Small G-proteins are structurally-conserved modules that function as molecular on-off switches. They function in many different cellular processes with differential specificity determined by the unique effector-binding surfaces, which undergo conformational changes during the switching action. These switches are typically standalone monomeric modules that form transient heterodimers with specific effector proteins in the ‘on’ state, and cycle to back to the monomeric conformation in the ‘off’ state. A new class of small G-proteins called “Roco” was discovered about a decade ago; this class is distinct from the typical G-proteins in several intriguing ways. Their switch module resides within a polypeptide chain of a large multi-domain protein, always adjacent to a unique domain called COR, and its effector kinase often resides within the same polypeptide. As such, the mechanisms of action of the Roco G-proteins are likely to differ from those of the typical G-proteins. Understanding these mechanisms is important because aberrant activity in the human Roco protein LRRK2 is associated with the pathogenesis of Parkinson’s disease. This review provides an update on the current state of our understanding of the Roco G-proteins and the prospects of targeting them for therapeutic purposes. The Ras/GTPase superfamily consists of monomeric small GTPases with molecular masses of 20–30 kDa. Small GTPases act as molecular switches that cycle between the active GTP-bound and inactive GDP-bound states. The functional cycle is regulated mainly by two different groups of regulatory proteins: (1) The Guanine nucleotide exchange factors (GEFs), which facilitate GDP dissociation from the small GTPases and exchanging with GTP leading to the transmission of various upstream signals; and (2) GTPase-activating proteins (GAPs), which accelerate the speed of the intrinsic GTP hydrolysis of the GTPase, thereby resulting in the inactivation of the molecular switch. The Ras/GTPase superfamily perform their cellular function by activating downstream protein kinase cascades. The Ras signaling pathway is a classic example in the mitogen-activated protein kinase (MAPK) pathway. Dictyostelium discoideum which possesses a complex protein termed GbpC (cGMP binding protein C) [In the past decade, a novel group of Ras/GTPase superfamily has been discovered, which is called the Ras of complex proteins (Roc). These Roc GTPase exist as a domain within a large multi-domain protein . Roc wasotein C) . P.J.M. Dictyostelium, plants, and animals, each consisting of a juxtaposition of the Roc and the COR domains; there are no proteins containing either Roc or COR domain alone. Based on their domain architecture, the identified Roco proteins are categorized into three groups. The largest group is found in Dictyostelium and metazoan, which is characterized by the central LRR-Roc-COR-Kinase tandem domains , and called proteins consisting of Roc and COR “Roco proteins” for their unique structures . The gro domains . In thes domains . The sec domains . The proDictyostelium and prokaryotes described above, have been studied for some time [Although the Roco proteins, such as DAPK1 and a few from ome time , they weome time ,10. Sincome time ,12. Althome time . This caome time . In receome time ,14. In tThe Roco family of proteins have been identified in a wide phylogenetic range, from bacteria to humans . This faD. discoideum. Although 11 Roco genes have been identified in D. discoideum so far [Some of the most studied Roco proteins have been found from m so far ,16, a prm so far  , LRRK2 (leucine-rich repeats kinase 2), and MASL1 . A common feature among these four proteins is that they all contain a central Roc-COR domain characteristic of Roco proteins; however, the variety of the different surrounding domains illustrates the diverse functions of these proteins. The smallest of the group, MASL1, is composed of only three domains, an LRR domain followed by the Roc-COR tandem domains. It lacks a kinase domain, thereby leaving GTPase as its sole catalytic activity, indicating that most likely the Roc domain is the main functional domain to carry out its cellular functions ,14. DAPKDAPK1 was first discovered as a protein involved in the process of cell death , and it The biological function of MASL1 is the least understood among the four human Roco proteins. MASL1 was originally identified as a novel gene which was amplified from a malignant fibrous histiocytoma, implying that MASL1 was involved in cell fate and division . Later cl-plastin [LRRK1 is closely related to LRRK2, and it was believed that they derived from the same ancient gene via DNA duplication . Both LR-plastin ,33. Among the human Roco proteins, LRRK2 has been the most studied. Human genetics has linked LRRK2 to PD, Crohn’s disease, multibacillary leprosy, and cancer . LRRK2 hTaken together, it appears that LRRK2 plays an important role in a number of different cellular processes involving membranous structures; however, its precise function(s) and mechanism in disease pathogenesis remain elusive.The typical small G-proteins function as molecular switches by cycling between the GDP-bound inactive and GTP-bound active conformations. This biochemical cycle is composed of a binary process; the GDP-bound inactive state is switched to the activated state upon GTP binding, and reciprocally, the GTP-bound active state is switched off by the intrinsic GTP hydrolysis activity of G-protein. These changes are intricately controlled by GEFs, which promote the exchange of GDP with GTP, and by GAPs, which accelerate the biochemical hydrolysis of GTP. Utilizing this highly-conserved functional G-protein fold, their different subcellular localization and specific downstream effectors enabled the small G-proteins to regulate a diverse set of biological processes in the cell. The small G-proteins in the Roco family of proteins are unique, in that they are intramolecularly-linked with a COR domain, and their nucleotide exchange process might not require GEFs or GAPs. However, the molecular details of their mechanism of action remain to be elucidated. D. discoideum GbpC, and they demonstrated that the nucleotide exchange process of Roc domain of Gbpc was specifically activated by its C-terminus GbpC-RasGEF domain [C. tepidum Roco in vitro by using purified recombinant Roc-COR domain, which showed that Roco has the low binding affinity for both GDP and GTP (in the micromolar range) [G-proteins are characterized by two main functions: guanosine nucleotides (GDP and GTP) binding and GTP hydrolysis. The guanosine nucleotide-binding property of the Roco proteins has been widely reported for proteins from prokaryotic to human ,28,54. WF domain . Katja Gr range) . Guanosir range) . There ir range) . Both LRr range) ,54,55, ar range) . Chlorobium tepidum Roco [Similarly, the GTP hydrolysis activity of the Roc domains of the Roco proteins has been well characterized, including DAPK1 , LRRK2 , and thedum Roco . Among tdum Roco , and thidum Roco ,55,59. Adum Roco ,60. A dudum Roco . These aD. Discoideum is uniquely regulated by its RasGEF domain in the same polypeptide chain. A number of studies have set out to identify the GEFs and GAPs for LRRK2. One of the potential GEFs identified for LRRK2 is ArhGEF, which is a GEF of CDC42 and Rac1, and its interaction with LRRK2 appears to increase GTPase activity in cells and mouse brain [C. elegans as a modulator of LRRK2 [in vitro [As mentioned above, the Roc domain of GbpC from se brain ,62. ArhGse brain . Interesse brain . The mamse brain ,65. Surpse brain ,65. If Aof LRRK2 . RGS2 isin vitro . It is uC. tepidum Roco protein model [C. tepidum Roco. A Roc-COR contiguous construct of the CtRoco has been crystallized as constitutive homodimer stabilized by the interactions between the two COR domains. Based on the structure, it was proposed that the dimerization mediated by the two COR domains would position the Roc domains in close proximity to each other, thereby exchanging an Arginine residue that would complement and constitute a productive catalytic site [C. tepidum Roc-COR does not exist in human LRRK2; therefore, it is still unclear whether the mechanism of action of CtRoco is the same as that of the human LRRK2. Another observation of LRRK2 that is inconsistent with the GAD model is that the binding of nucleotides does not regulate LRRK2 dimerization in cells. On the other hand, the human DAPK1 forms a homodimer in cells through interactions in the Roc and kinase domain instead of the COR domain.Interestingly, biochemical characterization of the LRRK2 protein reveals that the guanosine nucleotide affinity is much weaker than that of the canonical small GTPases ,55. Genein model . The nottic site ,69, its tic site , the isotic site , and thetic site ,72. Howetic site ,73, demoC. tepidum Roco protein is regulated by the conformational changes between dimeric and monomeric states during the process of GTP turnover [More recently, Egon Deyaert and colleagues proposed that the GTPase catalytic activity of the turnover . In contturnover ,75. The turnover . Additionally, emerging evidence has indicated that the intramolecular kinase activity might reciprocally regulate the GTPase activity ,76. For In addition to the cluster of sites located in the Roc domain mentioned above, autophosphorylation sites are also found in the region between the ANK domain and LRR domain, including S910, S935, S955, and S973 . Since tThe typical Ras-family GTPases undergo guanine nucleotide-dependent conformational changes that alter their interactions with various effectors in the cellular signaling cascades leading to changes in subcellular localization and/or oligomeric states. However, the Roco-family G-proteins are contiguously fused with a COR domain, and most of them comprise of an intramolecular kinase domain, among others. Given this modular arrangement that consists of a G-protein and a potential effector kinase in the same polypeptide chain, it is tempting to imagine that the Roc domain might interact with its intramolecular kinase domain in a fashion analogous to that of Ras with the MAP kinases. Indeed, in slime mold, the activation of the Roc domain of GbpC with GTP stimulates the activity of its kinase domain, which then goes on phosphorylates its downstream substrates ,18. SimiIt is still unclear how the nucleotide-dependent conformational changes in the Roc domain of the Roco proteins result in the activation of their kinase domain. However, several lines of evidence suggest the involvement of dimerization and/or overall conformational changes leading to rearrangement of the domains. For example, Rodrigo Carlessi and colleagues revealed that the Roc domain of DAPK1 mediates its homodimerization as well as its heterodimerization with other kinases, including LRRK2 and ZIPK (ZIP-kinase); however, the mechanistic significance of the dimerization event was unclear at the time . They alThe Roco family of G-proteins is the most recently characterized and least understood of the Ras-like small G-protein family. It is a class of G-proteins in which the G-domain functions within a multi-domain protein and is characteristically conjoined with a COR domain. Understanding the details of the functioning of the Roco proteins will have important applications. For example, LRRK2 is currently the most promising drug target for Parkinson’s disease, and it is being intensively investigated for that purpose. Because the aberrant kinase activity of LRRK2 is associated with disease pathogenesis, most efforts in drug discovery have been focused on inhibiting its kinase domain. This has yielded a number of highly specific and potential inhibitors of LRRK2 kinase activity ,93,94. HThe unique structure comprising of the Roc-COR domains might provide an alternative venue for therapeutic development. This is an attractive strategy because, in addition to the unique structure that might offer specific drug binding sites, it is an intrinsic modulator of the kinase domain; thus, modulating the modulator might offer a tunable regulation of LRRK2 kinase activity. To achieve this, we will need to understand the biochemical and structural details of the Roc-COR domains. The bottleneck in these endeavors has been obtaining sufficient amounts of correctly folded proteins suitable for the biochemical and biophysical studies. Even so, there are ongoing efforts to achieve precisely that.


