE) implementing those already designed and realized in a different project previously reported (del Gaudio, EDULEARN17 Proceedings). Flipping the virtual laboratory, online theoretical lessons and the wet laboratory, all students, working in team will stimulate to develop trasversal skills and they will learn and will regain knowledge with Biology teacher during electrohoretic separation of biomolecules (DNA or proteins). To be more clear, during the electrophoretic migration of biomolecules and/or molecular dyes high school students will have to rediscover and apply the gases’s law already learned with Physics teacher.

EXPERIMENTAL APPROACHES TO INVESTIGATE ORIGINS OF BUIDING BLOCKS OF LIFE

We report the gene characterization, the cDNA cloning and the temporal and spatial expression pattern of the relaxin receptor rxfp1 gene in the zebrafish Danio rerio. The zebrafish rxfp1 gene has the same syntenic genomic organization, and a similar exon-intron structure to the homologue human gene. Furthermore, the deduced Rxfp1 protein sequence shows a high degree of amino acid similarity when compared with the human protein and the conservation of all amino acid identity necessary for the binding with relaxin. Our results show that rxfp1 gene is active either during embryogenesis or in the adult organism, showing a wide expression pattern. Moreover, we provide the first description of rxfp1 spatial expression pattern during embryo development, showing that the transcript is already present at the early developmental stage and is distributed in all of the embryonic cells until somitogenesis. Starting at the pharyngula stage the gene expression becomes mainly restricted in the brain territories. In fact, at the larval stage, the transcript is detectable in the epiphysis, postoptic region, posterior tuberculum, hypothalamus, optic tectum, tegmentum ⁄ pons, medulla and also in the structure of a peripheral nervous system, the terminal nerve. The rxfp1 expression pattern in Danio rerio embryos is very similar to that reported in the adult mammalian brain, suggesting a pivotal role of this receptor in the neurophysiology processes already at very early developmental stages.