The generation of human dopaminergic neurons may serve as a tool of either restorative cell therapies or cellular models, particularly as a reference for phenotyping region‐specific human neural stem cell lines such as human embryonic stem cells and human inducible pluripotent stem cells. We cultivated 3 different midbrain neural progenitor lines at 10, 12, and 14 weeks of gestation for more than a year and characterized them in great detail, as well as in comparison with Lund mesencephalic cells. The whole cultivation process of tissue preparation, cultivation, and cryopreservation was developed using strict serum‐free conditions and standardized operating protocols under clean‐room conditions. Long‐term‐cultivated midbrain‐derived neural progenitor cells retained stemness, midbrain fate specificity, and floorplate markers. The potential to differentiate into authentic A9‐specific dopaminergic neurons was markedly elevated after prolonged expansion, resulting in large quantities of functional dopaminergic neurons without genetic modification. In restorative cell therapeutic approaches, midbrain‐derived neural progenitor cells reversed impaired motor function in rodents, survived well, and did not exhibit tumor formation in immunodeficient nude mice in the short or long term . We conclude that midbrain‐derived neural progenitor cells are a promising source for human dopaminergic neurons and suitable for long‐term expansion under good manufacturing practice, thus opening the avenue for restorative clinical applications or robust cellular models such as high‐content or high‐throughput screening. S For many years, human fetal midbrain‐derived neural stem or progenitor cells have been considered a potential source for restorative therapy in patients with Parkinson's disease. However, there have been considerable concerns with respect to limited expansion, early senescence, and loss of pluripotency. This study investigated such cells thoroughly and developed techniques that allow for long‐term stable expansion, overcoming the above concerns. The cells were found to be safe and potent, allowing clinical application.http://www.gforce-pd.com and http://www.transeuro.org.uk) Cell therapy is a promising treatment for Parkinson's disease, although its clinical application with primary tissue is limited because of restricted resources and minimum effective dosage. Hence, neuroscientists are focusing on new restorative approaches with human embryonic stem cells (hESCs) and fetal cell‐based treatments ‐lesioned adult rat brain. Karyotyping, DNA repair arrays, and in vivo teratoma formation assays, performed under strict good laboratory practice (GLP) compliance, confirmed preclinical safety of hmNPCs.All experiments were approved by the ethics committee of the University of Leipzig and the Technical University of Munich, Germany, or CHA University/Hospital, Korea, and were in accordance with all state and federal guidelines.Treponema pallidum, cytomegalovirus, and Toxoplasma gondii. In addition, endotoxin levels were evaluated. Only hmNPC lines from fetuses without any diagnostic findings were used for long‐term cultivation.hmNPCs were generated from central nervous system tissue of spontaneously aborted fetal human fetuses of female gender at gestational week (GW) 10, 12, and 14 with mother's consent. Fetal tissues were screened for relevant pathogens mentioned in Annex II of the EMEA Commission Directive 2006/17/EC, such as human immunodeficiency virus 1/2, hepatitis C, hepatitis B, http://www.roche.com) supplemented with 10 µg/ml DNase (Roche) for 30 minutes at 37°C, washed three times with Hanks balanced salt solution, and incubated in 50 µg/ml antipain solution (Roche) for 30 minutes at 37°C. After 3 further washing steps, samples were homogenized 20 times by gentle trituration. Propagation of the cell suspension was performed in Hanks balanced salt solution and cell culture flasks coated with poly‐l‐ornithine  and fibronectin . The expansion medium was based on Dulbecco's modified Eagle's medium/Ham's F12  supplemented with 2% B27 supplement minus‐AO , human recombinant epidermal growth factor, and human recombinant fibroblast growth factor 2 . Supplemented medium was changed every other day. A preparation time of ∼4 hours was needed from tissue preparation until cell seeding. Long‐term expansion of the cells (>6 months) was enabled in reduced atmospheric oxygen (2%–3%) Briefly, human fetal tissue at GW 10, 12, and 14 was prepared with a time delay of 1–3 hours after termination of pregnancy. The brain tissue was washed with sterile Hanks balanced salt solution and dissected into mesencephalic and nonmesencephalic primary tissue samples. The tissues were mechanically separated into small pieces, incubated in 