Creaserinus fodiens) and tertiary burrowing species (Faxonius rusticus). Individuals of both species were placed in an arena with artificial burrows embedded within the arena floor. The arena floor was fitted with a panel, which served as a treadmill belt to allow for translational displacement. Individuals were displaced after they had left the burrows. The movement pathways of displaced crayfish were compared with those in two control groups, one which underwent no displacement and the second in which the treadmill belt was displaced but returned to its original position almost immediately. Homing success for displaced individuals of both species was considerably reduced in comparison to the control groups. Moreover, displaced primary burrowers had significantly lower homing success in comparison to displaced tertiary burrowers. Primary burrowers exhibited greater homing error and significantly impaired homing behaviors compared with tertiary burrowers. Furthermore, heading angles in displaced groups (of both species) were significantly higher than the control group of both species. Species-specific differences in homing success and homing error indicate that primary burrowers were more negatively impacted by translational displacements. These homing differences indicate that these two species of crayfish have differing homing strategies.Crayfish have been model systems for examining complex behaviors and the underlying neural mechanisms that guide these behaviors. While spatial learning has been examined in a subset of crayfish species, homing behaviors remained largely unexamined. Here we examined homing behavior following translational displacements in a primary burrowing ( An essential tool for the survival of organisms is their ability to navigate between resource rich areas. Broadly defined, the movement of organisms between resource areas can be defined as movement toward a goal . As a suIn contrast, idiothetic homing uses internal cues as sources of information . Path inCreaserinus fodiens (the digger crayfish) build deep, complex burrows, and often spend a significant portion of their lives within the burrow (Faxonius rusticus (rusty crayfish). Consequently, primary burrowers invest more energy and effort into their burrows and the selective pressure to home to their burrow may be higher than tertiary burrowers who invest less energy into their burrows. This difference of investment in burrow use and construction between species creates a possible difference in the homing mechanisms.To further understand the complex underlying mechanisms of homing behaviors, we tested homing behavior in two sympatric species of crayfish. These two species of crayfish create burrows of differing complexity. Primary burrowers, such as e burrow . In contThe purpose of this study was to determine the homing mechanism used by primary and tertiary burrowing crayfish. To test which homing strategy animals are using, we intentionally induce an error in their walking paths by displacing the crayfish. If path integration is the primary mechanism of homing, we would predict that crayfish would exhibit a decrease in homing success and end their homing path at a distance equivalent to that of the displacement. For example, if the crayfish was displaced 10 cm away from a burrow, their path would end 10 cm before the burrow entrance. Altering the distance between the crayfish and the burrow, in this manner allowed us to determine if idiothetic information is used. Conversely, if the displaced crayfish were to successfully return to their burrow after displacement, there exists the possibility of the use of allothetic cues to supplement idiothetic cues.F.rusticus and C.fodiens. The F.rusticus were collected from branches of the Portage River, Wood County, OH, USA  while C.fodiens were collected from a local unnamed pond , in Wood County, OH, USA. Crayfish were sized matched as closely as possible with F. rusticus averaging a carapace length of 2.87 ± 0.4 cm and C. fodiens averaging 2.14 ± 0.12 cm (carapace) in length. All animals were housed in individual flow-through plastic containers to ensure visual and mechanical isolation. Animals were kept in a climate controlled chamber with a 12 h:12 h light:dark cycle and a constant temperature of 22.5 ± 0.5°C. All individuals were fed three times weekly with commercial-size rabbit food pellets . Animals were deprived of food for 10 days prior to the start of the experiment to increase motivation for foraging. Individuals were used only once during the trials.Two species of crayfish were used in this experiment, Trials were conducted in one of two identical homing arenas, each arena measuring 80 cm × 80 cm × 35.5 cm (L × W × H). Both arenas were placed in an environmental chamber in which temperature was maintained at 23 ± 0.9°C and kept on a constant 12 h:12 h light:dark cycle. An artificial floor made from egg grating (1.3 cm holes) and mesh wire (0.13 cm holes) was elevated 17 cm above the base of each arena and supported by PVC pipes. The artificial floor, sides of the arena, and walls of the environmental chamber were either white or painted white to enhance contrast against the crayfish and to eliminate possible visual cues. Previous trials have shown that the paint is not toxic to crayfish and does not alter their behaviors . The arePrevious work has shown that crayfish will readily use a PVC pipe as a burrow when provided . An artiFish gelatin was used in each trial to provide motivation for the crayfish to leave and return to the burrows during the trial . The geln = 5). A total of 120 crayfish were used and only used once. The summary and Ns of the treatments are:At the start of each trial, a single crayfish was placed at the center of the arena. Crayfish were allowed a period of 4 h to find the burrow and explore the arena. Crayfish were allowed 2 h to find the burrow initially and an additional 2 h to exit and return to the burrow. Preliminary analysis indicated that this time period maximized the number of trials to collect useable data. Longer time periods did not significantly increase the number of crayfish that performed the required homing task. After the crayfish found the burrow, a piece of fish gelatin, in a weighted mesh bag, was placed near the burrow entrance and slowly moved to the opposite end of the arena. The mesh bag was left in the arena for the entire trial period. Once the crayfish exited the burrow and located the fish gelatin, one of three treatments were performed. The control treatment had an experimenter, behind the black sheeting, but the treadmill remained stationary. During a second control treatment, deemed the displaced-control treatment, an experimenter would displace the treadmill at set distance (11.5 ± 1.0 cm) and then return the treadmill to the original location. The third treatment was the displaced group, where an experiment would displace the treadmill and the treadmill would not be returned to the original location. There were two instances of tail flips in the displaced group and three in the displaced control. These individuals were dropped from subsequent analyses  using EthoVision XT 8.5  and Tracker . The tracks were digitized at a single point per second using the center of the carapace as a reference point (t = 0) to the burrow and the line from the animals current position (t = 0) to the animals next position (t = +1). An angle of 0° indicated the animal is pointing and moving toward the burrow. The distance to the burrow as well as the initial location of the burrow prior to displacement and post-displacement in all three groups  were recorded. The distance between the location of the burrow and the end of a homing path is defined as the induced error. The return journey was defined as having begun when a crayfish had 10 consecutive points where the distance to the burrow decreased. This definition was used as a benchmark across trials to provide a starting point for the return pathway. Only the return pathways were used for homing behavior analysis as these pathways would most effectively indicate differences in homing behaviors between species and across treatment groups.All trials were video recorded with an overhead camera (SW PRO 530-4PK) using a SWANN DVR (model SWDVK-430004). The trials were recorded at one frame per second until the crayfish returned to the burrow or until the 2-h period following the fish gelatin placement expired. This temporal resolution was used as it has been used previously to describe the spatial parameters of movement pathways in crayfishes . A markece point . For a tce point . The heaposthoc test to determine where significant differences existed .There was no significant difference in the ability of either crayfish species to home under either control homing condition  = 4.34, P = 0.015). The heading angle only displayed a significant effect due to treatment  = 25.2, P < 0.001), but no interaction or species effect (P = 0.9 and 0.9). Finally, the statistics on walking speed only found a significant difference across species   = 12.1, P < 0.001), but not for treatment or the interaction between treatment