To minimize the potential consequences of an introduction of foot-and-mouth disease (FMD) in Europe, European Union (EU) member states are required to present a contingency plan. This study used a simulation model to study potential outbreak scenarios in Sweden and evaluate the best control strategies. The model was informed by the Swedish livestock structure using herd information from cattle, pig, and small ruminant holdings in the country. The contact structure was based on animal movement data and studies investigating the movements between farms of veterinarians, service trucks, and other farm visitors. All scenarios of outbreak control included depopulation of detected herds, 3 km protection and 10 km surveillance zones, movement tracing, and 3 days national standstill. The effect of availability of surveillance resources, i.e., number of field veterinarians per day, and timeliness of enforcement of interventions, was assessed. With the estimated currently available resources, an FMD outbreak in Sweden is expected to be controlled  within 3 weeks of detection in any evaluated scenario. The density of farms in the area where the epidemic started would have little impact on the time to control the outbreak, but spread in high density areas would require more surveillance resources, compared to areas of lower farm density. The use of vaccination did not result in a reduction in the expected number of infected herds. Preemptive depopulation was able to reduce the number of infected herds in extreme scenarios designed to test a combination of worst-case conditions of virus introduction and spread, but at the cost of doubling the number of herds culled. This likely resulted from a combination of the small outbreaks predicted by the spread model, and the high efficacy of the basic control measures evaluated, under the conditions of the Swedish livestock industry, and considering the assumed control resources available. The results indicate that the duration and extent of FMD outbreaks could be kept limited in Sweden using the EU standard control strategy and a 3 days national standstill. Picornaviridae, genus Aphthovirus) causes an acute vesicular disease in cloven-hoofed animals. Seven FMDV serotypes have been described, with cross-protection among serotypes not being observed: O, A, C, Asia1, and SAT1, SAT2, SAT3 (Foot-and-mouth disease (FMD) is described by the World Organisation for Animal Health (OIE) as “the most contagious disease of mammals” . The FMDIn 2000 and 2001, outbreaks in the Republic of Korea, Japan, Russia, Mongolia, South Africa, the United Kingdom, Republic of Ireland, France, and the Netherlands were caused by FMDV of serotype O (of a particular genetic lineage named the PanAsia strain) . The EurMathematical modeling was used extensively during the 2001 FMD outbreak, especially in the UK, which was most severely affected –10. SincBetween-herd spread is simulated using an agent-based model that simulates FMD spread through direct and indirect contact. Within-herd spread is modeled as a compartmental model based on the work of Carpenter et al.  is a stochastic simulation model developed at the University of Davis  and progr et al. , and parr et al. , as detar et al. . SeveralWe used a simulation model to study potential outbreak scenarios in Sweden in case of an introduction of FMD, assess their expected magnitude, and evaluate control strategy options. The model developed is a result of the partnership between epidemiologists from the Swedish National Veterinary Institute (SVA) and the Danish team that developed the spread model DTU-DADS at the Technical University of Denmark (DTU). SVA and the Swedish Board of Agriculture (SJV) worked together to define the main questions to be addressed, and the needed support to the decision-making process of drafting a contingency plan. Emphasis was given to the comparative effect of different control measures.The DTU-DADS spread model [version 0.15 ] was adaAn overview of the stochastic events simulated in the model is given in Figure Table All disease transmission parameters that were thought to be readily applicable from the Danish to the Swedish livestock population, or to be independent from the host population (intrinsic pathogen properties) were kept as set up in the DTU-DADS model, as explained individually for the parameters in Table To adapt the model to the Swedish livestock population, specific data were collected for all FMD susceptible herds in Sweden, including animal movement data, as shown in Table The model considers each group of animals from the same species, within the same farm, as one herd, and models herds individually; if a farm contains cattle and pigs, for example, cattle and pig herds are modeled individually. A farm ID is used to keep track of herds in the same farm, and enforce control measures in all herds within a farm equally. If for instance one of the herds is detected as infected, all herds belonging to the same farm are culled. A high probability of local area spread within 100 m is used to account for horizontal transmission between herds in the same farm.Outbreaks were modeled under different scenarios of disease introduction, to assess the effect of different population parameters in the development of the outbreak. In each of 21 base scenarios, outbreaks were set to start in a herd of a particular type, and in each iteration, the first infected herd was randomly selected among all herds of that type. Seven scenarios had infection seeded in the south of Sweden, in one specific herd type ; another seven scenarios were related to the same herd types, but seeded in the north of Sweden; and finally herds were chosen based on the frequency of direct animal contacts in a year . In addition, spread was also evaluated when 2, 3, or 4 initial seeds were set (number of infected herds to start the epidemic), all in cattle herds. The evaluated scenarios are listed in Table base control scenarios the mandatory conditions determined in the EU Council Directive 2003/85/EC were implemented, and in addition a 3-day national standstill:Culling of all animals in detected FMD-positive farms and their high risk contacts .Establishing of a 3 km protection zone and a 10 km surveillance zone around every detected farm. Susceptible animals’ movement prohibition is kept in these zones for 30 days, a period during which all herds are visited for clinical inspection twice, starting from the protection zone. More details are given in Presentation S1 in Supplementary Material .3-day national standstill, i.e., ban of all susceptible animal movements after first detection.Base scenarios were simulated using a fixed control strategy (here we use “control strategy” to denote a specified collection of “control measures”). In these A reduction in the number of indirect contacts among farms after detection of the outbreak was also enforced, as per parameters listed in Table base control scenarios was chosen to evaluate the effect of applying alternative control strategies. The choice was based on the evaluation of the base control scenarios and is described in the results. Alternative disease control measures were evaluated, using a range of parameters listed in Table Preemptive depopulation (ring culling), for different ring radii, and triggered when a determined number of detected infected herds was reached, or outbreak control was not reached after a number of days;Ring vaccination (vaccination to cull), using different ring radii, and also dependent on the number of detected infected herds reached, or the outbreak length.After the effects of different scenarios of disease introduction were evaluated with this base control strategy, one of the worst-case scenarios were chosen. Sensitivity analysis was carried out in these worst-case scenarios to ensure that the effect of different parameters could be more easily identified. Model sensitivity was evaluated against a range of values for the detection day and the effectiveness of the alternative control measures, and variation in the amount of surveillance resources available , and a cumulative number of failures in the effectiveness of all control measures applied. These conditions were intended to mimic an epidemic that starts and develops with a much greater magnitude than expected, compared to the typical outbreak scenarios modeled previously, or an epidemic that gets out of control. The effectiveness of specific control measures were challenged against this chaotic scenario ;Total number of herds infected during the course of the epidemic;Total number of herds visited by surveillance teams ;Total number of herds culled, and total number of animal in these herds;Total number of herds vaccinated, and total number of animals in these herds.Table Ten-thousand iterations of scenario 1 showed that output medians and interquartile ranges were stable after 500 iterations, but the maximum varied due to longer epidemics observed in individual iterations when more repetitions were run. As a compromise between achieving higher variability and keeping computational time manageable, 1,000 iterations were simulated for each scenario.The progression of scenarios described above focused on testing the model sensitivity to the control measures. To also evaluate the structure of the model, and the impact of the parameters that were imported from the Danish model, the transmission parameters listed in Table In general, the results showed that an FMD outbreak in Sweden would most likely be small and of short duration, and that base control measures as specified in the EU legislation, complemented with a 3-day national standstill of all susceptible animals movements, would be enough for bringing the outbreak under control. Considering the 24 base scenarios evaluated, the median epidemic duration (time from detection of the first infected herd to the day in which the last herd was detected) was 3–15 days, and the median number of infected herds was 2–19 . The 95% percentiles were for an epidemic of 20 days, involving 15 infected herds and culling nearly 5,000 animals.Summary statistics for all the scenarios evaluated are presented in Table S2-1 in Presentation S2 in Supplementary Material and Table The results of the base scenarios Figure  showed tThe effect of species and herd types seemed to be a direct effect of the contact network structure for each herd type. Epidemics starting in sheep/goats herds were generally smaller, due to a lower probability of direct  and indirect (people movement) contacts. Cattle herds had very different results depending on whether it was a dairy herd or not, reflecting the larger number of indirect contacts expected daily in herds with milking animals. Outbreaks starting in pig herds in general resulted in an average epidemic size between milking and non-milking cattle herds. The main impact of starting epidemics in pig herds was the higher number of animals that were culled, a reflection of their much larger herd size . As epidemics were generally small, with only a few herds being culled, the size of the seeding herd had high impact on the total number of culled animals. Only the number of animals culled is shown in Figure The number of pig herds in Sweden is very small, and as a result, epidemics that started in pig herds were ultimately driven by spread among cattle herds, as we could conclude from extensive analysis of the base control scenarios. Since cattle herds seemed to be driving spread, and the contact network (direct and indirect contacts) was the main driver of the epidemic size, a “typical outbreak scenario” was chosen as one starting in a cattle herd in the south of Sweden, with an average number of yearly direct contacts. This scenario was chosen to test the effect of alternative control measures, as shown in Table The main result for all scenarios designed to evaluate the effect of alternative control measures  showed that the number of herds to be visited per day only exceeded the capacity of surveillance immediately after detection in the median outbreak, with herds waiting at most a day to be visited in the cases when the outbreak was controlled within a week. Epidemics in iterations placed above the 90% percentile could take up to 19 days to be controlled. In those cases the number of days a herd would have to wait