0.1 mg/ml Papain solution  containing additives such as GlutaMAX , B‐27 supplement minus‐AO (2%), forskolin , picolinic acid derivative , and dibutyryl cyclic adenosine monophosphate . The cells were differentiated for 7 days. An immortalized cell line from fetal midbrain neural progenitor cells  were cultivated as a reference cell line according to the literature 2 Profiler polymerase chain reaction (PCR) array  according to the manufacturer's instructions.Karyotyping was performed by the Cytogenomic Services Facility of Sumkwang, Medical Laboratories, Korea. To analyze karyotype, cell division was blocked in metaphase by 0.05 μg/ml colcemid for 1–2 hours. The chromosomes were visualized by G‐band staining, and >100 metaphase cells were analyzed 5 of each sample) were resuspended in 75 µl phosphate‐buffered saline  and incubated with 0.1 µg of the primary antibody against CD15, CD133, and CD184  for 60 minutes at 37°C. As negative controls, undifferentiated and differentiated hmNPCs without incubation in primary antibody were used. Cells were washed with 1 ml PBS and 1% heat inactivated fetal bovine serum. After resuspension in 300 µl PBS, samples were analyzed by flow cytometry using FACScan . The sample flow rate during analysis did not exceed 300–400 cells per second.Undifferentiated and differentiated hmNPCs  was used. Gene regulation was statistically evaluated by subjecting the ΔΔCT values or CNRQ to a two‐tailed Student's t test, assuming equal variances.For quantitative real‐time PCR analysis (qPCR), SYBR Green Select qPCR Supermix (Thermo Fisher Scientific Life Sciences), 5 ng complementary DNA from total RNA, 0.2 μM forward and reverse primers, and 100 nM 5‐carboxy‐X‐rhodamine (passive references dye) were used. Oligonucleotide primers  were designed using Primer3 software to flank intron sequences if possible. PCR was performed in a Step One Plus instrument (Thermo Fisher Scientific Life Sciences) using the following protocol: 2 minutes at 50°C, 2 minutes at 95°C, and 40 cycles of 15 seconds at 95°C and 60 seconds at 60°C. Melting curves were recorded. The correct size of the respective single amplicons was assured by agarose gel electrophoresis, and the identity was verified by restriction enzyme cleavage or sequencing for at least 10% of the amplicons. Cycle threshold (CT) values were set within the exponential phase of the PCR. Data were normalized to five housekeeping genes, Cells were fixed with 4% paraformaldehyde. Fixed cells were permeabilized with 0.2% Triton X‐100. Unspecific binding was blocked in PBS supplemented with 2% bovine serum albumin and 3% chicken or donkey serum. Incubation followed with primary antibodies overnight at 4°C in blocking buffer. The primary antibodies are summarized in supplemental online Table 5. After washing, the cells were incubated with fluorescent secondary antibodies Alexa Fluor 488 conjugate or Alexa Fluor 594 conjugate  for 1 hour at room temperature. Nuclei were stained with 4′,6‐diamidino‐2‐phenylindole  for 5 minutes at room temperature.http://www.zeiss.com). Digital images were acquired with the AxioCam MRc camera using image‐analysis software AxioVision 4 (Zeiss). The percentage of labeled cells was determined by counting the number of positive cells in relation to the number of DAPI‐stained nuclei. Approximately 2,000–3,000 cells were counted within 6 randomly selected fields per well in a single‐blinded fashion by the German and Korean research teams. Neurite length was measured in a single‐blinded fashion using a Leica confocal microscope . Immunohistochemistry of postmortem brains was performed as previously described Coverslips were mounted onto glass slides and examined under a fluorescence microscope  was determined using a dopamine enzyme‐linked immunosorbent assay (ELISA) kit according to the manufacturer's instructions . As a positive control, DA release of PC12 cells was tested (data not shown).The concentration of dopamine released from cultured hmNPCs  were used in this study. The experimental procedure was carried out according to the animal care guidelines of the Institutional Animal Care and Use Committees in Germany and Korea.Female adult Sprague‐Dawley rats  were given 6‐OHDA as specified 5 cells per μl in PBS) was injected into the lesioned striatum using a KDS310 nano pump .Rats  was used in the present analysis 18F]N‐3‐fluoropropyl‐2β‐carboxymethoxy‐3β‐(4‐iodophenyl)‐nortropane ([18F]FP‐CIT) PET as described 18F]FP‐CIT PET scans with 1 mCi of [18F]FP‐CIT. One‐hour emission data were acquired with an energy window of 350–650 KeV and a coincidence window of 3.432 nanoseconds. Emission listmode data were sorted into a 3D sinogram and reconstructed using a 3D rapid prototyping reconstruction algorithm The Inveon positron