We estimate the amount of phonons caused by this transition applying precise quantum computing key tools, and resulting in a maximum value of 1.23 ± 0.02. Further applications of our microcantilever sensor were assessed using unambiguous mapping of the surface stress induced by the  With this phrase, Feynman exemplifies the implications of reaching a deep understanding of the physical and chemical ice crystal properties. These properties are related to its molecular morphology that depends on the order of the hydrogen–bonds configuration2. Hence, the ice presents stable configurations like cubic  of ice with photolysis and the formation of ionic radicals in comets and other astrophysical media; making it relevant in the understanding of the origins of the earlier solar system and life on Earth8. This underscores the need to understand the crystallization and SPT’s to unravel the still hidden secrets of the ice–related phenomena.14. Notably, the reactivity of water towards a metal oxide surface can be a fundamental tool in the understanding of these interactions16. Among the oxides, magnesium oxide (MgO) has been the subject of many studies due to its high ionic character and simple crystal structure17. Its abilities on catalysis, adsorption–desorption process, and its use as a lattice template for selective growth of thin films have been studied for different purposes21. MgO surface is an ideal platform for water interaction studies due to the formation of free OH group  for the 1.1 mK/s and 0.55 mK/s heating rates were employed. These experimental conditions contribute to maintaining constant the coverage factor and an amount of about two water monolayers on the MgO surface between the experiments.To understand the dynamics of the interstellar ice over which an Structural Phase Transition (SPT) can temporarily progress, we architecturally grew water monolayers on top of the (100)–oriented MgO substrates. We investigated the time evolution of the SPT by monitoring the intensity of the diffraction spots from the Low–Energy Electron Diffraction (LEED) patterns along with full control and discretization of an external perturbation energy. All of our intensity measurements were performed on the spot (10) of the LEED diffraction pattern. Figure entclass1pt{minimac caused by impurities in the MgO crystal lattice as a consequence of the SPT local perturbation within the lattice that is directly mapped on our sensor. Importantly, we show for the first time that the SPT in interstellar ice could be monitored by means of (100)–oriented MgO crystal lattices. To our knowledge, this versatile platform detecting mechanical changes via electron diffraction has not been explicitly examined before.We now turn to the characteristic shape of our findings in Fig. 29, we approximate the dynamics of the phonon in the MgO crystal by means of the following unperturbed Hamiltonian: h) divided by 2c. This model approximate the isotropic lattice strain in the MgO lattice, which is then used to estimate the lattice stress by analizing the coherent elastic scattering of electron diffraction. We explore isotropic stress generated by the water monolayer dynamics, which acts until a depth of about , D denotes the volume–weighted crystallite size, E is the isotropic Young’s modulus and The W–H analysis is a quantitative approach, which differentiates between size induced and strain induce peak broadening by considering the peak width as a function of the Bragg diffraction angle  of interstellar ice at 185 K. Here, we show for the first time that the use of a very precisely experimentally controlled disturbance is highly desirable for the characterization of SPTs in interstellar ice. By monitoring the intensity of the diffraction spot of the Low–Energy Electron Diffraction (LEED) pattern, we obtain the first experimental evidence of the existence of the SPT between the 35 and it was maintained at pressures of 35. The impurities had been monitored by an Ametek LC-Series quadrupole mass spectrometer coupled to our vacuum system that shows a low level of atmospheric impurities consisting of molecular nitrogen and oxygen, as well as the water characteristic ions in the ultra-hight vacuum system. Figure 35. Next, a cooling process of 4 hours was carried out to promote the formation of a predominantly ice with c(Our experimental setup is based on the apparatus used for previous workmentclass2pt{minimc(p(c(p(To induce the gradual ice mentclass2pt{minim


The purpose of this in-vitro study was to evaluate the effect of dynamic cyclic loading on screw loosening of retightened abutment screw versus new abutment screw in both narrow and standard implants.t-student, ANOVA, pair wise Tukey’s tests.Separate acrylic resin blocks containing implant assembly . Samples were divided into two main groups according to the diameter of implant: group 1 (GI 4.5-mm diameter) and group 2 (GII 3-mm diameter). Each group is subdivided into two subgroups according to the suggested option to manage screw loosening either by retightening  or using new screws . One hundred thousand cycles of eccentric dynamic cyclic loading (DCL) were applied before and after retightening or replacing the screw; then, removal torque loss (RTL) ratio was calculated, tabulated, and analyzed by There were differences between GI and GII regarding the incidence of screw loosening process. Removal torque loss ratio was higher in GIB and GIIB where the old abutment screws were replaced by new screws for both standard implants (SIs) and narrow diameter implants (NDIs). There was significant effect of retightening and replacing the abutment screws after exposure to DCL.Within the limitations of this in-vitro study, it can be conclude that screw loosening process occurred in both SIs and NDIs but with higher values in NDIs. It is better to retighten the screw of NDIs and SIs than replacing it with a new screw. Oral implantology has undergone a well-deserved rebirth or rediscovery and implants considered the treatment of choice in an increasing number of carefully selected cases.After osseointegration is achieved, long-term clinical follow-ups reported biological or mechanical complications . One of Screw loosening may cause implant or screw fracture, inadequate occlusal force distribution, and possible osseointegration failure. In addition, screw loosening would also lead to micro motion at the implant-abutment interface while chewing . Sones  reportedThe process of screw loosening was described in two stages . InitialMany factors related to screw design and fabrication may affect abutment or prosthetic screw loosening in a metal-to-metal screw system; these primarily are related to preload. Preload can be influenced by component and screw materials , 9, torqIn addition, factors that affect abutment screws loosening include hex height (or depth), diameter of the screw, platform diameter , surfaceOne of the variables that influence the joint stability is how the contacting parts change when the screw is tightened. After being tightened together by the screw, the micro-roughness of all the metal contacting surfaces slightly flattens, and the microscopic distance between contacting surfaces decreases . As a reWhen evaluating the screw loosening of new abutment screws and after successive tightening, it was found that the percentage of the initial torque loss is lower when screws that already suffered the application of an initial torque were used, remaining stable after application of successive torques  that is It was strongly recommended that retightening of implant abutment screw is important to decrease the possible screw loosening. Pardal-Peláez et al.  have staIf the abutment screw is exposed to excessive wear and still in place, screw replacement is a good option. Hum  has intrNDIs are considered a good treatment option replacing missing teeth where there is lack of adequate bone width and interdental space without need for bone augmentation procedures that aim to transform the deficient ridge into a ridge that is capable of receiving conventional tooth-form implants. These advanced augmentation procedures have disadvantages, such as a prolonged healing time, additional cost, and unpredictable complications including infection and wound exposure . Nearly So the purpose of this study was to evaluate the effect of DCL on screw loosening of retightened abutment screw versus new abutment screw in both NDIs and SIs and to test the null hypothesis that there are no differences between retightening screws and using new abutment screw in both NDIs and SIs.Total forty titanium fixtures  with conical hybrid connection were used in this study and divided into two groups, twenty each according to their diameter and so the diameter of the screw: group I (SIs), implants with 4.5-mm diameter; and group II (NDIs), implants with 3-mm diameter. Each group was divided into two subgroups A and B, ten each according to the suggested solution for screw loosening management, GIA and GIIA for retightening option, and GIB and GIIB for new screw option. For each sample, 12-mm length bone level implants were used with platform switching including conical hybrid connection (Morse Taper 5 degree).three dimensionally (3D) scanned to accurately get the desired design and dimensions of the metal tube that fit the abutment head; this was achieved by the Computer Aided Design Computer Aided Manufacturing (CAD/CAM) software (Dent create Exocad), with flat occlusal surface (10 mm in diameter) parallel to horizontal plane and perpendicular to implant fixture long axis to permit contact with the testing machine piston in a flat horizontal plane with small rounded hole exactly opposite to the abutment screw hole that facilitates screw driver accessibility. Using a special CAD/CAM wax type, forty identical wax patterns for metal tube of each group were made; these patterns were casted by a lost wax technique. The inner surfaces of the metal tube for each group were checked to the desired dimension using digital caliper and adjusted with a carbide bur, then checked to ensure that each tube fit accurately on corresponding abutment, and the screw driver inserted and removed easily.Forty stock straight titanium abutments were selected for this study with the same gingival height of 2 mm and the same post height for standardization. The titanium abutment was screwed to the fixture, sprayed, and Water balance device is adjusted over the top of the metal tube to ensure 180° surface just before cementation to the corresponding abutment; this procedure will be repeated after securing the specimens to the customized jig just before application of dynamic cyclic loading. Self-adhesive resin cement was used for cementation of metal tube to the abutment; the access of screw hole of each abutment was protected by cotton. Finally, all samples had been ready for the testing procedures.Before measuring the initial RTV, a customized rigid metal mounting jig was used to firmly fix the acrylic sample by tightening three metal screws Fig. .Fig. 1CAbutment screw was tightened to 30 Ncm according to the manufacturer’s recommendation using a digital torque gauge .Ten minutes after first torque application, a torque of 30 Ncm was reapplied again with the same digital torque gauge. Ten minutes later of screw retightening, the initial RTV was measured by rotating the screw driver in an anticlockwise direction and recorded.After measuring initial RTV, the abutment screw was tightened again to the recommended torque value (30 Ncm). Then, immediately the acrylic resin block was firmly mounted in a holder of the lower fixed compartment of a computer controlled universal testing machine , for 100,000 cycles of eccentric dynamic cyclic loading, 5 mm away from the center of the previously fabricated metal tube. The dynamic cyclic loading was performed with a metallic rod with round tip which was attached to the upper movable compartment of the machine, under load of 130 N perpendicular to the metal tube and 5 mm away from the center axis of the implant using a metallic rod with round tip which was attached to the upper movable compartment of the machine. The contact time between