Head direction cells are critical for navigation because they convey information about which direction an animal is facing within an environment. To date, most studies on head direction encoding have been conducted on a horizontal two‐dimensional (2D) plane, and little is known about how three‐dimensional (3D) direction information is encoded in the brain despite humans and other animals living in a 3D world. Here, we investigated head direction encoding in the human brain while participants moved within a virtual 3D “spaceship” environment. Movement was not constrained to planes and instead participants could move along all three axes in volumetric space as if in zero gravity. Using functional magnetic resonance imaging (fMRI) multivoxel pattern similarity analysis, we found evidence that the thalamus, particularly the anterior portion, and the subiculum encoded the horizontal component of 3D head direction (azimuth). In contrast, the retrosplenial cortex was significantly more sensitive to the vertical direction (pitch) than to the azimuth. Our results also indicated that vertical direction information in the retrosplenial cortex was significantly correlated with behavioral performance during a direction judgment task. Our findings represent the first evidence showing that the “classic” head direction system that has been identified on a horizontal 2D plane also seems to encode vertical and horizontal heading in 3D space in the human brain. Head direction (HD) cells in a network of brain structures including anterior thalamus, presubiculum, retrosplenial cortex (RSC), and entorhinal cortex (EC) are typically regarded as comprising the “neural compass” because they fire when an animal is facing in a particular direction in space . All had normal or corrected‐to‐normal vision and gave informed written consent to participation in accordance with the local research ethics committee.2.2www.fil.ion.ucl.ac.uk/Maguire/spaceship3D.The virtual environment was composed of two distinctive rectangular compartments, called here Room A and Room B for convenience, which were linked by a corridor Figure a. ParticThe virtual spaceship was rendered on two different mediums for pre‐scanning tasks and scanning tasks, respectively: a head‐mounted virtual reality (VR) display  and a standard computer screen (Dell Optiplex 980 with an integrated graphic chipset).The head‐mounted display provided participants with a fully immersive sensation of 3D space via its head motion tracking system, stereoscopic vision, and wide field‐of‐view (96°). A rotation movement in the VR display was made by a participant's physical head rotation and a forward/backward translational movement was made by a button press on the Bluetooth controller . For example, a participant could move up to the ceiling in the virtual spaceship by physically looking up and pressing the forward button on the controller. To rotate to the right, they physically rotated their head to the right or rotated their whole body when the required rotation was beyond the range of neck rotation. Participants could only move in parallel to their facing direction, and not straight up or down, or side to side. This allowed us to avoid a discrepancy between the HD and movement direction, because this can confound responses in HD cells . The video was a first‐person perspective that gave the participants the feeling of moving in the virtual spaceship (details of the tasks are provided in the next section). The stimuli were projected on the screen using a projector at the back of the MRI scanner bore , and participants saw the screen through a mirror attached to the head coil. The screen covered a field of view of ~19° horizontally and ~14° vertically.2.32.3.1Participants first familiarized themselves with the VR head‐mounted display and the controller during a simple “ball collection” task (duration = 5 min). Multiple balls were scattered in the spaceship and participants moved to the balls one by one. When they arrived at a ball, they received auditory feedback (a “ping” sound). The primary purpose of this task was to familiarize participants with controlling their movements in the virtual environment via head/body rotations and button presses on the controller. In addition, participants were asked to pay attention to the overall layout of environment for later tasks. This ball collection task also ensured that the participants visited every part of the virtual environment.2.3.2After the initial familiarization period, participants performed a spatial memory task which required a good sense of direction in the virtual 3D spaceship , testing took place in the same room where the ball was located during encoding. There were six additional trials where testing occurred in the other room; for example, participants encoded the ball's location in Room A but they were placed in Room B during the test phase, requiring them to point to the ball behind the wall. These “across‐room” trials were included in order to encourage participants to build an integrated map of the whole spaceship that was not limited to a local room. An integrated mental representation was important for the later fMRI analyses because we searched for direction information that was generalized across the two rooms.In the majority of trials  to confirm whether participants knew their 3D direction in the virtual environment. We then tested whether participants were better at knowing their vertical or horizontal direction. In comparing vertical and horizontal performance, it was more informative to consider how much a participant's response direction deviated from the true direction and not just whether they made a correct or wrong judgment. For example, when the true direction was 1  were acquired using a 3T Siemens Trio scanner  with a 32‐channel head coil. Scanning parameters optimized for reducing susceptibility‐induced signal loss in areas near the orbitofrontal cortex and medial temporal lobe were used: 44 transverse slices angled at −30°, repetition time (TR) = 3.08 s, echo time (TE) = 30 ms, resolution = 3 × 3 × 3 mm, matrix size = 64 × 74, z‐shim gradient moment of −0.4 mT/m ms . The first five volumes from each functional session were discarded to allow for T1 equilibration effects. The remaining functional images were realigned to the first volume of each session and geometric distortion was corrected by the SPM unwarp function using the fieldmaps. Each participant's anatomical image was then coregistered to the distortion corrected mean functional images. Functional images were normalized to MNI space.Data were preprocessed using SPM12  or horizontal (azimuth) direction information or both, we used a multivoxel pattern analysis similar to that used in previous studies . More importantly, this across‐room similarity analysis allowed us to detect relatively pure spatial direction information that was independent of view, which is naturally linked to HD. Figure r) into a dissimilarity value by inverting it (1‐r) for ease of later analysis.We then computed the neural representational similarities between each direction using Pearson's correlation using the multivoxel Finally, these neural dissimilarity measures were compared to the vertical and horizontal directional encoding models using multiple regression. We used encoding models in which neural dissimilarity is linearly dependent on the difference in pitch or azimuth between two directions Figure . For exat test. We also performed paired t tests to compare the betas of the vertical and horizontal models to ascertain whether the neural response was more sensitive to one model or the other.Regression coefficients (beta) of each participant were fed into a group level analysis to test whether the neural response in the selected ROIs was explained by vertical or horizontal encoding models. We tested whether the regression coefficient was significantly >0 using a 2.6.3r = .81, p < .001). We defined the direction information in individuals as the regression coefficient for the vertical and horizontal direction model in our ROIs. The Pearson correlation coefficient was used for the significance test.We also tested whether there was a relationship between the direction information represented in the multivoxel pattern in our ROIs and behavioral performance during the scanning direction judgment task. For the behavioral performance measure, we used the mean angular error pooled across the vertical and horizontal direction questions given that the vertical and horizontal errors were highly correlated  centered on each voxel across the whole brain. This generated regression coefficient maps for vertical and horizontal encoding models for each participant. These maps were fed into the group level analysis (one‐sample t test) in SPM. We report voxel‐wise p values corrected for our anatomical ROIs. For the rest of the brain, we report voxels that survived whole‐brain multiple comparison correction (family wise error rate of 0.05). We used SPM voxel‐level (peak‐level) inference which computes corrected p values using Random Field Theory.We performed the same representation similarity analysis using the multivoxel 33.1The pre‐scan pointing task involved participants wearing the VR head‐mounted display and looking at the remembered position of balls while they were positioned at random locations. The group mean angular error was 21 ± 9° for within‐room trials. Figure t(29) = −2.43, p = .021, Figure t(29) = 2.57, p = .015).During scanning, participants were moved in a preprogrammed 3D trajectory and were occasionally asked about their movement direction, either vertically or horizontally. The mean accuracy (74 ± 16%) was well above chance level 20%), suggesting that participants were able to keep track of their movement direction. We found that participants made significantly smaller errors for the vertical questions compared to the horizontal questions  = 3.69, p = .001; horizontal, t(29) = 2.05, p = .050, Figure t test, t(29) = 2.61, p = .014). In contrast, the left thalamus showed only horizontal direction encoding (t(29) = 2.81, p = .009, Figure t test, t(29) = −2.36, p = .025). The right thalamus and left subiculum also showed horizontal direction information  = 2.27, p = .031; subiculum, t(29) = 2.63, p = .013, Figure We investigated whether multivoxel patterns in our ROIs contained vertical and/or horizontal direction information. The right RSC showed both vertical and horizontal direction information .The above analysis revealed evidence of vertical and horizontal direction information in RSC, thalamus, and subiculum at the group level. We then tested whether direction information in these regions could explain the individual differences in behavioral performance during our direction judgment test. We found that vertical direction information in the right RSC was significantly correlated with angular error  = 5.04, p = .003, six voxels with T > 4.058 within RSC; small volume corrected for bilateral RSC masks, Figure t(29) = 7.29, p = .002, 10 voxels with T > 6.02; , t(29) = 6.87, p = .005, five voxels with T > 6.02) and cuneus  = 7.24, p = .002, 13 voxels with T > 6.02) also showed vertical direction information.A whole‐brain searchlight analysis for vertical direction encoding identified bilateral RSC  = 5.62, t(29) = 4.73, p = .016, three voxels with T > 4.313; small volume corrected for bilateral thalamus masks, Figure t(29) = 3.58, p = .039, one voxel with T > 3.483; small volume corrected for the bilateral subiculum mask). At the whole‐brain corrected level, horizontal direction information was also observed in the