In microbial communities, viruses compete with each other for host cells to infect. As a consequence of competition for hosts, viruses evolve inhibitory mechanisms to suppress their competitors. One such mechanism is superinfection exclusion, in which a preexisting viral infection prevents a secondary infection. The bacteriophage ΦX174 exhibits a potential superinfection inhibition mechanism (in which secondary infections are either blocked or resisted) known as the reduction effect. In this auto-inhibitory phenomenon, a plasmid containing a fragment of the ΦX174 genome confers resistance to infection among cells that were once permissive to ΦX174. Taking advantage of this plasmid system, we examine the inhibitory properties of the ΦX174 reduction effect on a range of wild ΦX174-like phages. We then assess how closely the reduction effect in the plasmid system mimics natural superinfection inhibition by carrying out phage–phage competitions in continuous culture, and we evaluate whether the overall competitive advantage can be predicted by phage fitness or by a combination of fitness and reduction effect inhibition. Our results show that viral fitness often correctly predicts the winner. However, a phage’s reduction sequence also provides an advantage to the phage in some cases, modulating phage–phage competition and allowing for persistence where competitive exclusion was expected. These findings provide strong evidence for more complex dynamics than were previously thought, in which the reduction effect may inhibit fast-growing viruses, thereby helping to facilitate coexistence. Inhibitory mechanisms have a large impact on virus population dynamics ,2. The iSuperinfection exclusion, a phenomenon in which a preexisting viral infection prevents a secondary viral infection, requires the dedication of resources to defense that often comes at a fitness cost. However, in the presence of competition, the advantage of defense can be substantial. This auto-inhibitory phenomenon has evolved independently in a wide variety of prokaryotic and eukaryotic viruses ,9,10,11.Microviridae family and has been used widely as a model system for experimental evolution [E. coli) surface, followed by the injection of viral DNA, replication inside the cell, and cell lysis to release the phage progeny. In virus replication, the phage’s single-stranded genome is first converted into a double-stranded circular replicative form  through the use of 13 host proteins [rep protein. Thirdly, single-stranded DNA (ssDNA) is synthesized and packaged into viral procapsids, necessitating viral proteins gpA and gpC. After the assembly of the capsid and insertion of the new viral genome, the new phages are released during cell lysis.The lytic bacteriophage ΦX174 is perhaps the best-known virus in the volution ,19. ΦX17volution ,21. The proteins ,21. NextThe viral pilot protein gpH is of particular interest, as it has several functions during the viral lifecycle ,23,24,25The ΦX174 reduction effect is an auto-inhibitory phenomenon specific to ΦX174 and its related phages. In this phenomenon, which was first described by van der Avoort et al. in 1982 [Microviridae phages is possible [Further work on the reduction effect has assessed the ability of ΦX174 to overcome inhibition by the reduction plasmid, indicating that recovery was possible via as little as a single point mutation in the phage genome within the H gene . Additiopossible , but notHere, we expanded this work to examine the reduction effect in wild ΦX174-like phages and to better understand the role that the reduction effect and superinfection inhibition may play to confer a selective advantage in microbial competition. First, we quantified how inhibitory the ΦX174 reduction effect is in wild ΦX174-like phages. Next, we assessed the reciprocal inhibitory capacity of various members of the ΦX174-like clade by determining the levels of inhibition that the reduction sequences from four wild ΦX174-like phages exerted upon each other. Finally, we sought to investigate the possible role of the reduction effect on the persistence of multiple viral species in microbial communities. To accomplish this aim, we measured the population frequencies of pairs of ΦX174-like phages that were grown together over time in continuous culture, and compared them to expectations based upon each phage’s fitness and the inhibitory strength of its reduction sequence. Our results revealed that some pairs of phage–phage competitions could be predicted based upon low MOI fitness. In others, however, the interplay between low MOI fitness and the reduction effect is more likely to govern the outcome, leading to coexistence rather than exclusion. Thus, the reduction effect may serve to inhibit superinfection by an identical or closely related phage, conferring a competitive advantage that maintains the coexistence of species with lower growth rates. The broader implication of this work highlights the complex dynamics governing bacteriophage competition in their natural environments.All isolates for the wild phage assays, including our laboratory strain of ΦX174, have been previously sequenced and described . AnnotatWe also engineered two phages, each with two missense mutations in the reduction sequence portion of the H gene, to create constructs of ΦX174 with the H reduction sequence of either WA11 (ΦXwa11) or NC16 (ΦXnc16). ΦXwa11 contained the H mutations I297F and I301V. ΦXnc16 contained the H mutations H275L and H299Y. Engineered phages were created by Golden Gate assembly of the phage genomes and transformation into host cells according to the methods described in Faber et al. .2. For solid plates, 15 g/L Bacto agar was added to ΦLB, and 7 g/L Bacto agar made the semisolid top agar.For liquid fitness assays and chemostat assays, a modified Luria–Bertani medium called ΦLB was used. ΦLB consists of 5 g/L yeast extract, 10 g/L tryptone, and 10 g/L NaCl, supplemented with 2mM CaClThe ΦX174 reduction effect plasmid described by van der Avoort et al. [Blind plaque assays were conducted initially to identify susceptibility to the reduction effect. Wild phage samples were coded and plated in parallel in cells containing either the control plasmid pACYC177 or the reduction plasmid pΦX174rs, and incubated at 37 °C for 4 h, after which plates were scored for visible plaques. Five replicates were conducted, and samples were decoded. Samples that did not show plaques in any replicates were deemed susceptible to the reduction effect. If five of five replicates showed plaques, the phage strain was considered insusceptible to the reduction effect. If at least one of five replicates showed plaques, the strain was considered semi-susceptible .To further quantify the level of inhibition of the reduction effect on different phages, liquid culture fitness assays were conducted. Results from the liquid assays indicated that the plaque assays were not always a good indication of the degree of inhibition. Due to this inconsistency, along with the binary nature of the plaque assay, this approach was abandoned in favor of liquid culture fitness assays.E. coli C cells containing the appropriate plasmid were grown in a 125 mL flask containing 10 mL ΦLB in a shaking water bath for 1 h at 37 °C to a concentration of ~2 × 108 cfu/mL. Phages were then added so that the flask contained between 103 and 104 phage/mL. The initial number of phages was chosen to assure that the final density of phages was not allowed to exceed cell density and keep the multiplicity of infection (MOI) < 1. Phages were grown for 40 min with shaking. Final time samples  were taken onto chloroform to stop bacterial growth. Samples from time points 0 and 40 min were titered, and doublings per hour were determined by the following formula:Fitness assays were designed to measure phage fitness as the number of phage doublings per hour in a homogeneous liquid culture. This low-density assay for growth rate has been described previously . High-deE. coli C, a fully permissive bacterial strain. We measured the inhibitory capacity of each wild phage’s reduction sequence as the percent reduction in fitness between the permissive host (pACYC177) and the reduction sequence assayed:Assays were conducted with both the control plasmid (pACYC177) and the ΦX174 reduction plasmid-containing (pΦX174rs) cells in triplicate unless otherwise noted. A number of phage types were assayed, including ΦX174, the related laboratory isolate S13, and 15 wild phage strains . For theE. coli C cells. Cells and media then flowed into the second test tube, which contained the phage population. From there, media, cells, and phage were pulled out using an aquarium pump into a waste container to maintain a continuous population volume [To examine the competitive fitness of each phage in a continuous culture, a two-stage chemostat was used . ΦLB medn volume ,33. Both8 cfu/mL) into the first tube. The chemostat was allowed to run for half an hour before phages were added to the second tube. Equal parts of both competing phages (~1 × 104 of each phage) were then added to the second tube. Samples were taken from the chemostat at hours 2, 4, 6, and, in some cases, 24. Samples were filtered at 0.2 µm to remove cells. At the end of each chemostat run, samples from the unused media and the cell tube were each taken and plated on ΦLB agarose plates to verify that contamination had not occurred. Twenty-four-hour samples were not available for all competitions, and due to the likelihood of evolution occurring by 24 h [Competition assays were conducted for six separate pairings of phages: ΦX174 vs. WA11, ΦX174 vs. NC16, WA11 vs. NC16, ΦX174 vs. ΦXwa11, ΦX174 vs. ΦXnc16, and ΦXwa11 vs. ΦXnc16. Each set of chemostats was run in triplicate for 6–24 h each. The chemostat was initially inoculated with 500 µL of cells growing at log phase . The MiSeq reads were mapped to each of the bases differing between the competing phages. The percentage of each unique base was averaged at each time point to give an estimate of the relative proportion of each phage. For any given time point, there was less than a 5% difference between the estimates of the phage proportions.P, of the dominant phage for each chemostat  was based on the growth data of