Close proximities between organelles have been described for decades. However, only recently a specific field dealing with organelle communication at membrane contact sites has gained wide acceptance, attracting scientists from multiple areas of cell biology. The diversity of approaches warrants a unified vocabulary for the field. Such definitions would facilitate laying the foundations of this field, streamlining communication and resolving semantic controversies. This opinion, written by a panel of experts in the field, aims to provide this burgeoning area with guidelines for the experimental definition and analysis of contact sites. It also includes suggestions on how to operationally and tractably measure and analyze them with the hope of ultimately facilitating knowledge production and dissemination within and outside the field of contact-site research. Given the recent growing interest in interorganelle membrane contact sites, the field will benefit from clear rules to define and study them. In this Perspective, a panel of experts aims to provide this growing field with guidelines for experimental definition and analysis. In Eukaryotes, intracellular membranes delimit organelles that have distinct biochemical functions. While for decades the organelle field was governed by studies aimed at identifying the unique characteristics of each compartment, the last years have seen a revolution in the field as more focus is being placed on the interactions between the organelles and their role in maintaining cellular homeostasis.2. However, the lack of a perceived physiological role made it hard to envision this as a general and functionally relevant phenomenon. The strong notion at the time was that the physical organization of the cytosol was performed solely by anchoring and movement on cytoskeletal elements. Moreover, it was believed that the transfer of small hydrophobic molecules between two organelles was catalyzed by freely diffusing cytosolic proteins, and that soluble metabolites and second messengers travelled long distances3. Together these two views delayed the appreciation of the importance of membrane tethering between two organelles.Published examples of interactions between two distinct organelles appeared in the late 1950s4, and several years later as the site of efficient Ca2+ transfer5. Similar roles were then ascribed to the ER–plasma membrane (PM) contact site7 and to ER–Golgi contacts8. The discovery of the nuclear vacuolar junction (NVJ) and its role in piecemeal microautophagy of the nucleus10, and later the assignment of a role for the ER–PM contact in autophagy regulation11 showed that the role of contacts could be diverse. In recent years more roles have been uncovered such as for the ER in regulating mitochondrial14 and endosomal15 fission in contact sites between these organelles16. Roles in controlling inheritance such as in the case of peroxisomes17 have also been described. Hence, it is rapidly becoming evident that organelles are highly interconnected and that there are multiple important functions for these physical associations at contact sites.The field started to expand and gain momentum when examples of functional apposition became evident and these areas were termed membrane contact sites. For example, when the juxtaposition between the endoplasmic reticulum (ER) and mitochondria was identified as the site of phospholipid biosynthesis and transfer20). Moreover, the uncovered molecular determinants are becoming implicated in a variety of cellular and pathophysiological processes22 demonstrating the importance of contact sites in normal development and physiology.In recent years additional contacts are being described and studied. More importantly, the previous functional observations are now being coupled by an increasingly clearer molecular understanding as tethers, molecules that bring and maintain the two membranes into close proximity, are being identified . In plants chloroplasts engage in contact sites with most other organelles20. Homotypic contact sites, that are not fusion intermediates, have been described between two peroxisomes25 or two LDs26 and potentially other multicopy organelles could also form them. Homotypic interactions between organelles that are intermediates for fusion, in our eyes, are characterized by different features, and operationally do not represent a contact site similar to the ones discussed here and hence will not be touched upon in this review. It should also be noted that membrane-less organelles can form contacts with membranous organelles: for example, inclusion bodies can interact with LDs27. However, since these contacts do not occur between two organelles bound by membranes they might be physiologically very different, and consequently we will also not discuss them here.Membrane contact sites are classically defined as areas of close apposition between the membranes of two organelles. There have been, to date, examples of both homotypic  and heterotypic (between two different organelles or two different membrane types) contact sites. Heterotypic contacts that have been well-studied originally all involved the ER. For example, the ER–mitochondria, ER–PM, ER–Golgi, ER–peroxisomes and ER-lipid droplets (LDs) contacts. Lately, contacts that do not involve the ER are being discovered such as: LDs–peroxisomes, mitochondria–vacuoles/endosomes/lysosomes, mitochondria–PM, mitochondria–LDs, mitochondria–peroxisomes, and mitochondrial inner and outer membranes  membrane-bound organelles. Many manuscripts define a “top limit” to the distance between the two organelles that can still be defined as a contact site . Obviously, the narrower the gap, the easier and more obvious it is to see a contact. However, it is not clear how the specific distance value has been defined as some contact sites have the capacity to be much larger (see below for example Num1 that can span over 300 nm28). We hence suggest that distance cannot be a sole measure and that simple juxta-positioning of organelles is not sufficient to be considered a contact site regardless of distance. What does define a contact site in all cases reported to date, is the presence of tethering forces that arise from protein–protein or protein–lipid interactions.We define contact sites as a do not include intermediates of an active, SNARE mediated or independent, fusion process. Fusion intermediates have been in the past referred to as “docking” events and this nomenclature, if kept, can help keep the two processes distinct. Limited vesicular trafficking between apposed organelles may, however, exist, but would follow established mechanisms and terminology.Contacts, in our view, must fulfill a specific function. Because the majority of contacts were initially described with the ER, this highlighted the role of contact sites in lipid and Ca2+ transfer29 and this dominated the view of the field for many years. It is now, however, becoming apparent, that virtually all organelles can contact each other and that the diversity of functions of these contacts is much wider. To date three types of functions have been suggested: (i) the specific bidirectional transport of molecules such as various ions, Ca2+, lipids, amino acids, and metals32. (ii) The transmission of signaling information or force important for remodeling activities, including regulation of organelle biogenesis, dynamics, inheritance, positioning, fission, and autophagy37. (iii) The positioning, in trans, of enzymes (such as the phosphatidylinositol (PI) phosphate phosphatase, Sac1; and the protein tyrosine phosphatase 1B, PTP1B39) so as to regulate their activity.Contacts Since all contacts must have a function, this requires that they be regulated and hence that dysregulation of contacts should impact cell function and contacts should therefore be selected for by evolutionary pressure.functional protein and/or membrane composition which is required for all of the above: tethering, function, regulation, and the maintenance of their architecture (see section below). The functions often benefit from concentration of specific proteins/lipids in the contact forming membranes, which creates a “quasi-synaptic” arrangement, where the organelles can locally and effectively cooperate with each other without altering the bulk cytosol40. In some cases, “moonlighting” may allow a protein to perform one function in the bulk of an organelle, but a contact-site-specific function when present there. At present, this may be harder to measure accurately. To date it has not been unequivocally shown that contacts have a unique lipidome but fractionation of ER–mitochondria contacts suggests that they are “raft-like” hence probably enriched in ceramides and sterols42.Contacts should have a 43, during ER–endosome contacts that control endosome motility35 following depletion of Ca2+ from ER stores44 or increase of intracellular Ca2+46. We suggest that period of existence is therefore not a defining characteristic of contacts. However, we recommend that the above four features all be experimentally characterized when a new type of contact is described.It seems that the extent of time that a contact exists is flexible and dependent on its function, regulation, and cell type. Indeed, dynamic and transient contacts exist as well as stable ones that are maintained over long periods. Dynamic contacts have been described during, for example, pulsatile insulin secretion29. However, it is now appreciated that pairs of molecules functioning at contacts can also drive tethering and that the sum of the forces exerted by all these pairs ultimately tethers the two organelles18. This explains why, to date, most contacts have had multiple tethering molecules described and why eliminating a contact by deleting any singular tethering pair has proven impossible. However, each protein at a contact should have at least one role and we define four possible roles that should be found and enriched at contact sites  that would establish/maintain the connection between the two membranes and by a second set of proteins that would fulfill functions specific to that particular contact site45. As revealed by cryo-electron tomography (ET), the intermembrane distance between bilayers tethered by E-Syts is ~20 nm, which is exactly the length of the cytosolic region of these proteins47.Structural proteins include tethers that hold the two organelles together and pillars/spacers that keep the two membranes at a defined distance and thus form the skeleton of a contact site as well as inhibit fusion. Spacers have not been well-studied to date. However, one example of active spacing is performed by extended-synaptotagmins (E-Syts) involved in ER–PM contacts in mammals48) and interact with proteins and/or lipids on the partner membrane through a second domain 50. Interaction with other proteins can also be homotypic53, but most tethers described to date are built on heterotypic interactions between two proteins on the opposing membranes. Some tethers like the VAPs have multiple interaction partners on the opposing membrane55. Generally, tethering pairs are structurally characterized by a protein domain composition that, ultimately, allows them to span the