Plant nucleotide-binding leucine-rich repeat receptors (NLRs) are intracellular pathogen receptors whose N-terminal domains are integral to signal transduction after perception of a pathogen-derived effector protein. The two major plant NLR classes are defined by the presence of either a Toll/interleukin-1 receptor (TIR) or a coiled-coil (CC) domain at their N-terminus (TNLs and CNLs). Our knowledge of how CC domains function in plant CNLs lags behind that of how TIR domains function in plant TNLs. CNLs are the most abundant class of NLRs in monocotyledonous plants, and further research is required to understand the molecular mechanisms of how these domains contribute to disease resistance in cereal crops. Previous studies of CC domains have revealed functional diversity, making categorization difficult, which in turn makes experimental design for assaying function challenging. In this review, we summarize the current understanding of CC domain function in plant CNLs, highlighting the differences in modes of action and structure. To aid experimental design in exploring CC domain function, we present a ‘best-practice’ guide to designing constructs through use of sequence and secondary structure comparisons and discuss the relevant assays for investigating CC domain function. Finally, we discuss whether using homology modeling is useful to describe putative CC domain function in CNLs through parallels with the functions of previously characterized helical adaptor proteins. Plants use intracellular immune receptors to perceive virulence proteins (effectors) secreted to the host cell by microbial pathogens during infection . These rPlant NLRs have a modular, multidomain architecture, and specific roles for each domain have been described that together contribute to the function of the full receptor .The C-teAlthough considered the predominant signaling units of NLRs, TIR domains and CC domains are structurally and functionally very different from one another. TIR domains adopt a conserved flavodoxin-like fold consisting of five α-helices surrounding a five-strand β-sheet, as observed in crystal structures of TIR domains from a variety of different plant, animal and bacterial species . Signal Here, we review current knowledge of CC domain- and CNL-mediated signaling in plant immunity and highlight some methods for investigating CC domain structure and function. We discuss the current classification of CNLs in the context of function, provide some guidelines on how to design CC domain constructs for structural and functional studies through analyses of sequence and secondary structure, and finally discuss putative functions for the variety of different CC domains found in CNLs.Nicotiana benthamiana; (ii) the need for self-association to signal cell death; and (iii) the presence/absence of CC-specific motifs, such as the EDVID motif. A result of these studies is that CC domains are frequently grouped into several classes: CCEDVID, CCR, CC   , and theclarity) . The CCEB domain . The CCR to RPW8 ; classicand RPS5 . CC domaand RPS5 . Finallyic clade . CNLs wiic clade .Recently, it has been found that many NLRs (both TNLs and CNLs) function synergistically either as pairs  or as paCAN and CCR subclasses, four CC domains from the I2-like subclass and the rest comprise CC domains of the CCEDVID subclass. While there have been multiple studies performed on the N-terminal SD-CC domains of Sw-5b and Prf SD-CNLs  capacity to induce cell death; (ii) ability to self-associate; and (iii) ability to interact with a cofactor , and I2-like subclasses, which distinctly segregate in sequence and function from other CNLs , despite belonging to the CCectively . Moreoveectively .Taken together, these observations highlight that the commonly used classifications of CC domains are not especially useful for confidently defining function. Therefore, care should be taken if these classifications are used when designing functional studies for CC domains from uncharacterized NLRs. The differences in CC domain function, coupled with the small sample size of proteins studied to date, serves to highlight the difficulties in forming classes, and consequently, prediction of putative functions for these proteins based on sequence. One limiting factor in characterizing the function of CC domains in plant NLRs is the difficulty in assigning domain boundaries, and therefore the appropriate design of constructs to analyze. Next, we give a short guide on how to analyze the protein sequences of CC domains with the goal of designing experiments to assay their function robustly.Due to a lack of knowledge, it is frequently necessary to make subjective decisions about the boundaries of protein domains when attempting to assess function. This can result in inappropriate constructs that complicate functional annotation. One potential point for error is to use domain boundaries based on homology or similarity to previously assayed proteins. For example, in TIR domains from plant NLRs, despite the core flavodoxin fold being conserved in the protein , there iAs, to date, the explicit function of CC domains in plant NLRs remains undefined, any initial constructs designed to assay activity should be overly inclusive, preferably spanning the region of the NLR from the N-terminus to the beginning of the NB domain (the boundaries of which can be better defined). These constructs should then be assayed for cell death signaling in planta, cofactor association and self-association to establish a functional reference point before, perhaps, trying to delimit minimal regions required for function . Not alTwo of the more useful, and extensively used, tools for assessing construct boundaries are secondary structure prediction and Pfam domain analysis . In manyPrograms such as COILS and PSIPRED, as well as 3-D homology modeling servers such as Phyre2 and I-Tasser, run a secondary structure prediction as a part of their pipelines . In geneGeneralizing plant NLR CC domain function has proven challenging. As discussed previously, the ability to cause cell death upon heterologous expression in model plants, self-association and whether the domain interacts with a cofactor are the most commonly assigned activities. Each of these activities are informative concerning CC domain function, but the conclusions can be subjective, in particular where a lack of activity is observed, as this could just be due to suboptimal experimental design.N. benthamiana or N. tabacum, is used as an indicator of cell death signaling consistent with plant immunity pathways. As it is less likely that negative (lack of cell death) responses will be reported in the literature, it is difficult to assess how common it is for CC domains to lead to cell death on expression. Good examples of how to use the HR assay to evaluate CC domain autoactivity are found in After assembling suitable constructs through incorporating best estimates of domain boundaries, assays to assess the CC domain functions described above can be employed. The in planta ‘HR assay’, often performed in heterologous hosts such as N. benthamiana) suffer from false negatives and positives due to the context of the assay. Y2H assays can positively report the interaction of two proteins, but this may not be biologically relevant, and false negatives can occur from inhibition of interactions by the reporter fusions, or because a partner of the interaction (e.g. a ‘bridging molecule’) is missing in yeast. While CoIP assesses associations derived from plant tissue , extraction conditions may both positively promote and negatively influence interactions between the proteins of interest. In cases where differences are observed between interactions in Y2H and CoIP analyses, further experiments should be conducted.To study CC domain self-association, or association with cofactors, yeast two-hybrid (Y2H), co-immunoprecipitation (CoIP) and analytical SEC  have all been extensively used. While powerful techniques, Y2H and CoIP  of interest, mostly commonly fidently . The natfidently , charactfidently  and invefidently . Furtherfidently .Overcoming the limitations of individual techniques to study plant NLR CC domain function requires multiple approaches. One of the best predictors of protein function is 3-D structure, and currently structural data on CC domains are limited. For example, to date, there is no structure of a plant NLR CC domain that fully covers a construct that induces cell death in model plants. In the next section, we assess the current understanding of CC domain structure, and ask whether homology modeling is useful to provide insight into plant NLR CC domain function, specifically to inform further experiments.The Sr33 CC domain [determined by nuclear magnetic resonance (NMR)], and the MLA10 and Rx CC domains , are the only structures of plant CC domains available to date . The Sr3As previously mentioned, none of the existing plant NLR CC domain structures represents functional signaling units, at least in the context of induced cell death in model plants . For botIn the absence of easy access to structural information, homology modeling offers an opportunity to gain insight into protein structure/function relationships, but should always be used with caution to prevent falling into the ‘functional homology trap’ . SpecifiCaenorhabditis elegans CED-4. The similarity between the CC domains of MLKL/CED-4 CARD and those of plant NLRs, as predicted by Phyre2, may be useful to inform further studies of plant NLR CC domain function.We took each of the CC domains detailed in R domains.MLKL is required in the activation of necroptosis, an auxiliary form of cell death thought to be triggered after suppression of apoptosis. The four-helix bundle region of MLKL was necessary for the insertion of the protein into the plasma membrane post-oligomerization, causing pore formation and resulting in the collapse of cell integrity . FurtherEDVID, CCCAN and I2-like subclasses) were generated using the CARD of CED-4 (PDB: 2A5Y) as a template by the prediction program. The structural folds of CARD domains, including that of CED-4  and maximize biological relevance. From the work presented here, it is clear that significant gaps remain in our knowledge of how CC domains of plant CNLs are involved in transducing defense-related signaling in cells in response to pathogens. Further studies are required to understand how these domains contribute to disease resistance in some of the world’s critical food crops.This work was supported by the Biotechnology and Biological Sciences Research Council ; the European Research Council ; and the John Innes Foundation.The authors have no conflicts of interest to declare.