each phage grown without the presence of the reduction sequence and was calculated as follows: t+1 = Pt × 2xP, where x is the control fitness, and t is time in hours. These predictions were based on the uninhibited growth rate only, and did not take competitive ability into account. The proportion of each competing phage in the population was then determined by calculating the percentage of each phage of the total.The predicted percentage, Sequences were aligned via the ClustalW method and divergences were calculated using the MegAlign program in the Lasergene suite .The sequences of the phage genomes and plasmid inserts were ascertained before use in fitness assays. Phage genomes were PCR amplified in overlapping halves and reactions were cleaned up on Qiaquick columns . Sequences of the PCR products were visualized on an ABI capillary sequencer using BigDye terminator chemistry  and assembled with the SeqMan program of Lasergene. Ambiguities and gaps were re-sequenced, using alternative primers when needed. Plasmid insert sequences were obtained by terminator labeling using forward and reverse primers to the plasmids.Phage populations from the chemostat competition assays were sequenced using MiSeq Illumina sequencing. To prepare the samples for sequencing, two rounds of PCR were run. The first round amplified an approximately 500-base segment of the C-terminal end of the H gene and added the adaptor sequence to each end necessary for the second round of PCR. The primers for the first round of PCR included 0–2 bases inserted as spacers between the adaptor and H portions. First round PCRs were column purified and products were checked by electrophoresis. The second round of PCR reactions involved further amplifying the selected genome fragment as well as attaching a unique Illumina adaptor to the ends of each sample. Samples were again cleaned and checked for concentration by electrophoresis. Illumina sequencing was performed in the IBEST Genomic Resources Core at the University of Idaho. All primers used for amplifying and sequencing phage DNA are given in To obtain a quantitative assessment of susceptibility to the reduction effect, liquid culture fitness assays were used to measure the phage growth rate (doublings per hour). Results from the fitness of phages in cells containing the control plasmid, pACYC177, were compared to those containing the reduction plasmid, pΦX174rs. A wide range of inhibition was observed .All bacteriophage strains exhibited reduction, i.e., a loss in fitness as measured in doublings/hour, when grown in the presence of the plasmid pΦX174rs. Assays of the strains grown in the presence of the reduction sequence plasmid showed an average fitness of 7.66 doublings/hour, compared to the average fitness of the strains with the control of 17.45 doublings/hour, representing a 56% average reduction in fitness. To visualize the fitness reduction as a function of a phage’s control fitness, To determine if there is a relationship between reduction sequence homology and fitness loss on the reduction sequence plasmid, sequence divergence of the wild phage strains from the ΦX174 reduction sequence was compared to fitness change using correlation analysis. Here, we show the average fitness change between control and the ΦX174 reduction sequence for each phage versus the percent divergence from the 495-base ΦX174 reduction sequence , Table 1Work by van der Avoort et al.  found anThe WA11 reduction sequence (pWA11rs), for example, almost completely inhibits all assayed phages (between 92.16% and 103.87% inhibition), while the NC16 reduction sequence (pNC16rs) inhibits virtually none (between −6.99% and 12.46% inhibition). The remaining reduction sequences  lie somewhere in between and largely depend on the infecting phage . The NC1As no relationship between sequence divergence and fitness loss was observed, we further explored a hypothesis in which specific mutations allow adaptation to inhibition. To determine if the inhibitory power of a reduction sequence plasmid is related to its specific genetic sequence, all five wild phages’ reduction sequences were aligned and analyzed . We obseTwo of these amino acid changes in protein H were unique to the strongly inhibitory plasmid pWA11rs: I297F and I301V. Being the only differences from ΦX174rs within this region, they are therefore associated with an increased inhibitory capacity. Another two protein H amino acid changes were unique to NC16, the particularly poor inhibitor: H275L and H299Y. Again, these two mutations are the only changes from the ΦX174rs within this region and are therefore associated with a diminished inhibitory capacity. Another H residue change, H275Q, is seen in our moderately inhibitory pID34rs plasmid and occurs at the same site as one of the pNC16rs mutations.In order to link the inhibition findings presented above with the ecological context of phage–phage competition, competition assays in chemostats were conducted using these phages: WA11 (strong inhibitor), NC16 (weak inhibitor), and ΦX174 (moderate inhibitor). Additionally, two engineered phages were used. Each engineered phage was identical to ΦX174, but with two genomic missense mutations to match the reduction sequence of either WA11 (ΦXwa11) or NC16 (ΦXnc16) . The resThe fitness values of the phages when grown alone were as follows: ΦX174, 25 doublings/h; WA11, 18.5 doublings/h; NC16, 15 doublings/h; ΦXwa11, 17 doublings/h; and ΦXnc16, 16.5 doublings/h . The twoPredictions for the outcome of each competition were based on two factors: low MOI fitness and the inhibitory properties of each phage’s reduction sequence . Based oAs expected based on both fitness and reduction sequence inhibition, when ΦX174 or WA11 were competed against NC16, the majority (>90%) of the resulting phages were either ΦX174 or WA11, respectively , Table 3When ΦX174 and ΦXwa11 were each competed against ΦXnc16, a modest majority (>60%) of the resulting phages were either ΦX174 or ΦXwa11, respectively, as predicted based on both fitness and reduction sequence inhibition , Table 3The ΦX174 reduction sequence was first observed by van der Avoort et al. in 1982 [We first quantified the fitness of wild ΦX174-like phages grown alone and in the presence of the ΦX174 reduction effect, and in each case we saw inhibition with the ΦX174 reduction sequence plasmid . A moderSequence divergence from ΦX174 was next examined to determine if reduction sequence homology plays a role in fitness loss on the reduction sequence plasmid . No relaWe then performed competition assays in continuous culture to assess the outcomes of competition between these phages. We chose the phages for pairwise competitions based upon the inhibitory properties of the wild phage reduction sequences along with each phage’s susceptibility to these reduction sequences. We identified certain trends : NC16 isWe expected ΦX174 as well as WA11 to dominate a competition with NC16, and this outcome was observed . With ΦXSimilarly, NC16 was nearly eliminated when competed against WA11, despite WA11 having only a slight fitness advantage, which is consistent with the strong inhibitory capacity of pWA11rs . Yet, inWhen the reduction effect plasmids are used in a lab-based system to mimic the primary infection of permissive cells, any productive phage infections are functionally secondary infections. Thus, we here interpret the fitness results as the response of a secondary infector to the presence of another genome at the time of infection. In this way, our lab-based system mimics the superinfection that is a product of high-density interaction.Indeed, previous work with the reduction sequence has hypothesized that the ecological function of the reduction effect is that of superinfection exclusion ,29. UndeIn summary, the outcomes of the competitions shown here reflect both the phages’ fitnesses without competition and their inhibitory capacities. Cases where the two predictions based on low MOI fitness and reduction sequence inhibition diverged tended to result in an outcome somewhere in the middle. Although we clearly demonstrated that fitness at low MOI does not necessarily predict phage performance at high MOI, more research is needed to understand the complex interactions that occur during high MOI competition. For example, an additional factor that may influence competition is the formation of chimeric phages. If two phages are replicating within the same host cell, it is possible that structural proteins from the different phages may incorporate into either phage during capsid assembly, resulting in phages whose genomes do not fully indicate their resulting protein structure. While these other proteins would not be passed along during the next infection cycle, the inclusion of alternate proteins potentially plays a role during capsid attachment, the insertion of the phage genome, or the start of DNA replication. As a result, a less fit phage may persist in the high MOI environment of the chemostat. While the current study does not evaluate these specific implications, it is worth noting the potential effects that co-infection may have on persistence despite poor estimated competitive ability.Overall, these findings provide an ecological context for the laboratory phenomenon of the reduction effect, and they provide strong evidence that the reduction effect modulates phage–phage competition. Further, these results support the hypothesis that the phenomenon proposed by van der Avoort et al.  is a mec


