Alzheimer’s disease is characterized by accumulation of amyloid plaques and tau aggregates in several cortical brain regions. Tau phosphorylation causes formation of neurofibrillary tangles and neuropil threads. Phosphorylation at tau Ser202/Thr205 is well characterized since labeling of this site is used to assign Braak stage based on occurrence of neurofibrillary tangles. Only little is known about the spatial and temporal phosphorylation profile of other phosphorylated tau (ptau) sites. Here, we investigate total tau and ptau at residues Tyr18, Ser199, Ser202/Thr205, Thr231, Ser262, Ser396, Ser422 as well as amyloid-β plaques in human brain tissue of AD patients and controls. Allo- and isocortical brain regions were evaluated applying rater-independent automated quantification based on digital image analysis. We found that the level of ptau at several residues, like Ser199, Ser202/Thr205, and Ser422 was similar in healthy controls and Braak stages I to IV but was increased in Braak stage V/VI throughout the entire isocortex and transentorhinal cortex. Quantification of ThioS-stained plaques showed a similar pattern. Only tau phosphorylation at Tyr18 and Thr231 was already significantly increased in the transentorhinal region at Braak stage III/IV and hence showed a progressive increase with increasing Braak stages. Additionally, the increase in phosphorylation relative to controls was highest at Tyr18, Thr231 and Ser199. By contrast, Ser396 tau and Ser262 tau showed only a weak phosphorylation in all analyzed brain regions and only minor progression. Our results suggest that the ptau burden in the isocortex is comparable between all analyzed ptau sites when using a quantitative approach while levels of ptau at Tyr18 or Thr231 in the transentorhinal region are different between all Braak stages. Hence these sites could be crucial in the pathogenesis of AD already at early stages and therefore represent putative novel therapeutic targets.The online version of this article (10.1186/s40478-018-0557-6) contains supplementary material, which is available to authorized users. Alzheimer’s disease (AD) is neuropathologically characterized by two hallmark lesions, which are extracellular amyloid-β (Aβ) plaques and intracellular accumulations of abnormally phosphorylated tau. Aβ plaques initially develop in neocortical regions and then progress to the limbic system, subcortical nuclei and reach the cerebellum at late stages of the disease . Tau patThe temporal and spatial phosphorylation pattern of tau residue Ser202/Thr205 has already been well characterized since staging of AD cases is based on labeling of ptau Ser202/Thr205 using the specific antibody AT8 , 20, 46.Phosphorylation at several residues during disease progression has been repeatedly investigated in tau animal models but barely in AD brain tissue , 20, 46.V/VI;  and conV/VI;   and 5 min in Tissue Clear/100% ethanol, washed for 5 min in 100% ethanol and then subsequently rehydrated with decreasing alcohol concentrations . Thereafter, sections were washed twice for 5 min in PBS. Sections for labeling of human total tau, ThioflavinS (ThioS), pThr231 tau, pSer199 tau, pTyr18 tau and pSer396 tau were treated for 15 min with citrate buffer  at 95 °C in a steamer and cooled down to room temperature (RT) for another 15 min. For ThioS staining, sections were then labeled with 0.5% ThioS  for 7 min under light protection. Afterwards, sections were pretreated with ice-cold sodium borohydride/PBS solution  for 4 min and tissue was then permeabilized with 1% Triton® X-100/PBS  for 10 min. Non-specific labeling was blocked by incubating sections for 30–60 min either with 10% donkey serum/PBS or 10% horse serum/PBS. For antigen detection, sections were incubated with primary antibodies , equipped with a Nikon DS-Qi1MC camera and controlled by the Nikon NIS-Elements AR software. For quantification of total human tau, pThr231 tau, pTyr18 tau and ThioS images were recorded using a Zeiss AxioImager.Z1 microscope with a high aperture lens and an AxioVision 4.8 software-driven AxioCam MRm digital camera .Quantitative image analysis was performed using Image-Pro Plus . Grey-scale single channel images were corrected for background intensities using lowpass filtering, and signal from autofluorescent objects (mostly lipofuscin and erythrocytes) was subtracted from the channel used for immunofluorescent labeling. Objects were identified by a combination of Edge+ filter, adequate thresholding, and size and shape restrictions  and centrifuged at 80,000 g for 15 min at 4 °C. The pellet was resuspended in extraction buffer 2  and centrifuged at 4000 g for 10 min at 4 °C. The Supernatant was transferred to a fresh tube and sarcosyl (30% aqueous solution) was added to a final concentration of 1% and incubated for 1.5 h at room temperature. After centrifugation at 80,000 g for 30 min at 4 °C, the supernatant was discarded and the pellet resuspended in buffer 3 . The suspension was aliquoted and frozen until used for Western blotting.Equal amounts of proteins of each brain extract were separated by molecular weight on a SDS-PAGE polyacrylamide gel. A protein marker visualized correct separation of the proteins and confirmed the correct protein band size. Subsequently, proteins were transferred onto a 0.45 μm nitrocellulose membrane using a wet blot chamber , blocked with 5% non-fat dry milk in 1 × TBS and incubated with primary antibodies overnight. To blot for total tau, pTyr18, and pSer199 the same primary antibodies as used for histological labeling were used in a concentration of 1:1000 Table . AntibodAll statistical analyses and preparation of graphs were conducted using Graph-Pad Prism . Descriptive statistical analyses were performed on all evaluated parameters including the evaluation of normal distribution using the Kolmogorov-Smirnov test.post-hoc analysis was followed. A detailed description of performed statistical analyses is given in the appropriate figure legend. Data were averaged and represented as mean + standard error of mean (SEM). An α-error level of p < 0.05 was considered significant.Group variances were calculated either by one-way or two-way ANOVA. If a significant interaction among groups was detected, Newman-Keuls or Bonferroni’s In order to evaluate the overall human tau expression levels, brain tissues were labeled with the anti-human total tau HT7 antibody. Quantification of the HT7 immunoreactive area in the isocortex and transentorhinal region (TEntR) revealed a progressive increase of total tau with advancing Braak stages. This increase in total tau signal was significant at Braak stage V/VI compared to healthy controls but also compared to both lower Braak stages I/II and III/IV Fig. . Data ofAfter evaluating the overall total tau and plaque load, we measured the phosphorylation levels of ptau at seven different residues. First, we show phosphorylation of pSer202/Thr205 tau since this phosphorylation site is already well characterized and thus can be utilized to validate the results.Phosphorylation of tau at Ser202/Thr205 was low in all isocortical brain regions up to Braak stage III/IV. Only at Braak stage V/VI Ser202/Thr205 tau phosphorylation significantly increased about 4- to 13-fold above control levels Fig. . At BraaPhosphorylation of tau at Thr231 was also low in all isocortical brain regions up to Braak stage III/IV. Only at Braak stage V/VI Thr231 tau phosphorylation significantly increased about 30- to 160-fold above control levels Fig. . At BraaOverall, phosphorylation of tau at Ser199 was low compared to other phosphorylation sites. In all isocortical regions Ser199 tau phosphorylation started late, namely at Braak stage V/VI Fig. . At BraaThe phosphorylation profile of pTyr18 tau in the isocortex and TEntR was very similar to pSer199 tau although the final phosphorylation levels in Braak stage V/VI were slightly higher and increased about 20- to 140-fold above control levels Fig. . The proPhosphorylation of tau at Ser396 shows higher variability than other ptau sites. In the isocortex overall Ser396 tau phosphorylation starts very late in Braak stage V/VI Fig. . When anPhosphorylation of tau at Ser262 was very low and showed almost no progression in higher Braak stages Fig. . Only inThe phosphorylation profile of tau at Ser422 was comparable in all isocortical regions and also the allocortical TEntR. In all analyzed regions phosphorylation significantly increased at Braak stage V/VI Fig.  and was When evaluating the signal increase relative to healthy control brain tissue, it became evident that ThioS labeling in the TEntR increased already at Braak stage III/IV and reached a plateau at Braak stages III/IV and V/VI. The phosphorylation at tau Tyr18 and tau Ser199 severely increased at the same time at Braak stage V/VI with already a slight increase at Braak stage III/IV Fig. . CompareTo validate results obtained by immunofluorescent labeling, Western blots of AD and control cases were performed Fig. . Cases sRepresentative images of total tau Fig.  and pSerIn summary, our quantitative analysis of AD brain tissue shows that a progressive increase of total tau and ptau expression can be observed in the transentorhinal region and in most of the analyzed isocortical regions. Staining of tissue with ThioS resulted only in a weak signal in the transentorhinal region and the occipital cortex. Labeling with