We demonstrate the advantages of optical coherence tomography (OCT) imaging for investigation of spontaneous retinal venous pulsation (SRVP). The pulsatile changes in venous vessel caliber are analyzed qualitatively and quantitatively using conventional intensity-based OCT as well as the functional extension Doppler OCT (DOCT). Single-channel and double-channel line scanning protocols of our multi-channel OCT prototype are employed to investigate venous pulsatile caliber oscillations as well as venous flow pulsatility in the eyes of healthy volunteers. A comparison to recordings of scanning laser ophthalmoscopy (SLO) – a standard en-face imaging modality for evaluation of SRVP – is provided, emphasizing the advantages of tomographic image acquisition. To the best of our knowledge, this is the first quantitative time-resolved investigation of SRVP and associated retinal perfusion characteristics using OCT. Before forming a single venous trunk just superior to the lamina cribrosa, the inferiorly descending retinal hemiveins are reported to present pulsatile luminal changes in up to 98% of the eyes of healthy human subjects6. The characteristics of SRVP are claimed to be determined by the transmural pressure  and venous compliance 1.Spontaneous retinal venous pulsation (SRVP) describes the phenomenon of rhythmic caliber oscillations of one or multiple major retinal veins at the site of the optic nerve head (ONH)7, the intraocular pressure (IOP) is commonly equated with the external venous pressure, while the internal venous pressure is equated with the intracranial pressure (ICP)8. Thus, reports suggest that the ratio between IOP and ICP may be reflected in the characteristics of SRVP10. More specific, the absence of SRVP is reported to be linked to an increase of ICP at constant IOP14. Hence, those reports emphasize that a better understanding of SRVP and its detection and characterization in the individual eye, in combination with standard-of-care IOP determination may enable non-invasive ICP assessment – one of the holy grails of neurology. As of today, methods for non-invasive ICP assessment are rare and error-prone16, thus, a more substantial and reliable approach would have immediate impact.In a simplistic scenario17. Thus, the significance of SRVP for potential ICP assessment is questionable and requires a more thorough analysis.However, SRVP shows strong inter-subject variability, in particular, due to the uniqueness of retinal vasculature features that govern venous compliance. Furthermore, SRVP seems very specific to any hemivenous section itself5, or via optical imaging modalities, such as fundus photography/videography18 or scanning laser ophthalmoscopy (SLO)20. These modalities are en-face plane imaging techniques, only providing 2D transverse projection images of the optic disc surface without depth information. However, as suggested by previous reports and demonstrated by mathematical simulations, the largest venous caliber change is expected to occur in axial and not transversal direction23. Only modified photo-plethysmography/densitometry tries to assess variations in the axial blood column over time by monitoring changes of retinal back reflection25.Previous studies of SRVP observed pulsatile venous caliber oscillations either directly, via examination using a conventional ophthalmoscope or a slit lamp26. OCT has revolutionized retinal diagnostics27 but until today only been used indirectly or with limited potential to study SRVP. First, the retinal nerve fiber layer thickness, determined by OCT, was reported to correlate with SRVP amplitude28. Second, Lam et al. investigated the structural characteristics of ONH tissue at the sites of SRVP using a commercially available OCT instrument17. However, the important aspect of time-resolved investigations to display SRVP at different cardiac phases was not pursued or mentioned in this article.However, there exists another highly successful optical imaging modality that enables high speed, high resolution, and, in particular, cross-sectional image acquisition of transparent or translucent samples such as the human retina – optical coherence tomography (OCT)29 to increase detection sensitivity of SRVP. DOCT’s additional functional image contrast enables qualitative as well as quantitative investigations of the retinal sites of venous collapse and the closely surrounding areas in previously unmatched detail.In this paper we present, to the best of our knowledge, the first quantitative time-resolved OCT investigations of the phenomenon of SRVP in the eyes of healthy human volunteers. In addition to purely intensity-based imaging, we exploit OCT’s functional extension Doppler OCT (DOCT)31. In short, the system features a tunable light source  and allows for switching of interferometer sample arms to enable sample illumination by up to three different channels within the same acquisition. However, in this study the system was only operated in single-channel and double-channel mode. Due to the applied switching scheme of inter-A-scan switching31, the double-channel mode only permitted an A-scan rate of 50 kHz per channel while for the single-channel mode the full 100 kHz were accessible. In contrast to previous multi-channel operation33, the individual channels were not probing a mutual sample spot but focused on separate sample locations to enable parallel line scanning34 – cf. Fig. All investigations were performed using a custom-built time-encoded multi-channel DOCT prototype described in greater detail elsewherein vivo investigations, informed consent was obtained from the healthy human volunteers. The laser safety limits in terms of maximum permissible exposures were met by a maximum beam power of 1.9 mW at the cornea.The presented study is in agreement with the tenets of the Declaration of Helsinki and was approved by the institutional ethics committee. Prior to the 35.Multiple consecutive linear B-scans were recorded at the same retinal location to enable visualization of changes over the cardiac cycle. Different B-scan lengths , sampling densities  and rotational orientations of the scanning line/lines were used throughout the study. Each data recording took between three and five seconds (depending on the exact measurement parameters). Including subject alignment, a single measurement session (consecutive acquisition of multiple data sets) did not exceed ten minutes. The OCT data processing pipeline to generate intensity as well as phase-difference (Doppler) tomograms from raw data, was similar to previously published workWithin this pilot study, we show exemplary image data of veins exhibiting SRVP of two eyes of two healthy human volunteers , that were imaged with our multi-channel OCT prototype. For comparison with previous SRVP reports, image data of standard ophthalmic imaging procedures (fundus photography and SLO) of the same eyes were recorded at the Department of Ophthalmology of the Medical University of Vienna. SLO movies were acquired using a commercial device  using a 15° × 15° scan field at 768 pixels squared and a frame rate of 10 frames per second (fps).The presented image data of the two eyes showing SRVP illustrate the advantage of cross-sectional image acquisition for investigation of SRVP. Data of the same eyes acquired with multiple ophthalmologic imaging modalities is shown. The OCT data sets in this sub-section were acquired at 100 kHz using the single-channel mode of our instrument.Figure In addition, we recorded a linear B-scan along the same partially collapsing hemivein – cf. Fig. Figure 1; full venous expansion – t2) are depicted. In Fig. 1 and t2. While the maximum transversal diameter change seems to be roughly 20% for both imaging modalities, the change of segmented area of flow decreases by roughly 50%. Since not every cardiac cycle is similar to the previous one in duration and strength, also the characteristics of venous collapses may change over time. However, in particular the DOCT data shows rather reproducible diameter and area changes over the inspected three consecutive cardiac cycles. For the SLO data especially the first collapse of the sequence seems not as pronounced as the latter two, and thus deviates from the transversal diameter DOCT curve. In general, the transversal diameter changes measured from DOCT and SLO seem to lie within a similar range.To compare SRVP quantification between SLO and DOCT, the image sequences of Supplementary Videos 1; full venous expansion – t2). Figure 1 and t2. The maximum change of transversal diameter is ~20%, while the maximum change of flow area is ~60% in this case. Thus, the detection sensitivity regarding SRVP is increased by a factor of ~3 by DOCT when comparing relative changes of transversal vessel diameter to relative changes of venous cross-sectional area.We also quantitatively evaluated Supplementary Video Besides caliber pulsations, we also found flow pulsations in the same retinal veins that exhibit SRVP, as illustrated in Fig. To obtain a reference regarding the flow evaluation of the collapsing vein, the artery was evaluated first. Figure In a second step, venous flow pulsations were evaluated. In comparison to the arterial pulsations, they were considerably weaker in amplitude and showed a more washed-out pulse profile. Figure Figure 1; highest amplitude of collapse – t2) and Fig. In case of venous collapse, we observed pulsatile intensity changes within the venous lumen. This effect is notable in Figs 23. (ii) The same venous caliber oscillations were imaged repeatedly in several imaging sessions on different days, tracked over multiple cardiac cycles and the detected venous flow pulsations were analyzed. (iii) Pulsatile changes of intensity, probably due to alterations of red blood cell (RBC) orientation, were detected, quantitatively evaluated, and compared to reports of modified photo-plethysmography/densitometry.Within this pilot study, we present first time-resolved quantitative OCT investigations of the phenomenon of SRVP in healthy subjects, using both the single- and double-channel imaging mode of our multi-channel instrument. The tomographic imaging approach and, in particular, the functional contrast enabled by DOCT, facilitate detailed investigations of the perfusion characteristics over time, unmatched by previous reports of SRVP. (i) The quantitative measurement of the SRVP amplitude was improved by a factor of ~3 when relative changes of transversal vessel diameter are compared to relative changes of venous cross-sectional area. Thus, the previous hypothesis of a much stronger caliber change in axial than in lateral direction could be confirmed42, and would add little additional value to this study. This observation stands in clear contradiction to previously reported occurrence rates of SRVP in up to 98% of the investigated eyes6. However, as pointed out in this work, these high occurrence rates were determined by use of enface imaging modalities such as fundus photography/videography and SLO. Slight intensity changes associated with fundus pulsations in the ONH area might be mistaken for SRVPs and potentially lead to overestimation, if the depth information is not available. We also observed such small intensity changes throughout the entire ONH area for all SLO recordings of the ten non-SRVP eyes, however those originated from fundus pulsations43 and not from SRVP. Future evaluations are needed to study SRVP using DOCT in a larger sample size to extend the findings presented in this pilot study. However, the lack of commercial DOCT instruments presently impedes clinical applicability for larger scale studies. In this regard SLO is a step ahead of DOCT, while also being the less complex approach. Nevertheless, given the recent success of OCT angiography that has already been implemented in several commercially available instruments, a similar development for quantitative DOCT in the near future does not seem unrealistic.The major limitation of this study is its small sample size. We present image data of only two partial retinal venous collapses in two healthy subjects. However, in the course of the study a total of twelve eyes of six healthy subjects  were imaged. Nevertheless, we did not find signs of partial or total venous collapse in any of the veins of the other ten eyes . We did not include imaging results of those eyes, since time-resolved DOCT investigations of non-collapsing veins have been reported multiple times45.A potential systematic error of the presented investigations may arise from the manual evaluation of cross-sectional flow area and transversal diameter of the vein. However, all reported area and diameter data were evaluated by the same expert reader. Therefore, such systematic segmentation errors would only have little impact on the results of relative changes, since it can be assumed that the reader’s systematic deviation would be similar for all phases of the cardiac cycle. Furthermore, manual retinal vessel segmentation and diameter evaluation in OCT data by an expert reader is state-of-the-art for ground truth determination and also reported to achieve accurate results in comparison to automated computational segmentation algorithms2. However, in cardiovascular research the term ‘pulsation’ is not necessarily restricted to caliber oscillations but equivalently used for rhythmical changes of blood volume throughput or