Face mask use offers an important public health tool for reducing the spread of coronavirus disease 2019 (COVID-19), yet the politicization of COVID-19 has resulted in uneven adherence. This study assesses the effects of setting characteristics and the sociodemographic composition of crowds on group-level masking rates.We conducted 123 site observations of masking behavior at public locations across Oklahoma (USA) between June and September 2020. We used analyses of variance and t-tests to examine variation in masking and ordinary least squares regression to model the effect of setting and sociodemographic characteristics on site-level masking rates.The masking rate across all sites averaged 34% but varied widely. Site-level masking rates were higher at metropolitan sites and sites with a store or municipal masking mandate. The masking rate at sites where women or older adults (60+) were the predominant group did not differ significantly from other sites. Ethnically diverse sites exhibited significantly higher masking rates compared with predominantly white sites. Findings indicate that setting characteristics explained a greater amount of variation in collective masking rates than sociodemographic differences.This study underscores the importance of place and policy for mask adherence. In the absence of state-level mandates, masking policies at a more local level may be effective. RUCA codes use population density, urbanization and daily commuting to categorize geographic areas on a 1–10 scale.retail , (2) food and beverage services , (3) travel and leisure sites , (4) outdoor recreation  and (5) other (e.g. non-profit and religious sites).Site types included (1) We gathered information about mask mandates at the municipal  and site (business or organization) levels through news outlets, government websites and company press releases, as well as signage at observation sites.predominantly women or predominantly men when 60% or more of observed persons presented as the respective gender category. Similarly, we categorized crowds as predominantly older or predominantly younger based on whether 60% or more of observed persons appeared to be over or under age 60.Observers assigned persons into relatively broad gender and age categories . We characterized sites as composed of predominately white and all other sites as ethnically diverse.Because of the difficulty simultaneously documenting multiple demographic categories, observers noted the approximate racial/ethnic breakdowns of crowds rather than individual persons. Racial categories included White, American Indian, Black, Latinx and Asian American. We classified sites with ~60% or more White people as n = 2). T-tests for independent samples were used to examine masking patterns by mask mandates and race/ethnicity. A paired t-test compared masking rates before and after the implementation of mask mandates using the subsample of repeat observation. Based on the findings from the ANOVAs, we narrowed down relevant geographic, site type, gender and age variables for inclusion in the regression analysis. We then performed multivariate ordinary least squares (OLS) regression to model the relationship between collective masking rates and site characteristics.We used one-way analysis of variance (ANOVA) to determine if collective masking rates varied by geography, site type, gender or age. The ‘other’ category of site types was excluded from the ANOVA because of its small sample size (N = 111). Prior to the regression analysis, we performed chained multiple imputation (m = 20) to fill the 12 missing values.P-value of 0.05 to determine significance and report 95% confidence intervals (CI). We performed all analyses using Stata 17.Errors in data collection resulted in 12 missing values on the race variable. The one-way ANOVA by race/ethnicity excluded observations with missing data (n = 66) of the sample, with micropolitan areas  and small town or rural areas  comprising the remainder. Most observations occurred at retail establishments , followed by food and beverage services , travel and leisure , outdoor recreation  and other sites .Research teams observed an average of 23 people per site over observations that averaged 7 min . The buln/N = 1,246/2,836). However, the collective masking rate for all 123 site observations averaged 34%  and varied considerably across locations, ranging from 0 to 100%. Observations largely occurred prior to the implementation of face mask mandates; only 29 sites (23.6%) had either a city or site policy at the time of the observation. Of these, 21 had both a city and site mandate, five had only a municipal mandate and four only a site mandate. Metro sites had higher rates of mask mandates: out of the metro sites, 27 (40.9%) had mask mandates compared with only two (3.5%) non-metro sites.On the individual level, 43.9% of total people observed wore masks  consisted of a predominantly white demographic. A total of 25 sites (20.3%) were ethnically diverse.A total of 28 sites (22.8%) consisted of predominately women and 29 sites (23.6%) of men. Older adults predominated at six sites (4.9%) and younger people at 106 sites (86.2%). Most sites  = 24.2; P < 0.001). A Tukey post hoc test revealed significantly higher masking rates at metro locations compared with micropolitan  and small town/rural locations . The difference between micropolitan and small town/rural sites did not reach significance.Metro locations had the highest collective masking rates at 51.4%. Small town/rural and micropolitan locations had dramatically lower masking rates at 15.7 and 10.3%, respectively. Using a one-way ANOVA, we found statistically significant differences based on geography  = 3.0; P = 0.04). Masking rates were significantly lower at outdoor recreation sites compared with retail sites . The differences between other site types did not reach significance.Retail sites averaged the highest masking rate at 38.2%, followed by travel and leisure (34.0%), food and beverage (24.2%), and outdoor recreation sites (3.9%). ANOVA reveals a statistically significant difference in masking between site types (t(121) = 7.2; P < 0.001). To further explore the relationship between policies and masking rates, we observed a subsample of nine retail stores in Norman and Oklahoma City before and after the enactment of mask mandates, including five grocery stores, a pharmacy, a liquor store and a farm/ranch store. Mask utilization increased from 58.5% in the pre-mask mandate observations to 86% following implementation a masking policy, a statistically significant change of 27.5%  = 4.5, P = 0.002).Sites with any mask mandate had dramatically higher masking rates (68.1%) compared with those without (23.0%). A t-test for independent samples showed that this difference is statistically significant  = 1.3; P = 0.28). Sites with large proportions of older adults exhibited higher rates of masking (50.9%) compared with those with younger people (33.9%) or mixed aged crowds (25.5%). The between-group differences based on age was not statistically significant  = 1.0; P = 0.37). Finally, ethnically diverse sites had higher masking (49.0%) compared with predominantly white sites (32.1%), a statistically significant difference of 16.9%  = 2.11, P = 0.04).Sites comprised of predominantly men had lower average masking rates (24.9%) compared with majority women (35.9%) or mixed gender (37.2%) sites. However, ANOVA shows that these differences were not statistically significant  on collective masking rates . None ofOur study found an average collective masking rate of 34% across public sites in Oklahoma between 13 June and 5 September 2020. Although we found differences in site-level masking rates based on the sociodemographic composition of crowds, these differences generally failed to reach significance. Notably, multivariate regression showed that the race, gender and age variables combined explained only a small amount of the variation in masking rates at sites (7%). The variables measuring setting characteristics  by contrast explained 42% of the variation in collective masking. These findings were robust to different model and variable specifications .,,Most prior studies on face mask utilization focus on the individual level and establish the importance of geographic, sociodemographic and policy factors on masking decisions. Both survey and observational studies find relatively high masking rates among women, older adults and people of color,,,,The New York Times survey data from July 2020 to explore predictors of county-level masking. Pro and colleagues found a significant relationship between collective masking and the percentage of residents who are female, older (65+) and non-white.A substantial amount of research examines demographic differences in individual masking behaviors and generally finds that gender, age and race significantly predict a person’s likelihood of masking.To our knowledge, this is the first masking study to employ direct observations to examine predictors of collective masking at the group level. Because social behavior is ‘collectively negotiated and context-dependent,’Our study has several limitations. First, this study has limited ability to shed light on the impact of mask policies/mandates in rural communities, as only two of our non-metropolitan sites had such policies. However, the average masking rate at these non-metro sites (21.7%) was still higher than non-metro sites without a mandate (13.5%). Second, because our data collection was observational only, the underlying motivations and meaning of masking remain unknown. Third, our study did not account for variation in COVID-19 levels which may have influenced Oklahomans’ likelihood of masking.The surge of the COVID-19 Delta and Omicron variants in 2021 and renewed calls for universal masking speak to the continued relevance of masking as long as the virus is linked to significant public health impacts.The datasets generated and analyzed during the current study are available from the corresponding author on request.This work was supported by a University of Oklahoma Vice President for Research and Partnerships COVID-19 Rapid Response Seed Grant.The authors have no relevant financial or non-financial interests to disclose.