and species . Both of these groups had higher induced errors than either species in either of the controls .When displaced, both distance . CreaserC. fodiens and F. rusticus exhibited significantly different heading angles relative to the burrow in the displaced trials when compared with either the control or the displaced-control trials (P < 0.001). There was no significant effect due to species or a species and treatment interaction (P > 0.5). In addition, the heading angles for the two crayfish species in the displaced treatments were not significantly different from each other (P > 0.5).Both P > 0.6: C. fodiens had an overall faster walking speed than F. rusticus across all treatments.There was no change in the walking speed of the crayfish during the return path under any of the treatments P > 0.6: , but C. not using cues directly associated with the burrow  or tertiary (F. rusticus) burrowers. Additionally, the reduced induced error in F. rusticus indicates possible compensatory mechanisms for translational error through a second homing mechanism.The results of this study demonstrate that homing success rate, in both species of crayfish, is significantly diminished when a displacement occurs . The pose burrow . Our resdistance . While dF. rusticus) displayed a reduced induction error. This reduced induction error indicates that F. rusticus may be possibly compensating for translational errors. As many animals employ secondary homing strategies to compensate for homing errors (F. rusticus also compensate through use of secondary homing strategies. Thus, when presented with conflicting sensory cues from the idiothetic information of a burrow location and the allothetic information about the burrow’s location, F. rusticus rely on allothetic information. This is consistent with conflicting cue studies in the desert ant, Cataglyphis fortis, which demonstrated that although path integration was the primary mode of homing, reliance on celestial compass took precedence over idiothetic cues (In our study, the tertiary burrower (g errors , it is ptic cues . Additiotic cues . Evidenctic cues .C. fodiens are a primary burrowing species and construct elaborate burrows they are likely to exhibit burrow fidelity, presumably due to the cost associated with the construction and maintenance of a burrow. Faxoniusrusticus on the other hand are tertiary burrowers which are more likely to occupy burrows constructed by others and thus display less of a fidelity toward specific shelters. Therefore, crayfish may use homing strategies that are a direct result of the time and energy investment in the construction of the burrow. Similar behaviors are found across species. Digger wasp’s ability to localize and defend a nest is tied to the investment in constructing the nest (As a result of these possible compensatory homing strategies, the primary burrowers were more negatively impacted than tertiary burrowers by the displacement. These findings are indicative of path integration as being a primary mechanism for short range homing in crayfish. Given that the nest . Speciesthe nest .Primary burrowers use their burrows more frequently and leave on smaller foraging trips . As suchWithin the landscape of these crayfish, numerous cues exist that may facilitate homing. For example, the burrows constructed by primary burrowers, and subsequently used by tertiary burrowers, often have structures such as “chimneys” associated with them . These cC. fodiens, these crayfish are less aggressive than F. rusticus (Other factors, beyond burrow construction and use, could play a role in the homing differences found within this study. While little is known about the biology and ecology of the crayfish, rusticus . Differerusticus . Underlyrusticus .C. fodiens appearing to rely on path integration, whereas the tertiary burrowing species, F. rusticus, relies on path integration as well as an unknown compensatory strategy. It is possible that the differences in homing strategies are related to their burrow use and energy investment in that burrow. Further investigation is needed to determine the secondary strategy that tertiary burrowers may be relying on.This study was designed to complement previous burrow displacement studies . The res


Like many other molecules playing vital functions in animals, the antibody molecule possesses a complex structure with distinctive features. The structure of the basic unit, i.e., the immunoglobulin domain of very ancient origin is substantially simple. However, high complexity resides in the types and numbers of the domains composing the whole molecule. The emergence of the antibody molecule during evolution overturned the effectiveness of the organisms’ defense system. The particular organization of the coding genes, the mechanisms generating antibody diversity, and the plasticity of the overall protein structure, attest to an extraordinary successful evolutionary history. Here, we attempt to trace, across the evolutionary scale, the very early origins of the most significant features characterizing the structure of the antibody molecule and of the molecular mechanisms underlying its major role in recognizing an almost unlimited number of pathogens.The vertebrate immune system provides a powerful defense because of the ability to potentially recognize an unlimited number of pathogens. The antibody molecule, also termed immunoglobulin (Ig) is one of the major mediators of the immune response. It is built up from two types of Ig domains: the variable domain, which provides the capability to recognize and bind a potentially infinite range of foreign substances, and the constant domains, which exert the effector functions. In the last 20 years, advances in our understanding of the molecular mechanisms and structural features of antibody in mammals and in a variety of other organisms have uncovered the underlying principles and complexity of this fundamental molecule. One notable evolutionary topic is the origin and evolution of antibody. Many aspects have been clearly stated, but some others remain limited or obscure. By considering a wide range of prokaryotic and eukaryotic organisms through a literature survey about the topic, we have provided an integrated view of the emergence of antibodies in evolution and underlined the very ancient origins. Receptors that are capable of recognizing non-self molecular structures are found in a diverse array of multicellular organisms. Most of them belong to the immunoglobulin superfamily (IgSF) as containing the immunoglobulin domain. With the explosion of adaptive immunity, about 500 million years ago, the antibody molecule, also termed immunoglobulin (Ig), has become the key player of the immune defense system in jawed vertebrates. The basic unit consists of four polypeptide chains, of which two are defined as heavy (H) and two as light (L) chains . The N-tV (variable), D (diversity), J (joining) gene segments, which contribute to encoding the IgV domain through a mechanism of “somatic recombination.” These gene elements precede the exons encoding the C region. In the jawed vertebrates, two different arrangements of the Ig gene locus are known. In cartilaginous fish, multiple “clusters” of V, D, J, and C segments are present [V, D, and J gene segments, followed by a single copy of the exons coding for the constant domains. The IgV domain possesses a distinctive feature, that is a region, encoded by a J gene segment, which forms a diglycine bulge and is important for the V domain dimerization. These types of V domains are known as VJ sets or VJ V domains [The structure of the gene locus encoding the H and L chains includes multiple copies of  present , whereas domains . IgV and domains . A distiThe main reason underlying the evolutionary success of the Ig domain certainly resides in its compact fold, which serves as the basic structure bearing the hypervariable loops involved in the formation of the antigen-recognition site. A key role in the folding dynamics is played by the clustering of the hydrophobic residues in strands B, C, E, and F. They are required for priming the folding nucleus of the Ig domain and for the formation of the disulfide bridge between strands B and F. In addition, to ensuring Ig domain compactness, this bridge in some way drives the formation of the Ig domain folding nucleus, thus preventing less stable or partially folded domains. The folding strategies that antibodies can adopt represent quality control checkpoints in Ig transport, ensuring a correct domain assembly .In eukaryotes, four different types of Ig domains have been grouped in IgI, IgV, IgC1, and IgC2, differing in the presence or absence of additional β strands.Here, we look through species that preceded the jawed vertebrates in an attempt to reconstruct the structural and functional properties that have resulted to be essential for the antibody assembly and to describe the most important steps that have led to its diversification in jawed vertebrates.Immunoglobulin-like domains have been detected for a long time in prokaryotic surface proteins belonging to the intimin/invasin family, playing a role