emission tomography (PET) scanner . Testing was performed 3, 6, 9, and 12 weeks after transplantation.Rotation behavior test by amphetamine  was measured for 90 minutes in automatized Rotameter bowls  were transplanted with 5 × 105 undifferentiated hmNPCs, differentiated hmNPCs, or tumor cells (positive control) in 20 µl PBS into the right testis. The left testis was used as sham control. Eight (n = 5) or 30 (n = 5) weeks after transplantation, mice were sacrificed, and each testis was fixed immediately in 10% buffered formaldehyde. All tissues were embedded in a paraffin block for histological analysis. The blocks were sectioned (10 µm thickness), stained with hematoxylin and eosin, and tested for nestin (immunocytochemistry [ICC]) and human chromosome 17 (fluorescent in situ hybridization [FISH]). Nestin‐immunoreactive (IR) cells were counted in sham control and hmNPC‐grafted testicles. To compensate for double counting in adjacent sections, Abercrombie's correction was used.The teratoma formation assay was performed according to the literature https://www.abbottmolecular.com) in mouse testis samples (n = 3) 30 weeks after hmNPC injection. Tissues were fixed in paraformaldehyde, embedded in paraffin, dewaxed, and rehydrated. After washing in saline‐sodium citrate (SSC) buffer at room temperature for 30 minutes, a mixture of probes was applied to each slide. The probes were denatured at 75°C for 5 minutes and then hybridized at 37°C overnight. After washing in 2× SSC at 60°C for 2 minutes, cells were counterstained with DAPI.Detection of aneuploidies for human chromosome 17A with one‐color FISH assay was performed , version Enterprise 4.0. For a behavioral assay, mean scores from the Rotation Test were used as the dependent variables, and treatment (sham control and undifferentiated and differentiated hmNPC transplantation groups) and time  were used as independent variables. Most of the statistical analysis was performed using analysis of variance or a mixed model analysis of variance procedure  with Fisher's least significant difference (LSD) post hoc tests to account for random effects. The number of injected cells was analyzed using one‐ or two‐way analysis of variance followed by LSD post hoc tests. An independent Student's t test was used for two‐group analysis. Results are expressed as means ± SEM. In all analyses, differences were considered significant at p < .05.Statistical analyses were conducted on a CHA University mainframe computer using the Statistical Analysis System  with poly‐l‐ornithine and fibronectin as an extracellular matrix. Stemness and early differentiation potential were studied in undifferentiated and differentiated hmNPCs in comparison with LUHMES cells, an immortalized clonal neuronal cell line derived from fetal human midbrain  as an example. Markers for early differentiation FERD3l, FOXA1, GATA6, and SOX17 were expressed at higher levels in differentiated hmNPCs compared with undifferentiated hmNPCs. In comparison with immortalized LUHMES cells (undifferentiated and differentiated), the expression levels of early differentiation markers were higher in hmNPCs (undifferentiated and differentiated)  , 1E.PAX5, EN1, EN2, PITX3, and TH consistent with midbrain identity FOXA2 and CORIN in long‐term‐cultivated hmNPCs  and regionalized expression patterns with marker genes specific for forebrain, midbrain, hindbrain, and floorplate d hmNPCs . Again, d hmNPCs , 2D. Thed hmNPCs . In addiF = 580.98, p < .0001], CD133 , and CD184 , indicating that a significant proportion of hmNPCs began differentiation after 7 days.To characterize neural stem cells, we used CD15, CD133, and CD184 To explore the dopaminergic potential of all three hmNPC lines, we compared undifferentiated and differentiated hmNPCs at different passages  using qPCR, ICC, and ELISA for dopamine content.TUBB3 transcription levels were significantly higher in differentiated hmNPCs compared with undifferentiated hmNPCs  . In addition, hmNPCs showed a significant increase of expression levels of TH in differentiated hmNPCs   compared with undifferentiated hmNPCs.F = 348.18, p < .0001] and TH‐IR cells with increasing passages .ICC analysis of hmNPCs for the neuronal and dopaminergic markers TuJ and TH, respectively , establiFor complete phenotyping with ICC, other markers for proliferation and neural and neuronal cell types were analyzed  in undifferentiated hmNPCs . We found ∼20% of proliferating Ki67‐IR cells in long‐term‐cultivated hmNPCs and ∼90% of SOX2 and PAX6‐IR cells. The GFAP‐IR hmNPC population was on average 23.4% ± 4.9% , and the percentage of O4‐IR oligodendrocytes was 0.9% ± 0.2% . In addition to those markers, we studied other neuronal phenotypes in differentiated hmNPCs in long‐term cultivation such as