contact-site width over considerable distances .The number of studied tethers, however, is much larger and is increasing rapidly. Tethers often possess double targeting determinants to bind and bridge the two membranes. Tethers are often directly anchored to one of the two bilayers through a transmembrane domain or by a lipid modification  to see a dramatic reduction in contact (and even then ER–PM contact is not ablated completely)66. It seems that for the major contact sites complete ablation is not viable, but this has to be better studied to be confirmed.The presence of multiple tethering pairs ensures maintenance of contact, with some local variation depending on local enrichment of components, under a diversity of functional conditions. Recently, even contact sites that for years were considered to be held by a single tethering pair, such as the NVJ, have new tethering proteins ascribed to them such as the tether Mdm159 and ER–PM60 contacts, different sets of tethers can form distinct sets of contact sites. Hence, in the future use of an umbrella term such as “the ER–PM” contact site may not be useful anymore and contact sites would be defined by both their opposing membranes and their component molecules or their specific functions61.Importantly, to date contact sites have been defined by the two organelles that they bring into proximity. However, recent reports suggest that in certain cases, such as mitochondria–vacuole50 has a role in sterol transfer between mitochondria and ER membranes128, and itself also exerts a tethering role62. Similarly three proteins out of the ERMES complex that tethers the ER and mitochondria in yeast have lipid transfer domains63 as does PDZD8 that performs a similar role in mammals64, and the tricalbins (in yeast)67 or extended synaptotagmins 68. Recently the Kv2.1 potassium channel has been shown to also have a role in tethering the ER to the PM54. Importantly, any functional protein that is proposed to also be a tether can be tested by re-expression of a version with mutation of the functional domain. Where this has not been done, even if partners on the opposing membrane exist, a role in tethering has to be taken as untested.Since each contact site must have a function, proteins that facilitate that function must reside in and be enriched at contacts. Such functional proteins can perform ion, protein, lipid, or metabolite exchange, for instance ion channels and pumps, lipid transfer proteins, or metabolite channels/transporters. It seems that in most cases described to date functional molecules also exert tethering capacity—for example, Lam670 and that palmitoylation serves as a signal to enrich proteins at these unique membrane patches71. Some of the sorter proteins would work in trans, serving to maintain the membrane properties on the partnering organelles. Proteins in this group may also recognize lipid properties to enrich tethers or functional/regulatory proteins to contacts. Examples for these are the ER proteins Rab32 and phosphofurin acidic cluster sorting protein 2 (PACS-2), which regulate the intra-organellar distribution of contact-site proteins72. This group also includes PI transfer proteins, as well as PI kinases and phosphatases that are often enriched in contact sites74, since they could in theory define a unique lipidome for these areas.Sorting and recruitment proteins work to define the contact-site proteome and lipidome. This can be done by active recruitment of proteins into the contact site or by repelling non-residents of contact sites. Sorting can occur by direct binding to proteins or indirectly by either altering lipids (to define curvature and charge) or the proteins themselves . For example, it has been suggested that the mitochondria–ER contact site has a unique lipid composition relative to either membrane75. Another example is p53 that changes the redox state of Ca2+ handling proteins, thus altering ER–mitochondria tethering76.These include proteins that regulate the extent of the contact site itself as well as the function of the active proteins in the contacts. For example, phosphorylation of the tether holding the mitochondria–vacuole contact site controls tethering77. Hence, this categorization is meant purely to aid in ordering the current knowledge and for ease of communication.Overall, the characterization of contact-site proteins has just begun and will likely continue to result in a plethora of mechanisms and players in the foreseeable future. However, we strongly suggest that a variable combination of these protein classes shall characterize any newly identified contact site. Importantly, the division of proteins into categories is purely synthetic as it is clear that many proteins have several capacities in the same molecule such as tethers that are also lipid transporters or regulators. Indeed, proteins belonging to these classes have all been identified in the several available proteomic studies of mitochondria–ER contacts78), thus preserving membrane identities. We propose fusion is inhibited at contacts by either:The two membranes engaged in contacts are stabilized by multiple tethering/functional complexes and kept in proximity without fusion -Syts. Interestingly, a nonfusogenic SNARE complex has been reported to bridge the ER and PM in neurons81 and a SNARE has been shown to regulate ER–mitochondria contact-site functions82. Due to their amphiphilic nature, SNARE motifs can also associate in an antiparallel configuration83. While the parallel alignment would lead to vesicle–membrane fusion, an antiparallel configuration would not. These finding support a novel additional function for SNARE proteins in stabilizing contacts beyond their well-established role in membrane fusion and in the future maybe more of these examples will be found.Despite the distinctions between these two defined processes, that we designate as mutually exclusive, structural similarities exist between proteins that operate at contact sites and at fusion sites, and analysis of these similarities could contribute to a better understanding of contact formation and maintenance. For example, Synaptotagmin (Syt)1 binding to the SNARE protein complex is an essential step for membrane fusionContact sites were first observed in electron micrographs (EM) as early as the 1950s, but for several years they were mostly overlooked given their transient nature or variable abundance within different cell types. In addition, to this day it remains challenging to visualize contact sites or purify them and therefore ascertain that they are present, understand their functions and study how environmental and genetic perturbations affect them. Hence, any study wishing to characterize contact-site components or functions faces first the issue of unequivocally identifying the contact, following its function and monitoring its changes upon perturbations. Below are approaches so far employed to study contacts as well as our recommendations for reliable contact-site visualization  accompanied by a second approach that is endowed with a resolution power amenable to detect distances in the range of contact sites; (ii) performed on live cells, using piezoelectric z-stepper or other similar approaches, to acquire the whole cellular volume in very short times; (iii) accompanied by careful experiments of reconstitution of organelle shape and number to exclude possible artifacts caused by variability in these traits.While tagging two organelles each with a different color is fast, easy and amenable to adaptation to high-content approaches, it suffers from intrinsic limitations. First, most contact sites are smaller than the optical diffraction limit: on the 60. Hence, different patterns for a contact site between the same organelles might result from using different markers. For example, using different tethering molecules to visualize the ER–PM contact gives rise to visualization of domains that are shaped as “patches” (when using E-Syt2/345) or “punctae” (when using ORP5/885). In addition, such approaches are clearly limited to the analysis of a subset of contacts whose components have already been identified.A different approach to visualize contact sites is based on imaging of characterized contact-site residents. Such residents often have a unique punctate appearance specific to the interface and are therefore very useful for accurate representation of the interaction space. While this approach has been powerful in enabling live imaging of contact sites in various cell types, one must remember that the various residents may not reside in the entire contact site and that it may even be that different contacts exist between the same two organelles86. This can be very powerful to study alterations that occur in response to genetic or environmental perturbations. While this approach can indeed give a sense of membrane proximities, it also suffers from a number of limitations: first, reductions in PLA signals can be caused by changes in expression or localization of one of the PLA partners; second, it requires that the PLA partners are unequivocally localized at the contact site; third, it might not recognize changes in contact-site extent not accompanied by changes in proximity between the two proteins measured in the PLA. We, therefore, believe that PLA can be a powerful tool to corroborate protein–protein interaction in situ especially in trans, but we do not recommend PLA as the tool to measure contact extent. If used it should be very carefully controlled.Proximity ligation assays (PLA) between two contact-site components on opposing membranes have been employed to extract quantitative information on the distance between two membranes or the extent of contactSimilarly, to PLA, fluorescence resonance energy transfer (FRET) and split fluorescence sensors can detect proximities between two membranes based on the effect of proximity on the fluorescence of the pair and can be used in live cells. Such probes can be broadly divided in three classes:FRET-based reporters: The transfer of energy between two fluorophores has been used to visualize organelle proximities such as ER–mitochondria juxtaposition87. Importantly, FRET reporters are exquisitely sensitive to the distance between the membranes as energy transfer is inversely proportional to the sixth power of distance between the fluorophores, and since no dimerization occurs. By adding a rapamycin-induced dimerization domain, maximal FRET measurements can be obtained, thus enabling quantitative measurements of contact distance at any given time point and cross-sample comparison. The greatest shortcoming of this probe is the requirement for equimolar expression of the two FRET pairs . A modified version of an ER–mitochondria FRET-based probe, overcame this complication by expressing the two fluorophores as a single mRNA with a self-cleavable TAV2a sequence between them88. By changing the targeting sequence of the individual fluorescent proteins, this approach can be adapted to measure any potential contact site. However, FRET measurements require proficient experimenters and dedicated equipment, thereby limiting the utilization of such probes.Irreversible split fluorescence probes: Split fluorescence