in host cell adhesion and pathogenicity . AlthougStudies in Eubacteria and Archaebacteria  have raiGiven objective difficulties in identifying which prokaryotic Ig fold is more closely related to the eukaryotic Ig domain, the prokaryotic one has been classified as bacterial Ig domain (BIg). Bateman et al.  underlin2+ binding module has been identified in BIg domains of Leptospira [Strepococcus pneumonia [2+ binding site has a secondary structure composed of β strands, but it shows low sequence homology and is distinguished by the absence of the core conserved disulfide bridge. BIg domains are frequently found in surface proteins as important structural and functional elements. A number of explanatory examples are given below. A component of the fimbrial structure of Streptococcus suis contains BIg domains that promote adherence to host cells [Porphyromonas gingivalis T9SS protein cover a role in the secretion of virulence factors [Salmonella BapA protein, which is important for biofilm formation, comprises tandem BIg repeats [Shewanella frigidimarina possesses ice-binding, protein-containing BIg domains that allow adsorbing their host bacterium onto ice [Structural analyses of BIg domains from various bacterial species reveal unique traits of these domains with respect to the eukaryotic Ig domain. A Captospira  and Streneumonia  proteinsst cells . BIg dom factors . Salmone repeats . The fla repeats . BIg con repeats . The Antonto ice   located in the middle of the domain .V) as that encoding the rest of the domain, but by a different gene segment (J).Another distinct feature of IgV domains is the presence of a 65–75-aa long stretch, between the two highly conserved cysteines involved in the formation of the disulfide bridge. Instead, the corresponding region in the IgC domain comprises 55–60 residues ; it can It has been argued that the Ig domain originated by duplication and divergence from an ancestral domain. A half-domain prototype has been hypothesized to consist of the β-strand ABCC’ or GFED, which is associated in a homodimeric form in the same manner as these β strands associate in the IgV domain. The presence of introns in the middle part of the sequence encoding the region between the conserved disulfide bridges is in line with the hypothesis of a half-domain origin . In addiGeodia cydonium (Porifera) [Branchiostoma floridae [Ciona intestinalis [J-like segment but show a particular folding in the strand G, which is a key constraint on the dimerization of IgV regions. The high-resolution structure of VCBP3 revealed that IgV domains are packed in a head-to-tail fashion, in contrast to all jawed vertebrate IgVs, found to pack in a head-to-head configuration [Botryllus schlosseri. This tunicate lectin is the first example of a soluble protein that uses also an Ig domain for recognition, in addition to the canonical carbohydrate-binding domain [Interestingly, a gene encoding an adhesion molecule (GSAM) that includes an Ig domain with high sequence similarity to the IgV domain of vertebrate L chains, has been identified in a living fossil, the marine sponge orifera)   . Immunolorifera) . Althougorifera) . Thus, tfloridae  and the stinalis  have beeguration . The preguration . This opg domain .B. floridae, an IgV domain containing a J segment-like structure has been described [In AmpIgVJ-C2, another molecule from escribed . The preescribed .At present, we can conclude that important features of the antibody V domain can be traced back to early chordates, before the appearance of the IgC1 domain.VJ segment continuity, such non-rearranging Ig-like genes would presumably have been served as primordial substrates that have led to the emergence of recombining antigen-binding receptors [Aside from Ig, the IgV domain is also present in several molecules of the innate immune system of jawed vertebrates. Of particular interest is the case of the novel immune-type receptors (NITRs), found in teleost fish ,37. NITReceptors ,39. Evoleceptors , a featueceptors . Apart feceptors .Looking across the tree of life over evolutionary timescales, we have accumulated in the previous paragraphs some clues about the non-vertebrate origin of the ancestral building blocks of the antibody molecule. Here, we have underlined that the IgV domain has been widely used by the recognition molecules of earlier vertebrates. However, only in jawed vertebrates, the V domains have acquired a structural significance to form the genuine antigen-binding site, along with the potential to vary in order to cope with a multitude of different antigens.V/D/J set to encode a complete V domain. In the H chain gene locus, it occurs in two steps, namely, D–J in pro-B cells and V–DJ in pre-B cells; in the L chain gene loci, it occurs in pre-B cells. The complex of the two endonucleases, encoded by the recombination activating genes RAG1 and RAG2, cut the DNA sequence of H and L chain gene loci at specific positions defined as recombination signal sequences (RSSs). RSSs are adjacent to V, D, and J gene segments and consist of conserved heptameric and nonameric sequences separated by 12- or 23-bp long spacers. RAG1 encodes a 1043-aa homodimeric protein, which carries the ability to recognize RSSs and cleave DNA; RAG2, encoding a 527-aa protein, cooperates in the recombinase activity together with other accessory factors. The recombination enzymatic complex induces the juxtaposition of the RSSs flanking the V, D, or J segments, cuts the DNA, and produces a circular fragment, consisting of the region between the two gene segments, and two truncated coding ends, which are ligated by the Artemis multi-enzymatic complex, including a kinase and the Terminal deoxynucleotidyl Transferase (TdT). The latter adds extra nucleotides to the rearranging junction.Somatic recombination is a complex genetic mechanism occurring during the different stages of B cell maturation. It consists of a random rearrangement of just one gene segment of each VDJ recombination has been found only in jawed vertebrates and it is specific for Ig and T-cell receptor (TCR) gene loci. However, precursors of the components of the VDJ combinatorial machinery have been identified in invertebrate species.The mechanism of the B. floridae the BfRAG1L gene has been shown to encode the functional central domain of vertebrate RAG1. Recombinant BfRAG1L contains a site motif of retroviral type II nuclease, able to cleave RNA and DNA. Moreover, it recognizes vertebrate RSS in presence of mouse RAG2 [VDJ recombination process [In the amphioxus use RAG2 . These r process .RAG1- and RAG2-like sequences have been identified in different invertebrates, e.g., the sea urchins Lytechinus variegatus and Patiria minata, and the mollusk Aplysia californica. Strongylocentratus purpuratus SpRAG1L and SpRAG2L genes show a genomic organization similar to that of vertebrates [RAG genes were already present in the deuterostome ancestor and that their role in adaptive immunity has been a recent acquisition.tebrates . They arIn addition, the Artemis nuclease, which participates in the recombination process to cleave the DNA hairpin extremities generated by the double-stranded breaks, possesses an ancient origin, as suggested by the identification of Artemis-like sequences in fungal and metazoan species .It seems reasonable to hypothesize that two evolutionary lines have driven precursors of recombination enzymes and immune receptors to meet each other at a given stage of jawed vertebrate evolution, providing the bases for the adaptive immunity.In order to fight an almost unlimited number of pathogens, the adaptive immune system, in addition to the recombination mechanism for the generation of the primary antibody repertoire, relies on another process, the somatic hypermutation (SHM), which drives further diversification of the antibody variable regions by generating higher affinity variants .The 24-kDa enzyme activation-induced cytidine deaminase (AID) initiates SHM by deaminating cytosine to uracil in the context of the so-called mutational hotspot motif RGYW  in single-stranded DNA. Thus, the C:G pair is converted to U:G mismatch; this is repaired by base excision repair enzymes, and error-prone DNA polymerase fills the gap creating mutations as well insertion/deletions. AID is a member of the APO lipoprotein B mRNA editing enzyme catalytic polypeptide-like (APOBEC) family, which edits messengers, also described as DNA mutators.AID/APOBEC genes across metazoans, underlining their involvement in several biological processes, with a primary role in immune defense [After discovery by Tasuku Honjo and Michael Neuberger in 1999 ,47,48, r defense . Identif defense . Gene ex defense , indicat defense . AID hom defense  and cart defense , which b defense , which d defense .V-elements shed light on the origin of Ig somatic hypermutation, dating it back before the radiation of bony fishes. After hyperimmunizing a nurse shark with hen egg lysozyme, IgNAR genes encoding specific antibodies with increased binding affinity were isolated, reinforcing the existence of an affinity maturation process [To date, the best molecular evidence for affinity