central sulcus  = 8.63, p < .001, six voxels with T > 6.02), supplementary motor cortex  = 6.10, p = .041, one voxel with T > 6.02), and visual cortex  = 6.22, p = .029, one voxel with T > 6.02; , t(29) = 6.04, p = .047, one voxel with T > 6.02; , t(29) = 6.24, p = .027, one voxel with T > 6.02). Of note, the number of voxels in a searchlight result should be interpreted with caution. Even when a single voxel is reported as significant, this result is driven by multiple neighboring voxels (6 mm radius) due to the nature of searchlight analyses. Furthermore, our data had a relatively small amount of smoothness compared to typical fMRI studies because we did not apply spatial smoothing during preprocessing or the group level analysis in order to retain the best spatial specificity.Horizontal direction information was observed in the anterior part of the left thalamus  are important subcortical structures for spatial navigation and memory . The dominance of vertical information in the RSC was concordant with our previous finding of vertical direction encoding when participants moved on a 3D rollercoaster  because of the views of the ceiling and floor, even though we also designed the side walls to provide clear polarization cues for the horizontal direction. Views are naturally dependent on HD, and the horizontal component of HD has less influence on views as the vertical tilt increases in 3D space. For example, if a participant looks straight toward East or West , the views can be very different due to distinct landmarks. In contrast, when the vertical tilt is 90°, the participant looks straight up in the sky and the views will be similar regardless of whether they face East or West. Although we tried to orthogonalize the view and HD by measuring the neural similarity between pairs of directional responses across different rooms in our virtual environment . Alternatively, cognitive tasks that test an explicit awareness of movement direction could be removed, given that HD cells are often recorded in rodents when animals forage in an environment without active navigation or a spatial memory test.In contrast, more spatially demanding tasks, such as 3D path integration with multiple pitch, roll, and yaw rotations  and physical gravity contributes to HD encoding in these brain structures. This could, perhaps, be facilitated by using the recently developed “mobile” magnetoencephalography brain scanner which allows head movements while measuring neural activity in humans, including from deep brain structures such as those implicated in the HD system (Boto et al., The authors declare no competing financial interests.SUPPORING FIGURE S1 3D movement trajectories during scanning. Different colors represent different scanning sessions.Click here for additional data file.


FUS or TLS) are the fourth most prevalent in Western European familial amyotrophic lateral sclerosis (ALS) populations and have been associated with causing both early and very late disease onset. FUS aggregation, DNA repair deficiency, and genomic instability are contributors to the pathophysiology of FUS‐ALS, but their clinical significance per se and their influence on the clinical variability have yet to be sufficiently investigated. The aim of this study was to analyze genotype–phenotype correlations and malignancy rates in a newly compiled FUS‐ALS cohort.Mutations in Fused in Sarcoma  was positively correlated with the disease duration. C‐terminal domain mutations were found in 90%. Among all, P525L and truncating/ frameshift mutations most frequently caused juvenile onset, rapid disease progression, and atypical ALS often associated with negative family history while the R521 mutation site was associated with late disease onset and pure spinal phenotype. Malignancies were found in one of 40 patients.We report the largest genotype–phenotype correlation of FUS‐ALS, which enables a careful prediction of the clinical course in newly diagnosed patients. In this cohort, FUS‐ALS patients did not have an increased risk for malignant diseases. Fused in Sarcoma (FUS) were found to be causative,FUS mutations were shown to have the highest proportion of all ALS‐related gene mutations in juvenile ALS patients in Germany.Amyotrophic lateral sclerosis (ALS) is recognized as one of the most severe neurodegenerative diseases. Progressive muscular paresis due to motor neuron (MN) demise leads to a rapid loss of autonomous mobility and usually culminates in death of patients after 3–5 years.FUS‐NLS mutations, which was shown to be associated with neuronal cell death.FUS knock‐out mouse embryonic fibroblasts display abundant chromosomal instability and enhanced radiation sensitivity would underline this reasoning.FUS exerts its function as a DNA‐/RNA‐binding protein primarily in the nucleus and is centrally implicated in splicing regulation, stress granule formation, and DNA repair.FUS mutations, a possible cancer hazard could be masked. Therefore, we identified via a multicenter approach 36 novel patients with FUS mutations and reviewed available medical records for the presence of malignancies. Furthermore, we analyzed individual disease parameters in context of previously reported FUS‐ALS cases to deepen our knowledge of genotype–phenotype correlations in this rare disease.Neurodegenerative diseases in general were reported to correlate with an altered risk for malignant diseases. Evidence exists for a lower tumor risk in Alzheimer's, Huntington's, and Parkinson's diseases,n = 8) analysis for the occurrence of neoplasms . We performed a multicenter cross‐sectional study to identify genetically proven FUS‐ALS patients according to El‐Escorial criteriaFurthermore, all available demographic and disease‐related data were obtained. To measure the individual disease course/survival more precisely, the term “onset to severe event”  was designed estimating the time from onset to the constant need for assisted ventilation, tube feeding, or death, which was also the censoring date for the Kaplan‐Meier curve in Figure FUS mutation (cohort 2) were collected by searching the pubmed database for the terms “FUS,” “ALS,” and “FUS MUTATION.” Only affected patients with both existing clinical data and known mutations status were included. Combined mean or median data were likewise excluded. Using this approach, a total of 186 patients from 44 studies were included in our analysis . The median AoO was 39 (CI 31–44) years and the median survival time from symptom onset until either permanent necessity for live‐prolonging measures or death (OTSE) was 25 months (CI 18–31) months. Sex did neither influence AoO  nor OTSE . Bulbar disease onset was observed in 13.8% of patients.In cohort 1, 34 patients (94%) had a C‐terminal mutation within the nuclear localization signal (NLS), whereas the two remaining patients showed mutations at position 23 in the Prion‐like domain (PLD) or at 254 in the Glycine‐rich repeats domain underlining the previously reported abundancy of C‐terminal mutations in FUS patients. Furthermore, our cohort included three cases with the P525L change, four patients were found to have a truncating mutation following inclusion of an early stop codon or frameshift alterations. Male sex was slightly more frequent with a ratio of 1.18:1 . Due to lack of data availability, the sex influence could not be completely evaluated.Cohort 2 included recently published FUS‐ALS cases for whom clinical information was available and genetic testing was done on the reported individual. By doing so, we collected 150 additional cases Table . C‐termiFigure As shown above, cohort 1 and 2 did not differ concerning AoO and OTSE Tables –S4. TherP < 0.001), which is contrary to sporadic ALS, in which late onset is associated with faster disease progression.Table Figure P < 0.001). Interestingly, the cumulative survival of the individual mutation carriers was significantly different  as indicated by the Kaplan‐Meier curves demonstrating the shortest survival for the P525L patients.Next, we grouped the most frequently reported amino acid changes at the loci P525L, R521, truncating/frameshift mutations, and others for deeper investigation. Figure P = 0.002, Mann‐Whitney U test). Surprisingly, however, the patients with bulbar course were additionally present with a significantly lower median age of onset . Considering that the study by Haberland et al. only covered a 5‐year prevalence, whereas our data can be understood as a lifetime prevalence, we recalculated with data from a register‐based study.P = 0.18).Recent reports show that FUS mutations cause impairment of proper DNA damage response,Interestingly, the affected patient, who had a severe FUS frameshift mutation R495Qfs*527 leading to truncation of the NLS, received prophylactic whole‐brain radiation during his ALL therapy. Later on, he was diagnosed with a meningioma and eventually with ALS.Conclusively, our data did not indicate an increased risk for malignancies in FUS‐ALS patients.In this study, we provide the largest so far reported cohort of 186 FUS‐ALS patients and thereby enable a careful prediction of the clinical course in newly diagnosed patients and their families carrying the more common amino acid changes. FUS‐ALS patients showed a younger disease onset (median 39 years) than sporadic ALS patients.Our work extends previous work on genotype–phenotype correlationsATM gene leading to insufficient DNA double‐strand break repair,FUS mutations.There is evidence for a lower risk of cancer in several neurodegenerative disorders like Alzheimer's, Parkinson's, and Huntington's disease.Fang et al. might be more suitable when comparing to FUS‐ALS patients. FUS‐ALS is otherwise regarded to be the most frequent ALS subtype in juvenile patients (<35 years)FUS mutations and to compare with younger control cohorts.Thus, we addressed the clinical relevant question if FUS‐ALS patients have a higher prevalence of malignant neoplasms. The retrospective assessment in our cohort revealed a lifetime prevalence of 2.5%. This was not significantly higherFUS mutations reported to influence DNA repair,Finally, there were only a few To our knowledge, our survey has assessed the largest FUS‐ALS patient collective so far. However, the rarity of the disease and the retrospective style has obvious limitations and warrants prospective validation. Still, we believe that our survey provides a sufficient cohort, which should be helpful when counseling patients and their families.M.N. and A.H. concepted and designed the study including statistics and data acquisition of previously published and newly reported patients and manuscript drafting. K.P. and R.G. contributed patient data and helped designing the manuscript. V.D., A.J.K., E.A., P.D., F.W., P.C., P.A., J.W., J.P., S.C., A.H., A.C.L., F.P., and A.G. contributed patient data, critically reviewed the manuscript, and helped designing it.The authors report no conflict of interest.Figure S1. Depiction of the individual age of onset of all patients (cohort 1 and 2) relative to the median (39), which illustrates the clinical heterogeneity of the disease.Table S1. FUS‐ALS patients identified by pubmed review and novel patientsTable S2. Descriptive data of newly identified patients (cohort 1).Table S3. Descriptive data of previously published cases (cohort 2).Table S4. Descriptive data of the combined cohorts (cohort 1 + 2).Data S1. Additional file providing raw data on published FUS‐ALS cases including their source and descriptive data. Figure S1 similarly to Figure 1 depicts the individual AoO but for all patients of the study.Click here for additional data file.