to be visited could be as high as 14, with daily medians ranging from 0 to 8.Sensitivity analysis were performed using three “worst-case scenarios” to magnify the observed effectiveness of control measures, which could have been hard to observe in the small outbreak sizes associated with the “typical outbreak scenario.” The sensitivity analysis showed that these results were robust for the range of parameters tested in the sensitivity analysis , the median number of infected herds in the 17 scenarios tested with detection on day 21 (but varying the efficacy of various control measures) ranged 10–11, and the median epidemic duration was always 13 days. Detection on days 28 and 35 increased the median number of infected herds to 30 and 61, respectively, and resulted in a median epidemic duration of 17 and 21 days.When the epidemic was seeded in four cattle herds at the same time (worst-case scenario C), but detection was not delayed, the median number of infected herds in all sensitivity analysis scenarios evaluated varied between 19 and 20 herds, with median epidemic duration varying between 15 and 16 days. Detection on days 28 and 35 increased the median number of infected herds to 40 and 87, respectively, and resulted in a median epidemic duration of 19 and 24 days.Sensitivity analysis for the transmission parameters showed that the results were very robust to changes in punctual transmission parameters. As for the previous analysis, this was particularly true in scenarios with low expected number of infected herds. In the “typical outbreak scenario,” for instance, changes of up to 20% in the probability of transmission following direct contact did not change the median number of infected herds. Evaluation of the percentage of all transmission events, over all iterations in that scenario, showed that about 45% were a result of direct contact, and 5% of movement to slaughter. This resulted in robustness of the model to changes in the probabilities of transmission associated with slaughter movements. About 28% of the transmission events were due to indirect contact (low and medium risk contacts), and 22% due to local spread. The probability of local spread within 100 m was kept high in all scenarios to ensure transmission between herds within the same farm. As expected, increases in the probability of transmission for other distances resulted in a higher number of infected herds, but a fivefold increase in the probability of transmission within 1 km, for instance, only increased the median number of infected herds in the typical scenario by about two herds.Based on the results of scenarios presented above, a cutoff of 10 detected infected herds was set as a decision point for when authorities should start considering that the outbreak was not being brought under control. In all base scenarios the expected number of infected herds was under 10, and only higher in scenarios with multiple starting seeds or failures in the effectiveness of control measures. The effect of deciding to implement ring culling or vaccination after this threshold was reached was evaluated in the chaos scenarios, and results are presented in Figure The scenario with infection seeded in four cattle herds in the south of Sweden at the same time, and detection after 4 weeks (base chaos scenario), resulted in a median number of 42 infected herds (95% percentile of 83 herds), and an epidemic duration of 20 days between detection of the first and last herd (95% percentile at 33 days). This is assuming that all base control measures would be applied, and the surveillance capacity would be at a regular level, but all applied control measures would be 15% less effective than in the base scenarios . Increasing the period of standstill was not effective in reducing the number of infected herds nor the epidemic duration. Ring vaccination was not effective in reducing the median number of herds infected, although the median epidemic duration was reduced by 1 day (median 19 and 95% percentile of 28 days).The implementation of preemptive depopulation of all susceptible animals, in a radius of 1 km around every infected farm, would reduce the median epidemic duration in the chaos scenarios by 4 days . The median number of infected herds was reduced to 38 (95% percentile at 72 herds). As a consequence of the reduction in the number of infected herds (fewer surveillance zones to be established), the median number of visited herds was reduced from 1,075 in the base chaos scenario to 875 when culling was applied . The median number of culled herds, however, was increased from 42 to 86 , and the median number of animals culled from 6,682 to 12,789 .A disease spread model was adapted to the Swedish livestock structure to evaluate the effect of different control strategies and inform FMD preparedness in Sweden. Results showed that an FMD introduction in Sweden will most likely spread slowly and be readily contained with adoption of a control strategy combining the control measures required in the current EU legislation for the control of FMD (Council Directive 2003/85/EC), and a national standstill. The detailed control strategy is: a 3-day prohibition of all movements of susceptible animals after first detection (standstill), 3 km protection zones and 10 km surveillance zones around every detected farm, and culling of all animals in detected farms and their high risk contacts.The results of the model are not meant to be interpreted as a strictly quantitative representation of reality. The application of models to decision-making, in general, should serve primarily as a means for comparing the effectiveness of different control measures, and assessing the comparative magnitude of various scenarios to understand the main outbreak drivers and the most important control targets . While wOur results are a direct contrast to those observed when the same model was applied in Denmark, where the adoption of additional measures such as protective vaccination and ring depopulation were concluded to be cost-effective on most scenarios of spread . The conAnother difference to the original Danish model is that a reduction in the number of indirect contacts between farms after outbreak detection was assumed. The assumption that people would reduce all unnecessary traffic from and to their farms, once an outbreak is known to be occurring in the country, is based on feedback from farmers . It is aThe model was scrutinized by individual evaluation of multiple iterations per scenario, and mapping of every modeled transmission event, including the mode of transmission (direct or indirect contact). This confirmed that epidemic size was mainly driven by infected cattle farms. It also confirmed the expected effect of long distance movements in keeping North and South of Sweden highly inter-connected .The choice of a predefined detection date (21 days after seeding the infection) was based on extensive review of information from previous outbreaks performed by the Danish team that developed the DTU-DADS model , 13. ComThe DTU-DADS model  did not allow adjustment of the surveillance capacity along the outbreak, that is, surveillance resources are fixed for the whole period of the epidemic. The surveillance capacity used in this model was based on what the Swedish Board of Agriculture considered feasible to gather in the first 1–3 days after detection of the first suspicion (and therefore arguably at the same time or shortly after confirmation). In reality, the number of surveillance resources could be increased after a few days of outbreak control. In the “typical outbreak scenario,” only in exceptional epidemics the number of herds that needed to be visited for clinical surveillance was greater than the number of teams available for field visits , and a herd queue was generated. The culling capacity, however, was never exceeded. Most of the farms in the model had herds smaller than the daily culling capacity per team declared by the Swedish Board of Agriculture and used in the model. Moreover, the number of herds that needed to be culled in the same day was very small.The relatively small expected outbreak size and low demand for surveillance resources in all scenarios resulted in a high observed efficacy of the base control measures. In all evaluated scenarios, even the most chaotic ones, an FMD epidemic is expected to be controlled within 3 weeks from the detection of the first case. The number of herds infected is small, and most of the surveillance effort needed will be to visit farms that fall into the surveillance zones around each infected farm, to rule out infection. Surveillance capacity was not often exceeded. In epidemics that took longer than 2 weeks to control, herds could eventually wait longer than 2 days to be visited by a surveillance queue. However, herds in queue were those that needed to be visited because they fell within the surveillance zone. Suspected farms and high risk contacts are given priority in the surveillance visiting list, and therefore can be visited on the day of detection/tracing, as long as the number of infected herds and their direct contacts is below the number of surveillance teams, as was the case in all scenarios evaluated.The base control measures were not only predicted to be effective, they were also robust. Reductions of up to 40% in the efficacy of a single measure can be compensated if everything else is assumed to be working properly. The number of infected herds was more sensitive to failures in control than the expected epidemic duration, due to the reasons discussed above.Direct contact and local spread were the main modes of disease transmission. The central role of direct contact transmission is expected . In thisThe worst-case scenarios observed were those related to multiple introductions at the same time, and delayed detection of introduction. Even in those cases, a reduction in the expected number of infected herds as a result of the application of vaccination could not be demonstrated. Preemptive depopulation had an effect in reducing the median number of infected herds when very large epidemics were modeled (multiple introductions and late detection). Considering, however, that this measure would double the median number of herds and total animals to be culled, cost–benefit analysis will be needed to determine whether the benefits of applying this measure would justify the costs both in resources and animal welfare. As the current results indicate that the effect of preemptive depopulation can only become relevant for very large epidemics, this measure should only be considered after a large number of infected herds have been detected. Models exploring scenarios of FMD spread in the UK and Denmark have shown that ring culling can have a positive effect in specific circumstances , 31. We While the base control strategy recommended based on this work is expected to be effective, it should be highlighted that the overall costs to the society and governmental agencies, as well as the workload, should not be overlooked. Effective control is associated with prompt implementation of a contingency strategy that would require deployment of 40 field surveillance teams per day and capacity to destroy thousands of animals per day. And for the control to be efficient, additional teams on central and regional level are needed working with contact tracing, data analysis, dissemination of information, logistics, etc., although these functions have not been included as a limiting factor in the model.In summary, a potential FMD outbreak in Sweden is expected to be small and controlled fast through a 3-day national standstill, application of surveillance zones around infected farms, and culling of all animals in detected farms and their high risk contacts. This result is based on the assumption that detection would not be delayed by more than 4–5 weeks after introduction, that these measures would be enforced quickly after detection, and that the effectiveness of these control measures can be expected to fall within the range of values evaluated in this work.FD adapted the infectious disease model to Sweden, parameterized and ran the model, summarized results, and wrote the manuscript. MN, JF, and KS helped parameterize the model, set relevant scenarios to be evaluated, and analyze and interpret results. SW helped adapt the model to Sweden, prepare Swedish data, and evaluate model behavior. AB and TH wrote the initial infectious disease model, trained the group into using it, and helped inspect model behavior and interpret results once the model was adapted to Sweden.