maturation in early gnathostomes comes up from the discovery of an Ig isotype exclusive to nurse sharks, that is IgNAR. The very limited number of IgNAR  process . Singula process ,58. It cTrematomus bernacchii, an Antarctic teleost species, have uncovered several peculiar features reminiscent of the mammalian hypermutation mechanisms. A high mutation rate in hotspot motifs, high accumulation of nucleotide substitutions in CDRs, a bias for transitions over transversions, and an unusual excess of insertions and deletions have been observed. These findings provide an example of higher CDR mutability in a cold-adapted fish than that observed in other actinopterygian species [Moreover, investigations on the diversity of the V domains of IgM H chains from  species ,60.The IgC domain comprises two β sheets, one formed by β strands A, B, E, and D, and the other one by strands G, F, and C, which are packed face to face with an angle of 30°; a disulfide bridge links strands B and F. There are two types of IgC domains—IgC1  and IgC2Very interestingly, while the IgC2 domain is present in numerous molecules in both vertebrate and invertebrate animals, such as lectins, cell adhesion molecules, connectis, major sperm, and zona pellucida proteins, the IgC1 domain is limited to jawed vertebrates and has been found only in the adaptive immune molecules, e.g., immunoglobulin, TCR, Major Histocompatibility Complex class I and II, and CD1. This observation supports the idea of the contemporary emergence of all the actors playing a major role in the adaptive immune response during the so-called immunological big bang, all using the novel IgC1 domain type .Since all these molecules possess at least a pair of IgC1 domains of different polypeptide chains facing each other, we launch the idea that this specific ability to dimerize might account for the evolutionary novelty introduced by the IgC1 domain. In contrast, IgC2 domains have not been found in a dimeric form in invertebrates. In this regard, the dimeric structure was confirmed for the IgV domain of the amphioxus AmpIgVJ-C2, but not for the IgC2 domain . In our An antibody is a complex molecule consisting of four polypeptides, two H and two L chains—the two H chains are linked to each other by disulfide bridges, and each L chain is covalently linked to an H chain, even though exceptions occur. For instance, the human IgA2m1 allotype has been reported to carry a disulfide bond between the constant regions of the two L chains, instead of the canonical H–L linkage . The IgVTakifugu rubripes IgH) [Danio rerio IgD) [H chain gene locus in cartilaginous fish consists of multiple mini-clusters, each composed of one V, two or three D, and one J, followed by one set of C region exons; some clusters are unique because carrying partially (VD or VJ) or completely (VDJ) recombined germline gene segments. A varying number of clusters (up to 100) has been found in different species [The number of C1 domains in the Ig H chain is particularly large in fish, ranging from 2 (pes IgH)  to 15 dorio IgD) . CartilaThree different H chain isotypes are present in these fishes—IgM, IgW  orthologous to IgD, which is present in all jawed vertebrates, and IgNAR . IgNAR eWe can try to formulate some hypotheses to explain the existence of these naturally occurring antibodies devoid of the L chain, despite its encoding gene is present and functional, as demonstrated by the presence of L chains associated with H chains in the other two isotypes. In IgNAR, the CH1-CH1 homodimer has been shown to have a dissociation constant Kd of about 600 nM suggesting a stronger interaction than that found for other CH dimers, e.g., CH3-CH3 dimers show a Kd of 188 μM . As a coMoreover, the absence of L chains could be due to a different mechanism adopted for assembly in cartilaginous fish. The release of newly synthesized H chains from the endoplasmic reticulum may not depend on the encounter with the chaperone BiP, as it happens in teleosts and other vertebrates. Generally, the CH1 domain does not automatically fold during synthesis but, to properly fold, it first needs to encounter the BiP peptide and subsequently associates with the L chain. BiP and IgL chains cooperate to control the folding of the H chain and ensure the fidelity of antibody assembly ,70.The shark IgNARs display another distinct feature compared to conventional antibody architecture. The V domain of IgNAR comprises a particularly long and structurally complex CDR3 loop, which ensures a higher sequence diversity of the antigen-binding site, compensating for the absence of VL domains E. Furthe2L2 dimerizes, thus resembling mammalian IgA, or polymerizes as tetramers such as teleost IgM, or exists as a monomer such as IgG, outlines the extraordinary intrinsic plasticity of the antibody architecture, permitting to exist as monomers or with different degrees of polymerization.An additional isotype, named IgT, has joined the IgM and IgD with the emergence of bony fish, but it is absent in tetrapods . It has A fundamental role in the evolution of the antibody architecture is played by the so-called hinge region—the key region that accounts for the functionality of the antibody, which is located in the middle of the molecule. Hinge presents a highly variable amino acid composition, particularly rich in cysteines and prolines—cysteines are used to bridge the two H chains; prolines confer conformational flexibility. Moreover, the length of the hinge greatly influences Fab–Fab flexibility .A flexible hinge allows (i) functional motions of Fab arms, the portions of the molecule containing the two L chains, and the two N-terminal domains of the H chain (IgV followed by the first constant domain) favoring the binding of antigens of various shapes and sizes, and (ii) triggers the effector activities performed by the region called fragment crystallizable (Fc), which consists of the remaining part of the molecule with the exception of the Fab arms .T. rubripes IgH chain [During vertebrate evolution, the hinge region first appeared in teleosts. A hinge region, encoded within the CH2 exon, has been highlighted in gH chain . This hiOnce acquired, the structural complexity of the hinge increased during evolution, along with antibody configuration. As a result, mammals exhibit the most diversified hinges within tetrapod species .Xenopus tropicalis IgF forms a loop connecting the CH1 and CH2 domains, possibly acting as a flexible spacer. The presence of a conserved cysteine, likely involved in a covalent bridge between the two IgF H chains, and of three prolines recall the characteristic features of the mammalian Ig hinge regions.The first example in the evolution of a hinge encoded by a distinct exon has been found in the amphibian IgF isotype . The hinPlacental mammals (Eutheria), including humans, possess five IgH chain isotypes— IgD, IgA, and the IgG; all consist of three CH domains and a hinge region that joins the Fab arms to the Fc region . ConversOver the past decades, different theories regarding the origins of the hinge have been formulated. One theory proposed that the hinge is encoded by an exon that evolved in parallel with the immunoglobulin genes ,81, and IgL genes across species remains elusive. Diversity has a major role in the antibody repertoire, and the multiple sets of V, D, and J gene segments encoding the V region of H chains have their own significance for antibody diversity. In striking contrast, the diversity of the L chain repertoire is very limited. This could be related to an important function the L chain plays to protect from self-reactivity [H and L chain genes may have co-evolved to ensure that appropriate L chains become available for each H chain that is generated during the early stages of B cell development.We will continue the long evolutionary history of the antibody molecule, covering another important building block, the L chain, although detailed knowledge of the evolutionary history of activity . It has IgL chain genes is difficult since these genes are short and rapidly evolving. However, IgL chains have been found in all vertebrate Ig isotypes, with the exception of camelid HCAbs and shark IgNAR, both lacking L chains, as stated above. Four isotypes have been identified— κ, λ (divided into sub-types in bony fish), σ1, and σ2. All isotypes are present in fish, but the isotype σ2 appears to have been lost in amphibians and σ1 in other tetrapods [Genes encoding the different L chain isotypes, unlike those of the H chains, are always found at separate loci. Understanding phylogenetic relationships among etrapods ,92, wheretrapods .2L2. During evolution, antibodies changed the polymerization degree in their secreted forms—IgM exist as monomers or pentamers in cartilaginous fish; the polymeric form in teleosts is tetrameric, while hexameric in amphibians, pentameric in birds, and pentameric or hexameric in mammals. Different arrangements of the basic unit depend upon the presence of an additional component, termed J peptide, and on the occurrence and the position of the cysteine residues that form inter-subunit disulfide bonds. Avian IgY, and mammalian