probes, such as split Venus or green fluorescent protein (GFP), are based on the appearance of a fluorescent signal when two nonfluorescent fragments targeted to the partner organelles bind each other once the juxtaposed membranes are in proximity91. Since it is now clear that a signal appears at the interface no matter which proteins on the two organelles are chosen to be tagged18, this approach does not require any pre-existing knowledge about a contact site and enables discovery of novel contact sites. However, these probes suffer from the fact that complementation of the two fragments is thermodynamically stable, leading to contact-site stabilization. In such approaches the dynamics of contact sites cannot be studied, and some contacts may become toxic under some conditions, when they cannot be eliminated. It is important to also remember that these approaches do not discriminate between close associations of organelles and tethered contacts and can cause synthetic expansion of the associations.Reversible or quasi-reversible fluorescent probes: This method is similar to the above method of bimolecular complementation but depends on usage of protein fragments that have very low intrinsic affinity of the two halves of the fluorophore. So far, two types of such probes have been utilized for contact-site detection: ddGFP92, which is based on a reversibly dimerizing dsRED fluorescent protein genetically modified to emit in the green zone of the light spectrum ; and split infra-red reporters93. However, the intrinsically low fluorescence of these probes might restrict their application. Brighter versions will be required for increased usage.Importantly, all above probes enable proximity measurements between two organellar membranes. While this can be highly important for detecting the presence of a contact site, it should be utilized carefully in measuring the effect of any single tether, functional molecule or spacer. To date, all contact sites described have more than a single tethering pair in them—hence loss of any one protein does not necessarily cause reduction in either the amount of contact or the distance between organelles. Moreover, increased distance between two organelles does not necessarily mean loss of contact and could also represent the formation of alternate, wider, contact areas by an alternate tethering mechanism as a compensation.94, or atomic force microscopy95 can be powerful tools to accurately visualize them and study the localization and the distribution of the proteins that reside within these structures. Super-resolution methods that can be used include structured illumination microscopy, stimulated emission depletion (STED) microscopy and single molecule localization microscopy, however, all such techniques require highly dedicated microscopes and technical expertise, and despite the ongoing development of sub-diffraction microscopy techniques, their application to contact-site analysis is still limited.Since contacts are below the diffraction limit of light microscopy, super-resolution96, which very often cannot be explored with other experimental approaches. Hence, TEM is often considered the “gold standard” for the study of the fine architecture of contact sites and to reveal their morphological and functional diversity. However, this approach is often only useful for either highly abundant contact -sites (rare contact sites will not be spotted in EM fields), or those that have very good marker proteins that can be used for immunodetection98 or correlative light electron microscopy (CLEM) approaches99. The latter two approaches can also both be used to pinpoint a protein specifically at a contact site and is therefore considered the best proof of it being a resident protein.Transmission electron microscopy (TEM) is an atomic-resolution microscopy system that provides static, high-resolution, ultrastructure information on contact sites within the cellular context100. We therefore recommend that TEM be used to provide qualitative and quantitative features of contact sites but not to study occurrence and changes in the extent, unless rigorous morphometric analyses are included.Unfortunately, TEM is a very low-throughput technique, affected by the commonly used chemical fixation procedures, limited to the analysis of a single plane, and therefore not suitable to measure the extent of contact in cells unless multiple parameters are taken into consideration  and computed into one index that is applied in extensive morphometric experiments101. ET has been successfully employed to study contact sites such as the ER–mitochondria96 or ER–PM47 as well as to generally map contact sites in small structures such as axons102. ET is the most reliable approach to validate the presence of linkers at the contacts104.An exciting “spin-off” of conventional TEM is ET. Conventional and cryo-ET offer the advantage of generating three-dimensional (3D) reconstructions of a cellular structure in a fully hydrated and unstained environment, providing more exhaustive and complete insights into its organization and possible functions. In ET multiple images are captured as the sample is tilted along an axis. The images are then aligned and merged using computational techniques to reconstruct a 3D picture, or tomogram. ET can be combined with immunostaining, allowing localization of proteins in the 3D reconstructionsHowever, ET often requires serial sectioning to reconstruct the 3D model of the entire structure, and the combination of such laborious approaches could be technically challenging. Also, it should be considered that the 3D reconstructions created from ET tilt series of images are not complete representations. This is due to the limited tilt range of the microscope holder in ET that leaves the 3D reconstruction with regions empty of information appearing as undefined cone shaped areas . Nonetheless, combined with advances in cryo-ET sample preparation, such as the introduction of focused ion beam milling to thin samples to ideal thicknesses for imaging, the potential for cryo-ET imaging is constantly increasing.105. However, despite recent improvements, 3D-SEM resolving power is still limited and requires extended research time and computer power for processing the large amount of datasets produced.Volume EM techniques based on scanning electron microscopy (SEM) enable high-resolution 3D imaging of large specimen volumes. 3D-SEM techniques overcome the artifact of the “missing wedge” in ET, and are therefore a potent tool for gaining insight into the 3D morphology of contactsThe biochemical characterization of contact sites occurred contemporaneously with their discovery by EM. Commonly, protocols for isolating membrane contacts utilized subcellular fractionation followed by sucrose gradient centrifugation. Any successful isolation of heterotypic contacts must consider that these have features of two organelles or maybe of neither. Moreover, purification of such contact regions can be challenging as they need to resist dilution and cell fractionation. Additionally, during the lengthy fractionation procedure, potential protein modifications and interactions (such as phosphorylation or dimerization) might be reversed, which could destabilize or alter contacts.106. The characterization of lipid-synthesizing membranes in the early 1970s assigned mitochondrial lipid synthesis to these contacts, although this was not formally recognized109. Following this insight, attempts to subfractionate domains of the ER led to further insight about this contact such as that it comprised only a specific portion of the rough ER membrane112. The real breakthough came with the identification of specific enzymes enriched in these domains, cementing the understanding that these were unique sites of lipid transfer between the two organelles115. These sites, from thereon termed mitochondria-associated membranes, became the first functional characterization of a contact-site function.Most of the early protocols that biochemically detected interorganellar contacts dealt with areas of strong association between ER and mitochondria. As early as the 1950s, it was observed that liver mitochondria preparations obtained from sucrose fractionation were “always contaminated with ER”116, but the significance of this finding, in particular for Ca2+ entry, was initially overlooked. PM-associated membranes were also shown to have unique lipid biosyntehtic capacities6.Similar to ER associations with mitochondria, the isolation of the PM via sucrose gradients from reticulocytes led to the identification of ER tethered to the PM119 takes advantage of the sustained attachment of ER tubules to peroxisomes under conditions that separate mitochondria from peroxisomes. Moreover, they have enabled proteomic analysis of these contacts117.The biochemical isolation of ER–peroxisome contacts (EPCONS)Hence, biochemical fractionation assays for contact sites are important tools for studying functional aspects of the contacts as well as identifying their unique lipid composition and resident proteins. Unfortunately, such protocols have not yet been developed for all contact sites.120 has been used to identify new contact-site proteins121. At the heart of this methodology lies an engineered version of ascorbate peroxidase (APEX), which can catalyze the oxidation of biotin phenol. This radical version of phenol is both short lived (<1 ms), has a small labeling radius (<20 nm), and can covalently react with electron-rich amino acids . Thus, by localizing the APEX protein to a contact site and then shortly exposing the cell to hydrogen peroxide and biotin phenol, all proteins within this compartment will be modified by biotin. Biontinylated proteins can then be easily extracted using the well-established streptavidin system, and identified via mass spectrometry. If no proteins are known for a specific contact it is possible to map the entire proteome of the outer membrane of two organelles and then look for overlapping proteins which may be residents of the contact sites122. APEX has also been used alongside biochemical fractionation to reach a better purity of contact residents121. In addition, a split APEX123 has recently been created, enabling each half to be expressed on one organelle and complementation to occur only at contact sites. Similarly, a nonspecific biotin ligase such as the BirA* can be used to biotinylate proximal proteins in a method called BioID125. A split-BioID has been described126 which, again, can be used to study contact sites by labeling two opposing membranes and retrieving biotinylation activity only at the areas of interface.A new way to define contact-site proteins is by using biotinylation approaches. APEX127 and Ca2+53 at ER–mitochondria or at ER–PM contacts. Such approaches should be developed to appraise if changes in contact extent or composition affects cellular physiology. Appropriate functional analyses need to be performed in combination with the imaging approaches detailed above, to draw conclusions on whether contact sites are present and/or changed in the studied system.Since each contact site has its unique function, different approaches will be required to measure the functionality of a contact. For example, a great deal of attention has been given to transfer of lipids2+ transfer).Care should be taken not to confuse a lack of effect on any singular function as an indication that a protein is not a tether or a functional contact-site protein. This is because deletion of a tether is often backed up by many other tethering molecules and its effect will therefore not necessarily be measurable if a function that it does not carry out is measured  function in both tethering as well as mitochondrial protein translocation. In such cases, it is necessary to generate separation-of-function alleles to inactivate just contact sites while maintaining the other function.Conversely, many tethers have functions outside of the contact site and thus their loss-of-function phenotype may not be due to their contact-site role. To give just one example, many of the mitochondrial contact-site proteins in yeast , does their molecular composition differ? What is the repertoire of functions carried out at contact sites? How does the loss of each contact affect cellular physiology and organismal function? How are contact sites regulated and co-regulated to maintain cellular homeostasis? And, given the apparent inability to completely deconstruct contact sites, what mechanisms exist that compensate malfunctions?131, interest in these structures will only increase. We anticipate that in the next decade the above questions will start to be addressed, calling for extended guidelines that help define good practices in these additional areas of contact-site research.As it is becoming clear that contact sites affect a variety of diseases


