Tau protein, the major player in Alzheimer’s disease forms neurofibrillary tangles in elderly people. Bramhi (Baccopa Monniera) is often used as an ayurvedic treatment for Alzheimer's disease. Therefore it is of interest to study the interaction ofcompounds derived from Baccopa with the Tau protein involved in tangle formation. We show that compounds such as bacopaside II, bacopaside XII, and nicotine showed optimal binding features with the R2 repeat domain of hyperphosphorylated tau protein forfurther consideration in the context of Alzheimer's disease (AD). Alzheimer's disease (AD) affecting mainly the elderly is associated with the nervous system. Two distinct pathologies of AD include amyloid plaque deposition and neurofibrillary tangles of hyperphosphorylated tau protein .PresentB. monnieri and W. somniferawas proven to be effective against neurological disorders [B. monnieri and W. somnifera has not been investigated from an in-silico perspective. Basically, W.somnifera is a nootropic agent which enhances cognition and is administered to improve mental health and immunity [B. monnieri, this perennial creeper helps to improve cognition and cures various nervous disorders [B. monnieri and W. somnifera.Tau protein, the major player in AD has eight domains classified into N-terminal domain , proline-rich domain (P1 and P2), and four microtubule-binding domains (MBD). These microtubule-binding domains have four repeat domains  . Apart fimmunity . As per immunity . Regardiisorders ,10.TherB. monnieri and W. somnifera were downloaded from the PubChem database [B. monnieri and five from W. somnifera were considered for docking. As all the downloaded chemical compounds were in 2D format, a stable conformer of each compound was generated using BioviaDiscovery studio [ptau) and hyperphosphorylation (hptau) usingand Vienna PTM 2.0 software in our previous study [ptau and hptau using the CDOCKER tool from Biovia Discovery Studio. The active sites were identifiedbased on the available protein crystal structure report. CDOCKER energy scores were generated for each docking. The conformer with the lowest CDOCKER energy was selected for further analysis.Phytochemicals of database  [Table 1y studio  based onus study . All tweADME  studies of all twenty-three chemical compounds and the control drugs were performed using SWISS-ADME online server (http://www.swissadme.ch/)  to predihptaucompared to ptau. Bacopaside II displayed eight and twelve hydrogen bonds with ptau and hptau respectively. With bacopaside XII, four and seven hydrogen bonds were observed between ptau and hptaurespectively. Nicotine showed two and a single hydrogen bond with ptau  and hptau respectively (Table 2 - see PDF). The phytochemicals bacopaside II and XII interacted with the R2 and R3 domain in ptau. Afterhyperphosphorylation, interactions were with R2, proline-rich domain2 and C-terminal domain. Importantly, there were no interactions observed between the phytochemicals and the R3 domain (Table 2 - see PDF). However, nicotine maintained its interaction with R2domain in ptau and hptau. Furthermore, the phytochemicals of W. somnifera showed stronger binding with the ptau compared to the hptau. In ptau, Anaferineinteracted with R2 and the R3 domains, which were further, confined to R1 and R2 domain in hptau. In essence, these phytochemicals were able to interact only with the R2 domain and not with the R3 domain after hyperphosphorylation due to themajor conformational changes within the repeat domain. The non-availability of the R2 repeat domain after the binding of phytochemicals could avert the fibril formation with the R3 domain. Control drug Cusohygrin showed no hydrogen bonds with ptaubut preferred R2 domain in hptau. Even Isopellenterine could not establish a strong binding with the hptau (Table 3 - see PDF) (Table S1 -see PDF).The modelled tau protein was phosphorylated and hyperphosphorylated from in silico perspective and later considered for active site identification based on the available tau crystal structure (PDB ID: 2MZ7) bound to microtubules .Thus, fB. monnieri irrespective of their strong interaction with the hptau showed poor flexibility, polarity, and size. However, their Log P values were within the permissiblelimit. Other derivatives of B. monnieri like bacopaside III, IV, V, XI, Bacopasaponin A, B, C, D, G, Bacoside A, Bacoside A1, and Bacoside A3 also followed the same trend. In contrast, the derivatives of W. somnifera likeAnaferine, Anahygrine, Withanolide, cuseohygrin, and Isopellenterine irrespective of their weak interaction with the hptau fared well with respect to their physicochemical space for oral bioavailability, pharmacokinetics, and drug likeliness.To confirm these findings, software like PKCSM and MOLINSPIRATION were taken into consideration. As per the PKCSM report, the Caco2 permeability score of BacosideII and XII were out of range < than 0.9. The intestinal absorption of II and XII was 25.49% and3.78%, which are less than the cut-off score of 30% for better absorption. Thus, the parameters associated with absorption, distribution, and excretion were out of the permissible limit for bacopaside II and XII.  MOLINSPIRATION report also confirms this throughtheir molecular weight, Hydrogen bond donor-acceptor, and drug-likeness (Lipinski rule) (Table 4 -see PDF). As per the ADME study, only nicotine showed permissible values.  Irrespective of favourable binding energy and the pharmacokinetics report, nicotine-basedtreatment needs to be taken with caution due to their significant role in enhancing tau phosphorylation [The phytochemicals Bacoside II and Bacoside XII of rylation -24. As rrylation .These nrylation . Poly .


The heterogeneous profiles highlight the importance of comprehensive assessments for adequate service provision. Individuals who are prelingually deaf and have intellectual disabilities experience great challenges in their language, cognitive and social development, leading to heterogeneous profiles of intellectual and adaptive functioning. The present study describes these profiles, paying particular attention to domain discrepancies, and explores their associations with quality of life and maladaptive behavior. Twenty-nine adults with prelingual deafness  and mild intellectual functioning deficits  were administered the Vineland Adaptive Behavior Scales-II (VABS-II) and an adapted sign language version of a quality of life scale (EUROHIS-QOL 8). Intellectual disability domain discrepancies were characterized as at least one standard deviation difference between the social domain and IQ and the practical domain and IQ, and a significant difference, according to the VABS-II manual, between the social and practical domains. Domain discrepancies were found between intellectual functioning and both the practical (58.6%) and social domain (65.5%). A discrepancy between intellectual and social functioning was significantly associated with a higher level of internalizing maladaptive behavior (T = 1.89,  Deafness is a heterogeneous condition that can impact communication, social–emotional development and cognitive development . Around Approximately one-third to one-half of individuals who are prelingually deaf or hard of hearing have additional disabilities ,5, most The diagnostic criteria for intellectual disability have been revised in the Diagnostic and Statistical Manual of Mental Disorder-Fifth edition  to encoIntellectual and adaptive functioning, the two aspects of intellectual disability, are related but separate constructs : a largeWith this more nuanced definition of adaptive functioning has come greater interest in intellectual disability domain discrepancy, in which one domain is markedly more deficient than another, as well as inquiry into whether different populations have unique, or at least specific, intellectual disability profiles. Sparrow, Cicchetti and Balla , authorsThere is a growing body of research on adaptive profiles in individuals with different neurodevelopmental disorders ,20,21,22Studies correlating adaptive profiles with such clinically relevant variables as quality of life (QOL) and problem behavior e.g., ,25) show show25])No research to date has investigated the intellectual disability profiles and the relationships between intellectual disability domain discrepancies, QOL and maladaptive behavior in a population with prelingual deafness and intellectual functioning deficits. Hence, the main aim of this study is twofold: (a) to describe the intellectual disability profiles and potential intellectual disability domain discrepancies in a sample of adults who are deaf with borderline and mild cognitive functioning impairment and (b) to explore how these intellectual disability profiles and domain discrepancies are related to maladaptive behavior and self-reported QOL in this population. We explored whether expressed intellectual disability domain discrepancies between cognitive potential and lower social and practical abilities are experienced as stressful barriers to unlock one’s potential and therefore may be linked with lower quality of life and increased rates of maladaptive behavior. This cross-sectional exploratory study was conducted within three therapeutic living communities (Lebenswelt) specifically developed to accommodate the needs of individuals with deafness and additional disabilities, focusing on supporting communication, social relationships, conflict resolution and work satisfaction. They are characterized by the constant use of sign language; one-quarter of the staff members are deaf themselves .We recruited 29 individuals (9 women and 20 men) who met the inclusion criteria of having at least moderate hearing impairment and an IQ score between 50 and 85 see . Of thesThis study was approved by the ethical committee of the hospital St. John of God in Linz, Austria. Consent was given by the participants themselves and/or by their legal guardians (if applicable).The Vineland Adaptive Behavior Scales-II  is a comTo assess participants’ intellectual functioning, we administered the Snijders-Oomen Non-verbal Intelligence Scale for individuals . The SOThe EUROHIS-QOL 8-item index  is a short version of the WHOQOL-BREF. It consists of 8 questions that are also included in the 26 questions of the WHOQOL-BREF . All fouThe VABS-II includes two subscales for internalizing and externalizing maladaptive behavior. The two subscales are reported as v-scale scores. A v-scale score below 18 indicates a non-clinical level of maladaptive behavior; a score between 18 and 20 indicates an elevated level; and a score between 21 and 24 indicates a clinically significant level of maladaptive behavior .Data collection took place between September 2017 and March 2018. Participants’ clinical characteristics  were extracted from the medical records of the individuals at the hospital St. John of God. The QOL self-reports were gathered through a structured interview between the resident and a sign language-competent staff member who was not directly involved in the care of the residents (non-involvement in direct care was considered important in order for the participants not to feel pressured to give answers that they thought would be preferred by the interviewer).VABS-II data were collected for each participant by the staff psychologist in consultation with either a family member or the participant’s primary caregiver. Primary caregivers serve the roles of coach, case manager, advocate and personal assistant for residents and were therefore thought to be particularly well-qualified to serve as informants for this study.intellectual disability domain discrepancy to indicate that there is a substantial difference between the level of intellectual, social and/or practical adaptive functioning. For the purpose of this study, when comparing intellectual