IgG, and IgE are present only in the monomeric forms.Antibodies are secreted by plasma cells in different degrees of polymerization of the basic unit HAt this point, it would be interesting to report the current knowledge of the main issues related to the evolution of tetrapod H chain isotypes and sub-isotypes. However, this goes far beyond the aim of the present review.The antibody molecule has been meticulously shaped by evolution in order to enable all its functions. It carries out them in two different steps, which are (i) antigenic recognition, performed by the Fab portions, which comprise the L chains and the two N-terminal domains of the H chain (VH and CH1) and (ii) activation of the cellular mechanisms that neutralize the antigen, conducted by the Fc region, which comprises the constant domains adjacent to the first one. The ability of the antibody molecule to assume different conformations to recognize different partners is ensured by its flexibility. Fab arms bind to antigen through the CDRs of both IgV domains of H and L chain, while the Fc region interacts with Fc receptors, bacterial antigens called “superantigens,” complement factor C1q.The antibody Fc region mediates diverse effector functions by engaging distinct classes of Ig receptors. These receptors are called FcRs because bind to the Fc fragment of antibodies that are attached to a bacterium or to an infected cell. FcRs appeared during the vertebrate evolution and are isotype-specific; they share protein and gene structure, indicating that originated from a common ancestor by gene duplications. . FcRs haThe interaction between IgG and Fcγ receptors occurs at the hinge region and the N-terminus of CH2 and is dependent on glycans bound to Asn 297, which stabilize an asymmetrical conformation of Fc; the stoichiometry of the binding is 1:1, differently from Fcα, which binds dimeric IgA [Apart from the classical FcRs, other receptors bind to the antibody Fc region—the polymeric Ig receptor (pIgR) and the so-called neonatal Fc receptor (nFc). pIgRs are integral single-pass transmembrane glycoproteins that facilitate the transcytosis of polymeric immunoglobulins and immunocomplexes . This mo2 microglobulin. To bind the antibody molecule, it uses a site other than the MHC class I groove that serves as the binding site for the antigen peptides presented by the TCR. The IgG region involved in the binding is at the boundary between the CH2 and CH3 domains; the FcRn region involved in the binding comprises the β2 microglobulin, which binds the C-terminus of CH2.nFc has a different structure and evolutionary history . It is sAn important effector mechanism involving antibodies is the activation of the complement cascade through the “classical”  pathway that leads to lysis of the bacterial cell. The first step of the cascade requires the contact between the antibody molecule (IgG or IgM) and the complement factor C1q, which appeared in the evolution in cartilaginous fish in parallel with IgM . An orthStaphylococcus aureus), Protein G (group C Streptococci), the M-protein (group A Streptococci) [Finegoldia magna) are some of the IBPs studied in more detail. In the case of soluble IgG, the superantigen interaction site is usually at the IgG interface between the second and third constant domains [Finally, we should mention the bacterial proteins called “B cell superantigens” or immunoglobulin binding proteins (IBPs), which bind the antibody protein at sites other than that of the recognition of common antigens. Protein A (tococci) , and the domains , whereas domains . The intIn an attempt to fit together the chapters of the antibody tale, we have provided an overview of the emergence of the structural elements that became, over a long evolutionary time, the most important building blocks to construct the antibody molecule (summarized in However, only with the emergence of innovative mechanisms generating diversity, antibodies have become a powerful shotgun for the complex adaptive immune system of vertebrates.Given the vast topic, we have placed a special emphasis on the basic structural features of the antibody molecule, which have made it the main character of the tale, ensuring its evolutionary success. In addition to a certain degree of structural conservation, we have also highlighted some of the most explanatory examples of the ways found by the antibody molecule to reinvent its own canonical structure through unique modifications, while preserving its function.We know that the picture of the evolutionary origins of the antibody molecule provided in this review is far from over, and we are also aware that it can become clearer as new species in the tree of life will be examined.


Lipid transfer between organelles requires proteins that shield the hydrophobic portionsof lipids as they cross the cytoplasm. In the last decade a new structural form of lipidtransfer protein (LTP) has been found: long hydrophobic grooves made of beta-sheet thatbridge between organelles at membrane contact sites. Eukaryotes have five families ofbridge-like LTPs: VPS13, ATG2, SHIP164, Hobbit and Tweek. These are unified into a singlesuperfamily through their bridges being composed of just one domain, called the repeatingbeta groove (RBG) domain, which builds into rod shaped multimers with a hydrophobic-linedgroove and hydrophilic exterior. Here, sequences and predicted structures of the RBGsuperfamily were analyzed in depth. Phylogenetics showed that the last eukaryotic commonancestor contained all five RBG proteins, with duplicated VPS13s. The current set of longRBG protein appears to have arisen in even earlier ancestors from shorter forms with 4 RBGdomains. The extreme ends of most RBG proteins have amphipathic helices that might be anadaptation for direct or indirect bilayer interaction, although this has yet to be tested.The one exception to this is the C-terminus of SHIP164, which instead has a coiled-coil.Finally, the exterior surfaces of the RBG bridges are shown to have conserved residuesalong most of their length, indicating sites for partner interactions almost all of whichare unknown. These findings can inform future cell biological and biochemicalexperiments. In the last two decades there has been a transformation in our understanding of howmembrane-bound organelles of eukaryotic cells interact with each other. Text books tend toemphasize the linear pathways of secretion and endocytosis, considering organelles apartboth from each other and from those that do not participate in vesicular traffic. This picture has increasingly beenfalsified by finding individual proteins that bind to two organelles at the same time,bridging the cytoplasmic gaps between them. A major activity that is found where twoorganelles interact is the transfer of lipids , which cin vitro ; and (3) Hobbit  to replace KIAA0100, in yeast: two paralogs Fmp27 andYpr117w newly renamed Hob1/2, in plants: SABRE, KIP and APT1).Subsequently, LTPs with an elongated rod-like structure ∼20 nm long were found in bacteria,with a “U”-shaped cross-section the internal surface of which is entirely hydrophobic, whilethe external surface is hydrophilic . This alin vitro , and thein vitro . A pivotin vitro   ong ATG2 , and aloong ATG2 , but it ong ATG2 , but preong ATG2 . Tweek hong ATG2  or by trong ATG2 . In contong ATG2 .Linkage of the two segments of VPS13 was examined by a ColabFold prediction of the regionencompassing both sides of the VAB repeats, with most of this central region beingomitted. The prediction tool placed the two VPS13 segments in direct continuity , with thEven though AlphaFold can reliably build 3D models from contact maps, the multimerpredicted by AlphaFold varies from solved cryo-EM structure in more subtle aspectsincluding both superhelicity and the extent to which the groove forms a sharp “V”,compared to a shallow groove . DespiteVPS13 has four major types of RBG domains: Sequence relationshipsbetween different RBG domains in VPS13 were examined to determine how the multimer of RBGdomains was formed. The tool used for this was HHpred, which has high accuracy and a smallnumber of known flaws ((II) wn flaws . A preliwn flaws . These dwn flaws . VPS13A wn flaws . All domwn flaws . The 12 wn flaws  and in mwn flaws , indicatwn flaws . The basCapsaspora, Trichomonas, Trypanosoma, Chlamydomonas andArabidopsis). One aspect of the method that was not based on biologicalobservation is that the six elements of RBG domains were defined as βββββ-loop. This waschosen in preference to any other permutation (for example βββ-loop-ββ) to maximize thepower of sequence analysis, because either including the whole loop that follows thehelix, the site of greatest variability, or making a deletion reduces the sensitivity ofMSAs (not shown). Indication that RBG domains may not be formed from the βββββ-loopbiological unit are highlighted in the descriptions of search results below.The next step was to identify homologies between individual RBG domains. When searchesare seeded with whole protein strongly homologous regions in some hits will cause falsepositive alignments of adjacent unrelated regions . This