antibodies against different tau phosphorylation sites showed that most ptau sites, namely pSer202/Thr205, pThr231, pSer199, pTyr18 and pSer422 were highly increased throughout the cortex at Braak stage V/VI. Only tau phosphorylation at Thr231 and Thy18 was already significantly increased at Braak stage III/IV. By contrast, pSer396 tau and pSer262 tau are only weakly expressed in all analyzed brain regions and only minor progression was observed. When comparing tau phosphorylation in percent relative to healthy controls, phosphorylation is specifically increased at tau Thr231, Ser199 and Tyr18. These data indicate that tau phosphorylation is a complex feature of AD progression, involving many but not all potential phosphorylation sites.The current study was designed to analyze spatial patterns of tau phosphorylation at multiple residues in discrete anatomical regions during AD progression. Our data revealed a very similar phosphorylation profile of most of the analyzed ptau sites in the allo- and isocortex while expression levels of phosphorylated tau at Tyr18 and Thr231 was distinguishable between Braak stages. When normalized to controls, phosphorylation of tau at Tyr18, Ser231 and also Ser199 was much more increased at Braak stage V/VI compared to other residues, suggesting a relevance of these sites for AD progression and a crucial role in pathogenesis.Since tau phosphorylation is a main characteristic of AD progression, several groups have already analyzed the temporal phosphorylation pattern of different tau sites by histological methods. According to Luna-Munoz and colleagues the phosphorylation of Thr231 tau is an early event in the neuronal pathology of AD . The temWestern blot analyses of total tau and the most prominent phosphorylation residues pSer202/Thr205, pThr231, pSer199, and pTyr18 overall validated the results obtained by immunofluorescent labeling. It has to be mentioned that tissue of not all human cases was available for blotting, so quantification and statistical analyses should be understood as preliminary. Blots for pSer202//Thr205 and pThr231 are not directly comparable, since different antibodies as used for immunofluorescent labelings had to be used for methodical reasons.post mortem AD tissue and in vivo imaging of AD patients should shed light on the impact of gender on tau phosphorylation.AD is known to affect women with a higher prevalence, which is among other reasons caused by hormonal differences. The higher frequency of AD cases in women is reflected in this study by the fact that only tissue of women was provided for Braak stage V/VI, while tissue of earlier Braak stages was predominantly from men. Although several in vitro and in vivo studies in rodents suggest an impact of sex hormones  and therefore gender on tau phosphorylation, there are so far no studies in AD patients validating these results in humans (for review see ). FutureIn addition to the severe increases in tau phosphorylation also the ThioS signal, that includes amyloid plaques, was highly increased in several allo- and isocortical regions. The ThioS signal increase partially already started at Braak stage III/IV and thus earlier than the increase of ptau. It has been shown that ptau residues, Tyr18, Thr231 and Ser199 can be phosphorylated by Aβ via different kinases like Fyn , 44, 45,Isocortical tau pathology is only sparse at early Braak stages I to IV and hence use of higher magnification and modified image analysis parameters may be necessary to reveal subtle changes in isocortical tau pathology at early Braak stages. Additionally, co-labeling of different markers to quantify ptau only in distinct areas or cell populations combined with an increased number of investigated brain regions may be helpful to gain a better understanding of earliest isocortical tau pathology. A tissue microarray method that was developed recently allows to examine over 35 brain regions on one slide could be used for such purposes . HoweverWe show here for the first time the phosphorylation profile of different tau sites in allocortical and isocortical regions of human brains with tau pathology ranging from Braak stages 0 to VI using quantitative rater-independent immunofluorescent labeling. Our data suggest that the profile of ptau in the isocortex is comparable between all of the analyzed ptau sites while expression levels of ptau at Thr231 and Tyr18 in the transentorhinal region are distinguishable between Braak stages combined with the highest tau phosphorylation relative to controls at these sites and Ser199.Additional file 1:Online Source 9 Double labeling of pSer262 and pSer202/Thr205 tau in the temporal cortex at Braak stage V/VI. Images show different labeling pattern of pSer262 (arrows) and pS202 (white arrowheads) as well as their overlay (yellow arrowheads) (a1) and single fluorescence images  of case 17. AF: autofluorescence. Scale bar: 20 μm. Online Source 10 Example of measurement procedure of tau pSer262. Objects in the unlabeled autofluorescence channel were detected by thresholding (red in a1). The resulting mask images (a2) were then subtracted from tau pSer262 images to remove autofluorescence (a3). The resulting images were Edge+ filtered (a4) to facilitate threshold-based detection of tau pSer262-positive objects (red outline in a5). These outlines were then loaded onto the raw images to quantify original tau pSer262 signal (red outline in a6). AF: autofluorescence. Scale bar: 20 μm. Online Source 11 Example of detecting ThioS-positive amyloid-β but not NFTs. Image a displays the co-labeling of ThioS (green) and HT7 (red), while images b and c, respectively, show single channel images. ThioS shows intense labeling of plaque-associated β-sheets  whereas tangles are only weakly labeled  (c). A combination of threshold-based identification of ThioS and size restriction  enables quantification of ThioS+ plaque labeling (red highlighted) but not tangles (d). ThioS: ThioflavinS. Scale bar: 20 μm. (PDF 599 kb)


In this research, the durability of skid resistance during the ice melting process with temperature increasing from −5 °C to 10 °C was characterized by means of a British Pendulum Skid Tester. Four types of pavement surfaces were prepared and tested. The difference between two antiskid layers prepared with bitumen emulsion was the aggregate. The detailed angularity and form 2D index of fine aggregates used for antiskid surfaces, characterized by means of the Aggregate Image Measure System (AIMS) with micro image analysis methods, were then correlated with British Pendulum Number (BPN) values. Results indicate that skid resistance has the lowest value during the ice-melting process. The investigated antiskid layers can increase the surface friction during icy seasons. In icy conditions, the skid resistance behavior first worsens until reaches the lowest value, and then increases gradually with increasing temperature. Results from ice-melting conditions on four investigated pavement surfaces give the same temperature range where there will be lowest skid resistance. That temperature range is from 3 °C to 5 °C. A thicker ice layer will result in a lower skid resistance property and smaller “lowest BPN”. Asphalt pavements, considered as flexible pavements, are widely used in both road and airfield engineering. It was reported that more than 90% of highways were constructed by asphalt materials ,2. A durAt all stages of pavement service life, the surface should have some sort of roughness to ensure sufficient friction between traffic wheels and the pavement surface ,4. Skid Typically, the Locked Wheel Skid Trailer (LWST) and British Pendulum Tester (BP Tester) are the most common and accepted methods of measuring pavement skid resistance. LWST, widely used in the United States, is standardized in ASTM E 274 specifications and measures the sliding force of a locked tire on a wetting pavement surface. The BP Tester is one of the most widely used devices to determine low speed related skid resistance in the laboratory. The value obtained is called the British Pendulum Number (BPN).However, one of the limitations of the current BP Tester is that it lacks any correction for temperatures. Previous research had concluded that BPN values are dependent on the pavement temperature . TemperaProf. Liang used a BP Tester to characterize the temperature effect on the measured frictional properties of a hot-mix asphalt mixture surface . He concHowever, cold winter conditions will influence the micro-texture of aggregates and stiffness of tire rubber itself. Hence, research on temperature effect in colder temperature range is important as well, typically in icy conditions. In a cold winter, a better understanding of skid resistance behavior will lead to safer traffic by ensuring logical deicing techniques and skid maintenance in asphalt pavements to ensure better skid resistance .Accordingly, this paper reports on the durability of skid resistance in very low temperature condition, especially in an ice-melting condition lower than 0 °C, has been developed and is discussed herein. BPN values under ice melting conditions were measured to examine the skid performance of antiskid surfaces under winter road condition. The angularity and surface texture index of the used fine aggregates were characterized by mean of Aggregate Image Measure System (AIMS) with micro image analysis methods. The detailed angularity and surface texture were then correlated with BPN values under ice-melting conditions. Conclusions from this research can be used as guidance for pavement maintenance in the winter time.Asphalt mixture gradation of AC-16  and SMA-16  were first prepared as a traditional asphalt pavement surface layer. The void content is 4.2% for AC-16 and 4% for SMA = 16. Their skid resistance at ice-melting conditions were first tested. Another two antiskid surfaces were prepared by applying bitumen emulsion and fine aggregates onto AC and SMA surfaces. The construction steps of the antiskid surface are as follows:2. 