serotonergic, cholinergic, GABAergic, and glutaminergic neurons  and founImmunoblots of all three long‐term‐cultivated hmNPC lines confirmed that TH and TuJ are expressed in increasing levels in undifferentiated and differentiated hmNPCs. The floorplate marker corin was also expressed in undifferentiated and differentiated hmNPCs .p < .01). As a positive control, PC12 cells were used (data not shown).The release of dopamine is a parameter for function of DA neurons and may thus be used as a potency assay. When stimulated with potassium, the differentiated hmNPCs exhibited a markedly higher DA release (643.53 ± 91.52 pg/ml) in contrast to undifferentiated hmNPCs (FOXA2 and CORIN) and midbrain‐specific  markers during long‐term cultivation. Fetal hmNPCs of various gestational ages  offer great potential to differentiate into dopaminergic neurons during at least the initial 20 passages.These data reveal that stemness and proliferation potential are preserved during prolonged proliferation of hmNPCs harvested at gestational weeks 10, 12, and 14. The generated hmNPC lines retained regional identity by expression of floorplate‐specific . In the controls with sham injection, the motor asymmetry increased over time to 15.0 ± 1.7 at 12 weeks after surgery.To address function and survival of the characterized hmNPCs, we grafted 2 × 10p = .36, p < .002, and p < .0001) and differentiated  hmNPCs compared with sham controls 6, 9, and 12 weeks after grafting. There was no significant difference in motor compensation between graft recipients of undifferentiated and differentiated hmNPCs  compared with sham control (right testicles). The presence of long‐term‐surviving intratesticular hmNPCs was confirmed by nestin staining . After survival times of 8 and 30 weeks, animals were sacrificed and testicles were processed for postmortem analysis. HE staining revealed no indication for tumor formation of hmNPCs after 8 and 30 weeks –5F. Therstaining –5K and Fstaining –5O.Although there is increasing knowledge about human fetal brain‐derived neural progenitor cells, standardized data on gene profiling of fetal tissue and primary hmNPCs during prolonged proliferation and differentiation are limited Chromosome stability and analysis of 84 genes involved in DNA repair revealed no indication for changes up to passage 20, which corresponds to a longstanding propagation period of approximately 1 year. The proliferation capacity was assessed via cell counts; ICC for KI67 and nestin; CD15, CD133, and CD184 FACS; and qPCR for markers of pluripotency and proliferation. Growth curves demonstrated that there was continued exponential growth in cultures derived from donors of 10 to 14 weeks of gestation. Tissue derived from the 14‐week‐old donor seemed to grow most efficiently, but all samples had satisfying growth rates and most importantly did not slow down at later passages. In the up‐scaled GMP‐compliant long‐term cultivation process, the yield per hmNPC line was high enough to generate a biobank with clinical samples for therapeutic purposes. We detected an increase in expression of stem cell markers over time; thus, establishing stemness and proliferation potential are preserved during prolonged proliferation in defined medium conditions. Our data provide novel and surprising evidence that stemness of tissue‐specific hmNPCs derived from primary tissue can be increased without genetic manipulation. The mechanisms by which such genes are upregulated remain obscure. One could argue that at least during the initial five passages, the dying off of primary cells could account for this increase. In addition, cell‐cell interactions could help to stabilize stem cell characteristics. However, we believe that low oxygen conditions and hypoxia‐inducible factor (HIF)‐1α‐dependent pathways play a prominent role. We have previously shown that low oxygen and activation of HIF‐1α are crucial for allowing long‐term proliferation of human and murine midbrain‐derived neural progenitor cells If detailed analyses of primary tissue provide the prerequisite for culturing hmNPCs that harbor predopaminergic cells, one still has to be concerned about the ability of such cells to maintain the potential to differentiate into dopaminergic neurons. In this study, we could clearly demonstrate that hmNPCs remain stable during the first 20 passages in vitro, which corresponds to a longstanding proliferation period of approximately 1 year. Moreover, all changes that we were able to identify pointed toward a significant increase of differentiation potential compared with early passages, despite continued growth. This is in line with reports that functional maturation of cultivated human cells mimics the timing of human brain development. Therefore, functional effects