isSupplemental Figure 1). Eight out of ten domains showed strong homology(predicted shared structure >90%) to one but not both of the penultimate N-terminal orthe penultimate C-terminal domain . The two domains not in this group were RBG10 withmoderate homology (predicted shared structure 77%), and RBG9 with weak and mixed homology(predicted shared structure 40% for N-terminus and 18% for C-terminus) .The observation that each RBG domain in VPS13 is to some extent unique led to a deeperstudy of homology relationships between the central 10 domains of VPS13A (RBG2 to 11),which found patterns of homology between the domains. This was seen in a second tranche ofhits  aligningeach domain to other regions of VPS13 (and in some cases to ATG2 and SHIP164). Thisindicated more distant relationships between RBG domains, which to date have only beendescribed approximately . To esta1–71 and 52–72 inside domains). Theinferred duplication events are located at a position similar to that previously suggested. RBG51+2, RBG61+2, andRBG71+2 from VPS13D were also homologous to each other, but much more weakly.This suggests that RBG5/6/7 duplicated in two independent events, more recently in VPS13Cthan in VPS13D.The same findings were made for VPS13C and VPS13D, except three of the central domains ofboth (RBG5–7) had very close homologs 3 domains distant in the multimer . Here opposite ends of the RBG domain are homologousto RBG2 and RBG11 (data not shown), which is evidence for inheritance of RBG domains inunits other than βββββ-loop. Structural features that differentiate between RBG2 and RBG11are described in Section D.Overall, this shows that VPS13A/C/D in their full extent consist of domains related toRBG1-2-11-12, which are therefore are the four major types of RBG domain in VPS13. In turnthis suggests that a very primitive ancestor of VPS13 may have consisted of just thesefour domains, with growth by repeated central duplication. Appearance of new domainscentrally like this is the typical pattern of duplication of multimeric domains in oneprotein . While dVPS13B diverged from VPS13A/C/D at the7 + RBG domain stage: VPS13Bis an outlier compared to the others in its VAB repeats (th domain is well conserved but not in sequence(RBG7 in VPS13B is similar to RBG3 in VPS13A). The remaining RBG domains in VPS13B followa quite different pattern . These findings indicate that VPB13B started todiverge from VPS13A/C/D at or after a 7 domain stage consisting of RBG1-2-3-4-10-11-12(numbering according to VPS13A). The timing of this divergence is addressed in the nextsection.(III)  repeats , and the pattern . Five ofPhylogeny of VPS13A/C/D and VPS13B indicates LECA expressed both of theseisoforms: looking across evolution to construct a phylogeny for VPS13, ininvertebrates the fruit fly D. melanogaster has three VPS13s: clearhomologs of VPS13B and VP13D, plus one protein called Vps13 that isrelated to both VPS13A and VPS13C. This is consistent with the VPS13A/C pair arising froma relatively recent (≥300 MYr) duplication (C. elegans has two VPS13s that resemble VPS13A/C (gene: T08G11) andVPS13D (C25H3.11)  has eleven RBG domains: in detail it lacks RBG3 and one set of RBG5-6-7. It alsodoes not contain the UBA domain found in human and fly VPS13D  lication . The dom25H3.11) . VPS13D y VPS13D . Other iCapsaspora owczarzaki, a free-living single cell organism related toanimal precursors . A key finding is that 8% of the VPS13B cluster werefrom SAR/Harosa protists and algae , with species such as Aphanomycescontaining full-length homologs of VPS13B (data not shown). By comparison, the VPS13Dcluster contained one amoebal protein and one algal protein, and the basis for suchclustering was unclear as each showed stronger BLAST hits to VPS13A/C than to VPS13D.Thus, in agreement with the RBG domain results , suggestingexpansion to 38 RBG domains with a hydrophobic groove >75 nm. It may be thatintracellular parasites have unique inter-membrane contacts that are wider than typicaleukaryotic cells.While the analysis here is based on assigning orthology, VPS13 in protists separated fromother extant organisms by long evolutionary branches cannot be assigned to any one group.They still merit study as they are examples of the general principle of plasticity in RBGproteins. Individual VPS13 proteins in VPS13X is a previously undescribed divergent plant homolog: Untilnow, plants have been thought to contain three VPS13 homologs (SHBY(At5g24740), named because mutations cause Arabidopsis to appear shrubby (SHBY) is proposed here to standardize the format across VPS13proteins in major clades of organism where possible. The two other identifiedArabidopsis VPS13 proteins (At4g17140 and At1g48090 (multiple) are used here. VPS13S and VPS13M1/2 have RBG domains homologous toVPS13A and yVps13 ((V) homologs . To descrubby . The namt1g48090 ) havenod yVps13 ,consistArabidopsis (At3g50380), which isannotated in databases as “Vacuolar protein sorting-associated protein”, named hereVPS13X, and which has close homologs in most land plants (data not shown). VAB repeatsnear the C-terminus identify it definitively as a VPS13 rather than ATG2, but it isvariant being shorter than other VPS13s with nine RBG domains, only six of which arehomologous to domains in other VPS13s: RBG1-2-4-7-8-9 follow the same pattern asRBG1-2-4-10-11-12 . Finally, one domain (RBG5) is homologous only to theorthologous domain in other VPS13X proteins, and has seven β-strands, confirmed byColabFold . This high level of divergence makes it impossible totell if VPS13X originated from VPS13A/C/D or from VPS13B or from their commonancestor.HHpred identified a fourth VPS13 in ATG2 shares six RBG domains with VPS13: Relationships between theeight RBG domains of ATG2 were traced as had been done for VPS13  SHIP164 lacks a specialized C-terminus: SHIP164 has homologs fromanimals to plants with six RBG domains, with homology confined to the their N-termini(RBG1/2/3), partially resembling the N-termini of both VPS13 and ATG2 , but RBG4 and RBG6 in plants are unique forms, andthe latter with 5 strands is considerably different from the same domain in animals, whichhas 2½ strands. The homology of the final domain of SHIP164 in animals to RBG2 of VPS13 issignificant because it groups the domain with those specialized to be in the middle of themultimer , suggestingthat the C-terminus is similar to the middle of an RBG multimer (see Section Ciii).(VII) and ATG2 . For RBGHob and Tweek proteins show more distant homologies to VPS13:Before mapping RBG domains in these proteins, a preliminary step was to check previousreports that homologs of Tweek exist only in fungi (Csf1) and animals, such as fly (Tweek)and human (BLTP1)  and Trichomonas, lengths within the range between yeast Csf1 (2958 aa) and flyTweek (5075 aa) or human BLTP1 (5093 aa). All length variation in this family results fromintrinsically disordered loops throughout the proteins. Although the presence ofTweek/Csf1 homologs in multiple protists might arise from horizontal gene transfer, itsuggests that Tweek/Csf1 was present in LECA, which is a more ancient origin than waspreviously considered.(VIII)  (BLTP1) . Using HTrichomonas. This indicates that acquisition of domains andtheir rearrangement all occurred before LECA.Hobbit and Tweek both start with transmembrane helices (TMHs) that anchor them in the ER Castro . A featuTo summarize the whole section on conservation: the majority of RBG domains haveconserved sequence across all eukaryotes. A minority of RBG domains  have changed so much that the individual RBG domain is not detectableacross all orthologs. This minority shows that primary structure can change to losedetectable homology without affecting secondary structure or lipid transfer function. Inturn this implies that the conservation present in all the other parts of RBG proteinsserves specific functions other than lipid transfer itself. Section D addresses theseconserved sequences in detail.VPS13 has a set of three widely conserved accessory domains near its C-terminus , ATG_C also being found in ATG2. After delineating the RBG domains, itbecame possible to identify all other accessory, folded, non-RBG domains using HHpred withconfirmatory modelling by ColabFold . Being aHelical bundles similar to those in VPS13 and ATG2 are found in Hobbit inTweek: The best known accessory domain in any VPS13 is the ∼45 aa helical UBAdomain in VPS13D ((I) n VPS13D . This isn VPS13D , and hasn VPS13D .UBA domn VPS13D . Secondan VPS13D   B repeat  . ColabFold modelled this as a β-sandwich  homology  . This is consistent with insertion in a membrane withmany packing defects and low levels of anionic headgroups, such as the ER where theN-terminus targets in almost all examples studied of VPS13 , which is consistent with these N-termini insertinginto tightly packed membranes with high levels of anionic headgroups, similar to thosethat insert in the plasma membrane ((I)  is weak . Inone  is weak , which i is weak . VPS13 sof VPS13 . Exceptiof VPS13 . Evenmomembrane .ATG2 differs from VPS13 and SHIP164 by commencing with an unstructured hydrophobic loopthat can enhance membrane