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Really interesting new gene (RING)-finger protein 52 (RNF52), an E3 ubiquitin ligase, is found in eukaryotes from yeast to humans. Human RNF52 is known as breast cancer type 1 susceptibility protein (BRCA1)-associated protein 2 (BRAP or BRAP2). The central catalytic domain of BRAP comprises four subdomains: nucleotide-binding α/β plait (NBP), really interesting new gene (RING) zinc finger, ubiquitin-specific protease (UBP)-like zinc finger (ZfUBP), and coiled-coil (CC). This domain architecture is conserved in RNF52 orthologs; however, the domain's function in the ubiquitin system has not been delineated. In the present study, we discovered that the RNF52 domain, comprising NBP–RING–ZfUBP–CC, binds to ubiquitin chains (oligo-ubiquitin) but not to the ubiquitin monomers, and can utilize various ubiquitin chains for ubiquitylation and auto-ubiquitylation. The RNF52 domain preferentially bound to M1- and K63-linked di-ubiquitin chains, weakly to K27-linked chains, but not to K6-, K11-, or K48-linked chains. The binding preferences of the RNF52 domain for ubiquitin-linkage types corresponded to ubiquitin usage in the ubiquitylation reaction, except for K11-, K29-, and K33-linked chains. Additionally, the RNF52 domain directly ligated the intact M1-linked, tri-, and tetra-ubiquitin chains and recognized the structural alterations caused by the phosphomimetic mutation of these ubiquitin chains. Full-length BRAP had nearly the same specificity for the ubiquitin-chain types as the RNF52 domain alone. Mass spectrometry analysis of oligomeric ubiquitylation products, mediated by the RNF52 domain, revealed that the ubiquitin-linkage types and auto-ubiquitylation sites depend on the length of ubiquitin chains. Here, we propose a model for the oligomeric ubiquitylation process, controlled by the RNF52 domain, which is not a sequential assembly process involving monomers. The modifier protein ubiquitin is structurally conserved in eukaryotic cells; post-translational modification with ubiquitin is known as ubiquitylation or ubiquitination –3. TargeRING-finger protein 52 (RNF52), a type of E3, is broadly expressed in eukaryotes from yeast to humans . The humThe central catalytic core region of BRAP has four subdomains, namely an NBP (nucleotide-binding α/β plait) motif, a RING zinc finger motif, a ZfUBP [ubiquitin-specific protease (UBP)-like zinc finger] motif, and a CC (coiled-coil) motif. The domain architecture of this region, encompassing these four subdomains, is especially conserved in RNF52 orthologs (Supplementary Data S1) and so can be referred to as the RNF52 domain. Nevertheless, the precise function of the RNF52 domain, comprising NBP–RING–ZfUBP–CC, remains unknown; thus, the underlying E3 activity of BRAP in the ubiquitin system has not been characterized.Here, we explored how the RNF52-domain functions with regard to the E3 activity of BRAP in the ubiquitylation process. We report on the characteristics of the RNF52 domain of BRAP with respect to ubiquitin chains (oligoubiquitin) and discuss a novel concept of ubiquitylation, utilizing oligoubiquitin, and catalyzed by the RNF52 domain.The approved HGNC [human genome organization (HUGO) Nomenclature Committee] name and UniProtKB (UniProt Knowledgebase) ID numbers are listed as follows: UBC (ubiquitin), P0CG48; UBA1 [also known as UBE1 (ubiquitin-activating enzyme E1)], P22314; UBE2D1 , P51668; and BRAP , Q7Z569. The accession numbers for the RNF52 orthologs from other species are shown in Escherichia coli, and for cell-free synthesis-coupled transcription–translation, the target cDNA fragments were sub-cloned into the pCR2.1-TOPO vector . Proteins, subjected to affinity purification, were N-terminally fused with a modified natural poly-histidine N11-tag (amino acid sequence: MKDHLIHNHHKHEHAHAEH) or a maltose-binding protein (MBP) tag, as well as with a TEV (tobacco etch virus) protease recognition site and a GSSGSSG linker sequence   .The N-terminal affinity-tagged recombinant BRAP 124–569), BRAP (1–592), and a RING-finger motif mutant (C264S), in which the first cysteine residue in the RING-finger consensus sequence was replaced with a serine residue ,16,17, w–569, BRAPurified protein concentrations were determined by measuring the absorbance at 280 nm using a NanoDrop spectrophotometer (Thermo Fisher Scientific).in vitro ubiquitylation assay of the RNF52 domain of BRAP was performed in a 25-µl reaction mixture containing 20 mM HEPES–NaOH at pH 7.5, 2 mM MgCl2, 50 nM UBA1 (for E1 activity), 0.5 µM UBE2D1 (for E2 activity), 1 µM BRAP (for E3 activity), 10 µM ubiquitin or ubiquitin chains, and 5 mM ATP (adenosine triphosphate). The reaction mixtures without ATP were preincubated on ice for 20 min; thereafter, the ubiquitylation reaction was initiated by adding ATP followed by incubation at 37°C for 60 or 90 min. To screen for BRAP activity in 34 different human E2s, we used E2scan Kit version 2 . The reactions were stopped by adding 20 µl of 2.5× SDS–PAGE (sodium dodecyl sulfate–polyacrylamide electrophoresis) sample loading buffer  and incubated at 95°C for 3 min.The The ubiquitylation reactions were resolved by SDS–PAGE on a 10–20% gradient gel and visualized by Coomassie Brilliant Blue (CBB) G-250 staining. For quantification of the protein band signals, the CBB-stained gel images were acquired using the LAS-3000 imaging system  with white light transillumination. The reaction products were also detected by western blot (WB) analysis using an anti-BRAP clone D-5 , which is a monoclonal antibody raised against amino acids 41–340 of BRAP and anti-multi Ub antibody clone FK2 , which recognizes both mono- and poly-ubiquitinylated species but not free ubiquitin. The WB signals for auto-ubiquitylated or non-ubiquitylated BRAP (124–569) were detected using an Immobilon Chemiluminescence Detection Kit , and the luminescent signals were acquired using the luminescent image analyzer in LAS-3000. The protein band signals from CBB-stained gels, and from WBs using the anti-BRAP antibody (clone D-5), were quantified using the ImageQuant TL software .For mass spectrometry analyses, gel regions, containing proteins, were excised and digested with trypsin (Merck KGaA) for 20 h at 37°C. The resulting peptides were analyzed by liquid chromatography–electrospray ionization tandem mass spectrometry (LC–ESI-MS/MS) at the RIKEN BSI Research Resources Center.N-hydroxysuccinimide; for immobilization, sodium acetate–acetic acid buffer solutions at pH 5.0 for ubiquitin chains and at pH 4.5 for the ubiquitin monomer; for deactivation, 1 M ethanolamine–HCl at pH 8.5. Mobile molecules  were serially diluted in running buffer . The buffer was filtered with a 0.22-µm vacuum filter and degassed prior to use. All experiments were performed at 25°C. The SPR sensorgrams, obtained for the control flow cell, were subtracted from the data for the flow cell immobilized with a ligand. Protein–protein interaction experiments, using monomer ubiquitin and M1-linked and K63-linked di-ubiquitin chains with BRAP (124–569) (RNF52 domain) or BRAP (1–592) (full-length), were repeated at least three times. The experiments using other ubiquitin linkages were performed once using full-length and the RNF52 domain, respectively. The binding affinity and interaction kinetics in each experiment were obtained from Biacore sensorgrams using the BIAevaluation software version 4.1.1  according to its instruction manual.SPR (surface plasmon resonance) sensorgrams, showing the profiles of protein–protein interactions, were obtained from ligand-binding experiments based on purified recombinant proteins (Supplementary Data S2), using the Biacore 3000 system . For the Biacore assays, we immobilized the ligand to a CM5 sensor chip  using an Amine Coupling Kit  under the following conditions: for activation, 0.4 M 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and 0.1 M Because the domain architecture of BRAP contains ZfUBP–CC motifs , and conBased on these observations, we first examined whether the RNF52 domain of BRAP recognizes ubiquitin monomers and/or ubiquitin chains. In the first experiment, we used K6-, K11-, K27-, K29-, K33-, K48-, K63-, and M1-linked di-ubiquitin chains  to examine whether the RNF52 domain of BRAP binds to all linkage types of oligo-ubiquitin. To perform this protein–protein interaction assay, we used purified protein samples of monomeric ubiquitin, ubiquitin chains, and a fragment containing the entire RNF52 domain of BRAP  (Supplementary Data S2). The RNF52 domain location of BRAP was determined based on multiple amino acid sequence alignment of RNF52 orthologs (Supplementary Data S1) and predictions for protein secondary structure .Kd (9.51 × 10−8 M) and the koff (8.99 × 10−3 s−1) of BRAP for M1-Ub were not significantly different from the Kd (8.00 × 10−8 M) and koff (8.11 × 10−3 s−1) for K63-Ub. Compared with the kon (9.45 × 10−5 M−1 s−1) for M1-Ub2, the kon (1.01 × 10−5 M−1 s−1) for K63-Ub was slightly higher for K63-Ub2 than for M1-Ub2. The binding affinity of BRAP (124–569) for K27-Ub2 was weaker than that for M1-Ub2 and K63-Ub2. In this experiment, the Biacore sensorgrams indicated that BRAP (124–569) may be bound to K29-Ub2 and K-33-Ub2; however, the response signals were too weak to determine binding affinity.Biacore sensorgrams (protein–protein interaction profiles)  showed tWe next examined the binding preference of full-length BRAP (1–592) for different ubiquitin-linkage types . The binin vitro ubiquitylation assay in the presence of a RING-finger motif mutant (RING-finger defective mutant) of BRAP (C264S) and confirmed that these polyubiquitylation reactions were dependent on the activity of BRAP. These results clearly show that the most suitable E2 partners of BRAP, for ubiquitin-to-ubiquitin ligation, were members of the UBE2D family . UBE2D proteins are Class I E2s and have a bare UBC fold , non-binding type [−], and faint-binding type [+/−]. K29- and K33-linked chains are categorized into the faint-binding type [+/−], rather than the binding type [+] or the non-binding type [−]  on the usage of M1-linked chains and found that BRAP (124–569) can use these phosphomimetic ubiquitin chains  for ubiquitylation and auto-ubiquitylation . HoweverUsing several chain lengths of M1-linked ubiquitin chains (ubiquitin oligomers), including the S65D-phosphomimetic forms, showed that oligomeric ubiquitylation with M1-Ub4 was hardly affected by the phosphomimetic mutation compared with those using M1-Ub2 and M1-Ub3. Therefore, we used a Biacore assay for detailed assessment of protein–protein interactions to examine whether the RNF52 domain of BRAP recognizes differences in these ubiquitin chains.The Biacore sensorgrams for the interaction between BRAP (124–569) and M1-Ub4, or the interaction between BRAP (124–569) and the S65D-M1-Ub4 mutant, differed from the ones for BRAP (124–569) binding to M1-Ub2/S65-M1-Ub2 and M1-Ub3/S65-M1-Ub3 . The binBased on these experiments using BRAP (124–569) and M1-linked ubiquitin chains of various lengths , we conclude that the RNF52 domain of BRAP recognizes the structural differences between these modified ubiquitin chains before it ligates them.We employed MS to analyze the reaction products of the oligomeric ubiquitylation using Ub2, Ub3, and Ub4, in order to explore ubiquitin-linkage types and auto-ubiquitylated sites on the RNF52 domain. A summary of the results is shown in n (n > 2) (Supplementary Data S8A), whereas K11 and K48 were detected in samples [M1-Ub3]n and [M1-Ub4]n .Peptide mass spectrometry, conducted on a gel slice obtained from a high molecular mass area of SDS–PAGE , detecteIn the SDS–PAGE analysis of the reaction products derived using M1-Ub2, we observed extra bands, indicating that two Ub2s had become connected (n (Supplementary Data S9A1 and SA2); K154, K246, and K507 were detected in sample BRAP (124–569)–[M1-Ub3]n (Supplementary Data S9B); and K381 was detected in sample BRAP(124–569)–[M1-Ub4]n (Supplementary Data S9C).Moreover, MS analysis of auto-ubiquitylated