Intermolecular aliphatic C–H functionalizations using nitrogen-centered radicals with reagent-dictated site selectivities are described, including reactions targeting primary C–H bonds across a range of simple and complex substrates. The site selectivities of intermolecular, aliphatic C–H bond functionalizations are central to the value of these transformations. While the scope of these reactions continues to expand, the site selectivities remain largely dictated by the inherent reactivity of the substrate C–H bonds. Herein, we introduce reagent-dictated site selectivity to intermolecular aliphatic C–H functionalizations using nitrogen-centered amidyl radicals. Simple modifications of the amide lead to high levels of site selectivity in intermolecular C–H functionalizations across a range of simple and complex substrates. DFT calculations demonstrate that the steric demand of the reacting nitrogen-centered radical is heavily affected by the substitution pattern of the starting amide. Optimization of transition state structures consistently indicated higher reagent-dictated steric selectivities using more hindered amides, consistent with experimental results. Recent advances in intermolecular aliphatic C–H functionalization have provided unique tools for the synthesis and late-stage derivatization of complex molecules.via the use of modified ligand architectures in catalytic C–H functionalizations proceeding via high-valent metal oxo or metal carbenoid intermediates.4Reagents or catalysts that override inherent substrate-dictated site selectivity hold significant promise in expanding the capabilities of intermolecular C–H functionalization. Noteworthy advances in this area have been achieved N-halo- and N-xanthylamides.We have previously developed practical systems for aliphatic C–H functionalization using readily accessed trans decalin, a benchmark substrate used in studies of intermolecular, aliphatic C–H functionalizations.trans decalin with bulky tetradentate ligands poorly discriminated between these sites (entries 1 and 2).N-chloroamide 3 proceeded with no selectivity between the methylene sites (entry 4).5We commenced our studies with the functionalization of N-chloroamide would offer improved site selectivities. We thus evaluated a set of N-chloroamides containing ortho trifluoromethyl substitution of the benzamide, which are readily available on scale (>10 grams). Reagents 4 and 5 containing a single ortho substituent increased the selectivity for the less hindered, distal methylene site, although the ratio of 1 : 2 remained close to 2.0 : 1 (entries 5 and 6). We next sought to prepare the highly hindered 2,6-bistrifluoromethyl reagent 6, which was obtained via addition of the aryl Grignard to tBu isocyanate.trans decalin with 6 proceeded with higher site selectivity than the other N-chloroamides or metal-catalyzed systems, with a 6.5 : 1 ratio of products (entry 7). Performing the reaction with 1 equiv. of substrate provided the products in a 7.2 : 1 ratio in 76% combined yield (entry 8). To the best of our knowledge, this is the most site-selective secondary C–H functionalization of trans decalin known using substrate as limiting reagent.We hypothesized that increasing the steric demand of the ld entry . To the n scale > grams. R6 across a range of substrates. As shown in 6 is a free-flowing white powder that is readily available on scale. The C–H chlorination of these substrates with N-chloroamide 6 also proceeded with unique site selectivities, and notably includes radical functionalizations selective for primary sites. Site selectivities with our first-generation reagent 3 are shown for direct comparison. For example, the chlorination of 3-methylpentane (7) with reagent 3 showed a preference for the 2-position (64%) over the 1-position (27%). However, reagent 6 provided a remarkable reagent-dictated switch in site selectivity, favoring the 1-chloro isomer . Free radical species demonstrating such selectivity for the strongest, primary C–H bond over secondary or tertiary C–H bonds in synthetic alkane functionalizations are unknown to the best of our knowledge.11We next evaluated the site selectivities of the C–H chlorination using reagent 8) and 2,2-dimethylbutane (9) demonstrate that fully substituted carbon centers enable steric discrimination between C–H sites, with 89% primary C–H selectivity in the reaction of substrate 9 with N-chloroamide 6. There was an increase in the level of primary functionalization of n-hexane (10) with reagent 6, however the 2-position remained the preferred site of functionalization.Our efforts continued with the C–H chlorination of diverse compounds . The chl11 and 12, respectively. The functionalization of 2-phenylbutyl benzoate (13) remarkably demonstrated a high selectivity for the primary position (89%) even in the presence of a benzylic C–H site. The fully substituted carbon centers in acetal 14 and ester 15 lead to a clear reagent-dictated change in selectivity between reagents 3 and 6; the primary C–H chlorination product is favored with up to 75% selectivity with ester substrate 15.The presence of electron-withdrawing functionality disfavors reaction at proximal methylene sites and increased the site selectivity for the primary positions to 88% and 89% for substrates 16 with reagent 6 only increased the level of primary functionalization to a modest 33%. In the case of substrate 17 containing chain branching, a higher level of primary functionalization was observed (45%). The shorter aliphatic chain in the norleucine derivative 18 electronically disfavored methylene functionalization, and the use of hindered N-chloroamide 6 resulted in good primary selectivity (80%).Phthalimide-protected amines undergo intermolecular C–H functionalizations using amidyl radicals with high levels of electronically-dictated site selectivities.N-chloroamide 6 is also evident in reactions with cyclic substrates 19–21. Each of these structures contain eight methylene and either three or six methyl C–H bonds for chlorination. In all cases C–H chlorination with N-chloroamide 3 was highly methylene selective. Switching to reagent 6 for the chlorination of these substrates significantly increased the levels of methyl group functionalization. This unique selectivity profile enabled the 1° C–H functionalizations of methyl groups in complex substrates (vide infra).The reagent-dictated site selectivity provided by 6 in the aliphatic C–H functionalization of complex natural products  in 59% combined yield  basis set, and Gaussian16 software . On the other hand, the aromatic ring of 6′ is out of the carbonyl plane  owing to the steric hindrance of the two ortho CF3 substituents. As a result, several hydrogen atoms of the tert-butyl group are within a <3 Å distance of the fluorine atoms in 6′, consistent with a highly congested chemical environment around the radical center.The calculated structures of amidyl radicals 3′ and 6′ involving select substrates, with the goal of identifying important factors that account for the observed site selectivities. We studied a diverse set of cyclic and acyclic substrates, including trans decalin, 3-methylpentane (7) and 3-pentyl acetate–a model for the benzoate substrate 12. Representative TS for the abstractions are shown in 7) involving amidyl radicals 3′ (left) and 6′ (right). The reactive site is much more crowded in the case of 6′  of the H-atom abstractions by se of 6′ , right, f 6′  and changes (ΔG) for the C–H abstractions evaluated are listed in G values), regardless of the substrate, position, or amidyl radical considered. Comparisons of the energy barriers involved indicate different trends for the two reagents, however. In particular, the last column of G‡ values quantifying the energetic difference between the two amidyl radicals for reaction at a given C–H site. For example, amidyl radical 6′ is more reactive than 3′ in the abstraction at the methylene C–H site distal from the ring fusion of trans decalin, as indicated by the negative ΔΔG‡ value  shows the largest (in absolute terms) calculated ΔΔG‡ value , which is consistent with experimental results; functionalization at this position involves the largest selectivity change upon switching from 3′ to 6′ (27% to 69%, see 6 consistently provided higher selectivities for the least hindered positions of substrates–either primary or distal—throughout our studies.In the case of acyclic substrates, amidyl radical N-functionalized amides has led to C–H functionalization of diverse substrates that are among the most site-selective intermolecular C–H transformations known. This strategy has also enabled the first radical-mediated aliphatic C–H functionalizations targeting primary C–H bonds across a wide range of simple and complex substrates. Computational studies revealed the different chemical environments of the reacting amidyl radicals, and established a direct correlation between the steric hindrance at the reacting site and the preference for functionalization of the least hindered substrate C–H bonds. Future studies will continue to develop unique reagents that offer increased levels of site selectivity in intermolecular C–H functionalization for applications in chemical synthesis.In conclusion, we have developed an approach to introduce reagent-dictated site selectivity to aliphatic C–H functionalizations using tuned nitrogen-centered radicals. The use of hindered There are no conflicts to declare.Supplementary informationClick here for additional data file.Crystal structure dataClick here for additional data file.