blood flow in non-collapsing vessels. Such flow pulsations are commonly observed throughout the arterial cardiovascular system and well described in the literature – also in the retina46. Venous flow pulsations are in general not observable50, and if occurring, restricted to specific locations within the human body. Venous flow pulsations have been reported in the internal jugular vein51, the brain52 and the retina39, although, reports are controversial in the latter case46. In contradiction to some of those studies, but in conformity to others, we did measure significant flow pulsations in retinal veins. However, we did not succeed in determination of the travel direction of the observed pulsation. Thus, the hypothesis of upstream pulse propagation of a wave that is generated in the optic nerve subarachnoid space and travels opposite to the direction of blood flow through the lamina cribrosa towards the retina, could neither be verified nor falsified53. The temporal resolution in our experiments was too low (or the maximum channel separation distance was too short) to reasonably evaluate the recorded pulse profiles.In the literature SRVP is termed a ‘pulsation’ due to the fact that pulsatile changes in venous vessel caliber were observed57 – using imaging parameters of 24 fps and ~0.9 mm distance between the measurements locations. This is in so far interesting, as a recently published article reports to do so, using similar measurement parameters but a different scanning method, called jump-scanning56. However, in that report the authors miss to explain how heart rate variability – the physiological variation in the time interval between two consecutive heartbeats – is taken into account. The results of their PWV measurements would be affected severely if this variation was not compensated for57. We believe, our technique of simultaneous data recording at separate vessel locations using multi-channel OCT, offers great potential to narrow the high range of previously published arterial PWV values down considerably56. In the future this could be enabled either by an increase of B-scan frame-rate or a larger separation distance between respective measurement locations.We were also unable to quantify arterial PWV – a recently critically discussed retinal perfusion parameter25. This technology measures the fluctuating light attenuation by blood during a cardiac cycle. The changes in attenuation are then directly related to changes of vessel cross-sections during SRVP using the Beer-Lambert law, assuming that the attenuation is caused by absorption of light by the hemoglobin of the RBCs. However, this assumption ignores a fundamental physical process that needs to be considered for such investigations – scattering. Modified photo-plethysmography/densitometry has no means to differentiate whether the observed pulsatile changes in backreflected intensity originate from changes in absorption or from orientation changes of the scattering particles – in this case RBCs. However, OCT enables such differentiation by measuring depth resolved scattering profiles. The scattering characteristics of moving RBCs that is of importance here, has been described in detail in the literature62. RBCs are reported to show preferential orientations for laminar flow inside a tubular lumen, as long as a sufficiently high flow velocity is maintained. This particular spatial arrangement results in a so-called hourglass signal pattern in the OCT intensity tomogram of a vessel cross-section. However, this signal pattern was only reported for laminar flow in static tubular organs. In Fig. et al.1 that variation in hemoglobin is the only pulsatile component is incomplete. The reason for the pulsatile intensity changes is not just the change in axial blood column. The change in orientation of scattering RBCs seems to have a larger effect and must be taken into account. Hence, the quantitative results obtained by modified photo-plethysmography/densitometry are questionable – this technique does not allow for reliable measurements of changing optical path length by consideration of absorption only.Modified photo-plethysmography/densitometry is described to assess variations in the axial blood column over time by monitoring changes of absorption during fundus videography16. The introduction of time-resolved DOCT for investigation of SRVP might assist the proposed optical methods for potential future non-invasive ICP assessment. That being said, certain doubts about whether the phenomenon of SRVP might eventually enable reliable ICP determination have to be expressed. We experienced a very strong inter-subject variability in terms of location, strength and characteristics of SRVP . Such strong variability may prohibit direct use of SRVP for ICP determination. However, the induction of non-spontaneous artificial retinal vein pulsations by means of ophthalmodynamometry11 may solve this problem. Besides potential ICP determination, monitoring of SRVP might proof beneficial for analysis of major ocular disorders such as glaucoma or venous occlusions8 that have been associated with vascular pathologies.A method to non-invasively assess ICP would denote a milestone for monitoring and management of numerous neurological disorder and conditionsIn this manuscript, we present first time-resolved investigations of SRVP using intensity-based OCT as well as functional DOCT. Both, the single- and the double-channel imaging mode of our multi-channel OCT prototype enable detailed qualitative as well as quantitative analysis of venous caliber oscillations. The presented OCT image data is compared to standard SLO en-face image data emphasizing the advantages of a tomographic imaging principle for SRVP investigations. Besides pure caliber oscillations also venous flow pulsatility is analyzed in the eyes of healthy volunteers and previously established theoretical hypotheses examined. We hope this first demonstration in combination with potential more elaborate future studies might assist to better understand the role of SRVP for non-invasive ICP assessment as well as for diagnosis and monitoring of certain retinal pathologies.Supplementary Video S1Supplementary Video S2Supplementary Video S3Supplementary Video S4Supplementary Video S5Supplementary Video S6


Records were reviewed of 247 patients with CMN who had had an MRI attempted, 161 under age 1 year. In total 114 of 247 (46%) were male, 208 of 244 (85%) had brain and whole‐spine imaging and 193 of 202 (96%) had contrast injection. The mean and median ages were 2·02 and 0·66 years, respectively (range 0–18·8 years).Information regarding sedation and anaesthetic was available in 208 of 247. Forty‐five of 208 (22%) were preselected for GA by an experienced sedationist, for example due to comorbidities, although a comparison of the demographic and phenotypic profiles of those who received sedation or GA showed no significant differences. Ten of 208 (5%) were awake, 151 of 208 (73%) were sedated and two of 208 (1·0%) underwent the ‘feed and wrap’ technique, all according to local protocols (Fig. The literature supports sedation for paediatric MRI as effective and safe,These data strongly support the use of sedation rather than GA as a first‐line approach for MRI in infants with multiple CMN, after triage by a sedationist or anaesthetist and with access to anaesthetic support if required.


Oryza sativa) is one of the main staple crops worldwide but suffers from important yield losses due to different abiotic and biotic stresses. Analysis of quantitative trait loci (QTL) is a classical genetic method which enables the creation of more resistant cultivars but does not yield information on the genes directly involved or responsible for the desired traits. Lectins are known as proteins with diverse functions in plants. Some of them are abundant proteins in seeds and are considered as storage/defense proteins while other lectins are known as stress-inducible proteins, implicated in stress perception and signal transduction as part of plant innate immunity. We investigated the distribution of lectin sequences in different QTL related to stress tolerance/resistance, morphology, and physiology through mapping of the lectin sequences and QTL regions on the chromosomes and subsequent statistical analysis. Furthermore, the domain structure and evolutionary relationships of the lectins in O. sativa spp. indica and japonica were investigated. Our results revealed that lectin sequences are statistically overrepresented in QTLs for (a)biotic resistance/tolerance as well as in QTLs related to economically important traits such as eating quality and sterility. These findings contribute to the characterization of the QTL sequences and can provide valuable information to the breeders.Rice ( Oryza sativa) is one of the major cereal crops worldwide and is the main food source for more than half of the human population around the world [Rice  and Q-TARO  database. While Gramene comprises QTL information for eight monocots and also integrates the data from Q-TARO [Multiple research groups focus their work and efforts on the investigation of QTLs related with different (a)biotic stresses but only a few publications address the combination of multiple stresses and search for combined tolerance/resistance . A largem Q-TARO ; Q-TARO m Q-TARO .Galanthus nivalis agglutinin (GNA)-related lectin Pi-d2 is one of the genes important for resistance against rice blast [Even though QTL analysis has been used widely in plant breeding for decades, the exact genes that confer or contribute to a particular trait are rarely known, cloned, or characterized, especially in complex traits such as drought tolerance . Neverthce blast . Additioce blast .Lectins are carbohydrate-binding proteins, they bind specifically and reversibly to sugars or more complex carbohydrates but their carbohydrate binding domain does not possess any enzymatic activity. The classification of plant lectins in different families is based on sequence relationships, rather than on carbohydrate-binding properties. Indeed, all lectin motifs are characterized by specific sequences, which after three-dimensional folding will yield a lectin or carbohydrate recognition domain with one or more carbohydrate-binding sites. In principle, each lectin family groups all proteins possessing one particular lectin domain based on sequence similarity. Different lectin motifs differ from each other in their sequences but can show reactivity towards similar carbohydrate structures, indicating that specificity is not linked to the occurrence of one particular lectin motif. Furthermore, the binding site for multiple lectin motifs was shown to be extended, enabling not only the binding of monosaccharides but allowing anchoring of elongated carbohydrate structures. Each lectin family contains at least one or a few lectins that have been studied in detail for their carbohydrate-binding properties. These data show that the specificity of some lectin domains is rather strict whereas other lectin domains can interact with a diverse range of carbohydrate structures .Agaricus bisporus agglutinin homologs, Amaranthins, Class V chitinase homologs with lectin activity (CRA), Cyanovirin family, Euonymus europaeus agglutinin family (EUL-related lectins), Galanthus nivalis agglutinin (GNA) family, Heveins, Jacalin-related lectins, Legume lectins, LysM domain lectin family, Nictaba family, and RicinB family [The large majority of the lectins studied consists of one or more lectin domains . Genome B family .Arabidopsis thaliana [Morus notabilis (mulberry) [Glycine max (soybean) and Cucumis sativus (cucumber) [Several reports investigated the distribution and phylogeny of plant lectin motifs in the model species thaliana , the medulberry) , and croucumber) ,24 or peucumber) ,25. All In this project, the occurrence of lectin genes in rice QTLs related to resistance/tolerance against biotic and abiotic stress as well as in QTLs associated with morphological and physiological traits was investigated. Furthermore, the genomic and phylogenetic analysis as well as the domain structure for different lectins in two rice subspecies are reported. This study contributes to the characterization of QTL regions and generates knowledge on the physiological role of lectins in plants and their importance for plant growth, development, and defense.http://rice.plantbiology.msu.edu/) and the Rice Annotation Project Database (RAP-DB) . A previous search for lectin families in rice using the MSU annotation database identified nine lectin families in O. sativa spp. japonica including homologs of the class V chitinases, Euonymus-related lectins, GNA-related lectins, Heveins, Jacalin-related lectins, Legume lectins, LysM domain lectins, Nictaba-like lectins, and RicinB lectins [The rice genome has been sequenced from two independent sequencing projects, in particular, the MSU Rice Genome Annotation Project  genome sequences with lectin motifs were mapped onto the 12 rice chromosomes, see To investigate the distribution of the lectin genes throughout the rice  contain at least one lectin gene, and at least 50% of the QTLs for other abiotic stress factors contain at least one lectin gene, see Salinity and drought are among the most devastating abiotic stresses for rice ,6; conseSaLT , a Jacalin-related lectin that confers salinity tolerance  is localRhizoctonia