and integration of hmNPCs may only be observed during extended time spans of many months and even years OTX2, PAX5, EN1, EN2, PITX3, TH, and floorplate markers FOXA2 and CORIN mRNA were detected in primary fetal human midbrain and hmNPCs during long‐term cultivation in GMP‐compliant conditions, in addition to corin and TH protein expression. Other neuronal phenotypes  constituted 2.4%–0.8% of the total population of neurons. We used TH as marker gene for late dopaminergic differentiation. TH could be detected in primary tissue as well as hmNPCs during proliferation and differentiation, supporting the notion that our cultures preserved the regional identity of the original midbrain tissue, which consists of neural progenitor cells with floorplate fate. If stemness and the proliferation potential are preserved during prolonged proliferation, one would also expect that differentiation into specific neurons of interest would not be impaired. Accordingly, we were able to show that hmNPCs not only maintain but rather increase their differentiation capacity into TH‐expressing neurons, thus mimicking human brain development. Although TH expression alone may not be sufficient evidence to prove dopaminergic differentiation, we could also show that other midbrain dopaminergic A9 markers, such as corin, were upregulated in parallel. Again, HIF‐1α could play a prominent role.Electrophysiological recordings of voltage‐gated ion channels and action potentials demonstrated the maturation of functional properties in hmNPCs so far only during 4 weeks of differentiation in vitro In vivo, partial reduction of motor deficits in graft recipients was paralleled by recovery of specific dopamine transporter binding (PET imaging). By using PET imaging, we found that grafted hmNPCs cells developed into DA neurons, consistent with postmortem dopaminergic immunocytochemical analysis. Behavioral compensation was significant ∼60%), but not complete. Graft recipients of differentiated hmNPCs showed a slightly better compensation of motor asymmetry, which could point to the fact that hmNPCs would also need more time for differentiation. These results are in line with previous transplantation experiments with unaltered NPC lines %, but noGrafted hmNPCs were immunoreactive for the human markers Stem121, MAP1B, GIRK2, and synaptophysin 1. In concert with significantly increased neurite length 12 weeks postgrafting, these data point to integration and synaptic plasticity of transplanted hmNPCs in the graft recipients. The survival and high differentiation rate of hmNPCs in vivo may indicate that the midbrain floorplate fate, which is required for the commitment of dopaminergic neurons from hES and IPS cells One important aspect of cell therapy is safety, because continued proliferation must be excluded to minimize the risk of tumor formation The data presented on hmNPC lines propagated in a GMP‐compliant long‐term cultivation process provide robust preclinical data on authentication of three different hmNPC lines, their midbrain regional identity, and evidence that cell replacement therapy using hmNPCs could be an option for patients with Parkinson's disease.J.M., S.C.S., and J.S.: conception and design, financial support, administrative support, provision of study material or patients, collection and/or assembly of data, data analysis and interpretation, manuscript writing, final approval of manuscript; H.‐S.L.: data analysis and interpretation, final approval of manuscript; J.M.K., Y.‐E.L., B.K., M.‐Y.S., and J.S.K.: collection and/or assembly of data, final approval of manuscript; G.H.: financial support, data analysis and interpretation, final approval of manuscript; F.W.: data analysis and interpretation, manuscript writing, final approval of manuscript; H.‐M.C., S.W.C., and K.Y.C.: financial support, administrative support, final approval of manuscript; K.‐S.K.: conception and design, financial support, administrative support, data analysis and interpretation, manuscript writing, final approval of manuscript.H.‐S.L. has uncompensated employment and stock options. J.S.K. has uncompensated employment, intellectual property rights, consultant advisory role, honoraria, research funding, stock options, and expert testimony. The other authors indicated no potential conflicts of interest.Supporting InformationClick here for additional data file.