We report a man with diffuse alveolar hemorrhage caused by multiple myeloma who was diagnosed with the aid of bronchoalveolar lavage and transbronchial lung biopsy. Multiple myeloma should be considered as an important differential diagnosis in patients with diffuse alveolar hemorrhage, and bronchoscopy may help to differentiate the cause. Chest X‐ray and computed tomography (CT) showed bilateral ground‐glass opacities  followed by oral prednisolone at 40 mg/d. However, the corticosteroid therapy was not effective: the inflammatory reaction increased, and his respiratory function continued to deteriorate. Therefore, we began another 3‐day course of intravenous methylprednisolone on the 9th hospital day. Tests for antibodies associated with connective tissue diseases, including MPO‐ANCA, PR3‐ANCA, and anti‐GBM antibody, were all negative. Although his IgA value was high, his IgG and IgM values were normal. Serum electrophoresis showed no M protein, but serum immunoelectrophoresis revealed the presence of IgA‐κ‐type M protein. Serum free light chain (sFLC) assay showed a κ/λ ratio of 21.4 . The patient’s β2‐microblobulin was 1.6 mg/L , and he was negative for urine Bence Jones protein. An abdominal CT performed to search for MM revealed a left iliac tumor  was obtained from the left B5 by bronchoscopy. We instilled 150 mL of 0.9% NaCl and aspirated 70 mL of bloody fluid containing 93.2% macrophages, 4.7% lymphocytes, 2.1% neutrophils, with a CD4/8 ratio of 0.5, and 25% hemosiderin‐laden macrophages. A transbronchial lung biopsy (TBLB) showed interstitial fibrosis but was negative for plasma cells. We found no amyloid deposition in the lung by direct fast scarlet staining. However, deposition of IgA was revealed on a blood vessel wall by immunofluorescence staining, but no deposition of IgG was present (Figure 3This case showed that IgA deposition due to MM might cause DAH. We used the diagnostic criteria of the International Myeloma Working Groupde Prost et alAmong five diagnosed cases of MM due to DAH, three were caused by PRSAfter treatment for MM was initiated in our patient, his serum IgA level and sFLC κ/λ ratio decreased, and his DAH and respiratory condition improved, although his iliac lesion remained unchanged. We are continuing to follow this patient to determine whether his MM has improved.In conclusion, we reported a case of DAH caused by IgA deposition associated with MM. In this patient, TBLB revealed deposition of IgA in the alveolar wall. MM should be considered as one possible cause of DAH as the initial symptom of MM might well be DAH. IgA deposition in the alveolar wall should also be considered as a potential cause of DAH in the absence of PRS.None declared.AF: is the guarantor of the paper, taking responsibility for the integrity of the work as a whole, from inception to published article. NK, TI, YT, KK, YS, and NT: aggregated the data and helped draft the discussion of the manuscript.


Investigating early white matter (WM) change in Huntington's disease (HD) can improve our understanding of the way in which disease spreads from the striatum.We provide a detailed characterization of pathology‐related WM change in HD. We first examined WM microstructure using diffusion‐weighted imaging and then investigated both underlying biological properties of WM and products of WM damage including iron, myelin plus neurofilament light, a biofluid marker of axonal degeneration—in parallel with the mutant huntingtin protein.We examined WM change in HD gene carriers from the HD–CSFcohort, baseline visit. We used standard‐diffusion magnetic resonance imaging to measure metrics including fractional anisotropy, a marker of WM integrity, and diffusivity; a novel diffusion model (neurite orientation dispersion and density imaging) to measure axonal density and organization; T1‐weighted and T2‐weighted structural magnetic resonance imaging images to derive proxy iron content and myelin‐contrast measures; and biofluid concentrations of neurofilament light  and plasma) and mutant huntingtin protein (in CSF).HD gene carriers displayed reduced fractional anisotropy and increased diffusivity when compared with controls, both of which were also associated with disease progression, CSF, and mutant huntingtin protein levels. HD gene carriers also displayed proxy measures of reduced myelin contrast and iron in the striatum.Collectively, these findings present a more complete characterization of HD‐related microstructural brain changes. The correlation between reduced fractional anisotropy, increased axonal orientation, and biofluid markers suggest that axonal breakdown is associated with increased WM degeneration, whereas higher quantitative T2 signal and lower myelin‐contrast may indicate a process of demyelination limited to the striatum. HTT). Given the certainty of onset in those that inherit the gene combined with genetic testing, we can examine brain changes from the earliest, presymptomatic disease stages.The ongoing development of novel therapeutics to treat Huntington's disease (HD) necessitates improved characterization of HD‐related brain changes. HD is a progressive neurodegenerative disorder characterized by a motor, cognitive, and neuropsychiatric phenotype and caused by CAG expansions in the huntingtin gene (Magnetic resonance imaging (MRI) measures of neuronal atrophy have characterized macrostructural brain changes associated with HD progression.However, the nonspecific nature of standard diffusion analysis techniques has limited our ability to identify underlying features of reduced WM organization in HD.The investigation of underlying biological properties and products of WM damage can potentially complement diffusion imaging of HD‐related WM changes. Myelin is a crucial component for axonal structure and efficient WM functionality. Early demyelination in HD may increase iron‐richmHTT (mutant HTT), the pathogenic agent in HD, and its concentration may be interpreted as a direct measure of the genetic–pathological burden in HD that appears to be neuronal in origin.Similarly, neurofilament light (NfL)—a key component of the neuronal cytoskeleton and marker of axonal degeneration—is elevated in HD gene carriers,mHTT), and measures of disease progression in HD gene carriers from the HD‐CSF cohort.Here we have attempted to characterize more fully white matter in HD by investigating the relationships between microstructural imaging markers, measures of myelin and iron content, biofluid markers  rather than simple categorical prediagnosis and postdiagnosis groups, which can exclude the subtle effects of heterogeneity. Following processing and visual quality control, our final analyses included 15 control and 39 HD gene‐carrier diffusion‐weighted datasets, 15 control and 38 HD gene‐carrier myelin‐contrast images, and 18 control and 36 HD gene‐carrier quantitative T2 maps.This study was conducted according to Declaration of Helsinki principles and was approved by the London Camberwell–St Giles Research Ethics Committee. All participants gave written informed consent prior to inclusion in the study.3. The diffusion‐weighted MRI data were acquired using a multiband spin‐echo EPI sequence with time‐shifted RF pulses2, with 72 slices collected at a resolution of 2 × 2 × 2 mm3. Two sets of multishell data were collected. The first consisted of no‐gradient images (b = 0) and gradient‐direction images of b = 100, b = 300, b = 700, b = 2000; the second set of data consisted of no‐gradient images (b = 0) and gradient‐direction images of b = 100, b = 500, b = 3000. A reverse phase‐encoding scan was acquired to correct for susceptibility‐induced distortion with the same acquisition parameters as the main diffusion scan.T1‐weighted (T1W), T2‐weighted (T2W) and diffusion‐weighted MRI data were acquired on a 3T Prisma scanner  using a protocol optimized for this study. The T1W images were acquired using a 3‐dimensional Magnetization Prepared Rapid Acquisition Gradient Echo (MPRAGE) sequence with a Relaxation Time (TR) = 2000 milliseconds and Echo Time (TE) = 2.05 milliseconds; inversion time of 850 milliseconds, flip angle of 8°, matrix size 256 × 240; 256 coronal slices of 1.0 mm thickness were collected. Parallel imaging acceleration was used  and 3‐dimensional distortion correction was applied to all images. T2W data for relaxometry were collected with a 3‐echo time Turbo Spin Echo sequence with TE1 = 13 milliseconds, TE2 = 81 milliseconds, TE3 = 121 milliseconds, and TR = 9400 milliseconds. Matrix size was 256 × 192 with 55 slices collected at a resolution of 1 × 1 × 2.5 mmhttp://www.fil.ion.ucl.ac.uk/spm/) on a MATLAB R2012B (https://in.mathworks.com). For the VBM analyses, after N3 bias correction (SLED)All scans underwent rigorous quality control, including metadata checks and visual quality control. T1W images were used for both voxel‐based morphometry (VBM) analyses and calculating myelin‐contrast maps using the MR‐ToolDiffusion data were analyzed using the whole‐brain approach, tract‐based spatial statistics (TBSS).mHTT levels in CSF were measured in triplicate using a single‐molecule counting immunoassay  as previously described.mHTT could not be detected in control participants. CSF levels of NfL were quantified in duplicate using the neurofilament (NF)‐ light enzyme‐linked immunosorbent assay (ELISA) according to the manufacturer's instructions . Plasma NfL concentration was measured in duplicate with ultrasensitive single‐molecule array (Simoa) technology—the commercially available NF light kits, as per the manufacturer's instructions .Sample collections were standardized to time of day, 12‐hour fast, plasticeware and processed within 15 minutes from collection as previously described.t tests and chi‐squared tests where appropriate in SPSS version 22 . For VBM analyses, group comparisons were performed for WM volume between control and HD gene carriers using linear regression models within SPM12. Regression analyses were then performed correlating WM volume with NfL plasma, NfL CSF, and mHTT levels independently in the HD‐only