Deposition of misfolded alpha-synuclein (αSYN) aggregates in the human brain is one of the major hallmarks of synucleinopathies. However, a target-specific tracer to detect pathological aggregates of αSYN remains lacking. Here, we report the development of a positron emission tomography (PET) tracer based on anle138b, a compound shown to have therapeutic activity in animal models of neurodegenerative diseases.3H]MODAG-001 were tested in in vitro binding assays using recombinant fibrils. After carbon-11 radiolabeling, the pharmacokinetic and metabolic profile was determined in mice. Specific binding was quantified in rats, inoculated with αSYN fibrils and using in vitro autoradiography in human brain sections of Lewy body dementia (LBD) cases provided by the Neurobiobank Munich (NBM).Specificity and selectivity of [3H]MODAG-001 revealed a very high affinity towards pure αSYN fibrils (Kd = 0.6 ± 0.1 nM) and only a moderate affinity to hTau46 fibrils (Kd = 19 ± 6.4 nM) as well as amyloid-β1–42 fibrils (Kd = 20 ± 10 nM). [11C]MODAG-001 showed an excellent ability to penetrate the mouse brain. Metabolic degradation was present, but the stability of the parent compound improved after selective deuteration of the precursor. (d3)-[11C]MODAG-001 binding was confirmed in fibril-inoculated rat striata using in vivo PET imaging. In vitro autoradiography showed no detectable binding to aggregated αSYN in human brain sections of LBD cases, most likely, because of the low abundance of aggregated αSYN against background protein.[MODAG-001 provides a promising lead structure for future compound development as it combines a high affinity and good selectivity in fibril-binding assays with suitable pharmacokinetics and biodistribution properties.The online version contains supplementary material available at 10.1007/s00259-020-05133-x. The molecular pathogenesis of all common neurodegenerative diseases is associated with the disease-specific aggregation of misfolded proteins. Aggregation of α-synuclein (αSYN) has been shown to play a crucial role in the pathogenesis of Parkinson’s disease (PD) , Lewy boNoninvasive imaging technologies such as magnetic resonance imaging (MRI) and positron emission tomography (PET) could serve as a valuable tool for the early and differential diagnosis of neurodegenerative diseases. Furthermore, they would allow for the monitoring of disease progression as well as evaluation of disease-modifying therapies. Whereas PET imaging of amyloid-β (Aβ) in Alzheimer’s disease (AD) is well established –9, PET tChallenges complicating their identification might be (i) the target structure, as pathological αSYN might be present mainly in an oligomeric form rather than as fibrillar structures; (ii) the target concentration, as the amount of aggregated protein in condensed fibrillar αSYN aggregates  are by far less abundant than, for example, Aβ plaques; (iii) the target localization, as pathological αSYN is located intracellularly, making the cell membrane an additional physical barrier for tracer accessibility; (iv) the target composition, as β-sheet-binding motifs might be shared between different pathological protein aggregates, therefore complicating differential diagnosis or leading to unselective binding in the case of common co-pathologies; (v) the highly complex LB and LN composition comprising lipid membrane fragments and distorted organelles as recently shown by , which m11C-labeling and showed high affinity towards αSYN aggregates in vitro; however, it showed suboptimal pharmacokinetics in vivo MODAG-001 at increasing concentrations (0.05–12 nM/24 nM) in 30 mM Tris-HCl, 10% ethanol, 0.05% Tween20, pH 7.4 in a total volume of 200 μL/well. Nonspecific binding of the radiotracer was determined by coincubation with 400 nM nonlabeled MODAG-001 dissolved in DMSO . To determine the potential binding site of MODAG-001, a competition binding assay using SIL26 previously evaluated by Bagchi et al. [3H]MODAG-001 and decreasing concentrations of a 1:4 serial dilution of nonlabeled SIL26 (4 μM-0.06 nM) in 30 mM Tris-HCl, 10% ethanol, 0.05% Tween20, pH 7.4 in a total volume of 200 μL/well.For saturation binding assays, the optimal fibril concentrations were determined according to Auld et al.  using a Filtration and readout of competition and saturation binding experiments were performed as described in the 3H]MODAG-001 for αSYN fibrils in brain homogenates, one C57BL/6 mouse was sacrificed with CO2, and its brain was surgically removed. PBS containing one tablet each of phosphatase inhibitors  and proteinase inhibitors  per 5 mL was added to the brain to reach a protein concentration of approximately 200 mg/mL. The brain was sequentially homogenized using large and small clearance pestles (approximately ten strokes per pestle), contained in a tissue grinder set , and the homogenate was stored at − 80 °C until further use. The protein concentration was determined using a BCA kit . Saturation assays were performed as described above. Homogenates were diluted in PBS to reach final protein concentrations of 0.5 mg/mL, 0.1 mg/mL, and 0.05 mg/mL, and fibrils were added to reach concentrations of 125 nM, 25 nM, and 5 nM.To determine the limit of detection of [11C]anle253b radiolabeling [Radiolabeling was performed in a manner analogous to [labeling . For a dRegierungspräsidium Tübingen). Twenty-two female C57BL/6 mice (25 ± 6 g) and four female Sprague-Dawley rats (278 ± 25 g) were obtained from Charles River Laboratories . All animals were maintained in our vivarium on a 12:12 h light-dark cycle at a temperature of 22 °C with 40–60% humidity and given free access to a standard diet and tap water.All animal experiments were conducted in compliance with the European directives on the protection and use of laboratory animals (Council Directive 2010/63/UE) and in addition with the German animal protection law and with the approval of the local authorities (11C]MODAG-001 (n = 3) or 12 ± 1 MBq (d3)-[11C]MODAG-001 (n = 5) 5 s after the start of the PET acquisition.PET imaging was performed on a dedicated small animal Inveon PET scanner , USA). Mice and rats were anesthetized with 1.5–1.7% isoflurane evaporated in 100% oxygen at a flow rate of 0.8 L/min, which was maintained during the entire experiment. After a tail vein catheter was placed, the animal was positioned in the center of the field of view (FOV) on an MR-compatible PET bed that was connected to a feedback temperature control unit set to 37 °C. Mice were injected intravenously (i.v.) with 17 ± 2 MBq [3)-[11C]MODAG-001.Rats were inoculated with αSYN fibrils  into the right striatum  definition, and determination of binding parameters are described in detail in the 11C]MODAG-001 (n = 4 per time point) or 120 ± 41 MBq of (d3)-[11C]MODAG-001 (n = 3 per time point) using a tail vein catheter. Five and 15 min after injection, plasma and brain homogenates were analyzed using radio-HPLC , two LBD cases (with pronounced αSYN and no to low Aβ and Tau pathology), one progressive supranuclear palsy (PSP) case, and one control case were kindly provided by Neurobiobank Munich , and sto3H]MODAG-001 showed a very high affinity towards αSYN fibrils (Kd = 0.6 ± 0.1 nM) with low nonspecific binding    Fig.  confirmeing Fig. . ComparenM) Fig.  and Aβ1–nM) Fig. . Of note3H]MODAG-001, a competition assay with SIL26 was performed. SIL26 displaced [3H]MODAG-001 binding in a dose-dependent manner with a Ki of 21 nM, indicating lower affinity for SIL26 than for MODAG-001  , prompting us to switch to direct methylation for subsequent experiments. Here, we achieved a Am of 98.6 ± 24.7 GBq/μmol but a radiochemical yield of only 3.6 ± 1.1% , a yield sufficient for our animal studies.MODAG-001 was readily radiolabeled by reductive methylation using in situ-generated formaldehyde with a radiochemical yield of 11.4 ± 3.7% (11C]MODAG-001 PET/MR images and time activity curves (TACs) of selected organs from one exemplary animal at different time points after i.v. injection, which resulted in rapid brain uptake with an SUV of 1.4  with only small regional differences across multiple brain regions and fast washout from the brain -[11C]MODAG-001, dynamic whole-body PET scans were performed in mice. Figure 3)-[11C]MODAG-001, which resulted in rapid brain uptake with an SUV of 1.7  with only small regional differences across multiple brain regions.After the successful synthesis of -[11C]MODAG-001 injection resulted in rapid brain uptake, with peak SUV values of 2.1 ± 0.1 in the left striatum of inoculated rats  and 2.1 in the non-injected rat, and fast washout from the brain . The non-injected rat showed no difference in mean SUV40-60min between the right and left striatum, with brain uptake comparable to that in the inoculated rats. In all rat brains, nonspecific binding was also observed in brain regions without fibrils, reducing the SNR in the inoculated striatum.Figure ain Fig.  with hig3H]MODAG-001 AR images of the cortex of two LBD cases, one AD case and one PSP case, and one control case. In all cases, [3H]MODAG-001 showed a nonspecific blocking effect, which was likely related to the high concentration of MODAG-001 (50 μM) used for blocking. For the AD case, clear binding of [3H]MODAG-001 in the cortex was observed. Binding values for the LBD 1 (80.5 ± 8.7 pmol/mg), LBD 2 (64.4 ± 9.8 pmol/mg), PSP (79.0 ± 13.3 pmol/mg), and AD (167.7 ± 40.0 pmol/mg) cases and the control (64.1 ± 19.1 pmol/mg) are shown in Fig. Figure Since the discovery in 1997 that the protein αSYN can be found in Lewy bodies in PD MODAG-001 was tested in saturation binding experiments against human recombinant fibrils and showed very high affinity to αSYN fibrils, with a Kd value of 0.6 nM in vitro. Compared to [3H]MODAG-001, all the compounds, which have been described in the literature as potential αSYN PET ligands, showed an at least one order of magnitude lower binding affinity  in vitro towards recombinant αSYN fibrils [125I]SIL23, (Kd = 148 nM) and its derivative SIL26 (Ki = 15.5 nM), [18F]BF-227 (Kd = 9.6 nM) [Kds in the micromolar and elevated nanomolar range) [18F-labeled 3-(benzylidine)indolin-2-one derivative [18F]46a (Kd = 8.9 nM) BF-r range) , 33, and1–42 fibrils, which was suggested for an ideal CNS PET tracer, as both structurally similar proteins have also been shown to be highly abundant in many patients with synucleinopathies MODAG-001 after i.v. injection into healthy mice revealed good BBB penetration with high uptake into the brain (SUV = 1.4) and relatively fast clearance from the brain. Our metabolite analysis revealed that [11C]MODAG-001 was degraded into the three metabolites, two of which were detectable in the brain, with 81% of the parent compound remaining at 15 min. We hypothesize that metabolite M3 might be the demethylated form of MODAG-001, as demethylation is a very common form of metabolic degradation. Fast metabolism resulting in BBB-penetrating radio-metabolites hampers tracer quantification. Previous studies have shown that improved metabolic