the rod and metal tube was adjusted to 0.2 s at a rate of 1 Hz which simulates the tooth contact duration of each masticatory cycle  was analyzed by measuring RTV before and after dynamic cyclic load by using the digital torque gauge. RTL can be an indicator of how much loosening takes place.Each RTL ratio was calculated using the following formula :Removal torque loss ratio before loading An indicator of how much loosening takes place before loading.Removal torque loss ratio after loading (% post load RTL)An indicator of how much loosening occurs after loading.Removal torque loss ratio between before and after loading .The screws of specimens of GIA and GIIA were retightened at the same initial torque (30 Ncm) using the digital torque gauge. Then, the procedures of dynamic cyclic loading were repeated for the second time.The screws of specimens of GIB and GIIB were changed with new screws, torqued to 30 Ncm, left for 10 min, and torqued again to 30 Ncm, and then the procedures of dynamic cyclic loading were repeated for the second time.After DCL, the loading machine was stopped. The acrylic resin block with fixture and abutment was transferred again to the metal jig to measure post load RTV using tire same digital torque gauge with procedures same as measuring initial RTV.An indicator of the degree of loosening caused by the DCL.t-test, ANOVA, and pair wise Tukey’s post hoc tests were used to compare mean of initial and post load RTL ratio between different groups and subgroups. Statistical analysis was performed by using SPSS program version 20 .Student’s 1) was recorded. The mean of initial percentage, post load percentage, and percentage of difference between initial and post load removal torque loss after second exposure to dynamic cyclic loading (RTL2) was also recorded for all test groups.The mean of initial percentage, post load percentage, and percentage of difference between initial and post load removal torque loss after first exposure to dynamic cyclic loading  is quite close to that of cancellous bone (3.6 × 105 lb/in.2).Implants were placed into acrylic resin as according to De Carvalho et al.  who usedConical hybrid connection was selected because it was reported that, among the different internal connection types, the conical hybrid connection showed the best stress distribution as it has a mechanical friction grip that enhance resistance to the lateral forces decreasing the probability of screw loosening, so it was the best connection to be used , 39.The desired design and dimensions of metal tube were designed by the CADCAM software  in wax , with flat occlusal surface (10 mm in diameter) which was parallel to the horizontal plane and perpendicular to the implant fixture long axis to permit contact with the testing machine piston in a flat horizontal plane. In the center of the flat occlusal surface, a small rounded hole was designed exactly opposite to the abutment screw hole that facilitates screw driver accessibility for easy tightening and removal. Then, this accurately designed wax pattern was casted to a nickel chromium alloy tube .CAD/CAM system was to ensure standardization as CAD/CAM system has the ability to produce physical models using digital methods instead of traditional impression techniques with high error rate, time consuming procedures, and lack of accuracy and standardization .Ten minutes interval was left after first torque application, and all screws were retightened to the same tightening torque (30 Ncm), to compensate for the preload loss due to settling effect of screw thus ensure achieving optimal preload.Tightening and removal of abutment screws in all the groups was done with a digital torque gauge instead of a hand torque wrench to eliminate the possibility of deviations from exact torque value which gives decimal precision for accuracy and standardization , 42. TheLoad was applied eccentrically at a distance of 5 mm away from the center of abutment , 44 to sFor all test groups, there were significant differences in RTL ratios before and after application of DCL. These findings emphasize on the occurrence of screw loosening process.The screws of NDIs are comparable with standard ones because the implant system used in this study provided narrow screws with narrow fixtures and standard screws for standard fixtures on contrast with most of implant systems that provide the same screw diameter for both narrow and standard implants. Standard screw within narrow fixtures  makes the force transmission more destructive and shows more biomechanical complications that were reported in most of studies worked on NDIs. This finding is supported by Patterson and Johns  who statThe results of this study showed that the screw loosening process occurred in both SIs and NDIs with non-significant difference; this can be due to use of various abutment screw diameter. The retained preload inside the standard screw provided more screw stability due to the relative increase in material thickness for the standard screw. In other words, when forces are greater than usual, a larger diameter screw will decrease the risk of loosening or fracture, so the standard screw has superior biomechanics over the narrow one; this finding matches the statements of Byrne et al.  who statThe results of this study revealed that mean percentage initial removal torque loss (% RTL) of NDIs is higher than  of SIs while mean percentage post load removal torque loss after exposure to dynamic cyclic loading is non-significantly different. This can be explained that the same tightening torque (30 Ncm) is applied for both diameters, so the initial tightening torque is relatively high for NDIs, and this is one of the suggested solutions for decreasing the chance of screw loosening process. This explanation is supported by Siamos et al.  who statAnother study has shown that retorque does not significantly interfere on the loosening torque when the titanium screws are used in dentures with passive fit. On the other hand, the retorque significantly increased the loosening torque when these screws were used in dentures with misfit .Replacing the old abutment screw with new one showed better results in SIs than NDIs with non-significant difference after two exposures of DCL; this is due that retaining much preload within standard screws will in turn increase the clamping force between screw threads and internal threads of screw channel making it more liable to surface flattening and excessive wear that is considered actual screw failure and requires its replacement with new functioning one. This finding is supported by Haak et al.  who discOther authors confined replacing the old screw with new one to the fractured abutment screw that no more performs function under loading. Fracture of the implant abutment screw can be a serious problem, as the fragment remaining inside the implant may prevent the implant from functioning efficiently as an anchor.Flanagan  had intrThis finding is supported by Barbosa et al.  who evalBut this finding is opposed by Bacchi et al.  who statAnother study has shown that retorque does not significantly interfere on the loosening torque when the titanium screws are used in dentures with passive fit. On the other hand, the retorque significantly increased the loosening torque when these screws were used in dentures with misfit .Screw loosening process occurred in both SIs and NDIs, but it had higher values in NDIs.Higher RTVs are obtained after retightening the old abutment screws of both SIs and NDIs after exposure to 100,000 cycles of DCL, in comparison with replacing the old screw with new one.Higher RTVs are obtained after replacing the old screws with new ones of SIs after exposure to 100,000 cycles of DCL, in comparison with NDIs.Within the limitations of this in-vitro study, it can be concluded as follows:


The prevalence of TW I-type infectious bronchitis virus (IBV) has been increasing rapidly, and it has become the second most common genotype of IBV in China threatening the poultry industry. In this study, 1-day-old specific-pathogen-free (SPF) chickens infected with TW I-type IBV were continuously observed for 200 days. TW I-type IBV affected the respiratory, urinary, and female reproductive systems, resulting in a mortality rate of 10% as well as a decrease in egg quantity and an increase in inferior eggs. During the monitoring period, serious lesions occurred in the female reproductive system, such as yolk peritonitis, a shortened oviduct, and cysts of different sizes with effusion in the degenerated right oviduct. The infective viruses persisted in vivo for a long time, and due to the stress of laying, virus shedding was detected again after the onset of egg production. Our findings suggest that TW I-type IBV is deadly to chickens and could cause permanent damage to the oviduct, resulting in the poor laying performance of female survivors and decreasing the breeding value and welfare of the infected flock. Gammacoronavirus, IBV has a remarkably high mutation and recombination rate, leading to numerous types and variants that differ from each other in pathogenicity  Kit . The primers and probe used for RT-qPCR were described in a previous study [The oral and cloaca swabs were washed in PBS, and suspensions of tissues were also prepared in PBS (20% w/v). Samples of 156-day-old hens were blind passaged three times in the allantoic cavity of 10-day-old embryonated SPF chicken eggs. Total RNA of the samples were extracted using the Ultrapure RNA Kit , and reverse transcription was performed with the EasyScriptus study . The reaFor haematoxylin–eosin staining, the collected samples of the trachea, lungs, kidneys, and oviduct were fixed in 10% neutral formalin for 48 h at room temperature. The fixed samples were processed, embedded in paraffin wax, and cut into 5 μm sections and examined by light microscopy for the presence of lesions.2 small squares. The squares were then fixed in 2.5% glutaraldehyde in 0.1 M PBS for 12 h at 4 °C. The samples were then washed with PBS and dehydrated via an alcohol gradient, followed by drying (CPD-30D) and conductive treatment (SCD 500). Finally, the ultrastructure of the tracheal cilia was observed by scanning electron microscopy (GeminiSEM 300) with a magnification of 5000×.For the examination of ultrastructural alterations, the cylindrical trachea were cut crosswise with two sharp blades into small strips of 1 mm wide and 3–4 mm long, and then the strips were cut into 1 mmp < 0.05 was considered as significant.All statistical analyses were performed with SPSS 26.0 software. Non-parametric Mann–Whitney U test was used to analyze the significant difference, and The clinical manifestations in the early infection stage caused by TW I-type IBV were identified as respiratory symptoms. Nearly half of the chickens in the challenge group exhibited gasping, tracheal rales or dyspnea. Seven chickens died from 9 to 30 dpi, accounting for 10% of the infected flock. Gross lesions such as light hyperaemia and serious catarrhal exudation in the tracheal mucosa and urate deposition in the tubules of the pale kidney, appeared in the infected flock from 5 to 30 dpi.The development of oviducts and ovaries was assessed at 140 days of age. The reproductive system of chickens in the control group was well developed. However, there were 4/9 chickens in the challenge group that showed delayed development of oviducts and ovaries (Table p < 0.05). These results indicate that TW I-type IBV has a significant effect on subsequent laying performance when the infection occurs at a very young age in chickens.The pathogenicity of TW I-type IBV in the laying period was characterized by a decrease in the quantity of eggs and an increase in the quantity of inferior eggs Table . During At the age of 200 days, various lesions were observed in the reproductive systems of the hens Table . In the 2.2 copies/μL, and two positive samples were blind passaged three times in SPF chicken eggs, 106.7 and 108.3 copies/μL were detected. At 200 dpi, one of the pooled caecal tonsil samples was detected containing 104.1 copies/μL : adhesion, lodging of cilia covered by mucus. C : shedding and remains of cilia.