functioning with the social or practical domain, a difference of at least 15 points (=1 SD) between the IQ score (SON-R 6-40) and the socialization and daily living skills (DLS) standard scores indicates an intellectual disability domain discrepancy. When comparing the social and practical domains, a difference between the socialization and the DLS standard scores with a significance level of 0.05 according to the VABS-II manual indicates a domain discrepancy between these two domains.Within the context of this paper, we use the term t-test was performed.First, univariate analysis of the key variables was applied to describe intellectual disability domains, QOL and maladaptive behavior, as well as domain discrepancies. Next, Spearman’s correlation was performed to test for a correlation between intellectual disability domains , QOL and maladaptive behavior. To investigate whether there were significant differences in the means of self-reported QOL and internal and external maladaptive behavior with and without intellectual disability domain discrepancies, an independent samples p = 0.000), as are the practical domain and intellectual functioning . The social domain and intellectual functioning are not significantly correlated (see p < 0.1).ated see . There aAlmost two-thirds of the individuals had an intellectual disability domain discrepancy between their intellectual functioning level and the social domain , and more than half had an intellectual disability domain discrepancy between their intellectual functioning and the practical domain . About one-quarter of the individuals had an intellectual disability domain discrepancy between the social and the practical domains , where in all cases the social domain was the weaker domain. Thus, participants’ social adaptive skills were often poorer than their intellectual functioning and practical adaptive skills.p < 0.05).When comparing participants with and without intellectual disability domain discrepancies, high mean QOL was endorsed across both groups, and no significant differences were found see . ParticiThe aim of this study was to describe profiles of intellectual disability and domain discrepancies in a sample of adults who are prelingually deaf with mild and borderline cognitive impairment and to explore how these domains of intellectual disability are related with each other and associated with self-reported QOL and maladaptive behavior. Our findings provide a first indication of possible intellectual disability domain discrepancies among individuals with deafness and intellectual disability and highlight the value—as well as potential challenges or limitations—of DSM-5′s definition of intellectual disability for the deaf population. Furthermore, we investigated differences between those with and without domain discrepancies with respect to QOL and internalizing and externalizing maladaptive behavior.Heterogeneous intellectual disability profiles were highly common among our participants, with only 24% showing no discrepancy between intellectual and adaptive functioning. In nearly two-thirds of the sample, intellectual disability domain discrepancies could be observed between intellectual functioning (65.5%) and both the practical (58.6%) and the social domain (65.5%). Domain discrepancy between the practical and social domain occurred in about one-fourth of cases (24.1%). Less than half of the sample had adaptive functioning levels in the practical and social domains that corresponded to their level of mild intellectual impairment, whereas severe levels of impairment were evident in the practical domain in 28% of the sample and in the social domain in 41%.In our sample, the results of the communication subscales of the VABS-II, which were adapted but not originally designed for use in deaf populations, indicated severe deficits in 65.5% of the sample. Due to pronounced floor effects, the values of this domain were used neither as an equivalent for the conceptual domain nor for further calculations. Language is an important driver of acquiring social and practical skills above a rudimentary basal level , and thep < 0.05) and in line with the results of the meta-analysis of Alexander and Reynolds )We must also note this study’s limitations. First and foremost, this is a small sample drawn from a highly enriched therapeutic residential care setting for adults who are deaf and have intellectual disabilities. Since this sample has only borderline-to-mild cognitive impairment, we make no claim to generalizability to the larger population of adults who are deaf with intellectual disability; much larger replication trials are needed.Second, we must query whether the VABS-II, widely regarded as the “gold standard” adaptive measure in the majority of cases, is the most appropriate measure for this population. We therefore encourage further research in the service of formulating an optimal assessment battery both to gauge and, we hope, discover how to unlock these individuals‘ full potential. We would emphasize the importance of developing and validating a communication scale that is independent of (or less conflated with) solely spoken and auditory modalities.Clearly, the assessment of intellectual disability transcends IQ, and we hope to inspire efforts toward an even higher level of measure refinement and collaborative research between investigators and participants. Underscoring the DSM-5′s incorporation of adaptive functioning into its definition of intellectual disability, our population of participants who were prelingually deaf with mild cognitive impairments had a broad array of strengths and challenges. Intellectual functioning emerged as a relative strength, whereas almost half our participants had severe deficits in the social domain. Critically, a higher level of internalizing maladaptive behavior was observed in those participants with a domain discrepancy between their intellectual and social functioning. We must acknowledge that, even with our best efforts in providing accessible therapeutic communities in adulthood, deficits in the social domain could not be fully compensated after histories marked by severe trauma and deprivation. This finding constitutes a strong case for the early prevention of communicative and social deprivation by providing full access to spoken and/or signed communication. Nevertheless, nuanced measurement of adaptive skills gives us a good opportunity to identify and target malleable factors to improve QOL in individuals who are deaf and have intellectual disability more broadly.


According to the thermal deformation data, the true stress-strain curve of TA1 was studied. Meanwhile, the constitutive model and processing map were established through the experimental data. These results indicate that the deformation temperature negatively affects strain rate and flow stress. The heat deformation activation energy of EB produced TA1 sample was lower than that of VAR produced TA1 sample in the studied range. The best processing areas of EB-produced TA1 were strain rates of 0.05–0.01 s−1, within 700–770 °C; or strain rates of 0.01–0.15 s−1; 840–900 °C. The results of this paper enrich the fundamental knowledge of the thermal deformation behavior of TA1 prepared by EB furnaces.The Gleeble-3800 thermal simulator was used for hot compression simulation to understand the hot deformation performance of TA1 prepared by the single-pass electron beam cold hearth (EB) process. The deformation degree is 50% on a thermal simulator when the temperature range is 700–900 °C, with a strain rate of 0.01–10 Titanium is an important metal applied in modern industry and has characteristics including high strength, lightweight, strong corrosion resistance, and good temperature endurance ,2. The THowever, in the actual production process, TA1 titanium slabs using EB furnaces usually adopt the same hot rolling process scheme as the TA1 titanium slab melted by the VAR furnace. Since the thermal deformation behaviors of TA1 slabs produced using EB and VAR are different, surface defects, including peeling and scratching, appear during the hot rolling process of TA1 slabs melted by EB furnace, which increases the edge cutting process of titanium coil and production cost.−1, a temperature at 700–750 °C, and the deformation of each pass is greater than or equal to 25%. Li et al. [−1 and 750–850 °C. However, few studies are devoted to the thermal deformation behavior of TA1 slabs melted by the EB furnace. Huang et al. [Many scholars have studied the thermal deformation behavior of TA1 slabs melted by a VAR furnace. Ma et al.  establisi et al.  conducteg et al.  researchTherefore, this manuscript researches the thermal deformation behavior of TA1 prepared by EB furnaces, focusing on the impacts of strain rate and deformation temperature on the flow stress. Meanwhile, the constitutive model and processing map are founded according to the stress-strain data, which provides a reference for the improvement of the EB furnace melting technique and the subsequent TA1 hot rolling process.The test material was an industrial TA1 slab produced by an EB furnace in a factory in Kunming, China. The chemical composition is indicated in the quality assurance certificate of the enterprise, and has been verified according to the GB/T4698 standard method . The cheThe heat treatment temperatures of the sample were selected at 700 °C, 750 °C, 800 °C, 850 °C, and 900 °C, according to the thermal simulation compression deformation temperature, combined with TA1 phase transformation temperature and empirical rolling temperature range. The technical details about mechanical testing are: The temperature is measured by a thermocouple fixed in the middle of the sample. The temperature was recorded by the data acquisition system of the thermal simulator. The heating rate is 20 °C/s. Since the technology of hot compression deformation test using Gleeble testing machine is relatively mature, using one sample for each specific temperature and strain rate can meet the research requirements. But it also depends on the ability of the tester to control the machine. When the sample deformation is not uniform, that is, the obtained sample has irregular “drum shape” or even non- “drum shape”, the collected data will also appear obvious anomalies. At this point, another sample will be made to correct the corresponding process.−1, 0.1 s−1, 1.0 s−1, 5.0 s−1, and 10 s−1, separately. 