stability can be obtained by incorporating deuterium into the molecule. In comparison to carbon-hydrogen bonds, carbon-deuterium bonds potentially decelerate metabolism by cytochrome P450 enzymes due to the primary kinetic isotopic effect MODAG-001 and [11C]MODAG-001 revealed that formation of the monodemethylated radio-metabolite M3 was reduced and that the ratio of M3 to M1  was increased for (d3)-[11C]MODAG-001. Moreover, total metabolism of the parent compound was reduced, resulting in increased levels of the parent compound in the brain at 15 min. This is emphasized in Table 11C]MODAG-001 was reduced from 10.5% at 5 min to 5.3% at 15 min, whereas the injected dose per gram for (d3)-[11C]MODAG-001 was reduced to a lower extent from 8.8% at 5 min to 7.2% at 15 min.These encouraging in vitro binding data were the prerequisite for further c effect . Since t3)-[11C]MODAG-001 can detect aggregated αSYN in the brain and thus inoculated the right striatum in rats with the same batch of αSYN fibrils used for the in vitro binding experiments. (d3)-[11C]MODAG-001 binding properties in three fibril-inoculated rats and one non-injected control rat were assessed using in vivo PET imaging. In the three fibril-inoculated rats, we observed higher binding in the right striatum compared to the vehicle-inoculated contralateral side, with mean DVR-140-60min of 0.14 ± 0.1 (whole striatum) and 0.44 ± 0.21 (70% automatic isocontour detection) 4 days after injection, respectively, indicating in vivo binding of (d3)-[11C]MODAG-001 to inoculated αSYN fibrils. Furthermore, similar overall brain uptake was observed in fibril-inoculated rats  and the non-injected rat , demonstrating that potential disruption of the BBB from the intracranial injection is unlikely to account for higher uptake into the inoculated striatum. A similar rat model was also used by Verdurand et al. [18F]BF227, [18F]2FBox, and [18F]4FBox to αSYN and Aβ1–42 fibrils using in vitro AR; however, no binding was observed in vivo despite good uptake into the cerebral tissue and high Am (68–543 GBq/μmol).We next asked whether  . The samDespite the presence of structural differences between recombinant fibrils and those found in human brain slices, homogenates, or αSYN brain extracts, the availability of the latter is very limited; as such, recombinant fibrils remain a widely used screening tool for the preselection of compounds before PET radiolabeling.postmortem tissue of 145 sporadic AD cases using immunohistochemistry for αSYN and observed LBs in 60.7% of all cases. Colo-Cadena et al. MODAG-001 competition assay using the tricyclic compound SIL26 as a competitor and observed a Ki value of 21 nM, indicating preferential binding to site 3/13 at fibrillar αSYN. However, the interpretation needs to be done with care, as preference to sites 2 and 9 were not tested.Determination of the αSYN fibril-binding sites of novel compounds is crucial for PET tracer development. Hsieh and Ferrie et al. identified three putative binding sites for fibrillar αSYN using a combination of in silico docking, photoaffinity labeling, and radiotracer binding studies . While sOver the past years, several attempts have been made to develop an αSYN-specific PET tracer , 24–31. ESM 1(DOCX 948 kb)


Syndesmotic injuries can occur with ankle fractures and can lead to destabilization of the ankle joint. As a result, it usually requires a transyndesmotic screw insertion to stabilize it. Currently, there is no consensus on the type, amount and diameter of screws used, the number of cortices needed to be engaged, the recommended time to weight-bearing, and whether the screw should be removed in these types of injuries. The aim of this study is to evaluate the evidence comparing the removal and non-removal of syndesmotic screws in open and closed ankle fractures that are associated with unstable syndesmosis in terms of functional, clinical, and radiological evidence. The study also looked at the evidence behind broken screw effects.The literature search was conducted on March 16, 2021, using the Ovid Medline and Embase databases. The literature was eligible if it aimed to compare syndesmotic screw removal and retention in ankle fractures. One study found that those with a broken screw had a better clinical outcome than those with an intact screw. The studies were excluded if they were biomechanical studies, case reports, or were relevant but had no adequate English translation.Initially, 53 studies were included but after scanning for eligibility, 11 were identified (including those added from references). Nine were cohort studies, seven of which did not find any difference in functional outcome between routine removal and retention of the syndesmotic screw. Two studies found there were better clinical outcomes in the broken screw group. Another study found that there were slightly worse functional outcomes in patients with intact screws as compared with those with broken, loosened, or removed screws. Two studies were randomized control studies that no significant functional outcomes between removed and intact syndesmotic screws. However, the majority of these studies had a high risk of bias.Overall, the current literature provides no evidence to support routine removal of syndesmotic screws. Keeping in mind the clear complications and financial burden, syndesmotic screw removal should not be performed unless there is a clear indication. Furthermore, removal in the clinic, with the use of prophylactic antibiotics should be considered if indicated in cases with pain or loss of function. Further research in a structured randomized controlled trial (RCT) to examine if there is any difference in short- or long-term outcomes between removed, intact, loose, or broken syndesmotic screws might be beneficial. A multinational protocol for randomized control trials  is an example of such a study to determine the usefulness of on-demand and routine removal of screws. Ankle fractures are one of the common presentations in the orthopaedics speciality. It is estimated that 75 per 100,000 adults who are <50 years old and 104 per 100,000 adults who are >50 years old presented with ankle fractures annually in the UK . It recoThe aim of this systematic review is to examine the latest evidence comparing the removal and non-removal of syndesmotic screws in open and closed ankle fractures that are associated with unstable syndesmosis in terms of functional, clinical, and radiological evidence. This study will also examine the effects of broken screws for the same parameters.MethodsThe systematic review literature search was conducted on March 29, 2021. The Ovid Medline and Embase databases were searched using the following search terms: syndesmosis, syndesmotic, fixation of syndesmosis, syndesmotic screw, syndesmosis screw, transfixing screw, trans-syndesmotic screw, ankle Injuries, ankle fractures, bone screws, remov*, device removal, bone screw, syndesmosis screw removal, syndesmotic screw removal, removal of syndesmotic screw, removal of syndesmosis screw, screw removal, removal of screw, implant removal, hardware removal, screw, retention, retain*, in situ, intact, not removed, retained screw, retention of screw, retaining the screw, intact screw, comparative study. The entire search string could be viewed in the appendix. There was no limit to the year of publication or language used. A list was created based on the title and the abstract of the search. Studies that were found to not be relevant to the review were excluded at the initial stage of screening. The literature was then evaluated further and excluded if they were biomechanical studies, case reports, or were relevant but had no adequate English translation. The reference lists of those studies were reviewed, and relevant studies included. A study was eligible if it aimed to compare syndesmotic screw removal and retention in ankle fractures using patient recorded outcomes measures (PROMs) . All theResultsFigure The majority of the studies compared elective removal versus retained screw (intact or broken). Functional and clinical outcomes were assessed using different scores, including Olerud-Molander ankle (OMAS) , AmericaIn an RCT by Boyle et al. , they coIn another RCT , they aiFigure DiscussionThe literature review did not identify a significant difference in functional outcomes, suggesting routine removal of syndesmotic screws. This is supported by the two RCTs -14 that The first randomized control study conducted by Boyle et al.  comparedThere were two retrospective case studies looking at the necessity of syndesmotic screw removal prior to weight-bearing ,23. BothKaftandzeiv et al.  and HamiTucker et al.  concludeRemoval of the screw has its associated risks and judging by the evidence produced by the literature, routine removal that is not clinically indicated cannot be recommended. Andersen et al.  found thTucker et al.  found thThere are several limitations to the current study. Firstly, several studies fell into the criteria of the study, however, they were not included due to inadequate translation to the English language. This could lead to missing significant results, which could add to the quality of evidence in the literature. Also, it was difficult to perform a meta-analysis due to the heterogenicity of the results of the studies. A more structured RCT would be required to carry out a full meta-analysis to determine the significance of these findings. Such an RCT protocol was designed by Dingmans et al. in 2018  with a rThe current literature provides no evidence to support the routine removal of syndesmotic screws. Keeping in mind the clear complications and financial burden, syndesmotic screw removal should not be performed unless there is a clear indication. Furthermore, removal in the clinic with the use of prophylactic antibiotics should be considered if indicated in cases with pain or loss of function. Further research in a structured RCT, to examine if there is any difference in short- or long-term outcomes between removed, intact, loose, or broken syndesmotic screws, might be beneficial. A multinational protocol for randomized control trials  is an example of such a study to determine the usefulness of on-demand and routine removal of screws.