group. Similarly, group analysis was performed in SPM12 to compare T1:T2 weighted (myelin contrast) and quantitative T2 maps between control and HD gene carriers. Regression analyses were then performed in the HD‐only group to investigate correlations between T1:T2 weighted (myelin contrast) and NfL plasma, NfL CSF, and huntingtin mHTT levels independently; these analyses were then repeated for the quantitative T2 maps in SPM12. All analyses are presented at the multiple comparison corrected level at P < 0.05 (family‐wise error [FWE]). Following TBSS processing, diffusion maps for each metric were analyzed by means of permutation tests using FSL Randomise. Similarly to the previous analyses, group comparisons were performed between control and HD gene carriers for each diffusion metric , and then correlations between each diffusion metric and NfL plasma, NfL CSF, and mHTT levels were performed independently. All TBSS results are presented at P < 0.05 Threshold Free Cluster Enhancement (TFCE) cluster corrected unless stated.Demographic and clinical information were analysed using independent mHTT.For demographic and clinical information, see Table P < 0.05 TFCE cluster corrected. FA was lower with congruent widespread higher MD, RD, and AD in the HD group when compared with controls  and higher NFL levels at a nonsignificant level.P < 0.05 (FWE).The HD group showed lower myelin contrast in the left caudate when compared with controls. In the HD‐only group, lower myelin contrast correlated with lower TMS in the bilateral caudate. DBS was not significantly associated with myelin contrast. There were no significant correlations with either biofluid marker.All analyses are presented at the corrected level at P < 0.05 (FWE). There was higher quantitative T2 (lower iron content) in the occipital lobe, the right motor cortex, and the caudate bilaterally in the HD group when compared with controls . WM volume was lower in premotor and motor areas in the HD group when compared with controls. In the HD‐only group, there was a negative association between bilateral WM volume around the striatum and NfL plasma levels, which survived adjustment for CAG and age–CAG interaction in the left striatum only. There was a similar negative association between left striatal WM volume and NfL CSF levels, but this did not survive adjustment for CAG and age–CAG interaction.All analyses are presented at the corrected level at Given the role of WM degeneration in HD pathology, we have used both neuroimaging and biofluid markers to provide a more complete characterization of WM microstructure during the course of HD. We examined MRI‐based changes in diffusivity as a measure of WM connectivity using both standard DTI modeling and a novel diffusion model designed to interrogate more explicity the biological properties underlying WM differences, plus proxy measures of myelin and iron content. We then looked at the relationships between these imaging metrics and biofluid markers of neuronal damage and genetic–pathological burden, namely, NfL (sampled from both blood plasma and CSF) and CSF concentrations of mHTT, the pathogenic agent in HD. We identified widespread higher diffusivity levels coupled with lower FA and neurite density in HD gene carriers when compared with controls. Furthermore, lower FA and higher diffusivity were strongly associated with clinical measures of HD progression, while lower FA was also associated with higher mHTT levels. Myelin contrast and quantitative T2 were higher (lower iron content) in the HD group when compared with controls and similarly both highly correlated with clinical burden. There were, however, no significant associations of myelin contrast or quantitative T2 with either biofluid marker.HD has largely been characterized by macrostructural changes, with neuronal loss occurring in the striatum and WM during premanifest stages, eventually spreading to the cortex. Despite extensive investigation of WM microstructure, the underlying biological bases of these macrostructural changes are still relatively unknown. By using multimodal imaging to investigate not only connectivity indexed by diffusion but also proxy measures of myelin contrast and iron content, in conjunction with biofluid markers of axonal degeneration, we can begin to formulate a more complete picture of the effects of HD pathology on the living human brain.P = 0.06, FWE corrected), which correlated with clinical progression at a similarly lower level of significance , indicating that axonal proliferation in striatal white matter tracts may be related to disease stage. Given that axonal dispersion in WM tracts surrounding the basal ganglia has been shown to be lower in preHD,Robust evidence across a number of HD cohorts has shown WM disorganization during the earliest disease stages.P = 0.06, FWE corrected—interesting given the evidence of early callosal degeneration in HD.While there is evidence for axon‐driven WM degeneration, in particular lower axonal density, we wanted to characterize these changes more fully by examining the relationship between this diffusivity‐based evidence of WM disorganization and NfL, a biofluid marker of axonal degeneration that is highly correlated with HD progression.Mutant HTT was also highly and robustly correlated with increased MD, AD, and RD, and although the specific nature of mHTT effects on WM microstructure is unclear, it would appear that higher levels of mHTT reflect some degree of WM degeneration. Our findings are of particular interest given that WM change in HD seems to occur considerably earlier than that of cortical matter atrophy and supports a hypothesis of axonal degeneration in HD. Interestingly, none of our correlations survived correction for the age × CAG interaction, signifying that axonal degeneration is unlikely to be independent of disease progression but, rather, is associated with the worsening effects of pathology.Myelin contrast (indexed by the T1:T2 weighting maps) and quantitative T2 changes in our HD cohort were less widespread than those associated with diffusivity. There was evidence of lower myelin contrast in the caudate and higher quantitative T2 (indicative of lower iron content) in the caudate and select cortical regions in the combined HD group when compared with controls. Furthermore, there was also lower myelin contrast and higher quantitative T2 (indicative of lower iron content) in the bilateral caudate as motor performance deteriorated in the HD‐only group. This combination of potentially lower iron content and myelin contrast suggests a reduction in the presence of iron‐based oligodendrocytes, which may indicate demyelination in striatal regions. Given the evidence for axonal degeneration, however, it would appear that demyelination is less associated with HD pathology than axonal breakdown, particularly given the lack of correlation between both iron and myelin and NfL or mHTT. However, we do acknowledge that both the myelin‐contrast ratio and quantitative T2 mapping are proxy measures of myelin and iron content, respectively. Quantitative T2 mapping, for example, may not be adequately sensitive to detect changes driven by changes in NfL levels, explaining the lack of association. Alternative MRI techniques, such as multiparameter mappingPlasma NfL increases appeared to show a greater association with increased diffusivity measures than CSF NfL. However, these patterns were only visible at a lower corrected threshold and require further investigation in a larger HD cohort.Using a HD cohort of both premanifest and manifest gene carriers, we have shown that WM diffusivity is higher in HD than control participants, correlating clearly with measures of disease burden, including clinical scores, such as DBS and TMS, and the toxic source of disease, mHTT itself. Proxy measures showed lower myelin contrast and iron content in the HD group when compared with controls, suggesting potential demyelination within striatal regions. By investigating white matter microstructure, underlying biological properties that may drive microstructural change, such as myelin and iron, and products of WM damage including NfL, we have presented a detailed characterization of WM change in HD.(1) Research Project: A. Conception, B. Organization, C. Execution; (2) Statistical Analysis: A. Design, B. Execution, C. Review and Critique; (3) Manuscript Preparation: A. Writing of the First Draft, B. Review and Critique.S.G.: 1C, 2A, 2B, 2C, 3A, 3BE.B.J.: 1C, 2B, 3BL.M.B.: 1B, 1C, 3BF.B.R.: 1B, 1C, 3BA.H.: 2B, 3BJ.M.: 2B, 3BD.T.: 1B, 3BH.Z.: 1B, 3BE.d.V.: 1B, 1C, 3BS.J.T.: 3BG.R.: 3BR.I.S.: 2B, 2C, 3BE.J.W.: 1A, 1B, 2C, 3BThis study was conducted according to Declaration of Helsinki principles and was approved by the London Camberwell–St Giles Research Ethics Committee. All participants gave written informed consent prior to inclusion in the study. We confirm that we have read the Journal's position on issues involved in ethical publication and affirm that this work is consistent with those guidelines.Funding Sources and Conflicts of Interest: This work was primarily supported by the Medical Research Council (MR/M008592/1). S.G., E.B.J., R.I.S., G.R., and S.J.T. receive support from a Wellcome Trust Collaborative Award (200181/Z/15/Z). F.B.R. is supported by Cure Huntington's Disease Initiative (CHDI) Foundation. E.D.V. is supported by the Wellcome Engineering and Physical Sciences Research Council Centre for Medical Engineering (WT 203148/Z/16/Z).Financial Disclosures for the Past 12 Months: Via University College London (UCL) Consultants, a wholly‐owned subsidiary of UCL, E.J.W. has participated in consultancies and scientific advisory boards with Hoffmann‐La Roche Ltd, Ionis Pharmaceuticals, Shire, Novartis, Mitoconix, PTC Therapeutics, and Wave Life Sciences and is an investigator on the IONIS‐HTTRx (RG6042) program. His host clinical institution, UCL Hospitals NHS Foundation Trust, has received funds as compensation for conducting clinical trials for Ionis Pharmaceuticals, Pfizer, and Teva Pharmaceuticals.