2. Two types of aggregates, basalt and dolerite, were used. Firstly, beams with 380 ± 5 mm length, 50 ± 6 mm height and 63 ± 6 mm width were cut from mixture slabs. Secondly, bitumen emulsion was uniformly sprayed onto the specimen surface with a ratio of 0.7 kg/mAfter bitumen emulsion is cured, loose aggregates were swept away and finally the antiskid layer was then obtained on the cut surfaces. A BP Tester was employed to measure the surface frictional properties during ice melting process in accordance with ASTM E303-93. The values measured, BPN, represent the frictional properties obtained with the apparatus. A BP Tester is a low-speed device, about 10 km/h, that measures the skid resistance related to surface micro-texture rather than macro-texture. Antiskid layers in this research used bitumen emulsion-based chip seal technology, which therefore lets the micro-texture of surface domains cause the pavement friction. Therefore, BP Tester can be used to correlate BPN during ice melting process. Skid resistance is a function of different factors including the micro-texture and macro-texture of the pavement surface. Micro-texture is highly dependent on the surface characteristics of aggregates, such as angularity and surface texture ,20. The Basalt aggregate with nominal size of 0.3 to 0.6 mm, and dolerite aggregate with nominal size of 0.6 to 1.18mm, were used to prepare the antiskid surface layer. AIMS has been employed to characterize the angularity and 2D form of these two types of fine aggregate. The ice melting process has three stages, initial stage, melting stage and end stage. At the initial stage, the pavement surface is completely covered with ice, while the melting stage has both ice and water. At the end stage, the pavement is then covered with cold water and no ice present at all. Ice thickness of 1mm and 2 mm was prepared under following steps. Firstly, the beams were immersed in water for 2 h at room temperature, and then placed in the refrigerating chamber at −5 °C for 5 h with plastic bag packed. Secondly, the top side of every beam was carefully fenced and a certain amount of water was then stored to create an ice layer with the expected thickness. The first step was needed to first fill voids in the beam and hence prevent the water from leakage when it was poured onto the beam surface. This process is explained with The ice-melting process was characterized by putting specimens under BP Tester at room temperature condition of 28 °C. The BPN values and surface temperatures were then continuously recorded every three minutes. In this research, the 1mm thick ice was completely melted at 7 °C, and 7.2 °C for the 2 mm thick ice layer. The surface temperature of specimens in this research was defined as the temperature of the entire specimen’s surface during the ice melting process, which means it was either temperature of the ice surface or temperature of the melted water. Therefore, the initial BPN value represents the skid resistance property of the pavement surface when it is fully covered with ice. The following BPN values illustrate the skid resistance behaves when the surface is influenced by ice and water. At the end, BPN values illustrate the surface friction characteristics under the water condition.The relationship between BPN and skid resistance properties on pavements is concluded in The ice-melting process results in a concave trend between friction and surface temperature (melting time as well). There is a temperature at which the minimum friction value at pavement surface is achieved during the ice-melting process. Antiskid-B on both AC and SMA surfaces presents the highest BPN values at the end stage, but with a far different trend during the ice-melting process. On the AC structure, friction property gets gradually enhanced when the surface temperature is higher than 4 °C, while it keeps decreasing until 8 °C and then increases significantly to the maximum value on the SMA surface. Antiskid-D presents the same BPN varying trend, as well as the maximum value at the final stage, both on AC and SMA structures.Angularity from AIMS analysis describes variation at the particle boundary that influences the overall shape. Higher angularity values illustrate more angular shape. A perfect circle has a small limited value of angularity, which is close to zero . The defFirstly, according to the definition of AIMS angularity, aggregates with values higher than 5400 fall into the extreme category. AIMS also defined Form2D and described the relative form from 2-dimensional images of fine aggregates, as Fujimoto conducted a quantitative evaluation of the relation between ice thickness on a road surface and the skid resistance property. He confirmed that the skid resistance decreases with increases in the ice film thickness . This reInvestigated surfaces have a concave trend between friction and surface condition. Lowest BPN value occurs during the ice-melting stage, and then increases gradually with increasing temperature. Melted ice will result in water flow from peak points to gorges between aggregates, leading to lower average depth. Hence the smallest macrotexture and then lowest friction property will be presented.The investigated antiskid layers can significantly increase the surface friction properties during icy seasons. The lowest BPN values of AC and SMA surfaces during the ice-melting process have been enhanced from 11.6 and 14.4 to a value of around 20, respectively, when an antiskid surface has been applied. Aggregate image analysis indicated that basalt fine aggregate had higher angularity values than that of dolerite aggregate, resulting in a better friction coefficient. But the lowest BPN values of the two studied antiskid layers are the same.A thicker ice layer will result in a lower skid resistance property and smaller “lowest BPN”. The lowest BPN for the 1mm condition is 20, while it is 18 for the 2 mm condition.The durability of skid resistance during the ice-melting process at temperature from −5 °C to 10 °C was characterized by means of a BP Tester. SMA-16, AC-16 and two types of antiskid surface layer, with 1mm thick and 2 mm thick ice layers, were studied. Based on the research results, the following conclusions can be drawn:


To compare the biomechanical characteristics of different posterior intermediate screw fixation techniques (ISFTs) with hybrid monoaxial pedicle screws (Mps) and polyaxial pedicle screws (Pps) used in thoracolumbar burst fractures.Fixation techniques are compared with regard to the von Mises stress (VMS) of the instrumentations and intradiscal pressures (IDPs) of the adjacent segments by finite element method (FEM).The redistributed ROM of the fixation models with Pps fixed at the lowest segment was twice of the other fixation models in flexion and extension. The largest value of maximal VMS of a pedicle screw was located at the lowest pedicle screws when Mps are fixed at the lowest segment. The largest value of maximal VMS of the rods was decreased when more Pps are fixed at the models. Maximal IDPs of the upper adjacent segments were all larger than those of the lower adjacent segments. The maximal IDPs of the fixation model with MPs fixed at the lowest segment were larger than the other fixation models in flexion and extension.Polyaxial pedicle screws could be placed at the upper or the median segment for the facilitated efficient application of the connecting rod. We should focus on the adjacent segmental degeneration especially the upper adjacent segment in the fixation model with Mps fixed at the lowest segment. Posterior short-segment pedicle screw fixation is widely used for the management of traumatic thoracolumbar burst fractures –3, posteThe focus of our research is to find how to provide sufficient biomechanical stability with hybrid Mps and Pps (how many Pps and location of the Pps to put) on the premise of ensuring convenient placement of rods. In the current study, biomechanical characteristics of fixation techniques including MMM (6 Mps fixated at three levels), PPP (6 Pps fixated at three levels), PMM , MPM , MMP, MPP, PMP , and PPM  were compared using finite element methods, redistributed ROM, VMS of instrumentations, and IDPs of the adjacent segment under displacement loading which was evaluated.A finite element model including 7 vertebrae and 6 discs between T9 and L3 of the spine obtained from 64 spiral computed tomography (CT) images of a 40-year-old healthy male (65 kg and 175 cm) without a history of spinal injury, osteoporosis, and radiographic evidence of degeneration was reconstructed and analyzed using finite element analysis software , 23. TheThis was a prospective study to assess the biomechanical characteristics of different posterior intermediate screw fixation techniques with