sites, produced during the oligomeric ubiquitylation reaction with M1-Ub2, -Ub3, and -Ub4, revealed that these auto-ubiquitylated sites differ from each other ]. The RNF52 domain preferentially binds to ubiquitin chains (oligo-ubiquitin), but not to the ubiquitin monomer. The RNF52 domain can utilize not only ubiquitin monomers, but also ubiquitin chains for ubiquitylation. Interestingly, use of oligo-ubiquitin was significantly higher than that of mono-ubiquitin in the RNF52 domain catalyzed ubiquitylation  and 5. FHowever, the molecular mechanism enabling this oligomeric ubiquitylation remained unclear. A recent crystal structural analysis of M1-linked chains demonstrated that ABIN-2, which belongs to the UBAN family , recognitrans-thioesterification reaction. While the E2-Ub complex is tethered transiently to the RING subdomain, E2 is released from the RNF52 domain. UBE2D1 has weak binding affinity for the RNF52 domain (Supplementary Data S4B); the RNF52 domain holds the oligo-Ub in place. In contrast, the RNF52 domain, bound to the donor oligo-Ub, recruits another RNF52 domain, which holds the acceptor oligo-Ub; these are then combined and form a ubiquitin link via covalent attachment of the C-terminal G from donor oligo-Ub to M  or K  of the acceptor oligo-Ub. In our study, the RNF52 domain bound preferentially to M1-Ub2 and K63-Ub2 , controlled by the RNF52 domain of BRAP . In this K63-Ub2 , but notIn the ubiquitin system, the conventional model for the mechanism of ubiquitin-chain assembly indicates that ubiquitin-chain elongation occurs one-by-one ,7. Sequein vivo M1-linked ubiquitin oligomer connected via K6, K11, K33, K48, or K63 linkages, or the biological function of oligomeric-ligated ubiquitin complexes generated by the combination of the RNF52 domain of BRAP and UBE2D1. From a biological perspective, BRAP was reported to be involved in the inhibitor-κB kinase/NF-κB signaling cascade [The present study did not examine the existence of oligomeric ubiquitylation  cascade ,15,42. B cascade , which c cascade . It has  cascade ,45. In a cascade . Based oThe existence and subcellular localization of ubiquitin system components, including E2, E3, pools of cellular ubiquitin, and ubiquitin chains, are very diverse in cells and tissues ,48. In o


T-TaS2 using scanning tunneling spectroscopy. We establish that the domain wall state decomposes into two nonconducting states located at the center of domain walls and edges of domains. Theoretical calculations reveal their atomistic origin as the local reconstruction of domain walls under the strong influence of electron correlation. Our results introduce a concept for the domain wall electronic property, the walls own internal degrees of freedom, which is potentially related to the controllability of domain wall electronic properties.Domain walls in interacting electronic systems can have distinct localized states, which often govern physical properties and may lead to unprecedented functionalities and novel devices. However, electronic states within domain walls themselves have not been clearly identified and understood for strongly correlated electron systems. Here, we resolve the electronic states localized on domain walls in a Mott-charge-density-wave insulator 1 T-TaS2 decomposes into two localized but nonconducting states at the center or edges of domain walls.The electronic states within domain walls in an interacting electronic system remain elusive. Here, Cho et al. report that the domain wall state in a charge-density-wave insulator 1 CDW with periodic modulations of charge/lattice at low temperature and their domain walls are widely observed in metallic layered transition metal dichalcogenides12. Among them, 1T-TaS2 shows a series of CDW transitions coupled to substantial electron-electron interaction. Especially, a metal-insulator transition occurs below 190 K through a transition from a nearly commensurate to a commensurate CDW state13. The insulating ground state is realized by electron correlation on narrow electron bands formed by the commensurate CDW lattice14. Recent experimental results have demonstrated that the correlated insulating ground state can be transformed into various quasimetallic and superconducting CDW states by external and internal control parameters, such as pressure8, optical  excitation20, and chemical doping23. These conductivity switchings are intrinsically very fast, which may make possible novel ultrafast devices based on correlated electrons.Conductive domain walls and their functionalities17 and/or to screen electron correlation within Mott-CDW domains21. Moreover, the emerging superconductivity out of the Mott-CDW insulating phase has been considered being directly related to conducting domain walls24. However, despite such long discussion, the electronic states of domain walls have not yet been clarified spectroscopically. Here, we show that the domain walls in 1T-TaS2 have two well-confined and non-metallic in-gap states above Fermi level (EF) using scanning tunneling microscopy and spectroscopy (STM and STS). They are located on the domain wall center and edges of neighboring domains, respectively. The theoretical calculations strongly suggest the substantial correlation effect in forming spatially decomposed and non-metallic domain wall states.In the metallic excited states, CDW domain walls are common objects and considered as the origin of the metallic property. They have been suggested as highly conducting channels themselvesT-TaS2 at 4.3 K. The unit layer of 1T-TaS2 consists of a Ta layer sandwiched by two S layers with each Ta atom coordinated octahedrally by S atoms. Unpaired 5d electrons of Ta form a half-filled metallic band which is unstable against the CDW formation driven by strong electron-phonon coupling. Ta atoms undergo a reconstruction into a so-called David-star unit cell which is composed of 13 Ta atoms; the 12 outer atoms pair up and shift toward the central one25, where a unpaired 5d electron is left over26. In the commensurate CDW phase, David-star unit cells  and the fourth (lower) one following the atomic indices of Fig. 19.In spite of the clear commensurability and the long range order of the undoped low temperature phase, there exist one-dimensional intrinsic defects, domain walls, across which the phase of CDW is abruptly shifted see Fig. . Since t1a19. In the present work, for example, STS spectra were measured at each pixel of the STM image containing two domain walls of different but most popular types  clearly show the zero conductance region at EF  within the band gap observed here is in line with localized states suggested in sharp phase kinks of order parameters in charge or spin ordered insulatorsT-TaS2 have well-localized non-metallic in-gap states along the center of domain walls and the edges of neighboring domains. Not only the lack of free carriers but also the lack of any substantial screening or doping effects by domain walls are disclosed unambiguously. These results request to rewrite most of the current scenarios on the metallic and superconducting phases emerging from this correlated insulator. The origin of the split non-metallic domain wall states is clearly understood as due to the reconstruction within domain walls composed of multiple atoms and electrons under the substantial influence of electron correlation. That is, the internal structural and electronic degrees of freedom within domain walls are indicated to be very important. The internal structural degrees of freedom of domain walls were recently noticed for one34 and two-dimensional30 systems, but to the best of our knowledge, the substantial electron correlation effect within a domain wall has not been observed before. These internal degrees of freedom might be exploited to provide controllability of domain wall electronic properties for the functionalization of complex materials with domain walls.In summary, we discover distinct electronic states of domain walls of a symmetry-broken correlated insulator for the first time. The domain walls of the Mott-CDW phase of 1Vs) applied to the sample. The standard lock-in technique with voltage modulation Vm = 10 mV and frequency f = 1 kHz has been adapted for acquiring dI/dV spectra. The high-resolution spectra were obtained with Vm = 2 mV. A single crystal of 1T-TaS2 was prepared by iodine vapor transport method, as described in ref. 18. The samples were cleaved at room temperature and quickly transferred to the precooled STM head.The STM and STS measurements have been carried out with a commercial STM (SPECS) at 4.3 K. Pt-Ir wires were used for STM tips. All STM images were acquired with the constant current mode with bias voltage  with the projector augmented wave method, Perdew-Burke-Ernzerhof exchange and correlation functional and GGA + The authors declare that the data supporting the findings of this study are available within the article and its Supplementary Information.Supplementary InformationPeer Review File


UBE3A) gene is imprinted with maternal-specific expression in neurons and biallelically expressed in all other cell types. Both loss-of-function and gain-of-function mutations affecting the dosage of UBE3A are associated with several neurodevelopmental syndromes and psychological conditions, suggesting that UBE3A is dosage-sensitive in the brain. The observation that loss of imprinting increases the dosage of UBE3A in brain further suggests that inactivation of the paternal UBE3A allele evolved as a dosage-regulating mechanism. To test this hypothesis, we examined UBE3A transcript and protein levels among cells, tissues, and species with different imprinting states of UBE3A.The ubiquitin protein E3A ligase gene  contains supplementary material, which is available to authorized users. Genomic imprinting is a rare epigenetic phenomenon that leads to the differential expression of paternally and maternally derived alleles of a gene in a parent-of-origin dependent manner , 54. It UBE3A) is located at the distal end of a cluster of imprinted genes on human chromosome 15q11-q13 and a homologous region on mouse chromosome 7C. In neurons of the central nervous system (CNS), UBE3A is imprinted with maternal-allelic expression, whereas in all other cell types, it is expressed from both alleles , ],UBEallele , with slightly higher, albeit not significant, Ube3a transcript levels in hippocampus compared to those in liver  and thymus  and significantly higher transcript levels than those in heart  and lung , with no significant effect of sex  or significant interaction between tissue and sex . Relative to cortex, UBE3A transcript levels were similar to those in bone marrow, esophagus, heart, kidney, liver, skin, and thyroid, and significantly higher than those in gallbladder, lung, spleen, and stomach  and maternal Ube3aYFP (Ube3amYFP/p+) protein levels in neural stem/progenitor cells (NSC) and in NSC-derived neurons over the course of 16 days of differentiation in vitro. In NSC cultures, maternal and paternal Ube3aYFP protein levels were approximately equal  or number of passages in culture (data not shown). In NSC-derived neurons, paternal and maternal Ube3aYFP protein levels were approximately equal at 1 day post-differentiation ; however, at 4, 8, and 16 DPD, maternal Ube3aYFP protein levels were significantly higher than paternal Ube3aYFP protein levels , with the protein levels produced by each parental allele diverging at a similar rate  = 1.3, p = 0.3  mammals suggests that imprinting of UBE3A evolved subsequent to divergence of the eutherian and metatherian  lineages. Expression data from unspecified tissues of tammar wallaby (Macropus eugenii) and platypus (Ornithorhynchus anatinus) brain show that UBE3A is not imprinted in these species, consistent with this evolutionary scenario  and laminin [Invitrogen] at a density of 380,000 cells per well in a 12-well, cell-culture plate; the plates were maintained in humidified incubators at 37 °C and 5% COYFP protein levels in differentiated neurons as previously described [YFP in individual neurons. Neurons and astrocytes were identified by immunoreactivity with the Tubb3 and GFAP antibodies, respectively.Immunofluorescence imaging was used to quantify Ube3aescribed . BrieflyYFP protein levels, a linear regression model was used with normalized