50% was the set value of compression deformation. The settled temperature is maintained for 5 min, and water-cooling was carried out immediately after compression treatment.The corresponding strain rates were 0.01 s−1 strain rate and 700 °C, the maximum flow stress is about 58 MPa. While the maximum flow stress is about 160 MPa at a 10 s−1 strain rate. At high strain rates, the strain hardening trend is obvious, and the degree of dynamic softening is low, so the flow stress is enormous. −1 strain rate. When deformed below 850 °C, α-phase equiaxed polyhedral grain structure is obtained, which is mainly different in grain size and uniformity. The non-uniformity of grains obtained after deformation at 750 °C is obvious. It can be seen that some grains have not grown up after recrystallization. Then, with the increase of temperature, the grains grow up gradually and distribute uniformly in the region. However, when the temperature reaches 900 °C for deformation, as the deformation temperature is already in the β-phase zone, the sample structure has a flaky nature, the grains are irregular, and the sawtooth structure of the β-phase grain boundary appears. So, when the strain rate is low, the flow stress is low, which is because the sample is affected by dynamic recovery and dynamic recrystallization softening [−1, while 30 MPa is the maximum flow stress at 900 °C. Therefore, when the deformation temperature grows gradually, the softening impact of TA1 is more obvious, and the trend of flow stress reduction is more significant.The friction effect in the process of thermal deformation is reduced by placing graphite sheets at both ends of the sample, the most important variables are the friction force and the geometry of the sample ,15. To rAt high-temperature compression deformation, the relationship between peak stress and strain rate as a function of temperature is presented in During the hot deformation of TA1, the hardening impact of strain hardening, dynamic recovery, and dynamic recrystallization softening mechanism will cause the variation of flow stress. The stress-strain curve has two stages: unsteady deformation and steady deformation. At the initial stage of deformation, namely unsteady deformation, the flow stress growths quickly within a very small range of strain increases; The strain endures to growth while the flow stress grows gradually in the steady-state deformation process. The flow stress rises slowly when the deformation temperature is at a low level, and the strain rate is high. The flow stress grows slowly to the peak value, then declines slowly, and finally tends to be stable under the condition of a low strain rate and high deformation temperature. Plastic deformation occurs in the stage of unsteady deformation. The interaction of epistatic faults in the slip system causes the dislocation density to increase speedily, resulting in dislocation entanglement and stacking, and finally forms cellular dislocation structure, resulting in higher stress for material deformation . HardeniAs the strain increases, the material accumulates more energy in the steady-state deformation stage. Cellular dislocations form substructures, and dislocations of opposite signs are canceled out. The softening influence of dynamic recovery rises continuously, and the growing trend of flow stress becomes gentle. The material will have enough time to accumulate more energy at a large deformation temperature and a small strain rate . Dynamic−1); σ represents the stress, (MPa); T stands for deformation temperature, (K); Q means the activation energy of thermal deformation, (J/mol); R means the gas constant, (J/mol·K), the value indicates 8.314 [A, A1, A2, n, n1, =When studying the mechanical properties of metal materials during plastic deformation, the constitutive model can frequently describe the relationship between strain, stress, temperature, and strain rate. As stated by stress-strain data of TA1 melted and cast in the EB furnace obtained in the experiment, the constitutive model was derived by using the constitutive equation. Arrhenius constitutive equation was exploited to derive, which was summarized and improved from various models, and can accurately express the relationship between flow stress, strain rate, and temperature . The thres 8.314 ; A, A1, 1 represented the average slope of the five straight lines –(3), and the equations were transformed into Equations (4)–(6). The T on both sides of Equation (6) was calculated, expressed as [The relationship of essed as : (7)Q=RnT when the true strain was 0.69. Under different strain rates, the average straight-line slope value was 16.55988, which was substituted into Equation (7) to reckon the thermal deformation activation energy (Q), which was 410.655 kJ/mol.In general, when characterizing the impact of temperature and strain rate on the deformation behavior of materials, the Zener-Holloman parameter of temperature-compensated strain rate factor is often used in Equation (8) .(8)ln ZAccording to Equation (3):lnA was equal to When The constitutive model can be obtained by replacing the above parameters with the hyperbolic sinusoidal constitutive equation:α, n, Q, and lnA are shown in Arrhenius’s constitutive model only thinks about the peak stress but ignores the influence of true strain . To obtaα, n, Q, and lnA in the process of fitting attempt, at least 70% of the calculated points fall on the fitting curve, and the points are evenly distributed on both sides of the curve, guarantee the accuracy of the fitting. Through fitting, it is found that the result of seven times fitting is more accurate. A polynomial of degree seven can express the relationship between α, n, Q, lnA, and true strain ε, as shown below:A polynomial fitting curve of the seventh degree between the strain and the material constants in the constitutive model is shown in The above parameters are substituted into the hyperbolic sinusoidal constitutive equation. The constitutive model of TA1 considering strain shadow is:To reflect the influence of deformation parameters on processing properties, the processing map is drawn to lead the hot deformation process of materials in actual production, where the dynamic materials model (DMM) is generally adopted ,27. WhenP represents the total energy absorbed [The dynamic material model treats the thermal deformation process as an energy dissipation element. During the plastic deformation of the workpiece, absorbed ,29. Duriabsorbed :(17)P=σ·G), mostly converted to heat; (2) The power dissipation covariance (J) is the energy consumed by microstructure evolution [The energy absorbed by materials is mainly scattered in two methods: (1) The energy dissipation affected by plastic deformation is expressed as power consumption  consumed by the evolutionary process of the microstructure and the linear dissipation energy is usually expressed by the power dissipation exponent η [During the material forming process, the proportional relationship between the energy  and (20). The thermal processing diagram of the dynamic material model is obtained by drawing the principle of superposition of the power dissipation diagram and instability diagram, as shown in −1, separately; The range of strain rate and deformation temperature is 0.01–0.15 s−1 and 840–900 °C, individually. Plastic flow instability region: the range of strain rate and deformation temperature are 3–10 s−1 and 860–880 °C, respectively; The range of strain rate and deformation temperature range are 1–10 s−1 and 700–840 °C, individually. To effectively reduce the rolling mill load, in combination with the actual industrial production, the processing area ④ is selected for the hot working deformation test of TA1 titanium billet. The preferred 860 °C open rolling annealing organization is shown in the following 0.2 is 275 MPa, elongation after fracture is 44%, and the strength and toughness are good.In The VAR process is difficult to eliminate high-density and low-density inclusions during the titanium melting process . In the (1)TA1 prepared by electron beam cold hearth melting in just one pass was very sensitive to strain rate and temperature. The strain rate and temperature of TA1 cast in the EB furnace were very significant. Rising the temperature and lessening the strain rate will reduce the flow stress. In the unsteady deformation stage, the flow stress increased rapidly; The flow stress raised slowly or even fell in the steady-state deformation stage.(2)The constitutive model of TA1 for EB furnace casting considering the effect of strain was Equation (15).(3)−1 at the deformation temperature range was 700–770 °C; When the deformation temperature range was 840–900 °C, the strain rate range was 0.01–0.15 s−1.Based on the processing map of the TA1 sample produced by the EB furnace, the suitable processing areas were obtained: The range of strain rate was 0.01–0.05 s(4)Compared with VAR produced TA1 sample, EB produced TA1 sample had lower activation energy of thermal deformation and a smaller suitable range for thermal processing.


The target search begins with the protospacer adjacent motif or PAM-interacting domain, recognizing it at the major groove of the duplex and melting its downstream duplex where an RNA-DNA heteroduplex is formed at nanomolar affinity. The rate-limiting step is the formation of an R-loop structure where the HNH domain inserts between the target heteroduplex and the displaced non-target DNA strand. Once the R-loop structure is formed, the non-target strand is rapidly cleaved by RuvC and ejected from the active site. This event is immediately followed by cleavage of the target DNA strand by the HNH domain and product release. Within CRISPR-associated protein 9, the HNH domain is inserted into the RuvC domain near the RuvC active site via two linker loops that provide allosteric communication between the two active sites. Due to the high flexibility of these loops and active sites, biophysical techniques have been instrumental in characterizing the dynamics and mechanism of the CRISPR-associated protein 9 nucleases, aiding structural studies in the visualization of the complete active sites and relevant linker structures. Here, we review biochemical, structural, and biophysical studies on the underlying mechanism with emphasis on how CRISPR-associated protein 9 selects the target DNA duplex and rejects non-target sequences.The CRISPR-associated protein 9 (Cas9) has been engineered as a precise gene editing tool to make double-strand breaks. CRISPR-associated protein 9 binds the folded guide RNA (gRNA) that serves as a binding scaffold to guide it to the target DNA duplex  Streptococcus pyrogenes or SpyCas9) is one of the most extensively studied systems for which crystal and cryo-EM structures are known in many functional states  is to recognize and destroy invading phages or plasmids during reinfection. Cas9  small local motions of the constituent domains responsible for rearrangements of catalytic residues required for activation and ii) large motions of domains necessary for the protein-nucleic acid complex assembly. Some of these dynamic properties have been addressed using MD simulations and NMR spectroscopy as well as single-molecule spectroscopy . We focuStreptococcus pyrogenes has 1,368 amino acid residues comprising the two catalytic domains of RuvC and HNH, three recognition (Rec) domains of Rec I, Rec II, and Rec III, a PAM-interacting domain, and a bridge helix  clearly shows that an extensive interface is formed between the HNH and Rec II domains so that the HNH catalytic active site is also buried in the HNH catalytically relevant complex.MD simulations including enhanced sampling have been applied to characterize large conformational changes in the HNH domain of Cas9 . It has 2+ ions with binding affinity of 1.6 mM and 5.9 mM, respectively, although these specific binding sites have yet to be structurally characterized. Structural biologists often use Mn2+ in crystallographic electron density maps or cryo-EM derived electrostatic potential maps for metal ion identification  plus a short α-helix (T769-R778) before entering the HNH domain while L2 includes an extended loop (S909-I917) and a long α-helix (I917-N940) connecting the RuvC domain. These two linker loops change local structures and swap positions in the HNH-relevant complex between the 5f9r and 6o0y complexes, related by a twisting domain motion . L1 becoKd)  . HoweverRecent MD simulations of an isolated HNH domain in the wild-type enzyme and three single Lys-to-Ala mutated enzymes show that the HNH domains remain in an inactive conformation in the absence of DNA substrate in an isolated HNH domain as determined by the N863 location and its backbone conformation . There iThe entire backbone of the HNH-RuvC region was reasonably well defined in the crystallographic electron density maps, although the loop containing K848 was built as a poly model and so were many other regions of the HNH domain in the 5f9r structure  Jiang e. This lovia the tDNA-induced activation of the HNH activation  and tDNA strand (involving a single Mg2+ ion), respectively. Crystallographic studies reported the most relevant catalytic state of Cas9 with Mn2+ as