Infectious bronchitis virus (IBV) induces respiratory and urogenital disease in chickens. Although IBV replicates in the gastrointestinal tract, enteric lesions are uncommon. We have reported a case of runting-stunting syndrome in commercial broilers from which an IBV variant was isolated from the intestines. The isolate, CalEnt, demonstrated an enteric tissue tropism in chicken embryos and SPF chickens experimentally. Here, we determined the full genome of CalEnt and compared it to other IBV strains, in addition to comparing the pathobiology of CalEnt and M41 in commercial broilers. Despite the high whole-genome identity to other IBV strains, CalEnt is rather unique in its nucleotide composition. The S gene phylogenetic analyses showed great similarity between CalEnt and Cal 99. Clinically, vent staining was slightly more frequent in CalEnt-infected birds than those challenged with M41. Furthermore, IBV IHC detection was more evident and the viral shedding in feces was overall higher with the CalEnt challenge compared with M41. Despite underlying intestinal lesions caused by coccidiosis and salmonellosis vaccination, microscopic lesions in CalEnt-infected chickens were more severe than in M41-infected chickens or controls, supporting the enteric tropism of CalEnt. Further studies in SPF chickens are needed to determine the pathogenesis of the virus, its molecular mechanisms for the enteric tropism, and its influence in intestinal health. Infectious bronchitis virus (IBV) belongs to the Gammacoronavirus genus and mainly causes respiratory and urogenital disease in chickens. Although IBV replicates and persists in the cecal tonsils and has been isolated from duodenum and jejunum, intestinal lesions are rare ,2,3. GenAn enterotropic IBV strain has been described for causing intestinal lesions, mainly in the rectum, consisting of the desquamation of epithelial cells on the villi tips and congestion of the intestinal submucosa ,10. ThisPreviously, we have described the detection and isolation of an IBV variant from the intestines of brown broiler chickens showing runting-stunting signs. Interestingly, the virus was not retrieved from the kidneys or respiratory tract. The enteric isolate was named CalEnt and was isolated from the intestines of chicken embryos, but not from the chorioallantoic membrane. Additionally, CalEnt induced runting-stunting syndrome-like lesions after experimental oculonasal infection of specific-pathogen-free (SPF) chickens. The S1 gene sequence of CalEnt showed nucleic acid sequence identities of 93.8% to IBV California 99  and of 85.7% to IBV Arkansas DPI (Ark DPI) . The aim4 EID50 and the dose of IBV M41 was 2 × 105 EID50 in a 200-µL inoculum. The challenge was performed either oculonasally or via crop gavage.IBV CalEnt and M41 strains were used in experimental challenges. Both IBV strains were grown and titrated in embryonated chicken SPF eggs  using standard procedures ,16. The Intestines from embryos infected with CalEnt were homogenized with PBS and RNA was extracted from a volume of 100 μL using TRIzol . DNA depletion was performed using the Turbo DNA-Free Kit  followed by rRNA depletion using the Terminator 5′-Phosphate-Dependent Exonuclease  as per the manufacturer’s instructions. The DNase reaction was ceased by adding EDTA at a concentration of 5 mmol/L. The RNA was purified using the QIAamp Viral RNA Mini Kit  without the addition of the carrier RNA, and eluted to a final volume of 30 μL. The RNA quality was evaluated using a 2100 Bioanalyzer . cDNA libraries were prepared using the NEBNext Ultra Directional RNA Library Prep Kit for Illumina . Libraries were sequenced using the Illumina at 100 bp paired end. Quality control of the reads, adaptor trimming, merging of paired end reads, and assembly were performed using Geneious Prime 2021.1.1. The sequences were assembled using IBV Cal99 as a reference (accession number AY414485). A second round of reference-based assembly was performed using the consensus sequence obtained in the first round as a reference. For accuracy verification, de novo assembly was performed using the SPAdes assembler. The two assemblies shared 100% nucleotide identity. The consensus was uploaded to GenBank (accession number MW556742).The obtained consensus sequence was aligned with several IBV variants and TCoV using the MAFFT plugin  in Geneiblast.ncbi.nlm.nih.gov, accessed on 20 July 2021) and the whole S gene from sequences showing the highest homologies was aligned using the MAFFT plugin [The S gene of CalEnt was submitted to the basic local alignment search tool , 1 , 2 , or 3 (severe respiratory sounds audible from a 20 cm distance) by a blinded investigator [At one, 2, 4, 6, 10, and 14 dpi, tears and cloacal swabs were collected from five chickens per group for detection of IBV by RT-qPCR. Tears were collected using granules of pure sodium chloride to stimulate lachrymation. Five birds per group were euthanized by carbon dioxide inhalation at 4 and 14 dpi, and tracheas and small intestine samples collected during necropsies were submitted for histopathology and immunohistochemistry (IHC).Sera collected at 10 days of age were tested for antibodies against IBV by ELISA . S/P ratios were transformed into titers as per the manufacturer’s recommendations. The cutoff titer for positivity is 396.RNA was extracted from tears and cloacal swabs in PBS using the QIAamp Viral RNA Mini Kit  as described by the manufacturer. A probe-based RT-qPCR using primers qIBf (5′-ATA CTC CTA ACT AAT GGT CAA CAA TG-3′) and qIBr (5′-GGC AAG TGG TCT GGT TCA C-3′) and probe qIBs (5′-CAA GCC ACT GAC CCT CAC AAT AA-3′) was performed, targeting a 134 bp fragment of the IBV M gene in 44 cycles of denaturation and combined annealing/extension steps ,22. ViraTissues were fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned at 4 μm, mounted on glass slides, and stained with hematoxylin and eosin (H&E) stains. IBV in situ antigen detection was performed using IHC staining as previously described . A combiViral loads (represented as 44-Cq) were compared using ANOVA followed by Tukey’s multiple comparison tests. Respiratory sign scores, vent staining, and uniformities were compared using Kruskal–Wallis (for non-parametric data) followed by Dunn’s multiple comparison tests. Data were analyzed using Prism 9 .The complete genome homologies revealed that the IBV strains most closely related to CalEnt were Cal99 (94.3%), Cal 56b (94.2%), M41 (94.1%), and ArkDPI (94.1%) . The whoDespite the similarities to Cal 99, Cal 56b, M41, and Ark DPI, CalEnt did not cluster with other IBV strains in the whole genome phylogenetic analysis A. The fuThe recombination analysis suggests that the S gene from CalEnt is the result of recombination events between Cal99, GA/1476/15, and IBS037A/14 . The inin = 50) at 10 days of age. The geometric mean titer and standard deviation was 172 ± 166.IBV maternal antibodies were detected in 10% of the collected serum samples  . With the exception of swabs collected at 1 dpi, the viral loads in cloacal swabs were overall higher in CalEnt-infected chickens, although only statistically significant at 2 and 10 dpi (p < 0.05). In general, the viral load in tears was higher in M41-challenged birds than in those challenged with CalEnt. At 1, 2, and 4 dpi, the highest viral load was seen in birds challenged with M41 oculonasally; at 6 dpi and onward, the viral load in tears was higher in birds challenged with M41 via crop . A few birds in each group had small numbers of coccidia. At 4 dpi, mild to moderate IBV IHC staining was present in the cytoplasm of enterocytes at the tips of the villi of the duodenum and ileum from birds challenged with CalEnt A. In conAlthough IBV is typically known for inducing respiratory and urogenital disease, few IBV strains have been described as being mainly enterotropic ,10,11,12The complete genomic sequence of CalEnt showed homologies higher than 94% to Cal 99, Cal 56b, M41, and Ark DPI . DespiteConsidering other genes, CalEnt also showed very high identity to other IBV isolates commonly used in vaccines in California, such as Mass, Conn, and Ark strains . These rClinically, the commercial broilers challenged with either M41 or CalEnt in our experiment presented with respiratory signs and lesions, regardless of the route of inoculation or IBV strain, although respiratory disease was more severe in M41-infected birds than those challenged with CalEnt . A similSalmonella at the hatchery were used in this study, and some baseline intestinal damage might have been caused by these live vaccines. Intestinal health in commercial poultry is extremely complex and involves numerous factors, including attenuated microorganisms provided in vaccines, the microbiota, co-infections with other pathogens, and the nutritional contents of the feed [Salmonella vaccines, the unchallenged control birds presented with less severe intestinal lesions compared to IBV-infected birds, and no IBV IHC staining was observed. The microscopic injuries observed in the CalEnt-infected birds appeared slightly more severe than those present in M41-infected birds, and the IBV IHC staining was more evident, supporting the enteric tropism of CalEnt as previously reported [Commercial broilers vaccinated against coccidia and the feed . In addithe feed . It is preported .Regarding the inoculation route, the crop gavage inoculation was performed in order to investigate if fecal–oral infection would have a different impact on CalEnt pathogenesis as opposed to the known airborne transmission of IBV. In general, it appeared that the crop gavage inoculation led to a late onset of clinical disease that persisted for a prolonged period compared with the oculonasal inoculation. It is possible that a portion of the inoculum was inactivated in the proventriculus or ventriculus of challenged chickens, reducing the viral load and consequently delaying the clinical signs and viral shedding.This study corroborates the evidence that CalEnt has a tropism for the intestinal tract, which is unusual for IBV. Its comprehensive pathogenesis and pathobiology in SPF chickens, its influence in intestinal health, and the underlying molecular mechanisms of this preferred replication site remain to be determined.