hybrid Mps and Pps used in thoracolumbar burst fracture model. Fixation models were described as MMM, PPP, PMM, MPM, MMP, MPP, PMP, and PPM Figs.  and 3 whThe screw diameter was 6 mm, and the screw length was 45 mm. The pedicle screws in the current study included Mps and Pps. The constraint was defined between polyaxial pedicle screw heads and shafts. However, a load limitation was defined. Surface-to-surface contact was defined between polyaxial pedicle screw heads and shafts. The screw tilt  was 25°, the static torque was 8 Nm which meant that the polyaxial pedicle screw heads will move relative to the shafts when the torque between the heads and shafts reached 8 Nm. These parameters are referred to as the polyaxial pedicle screw of Sofamor. The top surface of T9 was applied by a pure moment of 10 Nm combined with a pre-compressive load of 150 N, the inferior endplate of L3 was constrained in all degrees of freedom . The independent sample t test was used to compare the means.We used SPSS 15.0 software  to perform all statistical analyses, and P = 0.002) and extension , no significant differences in the axial rotation  and lateral bending .The fixation models presented with a decreased ROM than the intact normal spine model Table . The redP = 0.145).The largest and smallest value of maximal VMS of a pedicle screw was 382.6 MPa in the PMP model and 136.9 MPa in the PPP model, respectively (Table P = 0.000) and extension , no significant differences in the axial rotation  and lateral bending . With regard to the lower adjacent segments, there were significant differences between the fixation models with Mps fixed at the lowest segment or not in the flexion  and extension , no significant differences in the axial rotation  and lateral bending .Maximal IDPs of the adjacent segment was observed in the lateral bending. Maximal IDPs of the upper adjacent segments were all larger than those of the lower adjacent segments Table . The maxPosterior intermediate screw fixation at the fracture level can help improve and maintain the kyphosis correction, and the biomechanical stability also can be increased –18. HoweThe largest and smallest value of maximal VMS of a pedicle screw was 382.6 MPa in the PMP model and 136.9 MPa in the PPP model, respectively. The largest value of maximal VMS of a pedicle screw was located at the lowest pedicle screws when Mps are fixed at the lowest segment. These results may suggest that the PMP technique can increase the VMS of the pedicle screws. Upon suspecting that a pedicle screw is broken, we must focus on the median pedicle screws in PMP technique and the lower pedicle screws when Mps are fixed at the lowest segment. The largest and smallest value of maximal VMS of the rod was 439.9 MPa in the MMM model and 341.7 MPa in the PPP model, respectively. The largest value of maximal VMS of the rods was decreased when more Pps are fixed at the models. These results may suggest that the Pps technique can decrease the VMS of the rods. Upon suspecting that a rod is broken, we must focus on the MMM, PMM, and MPM fixation techniques.In our study, the maximal IDPs of the adjacent segment were observed in the lateral bending. Maximal IDPs of the upper adjacent segments were all larger than those of the lower adjacent segments in the fixation models. These results were consistent with previous studies –28, the This study has several limitations. It is necessary to discuss several factors, including different persons, muscle force, ribs, and length and diameter of pedicle screws, for a more clinically feasible conclusion because these factors can influence finite element analysis results.ROM of the fixation models with Pps fixed at the lowest segment was twice of the other fixation models in flexion and extension, and the largest value of maximal VMS of the rods was decreased when more Pps are fixed at the models. The largest value of maximal VMS of a pedicle screw was located at the lowest pedicle screws, and the maximal adjacent segmental IDPs of the fixation model were larger than the other models in flexion and extension when Mps are fixed at the lowest segment. Through the study, we can see that if the heads of the pedicle screws are not in a straight line, the polyaxial pedicle screws should be placed at the upper or the median segment for the facilitated efficient application of the connecting rod. Upon suspecting instrumentation failure, we must focus on the median pedicle screws in the PMP fixation technique and the lower pedicle screws when Mps are fixed at the lowest segment and the rod in the MMM, PMM, and MPM fixation techniques. We should focus on the adjacent segmental degeneration especially the upper adjacent segment in the fixation model with Mps fixed at the lowest segment.


Repetition suppression (RS) is the adaptation of the neural activity in response to a repeated external stimulus. It has been proposed that RS occurs at the thalamo-cortical level, hence activating a feedback loop to the cortex in order to counteract with the repeated motor cortical activation. In this study, to elucidate the common modulators between the RS and the inhibitory/facilitatory cortical networks, two TMS paradigms were applied, i.e. the characteristic long-interval intracortical inhibition (LICI) and the I1-wave timed short-interval intracortical facilitation (SICF). Since LICI is a local intracortical inhibitory phenomenon affecting cortical excitation over a long interval like the RS, the interaction between RS and LICI was tested. As the I1-wave timed SICF is likely not affected by inhibitory modulation, the appearance of the RS with respect to SICF was investigated. Non-linear interaction between LICI and RS was observed, while I1-wave timed SICF facilitated all MEP responses of RS by a common offset still preserving the RS. These findings implicate that the underlying mechanism for the observed interaction is likely contributed to the activation of the negative thalamo-cortical feedback loop represented by the RS, most likely at the cortical level. Pyramidal cells are most abundant in layers III and V. However, their horizontal and vertical extensions into other layers provide the motor cortical networks with a flexible synaptic organization3. This organization provides a cortical platform for neuroplasticity in the primary functions, such as movements. Neuroplasticity is a crucial characteristic in the central nervous system, enabling recruitment of the neuronal connections to adapt, as well as to maladapt, to modified requirements. Neuroplasticity is also mediated by the inhibitory GABAergic interneurons4.The primary motor cortex is a highly organized, five-layered convoluted sheet of neural cells, located on the precentral gyrus of the cerebral cortex. In the motor cortex, intermingled with the inhibitory GABAergic interneurons, the excitatory glutamatergic pyramidal neuron is the principal cell type5. TMS-evoked facilitation results in a number of cortico-spinal descending volleys in the pyramidal tracts: direct (D) and indirect (I) waves. D-waves, produced via direct activation of the pyramidal neurons of layer V, are the earliest of these descending volleys. While the origin of later I-waves is contributed to polysynaptic connections between the neurons in layer II/III and those in layer V, I1-wave is proposed to originate from the monosynaptic excitatory cortico-cortical projections on the cortico-spinal fibers6. Supporting the notion that the GABAergic inhibitory system is not involved in the formation of the I1-waves, insensitivity of these waves to GABAA agonists has been demonstrated7. To characterize the I-waves, short-interval intracortical facilitation (SICF) is a well-documented paired-pulse TMS paradigm. There is convergent evidence that the facilitatory interaction of the paired pulses in SICF occurs primarily at the cortical level8. SICF has been conventionally demonstrated using monophasic waveform8. However, it has been recently evoked applying biphasic waveform10. Repetition suppression (RS) refers to the adaptation of the neural activity in response to repeated external stimuli12. The recovery of the habituated response after the heightened initial response implies that the RS functions to prevent the brain from overreaction to a novel stimulus13. Impairment of the RS has been demonstrated previously in progressive myoclonus epilepsy and schizophrenia15. It has been suggested the RS occurs at the thalamo-cortical level, following TMS16. This phenomenon has been demonstrated in the motor system as a decrement in the amplitudes of the subsequent motor evoked potentials (MEP) following the initial one17. The decline in the MEP amplitudes indicates the adaptive behavior of the motor system. Adaptation has been associated with neuroplastic changes in the nervous system18, and therefore as an objective biomarker to assess the neuroplastic abilities. Several mechanisms have been postulated regarding the neurophysiological basis of the inhibitory phenomenon observed in the RS11. One such underlying mechanism might contribute to the increased activity of GABAergic inhibitory system, a mechanism that is also known to mediate long-interval intracortical inhibition (LICI)20. LICI is an intracortical inhibitory phenomenon that is demonstrated as suppression of neural activity, when a suprathreshold stimulus is followed by a second suprathreshold