Molecular interactions of plumbagin inclusion complexes with β-cyclodextrin (BCD), dimethyl-β-cyclodextrin (MBCD), and hydroxypropyl-β-cyclodextrin (HPBCD) were investigated by semi-empirical, Parameterization Method 6 and 7  in the aqueous phase using polarizable continuum calculations. The results revealed two different binding modes of the plumbagin molecule inside the BCD cavity with a negative value of the complexation energy. In conformation-I, the hydroxyl phenolic group of plumbagin was placed in the BCD cavity near the narrow-side of the host molecule. In the other model, conformation-II, the methyl quinone group of plumbagin was placed in the cavity of BCD near the narrow-side of the host molecule. The higher the negative value of the complexation energy, the more favorable is the pathway of inclusion-complex formation. Molecular modeling techniques are currently widely used in chemistry and pharmacology to obtain insight into information at the molecular level of systems of interest. The computational results help explain the molecular interactions and suggest the mechanisms that govern the processes when experimental techniques are insufficient. The calculation models are able to predict and screen the results when varying the compounds or the system conditions prior to laboratory tests. Semi-empirical quantum mechanical calculations have been successful for descriptions in organic chemistry because some parameters are approximated or generalized to simplify the calculation or to yield a result based on experimental data. Modern semi-empirical quantum mechanical models such as Parameterization Method 6 and 7 (PM6 and PM7) are often used to explore the electronic structure dependent properties of large molecules, where ab initio electronic structure methods (without approximations) are too expensive . The PM7β-cyclodextrin is a nontoxic cyclic oligosaccharide comprising seven α-D-glucoses . The innPlumbago indica root [Plumbagin  is an organic compound extracted from the ica root . Plumbagica root . There aica root ,10,11. UTo understand the properties of the complex geometries and the encapsulation process, we used semi-empirical PM6 and PM7 methods to describe the complexation of plumbagin/BCD, plumbagin/MBCD, and plumbagin/HPBCD systems in water. The complex energy, molecular interactions, and insertion pathway of plumbagin/BCDs were examined.All calculations were performed using GaussView 6.0 and Gaussian 16 software packages . The cryAutoDock 4.2.6  with theThe first step was to prepare the guest (plumbagin) and host (BCDs) coordinate files to include the information needed by AutoGrid and AutoDock. The non-polar hydrogens were deleted and their charges were merged with the carbon atoms. The atom types were assigned, defining hydrogen bond acceptors and donors and aromatic and aliphatic carbon atoms. The rotatable bonds of the guests were defined while the hosts were kept fixed. AutoGrid was used to calculate the grid maps, one for each atom type present in the guest being docked. The systems were investigated in a three-dimensional volume divided into many small grid boxes with a grid spacing of 0.375 Å. The grid center of the boxes were set at the center of the host molecules. The box has x × y × z dimensions of 14.25 Å × 14.25 Å × 7.50 Å, 15.75 Å × 14.25 Å × 9.75 Å and 18.75 Å × 14.25 Å × 9.00 Å for BCD, MBCD, and HPBCD, respectively. AutoDock used the Lamarckian genetic algorithm to calculate the conformational states of a flexible guest, using the grid maps generated by AutoGrid to evaluate the guest–host interaction at each point in the docking simulation. One hundred docking calculations were performed on each guest–host complex. The results were clustered to identify similar conformations based on all-atom root mean square deviation within 2 Å. At the end of molecular docking calculations, AutoDockTools was used to perform a cluster analysis of the different docked conformations. The lowest energy representative docked conformation from molecular docking was selected for further full geometry optimization.E) as being the difference between the heat of formation of the complex and the heat of formation of the involved free moleculesPL/BCDE, PLE, and BCDE represent the heat of formation of the complex, isolated plumbagin molecule, and isolated BCD molecule, respectively.The selected docked conformation of plumbagin/BCD inclusion complexes was then fully geometry optimized by the PM6 and PM7 methods. All atoms were allowed to move freely in an aqueous environment. The most stable conformation of the plumbagin/BCD, plumbagin/MBCD, and plumbagin/HPBCD inclusion complexes were selected by considering the complexation energy  from PM7  were considerably lower than PM6 . The PM7 method includes the description of the dispersion interaction and hydrogen bonding [The difference in ΔE between the two conformations (conformation-I–conformation-II), is also presented in (host)…H(OH-PL)). The second one, found only in plumbagin/BCD complexes, is from an oxygen atom of plumbagin’s carbonyl group and the hydrogen atom of the secondary hydroxyl group at O3 of BCD-I (O(CO-PL)…H(O3H-BCD)) and the primary hydroxyl at O6 of BCD-II (O(CO-PL)…H(O6H-BCD)). The third one, is found only in HPBCD-II, between an oxygen atom of plumbagin’s hydroxyl group and the hydrogen atom of the secondary hydroxyl at O2 of HPBCD (O(OH-PL)…H(O2H-HPBCD)). The molecular interactions of each host–guest system in an aqueous environment are further discussed below.(CO-PL)···H(O3H-BCD)), as shown in Two conformations of a plumbagin/BCD inclusion complex can be formed in an aqueous environment, as shown in The plumbagin molecule is located near the wide-side of the MBCD molecule in all complex conformations, as shown in The inclusion complex of plumbagin and HPBCD in conformation-I and conformation-II were stabilized in a water environment, as shown in (OH-PL)…H(O2H-HPBCD)), as mentioned in PM6 and PM7 calculations yield different HPBCD-II energy-minimized structures in a water environment. In HPBCD-II, for the PM6 calculation, the guest molecule is located near the wide-side of HPBCD b, due toThe complexation energy values of each system obtained by the PM7 method are significantly lower than those obtained by the PM6 method. The obtained results agree with the experimental data for a 1:1 guest:host ratio of plumbagin with BCD and HPBCD inclusion complexes. We predict that by using MBCD to increase the solubility and reduce the cytotoxicity of the plumbagin compound, a 1:1 guest:host inclusion complex can be produced. Our results revealed two different binding modes of the plumbagin molecule inside the BCD cavity. In conformation-I, the hydroxyl phenolic group of plumbagin was placed in the BCD cavity near the narrow-side of the host molecule. In the other model, conformation-II, the methyl quinone group of plumbagin was placed in the cavity of BCD near the narrow-side of the host molecule. The intermolecular hydrogen bond, van der Waals, and hydrophobic interactions play an important role in complexation process of plumbagin with BCDs.