Water ice exists in hugely different environments, artificially or naturally occurring ones across the universe. The phase diagram of crystalline phases of ice is still under construction: a high-pressure phase, ice XIX, has just been reported but its structure remains ambiguous. Ice, the solid phase of water, has always been in the focus of science. It was maybe even ice, that was a necessary substrate for first life on Earth to emerge1; Therefore, it is normal to be very curious about all possible structures of water ices under different conditions—twenty crystalline and further amorphous forms are now known to exist  ordering of hydrogen differs from what is predicted by computer simulation18.The ordering from ice I19, obtained, like the hydrogen ordered phase ice XV, upon cooling ice VI. At that time, experimental evidence was insufficient to attribute a Roman numeral to this phase – as is the convention for crystalline phases of ice – because its structure remained to be elucidated.Back in 2018, Gasser et al. reported on a ‘β-ice XV’ phase9 investigate this high-pressure phase and describe it as a hydrogen-ordered phase of ice VI, with a different hydrogen ordering as compared to ice XV, and assign to it a new Roman numeral, XIX. While they can show convincingly that ice XIX is different from ice XV and ice VI, by means of dielectric measurements and in situ neutron powder diffraction, its crystal structure remains ambiguous: several space groups remain possible according to the authors’ data on the deuterated in situ high-pressure samples of ice XIX… Ice XIX definitely forms a Now, in a recent paper, Yamane et al.10, again announced as a second (different from ice XV) hydrogen-ordered polymorph to ice VI. They performed ex situ high resolution neutron powder diffraction on recovered samples, providing better quality data as compared to in situ high-pressure data. The work also sheds more light on the phase boundaries, such as the order-order transition between ice XV and ice XIX. Yet, by a crystallographically different approach (filtering of eligible subgroups by compatibility with the oxygen lattice), an ambiguity between different possible space groups remains. To reduce the 109 possible solutions in five space groups to a smaller set of likely solutions, they referred to the preprinted paper of Yamane et al. and limited the quantitative tests to Yamane’s structure models in the three space groups both groups found to be eligible. Again, even a distinction of orthorhombic and tetragonal solutions remains ambiguous. The oxygen topology is found to be the same in ice VI, XV and XIX. The hydrogen order is only partial in both, ice XV and XIX. And in both cases, the ordering is found to be anti-ferroelectric . Two space groups are most likely, tetragonal PPcc2. It would be easy to distinguish the solutions as Pcc2 should exhibit pyroelectricity and non-polar PAt the same time, Gasser et al. have investigated the same phase of ice, and published a paper on the structure of ice XIXIt is not surprising that the phase, which had been called β-ice XV previously, which is found different from ice XV and therefore called ice XIX, is suspected to show hydrogen ordering, and a different one as compared to ice XV. How H atoms order and whether an ordered structure is ferroelectric or anti-ferroelectric remains difficult to predict, a fact which nourished the suspicion that not only one H-ordering could be possible. When, in the ordering of ice VI to ice XV, calorimetry suggested a second, underlying, process, and that both ordering processes would have entropy changes corresponding to only partial H-ordering, a different hydrogen ordering became the likely candidate. Yet, this expectation biases the scientist’s look at experimental results and may limit the possible interpretations of the experiment.In addition, as for the formation of other hydrogen-ordered phases, certain doping strategies were needed to obtain the new ice phase. And here we eventually run in some danger, as every group has its own recipe to obtain ice XIX, can we really be sure to look at the same phase in all three papers?8, published in Nature Communications only a few weeks later than the other two, comes from a team, Salzmann et al.17, which has quite some experience in the hunt for hydrogen-ordered ice phases. The structures of ice XIII, XIV and XV have already been determined as well by Salzmann et al. As in the previous investigations, upon cooling a doped sample at higher pressure, additional Bragg peaks appear which imply an increase of the ice VI unit cell to a Pbcn, allows for the observed additional Bragg peaks . The space group allows a good fit of the diffraction data — together with continued total hydrogen disorder in contradiction to the two previous papers. The lower symmetry with respect to the one of ice VI justifies the assignment of the Roman numeral ‘XIX’ to this phase, but concerning the hydrogen ordering, ice XIX remains a deep glassy state of ice VI with pressure-induced distortions as already suspected earlier by Rosy-Finsen et al.20.The third paper on ice XIXAlthough some weak hydrogen ordering cannot be excluded, it is not the main structural feature distinguishing it from ice VI (and ice XV). However, slightly different doping strategies have been applied, are we really looking at the same ice phase in all cases? Are the conclusions of Salzmann et al. compatible with Gasser et al.’s observations on the ice XIX to ice XV ‘order-order’ phase transition?Surely, we have not seen the last paper on this new crystalline polymorph of ice yet. There is still additional proof to provide to support the solution found by the paper hereafter. If the last solution offered, the deep glassy state, is the right one, one may wonder whether there is a hydrogen-ordered phase of this distorted phase. Also, might a second hydrogen-ordered phase, as it was claimed by Yamane et al. and Gasser et al. a few weeks ago, exist for any hydrogen-ordered phase?A remarkable common feature of all three papers is that they are based on neutron powder diffraction . I may conclude with this biased observation, the role of neutron diffraction in the investigation of ice structures remains crucial.


Clostridioides difficile, the extent and nature of phosphorylation events are poorly characterized, though a protein kinase mutant strain demonstrates pleiotropic phenotypes. Here, we used an immobilized metal affinity chromatography strategy to characterize serine, threonine, and tyrosine phosphorylation in C. difficile. We find limited protein phosphorylation in the exponential growth phase but a sharp increase in the number of phosphopeptides after the onset of the stationary growth phase. Our approach identifies expected targets and phosphorylation sites among the more than 1,500 phosphosites, including the protein kinase PrkC, the anti-sigma-F factor antagonist (SpoIIAA), the anti-sigma-B factor antagonist (RsbV), and HPr kinase/phosphorylase (HprK). Analysis of high-confidence phosphosites shows that phosphorylation on serine residues is most common, followed by threonine and tyrosine phosphorylation. This work forms the basis for a further investigation into the contributions of individual kinases to the overall phosphoproteome of C. difficile and the role of phosphorylation in C. difficile physiology and pathogenesis.Phosphorylation is a posttranslational modification that can affect both housekeeping functions and virulence characteristics in bacterial pathogens. In the Gram-positive enteropathogen IMPORTANCE In this paper, we present a comprehensive analysis of protein phosphorylation in the Gram-positive enteropathogen Clostridioides difficile. To date, only limited evidence on the role of phosphorylation in the regulation of this organism has been published; the current study is expected to form the basis for research on this posttranslational modification in C. difficile.   Proteins in organisms from all domains of life can be functionally altered by posttranslational modifications (PTMs) that are often species dependent . PTMs inTo date, phosphorylation has been identified on the side chains of different amino acids (aa): serine (Ser), threonine (Thr), tyrosine (Tyr), histidine (His), arginine (Arg), lysine (Lys), aspartate (Asp), and cysteine (Cys) . The cheS-phosphorylated form can interact with the transcription factor CcpA to regulate metabolic activity , also known as Hanks-type kinases . InteresThe exploration of bacterial phosphorylation has greatly been stimulated by the development of mass spectrometry (MS)-based phosphoproteomics techniques that provide a snapshot of the site-resolved phosphorylation state of proteins under given conditions , 11. TheEscherichia coli  as a result of the production of toxins that affect the integrity of the colon epithelium  from three independently grown cultures. Overall, this resulted in the identification of almost 3,000 phosphopeptides  from  species .10.1128/msphere.00911-21.5TABLE S1C. difficile. Download Table S1, XLSX file, 0.5 MB.Full data set of the phosphoproteome analysis and site assignment for Ser, Thr, and Tyr phosphorylation in Copyright © 2022 Smits et al.2022Smits et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/msphere.00911-21.1FIG S1C. difficile cultures. (A) Growth curve indicating obtained optical densities at 600 nm and sampling time points for this study. (B) Table summarizing protein yield from 100 mL (time point 1) and 50 mL (time points 2 and 3) of sample. Download FIG S1, PDF file, 0.4 MB.Overview of growth and sampling of the Copyright © 2022 Smits et al.2022Smits et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the To compare the different samples, we selected unique peptide sequences that were found to be phosphorylated, without considering phospho-isomers . In general, we observed a progressive increase in protein phosphorylation during growth. We identified most phosphopeptides at later growth phases, with the most prominent increase between the samples taken at the onset of stationary growth phase and the samples from the 24-h cultures .We assessed the reproducibility of the identifications between the different samples at the various time points. At the mid-exponential growth phase, 232 phosphopeptides were identified in all three samples, corresponding to 41% of the total number of peptides identified at this time point . For theWithin one culture, most phosphopeptides observed at the earlier time points were also observed at later stages  to identify potential kinases and evaluate the auto-phosphorylation or phosphorylation of substrates of these kinases on the basis of findings in other organisms. Here, we focus on the putative protein kinases that are expected to phosphorylate serine, threonine, and tyrosine residues: SpoIIAB (CD630_07710) (We used the UniProt annotation of the 0_07710) , RsbW (C0_07710) , HPr kin0_07710) , and CD6B. subtilis SpoIIAA (Ser58), which is known to be phosphorylated by SpoIIAB in that organism  spectrum  MS/MS spectrum of the tryptic phosphopeptide AMEPLYTSKPELDRpSGMGFTVMK from SpoIIAB, demonstrating the phosphorylation of Ser-15 . (B) MS/MS spectrum of the tryptic phosphopeptide NApSGLHARPAGMFVK from HPr, demonstrating the phosphorylation of Ser-3 . pS, phosphoserine. Ions with “-P” have lost H3PO4FIG S3, PDF file, 0.5 MB.Spectra of conserved phosphorylation in regulatory proteins of Copyright © 2022 Smits et al.2022Smits et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the