stimulus at an inter-stimulus interval (ISI) of 50–200 ms20. The inhibition time course of LICI and the pharmacological studies reflect the cortical origin and activation of the GABAB inhibitory receptors in this phenomenon22. In this study, we applied two stimulation paradigms with the RS: (1) SICF interaction in I1-wave and (2) LICI, to test the effects of immediate cortical facilitation and a cortical long-interval inhibitory modulation on the RS. The appearance of RS in relation to facilitatory I1-wave timed SICF, was applied as it is suggested to be unaffected by inhibitory modulation24, while LICI is known to be mediated by GABAB27. We hypothesized that I1-wave timed SICF would facilitate all MEP responses in the RS without affecting the inhibitory modulation, while the interaction of the LICI and RS was hypothesized to occur through the RS affecting the cortical inhibitory function through the thalamo-cortical feedback.Transcranial magnetic stimulation (TMS) is a non-invasive technique that allows us to study the facilitatory and inhibitory cortical networks by means of time-varying magnetic fieldsF = 19.24, p = 0.004), where the second, third and fourth MEP amplitudes were significantly lower than the first one . No difference in MEP amplitude was observed between the second and third, third and fourth, and fourth and second stimuli (p > 0.1). Further, to show the inhibitory effect of LICI and facilitatory effect of SICF at baseline, a Wilcoxon signed-rank test revealed a significant inhibition, on average of 92%, induced by the LICI paradigm (p = 0.012), and a paired samples t-test indicated facilitation effect on MEPs (273% on average), induced by the SICF paradigm (p = 0.013).The typical main effect of RS was observed in RS-baseline paradigm  = 4.12, p = 0.081 for the absolute MEPs and F = 15.04, p = 0.006 for the normalized MEPs) . No increase in the second and fourth MEP amplitudes was observed (p > 0.1). The Main effects of “Paradigm” and “Stimulus Order” on MEPs were also investigated  = 4.12, p = 0.082 (Paradigm) and F = 3.10, p = 0.127 (Stimulus Order) for the absolute MEPs and, F = 38.44, p = 0.001 (Paradigm) and F = 5.32, p = 0.054 (Stimulus Order) for the normalized MEPs).As opposed to MEP amplitude decline in the RS-baseline, the RS interacted with the LICI when combining the RS and the LICI paradigms, demonstrated as “Paradigm*Stimulus Order” interaction effect (Ps) Fig. . Post hoF = 7.22, p = 0.031) for the absolute MEPs and F = 8.34, p = 0.023 for the normalized MEPs), while the absolute RS effect remained unchanged (p > 0.5)  = 16.62, p = 0.005 for the absolute MEPs and F = 5.53, p = 0.048 for the normalized MEPs). Post hoc paired-samples t-test revealed the significant increase of the MEP amplitudes within the RS-SICF trains .Interaction between the RS and the SICF were observed through a combined RS-SICF paradigm. Using the absolute MEPs illustrates the common offset added to the RS by SICF clearly. Main effect of “Paradigm” revealed that the RS-SICF paradigm increased the MEP amplitudes by a common offset  the inhibitory tonus is impaired29, and the observed abnormal RS is weaker14. As the inhibitory tonus is prevailing and saturated, disabling the maintained RS and enabling an immediate recovery in adolescent patients, the mechanism described above could be a viable explanation for the observed recovery. Although several mechanisms have been proposed, the underlying neurophysiologic basis of the RS has remained elusive11. The heightened amplitude of the initial response as compared to the consecutive responses can be explained by the arousal effect; an effect originated in the brainstem ascending reticular activation system (RAS) in response to novel stimuli31. A potential model explaining the attenuation of the consecutive responses is the Sharpening model. According to this model, the population size of the firing neurons is optimized to a maximal capacity of the target organ by a feedback loop as the stimulus is repeated32.The RS is demonstrated by characteristic immediate drop in the MEP amplitude after the first stimulus, and a slight recovery of amplitudes may be observed towards the baseline19, a mechanism that is mediating the LICI22. Apart from the production mechanisms of these two inhibitory phenomena, it has been suggested that they occur at different levels of the central nervous system. The RS is thought to occur at the thalamo-cortical level, while the LICI originates at the cortical cortical level20. Therefore, most likely, RS is ruled out by LICI at the cortex, tapping into the thalamo-cortical feedback loop that is activated by the RS. To elaborate on this, one possible explanation for the underpinning mechanism of the observed interaction between the RS and LIC is the activation of the thalamo-cortical feedback loop. We speculate that similar to the baseline measurement, the negative feedback from the cortical areas such as tightly interconnected sensory areas close by may increase the GABAergic intracortical inhibition after the first stimulus, which in turn causes the suppression of the second MEP amplitude. To restore the balance between the inhibitory and excitatory states of the motor network, the negative feedback loop weakens the thelamo-cortical inhibition and disinhibition dominates, following the second stimulus, resulting in a greater MEP during the third stimulus. During the third stimulus, the feedback loop is attenuated again, allowing for a greater response to occur. Higher response causes the enhancement of the function of the negative feedback loop following the third stimulus, resulting in suppression of the fourth response. Even though, our results cannot directly pin-point the thalamo-cortical pathway as the interaction locus for RS and LICI, this possibility cannot entirely be ruled out either. Animal studies have demonstrated activation of the inhibitory thalamic reticular nuclei following stimulation of the cerebral cortex34. This activation can result in inhibition of the cerebro-thalamo-cortical pathway. It has been suggested that the conditioning stimulus of the LICI may affect the thalamo-cortical pathway at the thalamus35. Therefore, there is a possibility that the interaction site is at the subcortical level.Another plausible underlying physiological mechanism might contribute to the increased activity of the GABAergic inhibitory system36. Setting the time interval between the two consecutive pulses in SICF in such manner that I1-wave is targeted can enhance the effects of TMS by influencing the absolute momentary motor cortical activation, probably via synchronization of I2-wave in conditioning pulse with I1-wave in test pulse. Facilitatory effect of the I1-wave timed paired-pulse TMS has been recently demonstrated with biphasic TMS waveform10. In accordance with these data, our finding also suggests facilitation effect observed, using I-wave-timed paired-pulse TMS, where the inhibitory RS remains unchanged, i.e., the observed effect in the normalized MEPs was explained fully by the common offset added by the SICF to all absolute MEP responses in the RS paradigm.We observed that I1-wave timed SICF facilitated all MEP responses without modulating the absolute RS, meaning the inhibitory effect appeared not to affect the I1-wave SICF interaction. There is growing evidence on the stability of I1-wave, making it implausible to be modulated by inhibitory and excitatory mechanisms40. In the current study, ITI of 17 s was employed in all RS paradigms, since it has been depicted that RS effect is more pronounced at this ITI, probably due to more time to recover. No carry-over effects from trial to trial have been observed at this ITI28. Similarly, we optimized the parameters for LICI and SICF protocols to optimize the inhibitory and facilitatory effects confirmed as successful by our baseline measurements19. Altering the applied parameters for the SICF and LICI could have altered the results.Detrimental parameters that need to be taken into account to optimize the probability of the occurrence of the required paradigms, e.g. the RS and LICI, are the intensity, the location of the stimulus and the direction of the induced current, and ITIIn conclusion, in agreement with our hypothesis, the SICF did not interact with the RS, but facilitated all responses in the RS train. Again, in agreement with our hypothesis, the LICI demonstrated an interaction effect with RS, exhibiting a non-linear interaction partly reducing the common RS effect potentially interfering with the RS at the cortical level.41.Eight healthy right-handed volunteers with no history of neurological disorders were recruited in this study . Written informed consents were obtained from all participants. The study was approved by the research ethics committee of the Kuopio University Hospital (256/2017). The study was performed in accordance with the Declaration of Helsinki abiding the safety guidelines for TMS applications10. The subsequent TMS stimuli were administrated over the APB hotspot at an intensity of 120% rMT during three different paradigms used during this study: RS-baseline, RS-SICF and RS-LICI, utilizing the biphasic waveform  or a paired-pulse form (RS-SICF and RS-LICI). In RS-SICF a suprathreshold pulse was followed by a subthreshold one at an inter-pulse interval (IPI) of 1.4 ms10, whereas in RS-LICI, a conditioning suprathreshold pulse