Tripterygium wilfordii Hook F.) towards three important UGT isoforms UGT1A6, UGT1A9 and UGT2B7. The results showed that 100 μM of demethylzeylasteral exhibited strong inhibition towards UGT1A6 and UGT2B7, with negligible influence towards UGT1A9. Furthermore, Dixon and Lineweaver-Burk plots showed the inhibition of UGT1A6 and UGT2B7 by demethylzeylasteral was best fit to competitive inhibition, and the inhibition kinetic parameters (Ki) were calculated to be 0.6 μM and 17.3 μM for UGT1A6 and UGT2B7, respectively. This kind of inhibitory effect need much attention when demethylzeylasteral and demethylzeyasteral-containing herbs  were co-administered with the drugs mainly undergoing UGT1A6, UGT2B7-catalyzed metabolism. However, when extrapolating the in vivo clinical results using our present in vitro data, many complex factors might affect final results, including the contribution of UGT1A6 and UGT2B7 to the metabolism of compounds, and the herbal or patients’ factors affecting the in vivo concentration of demethylzeylasteral.Inhibition of UDP-glucuronosyltransferase (UGT) isoforms can result in severe clinical results, including clinical drug-drug interactions (DDI) and metabolic disorders of endogenous substances. The present study aims to investigate the inhibition of demethylzeylasteral (an important active component isolated from  Tripterygium wilfordii Hook F., commonly known in China as Lei-Gong-Teng, has been used to treat rheumatoid arthritis, chronic nephritis, ankylosing spondylitis and various skin diseases [Tripterygium wilfordii Hook F., has been demonstrated to exhibit some pharmacological activity, including immunosuppressive effects [diseases ,2,3. Dem effects . Ginkgo biloba [Herb-drug interactions have become a challenging problem in clinical application of herbs, and one of the major reasons is the influence of herbs on the pharmacokinetic behaviour of drugs . In prevo biloba , flavonoo biloba , and Dano biloba .UDP-glucuronosyltransferases (UGTs) are the most important enzymes catalyzing the conjugation of various xenobiotics and endogenous substances, which accounts for >35% of all phase II drug metabolism . Many imFor investigation of compounds’ inhibition towards CYP isoforms, many specific probe substrates and human liver microsomes have been developed. In contrast, few specific probe substrates exist for UGT isoforms. Therefore, when evaluating the inhibition of compounds towards UGT isoforms, the non-specific substrate 4-MU was the routine probe, and the recombinant UGT isoforms were commonly used as enzyme sources ,14.The aim of the present study is to investigate the inhibition of demethylzeylasteral towards three important UGT isoforms UGT1A6, UGT1A9 and UGT2B7. Furthermore, the detailed inhibition kinetic type and parameters were determined.50 values of 15.2 ± 0.6 μM and 62.5 ± 1.6 μM, respectively.At 100 μM of demethylzeylasteral, the residual activity of UGT1A6, UGT1A9 and UGT2B7-mediated 4-MU glucuronidation was 0.8 ± 0.1%, 61.3 ± 5.6% and 14.8 ± 1.1% of control group. Furthermore, inhibition kinetic analysis was carried out to determine the inhibition type and kinetic parameters for UGT1A6 and UGT2B7, which activities were inhibited by more than 50% at a concentration of 100 μM. The results showed that demethylzeylasteral exhibited a concentration-dependent inhibitory behaviour towards UGT1A6 A and UGTvs. the concentration of demethylzeylasteral was used to determine the inhibition kinetic parameters (Ki), and the values were calculated to be 0.6 μM and 17.3 μM for UGT1A6 and UGT2B7, respectively. These results were consistent with the previous literature in which another active component of Tripterygium wilfordii Hook F. celastrol  also exhibited strong inhibition towards UGT1A6 and UGT2B7 [Dixon B and Lind UGT2B7 .trans retinoic acid) [UGT1A6 is involved in the glucuronidation of various drugs, toxins, and endogenous substrates, including acetaminophen, benzopyrene, and serotonin ,16. The ic acid) ,18.in vivo clinical results using our present in vitro data, many complex factors might affect final results, including the contribution of UGT1A6 and UGT2B7 to the metabolism of compounds, and the herbal or patients’ factors affecting the in vivo concentration of demethylzeylasteral. It should be noted that the inhibition of demethylzeylasteral towards other UGT isoforms are not be included in this study, and the future experiments will be performed to examine this factor.Taken together, demethylzeylasteral showed strong inhibition towards UGT1A6 and UGT2B7, with negligible influence towards UGT1A9. This kind of inhibitory effect needs much attention when demethylzeylasteral and demethylzeyasteral-containing herbs are co-administered with the drugs mainly undergoing UGT1A6, UGT2B7-catalyzed metabolism. However, when extrapolating the Demethylzeylasteral with purity ≥98% was obtained from Weikeqi Biotechnology Co. Ltd. . 4-Methylumbelliferone (4-MU), 4-methylumbelliferone-β-D-glucuronide (4-MUG), Tris-HCl, 7-hydroxycoumarin and uridine 5′-diphosphoglucuronic acid (UDPGA)  were purchased from Sigma-Aldrich . Recombinant UGT1A6, 1A9 and UGT2B7 expressed in baculovirus were obtained from BD Gentest Corp. . All other reagents were of HPLC grade or of the highest grade commercially available.2, 50 mM Tris-HCl buffer (pH 7.4), and 4-MU in the absence or presence of different concentrations of demethylzeylasteral. The concentrations of 4-MU were as follows: 110 μM for UGT1A6, 30 μM for UGT1A9 and 350 μM for UGT2B7. Demethylzeylasteral was dissolved in methanol and the final concentration of methanol was 0.5% (v/v). After 5 min pre-incubation at 37 °C, the UDPGA was added to the mixture to initiate the reaction. Incubation time was 120 min for UGT2B7, and 30 min for UGT1A6 and UGT1A9, respectively. The reactions were quenched by adding 100 μL acetonitrile with 7-hydroxycoumarin (100 μM) as internal standard. The mixture was centrifuged at 20,000 ×g for 10 min and an aliquot of supernatant was transferred to an auto-injector vial for HPLC analysis.4-MU, the nonspecific probe substrate for UGT isoforms, was employed to investigate the inhibition of UGT1A6, UGT1A9 and UGT2B7 by demethylzeylasteral. The mixture  contained recombinant UGTs  5 mM UDPGA, 5 mM MgCl2O containing 0.5% (v/v) formic acid (A) and acetonitrile (B). The following gradient conditions were used: 0–15 min, 95–40% A; 15–20 min, 10% A; 20–30 min, 95% A. To determine the inhibition kinetic type and calculate the inhibition parameters, various concentrations of demethylzeylasteral were added to reaction mixtures containing different concentrations of 4-MU. Dixon and Lineweaver plots were adapted to determine the inhibition type, and the second plot of slopes from Lineweaver-Burk plot vs. demethylzeylasteral concentrations was utilized to calculate Ki value.The HPLC system  containing a SCL-10A system controller, two LC-10AT pumps, a SIL-10A auto injector, and a SPD-10AVP UV detector was used in the present study. C18 column  was employed to seperate the substrate and metabolites at a flow rate of 1 mL/min with UV detection at 316 nm. The mobile phase consisted of HAmong the tested UGT isoforms, UGT1A6 and UGT2B7 were inhibited by demethylzeylasteral, which indicated the necessity to monitor the potential clinical interactions between dimethyl- zeylasteral-containing herbs and clinical drugs mainly undergoing UGT1A6, UGT2B7-catalyzed metabolism.