Whole genome sequencing has identified the loss of certain parts of the Tn3‐type transposon typically associated with mcr‐5, providing a clue toward its mobilization. Phylogeny of MCR‐5 suggests that it is distinct from the MCR‐1/2 sub‐lineage, but might share a common ancestor of MCR‐3/4. Domain‐swapping analysis of MCR‐5 elucidates that its two structural motifs  are incompatible with its counterparts in MCR‐1/2. Like the rest of the MCR family, MCR‐5 exhibits a series of conservative features, including zinc‐dependent active sites, phosphatidylethanolamine‐binding cavity, and the mechanism of enzymatic action. In vitro and in vivo evidence that MCR‐5 catalyzes the addition of phosphoethanolamine to the suggestive 4′‐phosphate of lipid A moieties is integrated, and results in the consequent polymyxin resistance. In addition, MCR‐5 alleviates the colistin‐induced formation of reactive oxygen species in E. coli. Taken together, the finding suggests that a growing body of MCR family resistance enzymes are functionally unified.A growing number of mobile colistin resistance (MCR) proteins is threatening the renewed interest of colistin as a “last‐resort” defense against carbapenem‐resistant pathogens. Here, the comparative genomics of a large plasmid harboring  Apart from E. coli and Salmonella enterica, two additional bacterial reservoirs were detected for mcr‐5, namely, Pseudomonas and Aeromonas hydrophila.Aeromonas, the pathogens of aqua‐cultured fishes have been found to harbor the prevalent mcr‐3mcr‐5 in A. hydrophila raises crucial questions about its evolutionary origins.Since its first discovery in southern China, in late 2015,Though MCR‐5 shares low similarity with the paradigmatic MCR‐1 of the MCR family , it is still thought to function as a PEA‐lipid A transferase. However, little or no information is available about the functional, structural, and mechanistic aspects of MCR‐5. In this study, we aim to provide a composite picture of MCR‐5 from this perspective while also tracing its evolutionary relationships with the other members of the MCR family. The finding might allow us to potentially develop better strategies to manage its spread and to eventually develop therapeutic agents that can reverse colistin resistance.22.1mcr‐5 was identified by the whole genome sequence of strain WCHAH045096  and the resolvase (tnpR) genes share only 86 and 81% nucleotide identity, respectively, between the two transposons . This suggests that a resolvase‐mediated site‐specific recombination has occurred  on pMCR5_045096 flanked by a 5 bp DR (AGACG). In fact, the Tn3‐type transposon from pMCR5_045096 is 99% identical to that on the chromosome of Cupriavidus gilardii strain CR3, with mcr‐5 disrupting proP without IRL or IRR  with two distinct domains connected by a flexible linker, a periplasmic catalytic domain, and a transmembrane domain comprised of five α‐helices . The N‐terminal hexa‐histidine‐tagged MCR‐5 protein in full length was purified to homogeneity  with an apparent molecular mass of 63.34 kDa  and verified by peptide‐mass fingerprinting with a coverage of 91.04% . MCR‐5 behaves as a monomer in solution when examined using size exclusion chromatography . A circular dichroism spectrum of the purified protein shows a peak followed by a dip characteristic of an alpha helix rich protein . The presence of bound zinc was confirmed by inductively coupled plasma‐mass spectrometry . Further, the interaction of the physiological substrate lipid‐PE with MCR‐5 was predicted by molecular docking using the modeled structure of MCR‐5 . This reveals a cavity at the interface of the two domains that perfectly accommodates the PE head group of the substrate Figure4A. In E. coli host  measurements with S331 being resistant to colistin (2 µg mL−1), N112A and T116A having significantly reduced susceptibility to colistin (1 µg mL−1), T286A, H384A, E120A, and K334A having reduced susceptibility to colistin (0.5 µg mL−1) and E248A, D458A, H459A, H389A, and H471A being as susceptible as the negative control (0.25 µg mL−1) . Purified lipid A extracts were subjected to mass spectrometry. The strain expressing MCR‐5 has a peak  that is 123u more than that found in the wild‐type E. coli MG1655 . This corresponds to an addition of a PEA group to lipid A resulting in PPEA‐4′‐Lipid A. In agreement with the colistin MIC trials  when evaluated by mass spectrometry . A similar result is also observed for N112A and T116A . All other mutants could no longer modify lipid A in vivo and had wild‐type lipid A species . Together, the data suggest that the residues S331A, N112A, and T116A  might not be directly involved in the catalytic mechanism illustrated with a “ping‐pong” reaction model  and are at least partially dispensable to function. These residues might instead be involved in stabilizing the substrate.To further examine the functionality of MCR‐5 and its mutants, their ability to modify the lipid A of a susceptible host 2.5Figuremcr derivative  in the functional assay of colistin resistance with TM1‐MCR‐5, TM2‐MCR‐5, and TM5‐MCR‐2  analyses  condition , whereas the presence of MCR‐5 confers the ability of the recipient strain to resist bacterial killing by colistin . Intriguingly, in the presence of bipyridine, a chelator of ferric involved in Fenton reaction, the colistin‐susceptible E. coli MG1655 can be efficiently restored from colistin‐mediated cell death . In addition, the ROS scavenger, l‐cysteine alone (or together with bipyridine) also significantly rescue bacterial survival of E. coli stressed with colistin . Evidently, not only does the Fenton reaction participate in the formation of free hydroxyl radicals  but also functional expression of mcr‐5, an evolutionarily distinct genetic determinant of MCR family, quenches/interferes/prevents/terminates the entry of the recipient E. coli into the hydroxy radical death pathway . In general agreement with the statement of mcr‐1 by Yang et al.,mcr‐1/2  and mcr‐5 . In conclusion, we demonstrated that MCR‐5 modifies lipid A, an important component of bacterial surface structure, stops the penetration of polymyxin into cells, alleviates the formation of ROS, and in turn bypasses  antibiotic killing by colistin . This process in MCR‐harboring E. coli has an appreciable fitness cost, that is, linking of retarded growth caused by MCR “poisoning” .To further verify this question, we also applied chemical rescue trials . Obviously, no growth of 3E. coli, S. enterica, and Pseudomonas species. However, unlike the other members, MCR‐5 is associated with intact Tn3‐type transposition elements that would allow it to mobilize onto plasmids or chromosomes. Here, our study