solani) causing sheath blight disease.Among the QTLs related with biotic stress, sheath blight resistance showed the highest percentage of lectin sequences (80%), followed by blast resistance, insect resistance, and bacterial blight resistance, all of them having lectin genes in less than 50% of the QTLs for these traits, see Magnaporte grisea [Meloidogyne graminicola [Jacalin-related lectins containing a dirigent domain are considered as important players in plant innate immunity since the dirigent domain was reported to be involved in plant defense and experimental data show its involvement in the response to e grisea . OsJAC1 e grisea . Additiominicola , see SupThe appearance of the same lectins in QTLs for abiotic, as well as biotic stress, was observed a couple of times. For example, our analysis shows that 23 lectins which were represented in QTLs for cold tolerance also appeared in QTLs for insect resistance. In the case of bacterial blight disease this observation is even more striking because 12 of the 16 lectins present in these QTLs also appeared in QTLs for cold tolerance and 15 genes were found in QTLs for drought tolerance, see The stress response and the adaptation of a plant are intimately linked with the morphological and physiological traits of the investigated plant species or even cultivar. A large number of lectin sequences has been identified in QTLs for all six categories related to morphological traits , see A high percentage of lectin-containing QTLs (at least 50%) was observed in all categories of physiological traits, especially in the category lethality (83%). The presence of lectin sequences is statistically significant for the traits eating quality and sterility, see O. sativa japonica (Os06g0620200 and Os08g0230800). Os06g0620200 appeared in QTLs for dwarfs, roots, panicles, seeds, sterility, and eating quality and stress-related QTLs for drought tolerance and soil stress tolerance. Confirmation of the involvement of the legume lectins in some morphological changes such as growth inhibition and leaf senescence has been reported for OsSIT1 [The association between traits such as seeds and eating quality and panicle/flower and sterility is a good indication of the importance of lectins in plant reproduction and the marketing or seed quality and deserves further investigation at both a molecular and breeding level. An excellent example which illustrates these connections is a gene encoding a LysM domain protein called OsEMSA1 Os10g0524300). In our analyses, OsEMSA1 appeared in QTL for panicle, seeds, and sterility but not in any other QTL for morphological/physiological traits or QTL for tolerance/resistance. A GNA gene called OsLSK1 (Os01g0669100) and its six homologs in the rice genome  appeared in a QTL for seeds, and more specifically in a QTL responsible for grain yield, as well as in QTLs for drought tolerance and salinity tolerance, which is in agreement with the literature 0. In our, in O. sr OsSIT1 . The genO. sativa spp. japonica and indica. Second, phylogenetic trees have been constructed for the lectin domain sequences retrieved from O. sativa spp. japonica and indica. The phylogenetic trees shown in A detailed analysis of the phylogenetic relationships was performed for the two subspecies of rice, especially, the lectin families which appeared most frequently in our analysis and for which functionally analyzed lectins have been reported in literature. Therefore, the analysis focused on GNA-related lectins, Jacalin-related lectins, and lectins with LysM domains. First, the domain architecture of all lectin sequences within these families was analyzed for O. sativa. As shown in the The GNA family is the largest lectin family in O. sativa spp. japonica possesses 134 lectin sequences based on MSU while only 94 sequences with lectin motifs were identified in the genome of O. sativa spp. indica [A comparative analysis between the lectin sequences from rice subspecies japonica and indica revealed important differences in the numbers of lectin sequences retrieved from both genomes. The . indica . Some leO. sativa japonica and indica shows some grouping of the sequences based on the domain architecture, see (1–2)/PAN/protein kinase domain. All the sequences with GNA/protein kinase (PK) domains cluster together in the phylogenetic tree. A similar observation can be made for the DUF containing proteins from subspecies indica and japonica which cluster closely together as well as GNA/S-locus glycoprotein/PAN/protein kinase/SRK (S-locus receptor kinase). Some clades also cluster GNA domain sequences which are linked to different protein domains, resulting in different domain organizations. For example, the two single GNA domain sequences in japonica do not cluster together. While the one on chromosome 1 (LOC_Os01g72810.1) groups with its homolog in subspecies indica, the other one on chromosome 9 (LOC_Os09g02410.1) groups with GNA/S-locus glycoprotein/PAN/protein kinase and GNA/S-locus glycoprotein/PAN sequences. No clustering was observed for the combinations of GNA/S-locus glycoprotein and GNA/ S-locus glycoprotein/PAN, see Phylogenetic analysis of the GNA lectin family in O. sativa japonica. In total, 72% of the family members, or 97 genes in 24 duplication blocks, are expanded through this process while the segmental duplications are responsible for 13% (18 genes), see Tandem duplications have played an important role in the expansion of the GNA family in O. sativa, see The family of Jacalin-related lectins is the third largest lectin family in O. sativa spp. japonica and indica show some clustering based on their domain organization and are grouped into seven clades in the phylogenetic tree. The first clade comprises most of the multiple Jacalin domain sequences in japonica and the CTLH/Jacalin gene in indica rice. Clades 2, 3, and 6 group lectin sequences with similar domain organizations. Clade 2 combines the genes with a NB-ARC/single Jacalin domain structure, clade 3 includes proteins with single Jacalin domain in their sequence, and clade 6 consists entirely of sequences with a Jacalin domain combined with a dirigent domain. NB-ARC is a motif spread through pro- and eukaryotes and is involved in plant defense as a part of the R (resistance) genes [As shown in e) genes . The come) genes ,28, and O. sativa japonica can be explained by tandem duplications (14 sequences separated in six blocks). The Jacalin-related lectins are not influenced by any segmental duplication events. About 47% of the expansion in the Jacalins in The LysM domain has undergone several duplication events over time. The majority of the LysM sequences consists of a single LysM domain. Throughout evolution, the LysM domain has been duplicated or has been associated with other protein domains such as the F-box domain or the protein kinase domain.O. sativa spp. japonica and indica are grouped into 6 clades in the phylogenetic tree. Similar to the Jacalin-related lectin family, some clustering based on the domain organization can be observed but the domain architecture cannot fully explain the phylogenetic tree organization, see As shown in O. sativa japonica, since only 20% of the family members, or only four of the 20 genes, are involved in tandem duplications of the LysM family, see Tandem duplications did not have a major impact on the expansion of the LysM lectin family in Arabidopsis where 20% of the gene loci have been attributed to a particular function [+ concentration in the shoots and this presumably contributes to its higher salt tolerance [Pi-d2 (Os06g0494100) and encodes a GNA-related lectin that confers resistance to rice blast disease. The indica variety Digu, which carries a single substitution 441I, is resistant to M. grisea while the varieties with 441M are susceptible to this fungus [SalT and Pi-d2 are located in QTLs offering increased salt tolerance or rice blast resistance, respectively, suggesting that the lectin gene might contribute to the stress resistance and/or tolerance attributed to the QTL. Despite the fact that rice is the most investigated crop and multiple resources for research are available, only 1.6% of the rice loci are functionally characterized, in contrast to function . Althougfunction  but thisolerance . A secons fungus . The lecO. sativa. Our analysis revealed that lectin sequences are unevenly distributed in QTLs with the highest number of lectin genes on chromosomes 1, 4, and 6. According to Q-TARO, the QTL distribution is unequal over all the 12 rice chromosomes and occurs especially in regions with high gene density. Yonemaru et al. [The current study investigated the presence of lectin sequences in QTL regions in the crop and model species u et al.  reportedu et al. .SIT genes were identified in a QTL region according to the data in Q-TARO. This observation confirms the possibility for the potential presence of additional QTL(s) that have not yet been published or have not been included in the database.The putative link between the appearance of lectin sequences in QTLs and different traits for the morphology, physiology, and tolerance/resistance were investigated in detail and statistical analysis was applied. The data provided proof of the overrepresentation of lectin genes in some QTLs associated with important morphological, physiological, and tolerance/resistance traits. Rice is more susceptible to salt stress than other cereals. Since salinity can cause more than 50% yield losses, it is considered as one of the major limitations to grow this crop . There a2+, Al3+, etc.) or nutrient deficiency. Based on transcriptomic data, Oono et al. [2+ causes acute toxicity to rice seedlings and that cadmium stress shares some common signalization with drought stress. This was also in agreement with the observed phenotypic effect on the treated plants which was similar to the effect of drought stress [Drought is a trait which influences a broad range of other traits related not only with stress responses but also with grain yield, plant height, germination, fertility, etc. . For exao et al.  concludet stress .Pi-d2 sequence did not appear in any QTL for biotic stress resistance in our analyses does not exclude a role in pathogen defense since the conferred resistance is reported for a specific rice variety [Xanthomonas oryzae  and Magnaporthe oryzae (rice blast disease) [Even though the number of the lectin genes identified in QTLs for biotic stress is lower than for abiotic stress, some lectins have been reported as key players in the perception and the defense against these pathogens. This is especially true for members of the LysM family as well as Pi-d2 (GNA family). The fact that the  variety . OsCERK1 variety . Recentl variety  and play variety . Other mdisease) .M. grisea, gene expression shows a fast response reaching a maximum at 12 h after treatment. The transcript levels of the Jacalin-related lectin SaLT show an increase at 12 hpi with M. grisea and reach a maximum expression level at 24 hpi [Myzus persicae Sulzer and Acyrthosiphon pisum, and the beet armyworm Spodoptera exigua Hübner [OsJAC1 is one of the Jacalin sequences that contains a dirigent domain, a combination typical only for monocots ,28. The t 24 hpi . Jacalina Hübner .4 (abiotic stress) and incompatible necrotrophs (biotic stress), for example, and the cross-talk and shared molecules in ROS (reactive oxygen species) and MAP (mitogen-activated protein kinase) kinase signaling [The appearance of the same lectins simultaneously in QTLs for abiotic as well as biotic stresses could be indicative for the pleiotropic effect or crosstalk in the signal transduction and regulatory pathways. A dual role for one sequence/protein is also suggested in the literature because of the observed large overlap between molecular responses towards heavy metals as CdSOignaling .In recent years, there has also been evidence for the involvement of particular lectins in plant development, morphology, and physiology and in some cases, possible pleiotropic effects between the dual roles of lectins in defense and development processes have been suggested. The statistically significant appearance of lectin genes in QTLs for seeds is no surprise since many classical lectins have been reported as being abundant seed storage proteins . Few genM. grisea, Xanthomonas oryzae, and the insect brown planthopper (Nilaparvata lugens), to downregulation of some defense-related genes, and to a reduction in α-amylase enzymes during germination. These effects can be explained partially from the identified interaction between the kinase domain and actin-depolymerizing factors but most probably the gene acts through other interactors as well [Studies by Zou et al. and Cheng et al. ,31 showeArabidopsis [The already mentioned, OsSIT1, which influences salt tolerance/susceptibility through the ethylene signaling pathway, is linked to some morphological changes such as growth inhibition and leaf senescence when it is overexpressed. Delayed maturation and impaired productivity were observed in knockdown lines not only in rice but also in bidopsis , which iSome Jacalin-related lectins are associated with developmental functions in addition to their involvement in stress responses. Overexpression of OsJAC1 reduces the cell length which in turn results in shorter internodes, shorter coleoptiles, and a dwarf phenotype . AdditioO. sativa. Comparative analyses