RNA viruses are characterized by high mutation and recombination rates, which allow a rapid adaptation to new environments. Most of the emerging diseases and host jumps are therefore sustained by these viruses. Rapid evolution may also hinder the understanding of molecular epidemiology, affect the sensitivity of diagnostic assays, limit the vaccine efficacy and favor episodes of immune escape, thus significantly complicating the control of even well-known pathogens. The history of infectious bronchitis virus (IBV) fits well with the above-mentioned scenario. Despite being known since the 1930s, it still represents one of the main causes of disease and economic losses for the poultry industry. A plethora of strategies have been developed and applied over time, with variable success, to limit its impact. However, they have rarely been evaluated objectively and on an adequate scale. Therefore, the actual advantages and disadvantages of IBV detection and control strategies, as well as their implementation, still largely depend on individual sensibility. The present manuscript aims to review the main features of IBV biology and evolution, focusing on their relevance and potential applications in terms of diagnosis and control. Avian coronavirus, genus Gammacoronavirus, family Coronaviridae, order Nidovirales. IBV is an enveloped virus with a single-stranded positive-sense RNA genome of approximatively 27 kb, which displays the following gene organization: 5′UTR-1a/1ab-S-3a-3b-E-M-5a-5b-N-3′UTR. The RNA molecule has a 5′ cap and a 3′ poly(A) tail and acts as mRNA for translation of the replicase polyproteins, whose coding regions occupy the 5′ two-thirds of the viral genome. The two untranslated regions (UTR) play a key role in virus replication interacting with viral replicases and potentially other host proteins [Infectious bronchitis virus (IBV) is currently classified within the species The Spike (S) protein is the most described IBV structural protein. This transmembrane protein, organized in trimers, determines the typical crown-like aspect of coronaviruses, observable at electron microscopy . It is pThe other surface proteins, Membrane (M) and Envelope (E), are involved in virus morphogenesis and assembly. Besides E and S, the M protein interacts with the Nucleocapsid (N) one, which directly binds genomic RNA to form a helical ribonucleoprotein complex and probably participate in RNA packaging ,13.Mutation is the ultimate source of genetic polymorphism and represents the first fundamental substratum for evolution, since it generates the genotypic (and phenotypic) variants that spread and become fixed through genetic drift or natural selection ,15,16. −4 to 10−5 misincorporations per nucleotide position) because of the lack of RNA-dependent RNA polymerase (RdRp) proofreading activity coupled with the absence of RNA repair mechanisms [−1 replication−1, which is a 10-fold higher rate than retroviruses and 10,000-fold greater than most DNA viruses, probably justifying the limited genome size of RNA viruses. In fact, while the continuous production of mutants favors virus adaptability in the event of environmental changes, the accumulation of multiple mutations in the same viral particle would lead to a dramatic fitness reduction [RNA viruses are known for having an extremely high error rate  is still remarkable [Coronaviruses, including IBV, with their 30 kb long genome, can be considered an exception within the RNA virus group since their large replicase gene contains an ExoN domain in the nsp14, related to host proteins of the DEDD superfamily of exonucleases and involved in proofreading and repair activity. Nevertheless, the estimated substitution rate of IBV  is often implicitly stated in many manuscripts, the actual role of natural selection has been explicitly accounted less frequently. More recently, the relevance of natural selection in shaping IBV evolution has been determined for both vaccine and field strains. In longitudinally monitored birds vaccinated with live-attenuated Ark vaccines, for which subpopulations differed in the S protein sequence, minor viral subpopulations were rapidly selected and mutations emerged during passages in chickens, suggesting that the phenotype of these subpopulations was advantageous and/or favored adaptation to the chicken host ,24. AccoA study by Gallardo et al., based on S sequence analysis, highlighted that the predominant IBV phenotype contained in the vaccine became a minor population in the host, and also significant differences were detected in the incidence of some distinct IBV populations in the considered tissues and fluids. Therefore, in addition to the host role, the specific tissues could exert a certain pressure, selecting the variants able to replicate better in a particular microenvironment .Closely related strains sometimes demonstrate a relevant heterogeneity in cross-neutralization patterns, highlighting that mutations in specific amino-acid sites are responsible for immune evasion . AccordiImmune response is thus expected to be the main selective force acting on IBV evolution, at least in the antigenic regions. Some experimental evidences seem to further support this hypothesis. When field strains were inoculated in vaccinated and unvaccinated chicken groups, the emergence of non-synonymous mutations was described only in some of the vaccinated birds . EvidencHowever, it should be emphasized that vaccination is not “per se” a risk factor for viral evolution, but improper vaccination can be. Natural selection is expected to predominate when Ne∙S >> 1 . While vaccination clearly imposes a strong selection coefficient, it also drastically reduces viral replication and therefore the population size, preventing the emergence of more fit variants. On the other hand, improper vaccine selection or administration, as well as factors suppressing the bird immune response, can allow the virus to circulate in a partially immune environment, potentially leading to the emergence, selection and spreading of immune escape variants ,30,31.Current studies are essentially based on the S protein (or part of it), representative of less than 10% of IBV genome, whereas IBV virulence, pathogenesis and tropism are polygenic and also associated with genetic variants outside of the S gene ,33,34, eA second pivotal source of genetic variability is recombination. Coronaviruses are featured by a high recombination rate, mediated by a copy-choice mechanism . In thisBesides affecting viral biology, recombination represents a remarkable challenge for IBV classification. As previously mentioned, the current classification is officially based on the S1 gene sequencing , even thWhile zootechnical parameters and presence of symptoms are useful as preliminary evidences of infectious bronchitis (IB) outbreaks, IBV-induced signs are not pathognomonic. Laboratory assays are therefore mandatory to detect and characterize IBV strains. The most used diagnostic tests include viral isolation, serological and molecular assays. When attempting to isolate field viruses, samples should be promptly collected when IB-compatible signs are at first observed, since IBV titres peak in the first week post-infection, possibly before clinical manifestations . TracheaMethods based on antibody detection are used to study previous IBV circulation or assess the induced immunological response. A range of serological tests is available, including agar gel precipitation (AGP), enzyme-linked immunosorbent assay (ELISA), virus neutralization (VN) and hemagglutination inhibition (HI).AGP allows for quick and inexpensive responses but is not routinely adopted since it lacks in sensitivity and the main precipitating antibodies  are detected only for a few weeks after exposure .Commercial ELISA tests are the most commonly applied for routine serological monitoring because of their cost-effectiveness and rapid turnaround. Antibody titration is useful to monitor both the vaccination response, especially in layers and breeders, and exposure to field virus. For a proper interpretation, baseline values should be established based on a regular monitoring of the local situation since antibody titres depend on many factors like breed, type, age at sampling, vaccination program and schedule ,51. The observed results should be compared to the baseline in terms of average titres, within-group uniformity and persistence. Reasonably high, uniform and lasting titres are indicative of a proper vaccination, while low, uneven and short-lived titres suggest a defective vaccination, likely due to an improper administration or poor quality of the vaccine batch. When the observed titres are significantly higher than expected, a field infection should be suspected.The majority of the available ELISA kits are designed to detect polyclonal antibodies against the whole virion and do not allow serotypization. Several alternatives to commercially available ELISA kits have been developed ,54,55,56VN and HI tests can identify serotype-specific antibodies and are therefore useful to assess the specific response to both field and vaccine strains. However, only VN should be considered the method of choice for serotyping purposes, because of the more frequent occurrence of cross-reactions using HI . UnfortuAside from the described methods, other serological assays have been recently developed, including methods based on Unknown multi-analyte profiling (xMap) , microarCurrently, biomolecular assays are the most used tools for IBV detection, because of their sensitivity and quick response time. Besides viral RNA detection, they allow to characterize the detected strains from a genetic standpoint, allowing to properly plan and evaluate vaccination protocols and assess the presence of specific field strains. It is worth noting that the mere PCR positivity does not imply an active infection at the moment of sampling since genome traces can persist for a relatively long period after viral clearance. Thus, the results should be carefully interpreted. a priori subset of IBV strains or genotypes. In fact, the ideal diagnostic algorithm should comprise both generic and specific assays selected on the actually circulating variants in the field. A multitude of RT-PCR and qRT-PCR-based methods have been validated, either generic and targeting virtually all IBV subtypes, or genotype- or strain-specific ,66,67,68Restriction fragment length polymorphism (RFLP) has been used as an alternative to sequencing and specific probes development. The use of restriction enzymes represents a quick, inexpensive technique, but, unfortunately, not all strains can be differentiated in this way , and theQuantitative real-time RT-PCR (qRT-PCR) allows the quantification of the targeted genetic material present in the sample. Real-time assays targeting conserved regions of IBV genome cannot quantify the respective contribution of simultaneously present strains, limiting their usefulness to screening purposes. On the other hand, a properly chosen panel of strain-specific qRT-PCR assays enables their precise differentiation. Field strains quantification may help to discriminate an incidental detection from a disease primarily caused by IBV, while vaccine strain titres can be used to assess vaccine coverage, replication and persistence. The description of vaccine kinetics in longitudinal studies may be of great aid in characterizing different vaccination protocols, replication dynamics of the various vaccines and potential interactions when co-administered .Currently, the main issue of PCR-based methods used for IBV characterization is the lack of consensus on the genetic classification method. The results provided by different laboratories may differ because the considered genomic region differs in size or position, or because the same genetic subtype is referred to with different names by different laboratories . As prevThe absence of clear genetic markers also prevents the reliable discrimination between vaccine and field strains , althougSuitable specimens for PCR testing include dry swabs, tissues and FTA cards. These are paper substrates containing chemicals that protect nucleic acids and inactivate pathogens with no risk of contamination, requiring less stringent conitions for storage