insertion  Fugleba and distSupplemental Table 2A). Since this is unlikely to dominate over the TMH inER targeting, its role might be to select regions of curvature and/or to locallydisorganize the bilayer within the ER  the helices are amphipathic . These helices have biophysical properties similar tothe helices that immediately precede the β-sheet ((II)  β-sheet , and the β-sheet , so they β-sheet , or alte β-sheet .SHIP164 is the one RBG protein that has a different form. In opisthokont SHIP164 thedisordered region after the final RBG ends with a coiled-coil. This means that theC-terminus of SHIP164 is unique among human RBG proteins by lacking a amphipathichelix.Speculation about the C-terminus of SHIP164: might itdimerize end to end? In addition to the C-terminus SHIP164 having a coiled-coilbut no amphipathic helix ((III) ic helix , the RBGic helix . This suic helix . The sheic helix .in vivo forms tail-to-tail dimers. Although such dimers have beenreported for purified SHIP164 in vitro .These are also among the most highly conserved domains . What are the unique structural and sequence featuresof these domains? To start with the outermost RBG domains (RBG1/12) were examined,focussing mainly on VPS13 as this family has the richest information. In both RBG1 andRBG12 the amphipathic helices cap the groove by crossing it perpendicularly to the sheet(red arrows in (I) Conserved structural variations differentiating between the two archetypalcentral RBG domains VPS13 vary across orthologous domains: The two RBG domaintypes of greatest significance are RBG2 and RBG11 (VPS13A numbering), because theyrepresent archetypal forms in sequence terms that account for almost all central domainsin VPS13, ATG2 and SHIP164 and for ∼50% of domains in Hobbit and Tweek . Variability of the features is illustrated byRBG10, which has neither feature in any orthologous domain except for animal VPS13A/C,indicating that a loop may have been acquired just in that clade. Therefore, loops andbulges show too much variability to provide information about the events that created thedomains present in LECA.(II) Conserved residues in RBG domains VPS13 form a strip on the external,hydrophilic surface ideally placed for interacting with partners: As a finalstep to understanding RBG domain conservation, the most highly conserved residues in thecentral domains (RBG2–11) of VPS13A were mapped . This suggests that the latter group of helicesmight be mobile, however, countering this, in cryo-EM of VPS13 all the helices of RBGdomains 2 to 7 align with the long axis contacting the rim of the groove ((III) e groove .A second feature of the central domains is the presence of conserved residues along theentire outside convex surface of the groove, which is simplified here by showinghydrophilic residues only viewed from the convex surface and hydrophobic residues onlyviewed from the concave surface , and is Supplemental Figure 5A to C), and this applies across almost the whole ofHobbit . Overall, these findings indicate that a largemajority of the entire length of RBG multimers has conserved residues on the outside(mainly hydrophilic) surface. Conserved hydrophilic residues on the convex surface of RBGdomains tend to occur near the rim of the groove from which helices originate  sequence from1643–2840 was submitted to ColabFold after removing 2112–2542 (767 aa remaining) . This reD. melanogaster), nematode worm (C.elegans), Trichoplax adherens, S. cerevisiae,S. pombe, Capsaspora owczarzaki and Athaliana. For the one sequence that was incomplete, TrichoplaxVPS13A/C (1299 aa), additional sequence was assembled from adjacent genes that encodesequences homologous to the N- and C-termini of VPS13AC. This added the N-terminus, butleft a likely gap of 1500−2000 amino acids that is possibly be encoded in a genomic regionof 13891 bp. tBLASTn in this region identified 262 aa of VPS13A/C-like sequence in 6regions  in 5 statistically significant hits, suggestingthat an unannotated complete VPS13A/C is expressed in Trichoplax, ofwhich 1561 aa have so far been identified.Sequences were obtained from both Uniprot and NCBI Protein databases for VPS13 in 8organisms: human, fly . Based on these observations, all HHpred hits longer than 25 residues withpredicted shared structure ≥10% that contained any predicted sheet were considered truepositives.The form that RBG domains take in HHpred  was initth element, was submittedto HHpred with 5 iterations of HHblits to make multiple sequence alignments (MSAs), thenused to find homologs in target HMM libraries of key whole proteomes . Toidentify homology of a query domain to RBG2 and RBG11 in VPS13, four pair-wise alignmentswere made using the “Align two sequences/MSAs” option, aligning the query with MSAs fromthe penultimate domains both of VPS13A (RBG2 &11) and of VPS13B (RBG2 & 12). BothVPS13A and VPS13B were included here to capture the breadth of VPS13 sequences, and thefour MSAs of penultimate domains were made superficially (one iteration of HHblits) topreserve unique qualities of the domains.Each domain, defined as the strands plus 12–20 aa of the 6All regions with predicted secondary structure that did not align well with the typical 5strand + loop pattern of RBG domains were submitted to HHpred to identify their fold, andalsFor relationships between RBG domains, 229 domains from VPS13 proteins in the 8eukaryotes listed above were submitted to CLANS, using BLOSUM45 as scoring matrix . 162conp < 10−30.For relationships between whole VPS13 proteins, 1266 full length proteins sequences weresubmitted to CLANS, using BLOSUM62. This list was obtained by generating two separatelists of proteins homologous to the central portion of the RBG multimer (to avoid proteinsthat only are homologous to the VAB domain) seeding HHblits searches with eitherVPS13A/C/D (RBG2-9) or VPS13B (RBG2-10) . The resDrosophila (www.ebi.ac.uk). The first 9columns of this were visualized with WebLogo (https://weblogo.berkeley.edu).Sequences were analyzed for propensity to be amphipathic and coiled coils using Heliquestand Coils tools, respectively . A conseosophila . From 51Supplemental File 1) were submitted to Consurf Superfamily Reveal Past and Present Domain Variations Affecting Form, Function andInteractions of VPS13, ATG2, SHIP164, Hobbit and Tweek by Tim P Levine in ContactClick here for additional data file.Supplemental material, sj-docx-2-ctc-10.1177_25152564221134328 for Sequence Analysis andStructural Predictions of Lipid Transfer Bridges in the Repeating Beta Groove (RBG)Superfamily Reveal Past and Present Domain Variations Affecting Form, Function andInteractions of VPS13, ATG2, SHIP164, Hobbit and Tweek by Tim P Levine in Contact


In the past decade, a new generation of radar satellites have revolutionised our ability to measure Earth’s surface deformation globally and with unprecedented resolution. InSAR is transforming our understanding of faults, volcanoes and ground stability and increasingly influencing hazard management. The image featured on the cover of Nature and the results inspired a generation of scientists. Since then, the quality and quantity of available images has increased dramatically. The last decade has seen the first mission specifically designed for ground deformation monitoring (European Union’s Sentinel-1 constellation), a global Digital Elevation Model with unprecedented accuracy (TanDEM-X), and a constellation of small SAR satellites capable of acquiring imagery with 1 m resolution (CosmoSkyMed). These satellites, and others, now measure ground motion across the planet several times per day and can achieve accuracy of less than a millimetre per year allowing us to better understand the processes by which the Earth deforms and to provide relevant information for disaster risk reduction.The theory of Interferometric Synthetic Aperture Radar (InSAR) has been known for decades: after applying various geometric and atmospheric corrections, high resolution maps of surface displacements can be produced by comparing the phase of successive radar images Fig. . The firth Century we have understood that earthquakes occur because of the sudden, catastrophic release of strain energy that has slowly accumulated around tectonic faults2. However, at the time of the 1992 Landers earthquake, we had only measured the resultant coseismic deformation for a handful of earthquakes. InSAR data, mostly acquired by the European Space Agency’s (ESA) European Remote Sensing (ERS) and Envisat satellites in the 1990s and 2000s, have now allowed scientists to image deformation from more than 130 earthquakes3, a number that is increasing by 20–30 events per year thanks to systematic global acquisitions from Sentinel-1. These data have been used to help constrain detailed models of the fault slip that caused the earthquakes, confirming that the Earth does behave, as expected, like an elastic solid. However, InSAR also shows precisely which faults slipped during an event; and we have been surprised by how often earthquakes occur in unexpected locations and the complexity of fault ruptures. The best example of this surprising complexity is the 2016 Kaikoura