identifies mcr‐5 on a large nonconjugative plasmid from A. hydrophila that is missing the characteristic repeats flanking the transposon but retains the transposase (tnpA) and resolvase (tnpR) genes. During the progress of this work, another mcr‐5‐carrying A. hydrophila strain, I064‐2, with a pig origin was reported in China.mcr‐5 was carried by a small (7915 bp) ColE2‐type plasmid pI064‐2. Unlike the mcr‐5‐carrying Tn3 family transposon on pMCR5_045096 in this study, the tnpA and tnpR genes and one IRR of the Tn3 family transposon were missing but the IRL was present on pI064‐2 . Apparently, this is distinct from other chromosomal modifications that result in colistin resistance such as addition of cationic sugars to lipid AHowever, despite the evolutionary differences, the entire MCR family seems to share significant similarities in the architecture and composition of the active site cavity and a paralleled catalytic mechanism. This cavity is formed at the domain interface and is positioned close to the inner membrane, possibly to access the PE‐lipid substrate. Both the in vivo and in vitro data point toward a “ping‐pong” mode of catalysis, wherein the enzyme reaction proceeds in two steps , similar to MCR‐1/2/3 does.Taken together, the characterization of MCR‐5 action represents a functional proof for the rapidly evolving family of mobile colistin resistance. The proposal that an entire MCR family is functionally unified, constitutes a significant step toward curtailing the rapid evolution and spread of colistin resistance. In terms of detailed perspectives from genomic, evolutionary, structural, and mechanistic studies, we are allowed to be brought closer in the context of developing novel therapeutic agents and adjuvants that can address the whole MCR family.4Isolation and Identification of A. hydrophila: The strain WCHAH045096 of A. hydrophila was isolated from hospital sewage, which was collected from the influx of the wastewater treatment plant at West China Hospital, Sichuan University, Chengdu, China, in October 2014. Species identification was performed using partial sequencing of the gyrB gene as described previously.Strains, Plasmids, and Growth Conditions: Except for A. hydrophila, all the other strains referred to the derivatives of E. coli MG1655 . Primers were designed for gene cloning and/or PCR detection . The mcr‐5 gene was amplified with PCR from A. hydrophila, and then cloned into two expression vectors (an arabinose‐inducible vector pBAD24 and the IPTG‐inducible plasmid pET21a), giving pBAD24.8xHis::mcr‐5, and pET21::mcr‐5, respectively. As we recently describedmcr‐5 were generated using site‐directed mutagenesis with the Mut Express II fast mutagenesis kit V2 . Overlapping PCR experiments were performed to create the domain‐swapped versions of mcr‐5/2/1. Besides the two hybrid versions (TM1‐MCR‐2 and TM2‐MCR‐1) we developed earlier,E. coli strains were cultivated at 37 °C, in which either liquid Luria‐Bertani (LB) broths or LB agar plates are involved.Bacterial Conjugation: Conjugation experiments were carried out both in broth and on filters. The azide‐resistant E. coli strain J53 and a colistin‐susceptible azide‐resistant A. hydrophila strain both were used as the recipient. Possible trans‐conjugants were selected on LB agar plates containing 2 µg mL−1 colistin and 150 µg mL−1 azide.Determination of Colistin Susceptibility: MIC of colistin was determined using the microdilution method following the recommendations of the Clinical Laboratory Standards Institute (CLSI).http://www.eucast.org/) were applied. The strains tested here included A. hydrophila and the engineered versions of E. coli carrying mcr‐like genes . No less than three independent trials were conducted as recently described.Whole Genome Sequencing: Genomic DNA of strain WCHAH045096 was prepared using the QIAamp DNA mini kit  and was subjected to whole genomic sequencing using both Illumina HiSeq 2500 platform  and the long‐read MinION Sequencer . The de novo hybrid assembly of both short Illumina reads and long MinION reads was performed using Unicycler v0.4.3https://github.com/tseemann/abricate) to query the ResFinder database (https://cge.cbs.dtu.dk/services/ResFinder/). Plasmid replicon type and plasmid multilocus sequence type were determined using the PlasmidFinder (https://cge.cbs.dtu.dk/services/PlasmidFinder/) and pMLST tools (https://cge.cbs.dtu.dk/services/pMLST/). The chromosome of A. hydrophila WCHAH045096 and the complete sequence of pMCR5_045096 were separately deposited into GenBank under the Accession Nos. CP028568 and CP028567, respectively.Preparation and Identification of MCR‐5: The recombinant version of MCR‐5 integral membrane protein was overexpressed in the strain of BL21 (Rossetta) with pBAD24.8xHis::mcr‐5  as recently described with MCR‐1/2/3−3m Tris‐HCl (pH 8.0), 50 × 10−3m NaCl, 5 × 10−3m imidazole, 5% glycerol and 1% detergent DDM (m/v)] and then incubated with pre‐equilibrated Ni‐NTA agarose beads for 4 h at 4 °C for further affinity chromatography. The purified MCR‐5 protein was concentrated with a ultrafilter column , judged by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) (12%), and identified with A Waters Q‐Tof API‐US Quad‐ToF mass spectrometer.In Vitro Assays for MCR‐5 Catalysis Reaction: As initially reported by Anandan et al.Measurement of Cytosolic ROS Level: Prior to the challenge with colistin , the mid‐log phase cultures  of E. coli alone or carrying mcr‐1/5 variants were stained with the oxidant sensor dye DCFH2‐DA (Sigma)  to detect the intracellular ROS.6 CFU and subjected to confocal microscopy (and/or flow cytometry) based measurement of cytosolic ROS.ltures OD0, 0.5 ofltures OD0, 0.5 ofStructural Determination of LPS‐Lipid A: LPS‐lipid A pools were isolated and purified from the engineered E. coli with or without mcr‐5 (or its derivatives) as Liu et al.−3m sodium acetate buffer (pH 4.5) with aqueous 0.2% SDS was kept at 100 °C for 1 h to cleave the Kdo linkage, giving the purified lipid A species.Structural Modeling and Molecular Docking: The architecture of MCR‐5 in full length was modeled with Swiss‐Model (https://swissmodel.expasy.org/interactive/qMEvX5/models/),Neisseria meningitidis [PDB: 5FGN]The binding of MCR‐5 to its phosphatidylethanolamine (PE) lipid substrate was predicted through molecular docking with UCSF DOCK 6.7 software (version 6.7).Phylogenetic Analyses: The MCR family of colistin resistance enzymes was downloaded from GenBank database, which covers no less than five different subtypes (i.e., MCR‐1,N. meningitidisE. coli O157:H7 EDL933The authors declare no conflict of interest.SupplementaryClick here for additional data file.