and shipment but possibly entailing a loss in sensitivity . SamplesBesides being vastly adopted for diagnostic purposes, PCR-based methods followed by sequencing are of great use for research studies of IBV epidemiology. For example, phylodynamic projects allow to investigate the evolutionary history and spreading dynamics of the viral population, providing useful information to evaluate and improve IBV control measures on a large-scale perspective ,77.Other molecular techniques, such as loop-mediated isothermal amplification (LAMP) and padlock probes (PLPs) combined with rolling circle amplification (RCA), have been validated for IBV detection ,79. AddiNo technique can be considered fully conclusive by itself, usually requiring the combination of multiple tests to obtain a complete picture of the infectious status of single animals, flocks or larger populations. Rather than implementing a standard set of assays, the best diagnostic approach should probably be decided on a case-by-case basis to suit the particular demands and peculiarities of the epidemiological context. Different tests have different costs and response times, and they may require different types of samples, which are not always easily collected in certain situations . The purpose of the survey is also crucial to decide which tests to use. For instance, the investigation of a suspected IB outbreak may impose a different approach compared to the assessment of vaccination coverage.In a typical field situation, the common adoption of multiple live vaccines may complicate the diagnostic process and mask the simultaneous presence of field strains ,74,82. FThe most suitable procedures for each of the main diagnostic purposes are described in The remarkable economic impact of IBV on poultry production encourages the implementation of massive vaccination strategies, whose wide adoption requires to settle between ideal application and practical aspects. As for other pathogens, IBV control can be eased by good management, correct bird density, air quality, all-in/all-out period duration, etc. . NeverthThe first essential barrier against IBV is biosecurity, with the strict implementation of external and internal measures regulating the flow of animals, people, supplies and waste/manure ,87. Adeq0 < 1) and shedding (more than 1 log10 of excreted titre reduction) after homologous challenge in experimental conditions [To compensate for these aspects, vaccination is the most effective and frequently adopted option: even though it cannot fully prevent the infection, a decrease in clinical signs and infectious pressures can be achieved. For instance, H120-vaccinated animals showed a reduced viral transmission  and 793B-like (GI-13) strains, American strains such as Ark (GI-9), Conn (GI-1), DE072 and GA98 (GIV-1), QX (GI-19), Q1 (GI-16) and Var2 (GI-23), suggesting a variable protection degree depending on the challenge virus ,117,118.Although the likelihood of cross-protection is slightly higher among more similar strains, there is a weak correlation between genetic similarity and protection of two strains , which hAlternatively, an accessory evaluation of vaccine kinetics  and coveBesides vaccine type selection, the attempt of reducing labour and vaccine supply, inadequate mass administration, the use of fractional doses and wrong timing could significantly affect animal protection, coverage, vaccine-associated virulence and mixed vaccine-field virus circulation . IBV vacMass spray vaccination is an approach that clearly minimizes work whilst maximizing coverage, and it is usually applied at hatchery or farm by spray, which is considered analogous to the gold standard oculo-nasal vaccination in mimicking the entrance of respiratory viruses ,130, wheSpray vaccination could raise managerial problems when applied in a hatchery that supplies chicks to several farms requesting different vaccines. Nonetheless, the concern of chick contamination with different administered strains at the hatchery was diminished by Pellattiero et al., proposing that not yet replicating vaccines seem unable to spread to other non-vaccinated animals located in the same environment for several hours .Spray vaccination has been progressively replacing administration via drinking water, which is largely deprecated since it often implies the combination of vaccines and other substances, normally dissolved in the water or to be co-administered, that could lower the vaccine titre or viability, leading to low coverage, rolling reactions and reversion to virulence. Nevertheless, it is still used, mainly for booster administration of less attenuated vaccines in the field . Gel vacOn the other hand, many registered vaccines for broilers are declared to guarantee protection from day-old vaccination up to the end of the cycle, according to the supplementary protection certificates. An early combined vaccination at 1 day of age was proven to confer an efficient immune response , and epiIt should not be under-emphasized that inconsistencies between experimental and field results could be due to different conditions and better control of environmental, managerial and biological variables in experimental settings , which pAs extensively described, IB control complexity is determined by several factors, from field conditions to the biological properties of the virus, which need unceasing research efforts and studies. New vaccines and new control strategies have to overcome old problems, such as costs and management issues, cross-protection limitations and variant emergence, whose knowledge and evaluation must be considered as essential requisites for strategy planning. At the moment, IB eradication is a distant goal and all efforts in the field should aim at optimizing the implementation of measures of well-known efficacy. A steady and rigorous monitoring based on objective criteria and knowledge of the local epidemiological scenario should be also strictly implemented.


The use of robotic systems to aid in surgical procedures has greatly increased over the past decade. Fields such as general surgery, urology, and gynecology have widely adopted robotic surgery as part of everyday practice. The use of robotic systems in the field of spine surgery has recently begun to be explored. Surgical procedures involving the spine often require fixation via pedicle screw placement, which is a task that may be augmented by the use of robotic technology. There is little margin for error with pedicle screw placement, because screw malposition may lead to serious complications, such as neurologic or vascular injury. Robotic systems must provide a degree of accuracy comparable to that of already-established methods of screw placement, including free-hand, fluoroscopically assisted, and computed tomography–assisted screw placement. In the past several years, reports have cataloged early results that show the robotic systems are associated with equivalent accuracy and decreased radiation exposure compared with other methods of screw placement. However, the literature is still lacking with regard to long-term outcomes with these systems. This report provides a technical overview of robotics in spine surgery based on experience at a single institution using the ExcelsiusGPS  robotic system for pedicle screw fixation. The current state of the field with regard to salient issues in robotics and future directions for robotics in spinal surgery are also discussed. In the past decade, the role of robotic systems in surgical fields has expanded, and innovations have flourished . Early aTreatment of spinal pathologies often requires fixation via the placement of pedicle screws. Techniques for pedicle screw placement were first described in the late 1950s, and since that time, they have undergone a wealth of adaptation and methodological advances. These advances include the description of open and percutaneous approaches using a variety of navigated techniques –11. PediA key determinant of the widespread adoption of robotics in spine surgery is efficient and accurate screw placement. The accuracy of robotic systems must be similar to or better than that of well-established methods of pedicle screw placement, including free-hand, fluoroscopically assisted, or computer tomography (CT)–assisted screw placement. These systems offer the theoretical advantage of automating inherently repetitive tasks that are subject to human error. Early reports of the use of robotic technologies in spine surgery have shown equivalent accuracy compared with other methods of screw placement . MultiplHerein, we provide a technical overview of the incorporation of robotics in spine surgery based on our institutional experience using the ExcelsiusGPS robotic system for pedicle screw fixation. We also discuss salient issues regarding this technology based on our experience and consider future directions for robotics in spinal surgery.Exact operative technique and surgical workflow will vary on the basis of the specific robotic system that is used. This section will discuss the use of the ExcelsiusGPS system . Robotic systems may be considered for any spinal fusion procedure with planned placement of pedicle screw fixation. At our institution , robotics have most commonly been incorporated into lumbar fusion procedures, including anterior lumbar interbody fusion, lateral lumbar interbody fusion , and traPatient positioning and operating room set up are shown in First, a stab skin incision is made. The bovie electrocautery is used to dissect through the subcutaneous tissue and the fascia. Importantly, the fascia should be excised medial to the skin incision to allow for the appropriate trajectory toward the screw entry point. The bur is then placed on bone at the screw entry point to create a pilot hole for screw placement. This step is important to prevent skiving of the drill off of the cortical bone and facilitate smooth entry into the cancellous channel. Tapping is then performed under navigation for comparison with the planned trajectory. Finally, the pedicle screw is placed under navigation and is also compared with the planned trajectory . Once thIn our experience, screws have been most commonly placed in a percutaneous fashion. However, open screw placement is also done in appropriate scenarios, such as when accompanied by a spinal decompression for degenerative pathologies. In an open procedure, the dynamic reference base array and the intraoperative CT registration fixture are placed on a spinous process above and below the operative segments rather than on the posterior superior iliac spine. All subsequent steps may be performed in the fashion described above, with care taken not to disrupt the dynamic reference base array within the operative field. In the open exposure, the screw entry site may be directly visualized, and cortical bone may be drilled away to facilitate screw placement.The above-described technique is for patients who are placed in the traditional prone position. More recently, there has been increasing interest in performing single-position surgery in the lateral position for lumbar fusion, including both anterior lumbar interbody fusion and lateral lumbar interbody fusion, accompanied by pedicle screw fixation. We have used the robotic system for screw placement in this position as well . In thesWidespread adoption of robotic systems for pedicle screw placement must entail safe and reliable accuracy. The most common classification system used in the literature is the Gertzbein-Robbins classification . The repP < 0.01) as well as “clinically acceptable”   . Two oth < 0.01) , 24. A m < 0.01) .Another technique for pedicle screw fixation that has been widely adopted includes use of navigation with intraoperative CT scan. Some surgeons argue that accuracy of screw placement with use of navigation is high enough that they would not need the use of a robotic arm. Based on our experience, we support the use of a robotic arm for multiple reasons. First, with current technology a CT scan may now be performed pre-operatively. Screw planning is done prior to surgery and this can significantly decrease surgical time compared to the use of intraoperative CT scanning. The ability to plan the screw trajectory ahead of surgery maximizes the