B, an important regulator of the stress response in Gram-positive bacteria  are on the residues equivalent to the ones identified here in C. difficile RsbV  and lower-confidence phosphorylation of Ser30 (LP = 0.85) and Thr31 (LP = 0.82) (B. subtilis HPr (Ser12) was also identified in a phosphoproteomics experiment , which is known to be phosphorylated through the phosphotransferase activity of the PTS (HPr kinase (HprK/ScoC) can phosphorylate the phosphocarrier protein HPr (PtsH), a protein involved in the import of monosaccharides as part of the phosphotransferase system (PTS) . In B. son Ser46 , 39. We  = 0.82) . The equperiment . Importa LP = 0.8 and Thr3 the PTS . The MS/ the PTS . HoweverC. difficile proteins PrkC  throughout growth. Notably, the conserved residues Thr163 and Thr165 were also found to be phosphorylated in B. subtilis  were identified, suggesting that phosphorylation in this region is structurally conserved. We also found phosphopeptides that indicate phosphorylation close to the N terminus of PrkC   .prkC knockout strain  . For thit strain . The autfaecalis , though faecalis . In E. f residue . We conf residue .C. difficile DivIVA (CD630_26190), indicating phosphorylation of Ser75 (LP = 1.00), Ser82 (LP = 1.00), and Thr160 (LP = 0.98). The phosphorylation of DivIVA is one of the few examples of consistent phosphorylation at a specific site (Thr160) at early time points, but not after 24 h of culturing . In contrast, the other sites in DivIVA (Ser75 and Ser82) were found to be consistently phosphorylated in stationary growth phase but never at the mid-exponential phase. It therefore appears that the patterns of phosphorylation of DivIVA differ throughout growth. Exponential-growth-phase phosphorylation is not observed for all cell division proteins; for instance, ZapA (CD630_07910) and FtsZ (CD630_26460) are predominantly phosphorylated in stationary growth phase  in a process catalyzed by a PTS. This is of particular relevance for the import of monosaccharides into the cell, during which the sugars are converted to phospho-sugars . The iniC. difficile data, we observed the tryptic peptide ILVACGAGIATSTIVCDRVER from the PTS system IIB component  containing two cysteines (in bold and underlined). In total, more than 300 peptide spectral matches for this peptide were identified in all our LC-MS/MS analyses of C. difficile phospho-samples. In all these identifications, the second cysteine (aa 16 in the tryptic peptide) was found to be carbamidomethylated, while the first cysteine at position 5 was a free cysteine (As an example of a tryptic peptide with a free cysteine in our cysteine . Importacysteine . We ther10.1128/msphere.00911-21.4FIG S4CGAGIATSTIVCcamDRVERLVA from the phosphotransfer system (PTS) component IIB (CD630_10830), in which the first cysteine (underlined) is a free cysteine, while the second is carbamidomethylated (cam). Download FIG S4, PDF file, 0.4 MB.Indirect identification of cysteine phosphorylation. MS/MS spectrum of the tryptic peptide Copyright © 2022 Smits et al.2022Smits et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the In conclusion, it appears that a one-step Fe-IMAC enrichment procedure can indirectly detect at least a subset of cysteine-phosphorylated proteins.C. difficile at three different time points. We show that many proteins of C. difficile are phosphorylated in stationary growth phase, and an analysis of a subset of the identified phosphoproteins indicates that at least some of the phosphorylation sites are conserved between phylogenetically distinct organisms.In this study, we characterized the phosphoproteome of a laboratory strain of C. difficile. We identified Ser, Thr, and Tyr phosphorylation and provided indirect evidence for Cys phosphorylation  or phosphomimetic negatively charged amino acids (aspartate and glutamate) , 10. SucStaphylococcus aureus increased the number of phosphosite identifications over 20-fold compared to those of earlier analyses, thereby also increasing the number of processes affected  and temporally (between time points). Specifically, we observe low levels of phosphorylation in exponential growth phase, whereas phosphorylation increases markedly upon entry into stationary growth phase. Of note, we observed a highly variable number of phosphoproteins at the entry into logarithmic growth phase, with two out of three samples showing clearly elevated levels, whereas a single biological replicate resembled the profile observed in exponential growth phase . An initial preculture was grown until an optical density at 600 nm (OD600) of 0.36. Three independent cultures were started by adding 31 mL of the preculture to 400 mL fresh BHI-YE for each new culture. Samples were taken at the mid-exponential (3.25 h postinoculation), early stationary (5.25 h postinoculation), and late stationary (24 h postinoculation) phases , while 50 mL was taken at the two later time points .rm cells , 67 werel-glutamine. Both eukaryotic and bacterial cells were harvested by centrifugation  and washed three times with 25 mL of phosphate-buffered saline . Cell pellets were subsequently stored at −20°C until further use.JY cells were grown at 37°C in Iscove's modified Dulbecco's medium supplemented with 10% heat-inactivated fetal bovine serum and 2] and incubated for 20 min at room temperature. Cells were lysed by sonication . In between sonication steps, samples were cooled on ice for 30 s. Next, samples were centrifuged for 15 min at 7,200 × g at 4°C. The urea concentration was adjusted to 6 M by the addition of 1 mL of 50 mM Tris-HCl, pH 7.4, after which 1 μL Benzonase (250 U/μL) was added and samples were incubated for 2 h at room temperature.Cell pellets were resuspended in 3 mL lysis buffer ), equilibrated with Milli-Q H2O-acetonitrile-formic acid  (95/3/0.1 [vol/vol/vol]) . Following sample loading, the column was washed with solution A (3 × 1 mL), after which peptides were eluted with acetonitrile-water-formic acid (30/70/0.1 [vol/vol/vol]). Tryptic peptides were lyophilized  and stored at −20°C until use.Trypsin was added at a ratio of 1:25 (wt/wt), and overnight digestions were performed at 37°C. The next day, samples were centrifuged for 10 min at 11,000 × C. difficile cells, the total amount of peptides from each sample was applied, with an average of 11 (±1.4 standard deviations [SD]) mg protein per sample. Prior to each IMAC run, the column was stripped using 50 mM EDTA-0.5 M NaCl, pH 4, and charged with 25 mM FeCl3 in 100 mM acetic acid, according to the manufacturer’s instructions, using an offline pump system (Shimadzu). After this, the column was connected to an Agilent 1200 chromatography system running at a flow rate of 0.3 mL/min. Prior to sample loading, the column was equilibrated in solvent A (water-acetonitrile-trifluoroacetic acid 70/30/0.07 [vol/vol/vol]) for 30 min at a constant flow rate of 0.3 ml/min.A one-step phosphopeptide IMAC enrichment procedure was performed using a 4- by 50-mm ProPac IMAC-10 analytical column (Thermo Fisher Scientific). For the JY cells, an equivalent of 5 mg of protein was used for one IMAC purification, while for the 4OH). The complete peak fraction of phosphopeptides between 46 and 49 min was manually collected and lyophilized prior to mass spectrometric analysis.Tryptic peptides  were loaded, and nonphosphopeptides were removed by washing the column for 20 min with solvent A. Peptide separation was performed using a linear gradient from 0% to 45% of solvent B (0.5% [vol/vol] NH18 nano-high-performance liquid chromatography (nano-HPLC) MS/MS with a system consisting of an Easy nLC 1200 gradient high-performance liquid chromatography (HPLC) system  and an Orbitrap Fusion Lumos Tribrid mass spectrometer (Thermo Scientific). Samples  were injected onto a homemade precolumn  and eluted on a homemade analytical nano-HPLC column . The gradient was run from 2% to 36% solvent B  in 120 min. The nano-HPLC column was drawn to a tip of ∼5 μm, which acted as the electrospray needle of the MS source.Lyophilized peptides were reconstituted in 100 μL water-formic acid (100/0.1 [vol/vol]) and analyzed by on-line Cm/z was 300 to 1,500 at an AGC target of 400,000 at a maximum fill time of 50 ms. Dynamic exclusion was set after 1, with an exclusion duration of 60 s. Charge states 2 to 4 were included. For MS2, precursors were isolated with the quadrupole, with an isolation width of 1.2 Da. First mass was set to 110 Da. The MS2 scan resolution was 30,000, with an AGC target of 50,000 at a maximum fill time of 60 ms.The Lumos mass spectrometer was operated in data-dependent MS/MS mode for a cycle time of 3 s, with the higher-energy C-trap dissociation (HCD) collision energy at 32 V and recording of the master scan 2 (MS2) spectrum in the Orbitrap. In MS1, the resolution was 120,000 and the scan range C. difficile strain 630 database  or human canonical database . The mass tolerances for MS1 and MS2 were set to 4.5 and 20 ppm, respectively. Trypsin was selected as the enzyme with a maximum of two missed cleavages. Carbamidomethylation of cysteines was selected as a fixed modification and phosphorylation on serine, threonine, and tyrosine; in addition, oxidation of methionine and acetylation of the protein N terminus were selected as variable modifications. The match-between-runs option was selected with a window of 2 min. The false-discovery rate at the peptide level was set to 1%, and for modified peptides, a score cutoff of 40 was used.MaxQuant software (version 1.5.1.2) was used to process the raw data files, which were searched against the The MaxQuant output table “phospho (STY)Sites.txt”  was usedC. difficile as reported by the Gene Ontology database hosted by EBI (as of 15 August 2021). For the functional analysis and visualization, we used R 3.6.2 and the Bioconductor library Cluster Profiler.Gene ontology protein/gene set enrichment analysis was performed using a sorted list of all identified phosphoproteins at each analysis time point, i.e., mid-exponential, beginning stationary, and late stationary growth phases. The sorting was based on the abundance of the phosphoproteins identified and determined by the ion intensity, as reported by the mass spectrometer in the output of MaxQuant . We considered the phosphoproteins that were reported at least once in any of the three replicates. Each protein identification was considered only once in the sorted list, with a rank determined by the most intense results that we have obtained in any of the three replicates by any protein-associated phosphopeptide. The enrichment was performed using the weighted Kolmogorov-Smirnov-like statistic as implemented in the common gene set enrichment analysis. We limited our analysis to the top 30 enriched gene ontology terms. We generated enrichment maps that represent the relationship between the enriched terms as a network. The edges of an enrichment map correspond to the number of shared proteins between the associated terms. These maps give a higher-level overview of the enrichment analysis and allow identifying functional modules with ontology terms that are related to each other by the underlying protein set used. For the functional analysis, we used all gene ontology annotations of PXD029475.All data are contained within the manuscript or the associated supplemental material or are available from the authors upon request. The mass spectrometry proteomics data have been deposited into the ProteomeXchange Consortium via the PRIDE partner repository  with the