It is always difficult to assign the peaks of a vibrational spectrum in the far-infrared region. The two distinct peaks seen in many ice phases are still a mystery to date. The normal modes of ice XV were calculated using the CASTEP code based on first-principles density functional theory. On the basis of vibrational modes analysis, we divided the translational modes into three categories: four-bond vibrations, which have the highest energy levels; two-bond vibrations, which have medium levels of energy; and relative vibrations between two sublattices, which have the lowest energy. Whale et al. found that some intramolecular stretching modes include the isolated vibration of only one O–H bond, whereas the others do not vibrate in ice XV. We verified this phenomenon in this study and attributed it to local tetrahedral deformation. Analysis of normal modes, especially in the translation and stretching band of ice XV, clarified the physical insights of the vibrational spectrum and can be used with other ice phases. Ice VI consists of two interpenetrating yet independent hydrogen-bonded networks, and has thus been described as a “self-clathrate” [More than 17 ice phases have been experimentally identified at various temperatures and pressures . The onlathrate” .In 2009, Salzmann et al. found a new hydrogen-ordered phase by doping HCl in ice VI at a lower temperature, which is called ice XV. This hydrogen-ordered structure is antiferroelectric ow 130 K . Whale eow 130 K . In 2018ow 130 K .For theoretical investigations of vibrational modes, the most interesting results were those of Whale et al., who found exotic “rigid network” molecular vibrations in the translation band and “isolated stretching” modes of atomic vibrations in the stretching band . We also3, in which the three orthorhombic constants of a primitive cell are a = 6.23 Å, b = 6.22 Å, and c = 5.83 Å. Using the CASTEP [−9 eV/atom. The energy cutoff was 750 eV, and the k-point was 2 × 2 × 2. As the electron density has a large distribution in ice lattice, we chose generalized gradient approximation (GGA) for this work. To test the exchange-correlation (XC) functional, we tried RPBE, PBE, PW91, WC, and PBESOL separately for geometric optimization. The results showed that the densities are 1.21, 1.40, 1.41, 1.55, and 1.56 g/cm3, respectively. As the length of intermolecular hydrogen bond (H-bond) is relative to volume change and the vibrational frequency is very sensitive to H-bond length, the best selection is RPBE [We first constructed the oxygen structure of ice VI. Then, we used a self-compiled program to find two possible hydrogen-ordered structures. We selected the antiferromagnetic one that is the same as reported by Whale for this work. The density is 1.32 g/cme CASTEP  code bas is RPBE , which p−1 [−1 did not match any normal mode. We doubt that this is a false peak because a gap should be present between the translation and libration bands. There is a slight underestimation of the hydrogen bond (H-bond) by RPBE, but it is more accurate than Whale’s work by PBE [−1 . Howeverk by PBE .−1. Note that the peaks are matched with normal modes by the calculated intensities.In the translational band, the Raman peaks are from optic modes near the BZ that are detectable subject to selection rules. On the basis of harmonic approximation, 27 optic normal modes are present between 73 and 285 cmOur previous study of ice Ic revealed two kinds of intrinsic translational modes in the ice lattice . In the −1 shown in −1 is a two-bond mode, in which a molecule vibrates toward its two neighbors (please see −1 present vibrations of the two rigid ice XV networks. In this study, the three modes are at 73, 106, and 107 cm−1, respectively. They actually represent three orthorhombic vibrations in 3D space that originate from the interactions of two sublattices without H-bonds, and thus present the lowest vibrational energy. −1 (please see the dynamic process from Although the tetrahedral structure of ice XV presents deformation under pressure to some extent, these two kinds of modes can also be distinguished in the translation region. For the mode at 285 cm−1. There are three types of vibration modes: rocking, or rotation of the whole molecule around an axis perpendicular to the molecular plane; twisting, or rotation around an axis coincident with the HOH angle bisector; and wagging, or rotation of the molecule around an axis in the molecular plane, perpendicular to the bisector of the HOH angle. For an optic mode, the mass center of the primitive cell remains static while the molecules within vibrate. −1 is rocking, whereas the mode at 913 cm−1 is twisting. For the highest frequency at 936 cm−1, the vibrating mode of molecules is a mixture of wagging, rocking, and twisting.As shown in −1, as shown in −1, the vibrating mode of crystal lattice is in-phase. The lowest mode at 1609 cm−1 is out-of-phase. The mode at 1684 cm−1 shown in −1 has the greatest intensity of Raman activity, but no IR activity in this band. Because the intensities are very small compared with the stretching band, it is always difficult to detect signals for IR and Raman experiments.The bending band has 10 normal modes from 1609 to 1709 cm−1, including intramolecular symmetrical and asymmetrical stretching vibrations. For the lowest frequency at 3281 cm−1, all molecules show symmetric stretching. For the frequency at 3461 cm−1, the main vibrations show asymmetric stretching. The mode at 3280 cm−1 has the greatest intensity of Raman activity, but no IR activity, whereas the opposite is found in the mode at 3278 cm−1.There are 20 normal modes in the stretching band from 3278 to 3485 cm−1 (please see Whale et al. reported the normal modes in this band; some vibrational modes include isolated vibration of only one O–H bond, whereas the other does not vibrate . A typicIt is traditionally difficult to assign the peaks of a vibrational spectrum in the fingerprint region. We analyze the normal modes of ice XV via DFT simulation. On the basis of our series of investigations in the translational band of ice, we divide normal modes into three categories: four-bond vibrations that possess the highest energies; two-bond vibrations that possess medium energies; and cluster vibrations that have the lowest energy. In ice XV, VII, and VIII, the cluster modes are relative vibrations between two sublattices. Thus, we can analyze the translational modes in the far IR region of ice. This study presents physical insights into the vibrational spectrum and can be used with other ice phases.In addition, we reviewed the “isolated stretching” mode first reported by Whale et al. As we also found this phenomenon in other ice phases, we do not regard this mode as a third type in addition to symmetric and asymmetric stretching, and instead attribute it to a special asymmetric stretching due to local tetrahedral deformation by pressure and a hydrogen-disordered lattice.


Riemerella anatipestifer (R.anatipestifer) isolated from diseased ducks in China. For this purpose, a total of 48 genome sequences were investigated, which comprised 30 publicly available R. anatipestifer genome sequences, and 18 clinical isolates genomes sequences. Two ICEs, named ICERanRCAD0133-1 and ICERanRCAD0179-1 following the classic nomenclature system, were identified in R. anatipestifer through the use of bioinformatics tools. Comparative analysis revealed that three ICEs in Ornithobacterium rhinotracheale showed a high degree of conservation with the core genes of ICERanRCAD0133-1, while 13 ICEs with high similarity to ICERanRCAD0179-1 were found in Bacteroidetes. Based on the definition of ICE family, ICERanRCAD0179-1 was grouped in CTnDOT/ERL family; however, ICERanRCAD0133-1, which had no significant similarity with known ICEs, might be classified into a novel ICE family. The sequences of ICERanRCAD0133-1 and ICERanRCAD0179-1 were 70890 bp and 49166 bp in length, had 33.14 and 50.34% GC content, and contained 77 CDSs and 51 CDSs, respectively. Cargo genes carried by these two ICEs were predicted to encode: R-M systems, IS elements, a putative TonB-dependent receptor, a bacteriocin/lantibiotic efflux ABC transporter, a tetracycline resistance gene and more. In addition, phylogenetic analyses revealed that ICERanRCAD0179-1 and related ICEs were derived from a common ancestor, which may have undergone divergence prior to integartation into the host bacterial chromosome, and that the core genes co-evolved via a related evolutionary process or experienced only a low degree of recombination events during spread from a common CTnDOT/ERL family ancestor. Collectively, this study is the first identification and characterization of ICEs in R. anatipestifer; and provides new insights into the genetic diversity, evolution, adaptation, antimicrobial resistance, and virulence of R. anatipestifer.We report for the first time the occurrence of integrative conjugative elements (ICEs) in  Integrative conjugative elements (ICEs), a type of self-transmissible mobile genetic elements (MGEs) are widely distributed in bacterial genomes. ICEs are major mediators for horizontal gene transfer (HGT), which contribute to microbial evolution and adjustment to new niche , 2. ICEsR.anatipestifer, a member of the Flavobacteriaceae family, is a non-spore-forming and rod-shaped Gram-negative bacterium. It is a major contributor to acute and chronic septicemia associated with fibrinous pericarditis, perihepatitis, airsacculitis, and meningitis  for the first time. Analysis of coding sequences (CDS) within the accessory regions of the two ICEs identified several putative virulence factors and an antibiotic resistance gene, which can confer hosts with selective advantages beneficial for niche adaptation. Alignment and comparative analysis of those two ICEs revealed that homologous ICE-like elements are prevalent in other bacterial genomes, and shed light on their phylogenetic relationships and on their modular evolution.Here, we investigated 48 R.anatipestifer were obtained from the livers of infected ducks and identified using PCR. Among them, 9 strains obtained from Culture Collection of the University of Gothenburg (CCUG)  . All anide novo assembly was executed with MHAP (de novo assembly was conducted using Velvet version 1.2.09 (http://www.ncbi.nlm.nih.gov/genome/annotation_prok). The average nucleotide identity (ANI) of all the R. anatipestifer involved in this study was calculated by pyani  with the ANIb algorithm.The sequence of strain RCAD0133 was determined by applying a single-molecule real-time sequencing platform with a PacBio RS II sequencer. The raw data were filtered, and then, a ith MHAP . The remn 