fidelity of screw placement. Important considerations such as screw size, length, trajectory, and avoidance of the superior facet are all addressed prior to skin incision. Second, the use of the robotic arm mitigates the human error that is ever present in repetitive manual tasks. Lastly, the authors' experience has been that pedicle screw placement using the robotic arm is less taxing physically for the surgeon.Another important factor to consider with regard to the incorporation of robotics into spine surgery is the additional operative time required to use this technology. Multiple studies have found that increased operative times were associated with robotic systems. A commonly considered factor that may contribute to this phenomenon is the steep learning curve for use of this technology. The learning curve associated with robotics has been documented previously by multiple authors who have shown improved accuracy after an initial learning period , 27. MorA purported advantage of robotic systems is decreased radiation exposure. The surgeon is not exposed to the initial (preoperative or intraoperative) CT. Minimal intraoperative radiation is then required to register the robotic system to this scan using fluoroscopy. When intraoperative CT is used, no additional fluoroscopy is required for registration. Decreased radiation exposure to the surgeon during robotic procedures has been validated . Other sThe use of robotic systems in spine surgery is rapidly evolving. Continued adaptation will be important for future expansion in this field. These adaptations should include improvements in efficiency and surgical workflow to facilitate widespread adoption of these systems. Such improvements would involve enhanced imaging software to aid with patient registration, to minimize error, and to assist with trajectory planning both preoperatively and intraoperatively. The aim of robotic systems is to automate repetitive tasks that are subject to alteration and human error. As automation becomes more standardized, this may lead to more uniform patient outcomes. The indications for use of robotic systems will likely continue to expand. Currently, most literature involves fixation of the thoracolumbar spine. Both cervical and pelvic fixation may incorporate robotic systems in the future. In addition, applications for robotics may expand to include more complex spinal procedures, such as decompression, resection of neoplastic lesions, and complex deformity procedures. With more widespread use, head-to-head investigations that compare various robotic systems may help delineate the relative strengths and weaknesses of each system. Finally, the economic viability of these systems should continue to be addressed. Their use will remain limited to resource-rich settings if costs remain very high.Robotic systems have been widely adopted throughout the United States and in various surgical subspecialties. This innovative technology continues to permeate the field of spine surgery. These systems offer the potential advantages of increased accuracy of screw placement, decreased operative time, and decreased radiation exposure. However, there is a challenging learning curve, and various technical factors of these systems are continuously being reassessed to improve operative efficiency and to meet these goals. Maintaining clinical equipoise with established methods of screw placement, including freehand screw placement and various forms of navigation, will be important for further adoption of these systems in our field.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.JU receives consulting fees and royalties from NuVasive Medical, Inc., and is a consultant for Masonix, Inc., and SI Bone, Inc. JT receives consulting fees and royalties from NuVasive Medical, Inc. RP is the owner and founder of The Medical Memory, Inc. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Assessing the various anthropogenic pressures imposed on honeybees requires characterizing the patterns and drivers of natural mortality. Using automated lifelong individual monitoring devices, we monitored worker bees in different geographical, seasonal and colony contexts creating a broad range of hive conditions. We measured their life-history traits and notably assessed whether lifespan is influenced by pre-foraging flight experience. Our results show that the age at the first flight and onset of foraging are critical factors that determine, to a large extent, lifespan. Most importantly, our results indicate that a large proportion (40%) of the bees die during pre-foraging stage, and for those surviving, the elapsed time and flight experience between the first flight and the onset of foraging is of paramount importance to maximize the number of days spent foraging. Once in the foraging stage, individuals experience a constant mortality risk of 9% and 36% per hour of foraging and per foraging day, respectively. In conclusion, the pre-foraging stage during which bees perform orientation flights is a critical driver of bee lifespan. We believe these data on the natural mortality risks in honeybee workers will help assess the impact of anthropogenic pressures on bees. Variation in lifespan is generally linked to both intrinsic and extrinsic pressures acting separately or interacting with each other . While iApis mellifera) [Over the past decades, extrinsic pressures have greatly increased for many organisms due to anthropogenic alteration of ecosystems. This is especially true for insect pollinators –4, inclullifera) ,6, whichllifera) –9, but allifera) –13, climllifera) –16 and tllifera) –19. In tHoneybees are social insects that exhibit striking caste-specific differences in longevity. While honeybee queens can live up to 5 years, workers usually only live two to six weeks in the summer and about 20 weeks in the winter ,22. Beeset al. [et al. [Foraging activity provisions the colony with floral resources which are essential for the survival and reproduction of the colony, and is typically preceded by a few days in which bees perform exploratory orientation flights  enablinget al.  and Requ [et al. . TherefoOur aim was to document this particular and critical period of learning flight. For that purpose, we monitored, from emergence to death, the in-and-out activity of 3786 worker honeybees using automated lifelong individual monitoring devices. This was done in a broad range of conditions, consisting of multiple geographical, seasonal and colony contexts, to evaluate diverse life histories related to the well-known behavioural plasticity in honeybees . Variabi2.2.1.To date, two main techniques are available to monitor the in-and-out activity of honeybees at the beehive entrance, the radio-frequency identification device (RFID)  and the A. mellifera mellifera × caucasica strain) but were introduced into three monitored beehives (common 10-frame Dadant–Blatt model with sister queen of A. mellifera mellifera × caucasica strain) placed about 30 km from the source location. Moreover, the three monitored colonies were separated from each other by about 15 km to provide independent foraging landscapes of the tagged bees. In site B, tagged bees that were introduced each month in a single monitored colony came from three source colonies (A. mellifera mellifera × ligustica strain for the source and monitored colonies). Hence bees could be identified based on their colony of origin. These two different scenarios allowed the creation of variability in environmental and colony contexts with potential effects on bee life history.Beyond this inter-regional variability, we further introduced intra-regional variations in bee life-history traits . To do s2.2.To monitor individuals throughout their entire adult life, we collected newly emerged bees. Bees were selected from brood frames containing late-stage pupae, all adult bees were removed from this frame and placed in an incubator at 34°C and 50–70% humidity. Newly emerged bees were then collected after 2–10 h of incubation and tagged. In site A, tags consisted of RFID microchips of 1.0 × 1.6 × 0.5 mm . RFID tags weighed approximately 3 mg, i.e. 3% of an adult honeybee's body mass. This weight is considered low enough to not interfere with the individual life and tasks . In siteVarroa destructor each fall and no visible disease symptoms were observed.Tagged bees (less than 24 h old) were then introduced into colonies equipped with monitoring systems and monitored until death in both sites. All colonies were adequately treated against the mite 2.3.aof function developed in the aof R-package [aof function is a simple statistical procedure, derived from the behavioural change point analysis approach [aof function works at the individual level thus accounting for inter-individual variation to detect, assess and quantify shifts in the temporal pattern of time-activity budgets recorded by individual lifelong monitoring. Indeed, a clear breakpoint in duration, frequency and time of occurrence in the day was found between learning and foraging flights of honeybees, so-called AOF [aof function is freely available through the aof R-package that includes documentation, source code and examples using both simulated and empirical dataset [Of the 2100 and 1686 individually marked bees in sites A and B, respectively, life-history data were successfully recorded for 1867 (89%) and 1507 (89%) bees, i.e. with at least one exit and entrance sequence. This 11% loss  is attributable to the following: loss of barcode/RFID tag prior to leaving the hive, rejection from the host colony by nest-mates or death during the bee's first flight. Bees without at least one exit and entrance sequence were excluded from analyses, thus 3374 bees were available for the analyses. For the analysis of flight activity, exits with durations shorter than 2 s or longer than 180 min were excluded (not considered as flights). We then calculated the age of first exit and entrance sequence , the lifespan (i.e. the age at last exit) and AOF for each bee using the -package ,54. The approach , a well-lled AOF . Thus, A dataset ,54.2.4.2.4.1.To assess which factors were associated with lifespan we used Cox proportional-hazard models for datasets of site A and site B separately (given the differences in study sites and monitoring devices). First, we tested for autocorrelation of the residuals using a Durbin–Watson test on a linear regression of lifespan as a function of AFE, AOF and cohort. Once independence of the residuals had been established, we used the Cox model to investigate the association between bee survival and the predictors. Both models included AFE and AOF as predictors. The site A model included the landscape as a predictor and the replicate (monthly cohort) as a grouping cluster. The site B model included bee weight as a predictor and the replicate as a grouping cluster.To assess the distribution of lifespan frequency, we used Hartigan & Hartigan's dip test for unimodality . The dip2.4.2.We monitored the survivorship of forager honeybees and tested whether forager survivorship followed a type II log-linear curve with a constant mortality rate, as previously observed by Visscher & Dukas . For thiWe further analysed whether pre-foraging traits influence the foraging stage. We used a generalized linear mixed-effect model (GLMM) on the square root of the foraging stage for site A and site B separately (see above). The GLMMs had a Gaussian distribution of errors that considered AFE, AOF, the foraging intensity (mean min spent per day foraging), source colony (site B dataset), weight at emergence (site B dataset) and landscape (site A dataset) as the fixed effects. The cohort was considered as a random effect 3.3.1.n = 1867 bees) and 18.9 ± 10.6 days (n = 1507 bees) in site A and site B, respectively. Lifespan was, therefore, highly variable  and on the cumulative number of flights  did not present a normal distribution of their residuals so they were not considered. However, the cumulative hazard of foraging  could be explained as a function of the foraging tenure in both sites  at site A. The residual distribution of cumulative time spent foraging was close to normality at site B but failed to pass the test  and 8.7% per every hour of foraging at site A .The survivorship of