was delivered 100 ms before a suprathreshold test pulse42. An inter-stimulus interval (ISI) of 1 s within the trials and inter-train interval (ITI) of 17 s maintained throughout all the paradigms28. For confirming the induction of the LICI, baseline measurements were also conducted prior to the RS experiments applying 20 trials of paired pulses at 120% rMT at IPI of 100 ms at the ITI of about 5 s and comparing the first response amplitude to the second one in the stimulus pairs43. The occurrence of SICF was verified at the first stimuli of the RS-SICF protocol by comparing those to the first stimuli of the RS-baseline protocol.The measurements were performed using a customized NBS System 4.3  with an air-cooled figure-of-eight coil. Prior to the study, structural T1-weighted magnetic resonance images (MRIs) were acquired with a 3T MRI scanner . MRI data were further utilized in neuronavigation. The initial step in stimulation procedure was to determine the abductor pollicis brevis (APB) muscle “hotspot”, i.e. the cortical site capable of eliciting the maximal contralateral muscle response with minimal stimulator output intensity. Once the hotspot was identified, the resting motor threshold (rMT) was determined using system-integrated iterative threshold assessment toolEMG responses, i.e. MEPs, were recorded via an integrated EMG system at the sampling frequency of 3 kHz. A pair of disposable Ag-Cl electrodes were utilized with the active electrode placed over the belly of the APB muscle and a reference electrode placed over the joint distal to the active electrode Fig. . The MEPPost-hoc tests were performed to assess the effect of the paradigms on MEPs at each stimulus order. All tests were conducted on both absolute and normalized MEPs. Normalization was performed by dividing all average MEP amplitudes within the trials with the average amplitude of the first MEP. Post-hoc comparisons were conducted using paired t-test with Bonferroni correction. A p-value of <0.05 indicated statistical significance. Statistical analyses were conducted using SPSS  and Matlab .MEP data in each paradigm were first averaged over all the trains on the basis of their stimulus order in a train, for each subject. First, to assess the RS effect in the baseline condition, repeated measures ANOVA with “Stimulus Order” within a train  as fixed effect was applied. Also, to evaluate the inhibition effect in LICI baseline, Wilcoxon signed-rank test was conducted to compare the test pulses against the conditioning ones. A two-way repeated measures ANOVA with “Paradigm”  and “Stimulus order”  as within-subject factors was employed to evaluate the influence of the paradigms and stimulus order as main effects, as well as their interaction effect. Further,


It is known that the allele ancestral to the variation at a polymorphic site cannot be assigned with certainty, and that the most frequently used method to assign the ancestral state—maximum parsimony—is prone to misinference. Estimates of counts of sites that have a certain number of copies of the derived allele in a sample  made by parsimony are therefore also biased. We previously developed a maximum likelihood method to estimate the uSFS for a focal species using information from two outgroups while assuming simple models of nucleotide substitution. Here, we extend this approach to allow multiple outgroups (implemented for three outgroups), potentially any phylogenetic tree topology, and more complex models of nucleotide substitution. We find, however, that two outgroups and the Kimura two-parameter model are adequate for uSFS inference in most cases. We show that using parsimony to infer the ancestral state at a specific site seriously breaks down in two situations. The first is where the outgroups provide no information about the ancestral state of variation in the focal species. In this case, nucleotide variation will be underestimated if such sites are excluded. The second is where the minor allele in the focal species agrees with the allelic state of the outgroups. In this situation, parsimony tends to overestimate the probability of the major allele being derived, because it fails to account for the fact that sites with a high frequency of the derived allele tend to be rare. We present a method that corrects this deficiency and is capable of providing nearly unbiased estimates of ancestral state probabilities on a site-by-site basis and the uSFS. For example, H and E, that compare the numbers of high, intermediate, and low frequency derived variants, which can then be used to distinguish between different modes of natural selection and demographic change. A number of methods have also been developed to infer selection and demographic change based on the complete distribution of counts of derived alleles across sites, the unfolded site frequency spectrum (uSFS)  can be observed directly from sequence polymorphism data. In contrast, the derived cs e.g., . Of partThere is a related problem concerning the assignment of ancestral states, which does not seem to have been addressed. If ancestral states are assigned on site-by-site basis, potentially useful information is ignored. For example, consider the case of a single outgroup species that is uninformative about the ancestral allele of the variation in a focal species at a site. It is more likely, however, that the ancestral allele at the site is the low frequency allele, if sites with a high frequency of the derived allele are uncommon in the data set as a whole .Inference of ancestral states on a site-by-site basis has been problematic, but there has been progress in inferring the uSFS. Drosophila Population Genomics Project (DPGP) as a test case, and reinfer the ancestral state probabilities for a population of the 1000 Genomes Project in humans, which were previously inferred by a parsimony-related approach.In this article, we develop the approach of Following m orthologous gene copies at a set of sites from a population of a focal species. The uSFS we require to estimate therefore contains m − 1 elements, excluding the elements where the ancestral or derived allele is fixed. We assume that we have randomly sampled a single gene copy at each site in one or more outgroup species. We assume that the tree topology relating the species is known and does not vary among sites , along with the state for each outgroup . Let the number of outgroups = n (in n = 3), and denote the outgroups o1, o2...no. Assuming an unrooted tree  for each branch, K1...bK. The rates are the only parameters for the JC model. For the K2 model, an additional parameter, κ, specifies the rate of transition mutations relative to the rate of transversions. For the R6 model, there are six symmetrical relative mutation rates, r1...r6,r1...r5, require to be estimated.The JC model, K2 model, and a model allowing six symmetrical rates R6;  are cons∑i=16ri=1, so fiveb substitution rates and these, along with parameters of the substitution model , are estimated by ML using the simplex algorithm for likelihood maximization. We checked convergence by picking starting values for the parameters from wide distributions, restarting the algorithm when convergence had apparently been achieved, and checking that the same final maximum log likelihood was reached in multiple runs. Let ϕ be a vector specifying the model parameters, and let yi be a vector specifying the observed nucleotide configuration for the focal species and the outgroups at site i. Sites are assumed to evolve independently, so the overall likelihood of the data is the product of probabilities of the observed nucleotide configuration for each site:ntree = 4n− 1 possible unrooted trees, formed from all possible nucleotide combinations  at the unknown internal nodes along with the observed nucleotide configuration for the focal species and outgroups at the site.jc is a vector representing the observed nucleotide configuration for the focal species and the n outgroups along with the nucleotide states for the b − 1 internal nodes for tree j. If the focal species is polymorphic at a site, the probability for that site is computed as the average probability for each observed nucleotide , conditional on the nucleotide states x1,k and x2,k representing the ancestral and derived nucleotides of that branch, given the nucleotide states specified in jc:x1,k and x2,k differ from one another, the type of any difference (except in the case of the JC model), and the substitution rate parameters ϕ.The overall probability for a given tree is computed from the product of the probabilities of each branch : The algorithm to compute the probability of observing the same ancestral and derived base when two changes have occurred on a branch is illustrated by a simplified example where all relative rates in the model apart from two (r1 and r4) are zero  and k2 = 2kK(r1 + r4 + r5). In this example, the relative rates r2, r3, and r5 are all zero, but are included for completeness. Evaluation of the definite integral in (12) gives a closed form expression:For the case of x1,k ≠ x2,k: pbranch = p(1 change) + p(2 changes)2. p(1 change): Examine the example x1,k = A, x2,k = T.p(2 changes): Examine the example x1,k = A, b2,k = C.r1 and r4 are nonzero . We implemented this algorithm as follows, conditional on the ML estimate of the rate parameters, m elements, m/2 ML estimates require to be made. Assuming sites