an alternative to Mg2+ in the RuvC domain  showed a remarkable reduction in enzymatic activity .All-atom MD simulations, in combination with network models derived from graph theory, have shown that the binding of PAM induces a population shift and highly coupled motions of HNH and RuvC, showing a typical allosteric response that is in line with previous biochemical studies . PAM binB = 8π2|Δr|2, where |Δr| is root-mean-squares fluctuation). As a result of this analysis, the structure with the lowest free energy has the smallest mean atomic B-factors with the deepest free energy well. Unstable structures may never reach an equilibrium state and the corresponding MD-derived ED maps are often uninterpretable. The same computational approaches could be applicable to other domain-rotations problems such as DNA duplex strand exchange reactions within dimer of dimers in γδ resolvase and RuvC in resolution of Holliday junction  dynamics are critical for the signal transmission that mediates the communication of DNA binding information from the Rec domain to the nuclease sites . Surprisjunction . Domain junction . Enhancejunction . These fjunction .Divide-and-conquer methods, as have been demonstrated experimentally , could bCas9 is a highly dynamical biomolecule and exists in many different functional states in order to perform biological functions optimally and efficiently. To fully understand the specific function of each state, one must apply integrative approaches combining computational and experimental methods. Under a single experimental condition, three different functional states of Cas9 have already been detected to coexist simultaneously  , as is sExperimentally determined structures of macromolecules often correspond to equilibrium states under well-defined experimental conditions, which can be simulated by equilibrium structures obtained by MD simulations . Moreove


Picornaviridae. EV-D68 was initially considered a respiratory virus that primarily affected children. However, in 2014, EV-D68 outbreaks occurred causing the expected increase in respiratory illness cases, but also an increase in acute flaccid myelitis cases (AFM). Sequencing of 2014 outbreak isolates revealed variations in the 5′ UTR of the genome compared to the historical Fermon strain. The structure of the 5′ UTR RNA contributes to enterovirus virulence, including neurovirulence in poliovirus, and could contribute to neurovirulence in contemporary EV-D68 strains. In this study, the secondary and tertiary structures of 5′ UTR RNA from the Fermon strain and 2014 isolate KT347251.1 are analyzed and compared. Secondary structures were determined using SHAPE-MaP and TurboFold II and tertiary structures were predicted using 3dRNAv2.0. Comparison of RNA structures between the EV-D68 strains shows significant remodeling at the secondary and tertiary levels. Notable secondary structure changes occurred in domains II, IV and V. Shifts in the secondary structure changed the tertiary structure of the individual domains and the orientation of the domains. Our comparative structural models for EV-D68 5′ UTR RNA highlight regions of the molecule that could be targeted for treatment of neurotropic enteroviruses.Enterovirus-D68 (EV-D68) is a positive-sense single-stranded RNA virus within the family  Enterovirus and family Picornaviridae. When EV-D68 was first discovered in California in 1962 as a childhood infection, it was thought to only cause respiratory illness [Enterovirus D68 (EV-D68) is a positive-sense single-stranded RNA virus of the genus  illness ,4,5,6. I illness ,6,7. Fur illness . Since 2 illness ,12,13.The question is, what has caused contemporary strains of EV-D68 to cause AFM, which was previously unseen in historical cases? Possible virulence and tropism phenotypic changes that could result in contemporary EV-D68 strains causing increased rates of AFM include the gain of neurotropism, increased neurovirulence and increased neuroinvasion. These changes are not mutually exclusive and could coexist. Work in neuroblastoma cell lines has suggested that contemporary strains of EV-D68 have gained the ability to infect neuronal cells , and worSome general determinants of EV-D68 neurovirulence in later strains have been identified. For example, studies have suggested that viral capsid proteins VP1, VP2 and VP3 and nonstructural proteins 2A, 2C, 3A and 3D have an influence on neurovirulence . In addiThe 5′ UTR of enteroviruses is a highly structured RNA region that is responsible for cap-independent translation initiation and replication of the enteroviral genome ,23,24,25Our work compares the structures of the 5′ UTR of EV-D68 from the historic Fermon strain and the 2014 isolate KT347251.1. The secondary structure of the 5′ UTR RNA of each strain was determined using SHAPE-MaP and TurboFold II . The terEscherichia coli cells . Glycerol stocks of transformed cells were stored at −80 °C.For both the Fermon (NC_038308.1) and 2014 (KT347251.1) strains, a p2RZ plasmid containing the 5′ UTR sequence was designed, constructed and isolated . The strTo isolate the p2RZ plasmid constructs, an isolated colony from an LB plate containing 200 ug/mL of ampicillin (LBamp) was inoculated in 25 mL of liquid LBamp and cultured for 14 to 16 h at 37 °C. Plasmid DNA was isolated from 5 mL of this overnight culture using the Qiagen Spin Miniprep Kit  according to the manufacturer’s instructions. Plasmid concentration was evaluated using a Thermo Scientific NanoDrop 2000c while the DNA was visualized on a 1% wt/v agarose gel containing 2 μg/mL of EtBr following electrophoresis at 70 V for 60 min and imaged on an Axygen imager at a wavelength of 302 nm.The plasmids were linearized using Eco-RV  at 37 °C for 180 min and purified by extraction using phenol chloroform and chloroform and ethanol precipitation. The presence of the plasmid was once again confirmed using an agarose gel and imaging process as described above. The linearized DNA was transcribed into RNA using an Invitrogen MEGAscript T7 High Yield Transcription Kit  according to manufacturer’s instructions. RNA was isolated by two rounds of phenol extraction using phenol chloroform and chloroform and precipitated using isopropanol precipitation. The presence of RNA was confirmed using a 1.5% wt/v agarose gel with 2 μg/mL of EtBr electrophoresed at 70 V for 120 min and imaged on the Axygen imager. RNA was further purified using an Invitrogen MEGAclear kit, and concentration and purity were determined using a NanoDrop 2000c.2O, denatured at 95 °C for two minutes and cooled on ice for two minutes. The RNA was then refolded at 37 °C for 20 min after the addition of 6 μL of 3.3× folding buffer . A 300 mM stock of 1-methyl-7-nitroisatoic anhydride (1M7)  in DMSO  was further diluted to 100 mM of 1M7 in DMSO for the reactions. Each modification reaction was performed in duplicate from this point on. The positive sample was modified by the addition of 1 μL of 100 mM 1M7 to 9 μL of RNA solution and incubation at 37 °C for 85 s. For the negative sample, the same procedure is used with the use of neat DMSO rather than the 1M7 solution. Finally, to modify the DC sample, 10 pmol of RNA was diluted into 3 μL of ddH2O and mixed with 5 μL of 100% formamide  and 1 μL of DC buffer . This mixture was incubated at 95 °C for 1 min to denature the RNA. Finally, 1 μL of 100 mM of 1M7 was mixed with 9 μL of denatured RNA and incubated at 95 °C for 1 min. Once the positive, negative and DC sample sets were generated in duplicate, the two samples were pooled and purified using the Qiagen RNeasy Mini Kit  according to manufacturer’s instructions.SHAPE-MaP experiments were run according to the protocols of Smola et al. . Three RTo detect modified positions, the RNA was reverse transcribed and sequenced. Samples were reverse transcribed in duplicate using the Invitrogen Superscript II Reverse Transcriptase and New England Biolabs  NEBNext Ultra II Non-directional RNA Second Strand Synthesis module. Once cDNA was generated, samples were pooled and purified using the Invitrogen Purelink PCR Micro Kit  and sequenced at the University of Nebraska Medical Center Sequencing Core using Illumina MiSeq paired-end sequencing using a Nextera DNA library preparation kit.https://github.com/Weeks-UNC/shapemapper2, accessed on 23 February 2020). Sequencing runs were required to pass a minimum read depth of 2000 before moving forward with structure determination. Successful runs were advanced through TurboFold II and aligned against homologous sequence sets .The secondary structure of RNA from each virus was determined by running the FastQ files generated by sequencing through Shapemapper-2.1.5 . Then, a DCA file was generated using the Xiao Lab DCA Web Server  to guide the folding. The dot bracket file and DCA file were uploaded into the 3dRNAv2.0 webserver  and five potential tertiary structures were generated based on distance geometry and energy minimization constraints. Each of the five structures were evaluated based on the energy distribution between nucleotides using 3dRNAscore. The structure with the lowest score, symbolizing the highest probability of accurate base pairing and tertiary interaction, was then chosen for further analysis. The resulting tertiary structures were then visualized and pseudocolored in PyMOL 2.5 .Tertiary structure predictions were carried out by converting the ct file produced by TurboFold II to a dot bracket file using the University of Rochester CTtoDOT webserver  and TurboFold II . Figure Domain I is characterized by a cloverleaf structure that is highly conserved in enterovirus 5′ UTRs. The overall cloverleaf structure is conserved in both the Fermon and 2014 strains but a single nucleotide insertion at position 30, as well as two point mutations at positions 62 and 63, have led to structural changes in the secondary and tertiary structures . The mosIn contrast to the truncation seen in stem-loop 1b, stem-loop 1c expands from a two-nucleotide stem capped by a tetraloop in the Fermon strain to a four-nucleotide stem capped by an eight-nucleotide loop in the 2014 strain. Both strains, however, display an AGCU sequence in the loop. Stem-loop 1d is truncated in the 2014 strain, resulting in the loss of a stem with a bulge loop. However, as mentioned above, the internal loop of the apical stem-loop is maintained in both the Fermon and 2014 strains, and the tetraloop in stem-loop 1d changes from a CUYG tetraloop to a UNCG tetraloop as a result of a C to U substitution at position 63.The predicted tertiary structure of domain I also shifts between the Fermon and 2014 strains. As can be seen in Domain II undergoes significant structural change between the Fermon and 2014 strains . The relThe most significant change in the domain II secondary structure is the shift from a three-stem structure ranging from nucleotide 105–179 in the Fermon strain to a singular stem-loop ranging from nucleotide 136–182 and incorporating a large internal loop in the 2014 isolate. This reorganization results in the loss of structural features in the 2014 strain that were present in the Fermon strain. For example, stem 2a in the Fermon strain is not present in the 2014 strain. Instead, the entire span is part of the connecting region between domain I and domain II. The single domain II stem-loop in the 2014 strain is capped by a tetraloop. Certain structural features in stem-loops 2b and 2c of the Fermon strain are preserved in the 2014 strain. The six-nucleotide AAACCA hairpin loop present in stem 2b in the Fermon strain is also single-stranded in the 2014 strain, but it appears in the 5′ side of an internal loop. Similarly, the eight-nucleotide AGCACUUC hairpin loop present in stem 2c is also single-stranded in the 2014 strain, but it appears in the 3′ side of the internal loop.Just as the secondary