foragers followed a type II log-linear curve. The regressions of the cumulative hazard on the foraging stage (number of days between AOF and death) . The number of pre-foraging flights and the amount of minutes accumulated during the pre-foraging flights were slightly associated with a higher foraging stage at both sites  on bee lifespan and foraging performance. Specifically, the pre-foraging stage is associated with high mortality risks but contributes to shaping the individual's foraging performance and duration.aof procedure [During this pre-foraging stage, we observed a large portion of the worker bees dying; 40% of bees died before the age of 15 days and 50% of bees never reached the foraging stage. In fact, the distribution of bee lifespan was mostly bimodal, with a short-lived population of worker bees that never became foragers, and a second population of bees living longer and becoming foragers. This high mortality of bees occurring before they become foragers was previously noticed   had a small but positive effect on the foraging stage in site B. This goes against the rate-of-living theory, according to which, the higher an organism's metabolism, the shorter its lifespan , as wellFinally, we found that the main mortality factor of foragers was not associated with senescence (type I survivorship curve), or high early mortality (type III survivorship curve), but rather forager bees faced a constant probability of death during their foraging activity (type II curve). This confirms previous work based on the visual recording of 33 individuals , as well5.In conclusion, the pre-foraging stage is highly critical to honeybee survival. During this transition, bees learn their environment (orientation flights) and develop a physiology adapted to flight, but also get exposed to extrinsic mortality risks (predation and weather conditions). This leads to a cost for the colony with a sharp reduction in the forager population. However, this learning stage is essential to accumulate flight experience and improve foraging performance and duration. Another aspect of our study is the remarkable consistency of results between the two datasets obtained in different environmental conditions and years, and using two different automatic monitoring systems, highlighting the robustness of the pre-foraging influence on bee lifespan. Finally, we hope the improved characterization of bee mortality pattern , will inform colony dynamic simulations –74 and f


Rauwolfia serpentina roots. Novel LCMS and HPLC methods were developed for identification and quantification of reserpine and ajmalicine. In vitro enzyme inhibition assays were performed to evaluate anti-cholinesterase, β-site amyloid cleaving enzyme (BACE-1) inhibition and monoamine oxidase-B (MAO-B) inhibition, further analyzed with in silico analysis. Anti-amyloidogenic potential was studied using anti-aggregation studies along with TEM and circular dichroism (CD) analysis. In vitro neuroprotective potential against Aβ toxicity and anti-oxidative stress was demonstrated using PC12 cell cultures. Reserpine is a more potent dual cholinesterase inhibitor than ajmalicine  and 2.8 μM (BuChE)). The anti-aggregation activity of reserpine (68%) was more than ajmalicine (56%). Both compounds demonstrated neuroprotective activity against Aβ42 (92%) and H2O2 (93%) induced toxicity in PC12 cells against controls. Phytocompounds also inhibited MAO-B and BACE-1 enzymes in concentration dependent manner. Molecular docking studies indicated the strong binding of compounds to the catalytic site of targets. This novel study demonstrated that reserpine and ajmalicine as a multi-target directed ligand that have disease modifying potential for amelioration of AD.Alzheimer’s disease (AD) is a multifactorial disorder characterized by exponential loss of memory and cognitive deficit involving several disease modifying targets . The present study explores multi-target directed ligand approach using secondary metabolite reserpine (RES) and ajmalicine (AJM) obtained from  N-methyl-D-aspartate) antagonists (memantine)  and [M − H] ions were identified and matched with literature database and identity of metabolites was established  method for sensitive and rapid detection (or quantification) of RES and AJM was optimized for each, using economic solvents. The methods were validated according to “International Council for Harmonisation (ICH) of technical requirements for pharmaceuticals for human use” for stability, linearity, precision, limit of detection (LOD), and limit of quantification (LOQ) levels  13]..13].2) greater than 0.999 indicates good linearity thus the results showed an excellent correlation exists between the peak area and respective concentration of the compound (RES and AJM). Peak asymmetry greater than ‘1′ indicates peak tailing and less than ‘1′ indicates peak fronting. In the current study, both the methods developed have their peak asymmetry ratio around 1, which is a good measure for peak shape. Relative standard deviation (RSD) determination gives a validation for method precision. The RSD percentages for both the methods are low (RSD < 3%) therefore the RP-HPLC method precision is good. LOD and LOQ are calculated from signal-to-noise (S/N) ratio in multiples of 3 and 10, respectively. As a measure of sensitive method development, it was found that as minimum as 2.8 ppm for RES and 3.7 ppm for AJM could be successfully detected in the sample. Moreover, a least amount of 9.76 ppm for RES and 10.5 ppm for AJM could be successfully quantified in the sample using the developed method.The calibration curve was plotted between absorbance and different concentrations (0.5–3.5 ppm) of test compound and linear regression equation was obtained. A correlation coefficient  and (51% at 100 μg/mL), respectively, as compared to control samples (Aβ42 only). An 85% inhibition of fluorescence was observed when Aβ42 incubated with tannic acid (positive control). The results indicate statistically significant anti-aggregation effect of RES and AJM.Amyloidogenesis involves aggregation of Aβ monomers into oligomers and proto-fibrils that leads to formation of plaques in CNS. The anti-aggregation potential of RES and AJM was analyzed through thioflavin T (ThT) fluorescence assay along with evaluation of red shift in Congo red (CR) dye binding assay. ThT probe gives bright fluorescence upon binding exclusively to Aβ42 proto-fibrils at 480 nm (emission) whereas free ThT quenches at the same excitation wavelength (450 nm), which makes it the most widely used probe against amyloid fibril detection. RES and AJM significantly inhibited the formation of Aβ42 fibrils in concentration dependent manner (11–44 μM), with 69% inhibition of fluorescence by RES (at 44 μM)  as compaThT fluorescence emission spectrum (400–650 nm) showed concentration dependent percentage inhibition of aggregation by test compounds A,C,E supR. serpentina extract incubated with Aβ42 fibrils demonstrated reduced CR red shift close to 510 nm indicating strong anti-aggregation property of test compounds.Congo red dye absorption spectra also support ThT determination of Aβ42 fibril inhibition in spectral shift assay. Upon Aβ42 interaction, CR undergoes a red shift in absorbance from 480nm to 500–550 nm. CR assay showed a typical absorbance at 490 nm whereas when Aβ42 added to CR the absorbance wavelength is shifted to 530 nm B,D,F. REAβ42 undergoes conformational changes during the process of oligomerization from random coil α-helix to structured β-sheets . This chThe secondary structure content  was determined using CD spectra analysis software . During The influence of RES and AJM on fibril formation was studied by analyzing the morphologies of Aβ42 aggregates by TEM. After 48 h of aggregation process, Aβ42 control showed abundant long, branched, and dense fibrils with peptide nucleation, which is characteristic of amyloids whereas RES and AJM shows shorter, loose, and less Aβ42 fibrils along with small oligomers .Further investigation was done to study the cytotoxic effect of Aβ42 and protection against its neurotoxic effect by RES and AJM towards PC12 cells using MTT assay. PC12 cells resemble the phenotype of sympathetic ganglion neurons and are therefore used as model system to study neuroprotection against damages in AD . Aβ42 isR. serpentina extract also imparted 55% protection against Aβ42 cytotoxicity at 100 μg/mL which was comparable to the protection given by homotaurine (58%), used as positive control in the study  and AJM (67%) at equimolar concentration of Aβ42 (40 μM). he study .2O2 induced oxidative stress towards PC12 cells was evaluated by MTT assay. 2O2 cytotoxicity was observed in PC12 cells after pre-incubation with R. serpentina extract and its indole alkaloids RES and AJM. At extract concentration of 100 µg/mL highest viability was observed (86.4%) as compared to 31% viability in negative control group . While PC12 cells had statistically significant results, surviving to 93% with RES (40 μM) treatment and to 89% with AJM (40 μM) treatment. The positive control glutathione (GSH) used in the study imparted 91% protection against H2O2 induced oxidative stress.Oxidative stress and generation of ROS have been shown to induce lipid peroxidation in cell membrane, which leads to cellular damage and subsequent cell death. Thus, the protective effect of RES and AJM against HR. serpentina extract in concentration dependent manner . RES at 50 µM concentration gave highest relative inhibition (96%), which is slightly higher than positive control donepezil inhibition percentage of AChE (94%). AJM showed 90% inhibition at 50 µM concentration. R. serpentina extract showed IC50 value of 14 µg/mL and 22 µg/mL for AChE and BuChE, respectively.Cholinergic neurotransmission is severely affected in AD, thus, to prolong neurotransmission a suitable drug compound must have anti-cholinesterase effect. RES and AJM achieved statistically significant dual anticholinesterase inhibition along with t manner . R. serp50 values along with the selectivity of the inhibition by respective compounds for AChE and BuChE. Selectivity is the measure of inhibition for a particular enzyme over the other. In later stages of AD, BuChE levels increases in brain. BuChE catalyzes acetylcholine as it’s substrate. Therefore, the BuChE inhibition becomes important along with AChE inhibition. Although RES and AJM have selectivity for AChE, the results suggest almost equivalent inhibition for BuChE.The BACE-1 enzyme inhibition has been shown to reduce the amount of pathogenic Aβ42 formation, which makes it one of the important targets for AD . The resMAO-B is responsible for production of toxic oxygenated free radicals, which is responsible for neurodegeneration in AD. RES and AJM significantly inhibited the MAO-B enzyme in a concentration dependent manner, at 10 μM, both the tested compounds gave comparable inhibition for MAO-B enzyme, 89% (40 μM) inhibition  was showMolecular docking revealed interaction of RES and AJM with catalytic site residues of studied AD targets  giving insight of the mechanism of inhibition of respective enzymes and peptide that correlate with in vitro studies. Molecular docking analysis  clearly Pharmacokinetics analysis for ADMET, of identified phytoconstituents showed that AJM proves to be a more ideal drug molecule as it obeys Lipinski rule of five along with suitable profile to cross the blood brain barrier (BBB) . While RR. serpentina  for their MTDL properties.Pathophysiology of AD is attributed to diverse, complex factors such as cognitive decline, memory loss, and extracellular plaque formation along with neurofibrillary tangles. From the inception of amyloid cascade hypothesis to its reappraisal, it is considered to be the primary cause of this form of senile dementia ,18. WithR. serpentina is abundant in many indole alkaloids such as reserpine, serpentine, rauwolfine, sarpagine, ajmaline, yohimbine, and ajmalicine [R. serpentina by UPLC-UV [Naturally occurring alkaloids have long been used in therapeutic strategies against AD, such as galanthamine and huperzineA that are used in providing symptomatic relief to AD patients . R. serpmalicine . In the  UPLC-UV ,20. RES  UPLC-UV ,22. The R. serpentina. The exact mechanism of Aβ42 