intensity values of Ube3aYFP as the dependent variable and days post-differentiation (DPD) and allele  as fixed effects. To compare maternal and paternal Ube3aYFP protein levels at each time point, a least square means contrast linear regression model was used with normalized gray values of Ube3aYFP protein levels as the dependent variable and DPD (dummy variable) and allele as fixed effects.To examine the effect of day on Ube3aMonodelphis domestica) tissues  samples were obtained from a colony at the Comparative Medicine Program facilities at Texas A&M University. All animals were derived from an outbred stock (LL1) that was founded using wild animals captured in Eastern Brazil. All procedures involving animals were approved by the Texas A&M Institutional Animal Care and Use Committee. For the allelic expression analysis of UBE3A, opossum fetal brain (cortex) samples were obtained at 13 days post-copulation (d.p.c.) from F1 animals derived from reciprocal crosses of two semi-inbred stocks (LL1 and LL2) [For the analysis of UBE3A protein levels, opossum (and LL2) .The brain tissues were homogenized in TRI Reagent (Invitrogen) and total RNA was extracted using BCP (1-bromo-2 chloropropane), precipitated with isopropanol, and resuspended in RNase-free water. Potential DNA contamination was removed by both DNase I treatment and DNA removal columns in Qiagen RNeasy Plus Mini kit . RNA-seq libraries were made using the Illumina TruSeq RNA Sample Prep Kit , and sequenced on an Illumina HiSeq 2000 instrument . Details on RNA-seq data analysis could be found in Wang et al. .UBE3A were equal .A Pearson’s two-sided Chi-square test of the allelic counts of each SNV was used to determine the whether the allelic ratios of n = 4) and adult (8-week-old) mouse cortex (n = 4) as described above. For the analysis of UBE3A protein levels among opossum tissues, a single western blot was performed for each animal for all tissues. UBE3A protein levels were estimated and normalized as described above. For the comparison of Ube3a/UBE3A protein levels between opossum and mouse cortex, samples were run on a single western blot, and Ube3a/UBE3A protein levels were normalized and compared as described above.Total protein was isolated from adult (21-week-old) opossum tissues  were anesthetized with 0.5–1.0 mL of 20 mg/mL Avertin  via intraperitoneal injection. Mice were perfused with ice-cold phosphate-buffered saline (PBS) and 4% paraformaldehyde. Dissected brains were post-fixed in 4% paraformaldehyde solution for approximately 12 h and then cryoprotected in 30% sucrose solution. Fifty µm sections  were cut on a cryostat and stored in PBS. For immunostaining, sections were blocked in 0.3% Triton-X100 in PBS (T-PBS) plus 5% serum (goat or donkey) for 1–2 h at room temperature in a humidified chamber with gentle agitation. Primary antibodies  were processed and immunostained as described above. Confocal images of Auerbach’s ganglia  were obtained using a 43× (oil) objective. Images were imported into ImageJ and converted to the RGB color format. The Plot Profile feature was then used to determine the median gray value of Ube3aYFP in individual neurons (RBFOX3 positive) as described above.YFP protein levels, with sample ID as a random effect to account for repeated measurements of neurons within an individual animal.A mixed-effect model was used to examine the effect of allele  on Ube3a® (version 12). Mixed-effect models were used for analyses involving repeated measures of cells/tissues from an individual animal. The degrees of freedom were calculated using the Kenward–Roger correction.Inferential analyses were performed using JMP Pro® (version 12) and formatted in Adobe Illustrator.Charts were generated using JMP ProAdditional file 1: Table S1. Pair-wise comparisons of maternal and paternal Ube3a transcript levels.Additional file 2: Table S2. Pair-wise comparisons of UBE3A transcript levels.Additional file 3: Table S3. Pair-wise comparisons of UBE3A transcript levels.Additional file 4: Table S4. RNA-seq analysis of UBE3A allelic expression in opossum brain.Additional file 5: Figure S1. Alignment of mouse, opossum, and human Ube3a/UBE3A amino acid sequences. (A) ClustalW alignment of amino acid sequences of human UBE3A isoform 3, mouse Ube3a isoform 2, and opossum UBE3A. (B) Percent identity values of pair-wise comparisons of UBE3A/Ube3a among human, mouse, and opossum. Values represent conservative, semiconservative, non-conserved, and insertion/deletion percent identity.Additional file 6: Figure S2. (A-C) Immunofluorescence images of paternal Ube3aYFP protein expression in the lateral ventricle of adult Ube3a+/YFP mice. (D) Immunofluorescence image of maternal Ube3aYFP protein expression in the lateral ventricle of adult Ube3aYFP/+ mice. (E) Immunofluorescence images of maternal Ube3aYFP protein expression in the lateral ventricle, subventricular zone, rostral migratory stream, olfactory bulb, and adjacent cortical neurons in Ube3aYFP/+ mice. (F) Immunofluorescence images of maternal Ube3aYFP protein expression in the granular cell layer and subgranular zone of Ube3aYFP/+ mice.Additional file 7: Table S5. List of antibodies used for western blot and immunofluorescence analyses.


Dear Editor,CD147 (cluster of differentiation 147), also known as basigin or extracellular matrix metalloproteinase inducer (EMMPRIN), is a cell-surface type I transmembrane glycoprotein expressed at different levels in various cells and tissues, especially at high levels in tumor cells  with two Ig domains separated by a 5-residue flexible linker, a single transmembrane domain and a short intracellular domain. There are three conserved N-linked glycosylation sites on CD147EC, Asn44 on the N-terminal domain, Asn152 and Asn186 on the C-terminal domain , CD147 is composed of an extracellular portion  was overexpressed and purified from the E. coli strain origami B (DE3) . The recombinant CD147EC protein was eluted in a single fraction peak as a monomer on size-exclusion chromatography  and five related monosaccharides  were used in NMR titration experiments. Comparison of 2D 1H-15N HSQC spectra of CD147EC, with or without 10-fold excess of glycans, showed that there was no NH signal chemical shift perturbation caused by all the glycans , except sialic acid . Although NH signals of several CD147EC residues were significantly perturbed by sialic acid titration, however, this could be explained by the effect of small pH change due to the addition of sialic acid, as revealed by comparing the 2D 1H-15N HSQC spectra of CD147 at pH 6.5 and pH 7.0 . Taken together, our titration data indicate no direct interaction between the glycans and CD147EC.As glycosylation was suggested to play a role in the self-association of CD147, we examined the interaction of CD1471H-15N HSQC spectra of CD147EC , the N-terminal domain of CD147EC showed an average of ~1.7-fold decrease in NH signal intensity, and the C-terminal domain of CD147EC showed an average of ~4.5-fold decrease. While the changes of NH signal intensity are relatively uniform for residues in the N-terminal domain, some NH signals had over 10-fold intensity decreases for residues in the C-terminal domain. NH signals of residues Lys111, His115-Asn117, Gly119, Thr121, Phe159, His170-Ile171, Asn175, and His205 were even missing, while residues Val110, Ser113, Glu118, Glu120, Ala122-Leu124, Glu155, Ser156, Phe158, Ser161-Ser162, Glu172-Leu174, Met176, Asp179, Arg201-Ser204 had over 5-fold NH signal intensity decrease . Indeed, strong protein bands corresponding to CD147EC dimer and even high-order oligomers could be detected on the SDS-PAGE , the amounts of cross-linked dimer and oligomers were significantly increased in the presence of Zn(II) for C-CD147EC, with only a slight increase of dimer for N-CD147EC  may mediate self-association of CD147EC was immobilized on SA sensor chips. Sensorgrams with clear association and dissociation processes were observed with the flow though of different concentrations of CD147EC in the presence of Zn(II)  analysis, in which biotinylated C-terminal domain of CD147 EC, residues 99–205) using NMR spectroscopy. Detailed restraints and structural statistics of C-CD147EC are summarized in Table S1. The solution structure has been deposited into protein data bank (PDB ID: 5XF0). The solution structure of C-CD147EC comprises nine β-sheets: β1, residues 107–109; β2, residues 113–117; β3, residues 122–128; β4, residues 136–142; β5, residues 147–150; β6, residues 158–163; β7, residues 166–172; β8, residues 181–188; β9, residues 193–203 , with a RMSD of ~0.7 Å for the backbone heavy atoms in the secondary structure regions . Our results demonstrate that C-CD147EC adopts a rigid structure as reflected by the high {1H}-15N NOE values (>0.8) for most residues in the secondary structures. The N- and C-terminal regions, loops linking β3 and β4, β4 and β5, and β5 and β6 are relatively more flexible, as reflected by the relatively low {1H}-15N NOE values (≤0.75).We measured EC mediated by Zn(II). Based on R2/R1 ratios, the correlation time τc for C-CD147EC was calculated to be 7.1 ns, which can be translated into a molecular weight (MW) of ~12 kDa according to previous studies  is monomeric in solution. In the presence of Zn(II), τc of the C-CD147EC derived from the R2/R1 ratio is increased to 9.3 ns , corresponding to a molecular weight of over 15 kDa, suggesting that the apparent molecular weight is higher due to Zn(II).Model-free analysis shows that residues His102, Lys111, Phe159, Ser161, Gln164, Asn175, and Asp179 display conformational exchanges on the microsecond to millisecond timescales, and some of these residues are just located on the self-association interface of C-CD147EC, as NH signals of residues His115, His170, and His205 were missing with the addition of Zn(II), we analyzed the tautomeric states of histidines on CD147EC . The 1H and 15N resonances of all histidine sidechains were assigned based on multi-bond 1H and 15N imidazole signals of 2D 1H-15N HSQC experiment data, with the help of His to Ala substitutions . Based on the positions and intensities of the four multi-bond correlation 1Hɛ1-15Nɛ2, 1Hɛ1-15Nδ1, 1Hδ2-15Nɛ2, and 1Hδ2-15Nδ1 signals . We further monitored the signals of the histidine side-chain in 2D 1H-15N HSQC when CD147EC was titrated by Zn(II). It is obvious that signals of His115, His170, and His205 are also missing due to the addition of Zn(II), while His53 and His102 are not affected by Zn(II) . As His205 sidechain exists in charged state which can not provide coordination for Zn(II), His115 and His170 in the C-terminal domain of CD147EC are likely to be candidates for the coordination of Zn(II).Zn(II) coordination is generally occurring through the side chains of cysteine, aspartate, glutamate or histidine residues of Zn(II)-binding protein. For CD147EC mutants. NMR titration experiment data revealed that H115A mutation only decreased the NH signal responses to Zn(II) for residues around His115 and His205  show minor shift  and self-association of CD147EC. As the three histidines are all located on the C-terminal domain, it indicates that only the C-terminal domain is responsible for the self-association of CD147EC, while the N-terminal domain is not involved.We then generated a series of CD147EC is incapable of self-association and exists only as a monomer in solution, confirming the previous report  can directly interact with the extracellular C-terminal domain of CD147 and mediate its self-association. While the solution structure of the extracellular C-terminal domain alone of CD147 is roughly the same as the crystal structure. We identified that His115 and His170 are critical to the Zn(II) interaction and self-association, and very likely participate in Zn(II) coordination. As the two histidines are far away from each other, and our mutagenesis study results suggest that their interactions with Zn(II) are independent of each other, there probably exists more than one Zn(II) mediated dimer interface on the extracellular C-terminal domain of CD147.As an essential trace metal for living organisms, zinc is involved in many cellular processes, and the recent studies have revealed that zinc ion plays important signaling roles both intracellularly and extracellularly Below is the link to the electronic supplementary material.