1.2.09 . The 18 R.anatipestifer genome sequences (18 were newly sequenced and 30 sequences were publicly available in the NCBI database) were analyzed using bioinformatics tools. Islandviewer 4 was used to predict genomic islands; also the secretion systems were identified via MacSyFinder, then the genomic islands containing a type IV secretion system (T4SS) (Ran elements were standardized using the PATRIC annotation tool (All 48 m (T4SS)  and a rem (T4SS) –14. ICEsion tool , 16. BLAion tool , 18. Easion tool . For amiion tool .Riemerella taxon.Searching homologous regions (ICE-like elements) for the putative ICE were performed by NCBI BLASTn comparison analysis using the original confirmed ICE sequence as a probe against the NCBI non-redundant nucleotide sequences(nr) database and the whole-genome shotgun contigs (wgs) database limited by infB, gyrB, and rpoB; ICE elements core genes and the analogous ICE-like elements core genes were extracted, concatenated, and aligned using MUSCLE  and inserted into the vicinity of hypothetical protein at position 991320-1062210, yielding 13 bp direct repeats (5'-AAGTTGAGTTACT-3') representing attL and attR sites. Similarly, ICERanRCAD0179-1 was a 49166 bp genomic region with 51 CDSs and inserted into position 54705-103871 of contig (QXQM00000004) of isolate RCAD0179 near an Arginyl-tRNA synthetase, where it yielded 7 bp direct repeats (5'-GTAACTT-3') located on either ends. The overall GC content of ICERan elements was 33.14% (ICERanRCAD0133-1) and 50.34% (ICERanRCAD0179-1). The G+C content of ICERanRCAD0133-1 (33.14%) was slightly lower than that of the rest of the chromosome (34.85% for the complete chromosomes of RCAD0133). However, the overall GC content of ICERanRCAD0179-1 was 50.34%, which was dramatically higher than the genomic GC content (34.58%) of R. anatipestifer RCAD0179. Comparison analysis of ICERanRCAD0133-1 and ICERanRCAD0179-1 was carried out through the BLASTn and BLASTp, which indicated that these two ICEs were not similar in DNA sequences or protein productions encoded by core genes and therefore were not classified into the same ICE family , which was found in different mycobacterium phage genomes, in Streptomyces coelicolor plasmid SCP1, and in bacterial genomes near various markers that suggest lateral gene transfer. Five insertion sequences (IS) were encoded by this ICE, which were all closely related to ISRa1, first found on the R.anatipestifer plasmid pCFC2, that belongs to the IS982 family; and is widely distributed in various R.anatipestifer strains with 2–20 copies  was carried by this ICE, which is an important contributor to the wide distribution of antibiotic resistance loci. A gene containing a lysozyme_like domain (cl00222) was found on ICERanRCAD0179-1, which is involved in the hydrolysis of β-1,4-linked polysaccharides. Bifunctional deaminase-reductase is involved in catalyzing the second and third steps in the biosynthesis of riboflavin. One such protein was present in ICERanRCAD0179-1, which contained a bifunctional deaminase-reductase domain  within the conserved region of ICERan elements confirmed the presence of core genes involved in ICE integration and excision, regulation, and maintenance, replication, and conjugative transfer.The two ICERanRCAD0133-1 and ICERanRCAD0179-1, respectively. An alignment of these two integrases with the characterized recombinases of the tyrosine integrase family suggested that they both contain a characteristic RHRH tetrad with conserved nucleophilic tyrosine at the C-terminus , regulators of excision and conjugative transfer , A single-stranded DNA-binding protein and a DNA topoisomerase that are involved in DNA replication, recombination and repair, and often participate in single-stranded DNA recombination and replication intermediates, respectively, were also found in this ICE  domain, a member of restriction-modification (R-M) systems. These solitary methylases might provide a broad protection from the host R–M systems  were identified. Moreover, this ICE had a series of two-component systems for ICE maintenance and regulation   , 40. A pRan elements among the sequences of ICE-like elements available in the GenBank database, using the nucleotide sequence of ICERan elements as a probe. BLASTn analysis of the ICE in RCAD0133 against the NCBI nr database, suggested that three ICE-like elements similar to ICERanRCAD0133-1 were identified in Ornithobacterium rhinotracheale specie  encoded by ICERanRCAD0179-1 and the closely related ICE-like elements to speculate on the evolution of these ICEs. Comparison of the phylogenetic tree topologies produced by neighbor-joining analysis of concatenated amino acid sequences of the conserved ICE CDS and protein products of ICE hosts chromosomal housekeeping gene  showed that ICERanRCAD0179-1 was clustered with most ICE-like elements in Bacteroides, and the ICE-like element in Riemerella columbina DSM 16469, and the evolutionary relationship of ICEs wasn't consistent with that of the ICE host genes  corresponding to significant ICE function, such as integration, exclusion determination, transfer, and regulation, respectively. Our analysis showed that ICERanRCAD0179-1 and homologous ICE-like elements in Bacteroides were very closely related and clustered into one branch, while the ICEPinkCOM1741-1 always formed a separate branch  in the R.anatipestifer genomes of RCAD0133 and RCAD0179, respectively. The ICE family is generally determined by the definition that they should have an integrase homology of ≥60% and significant structural synteny. Based on the significant similarity of ICE core modules, ICERanRCAD0133-1 and its related ICEs were grouped in one novel ICE family, due to lack of similarities to known ICE families (RanRCAD0179-1 and its 13 related ICEs were classified into the same ICE family (CTnDOT/ERL). The high prevalence of ICE-like elements similar to ICERanRCAD0179-1 indicated that interspecies genetic exchange also occurred via ICEs, thus broadening the host range, and dissemination of combined cargo genes across different strains. In accordance with the phylogenetic tree of the integrases of these two ICEs, the integrase of ICERanRCAD0133-1 was grouped in a cluster with Ornithobacterium rhinotracheale specie, and the integrase of ICERanRCAD0179-1 had a close phylogeny relationship with Bacteroides phylum because they were in one clade of the phylogenetic tree. Interestingly, integrases of the two ICEs were both classified in the tyrosine integrase family. Therefore, it can be envisaged that the two ICEs may have a similar integration or excision behavior for mobile element DNA because integrases of the ICERan elements are in the same family.Genomic sequence analysis first revealed two ICEs  of the host. The presence of arylsulfatase may contribute to the ability of the pathogen to cross the blood-brain barrier leading to meningitis (R.anatipestifer through participation in the regulation process of arylsulfatase. Based on previous research, we conferred that the putative TonB-dependent receptor encoded on this ICE may significantly influence R.anatipestifer pathogenicity via hemin and iron acquisition. Bacteriocins are synthesized ribosomally, and always display highly potent antimicrobial activity, their production relies on genes encoding a bacteriocin, a bacteriocin ABC transporter, and bacteriocin immunity proteins (ICE systems . IS elem systems . Therefoningitis –32. Inteproteins . The bacBacteroides group (RanRCAD0179-1 will be an important vehicle for the wide distribution of antibiotic resistance loci. It provided a competitive advantage for the host to rapidly overcome harsh environment. Comparison of the phylogenetic trees of ICERanRCAD0179-1 and related ICE elements against their hosts demonstrated that the compared ICEs were derived from a common ancestor that underwent divergence prior to integration into the host bacterial chromosome. Furthermore, through analysis of the phylogenetic trees based on the single core gene of these ICEs, it was identified that they shared almost the same topology structure of phylogeny, which indicated that the individual core genes most likely have not evolved independently; rather; these ICEs underwent a related evolutionary process or experienced only a low degree of recombination events during spread from a common CTnDOT/ERL family ancestor.The ICE element identified in isolate RCAD0179 had a significantly higher GC content than the host genome, which implied that this region was not native and had been adopted by HGT. Comparative analysis confirmed that it belonged to the CTnDOT/ERL family. The spread of resistance determinants was mostly contributed by the conjugative transfer of the CTnDOT/ERL element within the es group . A tetraRan elements in R.anatipestifer for the first time. In silico characterization of the ICERanRCAD0179-1 elements showed that similar ICE-like elements were found in other Bacteroides and that these ICEs are likely derived from a common ancestor. The two ICERan elements possess the core elements required for ICE DNA integration, maintenance and regulation, and dissemination. The presence of these core modules, however, can't prove that the ICERan elements can excise, circularize, conjugative transfer and then integrate within the chromosome. Genes encoding resistance to tetracycline antibiotics, DNA repair and recombination systems, virulence factors, toxin-antitoxin system, and regulation of conjugative transfer, were found to be present within cargo genes of ICERan elements. In particular, the putative antibiotic biosynthetic loci encoded on ICERan elements provide potential targets for the exploration of R.anatipestifer antibiosis phenotypes. ICERan elements could therefore play a significant role in providing beneficial phenotypes to their hosts. With the emergence of massive amounts of available genome sequences, it can be reasonably envisaged that more novel ICE elements will be identified and novel ICE families will be described. It may assist in studying the special mechanisms by which ICE impacts host metabolism and physiology.In conclusion, genomic analysis revealed two ICEDZ and AC conceived and designed the project. JW, JX, LZ, YL, and XC cultured bacteria and extracted genomic DNA. ZY, ML, RJ, SC, JX, and MW assembled and annotated the genomes. JW, XZ, QY, YY, and YW performed genomic islands analysis. JW, ZY, and SZ performed ICE identification and related ICE search. JW, DZ, and ZY performed the construction of the phylogenetic tree. JW and DZ drafted and revised the manuscript. All authors have read and approved the final version manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.