oligomerization to insoluble plaque formation is not clear but the secondary structure conformation studies suggest that β-sheet content is increased in Aβ42 oligomerization [Aβ42 is a 4KDa fragment of mature APP, which self-aggregate to form toxic Aβ42 oligomers and finally proto-fibrils that are considered as a main pathological factor implicated in AD . Compounrization . CD analrization . The indIn advanced cases of AD, BuChE secretion increases responsible for hydrolysis of AChE as it’s substrate. This accelerates the cholinergic decline process further. RES and AJM counteract the decrease in neurotransmitter levels (AChE) by significantly inhibiting BuChE in concentration dependent manner evident from in vitro enzyme inhibition assay. The molecular interactions showed strong binding energies of RES and AJM with BuChE, suggesting stable protein–ligand complex. Tacrine (known inhibitor) had lower docking score then RES and AJM. RES forms strong hydrogen bond with BuChE CAS residue  and occupies the PAS by interacting with a residue (Asn68) which lies near PAS (Asp70). Whereas AJM binds directly to CAS and PAS residues . Thus, RES and AJM could prove to be better anti-cholinesterase drug ligands for therapeutics towards AD.BACE-1 enzyme structure is found to exist in three different conformations based on a flap like secondary structure, which covers the active site of the enzyme. The BACE-1 structure (PDB ID 4D8C) used in the current study is closed flap structure, which is the dominant form exists upon binding to its substrate. Molecular docking analysis showed that the indole ring of RES interacts with the main catalytic site residues of the BACE-1 enzyme. The indole ring of RES acts as a hydrogen bond donor for Asp32, forming double hydrogen bond with catalytic site residue of BACE-1 while the isoprene tail of RES forms hydrophobic interaction in the catalytic gorge of BACE-1. On the other hand, AJM binds more strongly to BACE-1 with all hydrophobic interactions as it gave a high binding score with BACE-1. Probable explanation could be the stability AJM provides to the hydrophobic catalytic gorge of BACE-1 due to its compact size and orientation then RES. These molecular interactions are complemented with in vitro results showing statistically significant inhibition of BACE-1 enzyme, where observed potency of AJM is more than RES.MAO-B enzyme plays an important role in generation of free radicals thus imparting oxidative stress at cellular level. RES and AJM inhibited MAO-B enzyme in concentration dependent manner comparable to positive control (selegiline). The in vitro results are complimented by molecular docking analysis where RES was observed to interact with the key catalytic site residues of MAO-B while AJM occupies the catalytic site by interacting with other residues there. RES interacted with Tyr326 and Ile199 by hydrogen bonding. AJM gave high binding score then RES probably because AJM formed more strong hydrogen bonds with residues surrounding catalytic site covering the main residues involved in hydrolysis of the substrate. The ADMET profile of AJM showed promising results of being able to act as ideal drug molecule with BBB permeability while RES structure could be utilized to develop active MTDL drug candidate with small molecular size.18 Silica Column, Agilent 6520 QTOF instrument, acetylcholinesterase from electric eel, butyrylcholinesterase from equine serum, amyloid beta protein (Link biotech), thioflavin T, Congo red dye, reserpine, and ajmalicine were purchased from Sigma Aldrich, India. MAO-B inhibition kit by Biovision. BACE-1 activity assay kit by Sigma Aldrich. RPMI 1640 medium, FBS, PBS, and other tissue culture grade chemicals were purchased from Himedia, India. PC12 (rat pheochromocytoma) cell line was obtained from NCCS, Pune, India. All other reagents used were of analytical grade. All preparations used in tissue culture experiments were filtered through 0.45 µm Axiva 25 mm CA filter. Ethical approval was obtained from GGS Indrapratha University for all the cell culture work done in proper confined facilities.LC-MS grade water, methanol, acetonitrile, formic acid, Zorbex UPLC CR. serpentina root sample  was infused in 1:1 ratio of ethanol and freshly boiled distilled (de-ionized) water. The infusion was kept in shaker at 40 °C for 48 hrs and filtered. The supernatant was freeze dried (lyophilized) in lyophilizer for 24 h. The yield obtained was calculated and freeze-dried aliquots were sealed properly to avoid any moisture inside the vials and stored in deep freezer (−20 °C) till further use.The NISCAIR authenticated dried R. serpentina hydro-alcoholic root extract was analyzed using various mobile phase combinations for best possible resolution of chromatographic peaks. The LCMS run condition was tested four times for a standardized protocol development to estimate secondary metabolites profile  run under same chromatographic conditions.RES and AJM present in 44 µM of Aβ42 was dissolved in milliQ water and mixed with various concentrations of compounds to study. Aβ42 alone sample was used as control. Samples were incubated at 37 °C at 260 rpm for 48 h in the shaker incubator. Samples were taken out at their stipulated time interval and various experimentations  were performed to study the inhibition of amyloid fibril formation by the compounds under study.Aβ42 samples were incubated with or without compounds at different concentrations, after 48 h of fibril formation samples from each experimental set were withdrawn and mixed with ThT in opaque micro titer plate. The reactions were then incubated in dark at 37 °C for 30 min for ThT probe to bind the formed amyloid fibrils. The ThT was excited at 450 nm and spectra were recorded from 400 to 650 nm . All meaCongo red (200 μM) was dissolved in a 50 mM phosphate buffer (pH 7.4) consisting of 50 mM NaCl and 10% ethanol and filtered through 0.45 µm CA membrane filter. The Aβ42 concentration was fixed at 10 µM in the reaction mixture. CR and protein were mixed at a molar ratio of 1:1 for various experimental setups and kept for 30 min incubation at room temperature. The absorbance spectra (400–650 nm) of the samples were recorded with a UV-visible spectrophotometer . All expSecondary structure of Aβ42 peptides were recorded as far UV circular dichroism spectra using Chirascan  instrument and software. 22 μM Aβ42 samples  were mixed well ensuring a homogenous suspension and transferred to quartz cell for CD spectra scanning. CD spectra was recorded from 200–260 nm at RT using a bandwidth of 1.0 nm, times per point 0.5 sec, a step interval of 1 nm, and 10 mm path length. Five scans of each sample were measured and averaged. Averaged control buffer scans were subtracted from the sample spectra. The results of CD measurement were plotted as graph between measured ellipticity (millidegree) and wavelength (nm). The secondary structure contents  of the Aβ42 peptide samples were estimated from the CD spectra using the CD Pro software .5 μL Aβ42 prepared fibril samples (22 μM) Aβ42 alone and Aβ42 with RES and AJM) were loaded on carbon stabilized, formvar coated grid for 40 sec. Ashless filter paper was used to wipe out extra sample from the edges of the grid. Loaded grid was negatively stained with 1% uranyl acetate for 20 sec and air-dried. Grid was then examined and photographed on JEOL 2100F transmission electron microscope.5 cells per well. Pre-incubation was given with homotaurine , RES , AJM , and R. serpentina extract . Cytotoxicity with preformed Aβ42fibrils aged 48 h at 40 μM  was induced to compounds pre-acclimatized PC12 cells for 24 h. MTT solution was added to the wells and incubated in dark, for the live cells to convert the tetrazolium salt in purple formazan crystals. The formazan crystals were dissolved using DMSO and absorbance was measured at 570 nm. Relative survival percentage of PC12 cells was calculated compared to control (PC12 cells only). All measurements were performed in triplicates and all experiments were repeated thrice individually.Undifferentiated PC12 cell line used as an AD model to study neurotoxicity in the current study, was maintained as monolayer adherent cultures in RPMI 1640 medium supplemented with 5% FBS and 1% antibiotic . Cells w50 200 μL) and concentrations of glutathione (positive control), RES, AJM , and R. serpentina extract  were used to analyze neuroprotective effects against oxidative damage through MTT assay. Cell viability was determined through trypan blue dye exclusion assay and cells were seeded to 96 well tissue culture plate at a 5 × 105 cells/well concentration. Pre-incubation with compounds were given to PC12 cells prior to inducing H2O2 cytotoxicity for 18 h. MTT was added to the well at 5 mg/mL concentration and incubated in the dark for enzymatic reaction by live cells for 2 h. DMSO was added to well to dissolve formazan crystals and absorbance was recorded at 570 nm. PC12 cells relative survival was calculated compared to control (PC12 cells only).Oxidative stress was induced to cultured PC12 cells using hydrogen peroxide (ICR. serpentina extract [Ellman assay was performed to assess the inhibition of the enzyme AChE (EC 3.1.1.7) and BuChE (EC 3.1.1.8) by donepezil (positive control), RES, AJM, and  extract . Five miBACE-1 (EC 3.4.23.46) activity inhibition was assayed using FRET (fluorescence resonance energy transfer) kit . With all reaction components at RT, 50 μM substrate was added to 75 μL assay buffer containing RES and AJM  in the microtiter plate. Reaction was initiated by the addition of 0.03 U BACE-1 enzyme in the well and zero minute fluorescence was recorded . After 2 h incubation in dark at 37 °C, reaction fluorescence was recorded every one hour for next three hours. Relative fluorescence inhibition was calculated from zero minute fluorescence reading as control. Three individual experiments were performed.MAO-B (EC 1.4.3.4) activity inhibition was assayed using Biovision inhibition screening kit (fluorometric). With all reaction components at room temperature, 0.2 U enzyme was pre-incubated with compounds under study and incubated for 10 min. Working substrate was prepared fresh by adding developer and oxired probe with assay buffer according to kit instructions. Substrate was added to the reaction and incubated for 10 min; zero-minute fluorescence was recorded  to serve as control for relative fluorescence inhibition calculation. All measurements were performed in triplicates and all experiments were repeated thrice individually.Molecular interactions of RES and AJM with selected AD targets  were studied using Autodock 4.2 . Respectwww.niper.gov.in/pi_dev_tools/DruLiToWeb/DruLiTo_index.html) to study ideal pharmacokinetics profile of RES and AJM for drug development. Two filters were used for screening; Lipinski rule and blood brain barrier. Lipinski rule states that for an ideal drug molecule should weigh below 500 g/mol, hydrogen bond donor should be less than or equal to 5 and number of hydrogen bond acceptor should be less than or equal to 10 along with a partition coefficient of 5 or less. Such compounds would pass the BBB if the number of hydrogen bonds present is below eight and there should not be any acidic group present in the molecule. Total polar surface area (TPSA) showed bioavailability of the drug molecule as per Veber’s rule the TPSA less than or equal to 140Å indicates good oral bioavailability.ADMET analysis was carried out using Drulito software (n = 4 runs were used. Statistical analysis was performed using one-way ANOVA for comparing independent means, followed by Bonferroni post test for multiple comparisons of experimental setups with control groups. Level of significance was determined as p ≤ 0.05 and p < 0.01, until otherwise stated.Three independent experiments and each experiment with three replicates, were used in each experimental setup to present data as mean ± standard error mean but for LCMS data In conclusion, the novel study showed that RES and AJM could act as multi-target directed ligands and can be used to develop into novel compound that can be used against several targets Aβ42, AChE, BuChE, MAO-B, BACE-1, and ROS that are implicated in AD and thus, help in alleviation of symptoms and having disease modifying effect in AD.