evolve independently (cf. Equation 1), the likelihood of jπ for the subset of sites (numbering sitesj) having j copies of the minor allele in the focal species is:The ML approach described by site see .i vs. allele Yi being ancestral at site i could be computed from their relative probabilities, i.e.,vs. minor copies at the site. For example, if the outgroup information were uninformative, we would assign p1 = p2. If there are few sites in the data set as a whole where the derived allele is at a high frequency, however, the estimated uSFS would tell us that A is more likely to be ancestral.The probability of allele Xi, information from the estimated rate parameters is augmented by the nearly independent information from the estimated uSFS  and extracted the 99 unrelated individuals from the Luhya in Webuye, Kenya (henceforth LWK) population. First, we restricted our analyses to sites that were fourfold degenerate in all autosomal transcripts of protein-coding genes in humans according to Ensembl release 71. We used the six-way EPO multiple alignments of primate species (available from ftp://ftp.ensembl.org/pub/release-71/emf/ensembl-compara/epo_6_primate/) to determine the alleles in orangutans and macaques at each fourfold degenerate site, and to determine whether those sites were within a CpG in humans or either of the outgroup species. We used orangutan and macaque as outgroups in our analysis. Chimpanzee and gorilla are closer and potentially more informative, but they share a high proportion of polymorphism with human and this violates an assumption of our analysis. The EPO multiple alignments were first converted from .emf format to .maf format, and then specific regions were accessed using the WGAbed package (https://henryjuho.github.io/WGAbed/). The data for the human ancestral alleles, as used by the 1000 Genomes Project , orangutan, or macaque. Non-CpG sites were defined as sites that were never CpG in their context in any of the same species, including both REF and ALT alleles in the human sample. Alleles at polymorphic sites were used to populate the uSFS following two methods: (1) using the ancestral allele provided by the 1000 Genomes Project to polarize derived and ancestral variants, and (2) using the ML method described in the present study.https://sourceforge.net/projects/est-usfs/. Supplemental material available at Figshare: https://doi.org/10.25386/genetics.6275915.Software are available for download from e.g., element 19 of a 20-element uSFS). Our measures of bias and accuracy were the average deviation and root-mean-squared error (RMSE) for this element.The uSFS inference method allows several outgroups to be included, but the extent of any benefit from additional outgroups has been unknown. To investigate this, we simulated unlinked sites according to the tree topology shown in i.e., high frequency uSFS elements tend to be slightly underestimated; Material and Methods. RMSE is reduced somewhat if a second outgroup is added, but there is little benefit from adding a third outgroup . This is relevant for inferring the uSFS for nonneutral sites, such as nonsynonymous sites of protein-coding genes, and for cases where there is variation in the mutation rate among sites, leading to variation in the rate of substitution. Such variation violates an assumption of the uSFS inference method and is therefore expected to cause the method to break down to some extent. As we previously observed . We compared the inferred uSFSs obtained using h better , largelyD. yakuba) perceptibly reduced the high derived allele frequency uSFS elements, compared to using a single outgroup (D. simulans)  . There ie.g., Mondal et al. 2015; SNP ancestral states inferred by the For non-CpG sites, the uSFS produced using the 1000 Genomes Project’s ancestral states and the uSFSs produced by our ML method broadly agreed . In contκ) at the two classes of site. Under the R6 model, which is the best-fitting model for both classes of site, the length of the branch between the human–orangutan common ancestor and humans was 0.0083 for non-CpG sites and 0.092 for CpG sites. Estimates of κ under the K2 model were 4.2 and 8.3 for non-CpG and CpG sites, respectively, which are broadly in agreement with previous studies  and r1 = r2 = r5 = r6 (transversion mutations)  see , the sama priori that change is unlikely , p = 0.98. Taking into account the fact that high frequency derived allele sites are rare in the data set as a whole, and applying Equation 18, base A is even more strongly supported as the ancestral allele, i.e., p > 0.99. This illustrates that parsimony is a good approximation for sites likely to have a low number of derived gene copies. The outcome is different for p = 0.016. Taking into account other sites in the data, which tell us that sites having 19 derived allele copies are uncommon, and applying Equation 18, p = 0.14. Thus, we are much less certain that the derived allele is A at this site. This probability increases (decreases) if the outgroups are more distant (closer) to the focal species.We have also addressed the problem of calculating ancestral state probabilities for polymorphic sites on a site-by-site basis. In doing so, we take into account both the nucleotide substitution parameter estimates (which determine the frequencies of multiple hits) and the frequencies of derived (e.g., 1  is 0.05,


Mycobacterium tuberculosis complex (MTBC). Previous studies have shown that there are at least nine members of the MTBC infecting animals other than humans; these have also been referred to as ecotypes. However, the ecology and the evolution of these animal-adapted MTBC ecotypes are poorly understood. Here we screened 12,886 publicly available MTBC genomes and newly sequenced 17 animal-adapted MTBC strains, gathering a total of 529 genomes of animal-adapted MTBC strains. Phylogenomic and comparative analyses confirm that the animal-adapted MTBC members are paraphyletic with some members more closely related to the human-adapted Mycobacterium africanum Lineage 6 than to other animal-adapted strains. Furthermore, we identified four main animal-adapted MTBC clades that might correspond to four main host shifts; two of these clades are hypothesized to reflect independent cattle domestication events. Contrary to what would be expected from an obligate pathogen, MTBC nucleotide diversity was not positively correlated with host phylogenetic distances, suggesting that host tropism in the animal-adapted MTBC seems to be driven by contact rates and demographic aspects of the host population rather by than host relatedness. By combining phylogenomics with ecological data, we propose an evolutionary scenario in which the ancestor of Lineage 6 and all animal-adapted MTBC ecotypes was a generalist pathogen that subsequently adapted to different host species. This study provides a new phylogenetic framework to better understand the evolution of the different ecotypes of the MTBC and guide future work aimed at elucidating the molecular mechanisms underlying host range.Tuberculosis (TB) affects humans and other animals and is caused by bacteria from the  Mycobacterium tuberculosis complex (MTBC)  and two lineages traditionally known as Mycobacterium africanum  (Mycobacterium microti (a pathogen of voles)  (Mycobacterium orygis (antelopes) (Mycobacterium mungi (mongooses) (Mycobacterium suricattae (meerkats)  remains a major concern both from a global health and economic point of view. With an estimated 10 million new human cases and 1.4 million fatalities in 2017, TB kills more people than any other infectious disease . Moreovex (MTBC) . The hum5 and 6) . Among t5 and 6) . These if voles) , Mycobaca lions) , Mycobactelopes)  and the telopes) , which hngooses) , Mycobaceerkats)  and the eerkats) . Mycobacservoirs . Mycobacn humans . We therive oryx  but has ive oryx . Thus, t-defined . Similar-defined . For exato human  and is oto human . Howevern cattle  and trann cattle . Conversraw milk  or closeuncommon . In cont possums .The different members and phylogenetic lineages of the MTBC share a high nucleotide identity (>99.9%), and it has recently been suggested that they should be regarded as part of the same bacterial species . The facM. bovis we added to that dataset M. bovis genomes from different geographic locations  repository by December 2017 as in . To incrocations  . Libraries were also prepared with the NEXTERA XT DNA Preparation Kit and sequenced on an Illumina MiSeq using the Miseq Reagent Kit v2, 250-cycle paired-end run .The MTBC isolates were grown in 7H9-Tween 0.05% medium (BD) ±40 mM sodium pyruvate. We extracted genomic DNA after harvesting the bacterial cultures in the late exponential phase of growth using the CTAB method . Sequenc1. We used BWA v0.7.13  -(read_length∗4∗0.07), corresponding to more than 7 miss-matches per 100 bp. SNPs were called with Samtools v 1.2 mpileup (M. tuberculosis H37Rv reference annotation (NC_000962.3). SNPs falling in regions such as PPE and PE-PGRS, phages, insertion sequences and in regions with at least 50 bp identities to other regions in the genome were excluded from the analysis (M. canettii (SRX002429) and annotated using as reference NC_015848, following the same steps described above   to clip gorithm)  to aligngorithm) . There i mpileup  and VarS mpileup  using th mpileup , in accoanalysis . SNPs knM. canettii. The topology was annotated using