UBE3A gene, which is located in the 15q11.2-q13 imprinted region. Functional loss of UBE3A is due to 15q11.2-q13 deletion, mutations in the UBE3A gene, paternal uniparental disomy and genomic imprinting defects.Patients with Angelman syndrome (AS) are affected by severe intellectual disability with absence of speech, distinctive dysmorphic craniofacial features, ataxia and a characteristic behavioral phenotype. AS is caused by the lack of expression in neurons of the UBE3A intragenic deletions. Subsequently, the MLPA P336-A2 kit was used to confirm and determine the size of the UBE3A deletion in the two patients. A review of the clinical features of previously reported patients with whole UBE3A gene or partial intragenic deletions is presented here together with these two new patients.We report here two patients with clinical features of AS referred to our hospital for clinical follow-up and genetic diagnosis. Methylation Specific-Multiplex Ligation-Dependent Probe Amplification (MS-MLPA) of the 15q11.2-q13 region was carried out in our laboratory as the first diagnostic tool detecting two novel UBE3A intragenic deletions may represent a small fraction of AS patients without a genetic diagnosis. Testing for UBE3A intragenic exonic deletions should be performed in those AS patients with a normal methylation pattern and no mutations in the UBE3A gene.Although rare,  Angelman syndrome (AS) is a neurogenetic disorder characterized by a severe intellectual disability with absence of speech, distinctive dysmorphic craniofacial features such as microcephaly with flat occiput and occipital groove, wide mouth, ataxia related neurological problems and/or tremor in the limbs and seizures with specific EEG pattern abnormalities that persist into adulthood. The behavioural phenotype is characterized by happy disposition, hyperactivity, attention deficit and frequent disruption of sleep cycles. Other clinical problems are gastrointestinal difficulties with feeding problems, gastroesophageal reflux, constipation, scoliosis, and an increased sensitivity to heat. Its prevalence is about 1/15000 births .UBE3A gene, which is located on chromosome 15q11.2-q13 region. This region includes a cluster of genes under imprinting control which show differential expression depending on the parental origin, in a tissue-specific manner. UBE3A shows a specific expression of the maternal allele in neurons while the paternal allele is silenced by the synthesis of an antisense transcript from the SNURF-SNPRN gene (UBE3A-ATS). The absence of the specific maternal expression of UBE3A is the main cause of AS [The cause of AS is the loss of function in neurons of the ubiquitin protein ligase E6-AP (E6-Associated Protein) encoded by the se of AS .UBE3A in the maternal allele is due to different genetic mechanisms: (i) deletion of the region 15q11.2-q13 in 70–75% of patients, (ii) mutations in the UBE3A gene in 10–15% of the patients, (iii) paternal uniparental disomy in 1–3% of patients, and (iv) genomic imprinting defects in 2–4% of the patients. The genetic cause remains unknown in approximately 10% of patients presenting clinical features characteristic of AS [UBE3A gene [The functional loss of E3A gene –10.UBE3A gene spans 120 kb of genomic DNA and consists of 10 exons (NM_130838.1). The ubiquitin protein ligase E6-AP encoded by the UBE3A gene is necessary for the ubiquitination of proteins targeted for degradation. E6-AP belongs to the HECT (homologous to E6-AP COOH terminus) class of E3 enzymes that share a 40 kDa COOH-terminal catalytic domain. This domain is encoded by exons 3–10 [The ons 3–10 . Exons 1UBE3A gene are widely distributed and have been detected throughout all regions of the gene [Mutations in the the gene , 14. Rarthe gene .UBE3A gene.Here, we report two patients with AS who present two novel intragenic deletions within the Patient 1 is a 5 year-old girl born at 37 weeks of gestation. Birth weight was 2850 g. Neither perinatal problems nor hypotonia was reported. Initially, she didn’t show feeding problems but when solid food was introduced into her diet, she had difficulties to swallow. Psychomotor development was delayed. She sat unsupported at 9 months, walked independently at 24 months and pronounced only two words which she lost later. She came to the clinical geneticist when she was 2-year-8-month old. Phenotypic features included relative microcephaly , small and wide-spaced teeth and protruding tongue. Her weight was 12 Kg (25th percentile) and her height was 80 cm (50th percentile). Neurological examination detected severe intellectual disability, ataxia of gait, receptive and non-verbal communication skills higher than verbal ones, hyperreflexia of the lower extremities, tremulous movement of limbs and frequent drooling. She also had abnormal sleep-wake cycles and by the age of 20 months she developed seizures. EEG showed generalized slow wave activity with paroxysmal activity. The behavioural phenotype included frequent laugher, happy demeanour, easily excitable personality, hyperactive behaviour, attention deficit and exploration of objects throughout the mouth. She also presented attraction to water and an increased sensitivity to heat . This kit contains 46 probes, 32 of which are specific for sequences in or close to the PWS/AS critical region on 15q11.2-q13 which can be used to detect copy number changes in this region. As a control for copy number changes, 14 probes outside the PWS/AS region are included. Among the PWS/AS specific probes, seven probes are methylation-sensitive and contain a HhaI restriction site. Five probes are located within the UBE3A gene corresponding to exons 1, 2, 3, 4 and 9 (NM_130838.1). In this paper, we use exon numbering according to NM_130838.1 sequence which is the standard reference sequence for the UBE3A gene where exons 1 to 10 correspond to exons 5b to 14 of the NM_000462.3 sequence. MLPA was performed according to manufacturer’s instructions (MRC Holland). Amplification products were run on an ABI3130 analyser  and analyzed using the GeneMapper software (Applied Biosystems).Methylation PCR and UBE3A exon 2 while probes corresponding to UBE3A exons 1, 3, 4 and 9 (NM_130838.1) showed a normal diploid dosage  was used. This kit contains 42 MLPA probes, including at least one probe for each exon of the UBE3A gene.Methylation pattern of the 15q11.2-q13 region was normal. However, dosage analysis showed a reduction of 50% in the relative peak height of the probe corresponding to age Fig. . To confUBE3A exonic probes presented normal dosage confirming that the deletion only involved exon 2  and the head circumference was 48 cm (−1.5 SD) (Table UBE3A gene (data not shown). Subsequently, a DNA sample of the patient was sent to our laboratory for further studies. MS-MLPA testing was performed and showed a normal methylation pattern. However, a reduction of 50% in the relative peak height of the probe corresponding to the UBE3A exon 9 was observed while probes corresponding to UBE3A exons 1, 2, 3, 4 (NM_130838.1) showed a normal diploid dosage  of the age Fig. . To conf 10 Fig. . The gen 10 Fig. . In patiUBE3A gene due to four different molecular etiologies: Deletion of the 15q11.2-q13 region on the maternal chromosome, mutations in the maternally-inherited copy of UBE3A, paternal uniparental disomy for chromosome 15 and an imprinting defect causing lack of expression of the maternal copy of UBE3A [Angelman syndrome is caused by the lack of expression of the maternal copy in neurons of the of UBE3A .UBE3A mutation or paternal uniparental disomy present significantly better linguistic properties than deletion patients, in particular the expressive area, where 6 out of 9 children with UBE3A mutation or paternal uniparental disomy could use 2–7 words while it was just the case in 3 out of 30 children with a 15q11.2-q13 deletion [Genotype-phenotype correlations among molecular subclasses have shown that deletion patients show a more severe phenotype in all aspects of neurodevelopmental delay except for expressive language skills that are extremely poor regardless of their molecular subclass –21. Howedeletion . In addideletion . In partdeletion .UBE3A mutations show a phenotype somewhere in the middle between deletion and paternal uniparental disomy carriers. They present higher incidences of seizures and microcephaly, similar to deletion patients, while their neurodevelopment delay is similar to paternal uniparental disomy and imprinting defect patient carriers [It has been suggested that AS patients carrying carriers , 19. A rcarriers .UBE3A exonic deletions that affect the UBE3A open reading frame. Exon 2 deletion in patient 1 is predicted to eliminate the start codon and consequently no protein will be produced (p.0?). Deletion of exons 9 and 10 in patient 2 is predicted to eliminate the last 88 aminoacids (p.Arg765_Leu852del88) disrupting the ubiquitin ligase catalytic domain located at the 3′ end of the protein  while seizures are present in all except one (7 of eight patients available). The high incidence of seizures and microcephaly is in accordance to what has been reported before by Lossie et al., 2001 and Moncla et al., 1999. Associated clinical features show variability between patients. A comparison of AS patients with mutations and intragenic deletions has not been reported before and from our observations there are no differences between the two groups.UBE3A gene is performed, as 10% of Angelman patients harbor UBE3A mutations.In our laboratory, array Comparative Genomic Hybridization (aCGH) is the first diagnostic tool in neurodevelopmental disorders. Methylation Specific-Multiplex Ligation-Dependent Probe Amplification (MS-MLPA) of the 15q11.2-q13 region is carried out when there is a strong clinical suspicion of Angelman or Prader Willi syndrome. MS-MLPA analysis simultaneously assesses the methylation status and genomic dosage changes at the 15q11.2-q13 region and can confirm the diagnosis and identify the presence of a causative deletion in 70% of cases. In the case of a normal methylation pattern and if the clinical suspicion remains high, sequence analysis of the UBE3A gene should be taken into account, once the MS-MLPA test and the sequencing of UBE3A have been carried out with a negative result. Although rare, intragenic deletions may account for a small proportion of AS patients without a genetic diagnosis and if present, they have a recurrence risk of up to 50%, depending on the carrier status of the mother. In the two patients reported here the mutation has appeared de novo but in the other intragenic deletions previously reported they were transmitted by healthy mothers to their affected children .In addition, we propose here that the analysis of the copy number variations within the en Table . MoreoveUBE3A mutations. Here, we report two patients with AS who present two novel intragenic deletions within the UBE3A gene that together with those reported in the literature show that UBE3A intragenic deletions may represent a small fraction of AS patients. Testing for UBE3A intragenic exonic deletions/duplications should be included in those AS patients with a normal methylation pattern and no mutations in the UBE3A gene.To date, few reports have evaluated the contribution of exonic deletions to the spectrum of