PLB phosphorylation at residue Ser16 (pSer16) enhances SERCA activity in the heart via an unknown structural mechanism. Here, we report a fully atomistic model of SERCA bound to phosphorylated PLB and study its structural dynamics on the microsecond time scale using all-atom molecular dynamics simulations in an explicit lipid bilayer and water environment. The unstructured N-terminal phosphorylation domain of PLB samples different orientations and covers a broad area of the cytosolic domain of SERCA but forms a stable complex mediated by pSer16 interactions with a binding site formed by SERCA residues Arg324/Lys328. PLB phosphorylation does not affect the interaction between the transmembrane regions of the two proteins; however, pSer16 stabilizes a disordered structure of the N-terminal phosphorylation domain that releases key inhibitory contacts between SERCA and PLB. We found that PLB phosphorylation is sufficient to guide the structural transitions of the cytosolic headpiece that are required to produce a competent structure of SERCA. We conclude that PLB phosphorylation serves as an allosteric molecular switch that releases inhibitory contacts and strings together the catalytic elements required for SERCA activation. This atomistic model represents a vivid atomic-resolution visualization of SERCA bound to phosphorylated PLB and provides previously inaccessible insights into the structural mechanism by which PLB phosphorylation releases SERCA inhibition in the heart.Sarcoplasmic reticulum Ca SERCA uf the SR . PLB inhactility .T state in favor of the disordered, non-inhibitory R state [R state, PLB physically separates from SERCA to relieve inhibition [T-to-R state structural shift of PLB induced by phosphorylation relieves inhibition while preserving the SERCA–PLB heterodimer [PLB phosphorylation at residue Ser16 (pSer16) induces an order-to-disorder structural transition of the cytosolic domain of PLB, destabilizing a inhibitory ordered  R state ,6. Two mhibition ,8 whereaerodimer ,10. The erodimer ,12,13,14erodimer . More reR state is intrinsically disordered, so the high-resolution structure of the of phosphorylated PLB bound to SERCA has remained inaccessible to experiments. Based on structures of the inhibitory SERCA–PLB complex and the solution structure of PLB, here we report a fully atomistic model of SERCA bound to PLB phosphorylated at residue Ser16 (SERCA–pPLB) and study the structural dynamics using microsecond-long all-atom molecular dynamics (MD) simulations in an explicit lipid bilayer and water environment. Here, we studied the complex under Ca2+-free conditions to study the direct effects of PLB phosphorylation alone on the functional dynamics of SERCA. The result is a realistic atomic-resolution model of the molecular interactions and mechanism by which PLB phosphorylation releases SERCA inhibition in the heart.Despite these advances in the field, there are major gaps in our understanding of SERCA regulation by phosphorylation of PLB: (i) What is the atomistic structure of the regulatory complex between phosphorylated PLB and SERCA? (ii) Does SERCA have a well-defined binding site for the disordered cytosolic domain of phosphorylated PLB? (iii) What is the mechanism for control of SERCA structural dynamics by PLB phosphorylation? Answering these questions in high spatiotemporal resolution is much needed in the field to understand SERCA regulation in terms of intermolecular interactions and activities. The structure of SERCA bound to the TM domain of PLB was solved recently , but theα atoms of the phosphorylation domain showed that this region exhibits high flexibility in the bound complex  drift in the RMSD ; this sm0 ns drifThe structure of the SERCA–pPLB complex shows that the disordered phosphorylation domain of PLB interacts with the flexible cytosolic headpiece of SERCA. However, the simulations revealed that the N-terminal phosphorylation domain of PLB binds unspecifically to SERCA, thus contradicting previous studies suggesting that PLB phosphorylation is required to produce a relatively well-defined structure of the complex that is non-inhibitory . AlthougThe analysis showed that SERCA residues Arg324 and Lys328, as well as PLB residue pSer16 have substantially higher occupancy values (OF > 0.75) compared to other residues located at the SERCA–pPLB interface . Based o2+ may occur without changes in the binding mode of PLB in the canonical site [The crystal structure of the inhibitory SERCA–PLB complex, the TM domain of PLB binds to a canonical site formed between TM helices M2, M6, and M9 , and precal site . To detecal site . TherefoSite-directed mutagenesis, X-ray crystallography, and molecular simulations have shown that residue Asn34 of PLB plays a central role in SERCA inhibition ,29,30. O2+, the headpiece in SERCA–pPLB populates structures that are distinct from those populated by the unphosphorylated SERCA–PLB complex. More importantly, this structural shift leads to a reduction in the distance between A-domain and the loop Nβ5-β6, in agreement with the notion that PLB phosphorylation stabilizes a more compact structure of SERCA’s headpiece [The changes in the hydrogen bonding pattern of Asn34–Gly801 are consistent with release of SERCA inhibition and suggest that the structural ensemble of SERCA–pPLB represents the non-inhibitory state of the complex. Therefore, we asked whether disruption of the inhibitory contacts by PLB phosphorylation induces functional structural changes in the cytosolic headpiece that are associated with SERCA activation. To address this question, we first measured Cα-Cα distances between SERCA residues Arg139 (A-domain) and Asp426 (loop Nβ5-β6) a. We choeadpiece .2+/nucleotide-bound state of the pump . The absence of a Ca2+-bound-like orientation of the A-domain in the trajectories of SERCA–pPLB is consistent with binding of Ca2+ ions and correct charge neutralization of the transport sites is required for the formation of a competent orientation of this domain [We charted changes in the rotation and displacement of the N-, P-, and A-domains to gain further insight into the functional transitions of SERCA that occur in response to PLB phosphorylation. We found that both N- and P-domains, the catalytic elements of SERCA, occupy the structural space defined by the Cathe pump a,b. Thispump  obtained using solution NMR  bilayer. The stereochemical quality of the initial models was verified with PROCHECK [We used the structure of the full-length structure of the B: 2lpf, ) as the , [4kyt, ). Upon aPROCHECK ,45, show2+-free conditions. We modeled residue pSer16 of PLB, and SERCA residues Glu309 and Asp800 as unprotonated and residues Glu771 and Glu908 as protonated. In addition, we adjusted the pKa of other ionizable residues to a pH value of approximately 7.2 using PROPKA [+ and Cl- ions were added to neutralize the system and to produce a KCl concentration of ~100 mM.We used the atomistic model of the full-length PLB bound to SERCA generated here study to simulate the SERCA‒pPLB complex under Cag PROPKA ,47. The g PROPKA  as a ref−1 Å−2. We performed 8 independent MD simulations of SERCA–pPLB for a total simulation time of 22.7 µs.MD simulations of all systems were performed with NAMD  using pedssp [select built-in tool implemented in GROMACS [HydrogenBondAnalysis library of MDAnalysis. We mapped MD structures onto the rotation-displacement map created using 74 crystal structures of SERCA to determine the effects of PLB phosphorylation on the structural dynamics of the cytosolic domain [2c9m [2c9m [draw_rotation_axis.py script running on PyMOL (http://pymolwiki.org/index.php/RotationAxis). The programs PyMOL and VMD [We calculated the backbone root mean square deviation (RMSD) for SERCA, cytosolic, and TM domains, and PLB, N-terminal phosphorylation, and TM domains. Changes in the secondary structure of PLB was computed employing the program dssp  includeddssp . We also GROMACS . The prec domain . All SERin [2c9m  using th9m [2c9m  as refer and VMD  were use