the package ggtree  based on SNP alignments using the R package adegent . Geneticent   . Our newneage 6) , and givneage 6) , the jumM. mungi , but no human-to-human transmission has been documented to date. M. microti was originally isolated from voles in the 1930s  (M. bovis BCG vaccine (RD1BCG) , we detected only one infection with M. microti  , which i(RD1BCG) . In supp patient  among alM. orygis. Even though M. orygis has been isolated from many different wild and domestic animals , we extend this notion, and hypothesize that M. orygis is primarily a pathogen of cattle in South Asia, leading to zoonotic TB in humans through e.g., the consumption of raw milk. This hypothetical scenario offers a parsimonious explanation for why M. orygis has repeatedly been isolated from South Asian migrants living in low TB-endemic countries in Europe, United States, and Australia  are the sub-species Bos taurus and Bos indicus. Hence, M. bovis might have adapted to B. taurus whereas M. orygis might be better adapted to B. indicus. While highly speculative at this stage, this hypothesis could be tested experimentally for instance by comparing the virulence of M. bovis and M. orygis in macrophages from B. taurus or B. indicus (We end this section by speculating that if our hypothesis regarding the host range of ectively . The cor indicus .n = 367)  Figure . The res) Figure .M. bovis  of human versus animals isolates. The estimates indicated that two randomly picked human isolates differed on average by 0.0345% nucleotide differences (95% CI: 0.0337–0.0352%) whereas animal isolates differed on average by 0.0313% (95% CI: 0.0305–0.0321%). Despite non-overlapping confidence intervals, the difference between our π estimates was small (0.003%). A higher genetic diversity of human-adapted MTBC relative to animal-adapted MTBC was also uncovered by  using otM. canettii as a reference, we found no regions that would be present in all representatives of each of the different animal ecotype genomes and absent from human-adapted MTBC genomes. Several genomic deletions have been described in the genomes of animal-adapted MTBC members which we could confirm here  expn cattle , M. mungmongoose , and trauggested .There are several reports about animal-adapted members of the MTBC infecting humans, wild and domestic animals, but an overarching analysis of all information available is required. In this study, we have combined all available information about animal-adapted MTBC strains and expanded it by sequencing more animal-adapted MTBC strains gathering the most comprehensive whole genome dataset of animal-adapted MTBC to date. We have used genomic analysis to elucidate the evolutionary history of the animal-adapted MTBC and have confirmed that the former are paraphyletic and that at least four different main clades can be defined. The phylogeny presented together with the known host range would be compatible with two scenarios during the evolutionary history of the non-human MTBC, both involving more than one host jump. One scenario would present the ancestor of the group including L6 and all animal-adapted clades as a generalist capable of infecting a wide group of mammals, and different host adaptations would have occurred thereafter. An alternative scenario proposes that the ancestor of L6 and animal-adapted MTBC was adapted to humans, and subsequent host jumps lead to the host specificity of the four clades.We found no correlation between genetic diversity of the pathogen and the phylogenetic distance of the host, as animal-adapted MTBC strains are not more diverse in average than human-adapted strains. Based on the current known host-ranges and geography of the animal-adapted MTBC, we propose that host expansion has been driven to a great extent by host geographical proximity, i.e., by contact rates among different species of mammals, and by high host population densities rather than by host genetic relatedness.DB, CL, MC, and FM have analyzed the data. DB, SG, and MC wrote the manuscript. MBB, RW, AD, SP, MB, CB, SB, and JF contributed reagents and performed the experiments.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.


Oxygen consumption (VO2) was measured at rest, and after 45 and 88 min of ice immersion, in order to calculate the metabolic heat production. Tskin dropped significantly (28–34°C to 4–15°C) and VO2 doubled (5.7–11.3 ml kg−1 min−1), whereas Tgi (37.6°C), HR (72 bpm), and mean arterial pressure (106 mmHg) remained stable during the first 30 min of cold exposure. During the remaining of the trial, Tskin and VO2 remained stable, while Tgi gradually declined to 37.0°C and HR and mean arterial blood pressure increased to maximum values of 101 bpm and 115 mmHg, respectively. Metabolic heat production in rest was 169 W and increased to 321 W and 314 W after 45 and 80 min of ice immersion. Eighty‐eight minutes of full‐body ice immersion resulted in minor changes of Tgi and cardiovascular responses, while Tskin and VO2 changed markedly. These findings may suggest that our participant can optimize his thermoregulatory, metabolic, and cardiovascular responses to challenge extreme cold exposure.Exposure to extreme cold environments is potentially life‐threatening. However, the world record holder of full‐body ice immersion has repeatedly demonstrated an extraordinary tolerance to extreme cold. We aimed to explore thermoregulatory, metabolic, and cardiovascular responses during 88 min of full‐body ice immersion. We continuously measured gastrointestinal temperature (T We investigated the thermoregulatory, metabolic, and cardiovascular responses of the world record holder of full‐body ice immersion when exposed to 88 min of ice immersion. Ice immersion resulted in minor changes of Tgi and cardiovascular responses, while Tskin and VO2 changed markedly. FurtherThe purpose of this case study was to examine the thermoregulatory, cardiovascular, and metabolic responses of the world record holder of full‐body ice immersion during prolonged full‐body contact with crushed ice. We expected to observe a drop in Tgi and concomitant drop in skin temperature (to minimize heat loss) and an elevation of the metabolic rate  to increase heat production Sessler, .22 and BSA = 2.07 m2). He was positioned in an open‐top container filled with crushed ice to the shoulder level (2.45 p.m.). The participant was wearing shorts only, whereas his body was in direct contact with the crushed ice. Core temperature , skin temperature (Tskin), blood pressure, and heart rate (HR) were measured continuously before, during and after ice exposure. Oxygen consumption was used to determine metabolic activity at rest and after 45 and 88 min of crushed ice immersion. According to prior mutual agreement, ice immersion was finished after 88 min. Signed written informed consent was obtained prior to the study. Ethical approval was waived as ice exposure was part of a demonstration session of the participant, so no additional burden was present. All measurements were conducted in line with the ethical standards of the Declaration of Helsinki.The world record holder of full‐body ice immersion is a Caucasian male (50 year), with a height of 183 cm, and a body mass of 85 kg   using a telemetric temperature capsule  and an external recorder, which has been demonstrated to be safe and reliable  using wireless temperature recorders  . A pulse oximeter  and a manual sphygmomanometer were used every 10 min as a control for the continuous Nexfin measurements.2.32) and carbon dioxide (VCO2) production were measured by indirect calorimetry using a calibrated gas exchange analyzer  with a breathing mask. The mask was carefully placed over the mouth and nose to avoid of air leakage. Metabolic heat production (M in Watt) was subsequently calculated using the following formula:Oxygen consumption , and Ec and Ef are the caloric equivalents per liter of oxygen for the oxidation of carbohydrates (21.13 kJ) and fats (19.62 kJ), respectively  demonstrated a gradual decrease from 34°C (pre‐immersion) to 32°C at the end of ice immersion Figure b. Tskin 3.2Resting HR before ice immersion was 77 bpm (range: 69–88 bpm) and HR remained relatively stable at 72 bpm (range: 66–85 bpm) during the first 40 min of ice immersion . In the second part of crushed ice immersion, our participant was still able to maintain a Tgi above 37°C, whereas HR and blood pressure were only modestly increased. There was, however, a drop in Tskin immediately upon full‐body ice immersion. Despite 88 min of full‐body contact with ice, clinically relevant complications or signs of  discomfort were not observed. Our study describes the physiological responses which counteract an extreme cold exposure in humans.2) decreased with 1.1 ± 0.1°C after 60 min of water immersion at 26°C in normal swim suit. Although clear differences exist between ice immersion and cold‐water immersion , the ability to sustain the cold tolerance and maintain a relatively stable Tgi is remarkable. One explanation for this finding may be the ~2‐fold increase in metabolic heat production in the absence of shivering or any other voluntary bodily movement. The level of oxygen consumption achieved during ice immersion is, in daily living and under normal conditions, comparable to light intensity exercise such as walking  vasoconstriction and marked elevation of metabolism.The authors report no conflict of interest.