Acidovorax citrulli. The crystal structure at 2.19 Å resolution shows a large displacement of the catalytic lysine (Lys163) in domain 2 away from the active site core, whereas the two other active site lysines from the two other domains are not able to move. The lysine displacement is proposed here to open up a channel for product release. Consistent with that, the structure also showed two molecules of the co-product, carbon dioxide, one in the active site and another trapped in the proposed exit channel. Previous data indicated that the domain 2 lysine residue plays a role in activating an adjacent serine residue carrying out nucleophilic attack, opening the cyanuric acid ring, and the mobile lysine guides products through the exit channel.Cyanuric acid hydrolases are of industrial importance because of their use in aquatic recreational facilities to remove cyanuric acid, a stabilizer for the chlorine. Degradation of excess cyanuric acid is necessary to maintain chlorine disinfection in the waters. Cyanuric acid hydrolase opens the cyanuric acid ring hydrolytically and subsequent decarboxylation produces carbon dioxide and biuret. In the present study, we report the X-ray structure of TrzD, a cyanuric acid hydrolase from  Cyanuric acid, or 1,3,5-triazinane-2,4,6-trione, is a building block for commercial triazines and it is used directly, principally to stabilize hypochlorite for chlorine-based disinfection processes.Cyanuric acid hydrolases are rare, ancient enzymes undergoing a modern renaissance due to the annual production of more than one billion pounds of The use of more than 100 million pounds of cyanuric acid as a chlorine stabilizer in outdoor pools, spas and fountains has provided new impetus to study the cyanuric acid hydrolases that can enzymatically remove the compound from those waters3s-triazine ring via a nucleophilic serine residue that is activated by a nearby lysine, a mechanistic motif known as a serine-lysine dyad79Cyanuric acid hydrolases are members of a protein family containing only themselves and the barbiturase enzymes, they are found only in microorganisms, and are present in approximately 0.3% of sequenced prokaryotic genomesAcidovorax citrulliPseudomonas sp. ADPAzorhizobium calindulanskcat that is an order of magnitude greater than other known cyanuric acid hydrolaseskcat enzyme in commercial applications, and for obtaining insights into the outstanding mechanistic questions, the present study focused on determining the X-ray structure of TrzD. In the course of the study, we observed an unusual orientation of the second-domain lysine that interacts with the proposed catalytic second-domain serine. This observation simultaneously provided support that the second domain serine serves as the active site nucleophile and it gave evidence for a product exit channel. Moreover, the presence of trapped carbon dioxide in the enzyme is consistent with the view that the enzyme assists in the decarboxylation of carboxybiuret, and the cell does not rely on a spontaneous decarboxylation to produce the next intermediate, biuret, in the cyanuric acid biodegradation pathway vectorEscherichia coli BL21 (DE3) that was grown in LB medium  supplemented with 50 ug/ml kanamycin. Cells were grown at 37 °C and induced with 0.5 mM of isopropyl β-D-1-thiogalactopyranoside (IPTG) for 30 hours at 20 °C. After collection by centrifugation , the cells were resuspended in buffer A . Cells were lysed by three passes at 1000 p.s.i. through a French press, and the cell lysate was centrifuged at 48,000 × g for 45 min at 4 °C. After centrifugation, the cell lysate was applied onto a column packed with a Ni–NTA resin (Thermo Scientific) that had been equilibrated with the binding buffer. After washing, the bound protein was eluted from the column with a gradient using elution buffer B . For further purification, gel filtration chromatography was performed using a HiLoad™ Superdex-200 16/600 gel-filtration column  equilibrated with Buffer A. The enzyme was concentrated to 12 mg/ml and stored at 4 °C until used.The full-length The protein was crystallized with its natural substrate cyanuric acid by hanging drop, vapor-diffusion experiment in which equal volume of protein (12 mg/ml concentration) and reservoir solution were mixed and allowed to equilibrate against the reservoir at 20 °C. The final concentration of cyanuric acid was 10 mM in the crystallization drop. The initial crystallization hit contained 0.05 M ammonium sulfate, 0.05 M BIS-TRIS pH 6.5, and 30% pentaerythritol ethoxylate (15/4 EO/OH). Needle-shaped crystals appeared in 48 hours. Two week old crystals were harvested and flash-cooled in liquid nitrogen without additional cryoprotectant, since 30% pentaerythritol ethoxylate (15/4 EO/OH) serves as good cryoprotectant. A single crystal was used for data collection.The flash-cooled crystal was diffracted at beamline 23-ID-B in the Advanced Photon Source (APS) at 1.03322 Å wavelength with 0.5 degree oscillation per frame. A MAR CCD300 detector was used to collect 270 frames at a distance of 250 mm from the crystal. All data were processed and reduced using the HKL2000 packageMolecular replacement was performed using the software MOLREP17Pseudomonas sp. ADP) (4BVQ). The crystallographic asymmetric unit contained one TrzD molecule. The product-bound structure was refined to a resolution of 2.19 Å, with Rwork and Rfree values of 0.15 and 0.20, respectively, yielding high quality electron density maps. We could model the N-terminal His-tag. The crystallographic data collection and refinement statistics are listed in Azorhizobium calindulans). The next nearest structural homolog is chorismate mutase (4BPS) with 11% sequence identity and 4.0 Å rmsd (using the DALI server)o − Fc) clearly showed electron density corresponding to two CO2 molecules and Lys163 within the TrzD structure , domain 2 is composed of 137 (113 to 249) and domain 3 is composed of 115 (255 to 369) residues. Each domain starts and ends with two antiparallel β-strands. Domain 2 has a 15 residue long extra helix, which is not present in the other two domains, making it slightly bigger. Residues important for catalysis, i.e., Ser, Lys and Arg residues from each domain, showed similar spatial disposition, when superimposed. The three structurally homologous domains form a pseudo-three-fold internal symmetry in the TrzD monomer . The lat2 respectively, supporting the conclusion that TrzD exists largely as a tetramer in solution. Domains 1 and 2 do not take part in tetramer assembly, nor are they in crystal contact. Domain 1 and domain 3 form six hydrogen bonds between them. Arg314 of domain 3 forms three hydrogen bonds, one with the Met10 main chain carbonyl oxygen and the other two with side chain oxygens of Glu41. Ser321 forms two hydrogen bonds with Val46 and Asn47 of domain 1. The Asp322 side chain oxygen is hydrogen bonded with Asn43. Domain 2 and 3 form three hydrogen bonds. Arg147 and Arg250 from domain 2 form hydrogen bonds with Glu309 and Asp290 side chain oxygen atoms. Arg147 forms another hydrogen bond with the Ala370 main chain carbonyl oxygen. Crystal contacts were analyzed using the LIGPLOT programBased on size exclusion chromatography and dynamic light scattering, TrzD forms a stable tetramer in solution. An analysis of present structure by the PISA server (PISA v1.51 22/09/2014]) predicted an average solvation free energy of −70 kcal/mol for tetramer formation. The average solvent-accessible surface area of monomeric units buried upon assembly formation and solvent-accessible surface area for a tetramer is 12250 and 51030 Å/09/2014)GGxEHQGPxGG is identified throughout the cyanuric acid hydrolase/barbiturase protein family. Ser/Ala/Gly residues can substitute in the first X and the second X can be a Ala/Asp/Pro/Ser residue (+2 bound and makes 6-contacts with neighboring residues and one with a solvent molecule (2.8 Å). Mg+2 interacts with the backbone carbonyl oxygen of Ser352 (2.5 Å), Gln355 (2.7 Å), Pro357 (2.6 Å) and Gly360 (2.9 Å) and the side chain oxygens of Glu303 (2.8 Å) and Ser352 (2.9 Å). Glu303 is the only residue to interact with the metal ion that is not in the conserved loop , and the backbone amide nitrogen of Gly350 (3.3 Å), backbone carbonyl oxygen of Gly83 and also with a side chain nitrogen of Arg330 .Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