Matrix metalloproteinases‐20 (MMP20) expression is widely regarded as tooth specific, with expression limited to dental hard tissues. Recently, we reported MMP20 expression and interaction with dentin sialophosphoprotein (DSPP), a member of the Small Integrin Binding Ligand N‐linked Glycoproteins (SIBLINGs), in human oral squamous cell carcinoma (OSCC) and dysplastic oral premalignant lesions (OPLs), suggesting a role for MMP20‐DSPP interaction in oral carcinogenesis.This study aimed to survey the expression of MMP20 and its cognate DSPP partner in the breast, colon, prostate, thyroid, and cervical neoplasms. Using commercially available tissue microarrays (TMAs) and cell lines, we performed immunohistochemistry, immunofluorescence, proximity ligation assay, and western blot experiments to determine the expressions of MMP20 and DSPP in the breast, colon, prostate, thyroid, cervical neoplasms, and their normal counterparts.Significantly high expression levels of MMP20 and DSPP were observed in the malignant breast, colon, prostate, thyroid, and cervical neoplasms compared with their benign and normal counterparts. Furthermore, MMP20 levels increased with advanced stages of colon and thyroid cancers. DSPP expression increased significantly with tumor stage in all cancers examined.The co‐localization and potential MMP20‐DSPP interaction previously reported in oral cancers are present in other cancers. These results suggest MMP20‐DSPP pairing as a potential marker of disease activity in some epithelial cancers with diagnostic and prognostic implications. In humans, the MMP20 gene is localized on chromosome 11q22.3‐q23 with 10 coding exons.Dentin sialophosphoprotein (DSPP) is a member of the Small Integrin Binding Ligand, N‐linked Glycoprotein (SIBLING) family. Along with other members of the family, the DSPP gene is clustered on a narrow region of approximately 375,000 base pairs on chromosome 4 in human and chromosome 5 in mice. Other members of the family are bone sialoprotein (BSP), osteopontin (OPN), dentin matrix protein 1 (DMP1), and matrix extracellular phosphoglycoprotein (MEPE).Reports of the upregulation of DSPP and other members of the SIBLING family with their cognate MMP partners in human cancers, along with their diagnostic and prognostic significance, have been published.22.1Human TMAs were purchased from Protein Biotechnologies . Different TMAs used in the study include human breast cancer (cat # TMA‐1001), cervical cancer (cat # TMA‐2220 and TMA‐2218), thyroid cancer (cat # TMA‐1502), prostate cancer (cat # TMA‐2208), and colon cancer (cat # TMA‐1414).2.2l‐Glutamine, Extract P, epinephrine, rhTGF‐α, hydrocortisone, rh‐insulin, and apo‐transferrin. The cells were maintained with 5% CO2 under standard conditions. Antibodies used in this study were anti‐MMP20  and anti‐DSPP . The use of these commercially available antibodies has been published in previous studies.Epithelial HPV‐16 E6/E7 transformed cells (Ect1/E6E7), cervical carcinoma cell line DoTc2, and squamous cell carcinoma (SCC) cell line SiHa were purchased from ATCC and grown in keratinocyte‐serum free media (Gibco), ATCC‐formulated DMEM, and ATCC formulated‐EMEM, respectively. Breast cancer cell lines, MCF12F and MCF7, were purchased from ATCC and grown in complete DMEM and Ham's F12 growth medium containing 20 ng/mL epidermal growth factor (sigma), 100 ng/mL cholera toxin (sigma), 0.01 mg/mL human insulin (sigma), 500 ng/mL hydrocortisone (sigma), and 5% chelex‐treated horse serum for MCF12F. For MCF7, the medium was ATCC formulated EMEM to which 0.01 mg/mL human recombinant insulin and fetal bovine serum were added to a final concentration of 10%. Human primary prostate epithelial cell line (HPEpiC) was purchased from ATCC and was grown in prostate epithelial basal medium (ATCC) to which the following supplements were added. 2.3β‐actin, and quantified.Western blot was performed on commercially available tissue lysates and whole cell extracts from cell lines to determine the expression of MMP20 and DSPP. Tissue lysates for the normal and cancer breast , colon , cervix , prostate , and thyroid  tissues were purchased from Protein Biotechnologies . Whole cell extracts from different cell lines to the breast (MCF7 and MCF12), colon , cervix  and prostate  were made using RIPA buffer containing protease and phosphatase inhibitors. Total amount of protein was quantitated, and equal amounts loaded and resolved with 4%‐20% Criterion TGX  SDS‐polyacrylamide gel electrophoresis. This was followed by electrophoretic transfer using a Trans‐Blot Turbo Transfer System  to a low‐fluorescence PVDF membrane . Assay was performed in triplicate (N = 3). Proteins were detected using respective primary and secondary antibodies, and imaged using a LiCor Odyssey scanner. Band intensity was normalized using 2.4Standard immunoperoxidase techniques were carried out on ~5 μm sections of formalin‐fixed, paraffin‐embedded TMAs of the breast, prostate, colon, and cervix neoplasms, and their normal counterparts as previously described.2.5We have previously reported a semiquantitative scoring method for immunostains on tissue sections. Briefly, immunoreactivity for MMP20/DSPP was scored as “negative” ; “1” (10%‐50% immunoreactive tumor cells); “2” ; and “3” .2.6Cells plated on coverslips in a 6‐well dish overnight, were washed with 1XPBS and fixed in 4% paraformaldehyde  for 20 minutes, before permeabilization in 0.1% triton X‐100 for 10 minutes. Cells were then treated with blocking buffer  for 1 hour at room temperature, followed by overnight incubation at 4°C with primary antibodies to DSPP and MMP20 diluted in blocking buffer. Coverslips were washed 3X with 1XPBS and incubated with secondary antibodies for 1 hour before mounting with Prolong Gold anti‐fade reagent with DAPI .2.7In situ proximity ligation assay (iPLA) was used to verify specific cellular interactions of MMP20 with DSPP in the fixed human breast, colon, prostate, cervix, and thyroid tissue sections. Prior to iPLA, paraffin‐embedded tissue sections were deparaffinized and antigen retrieved. iPLA was performed using the HRP‐detection kit from Olink Bioscience according to the manufacturer's protocol . Briefly, tissue sections were incubated with primary anti‐MMP20 polyclonal (rabbit) antibody and anti‐DSPP monoclonal (mouse) antibody or with normal rabbit/mouse IgG. They were then incubated with corresponding secondary antibodies conjugated to oligonucleotides PLA probes (MINUS and PLUS) for 1 hour at 37°C. Rolling circle amplification (RCA) was performed using T4‐ligase as described by the manufacturer . HRP oligonucleotides  were used to detect RCA products in tissues sections. The protein interactions were observed as brown punctate signals and were captured using Nikon Eclipse Ni‐E microscope and NIS Elements AR software .2.8t test or one‐way analysis of variance (ANOVA) with subsequent post hoc Tukey's pairwise analysis using GraphPad Prism . The correlation between expression of MMP20 and DSPP in normal and tumors tissues was tested by regression analysis. Data are presented as means ± SEM with statistically significant differences as P‐values less than or equal to 0.05.Results were analyzed using either Student's 3We employed immunohistochemistry (IHC), immunofluorescence (IF), and in situ proximity ligation assay (iPLA) to survey five common human neoplasms, along with their normal tissue/nonneoplastic counterpart, for the expression of MMP20 and DSPP, using TMA, lysates, and cell lines. The advent of TMA technology allows for the screening of protein expression by IHC and iPLA on hundreds of tissue samples on a single slide.3.1As shown in Figure With respect to breast tissues, levels of MMP20 in invasive ductal carcinoma of the breast were notably higher than that in hyperplasia, invasive lobular carcinoma, and Paget's disease of the breast Figure B. Simila3.2Mirroring MMP20 expression, DSPP levels in all five tissue types and their corresponding whole tissue lysates were expressed in normal and neoplastic counterparts Figure . As show3.3Our recent report indicates that MMP20 and DSPP co‐express and potentially interact with each other in human OSCC.3.4Our recent reports show MMP20‐DSPP co‐localization and evidence of interaction in OSCC by iPLA.4Although reports on the expression and potential role of DSPP in the biology of a number of human epithelial cancers have mounted in recent years,Fisher et al,Our previous reports established the expression of DSPP and its cognate MMP20 partner in duct systems of metabolically active epithelial cells of normal salivary glands, and nephrons.P = 0.023). However, there was no significant correlation between MMP20 expression and disease stage (early versus late stage). The authors reasoned that, since MMP20 has a broad substrate specificity that included several proteins , it also may play a role in the pathogenesis of certain cancers.In a retrospective study of 73 laryngeal squamous cell carcinoma (LSCC) tissue specimens from patients by Liu et al,Although, the mechanisms of DSPP‐MMP20 interactions in cancers have yet to be fully deciphered, our published data showing a ninefold enrichment of DSPP at the MMP20 promoter in oral cancer cell lines suggest a regulatory role for DSPP in the transcription of MMP20. DSPP, directly and specifically binds MMP20 promoter‐proximal element, and in the process MMP20 peptides (residues 107‐153) within its catalytic domain interact with DSPP peptide (residues 34‐120).Nevertheless, it is speculated that MMP20‐DSPP interactions may involve one or more of the mechanisms already identified for the other SIBLING‐MMP pairing.In summary, we report MMP20 and DSPP expression and interaction in five common human epithelial neoplasms and their normal tissue counterparts: the breast, colon; cervix; prostate; and thyroid. This report also represents the first on MMP20 expression in these tissues. Whereas, DSPP‐MMP20 expression in the normal and neoplastic breast, colon and cervix epithelial suggests that these epithelia function as highly metabolic tissue, expression in thyroid and prostate neoplasia with minimal expression in their normal counterparts casts MMP20‐DSPP cognate pair as potential diagnostic and prognostic markers for neoplasm in these tissues. Retrospective and prospective cohort studies beyond the scope of our study will aim to decipher the prognostic and diagnostic utility in these cancers. Furthermore, we anticipate our data will serve as a major reference and baseline document for studies on the role and significance of MMP20‐DSPP expression in these and other major human epithelial neoplasms.The authors declare no conflicts of interest.KUEO: conceived the manuscript; designed all experiment; analysis of results of study; produced initial draft of manuscript; and reviewed final manuscript; JA: contributed to design of immunohistochemistry (IHC) experiment, carried out IHC; analysis of results of study; contributed to draft manuscript; and reviewed final draft of manuscript; KK: contributed to design of IHC experiment, carried out IHC; analysis of results of study; contributed to draft manuscript; GS: contributed to design of in situ proximity ligation assay (iPLA) and immunfluorescence experiment; analysis of results of study; and contributed to draft manuscript; CCA: contributed to design of IHC experiment; analysis of results; draft manuscript. Click here for additional data file.