structure changes significantly between the Fermon and 2014 strains, so does the model of the tertiary structure . Within Domain III is nearly identical in secondary structure in the Fermon and 2014 strains of EV-D68 a,b. BothDomain IV undergoes significant changes between the Fermon and 2014 strains . Within Whereas domain IV in the two strains shows common elements that are important for IRES function, several details of the long, extended helix differ markedly between the strains. Most prominently, two stem-loops that are generally grouped in domain V, and do indeed appear in domain V in the Fermon strain, have been subsumed into domain IV in the 2014 strain. This creates a IV/V super domain in the 2014 strain that greatly extends the length of domain IV.As would be expected, the formation of the IV/V super domain significantly impacts the model of the tertiary structure of the domain IV elements . Domain The structure of domain V in the 2014 strain is significantly impacted by the formation of the domain IV/V Super domain. In the Fermon strain, domain IV ends near position 440, which begins the transition into domain V. In the 2014 strain, position 440 is part of the stem-loop we label as 5a. A second stem-loop, labeled as 5b, as well as nucleotides that form the closing helix of the super domain, are part of the expanded domain IV. This leads to an overlapping of domains IV and V, causing a significant expansion of both domains in the 2014 strain.A three-stem-loop organization is present in domain V of both strains but undergoes significant remodeling. Stem 5a shifts from nucleotide 450 in the Fermon strain to nucleotide 414 in the 2014 strain and shares minimal sequence similarity between the two strains. Stem 5a is mostly composed of the nucleotides that make up the 3′ end of the initial extended helix of domain IV in the Fermon strain. Stem 5b in the 2014 strain is composed of the sequence that makes up the end of stem 5a and beginning of stem 5b of the Fermon strain. Because of this, the internal stem-loop present in stem 5b of the 2014 strain at position 459 maintains the AAU nucleotides present in the hairpin loop at the end of stem 5a in the Fermon strain. Stem 5c is shortened from 24 nucleotides in the Fermon strain to 15 nucleotides in the 2014 strain, and the hairpin loop at the end of the stem is reduced from nine to three nucleotides; however, the UUG motif of the nine-nucleotide loop is preserved in the three-nucleotide loop of the 2014 strain.As would be expected, the secondary structure changes described above led to significant shifts in the tertiary structure predicted by 3dRNAv2.0 . The movDomain VI is highly conserved between the Fermon and 2014 strains . It occuThe intrinsically disordered RNA region (IDRR) of the IRES is the most highly conserved region of the IRES, maintaining a length of 19 and 21 nucleotides in the Fermon and 2014 strains, respectively, and is flanked by domains V and VI. There are no point mutations within this feature of the IRES, making it nearly identical in each strain. It is very similar in the model tertiary structure between the two strains as it is completely disordered and positioned on the outside of the molecule in both. However, it is closely associated with stem 4a of domain IV in the 2014 strain.Enteroviruses are ubiquitous and prevalent infectious agents with an incredible diversity of viral types, disease manifestations, tissue tropisms and virulence phenotypes ,36,37. DThe secondary structure of every 5′ UTR domain changes in some fashion in the comparative models proposed here. Some domains, such as domains I, III and VI, experience minor changes, while other domains, such as domains II, IV and V, undergo significant structural changes. In the tertiary models predicted by 3dRNAv2.0, the secondary structure differences produce differing overall 5′ UTR conformations. In the 2014 strain, domains I, II, III, V and VI, together with the IDRR, are collected into a compact globular arrangement, with domain IV and the domain IV/V super domain extending away from the globular region, whereas the Fermon strain displays a more extended conformation where domains I, II and III are together on one end and domains IV, V and VI with the IDRR are together on the other. The changes in 5′ UTR structure and organization may contribute to increased neurovirulence in contemporary strains of EV-D68 by improving the efficiency of translation and/or replication in neural tissue.Our models of the 5′ UTRs of the EV-D68 Fermon and 2014 strains both show common features of enterovirus genome organization. The genome begins with a highly conserved cloverleaf structure in domain I. The cloverleaf is known to play a role in both the translation and replication of the viral genome in enteroviruses ,46,47,48Changes in the secondary structure also have the potential to impact the functions of the 5′ UTR by changing the orientation of the domains in relation to one another. In the Fermon strain, stem-loop 1d is orientated away from the rest of the molecule. However, within the 2014 isolate, stem-loop 1d is orientated towards domain II and above the oligo(rC) tract that binds PCBP2 in the connecting region between domains I and II. PCBP2 binding to the connecting region oligo(rC) helps to recruit 3CD to stem-loop d . This shBoth strains maintain the long single-stranded pyrimidine-rich connecting region containing the oligo(rC) tract between domains I and II that is common to enteroviruses. Domain II has been previously suggested as a determinant of virulence in other enteroviruses such as CVB3, poliovirus and EV-A71 ,54,55,56Domains IV and V, as well as the pyrimidine-rich IDRR in the connecting region downstream of domain V, are arguably the most active regions of the type 1 IRES. Binding sites for canonical initiation factors, noncanonical IRES trans-acting factors (ITAFs) and the small ribosomal subunit are clustered in these regions ,67,68,69The positioning of domain IV within the tertiary structure models of the Fermon and 2014 strains shifts drastically. Within the Fermon strain, domain IV is largely separate from the rest of the molecule, and only associated with domain VI, meaning it must travel greater distances to interact with other portions of the IRES and the factors resident there. In both the Fermon and 2014 models, stem-loop 4b and the apical bulge loop extend away from the molecule potentially available to recruit incoming initiation factors and ITAFs. In the 2014 model, domain IV is closer to the majority of the domains in the molecule as a whole, particularly domains I through III, thereby reducing the distance between these domains and potentially increasing the efficiency of viral translation. This increased kinetic efficiency of domain IV is also supported by work previously performed by Furuse et al., which shows that exchanging domain IV sequences between the Fermon strain and a contemporary EV-D68 strain increases IRES activity in neuronal glioblastoma cells .Domain V recruits factors such as eIF4G, eIF4A and PTB, which in turn aid in the recruitment of ribosomal subunits to the IDRR. Additionally, domain V has been shown to be a determinant of virulence in poliovirus ,72,73,74The tertiary structure model shows that the orientation of stem-loop 5c is conserved in both strains. However, the inclusion of stem-loops 5a and 5b into the IV/V super domain separates these elements from the IDRR and the rest of domain V. It was demonstrated that replacement of the contemporary domain V with the Fermon domain V increased IRES activity in neuronal glioblastoma cells, while replacement of the Fermon domain V with a contemporary domain V leads to no statistically significant change in IRES activity . This sun-Xm-AUG  [The polypyrimidine region between domain V and domain VI, identified as Yt codon) ,77, has t codon) . This set codon) ,78. Motit codon) .Enterovirus D [Similar to domain III, the secondary structure of domain VI is highly conserved between the Fermon and 2014 strains, with only minor shifts in the base pairing of the internal loops. The cryptic AUG, which is utilized to promote viral translation and act as an alternative start site of translation of an upstream ORF (uORF) in many enterovirus species, but not in ovirus D ,80, is lOur work establishes comparative secondary structures and tertiary structure models for the 5′ UTR of the prototypic Fermon strain, as well as a 2014 outbreak strain of EV-D68 with a neurovirulent phenotype. Recent research that explores virulence determinants in EV-D68 shows that newly acquired neurovirulence is primarily, but not exclusively, a result of changes in the 5′ UTR ,81. ThesWe show that the structure of the 5′ UTR undergoes significant changes in overall organization in two strains of EV-D68 that differ in neurovirulence. These changes may underlie the shift in neurovirulence in contemporary EV-D68 strains. Specifically, the tertiary structure changes provide room to speculate about the potential for easy and efficient interaction of key functional 5′ UTR domains in the 2014 strain. The most active and important RNA elements in domains I, IV and V are clustered together with the pyrimidine-rich connecting region downstream of domain I and the IDRR downstream of domain V in the 5′ UTR of the 2014 strain. In contrast, the tertiary structure of the 5′ UTR from the Fermon strain is much less compact with greater separation of functional domains. Perhaps the potential for increased efficiency in the 2014 strain enables the virus to replicate efficiently in the cellular environment of neurons.One pattern that emerges from our structural comparison involves changes that make the 2014 structure more similar to the poliovirus structure. While these changes are driven by relatively few sequence differences between the Fermon and 2014 5′ UTR, virulence determinants in enteroviruses are often localized to short regions or even single nucleotide positions ,56,75,82EV-D68 is an enterovirus that was initially thought to only cause mild respiratory illness but during an outbreak in 2014 also became associated with AFM and has since been shown to be a causative agent of AFM ,8,9,10. In this study, we utilized SHAPE-MaP to generate comparative secondary structures of the 5′ UTR between the prototypic Fermon strain and a 2014 outbreak isolate of EV-D68. These secondary structures then informed the formation of a predictive tertiary structure model of both strains using 3dRNAv2.0. The structures generated show shifts in the secondary structure of every domain within the 5′ UTR; however, within these domains, many elements shown to be important to the function of the 5′ UTR are maintained. The tertiary structure models show a shift from an extended conformation within the Fermon strain to a more compact globular structure within the 2014 strain. This change toward a more compact shape could increase the kinetic efficiency of the 5′ UTR by reducing the distance between sections that interact with one another. The structures we generated highlight domains that change drastically between the Fermon and 2014 strains and could serve as potential targets for therapeutics against EV-D68 disease. Small-molecule treatment targeting domain II of EV-A71 has been shown to be effective in attenuating virulence by stabilizing a tertiary structure that decreases the efficiency of viral multiplication . With de