The skeletal remains of Late Pleistocene-early Holocene humans are exceptionally rare in island Southeast Asia. As a result, the identity and physical adaptations of the early inhabitants of the region are poorly known. One archaeological locality that has historically been important for understanding the peopling of island Southeast Asia is the Niah Caves in the northeast of Borneo. Here we present the results of direct Uranium-series dating and the first published descriptions of three partial human mandibles from the West Mouth of the Niah Caves recovered during excavations by the Harrissons in 1957. One of them (mandible E/B1 100") is somewhat younger than the ‘Deep Skull’ with a best dating estimate of c30-28 ka (at 2σ), while the other two mandibles (D/N5 42–48" and E/W 33 24–36") are dated to a minimum of c11.0–10.5 ka (at 2σ) and c10.0–9.0 ka (at 2σ). Jaw E/B1 100" is unusually small and robust compared with other Late Pleistocene mandibles suggesting that it may have been ontogenetically altered through masticatory strain under a model of phenotypic plasticity. Possible dietary causes could include the consumption of tough or dried meats or palm plants, behaviours which have been documented previously in the archaeological record of the Niah Caves. Our work suggests a long history back to before the LGM of economic strategies involving the exploitation of raw plant foods or perhaps dried and stored meat resources. This offers new insights into the economic strategies of Late Pleistocene-early Holocene hunter-gatherers living in, or adjacent to, tropical rainforests. While researchers have investigated the prehistory of the region for over 130 years [Island (or ‘maritime’) Southeast Asia (iSEA) spans a geographic area of approximately 3 million km30 years ,5; 2) th30 years –8; and 330 years ,10. The 14C dating of charcoal to be 22,000–24,000 B.P. [Despite the long history of archaeological research in iSEA, the remains of Late Pleistocene AMH have been found at only four localities. These comprise partial skulls and postcranial elements from Wadjak in Indonesia discovered in 1888 (Wadjak-1) by B.D. van Rietschoten and in 1890 (Wadjak-2) by Eugene Dubois ; and mos000 B.P. . More re000 B.P.  and an u000 B.P. .14C of charcoal and direct U-series dating of human bone c39-30 ka (at 94.5 per cent probability) [In the centre of iSEA lies the Niah Great Cave complex (Batu Niah) on the island of Borneo in East Malaysia (Sarawak)  where thability) .The Niah Great Cave is a spectacular karst system located about 80 km southwest of Miri in the northeast of Sarawak . It was 2. They were exposed during excavations in the ‘Hell Trench,’ where the Deep Skull was recovered, and adjoining deposits, as well as in Areas A and B (as designated by the Niah Caves Project) of the Harrisson archaeological zone [In the West Mouth, the Harrisons also excavated more than 250 isolated human bones and partial skeletons designating many of them ‘burials’ . Late Plcal zone –31. The cal zone –31 makincal zone , while Bcal zone . In totacal zone . Howevercal zone , have recal zone –31 they It is within this context that we present the results of direct U-series dating and the first published descriptions of three partial human mandibles Figs  from AreAll of the human remains studied in the present research are stored at the Sarawak Museum Department, Kuching, Sarawak . Permission to collect mandibles from the Neolithic cemetery at Niah Caves  in the Radiogenic Isotope Facility (RIF) at the School of Earth and Environmental Sciences, The University of Queensland (UQ) following chemical treatment procedures and MC-ICP-MS analytical protocols described elsewhere ,35. Fossisotopes ,36. The  Program .Isoplot program [For laser ablation U-series analyses a cut was made perpendicular to the bone surface using a rotatory tool equipped with a thin (100 μm wide) diamond saw blade. The cut sample was mounted into an aluminium cup, aligning the cross-sectioned surface with the outer rim of the sample holder to position the samples on the focal plane of the laser in the sampling cell. Sequential laser spot analyses were undertaken along two parallel tracks, starting from the interior of the cross-sectioned bone. The analyses were made at regular spacing  along each track, using a laser spot size of 265 μm and a 5 Hz pulse rate. The samples were initially cleaned for 5s and ablation pits were measured for 50s. U-series ages were calculated using the  program . Closed  program .in situ exposed burials was collected by us from the West Mouth of Niah Caves in 2017. These were collected from the surface of graves originally excavated by the Harrisons and left exposed in the cave. As we did not undertake additional excavations to recover these bones, the size of the sample is necessarily small. Morphological observations on all of the Niah Cave mandibles were made using standard methods [A sample of late Holocene (Neolithic) mandibles from  methods ,39. Mand methods . Data fr methods ,41–53 . In this case, the U-Th date would approximate the depositional age of the fossil . Howevertimation . Such vaThe results of U-series dating are summarised in 232Th, which may derive from either sediment contained within pores or diffusion from external sediment, can also compromise U-series dating results. When the elemental U/Th ratios drops below 300 the resulting U-series result may be influenced by detrital 230Th. This was the case for only two spots in Track 2 (Spot-16 and Spot-29) where the U/Th ratios were 265 and 233 respectively  of the body as a result, but it is confined to this region . The right body is preserved from just behind P1 posteriorly, P2-M1 alveoli being intact, and preserving the mesial alveolar wall and septum between the mesial and distal roots and partial mesial root alveoli of M3.Mandible E/B1 100" comprises two partial (left and right) fragments of an adult mandibular body lacking completely the dentition . The lef1. On the inner margin of the remaining symphysis the superior and inferior transverse tori are observed to be enlarged with the superior torus forming a small shelf. The digastric fossae are incised and face posteriorly, while the sublingual fossae are broad and relatively deep structures.The body of E/B1 100" is relatively ruggedly built indicating well-developed musculature associated with the oral handling and processing of food and accompanying broadening or buttressing of the body. There is a deep sublingual fossa accompanied above by a prominent alveolar shelf along the course of the molars. In addition, the mylohyoid line is clearly demarcated and runs parallel to the alveolar margin, fading out by about mid-M2 in E/B1 100" (left and right), which is the most common state for mainland and island Southeast Asian and Northeast Asian mandibles (83/100%), although, it is less frequent in European mandibles (60%), the small sample of Neolithic mandibles from the West Mouth (33%) and Sahul mandibles (10%)  sits within a broader hollowing of bone and is surrounded by a bony rim (left and right). There is observable pitting around the superior section of the rim of the foramen on both sides suggestive of hypervascularization. The MF is accompanied by a small accessory foramen which is located within the anterior margin of its rim (both sides). The MF is located beneath the Pes (10%) . E/B1 10es (10%) . Althoug1 to M1 alveoli. The left minimally worn I2 and C crowns were preserved in situ but have subsequently dislodged during storage. The body is complete anteroposteriorly from the symphysis to beneath the mid-alveolar septum separating the medial and distal M1 root alveoli, where after it is missing. On the right side, the I1 alveolar is complete but the body is broken from just lateral to the incomplete I2 alveolus.Mandible D/N5 42–48"  comprise2/M1 and opens in the superior direction. This location is most frequent in Niah Caves Neolithic (67%) and Sahul mandibles (90%), common in European LPH mandibles (40%), but far less common among mainland and island Southeast Asian (0/17%) and Northeast Asian (11%) individuals  is also very small, being 1.8σ below the pooled LPH sample mean , and well below the ranges (minima) of Southeast/Northeast Asian  and Neolithic mandibles from the West Mouth . However, the body area of the larger D/N5 42–48" mandible (330.1 mm2) lies well within the range of all LPH samples  and shape index (52.5%) are unremarkable, their values sitting within 1σ of the pooled LPH sample mean, and within 1σ of sample averages where it can be compared (compared .2 is slightly narrowed (6.5 mm), but identical to average values for recent Negrito and Malay samples, as well being within the range of all other comparative samples  [Of the three mandibles from the West Mouth of the Niah Caves we have analysed here, E/B1 100" stands out owing to its secure dating to the Late Pleistocene and unusual morphology. Given that U-series dating of bone provides only a minimum age, the conventional date for the specimen of c26-25 ka (at 2σ) should be viewed as a minimum age only. However, as the maximum laser ablation date should probably be closest to the true age of the specimen, the age for E/B1 100" is more likely to be within the range c30-28 ka (at 2σ). This is somewhat younger (by up to 9 ka) than the estimated age for the oldest human remains from the site (the Deep Skull) as determined through Bayesian modeling combining AMS ability) .The scarcity of Pleistocene AMH remains across iSEA brings into focus the importance of the Niah Caves jaws, especially E/B1 100". It is similar in age to human mandibles from Wadjak (Indonesia) and Tabon Cave (Philippines), and along with the Deep Skull, they together provide direct evidence of penecontemporary human populations living across a broad segment of the region prior to the Last Glacial Maximum. The Wadjak and Tabon mandibles are clearly from very large bodied individuals ,60, howeThe morphology of the mandible preferentially over the remaining skull reflects masticatory pressure rather than neutral population history . There aOther morphological indicators of hypertrophy of the muscles of the oral cavity seen in E/B1 100" might include deep sublingual fossae and molar shelving, thickening of the superior and inferior transverse tori and hollowing of the digastric fossae. These would also reflect masticatory strain under a model of phenotypic plasticity, perhaps involving a diet that included hard and brittle foods with minimal preparation or even the use of the teeth as tools. In the case of the Niah remains, possible dietary causes might include the consumption of tough or dried meats or underground storage organs ,65. The We have provided for the first time the results of direct-dating and morphological investigations of three human mandibles found more than 60 years ago by the Harrissons in the archaeological zone of the West Mouth of Niah Caves. The rarity of Late Pleistocene-early Holocene human remains from across island Southeast Asia underscores their importance in helping to fill a major gap in understanding about the early hunting and gathering populations of the region. Our work also shows that Niah Caves might document a long history stretching back to before the LGM of economic strategies involving the exploitation of raw plant foods or perhaps even dried and stored meat resources. This potentially also offers some new insights into the economic adaptations of Late Pleistocene-early Holocene hunter-gatherers living in, or adjacent to, tropical rainforests, the economic difficulties of which are well known .S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S1 Fig(DOCX)Click here for additional data file.S2 Fig(DOCX)Click here for additional data file.