revealed some unique domain combinations as well as quantitative differences between the subspecies indica and japonica, mainly for the lectin motifs in the largest lectin families . Most lectin motifs are linked to other protein domains with known function. GNA and Legume lectins appeared predominantly in combination with protein kinase domains. Taking into account the localization of many of these sequences in the cell wall, these proteins probably play an important role in the perception of extracellular signals and are the first players in signaling cascades as part of plant immune response. Such functions have already been reported for Pi-d2, OsSIT1, and OsLSK1 [In addition to the in-depth analysis of lectin sequences in QTL regions, a detailed study was performed on the presence and the evolution of lectin sequences in two subspecies of d OsLSK1 ,31,32. AN-glycan structures. Within the Jacalin family, two subclasses of lectins with specificity towards mannose and galactose have been reported. The legume lectin motif can interact with very diverse carbohydrate structures since the shape of the carbohydrate binding site will change due to different positioning of a flexible loop. These data illustrate that it is impossible to predict the carbohydrate binding properties based on sequence information. Ultimately the carbohydrate binding specificity for each lectin should be analyzed experimentally. In recent years, it has become clear that most lectin sequences are composed of multiple protein domains and can be considered chimeric lectins in which a lectin domain is linked to one or more protein domains, indicating that the biological activity of these proteins will depend on the activity and possible interplay of multiple protein domains. Future experiments will need to investigate the physiological importance of the lectin domains in rice.The presence of a large collection of lectin motifs distributed over QTL regions raises questions on the carbohydrate-binding activity and how protein–carbohydrate interactions contribute to the biological significance of the lectin domains. Different lectin domains can recognize very diverse mono- and oligosaccharides. For instance, the hevein and the LysM domain both recognize and bind chitin and chito-oligosaccharides. Lectins from the GNA family are mainly mannose-specific but also interact with mannosylated In conclusion, at present, little is known with respect to the physiological role of lectin sequences. Our statistical analyses showed that lectin sequences are overrepresented in several QTLs, suggesting that the lectins sequences could play a role in the trait(s) attributed to the QTLs. Since one gene can have pleiotropic effects, more in-depth studies are needed to provide more insights regarding the physiological role of the lectins, possible pleiotropic effects, and how to integrate and use these data in practice. Furthermore, the possibility that lectins are involved in or are important for many more traits cannot be ruled out since this research was based on a database that is probably incomplete, simply because not all QTLs are known at present. Our data contribute to the knowledge of lectin functionality and the importance of lectins for plant growth and development. By combining QTL analyses and genome analyses, we could link some genetic information with physiological and biochemical studies, aiming for a better functional understanding of plant lectins.O. sativa spp. japonica genome. The lectin sequences were screened for the presence of conserved protein domains to confirm the lectin domain and determine the domain organization. Only sequences with the conserved lectin domains were retained in the analysis. The protein sequences retrieved from the O. sativa spp. japonica were used to identify orthologs in the genome of O. sativa spp. indica using BLAST searches [https://rapdb.dna.affrc.go.jp/tools/converter).The identification of putative lectin genes in the rice genome was previously described by De Schutter et al. . In shorsearches . The MSUhttp://chibba.agtec.uga.edu/duplication) [Gene expansion through segmental and tandem duplications was analyzed for the japonica sequences . Tandem ication) . The datO. sativa japonica were mapped on the chromosomes using the MapChart software  [http://qtaro.abr.affrc.go.jp/) and mapped with the same tool using RAP-DB gene annotations since Q-TARO is based on this database. Overlaps of genomic loci between QTL and lectin genes were determined for every combination of lectin families and QTL group using the intersect function of the bedtools utilities  [Meloidogyne graminicola resistance was retrieved from Dimkpa et al. [The lectin genes from art.htm) . The inflatest/) . The infa et al.  since thhttps://rapdb.dna.affrc.go.jp/tools/dump). An additional step involving the filtering of the redundant transcripts was applied to provide the total number of the unique gene IDs. During our analysis, QTLs which do not contain any lectin or another type of gene were identified and excluded from all subsequent computations.Every trait was analyzed for the presence of redundant QTL. All overlapping QTL were merged into a non-redundant single QTL and all redundant QTL were excluded from further analysis . The idez-score was calculated as the proportion of lectin genes in the genome (gp) subtracted from the QTL proportion of lectin genes for a specific trait (traitp), divided by the standard error on traitp. The latter can be computed from the hypergeometric distribution due to the fact that lectin genes occur once in the genome. Hence,The significant overrepresentation of lectin genes was determined with the use of a Wald test in which the null hypothesis states an absence of lectin overrepresentation, i.e., lectins are not preferentially located on the QTL. The n—number of the genes in QTL; s—number of the lectins in the genome; r—number of the non-lectin genes in the genome; N—total sample ; x—number of lectin genes (in the trait).N as being all protein coding sequences in the genome of O. sativa spp. japonica according IRGSP-1.0 published in 2016 (https://www.ncbi.nlm.nih.gov/genome/annotation_euk/Oryza_sativa_Japonica_Group/101/#FeatureCountsStats) with the total number of genes in QTL (N) equal to 27,912 and the total number of the lectin genes in the genome (s) equal to 291 (according to RAP-DB).We considered p < 0.001, where the significance level, α, for a single two-sided test was defined as 0.025 and was divided by the number of performed tests, i.e., 25.A Bonferroni correction was applied that considered multiple testing. A p-value was considered as significant if https://www.megasoftware.net/) with MUSCLE using default settings [http://trimal.cgenomics.org/) [http://tree.bio.ed.ac.uk/software/figtree/).Maximum-likelihood phylogenetic trees were generated using the lectin domain sequences identified for different lectin families according to the MSU annotation project, as described by . For seqsettings . Consecucs.org/) . Phylogecs.org/) . RAxML u


In this work, the effect of microwave (MW), ε‐poly‐L‐lysine (ε‐PL), calcium propionate (CP), and their combinations on the shelf life and quality characteristics of highland barley fresh noodle (HBFN) was studied. Firstly, the mixed flour was treated by MW (800 W) for different time. Subsequently, HBFN was prepared by mixing flour and sterilized water with addition of 0.04% ε‐PL combined with different concentrations of CP  and stored at 25 ± 1°C. Changes of total plate count (TPC), moisture content, water state, textural properties, color, and pH in HBFN were monitored during storage. The results indicated that microwave treatment (40 s) provided 30% reduction of initial TPC of the flour without decline of noodle quality. Shelf life of HBFN treated with MW + ε‐PL (0.04%) + CP (0.02%/0.025%) reached 80 and 88 hr, respectively, and had good edible qualities during storage. It has a good freshness, unique flavor, and taste. Nowadays, with changing in dietary habits and the development of “functional” foods, there are increasing interests in searching for new foods, which contain functional ingredients  and calcium propionate (CP) with different concentrations on shelf life extension and quality improvement of HBFN was investigated.ε‐poly‐L‐lysine is a natural antibacterial peptide and decomposes into lysine in the human body, which can be used as a source of lysine  was purchased from Yonghui supermarket, Beijing, China. There were two kinds of highland barley flour, which were extrusion flour and unextrusion flour. Unextrusion highland barley flour was obtained by grinding the highland barley grains using high efficiency pulverizer , and the extrusion highland barley flour was obtained by extruding unextrusion flour through twin screw extruder  at the moisture content of 17% and grinding using WF‐20B high efficiency pulverizer. ε‐PL was purchased from Silive Biological Co., Ltd., Zhejiang, China. CP was purchased from Hengde Biotechnology Co., Ltd., Beijing, China. Other chemicals used in the experiment were of analytical grade. Highland barley flour was prepared according to highland barley extrusion modification method  at 800 W for 0, 10, 20, 30, 40, 50, and 60 s, respectively. Then, the reduction effect of microwave treatment on total plate count (TPC) and moisture content of mixture flour were investigated.2.3The moisture content was determined following AACC method . A dough maker was used to prepare noodle crumbles . The speed of dough maker was 90 rpm, and mixing time was 8 min. The obtained dough crumbles were passed through the roller unit of experimental pasta machine  with the roller gap of 1.5 mm for three times, then placed into a sterilized plastic bag, and rested for 30 min at 30°C. Then, the dough sheet was pressed through the pasta machine with the roller gap gradually reduced from 2.5 to 1.0 mm to get the dough sheet with 1.0 mm in thickness. Subsequently, the dough sheets were cut into strands with 20 cm in length and 1.5 mm in width and put into the plastic bags. For the noodles, the addition ratio of ε‐PL and CP includes ε‐PL (0.04%) + CP (0.015%), ε‐PL (0.04%) + CP (0.020%), and ε‐PL (0.04%) + CP (0.025%), respectively.2.5Before testing textural properties, the optimal cooking time (OCT) of HBFN was measured according to the method described by Bai et al. . Briefly2.6T2 was determined using a CPMG pulse sequence. Uncooked HBFN was scanned at 32 ± 0.01°C. The number of sampling points (TD) was 10,106, number of echo (NECH) was 500, TW was 1,000, and repeated time was 32. Each sample was scanned in triplicate.The water state of fresh noodle sheets was analyzed with a LF‐NMR system . The transverse relaxation time 2.7Uncooked HBFN sample (10 g) was put into 90 ml of distilled water and homogenized by stomacher machine  for 2 min and obtained the uniform mixture. A pH meter  was used to measure the Ph of HBFN.2.8HBFN sheets were cut into pieces of approximately 5 cm in width and length, and placed in the plastic bags at 25°C. The color was measured every 12 hr used a chroma meter  with the CIE 1976 L*, a*, and b* color scale.2.9p < .05. All tests were represented as the mean of three replicates and standard deviation (SD). TPC data in figures were transformed into logarithms of the number of colony forming units (CFU/g).Analysis of variance (ANOVA) was performed using Duncan's multiple range test by SPSS version 20.0, and significant differences were defined at 33.1p < .05) reduction in initial TPC of the mixed flour, especially from 30 to 60 s. Considering from the point of TPC reduction, 60 s was chosen for microwave treatment sterilization. However, it should be noted that the temperature of mixed flour was increased rapidly with the microwave‐treated time and then appeared serious caking phenomenon in 60 s. It had been investigated that there was a strong correlation between the caking phenomenon and temperature  is considered as the most important factor leading to the perishability of fresh noodles  were investigated Figure Relatively speaking, 10 and 20 s were not very effective on reducing initial TPC. Fifty seconds and sixty seconds had a good effect on reducing initial TPC, but both flour quality and noodle quality were also declined. Therefore, in terms of bactericidal efficiency and noodle quality, the optimum microwave treatment time of flour was 40 s.3.35 CFU/g was defined as the upper limit of microbe in fresh noodles. Hence, it is terminated to test the microbe quantity when TPC overtake 3 × 105 CFU/g, and changes of TPC in HBFN with different treatment during storage at 25°C are showed in Figure According to the Chinese standard of NY/T1512‐2014 “green food—uncooked processed food made of cereals,” the TPC value of 3 × 103.4The texture of noodles is the most important issue for consumers  for 40 s provided 30% reduction of initial TPC of the mixed flour without decline of noodle quality. Shelf life of HBFN treated with MW + ε‐PL (0.04%) + CP (0.020%/0.025%) reached 80 and 88 hr, respectively, while HBFN had good edible and color qualities during storage at 25°C. The results point out that the “hurdle effect” is a practical and economical approach, promising technique to prolong the shelf life of highland barley fresh noodles.The authors have no conflict of interest to declare.This article does not involve any human or animal testing.None.