Nucleotide-binding and leucine-rich repeat receptors (NLRs) are intracellular plant immune receptors that recognize pathogen effectors secreted into the plant cell. Canonical NLRs typically contain three conserved domains including a central nucleotide binding (NB-ARC) domain, C-terminal leucine-rich repeats (LRRs) and an N-terminal domain. A subfamily of plant NLRs contain additional noncanonical domain(s) that have potentially evolved from the integration of the effector targets in the canonical NLR structure. These NLRs with extra domains are thus referred to as NLRs with integrated domains (NLR-IDs). Here, we first summarize our current understanding of NLR-ID activation upon effector binding, focusing on the NLR pairs Pik-1/Pik-2, RGA4/RGA5, and RRS1/RPS4. We speculate on their potential oligomerization into resistosomes as it was recently shown for certain canonical plant NLRs. Furthermore, we discuss how our growing understanding of the mode of action of NLR-ID continuously informs engineering approaches to design new resistance specificities in the context of rapidly evolving pathogens. This recognition often leads to a programmed cell death in plants known as the Hypersensitive Response (HR) to limit pathogen spread in neighboring cells, tissues, and/or organs ..leucine-ke)-NLRs  proposeske)-NLRs .3-Induced) domain binds the host protein Exo70-F3, which is a target of the pathogen effector AVR-Pii from Magnaporthe oryzae [OsExo70-F3 and detect AVR-Pii [A subcategory of NLRs carry additional noncanonical domain(s) and can make up to 10–15% of the NLRome of a given plant species . Functio AVR-Pii . Another AVR-Pii . It is uIn this review, we report on the latest advances in the understanding of NLR-ID mode(s) of action and how it continuously informs NLR-ID engineering. We speculate on the activation mechanisms of NLR-IDs in regard to the recently solved structures of plant NLR resistosomes.M. oryzae RGA5/RGA4 [A. thaliana, conferring resistance against R. solanacearum, P. syringae pv. tomato, and C. higginsianum [Among all characterized NLR-IDs, three NLR pairs have been extensively studied: the rice CNLs conferring resistance against GA5/RGA4  and Pik-GA5/RGA4 ,25 and tinsianum . Hence, M. oryzae [M. oryzae isolates expressing AVR-Pia or AVR1-CO39 and interacts with these effectors via its HMA domain [M. oryzae AVRs and ToxB-like (MAX) effector family and bind RGA5HMA at the same interface [N. benthamiana. However, this HR is repressed when RGA4 is co-expressed with RGA5. The present study suggests that upon direct interaction of the effector protein AVR-Pia with the HMA domain in RGA5, repression mediated by RGA5 on RGA4 is relieved and HR occurs [RGA4 and RGA5 recognize two sequence-unrelated effectors AVR-Pia and AVR1-CO39 from rice blast pathogen . oryzae . NLR-ID A domain . Althougnterface . Furthernterface . The NLRR occurs .M. oryzae following recognition of the effector protein AVR-Pik [HMA/AVR-PikD is different from that of RGA5HMA/AVR-Pia or AVR1-CO39 [The NLR pair Pik-1 and Pik-2 from rice confers resistance to  AVR-Pik ,34,35. A AVR-Pik . Similar AVR-Pik ,38. AVR-VR1-CO39 ,38. AddiA. thaliana RRS1/RPS4 immune receptors pair provide further insights into the immune activation by NLR/NLR-ID pairs. RRS1 and RPS4 confer resistance to the bacterial pathogens P. syringae and R. solanacearum through the recognition of the effectors AvrRps4 and PopP2, respectively [A. thaliana, two different RRS1 alleles show different recognition specificities. RRS1-S (in ecotype Col-0) recognizes AvrRps4 but not PopP2, while RRS1-R (in ecotype Ws-2) can recognize both AvrRps4 and PopP2. RPS4 is a canonical TNL type of resistance protein, while RRS1-R is a TNL protein with an integrated WRKY-like domain near its C-terminus. Interestingly, there is a paralogous RPS4B/RRS1B pair in A. thaliana (ecotype Ws-2) that also recognizes AvrRps4 but not PopP2. The WRKY domains of RRS1 and RRS1B phylogenetically cluster with different WRKY groups, suggesting an independent integration event [Studies involving ectively ,29,40,41on event .AtWRKY41, a host WRKY transcription factor, share the same binding interface as RRS1WRKY/AvrRps4 or PopP2 and effector binding reduces the DNA-binding activity of AtWRKY41 [WRKY triggers a constitutive RPS4-dependent immune activation, suggesting that the RRS1-RWRKY maintains the complex in an inactive state [Effector recognition occurs by direct binding of structurally distinct AvrRps4 or PopP2 effectors to the integrated WRKY-like domain ,13. The AtWRKY41 . This suve state . Intramove state . PopP2-mve state .TIR from RRS1TIR inhibition [Furthermore, multiple sites within the region harboring C-terminal and the WRKY-like domains of RRS1-R but not RRS1-S are phosphorylated . Phosphohibition . This cohibition ,48. Finahibition . The autN. benthamiana [P-loop  and MHD motifs associated with the NB-ARC domain contribute to NLR activation ,51. Alththamiana . With ththamiana ,48,52,53thamiana , it woulthamiana ,59. In tPlant pathogens rapidly evolve virulent races that can nullify new resistance specificities shortly after being deployed in the field. To address this, we need to continuously identify new resistance genes and alleles targeting the pathogen genotypes associated with plant disease outbreaks and transfer these into commercial elite cultivars in a timely fashion. With our growing understanding of NLR mode of action, fine-tuning resistance mediated by characterized NLRs and adapting it to current epidemics might become possible. Recent work showed that engineering new resistance specificities in NLR-IDs is achievable, although it involves considerable prior knowledge of the system. The two main avenues for engineering IDs in NLR-IDs are mutagenesis or domain shuffling .HMA/AVR-PikD [HMA/AVR-PikD, E, A [HMA variant able to bind AVR-PikD, E, A, C and trigger HR in the presence of AVR-PikD, E, A in N. benthamiana when co-expressed with its executor Pikp-2. Cesari et al. [HMA (aka Pia-2 HMA), recognizing AVR-Pia and AVR1-CO39, to bind AVR-PikD. The authors combined the binding interfaces of RGA5HMA/AVR1-CO39 and Pikp-1HMA/AVR-PikD into RGA5HMA to design a RGA5HMA variant able to bind AVR-PikD and retain binding to AVR1-CO39 and AVR-Pia. Co-expressing this RGA5 variant with its executor RGA4 in the presence of Avr-PikD, Avr-Pia or AVR1-CO39 triggers HR in N. benthamiana. However, the engineered Avr-PikD recognition did not translate into resistance in rice transgenics infected with M. oryzae strains expressing this effector. Finally, Liu et al. [HMA/AVR1-CO39 complex to the modelled structure of RGA5HMA/Avr-Pib [HMA lost AVR-Pia binding and recognition and triggers HR in responses to AVR-Pib in the presence of RGA4 in N. benthamiana. Additionally, RGA5HMA variant/RGA4 conferred resistance against M. oryzae expressing AVR-Pib in rice transgenics.The integrated HMA domains of rice NLRs Pik-1 and RGA5 were engineered using structure-guided mutagenesis to expand their recognition spectrum or switch effector specificity. De la Concepcion et al.  combinedAVR-PikD  and PikmkD, E, A  complexei et al.  engineeru et al.  modified/Avr-Pib . The engOsHIPP19, including the two variants AVR-PikC and F that are not recognized by any known Pik alleles. The structural information of the OsHIPP19/AVR-Pik complexes resolved in the present study could thus inform on HMA domain engineering to expand Pik alleles spectrum of recognition to these AVR-Pik variants. Recently, Wang et al. [slh1, RPS4, and RRS1-R fused with the GATA domain that is recognized by SAP05 in this system. In the absence of SAP05, RRS1-Rslh1 autoactivity is repressed by RRS1-R-GATA. In the presence of SAP05, RRS1-R-GATA is degraded, re-enabling RRS1-Rslh1 autoactivity that is dependent on RPS4. The tripartite system was able to induce HR in the transient system of N. benthamiana in the presence of the SAP05 effector. Transgenic Arabidopsis lines expressing RRS1-R-GATA, RRS1-Rslh1, and RPS4 showed delayed symptoms in the presence of the pathogens but were not fully resistant, however.Effector/host target interactions could provide valuable information on how to engineer new resistance specificities to bait effectors ,64. Maidg et al.  developeg et al. . The autN. benthamiana in the presence of Pikm-2 and the corresponding fluorescent protein. Furthermore, Pikobodies conferred resistance against a Potato Virus X variant expressing free GFP or free mCherry. It remains to be shown whether Pikobody-mediated resistance is functional in stable transgenics, however. Given that nanobodies can be raised against virtually any molecule, Pikobodies have the potential to generate resistance against all major plant pathogens and pests secreting effectors in the plant cell.More recently, Kourelis et al.  swapped Challenges can arise at each step of the NLR editing process . For exaAn additional perspective connected to NLR-IDs is the identification of essential host’s components that are targeted by pathogens and pests to promote susceptibility. All the identified and studied IDs potentially reflect the original targets of the pathogens at the subcellular level so far. This indicates that these integrated domains can be used as a toolbox toward the identification of the original effector targets in the host cells as well as novel host susceptibility components that could be utilized further ,71.Combined with the increasing quality of genome assemblies and annotations available for a wide range of plant species and varieties, NLR discovery pipelines , improveNLR-IDs are present across the plant kingdom.Functional studies showed that these extra domains are involved in direct or indirect effector recognition.Most functionally characterized NLR-IDs work in pairs.It is still unclear how NLR-IDs are activated upon effector recognition.Integrated domains in NLRs can be engineered to expand or modify their spectrum of effector recognition.


We studied their orthologs, domain architecture, conserved motifs, and amino acid variations. Our study also adds a timeframe to infer the duplication of TICAM protein from Leptocardii and later divergence into TICAM1/TRIF and TICAM2/TRAM. More evidence of TRIF proteins was seen, but the absence of conserved co-existing domains such as TRIF-NTD, TIR, and RHIM domains in distant relatives hints on diversification and adaptation to different biological functions. TRAM was lost in Actinopteri and has conserved domain architecture of TIR across species except in Aves. An additional isoform of TRAM, TAG (TRAM adaptor with the GOLD domain), could be identified in species in the Mesozoic era. Finally, the Hypothesis based Likelihood ratio test was applied to look for selection pressure amongst orthologues of TRIF and TRAM to search for positively selected sites. These residues were mostly seen in the non-structural region of the proteins. Overall, this study unravels evolutionary information on the adaptors TRAM and TRIF and how well they had duplicated to perform diverse functions by changes in their domain architecture across lineages.Toll/Interleukin-1 receptor (TIR) domains are cytoplasmic domain that mediates receptor signalling. These domains are present in proteins like Toll-like receptors (TLR), its signaling adaptors and Interleukins, that form a major part of the immune system. These TIR domain containing signaling adaptors binds to the TLRs and interacts with their TIR domains for downstream signaling. We have examined the evolutionary divergence across the tree of life of two of these TIR domain containing adaptor molecules (TICAM) The online version contains supplementary material available at 10.1186/s13062-022-00335-9. Toll-like receptors play a major role in the innate immune system by recognizing diverse exogenous and endogenous biomolecules  as their ligands and produces cytokines along with other inflammatory mediators during infections. Structurally they have an extracellular Leucine-rich repeat (LRR) domain, a transmembrane domain, and an intracellular Toll/Interleukin-1 receptor (TIR) domain .Amongst these TLR proteins and its signaling adaptors, TIR domains are found in both. Apart from that, these domains are also present in Interleukins receptors as well as some accessory