Earthquake in New Zealand, which ruptured a network of a dozen or more faults4 . InSAR has been used to identify aseismic slow earthquakes (transient creep events) on both continental faults9  to map interseismic strain over large regions9 Fig. . Althougth century. However, it was not until the satellite era that is was possible to measure remote or dangerous volcanoes, or to survey large regions12 (Fig. 13. Ongoing improvements in satellite technology and data processing have reduced detection thresholds while increasing coverage, causing a dramatic rise in the number of volcanoes known to be deforming and providing the first detailed information on the spatial and temporal characteristics of volcanic deformation. The first satellite-based catalogue recorded 336 deformation events at 160 different volcanoes, revealing a remarkable diversity in the patterns, rates, durations and extents14. Statistical analysis shows that roughly half of these deformation events are linked to eruptions15 and satellite data is increasingly used by volcano observatories. Perhaps the best example of the use of satellite data for decision-making is the radar observation of rapid dome growth at Merapi, Indonesia – the subsequent decision to evacuate an additional 400,000 people is credited with saving 10,000–20,000 lives16.Another source of surface movement is magma moving underground: visual observations of bulges or changes in relative sea level preceding eruptions have been reported for centuries, and ground-based surveying techniques measured deformation at a handful of well-studied volcanoes during the 20s12 Fig. . The pas17. The simple model of liquid magma pressurising an elastic-sided chamber until failure can no longer explain the wealth of observations. Instead, the prevailing view is of a transcrustal system composed of multiple lenses of crystals, melt and gases17. Translating this new view of magmatic systems to the interpretation of volcano deformation will require a multidisciplinary approach, but through improved eruption forecasts, will ultimately benefit the 800 million people globally who live within 100 km of a volcano.InSAR data, together with information from geochemistry, petrology, and other geophysical methods, are contributing to a multidisciplinary paradigm shift in our conceptual understanding of magmatic systems18. Now that Sentinel-1 is providing a reliable and long-term archive of InSAR ground measurements, satellite data will likely play an increasing role in monitoring and mitigating against the impacts of anthropogenic sources of ground motion. Several national ground motion products are already available and pan-national services are under development with potential impacts on the civil, geotechnical and structural engineering sectors.The growth of satellite deformation data has made us increasingly aware that human activity also causes surface motion. Fluids, including hydrocarbons and ground water, are extracted from and injected into the subsurface by various industries, while poorly-constructed buildings and infrastructure can deform and collapse. Even in the UK, where tectonic activity is low, the average cost of ground movement is estimated to be £300–500 million per year, while a single earthwork failure on a mainline railway could cost over £10M19 are likely to be important for maximising the benefit from the data (Fig. By combining data from several existing SAR satellites, daily monitoring of ground motion is now possible, whether that be the deformation caused by an earthquake, the evolution of the magmatic system during a volcanic crisis, or the failure of an embankment. In the future, InSAR monitoring will become ever more routine, and the launch of several new SAR missions with new capabilities is already scheduled. Handling the large volumes of data generated is already challenging, and machine learning algorithmsata Fig. . Over th


The autoinhibited Parkin catalytic domain (PDB ID: 4BM9) receptor has been described to have a role in the ubiquitination of α-syn in Parkinson's disease. Therefore, it is ofinterest to discuss the molecular docking analysis data of phytochemicals from ethanolic extract of Crescentia cujete with the auto inhibited Parkin catalytic domain. We report thedocking features of the phytochemical named 1, 2-Ethanediamine, N-(2-aminoethyl) with the target protein for further consideration towards the design and development of anti-Parkinsonagents. Neurodegeneration is a process, which involves neuropathological condition, and brain aging due to lack of physical movement and mental relaxations lead to all the stress relateddisorders . The mosSeveral research studies have been identified the autoinhibited Parkin catalytic domain (PDB ID: 4BM9) is an E3 ubiquitin (Ub) ligase found in Homo sapiens .MutatioThe available drugs are levodopa, carbidopa, apomorphine, amantadine, orphenadrine, benzhexol, benztropine, selegiline, pergola and many more. These drugs are effectively reversingthe symptoms of Parkinson and improve the level of dopamine. The greatest disadvantage at present is available potent synthetic drugs lies in their adverse effects like constipation,ulcer, respiratory depression, hypertension, toxicity and reappearance of symptoms after discontinuation . To overCrescentia cujeteis a small tree belongs to the family Binoniacea, which grows about 6–10m tall with a wide crown and long branches covered with clusters of tripinnate leaves andgourd-like fruit and these branches are arranged as simple elliptical leaves clustered at the anode. C.cujete leaves are used to treat cold, bronchitis, cough, asthma, urethritis andalso to cure haematomas and tumours . The leaThe leaves of Crescentia cujete were collected from different localities of Coimbatore District and authenticated by the Botanical Survey of India (BSI), Southern Regional Centre,Tamilnadu Agricultural University campus, Coimbatore. A voucher specimen (No: BSI/SRC/5/23/2017/Tech 2021) has been deposited at the Herbarium of the Botany department.The leaves were cleaned and shade dried for 7 days, then grounded well to a fine powder. About 600 g of dry powder was extracted with ethanol (80%) at 70°C by continuous hotpercolation using soxhlet apparatus. The extraction was continued for 2 days. The ethanolic extract was then filtered and kept in a hot air oven at 40°C for 1day to evaporate theethanol from it. A greenish brown residue was obtained.Ethanolic extract of Crescentiacujetewas lyophilized and the sample was subjected to GC-MS equipment Thermo MS DSQII  for the analysis. The equipmenthas a DB 35 - MS Capillary Standard non-polar column with dimensions of 30 mm x 0.25 mm ID x 0.25 µm films. The carrier gas used is Helium with at a low of 1.0 ml/min. The injectorwas operated at 250°C and the oven temperature was programmed as follows: 60°C for 15 min, then gradually increased to 280°C at 3 min. The identification of components wasbased on Willey and NIST libraries as well as the comparison of their retention indices . IdentifADMET properties  were calculated by using the QikProp program in Schrodinger. QikProp generates physically relevant descriptors and overall ADMEproperties and drug-likeness parameter, which were used to assess the druggability of the compounds as shown in  18]..18].Molecular docking is a process to predict the binding sites of the molecules. Maestro modules  in SchroThe crystal structure of the autoinhibited Parkin catalytic domain was downloaded from Protein Data Bank (PDB) with the PDB ID: 4BM9. The protein preparation was carried out usingthe protein preparation wizard of Schrodinger Suite 2018. In general, the protein preparation consists of fixing structures, deleting unwanted chains and waters, fixing hetero groupsand finally optimizing the fixed structure. The 3D structure of the autoinhibited Parkin catalytic domain (PDB ID: 4BM9) was shown in .The structures of the ligands from the results of GCMS analysis were retrieved from PubChem database. Retrieved ligands were prepared by using LigPrep module in SchroOur study is focusing on the inhibition of Parkin catalytic domain. We want to enhance inhibitory activity of autoinhibited parkin catalytic domain, so we are not considering theactive form of protein. GlideScore is an empirical scoring function that approximates the ligand binding free energy. It has many terms, including force field  contributions and terms rewarding or penalizing interactions known to influence ligand binding. It has been optimized for docking accuracy, database enrichment, and bindingaffinity prediction and be used to rank poses of different ligands, for example in virtual screening. As it simulates a binding free energy, more negative values represent tighterbinders. The Crescentia cujete compounds that were used for the In-silico study are listed . MoleculWe report the docking features of the phytochemical named 1,2-Ethanediamine, N-(2-aminoethyl) with the target protein for further consideration towards the design and development ofanti-parkinson agents. The lead compound satisfying the Lipinski rule of 5 and ADMET properties, to be act as a drug.