Bacillus subtilis involved in making sporulation killing factor (SKF). We found that SkfB is a modular enzyme with an N-terminal peptide-binding domain comprising a RiPP recognition element (RRE), a middle domain that forms a classic AdoMet radical partial (β/α)6 barrel structure and displays AdoMet bound to the [4Fe-4S] cluster, and a C-terminal region characteristic of the so-called Twitch domain housing an auxiliary iron-sulfur cluster. Notably, both crystallography and Mössbauer analyses suggest that SkfB can bind a [2Fe-2S] cluster at the auxiliary cluster site, which has been observed only once before in a SPASM/Twitch auxiliary cluster site in the structure of another AdoMet radical enzyme, the pyrroloquinoline quinone biosynthesis enzyme PqqE. Taken together, our findings indicate that SkfB from B. subtilis represents a unique enzyme containing several structural features observed in other AdoMet radical enzymes.Sactipeptides are a subclass of ribosomally synthesized and post-translationally modified peptides (RiPPs). They contain a unique thioether bond, referred to as a sactionine linkage, between the sulfur atom of a cysteine residue and the α-carbon of an acceptor residue. These linkages are formed via radical chemistry and are essential for the spermicidal, antifungal, and antibacterial properties of sactipeptides. Enzymes that form these linkages, called sactisynthases, are AdoMet radical enzymes in the SPASM/Twitch subgroup whose structures are incompletely characterized. Here, we present the X-ray crystal structure to 1.29-Å resolution and Mössbauer analysis of SkfB, a sactisynthase from  Sulfur to alpha-carbon cross-linked peptides (sactipeptides)2–A) (–S-adenosyl-l-methionine (AdoMet) radical enzyme superfamily, which utilize a canonical CX3CXφC motif (where φ is a conserved aromatic amino acid) to coordinate a [4Fe-4S] cluster, thereby named the AdoMet radical cluster (•) species. 5′-dAdo• subsequently abstracts a hydrogen atom from the substrate, forming 5′-deoxyadenosine and a substrate radical, which undergoes further transformation to form the desired product. In the case of the characterized sactisynthases, H-atom abstraction has been shown to occur from the α-carbon of the acceptor residue  –10. KnowA) –, sporulaA) –, and theA) –, respectA) –. These s cluster . AdoMet  residue , 13–15.In addition to a characteristic AdoMet radical core, which houses the active site, sactisynthases are predicted to contain an N-terminal peptide-binding domain called a RiPP recognition element (RRE)  and a C-Fig. S1). SPASM domains are characterized by the presence of a conserved seven-cysteine motif (CX9–15GX4CgapCX2CX5CX3CgapC) that is used to coordinate two iron-sulfur clusters, Aux I and Aux II . Thus, the role of the Aux I cluster in CteB is not clear at this time.The first set of structures of SPASM and Twitch domain–containing enzymes showed variations in the cysteine ligation of the Aux clusters. anSME (SPASM subclass) and BtrN (Twitch subclass) displayed Aux clusters that were fully ligated, whereas the molybdenum cofactor biosynthetic enzyme MoaA displayed an open coordination site for the binding and positioning of the substrate GTP in the active site –21. For –Fig. 112 of the precursor peptide SkfA (B) during the maturation of the sactipeptide sporulation killing factor in Bacillus subtilis . This architecture is characteristic of AdoMet radical enzymes and provides a binding site for cofactors and substrates in a protected cavity that facilitates the corresponding radical mechanisms of members of the superfamily. Herein, we will refer to the partial (β/α)6 TIM barrel architecture as the AdoMet radical domain. Residues from the canonical CX3CXφC motif, Cys117, Cys121, and Cys124, are located on the loop following β1 and ligate an essential [4Fe-4S] cluster, the AdoMet radical cluster, leaving a unique iron for direct coordination of AdoMet .The central domain of SkfB (residues 106–322) folds into a partial (β/α)itecture , A–C. Th160, Gly161, and Glu162, and Arg223 orient the α-amino and α-carboxyl moieties of AdoMet. The ribose motif, Ser211, and residue Arg223, located adjacent to helix α4a, serve to position the hydroxyls of the ribose ring in the active site. The adenine moiety of AdoMet is oriented in the active site through hydrophobic interactions from Thr251 of the “GXIXGXXE” (or β5 motif) and from Phe123 of the CX3CXφC motif. Polar backbone interactions from the β6 motif, Leu258, and from the hydrophobic residues of the CX3CXφC motif, Phe123 and Tyr125 , 2322, 2d Tyr125 , furtherB). The majority of the linker between the AdoMet radical domain and the C-terminal domain (residues 322–329) is disordered in SkfB. Following the first visible residue of the C-terminal region (Thr330), the structure folds into a three-stranded antiparallel β-sheet, β1′–β3′, followed by two α-helices, a short α1′ and a longer α2′ . This C-terminal domain ligates Aux I, which in this structure is a [2Fe-2S] cluster (B). Cluster ligation involves one cysteine from the loop following β2′, Cys351, and two cysteines from the loop following α2′, Cys385 and Cys387 (B). This architecture is as expected for a Twitch domain (A–D) (A and E–G), but the identity of the auxiliary cluster as a [2Fe-2S] cluster was a surprise. Although a [2Fe-2S] cluster is observed in the Aux I–binding site of PqqE (E), all other structurally characterized SPASM- or Twitch-containing AdoMet radical enzymes have Aux I clusters that are [4Fe-4S] clusters (Table S1)  , 20 and  of PqqE E, all otable S1) –26.XC sequence, Cys385 and Cys387 and Cys323 and Cys325, respectively, following helix α2 to ligate the visualized [2Fe-2S] cluster where each cysteine coordinates a different iron of the cluster. A CXC cluster-binding sequence has not been seen in the other SPASM/Twitch enzymes structurally characterized . The fourth conserved cysteine, Cys333, was expected to ligate the cluster from its position in the sequence before the β-hairpin in SkfB; coordination of the cluster by a cysteine in this position has been observed in all other characterized SPASM/Twitch family members. However, in this structure, Cys333 is visible about 12 Å away from the cluster on the linker region that connects the AdoMet domain to the Twitch domain .In terms of cluster ligation, the SPASM enzymes anSME, SuiB, and PqqE and the Twitch enzyme BtrN have fully ligated auxiliary clusters , whereasFig. 557Fe Mössbauer spectroscopy. The 4.2 K, 53-mT Mössbauer spectrum of reconstituted SkfB  can be simulated with two quadrupole doublets with parameters typical of [2Fe-2S]2+ clusters  and [4Fe-4S]2+ clusters . Together with the 57Fe/SkfB stoichiometry of 5.41 (determined by ICP-MS), these results indicate the presence of 1.0 [2Fe-2S]2+ and 0.8 [4Fe-4S]2+ clusters per polypeptide, consistent with the notion that SkfB harbors one [2Fe-2S] and one [4Fe-4S] cluster. Addition of the cosubstrate, AdoMet, to the sample leads to a noticeable broadening of the high-energy line of the [4Fe-4S]2+ cluster . The observed perturbation is consistent with binding of AdoMet to the unique iron site of the [4Fe-4S]2+ cluster and can be rationalized with a site-differentiated mixed-valent (Fe2)5+ pair as was observed for other AdoMet radical enzymes previously  exhibits only one quadrupole doublet with parameters virtually identical to those of the [2Fe-2S]2+ cluster observed in WT SkfB, consistent with notion that the [2Fe-2S]2+ cluster resides in the Aux I site and that the AdoMet radical cluster is a [4Fe-4S]2+ cluster.We further investigated a reconstituted sample of the SkfB variant in which the three cysteines coordinating the AdoMet radical cluster were substituted with alanine residues (ΔRS SkfB). The Mössbauer spectrum of this variant (A and B). It folds first into a three-stranded antiparallel β-sheet, β1″–β3″, followed by a helical bundle comprising four consecutive α-helices, α1″–α4″, that stacks against the lower face of the antiparallel β-sheet (E). Following α4″, SkfB folds into a fourth β-strand, β4″, before connecting to the AdoMet radical domain. It is the upper face of the antiparallel β-sheet that forms the interface with the AdoMet radical domain and Twitch domain.The N-terminal domain of SkfB (residues 12–105) sits below the N-terminal region of the AdoMet domain and the C-terminal Twitch domain , A and B β-sheet E. FollowE and 8A) is reminiscent of a peptide-binding protein that is involved in the biosynthesis of pyrroloquinoline quinone, PqqD (B), the peptide-binding domains of a nisin biosynthetic enzyme, NisB (C), and a cyanobactin biosynthetic enzyme, LynD (D), an architecture termed the RRE (30–C and D). In SkfB, the antiparallel β-sheet, β1″–β3″, and α1″–α3″ constitute the RRE motif .The N-terminal domain architecture of SkfB E and 8A ne, PqqD B, the peme, NisB C, and a me, LynD D, an arc the RRE , 30–33. e RRE 30–, C and DA) and the enzyme SuiB (B), which is involved in cross-linking two amino acid side chains . Similar to SkfB, both CteB and SuiB are members of the SPASM/Twitch subfamily and adopt a modular overall architecture comprising three domains: an N-terminal peptide-binding RRE domain, a central AdoMet radical domain, and a C-terminal SPASM domain in place of the C-terminal Twitch domain visualized