proteins. Overall there are 25 genes in the human genome that contain TIR domains as per PROSITE database—. A figure depicting the domain architecture of these TIR domain containing proteins are shown in Fig. Amongst these TIR domain containing proteins, Toll-like receptors, MyD88, SIGIRR, and Interleukin receptor proteins share similar kind of TIR domain structure (Pfam ID: PF01582.22), but the adaptor proteins TIRAP, TRAM, TRIF and SARM1 share a different Pfam TIR_2 domain (PF13676.8). Besides these PIK3AP1 and BCAP have TIR_3 domain (PF18567.3).These TIR domains are cytoplasmic in nature and consist of approximately 200 amino acids. They in general promote the assembly of signaling complexes via protein–protein homotypic or heterotypic interactions. The TIR structure contains a central five-stranded parallel β sheet surrounded by five helices. Although TIR domains from different proteins have similar structure, their amino acid sequence identity is less than 30% when compared amongst each other. This makes them significantly diverse in sequence and structure amongst Toll-like receptors (TLRs), TLR adaptors, and Interleukin receptors .MyD88 in older taxa explains the origins of MyD88 dependent pathways in invertebrates, although the TRIF and TRAM have resulted from an early duplication event in vertebrate TLR phylogeny . The motif sequence and residue positions of human TRIF protein for typical TRAF6 binding sites are PEEPPD (86–91), PEEMSW (250–255) and PVECTE (301–306), whereas the same for atypical sites are PLESSP (491–496) and PPELPS (264–269). Amongst these, the second typical TRAF6 binding site [PEEMSW (250–255)] is one of the most important sites for TRIF interaction with TRAF6 and NF-κb induction {P}], which is important for signal transduction [Aves, suggesting they may not have a functional TRAM protein. Additionally, Aves taxa has the highest branch lengths and highest amino acid distances as shown in Additional File Aves taxa may have undergone a higher divergence and genetic changes over evolutionary time. Previously reported studies with representative sequences across taxa claims TRAM to be lost in fishes, birds, and amphibians. But interestingly by our genome-wide search, we found TRAM orthologues for Aves [Actinopteri (bony fishes).We found TRAM ortholog sequence in Callorhinchus mili from Chondrichthyes (cartilaginous fish), it also shows a concentional TIR_2 domain architecture along with conserved motif. This may be the oldest organism with functional motifs conserved with TIR_2 domain. Also, amongst members of Amphibia, all the hits have conserved TIR domain along with conventional N-Myristoylation motif except for Xenopus laevis. On combining all of our observation we observe that TRAM may have diverged from TRIF and first appeared in Chondrichthyes (399 mya), was then lost in Actinopteri, and then reappeared in Amphibia (323 mya), Cryptodira, Crocodylia, Bifurcata, Aves, and Mammalia.Unlike TRIF, human TRAM (Uniprot ID: Q86XR7) consists of a putative e to LPS , 19. Thesduction . TRAM prsduction . Upon obfor Aves . None ofBifurcata, one Crocodylia, a few Aves, and Mammalia taxa. All of them depicted the well-conserved domain architecture of EMP24_GP25L  with GOLD domains was also seen across one  complex ) and TIRA representative phylogeny from each category is shown below showing the proportion of organisms. The evolutionary timeline for the divergence of TICAM is also shown below in Fig. Homo sapiens , Falco peregrinus (Aves), Gekko japonicus (Bifurcata), Alligator mississippiensis (Crocodylia), Chelonia mydas (Cryptodira), Xenopus laevis (Amphibia), Callorhinchus milii (Chondrichthyes), Danio rerio (Actinopteri), Latimeria chalumnae (Coelacanthimorpha) and Branchiostoma belcheri (Leptocardii) were selected. The accession ID and domain architecture for these representatives can be seen in Additional Files To focus on the orthologous relationships of TRIF and TRAM, we next examined the syntenies and check the preservation of the order of genes amongst the species and check for the neighbour genes. One representative from each taxon e.g., Amphibia (lacks the gene neighbours), and TRAM of Aves (does not have a full one-to-one correspondence) as seen in Fig. Chondrichthyes and other TICAM2 orthologs. Due to the event of whole-genome duplication occurring at Actinopteri, two forms of TICAMs are generated. Since these duplicates are preserved across the lineages, there may be subfunctionalization or neofunctionalization [Coelacanthimorpha and Actinopteri.There is overall good correspondence amongst the neighbours of TRAM and TRIF orthologs, except TRIF of lization . To asceFrom the previously obtained TRIF and TRAM phylogeny we observed the variation among branch lengths, and also the varied domain architecture amongst the sequences.N/dS, ratio of nonsynonymous/synonymous substitution rates) to vary among codon sites in protein. Based on the Likelihood ratio test, using the Bayes empirical Bayes (BEB) method or posterior probabilities of model M8 for 11 site classes (k = 11) along the sequence of the protein was plotted. The values from 11 site classes were grouped into two categories as ω > 1 and ω < 1. Also, another graph depicting the mean probabilities for each site was plotted in Fig. We were further interested to look into the selection pressure of TRIF and TRAM protein. We used the orthologues sequences and used site model from Codon substitution models (codeml) of PAML4.9 package. The implemented site models  allowed ω ratio  ones, provide an alternate pathway for the production of inflammatory mediators. Previous studies have concentrated on the evolution of TIRs and adaptors involved in the MyD88-dependent pathway. However, the evolutionary lineage of adaptors that are known to operate in MyD88-independent pathway have not been studied in detail. The prime objective of our study has been to look into the divergence of TICAM to TRIF and TRAM. Unlike the MyD88 pathway, which seems to have emerged in early invertebrates, TRIF and TRAM related pathway trace back and emerge with the duplication event in vertebrates. A previously reported study has shown these adaptors in 25 metazoans [Leptocardii appears as the oldest ancestor for these proteins. This was referred as TICAM in basal chordates for the MyD88 independent pathway [Homo sapiens. Further, with our sequence search approaches, we found TAG (an isoform of TRAM with GOLD domain) which is seen in some species of Bifurcata, Crocodylia, Aves, and Mammalia. We also observed, through synteny analysis, that TRAM lineages and their immediate gene neighbours to be more highly conserved, as compared to TRIF where some ambiguity was seen for Actinopteri and Amphibia. TIR domains within TRAM are more conserved than in TRIF .We initially curated the human TIR domain sequences from Uniprot and filtered the hits to include Swiss-Prot reviewed sequences. Further, amongst these, those which followed PROSITE-ProRule annotation: PRU00204, were only selected. This PROSITE profile is specific to the pattern of TIR domains’ scaffold that promotes assembly of signaling complexes via protein–protein interactions. Motif search was performed using MEME suite by classic search method and gapped local alignment was performed by the GLAM2 module using protein seqeuences as input structure –28. SuchOrnithorhynchus anatinus), Didelphimorphia (Monodelphis domestica), Dasyuromorphia (Sarcophilus harrisii), Diprotodontia (Phascolarctos cinereus), Cingulata (Dasypus novemcinctus), Proboscidea (Loxodonta africana), Afrosoricida (Echinops telfairi), Tubulidentata (Orycteropus afer afer), Rodentia (Marmota flaviventris), Primates (Pan troglodytes), Eulipotyphla (Condylura cristata), Chiroptera (Pteropus vampyrus), Artiodactyla (Sus scrofa), Cetacea , Perissodactyla , Carnivora (Felis catus), Lagomorpha (Ochotona princeps), Macroscelidea (Elephantulus edwardii), Scandentia (Tupaia chinensis), Dermoptera , Sirenia (Trichechus manatus latirostris), Pholidota (Manis javanica) orders along with two model organisms  protein and Homo sapiens.In order to search the homologues of TRAM and TRIF, we enriched our dataset to include organisms from 22 orders across the Mammalian class. TRAM and TRIF proteins of different organisms from the following orders were included. Monotremata  [Later, the TRAM and TRIF protein from 25 organisms were used as a query to perform CS-BLAST  against ignment) . The lisignment) . These hDomain architectures were searched for these sequences using Hmmscan modules from the HMMER suite package (version 3.1b2) against Pfam database (version 31.0)  with an The fasta sequences were aligned using MUSCLE v3.8.31  and a phGenome assembly for one representative organism from each taxon was used to look into the gene neighbours. The sequence ID corresponding to TRAM and TRIF protein were searched in NCBI and the neighbours were found using the Genome Data Viewer .M0-one ratio, M1-nearly neutral, M2a-positive selection, M3-discrete, M7-beta, M8-beta, and ω > 1, M8a-beta and ω = 1) were used for these sequences and individual dN/dS ratio was also calculated for each branch. The hypothesis of different rates of evolution amongst different groups  were also tested. The phylogeny used considered branch lengths obtained from the Maximum Likelihood. Also, species from the Leptocardii group was included in both TRIF and TRAM phylogeny as an outgroup to root the phylogeny tree.Nucleotide codon sequences were retrieved for each protein ID using the Batch Entrez mode of NCBI . They weThe parameters used for codeml run of site model were as shown in the table in Additional File M3 versus M0, M1 versus M2a, and M7 versus M8 for comparing positive selection. For identifying the potential amino acid residues that would have been under selection, we performed a Likelihood ratio test calculation for pairwise comparison of codon models using the Bayes empirical Bayes (BEB) method. The residues with probability above 0.95 and above were documented. Additionally, values for BEB or posterior probabilities of model M8 for 11 site classes (k = 11) along the sequence of protein was plotted. The values from 11 site classes were grouped into two categories as ω > 1 and ω < 1. Also, the mean probabilities for each site were examined.The site model was performed for both protein family (TRAM and TRIF) using three models; Additional file 1: Figs. S1–S6.Additional file 2: Amino acid sequence of TLR family proteins from 10 different orthologues.Additional file 3: A maximum likelihood tree for TLR family proteins from 10 different orthologues.Additional file 4: A detailed phylogeny with amino acid distance, domain architecture and motif conservation of TRIF orthologues.Additional file 5: A detailed phylogeny with amino acid distance, domain architecture and motif conservation of TRAM orthologues.Additional file 6: Protein Sequence ID’s and respective organism name of TRIF orthologues.Additional file 7: Multiple Sequence Alignment of TRIF orthologues in FASTA format.Additional file 8: Protein Sequence ID’s and respective organism name of TRAM orthologues.Additional file 9: Multiple Sequence Alignment of TRAM orthologues in FASTA format.Additional file 10: Protein Sequence ID’s and respective organism name of TAG orthologues.Additional file 11: Multiple Sequence Alignment of TAG orthologues in FASTA format.Additional file 12: The parameters and values used for codeml run of site model.Additional file 13: Table showing domain architecture of TRIF and TRAM orthologue along with domain boundaries and e-value.Additional file 14: List of hits obtained from genome wide search of TRIF orthologues using CS BLAST after filtering.Additional file 15: List of hits obtained from genome wide search of TRAM and TAG orthologues using CS BLAST after filtering.