Context: There is growing interest in the pharmacological evaluation of Rue due to its potential to treat a variety of clinical diseases. The plant seems to present potent endocrine disrupting effects, and its excretion and disposal are not a concern.Objective: The effects of Ruta graveolens L. (Rutaceae) ethanol extract (RE) on reproductive behaviour, fertility, and steroid and thyroid hormone levels in zebrafish were investigated.Material and methods: We exposed subjects to varying concentrations of RE, and one-tenth the LC50 concentration (2.37 ppm) was established as the sublethal dose. After 2 weeks exposure, reproductive behaviour, cumulative number of eggs laid, percentage of fertilized eggs, and whole body steroid and thyroid hormones were measured.Results: Reproductive association behaviour did not differ between control and RE-exposed animals, but spawning attempts were reduced in RE exposed animals. Cumulative egg production between days 9 to 14, RE exposed fish laid 672 eggs while control fish laid 1242 eggs. Also, percentage of fertilized eggs was higher for the control than for the RE exposed fish. Estradiol-17β (E2) levels were reduced in females exposed to RE and testosterone (T) was statistically lower in both males and females treated with RE. Furthermore, thyroid hormones (T3 and T4) declined in fish treated with RE.Conclusion: RE has endocrine disrupting potential in fish, which has important implications for studying the effects of unintentional pharmaceutical exposure. Moreover, the results demonstrate that drug exposure may affect more than just the overall level of behaviour, emphasizing the relevance of examining the effects of individual exposure. We reinforce the use of zebrafish as a model organism in physiology and behaviour, and raise concerns about the toxic effects of RE in non-target organisms such as aquatic vertebrates, which may ultimately affect human health. Throughout human history, plants have been the basis of medical treatments. While many consider herbal infusions, ointment and balms alternative medicine, scientific evidence of active plant compounds contributes to increasing knowledge of traditional medicine. It is known that more than three thousand substances are used for human medical treatment , an odoriferous herb, is more important  were purchased from a local fish distributor and transferred to a storage system (140 L tanks). Animals were housed (one fish/L) with aerated and filtered water, and temperature (27 ± 1 °C), pH (7.3–7.8), hardness (180–245 mg/L as CaCO3), ammonia <0.1 mg/L, nitrite <0.05 mg/L, and dissolved oxygen (7.3–8.1 mg/L O2) were measured regularly. Fish remained under a 14:10 h light:dark cycle, with zeitgeber time (ZT) 0 corresponding to lights on time (8 am–10 pm). Zebrafish were fed twice a day (3% body weight) with BioMar dry pellets  and frozen Artemia. Before the experimental procedures, male and female zebrafish were separated into two 140 L tanks for 7-days’ acclimation.Adult zebrafish  in system water. We calculated these concentrations based on a previous study on Artemia salina L. and extrapolated to an approximate body mass of 650 mg of the weight of present zebrafish , ammonia <0.1 mg/L, nitrite <0.02 mg/L, and dissolved oxygen (7–7.9 mg/L O2). Fish were exposed to RE for 96 h (4 days), 24 h per day and were not fed during the exposure procedure. Mortality was checked three times daily, and the dead fish removed.RE concentrations were prepared to determine the LC50 values (GraphPad Prism 6)   and assigned to one of two conditions: exposure to 0 or 2.37 ppm of RE for 2 weeks.Ruta powder dissolved in system water) were prepared in 5 L glass tanks . Female zebrafish were held in groups of 6 in each tank, at the density of 1 fish/833 mL solution , and male zebrafish were kept in groups of 4 in each beaker, at the density of 1 fish/1250 mL solution . Solutions containing the proper concentration of RE were prepared fresh daily and the volume of the beakers was completely replaced (every day at 9 am) to maintain appropriate water quality and desired RE concentration. Control beaker water was also replaced to equalize handling stress.Males and females were kept isolated during the first 7 days of RE exposure . The control solution (system water) and the solution containing RE  was added to each beaker, 3 cm from the bottom. Plastic mesh served as a porous barrier to prevent spawned eggs from being eaten by the adults. Fish from the two conditions were held in groups of 10/beaker for another 7 days; RE solution was replaced daily and spawned eggs were collected during water renewal  were randomly chosen and used to measure testosterone (T) and estradiol-17β (E2) levels. Fish were not fed for 24 h before sampling. Steroid whole-body extraction was performed using the method described by Arukwe et al. , with soWhole-body thyroid hormone (THs) levels, triiodothyronine (T3) and thyroxine (T4) were measured by PishtazTeb diagnostics ELISA kits using the method described by Chen et al. . To thatMale zebrafish sexual behaviour was assessed after 14 days of RE exposure. Only males were evaluated because of the anti-androgenic properties of rue described in mammals and fish  from the stock population were divided into two groups: 10 fish were exposed to 2.37 ppm of RE and another 10 to 0 ppm of RE (control) in 5 L beakers for 14 days. Complete solution exchange was carried out daily for both groups. Fishes were fed twice a day with BioMar dry pellets and frozen −1) using a KM-media player and sexual behaviours in terms of female association and spawning attempts were counted. As described by Colman et al.  and a non-exposed female (mature fish from the stock) was inserted into a separate partition of the tank. This procedure ensured that fish maintained visual and chemical contact but prevented physical interaction. The two fish were held in the tank overnight, and on the following morning, immediately after the lights were turned on, the barrier was lifted and the fish were allowed to have contact with each other. After 15 min of interaction, fish behaviour was recorded with a Sony handy camcorder (DSC-W800) for 15 min. Video recordings were converted into images , testosterone (T), T3 and T4, as well as female association behaviour and spawning attempts between RE-exposed animals and control animals. For all these comparisons we used independent sample Student’s t-tests, since data were normally distributed according to the Kolmogorov–Smirnov test. A p value <0.05 was considered for statistical significance.Data were analyzed using SPSS v22.0 software . GraphPad Prism 6 was used to determine the LC50 for 96 h of RE exposure in adult zebrafish was 23.71 ppm. The limits 95% confidence (mg/L) was determined as 16.58-25.96, and the slope was 1.87.The value of LC4 = 3.55; p = 0.024). There was also a significant difference in egg production between control and RE exposed fish on days 11 , 12 , 13 , and 14   .40 = 2.52, p = 0.016; 40 = 3.57, p = 0.001; Steroid (T and E2) and thyroid (T3 and T4) hormones were measured after 14 days of RE exposure.4 = 3.49, p = 0.025; females: t4 = 3.96, p = 0.017; 4 = 4.64, p = 0.01; 4 = 1.98, p = 0.118; Total T level was statistically lower in both males and females treated with RE  influences reproductive behaviour, fertility, and steroid and thyroid hormone levels in zebrafish. We observed that RE exposure decreases both steroid (testosterone and estradiol) and thyroid (triiodothyronine and thyroxine) hormone levels, reduces spawning attempts and lowers the number of eggs spawned and fertilized.Endocrine disruptors that enter the environment have the potential to affect the life cycle and development of non-target organisms, disrupting normal functioning and interfering in physiological and behavioural responses. This study examined how exposure to The effects of RE on egg production and fertilization seem to be related to its action on gonadal steroids. It is well established that control of reproductive activities is mediated by the endocrine system, whereby each hormone plays a specific role in different stages of the reproductive process (Kobayashi et al. Betta splendens (Forsatkar et al. Other studies investigating rue have shown adverse effects on the aggressive and reproductive behaviour of mammals. For instance, rue extract decreased territorial defence and sexual behaviour in male Albino rats (Khouri and El-Akawi In our study, we report that not only steroid hormones but also the production of thyroid hormones were inhibited in zebrafish in response to RE exposure. The metabolic, reproductive and behavioural functions of thyroid hormones are well documented in fish (reviewed in Carr and Patiño In this study, we observed the effects of RE exposure on both hormone levels and behavioural expression. Sexual behaviour is the ultimate arbiter of animal reproductive output (Huang et al. 50, and one-tenth the LC50 value as the sublethal concentration to be tested. The mortality of zebrafish due to RE exposure in the present study suggests the toxic action of RE in physiological processes such as enzyme activity (Khouri and EL-Akawi R. graveolens could link physiological functions and behavioural aftereffects in a translational model. However, further studies on this issue are needed to better understand the effects of RE on other relevant aspects regarding the proper functioning of physiology and behaviour.Overall, our results indicate RE’s potential as an endocrine disrupting chemical in fish, and raise concern about its toxic effects in non-target organisms such as aquatic invertebrates and vertebrates, which may ultimately affect human health. While there is still no report about the environmental concentrations of rue, precluding extrapolation of these results to environmental levels, the lack of information is partly related to the fact that rue is an herb traditionally used in developing countries (Harat et al.