in SkfB. CteB and SuiB structures contain three [4Fe-4S] clusters, including the AdoMet radical cluster, bound by the canonical CX3CXφC motif, and two auxiliary clusters, Aux I and Aux II, bound by the SPASM domain. In addition, the linkers between the AdoMet radical domain and the SPASM domains were visualized in these structures. In SuiB, this linker region donates the first cysteine ligand to Aux I, resulting in two fully ligated auxiliary clusters (G). In CteB, Aux I has an open coordination site, which is ligated by a cysteine from the N terminus of the peptide substrate CteA (F). Structural comparisons show that the AdoMet radical domain and SPASM domains of CteB and SuiB overlay well with the AdoMet radical and Twitch domains of the SkfB structure (C and D). The main differences between SkfB and CteB or SuiB are that SkfB has a Twitch rather than SPASM domain, the ligation and nature of the Aux I cluster, and the location of the N-terminal peptide-binding domain.In addition to SkfB, the structures of two other AdoMet radical enzymes involved in the biosynthesis of RiPPs have been determined: the sactisynthase CteB A and theyme SuiB B, which clusters G. In Cteate CteA F. Structtructure , C and DA). Surprisingly, the leader peptide of SuiA binds to SuiB without interacting with its RRE domain. Instead, SuiA interacts with the AdoMet radical domain, the SPASM domain, and the linker between these two domains (B). There is considerable variability in the placement of the RRE domain with respect to the other domains. CteB has a long flexible linker between α3″ of the RRE domain and β1 of the AdoMet radical domain (A), which could allow flexibility in the positioning of the RRE domain with respect to the rest of the protein structure. In fact, the RRE domain of CteB and the RRE domain of SkfB are observed on the opposite sides of the AdoMet radical domain (C). The placement of the RRE domain of SuiB is more similar to that observed in SkfB (D). Shorter linkers between the RRE domain and the AdoMet radical domain are observed in SuiB (five residues) and SkfB (nine residues) than is seen in CteA (22 residues).The N-terminal domains of both CteB and SuiB adopt typical RRE folds. As aforementioned for NisB and LynD, the leader peptide, CteA, adopts a β-strand conformation and interacts with β3″ of CteB, extending the antiparallel β-sheet of the RRE domain A. Surpri domains B. There l domain A, which l domain C. The pl in SkfB D. ShorteHere, we present the structure of an AdoMet radical Twitch enzyme, SkfB, involved in maturation of a sactipeptide natural product. The structure of SkfB allows us to compare and contrast structural features with the previously solved SPASM domain–containing RiPP biosynthetic enzymes, SuiB and CteB, as well as other SPASM and Twitch domain–containing AdoMet radical enzymes for which structures have been determined, anSME, BtrN, MoaA, and PqqE.X33CXC sequence. This cluster identity was unexpected as the initial biochemical and spectroscopic characterization proposed that SkfB contained a [4Fe-4S] auxiliary cluster ligated by a CX4CXC sequence  from the N-terminal fragment of CteA cocrystallized with CteB. The N-terminal fragment used in cocrystallization encompassed the leader peptide sequence (residues −18 to −1) and the first three residues of the core peptide (residues 1–3) of CteA  but did not include the cysteine involved in thioether bond formation. This cysteine ligation to Aux I could foreshadow binding of the reactive cysteine (Cys14) in the presence of full-length CteA.The first structure of a sactisynthase, CteB, revealed that there is an open coordination site on Aux I of the SPASM domain, whereas Aux II is fully ligated . InteresH and I). Interestingly, the fourth cysteine residue, Cys333, was observed in the structure a distance away from Aux I. Because of the disorder in this region, however, we cannot rule out that Cys333 could serve as a cluster ligand in some enzyme states. A structure of SkfB in which the linker is ordered will be important for making a final determination. Regardless, the structure of SkfB leaves open the possibility that substrate will interact directly with Aux I. Recent studies  is short, about 20 Å, compared with 33 Å in CteB. To position the 55-mer SkfA precursor peptide in a catalytically competent orientation, it is possible that SkfA will adopt an ordered secondary structure, the RRE domain of SkfB will undergo a conformational change upon SkfA binding, or a combination of both. How the full-length precursor peptides bind to sactisynthases to allow for sactionine linkage formation and possible conformational changes associated with this interaction still remains to be determined.Overall, SkfB represents a unique hybrid of features observed in other AdoMet radical enzymes. It has the same domain organization as CteB and SuiB but has a Twitch, not a SPASM, domain. SkfB's Twitch domain is like that of BtrN and MoaA, but its Aux I cluster is most like the Aux I cluster found in PqqE and not in BtrN or MoaA. A sequence similarity network  shows thskfB gene was previously amplified from the B. subtilis subsp. subtilis strain 168 genome and cloned into a pET28a vector containing a tobacco etch virus cleavable N-terminal His6 tag . To obtain data-quality crystals, 1.5 μl of SkfB  was mixed with 0.5 μl of precipitant (0.25 m magnesium formate and 15% PEG 3350) and equilibrated over a 500-μl reservoir. Brown crystals grew within 24 h and were transferred to a Coy Scientific anaerobic chamber (95% argon and 5% H2 atmosphere) for harvesting. To cryoprotect SkfB crystals, the crystals were transferred in four steps into a cryoprotectant solution containing 0.2 m magnesium formate, 20% PEG 3350, and glycerol. Upon each transfer, the glycerol concentration was increased to a final concentration of 20%. Crystals were then flash frozen in liquid nitrogen within the Coy chamber.SkfB was crystallized within an MBraun anaerobic chamber at 21 °C and under a Nα rotating anode source (Rigaku) with an imaging plate . To obtain phasing information, the data set was collected using a pseudo inverse beam in four 90° wedges with 1° increments. Following the home data collection, the crystal was shipped to the Advanced Photon Source  where a native data set was collected to a 1.29-Å resolution at beamline 24-ID-C using a Pilatus 6M pixel detector at a temperature of 100 K and a wavelength of 0.9792 Å. All data were processed in HKL 2000.An anomalous data set and a native data set of SkfB were collected on the same crystal. The anomalous data set was collected to a resolution of 1.9 Å using an in-house CuKC2 space group that contained one molecule in the asymmetric unit. Using anomalous data trimmed to 2.5-Å resolution, six iron sites  code 4M7T) and MoaA (PDB code 1TV8) as guides. This initial model was used to define the solvent boundary during a subsequent round of solvent flattening in SOLOMON  refinements in Phenix  using rigid-body atomic coordinates and atomic displacement  cells were cotransformed with pNB529  and pDB1282 . A starter culture was incubated overnight in LB at 37 °C and 200 rpm. The next day, 15-ml aliquots of starter culture were pelleted by centrifugation (5000 × g) and gently resuspended in 15 ml of a modified M9 minimal medium containing 2% (w/v) glucose. Large-scale growth cultures containing M9 mineral medium (12 × 1.5 liters in 2.8-liter Fernbach flasks) supplemented with ampicillin (0.1 mg/ml), kanamycin (0.034 mg/ml), and 57FeSO4 (25 mm) were inoculated with 15 ml of resuspended starter culture cells. The large cultures were incubated at 37 °C and 175 rpm until the A600 nm reached 0.3, and arabinose and 57FeSO4 were added to final concentrations of 0.07% (w/v) and 0.05 mm, respectively, to induce pDB1282. After the addition of arabinose and 57FeSO4, the cultures were gradually cooled to 18 °C and slowed down to 150 rpm. After the cells reached an A600 nm of 0.6, expression from the skfB gene was induced upon addition of isopropyl β-d-1-thiogalactopyranoside to a final concentration of 0.1 mm. Cells were allowed to express SkfB at 18 °C for 18 h, harvested by centrifugation at 5000 × g, flash frozen in liquid nitrogen, and stored at −80 °C. SkfB and variant were purified and reconstituted as described previously  in 1% (v/v) trace-meta-grade nitric acid. Analyses were conducted by the ICP-MS Metals Lab of the Department of Geology and Geophysics at the University of Utah.The Mössbauer spectra were measured on a spectrometer from WEB Research  with an SVT-400 cryostat from Janis  to maintain the temperature at 4.2 K. A 53-mT magnetic field was applied parallel to the propagation direction of the γ-beam. Isomer shifts are reported with respect to the centroid of the spectrum of α-iron metal at room temperature. Simulations of Mössbauer spectra were carried out with the program WMOSS .T. A. J. G., W. M. K., N. A. B., E. J. B., C. K., V. B., and C. L. D. conceptualization; T. A. J. G., W. M. K., N. A. B., E. J. B., C. K., V. B., and C. L. D. investigation; T. A. J. G., V. B., and C. L. D. writing-original draft; T. A. J. G., W. M. K., N. A. B., C. K., V. B., and C. L. D. writing-review and editing; C. K., V. B., and C. L. D. funding acquisition; V. B. and C. L. D. supervision.