Streptomyces lavendulae with those of three multidomain dinoflagellate transcripts and coexpressed these constructs with each of three dinoflagellate PPTases looking for specific pairings that would identify distinct pathways. Surprisingly, all three PPTases were able to activate all the thiolation domains from one transcript, although with differing levels of indigoidine produced, demonstrating an unusual lack of specificity. Unfortunately, constructs with the remaining thiolation domains produced almost no indigoidine and the thiolation domain for lipid synthesis could not be expressed in E. coli. These results combined with inconsistent protein expression for different PPTase/thiolation domain pairings present technical hurdles for future work. Despite these challenges, expression of catalytically active dinoflagellate proteins in E. coli is a novel and useful tool going forward.Photosynthetic dinoflagellates synthesize many toxic but also potential therapeutic compounds therapeutics via polyketide/non-ribosomal peptide synthesis, a common means of producing natural products in bacteria and fungi. Although canonical genes are identifiable in dinoflagellate transcriptomes, the biosynthetic pathways are obfuscated by high copy numbers and fractured synteny. This study focuses on the carrier domains that scaffold natural product synthesis (thiolation domains) and the phosphopantetheinyl transferases (PPTases) that thiolate these carriers. We replaced the thiolation domain of the indigoidine producing BpsA gene from  Dinoflagellates make a variety of natural products that have largely been identified based on their impact to human and animal health ,2,3,4. TNatural product synthesis has been extensively studied in bacteria and fungi, yielding a mechanistic framework that operates as a series of modules with repeated chemistries followed by some modifications resulting in the final molecule. Essentially, small carboxylic acids are added to the thiol end of a phosphopantetheinate group attached to the serine of a carrier protein  via a coResearch into the biosynthesis of many natural products has relied heavily on the fact that gene arrangement is strongly predictive of a given natural products’ final structure. Unfortunately, dinoflagellate genomes are large and heavily duplicated , althougE. coli [Streptomyces lavendulae [The aim of our project is to extend the current sequence-based knowledge into a biochemical based understanding of natural product synthesis by expressing dinoflagellate proteins in a heterologous system. An attractive target is the carrier protein called the thiolation domain that is activated by the attachment of the phosphopantetheinate group of coenzyme A by a phosphopantetheinyl transferase creating a free thiol moiety. This is the first rate limiting step in natural product synthesis and provides the substrate upon which the actual anabolism is performed by all of the catalytic enzymes. Generally, the activation of a thiolation domain by any phosphopantetheinyl transferase is highly specific and separates specific biosynthetic pathways, although the actual transfer of a phosphopantetheinyl group is not required for recognition of the transferase to a thiolation domain . The thiE. coli  along wivendulae  that hasvendulae . The ratvendulae ,47 and hvendulae ,49. ThisThe BpsA reporter described in Owen et al.  was kindThe HindIII and AflII sites were incorporated into the vector in a two-stage process. First, the HindIII site was created via two amplifications using “BpsA_outF2” with “BpsA_hindiiiR1” and “BpsA_outR2” with “BpsA_hindiiiF1” with the same reaction conditions as the thiolation domain amplification. The resultant products were purified using a DNA Clean and Concentrate-5 kit from Zymo research  and eluted into 10 ul of distilled deionized water. Approximately 2.5 µg of product was digested with the HindIII-HF restriction enzyme from New England Biolabs for four hours at 37 °C, separated on an ethidium bromide impregnated 1% agarose gel in 0.5× TBE at 15 V/cm for 50 min, excised under ultraviolet illumination, and purified using a Monarch DNA Gel Extraction kit from New England Biolabs  as directed. The two digested fragments were then combined and ligated using a T4 ligase from Promega  overnight at 18 °C. This product was then used as template for the second stage amplification using primers “BpsA_outF2” with “BpsA_afliiR1” and “BpsA_outR2” with “BspA_afliiF1” using the same conditions as the HindIII site amplification. This was purified, digested with AflII restriction enzyme from New England Biolabs, agarose gel purified, and combined and ligated in the same manner as the HindIII products resulting in “BpsA_insert1” .Streptomyces lavendulae originally published in Takahashi et al. [Shown above is the BpsA gene from i et al.  showing E. coli JM109 from Promega  according to the manufacturer’s directions. A selection of the resultant colonies was grown and the plasmid extracted for co-expression with each of the PPTases from Amphidinium carterae as in [BpsA_insert1 was amplified using the same conditions as the original thiolation domain and purified using the DNA Clean and Concentrate-5 kit. This product as well as the original BpsA vector were double digested with the NotI-HF and FspI restriction enzymes from New England Biolabs at 37 °C overnight in cutsmart buffer followed by agarose gel purification and ligation as with the HindIII and AflII amplicons resulting in the BpsA2.1 vector. This was amplified using the Templiphi 100 kit from Cytiva  and cloned into ae as in  to confiA. carterae were chosen for complementation in E. coli with the three A. carterae phosphopantetheinyl transferases (PPTases) that could activate them .The natural product associated thiolation domains  in threeate them . These w species , and “Zm cluster . Each inIndividual modular synthase domains are shown at the top with example products for their reaction. In addition, Adenylation (A) and FSH1 serine hydrolases (FSH1) are shown for the multi-domain transcripts with examples of potential products included. The phosphopantetheinate group is shown as “P~P” with a single bind to a sulfur. “SL” refers to the dinoflagellate spliced leader sequence and is present if a spliced leader sequence has been verified.Each oligonucleotide was synthesized with common linker sequences containing the AflII and HindIII restriction sites in the BpsA2.1 plasmid, one for the 5′ end, and one for the 3′ end . Thus, eE. coli JM109 from Promega according to the manufacturer’s directions. JM109 clones were sequenced to verify the presence of the dinoflagellate insert in the plasmid followed by alkaline extraction [E. coli  along with one of the three PPTase activators , and proteins were extracted in a French press at 20,000 local PSI followed by centrifugation at 10,000× g for 10 min at 4 °C to separate soluble and insoluble material. Insoluble proteins were recovered from the pellet by the addition of 6 M urea in equal volume to the supernatant. Heterologous proteins were purified with a 1 mL HiTrap Talon crude column  on an AKTA chromatography system with elution into 50 mM Tris with 250 mM imidazole. Proteins were separated by SDS-PAGE electrophoresis with 4–12% bis-tris gels  and imaged with Imperial Coomassie stain .For each thiolation domain, the synthetic oligonucleotide as well as the BpsA2.1 plasmid were double digested with HindIII and AflII overnight at 37 °C in cutsmart buffer followed by agarose gel purification using a Monarch DNA Gel Extraction kit from New England Biolabs. The cut insert and plasmid were combined and ligated with a T4 ligase (Promega) at 18 °C overnight. Each ligated plasmid was amplified with the Templiphi 100 kit from Cytiva and cloned into traction . Plasmidtivators  from A.  plasmid  accordinE. coli clones containing one of the three A. carterae PPTases and one of the eight BpsA reporters with dinoflagellate thiolation domain sequence were each grown onto agar plates containing “autoinduction” media [https://imagej.net/, accessed on 1 December 2018) with the space in between colonies as a baseline for background subtraction.The n” media . ColonieAmphidinium carterae have previously been shown to interact with the wild type BpsA vector when co-expressed in E. coli [E. coli host by French press. PPTase 2, however, was only visible in the insoluble fraction, and PPTase 1 had low expression in general.Following the generation of the BpsA2.1 vector with restriction sites flanking the phosphopantetheinyl transferase (PPTase) binding site of the thiolation domain, each of the eight dinoflagellate thiolation domain oligonucleotides were successfully inserted and verified by Sanger sequencing in both directions (not shown). Although each of the PPTases from  E. coli , indepenE. coli clones containing the three Amphidinium carterae phosphopantetheinyl transferases following induction of protein expression with IPTG. Both the soluble (supernatant following French press isolation) and insoluble (Proteins retrieved from the pellet with 6 M urea) fractions are shown with arrows indicating the expected size of each protein based on the molecular weight marker designated as “Ladder” with kiloDaltons indicated.An SDS-PAGE gel is shown for three E. coli, co-expression of PPTase 2 with both the BpsA2.1 vector without a heterologous insert as well as with the ZmaK1 insert was his-tag purified  on separate vectors. The thiolation domain is indicated on the x-axis including wild type sequence (BpsA), as well as the Amphidinium carterae triple KS (KS), ZmaK-like (ZmaK), and BurA-like (BurA) transcript sequences with a numeral indicating the particular thiolation domain from the N to C terminus. The z-axis indicates which clade of PPTase sequence was co-expressed from Williams et al. [x and z-axes for reference.The graph shows the relative darkness of each colony to a black pixel on the s et al. . The actGenetic tools such as knockdowns, knockouts, and knockins can be powerful means to determine gene function by looking for phenotype changes following a change in the expression of that gene. While these techniques are well established in many bacteria, fungi, and vertebrates, there are many branches of the tree of life where genetic techniques are not well developed for a variety of reasons. Protists, in particular, representing a huge amount of eukaryotic evolutionary diversity, have lagged behind, although new sequencing technologies have provided a much-needed boost in investigative power , resultiCrypthecodinium cohnii [In many ways, this study is a bridge between dinoflagellate biology, focusing on ecology and species diversity, and natural product research, a biochemical approach to discovering and harnessing useful compounds. This marriage seems a foregone conclusion given that dinoflagellates make compounds that affect human health negatively  but potem cohnii  and the m cohnii  being a m cohnii ,74,75,76m cohnii ,78,79,80Elucidating the function of genes involved in the synthesis of dinoflagellate toxins is especially tricky, since, like most natural products, the synthesis is inherently modular in nature , resultiE. coli strain has the same plasmid copy number and expresses each protein equivalently. While the former is likely true, given that equivalent amounts of plasmid were used in the transformation, the latter is certainly false given the differential expression based on PPTase type and reporter pairing (E. coli colonies were allowed to induce expression and produce indigoidine for 48 h, and, not shown here due to a loss of quantitation, would eventually become dark in color for most insert/activator combinations. This immediately leads to the conclusion that all forms of the reporter are capable of producing indigoidine and that there is some possible phosphopantethienation by each dinoflagellate PPTase for any given thiolation domain, another example of promiscuous PPTase binding in protists [E. coli sequence at the insert site (E. coli likely due to toxicity. Thus, comparisons between the thiolation domains presented in this study and the acyl carrier protein would be more appropriate for in-vitro based studies or co-expression studies in another host assuming that the issue of toxicity does not still exist. In contrast to the previous study of dinoflagellate PPTases [Technically, this study represents a step forward as the first example of a catalytically active reporter containing dinoflagellate genes. Natural product synthesis can be quite complicated and dinoflagellate toxins are much larger than most natural products . It is a pairing . The E. protists . This alprotists . In termert site . This co PPTases , PPTase Hematodinium sp. [Amphidinium carterae PPTases, the ability to activate the BurA and ZmaK inserts is sub-optimal. This was suggested in the Williams et al. 2021 study on the thiolation domains of dinoflagellate domains based on the observation that many of the ZmaK sequences lie outside the cluster of natural product associated domains with the more basal sequence the furthest away, indicating that convergent evolution may be an active force. Thus, PPTases from more distal species of dinoflagellates may be better at activating the BurA and ZmaK insert containing reporters than the A. carterae based PPTases used here. It could also be that the BurA and ZmaK based sequences themselves are interfering with the reporter’s ability to produce indigoidine given that the thiolation domain acts as an intermediary for all other domains. The BurA and ZmaK sequences may be sterically interfering with the other domains of the BpsA protein, reducing its overall efficiency.There were some obvious differences observed between the triple-KS inserts and the ZmaK or BurA inserts in terms of the indigoidine produced . The trinium sp. , a parasE. coli heterologous host, some dinoflagellate genes simply cannot be expressed in this system, likely due to host interactions and resultant toxicity. This is both intriguing and frustrating that genes involved in lipid synthesis can be so conserved in such a dynamic group of organisms like dinoflagellates. Perhaps some comparisons to syndinian dinoflagellates that parasitize and absorb lipids from dinoflagellate hosts can shed light on how these genes function and have been modified over time. A second consideration is the dynamic nature of expression of the PPTases depending on the thiolation domain it is paired with. This renders these results qualitative at best and implies that in vitro methods, while more challenging, are likely more accurate comparisons of PPTase activity. Finally, the lack of indigoidine synthesis for the BpsA vectors containing the ZmaK and BurA inserts is suspicious and may be the result of steric hindrance rather than a lack of PPTase activation. A general conclusion from the activation of all the thiolation domain sequences used here by all three PPTases is that the substrate specificity observed in bacteria and fungi may be the exception in protists rather than the rule. The mechanism that dinoflagellates use to regulate the initiation of lipid and natural product synthesis is an important topic for future study. Targeted knockdowns are another avenue going forward to validate these results since broad substrate recognition shown here does not mean that specific natural product pathways are not physically separated in situ. In addition, knockdowns of acyl transferases and thioesterases that join and terminate major portions of natural product synthesis as proposed in Van Wagoner et al. 2014 [The modification and deployment of this indigoidine synthesizing reporter for dinoflagellate genes allows for new approaches to studying genes that may be involved in toxin synthesis. Specifically, methods that can validate suspected interactions during natural product synthesis are crucial in overcoming the limitations of sequence-based annotations. Here, domain substitution has been used to demonstrate the broad substrate recognition of dinoflagellate PPTases that can help explain gene losses and gains throughout dinoflagellate evolution that do not correlate with a loss or gain in toxin synthesis. There are also several not unexpected lessons to be learned from these results. First and foremost is that, despite the huge evolutionary distance between dinoflagellates and the al. 2014  can help