Here we explore the possibility that the use of T2 relaxometry may provide information complementary to that provided by diffusion tensor imaging (DTI) in ageing of healthy controls (HC), Alzheimer’s disease (AD) and mild cognitive impairment (MCI). T2 and diffusion MRI metrics were quantified in HC and patients with MCI and mild AD using multi-echo MRI and DTI. We used tract-based spatial statistics (TBSS) to evaluate quantitative MRI parameters in white matter (WM). A prolonged T2 in WM was associated with AD, and able to distinguish AD from MCI, and AD from HC. Shorter WM T2 was associated with better cognition and younger age in general. In no case was a reduction in T2 associated with poorer cognition. We also applied principal component analysis, showing that WM volume changes independently of  T2, MRI diffusion indices and cognitive performance indices. Our data add to the evidence that age-related and AD-related decline in cognition is in part attributable to WM tissue state, and much less to WM quantity. These observations suggest that WM is involved in AD pathology, and that T2 relaxometry is a potential imaging modality for detecting and characterising WM in cognitive decline and dementia.In MRI, the coherence lifetime T2 is sensitive to the magnetic environment imposed by tissue microstructure and biochemistry  Typically, AD pathology is considered predominantly to affect grey matter (GM), in particular the limbic system  or radial diffusivity (RD) are attributed to a degradation of the myelin sheath, whilst axial diffusivity (AxD) is considered an index of axonal damage. A similar argument applies to increases in fractional anisotropy (FA). Of course, many other structures impede the Brownian motion of water. Coming largely from tract-based spatial statistics (TBSS) analyses of DTI data, it has been consistently observed that MD, as well as RD and AxD are regionally increased relative to HC in both AD and MCI, whilst FA is generally reduced , 12 participants with MCI  and 9 with mild AD . MCI diagnosis was based on the criteria given by Petersen et al . Particial et al. This ta2.2.3, acquired matrix size 152  ×  320  ×  144, reconstructed resolution 0.34  ×  0.34  ×  1.60 mm3 (after 2-fold interpolation in-plane by zero-filling in k-space), reconstructed matrix size 540  ×  640  ×  144, GRAPPA factor 2 , reconstructed matrix size 540  ×  640, 34 slices, GRAPPA factor 2, time 11:07. DTI: axial, TR 3800 ms, TE 85.2 ms, Bval 1000 s mm−2, number of gradient directions 60 (full-sphere), acquired and reconstructed resolution 2  ×  2  ×  2 mm3 inclusive of 10% slice gap, matrix size 122  ×  122, 60 slices, GRAPPA factor 2, multi-band factor 2  WM tractography atlas  was used, as implemented in Matlab 2013b. The median T2, FA, RD and AxD in the 7 WM tracts for which it was calculated were included, as well as age, total WM volume, and two measures of cognitive function, namely the total accuracy and mean reaction time from the PAL task of the CANTAB toolbox. Therefore, in total, 32 variables were included in the PCA. Variables were discarded if the magnitude of their PCA score vector in the space of the first four principal components was below the mean magnitude. Before analysis, variables were demeaned and normalized by their standard deviation.3.3.1.An example R2 =1/T2) map is shown in figure /T2 map istacks.iop.org/PMB/61/5587/mmedia). We did not detect group differences using DTI at the 95% significance level. Whilst slightly higher diffusivities and slightly lower FA were associated with AD and MCI, these were significant only at thresholds below 95% and are not presented in our main results.The main group differences in which T2 was increased in the AD group relative to both NC and MCI were in the FMi, IFOF, ILoF, SLoF and TSLoF. The ATR and CST were also affected, though to a lesser extent. The remaining tracts of the JHU WM tract atlas were negligibly affected, if at all. The number of voxels in the FA skeleton which distinguish groups in each WM tract is plotted in figure 3.2.By pooling the observations in each tract and calculating the tract-wise median for each imaging parameter , we were able to delineate age effects in different WM tracts. Figure p  =  0.021) and lower than average in the ILoF (p  =  0.026) (inclusive of Bonferroni correction). The ageing rates of the ILoF and ATR were also distinguishable at the 95% confidence level. Ageing rates are shown in figure The effect of age on T2 could be resolved at the 95% confidence level by both slope and intercept for each tract, whilst for DTI measures FA, RD and AxD we could only separate the intercepts at the 95% level. Therefore a common gradient was used for all tracts in those analyses. ANCOVA tables may be found in the supplementary information. T2 in all tracts increased with age, whilst FA always decreased. RD and AxD also increased with age. CST showed the longest T2 on average, figure , the hig3.3.The results of our PCA, using the median T2, FA, MD, RD and AxD in all 7 major WM tracts as well as age, WM volume, accuracy and reaction time in the PAL task are shown in figures We found that 4 principal components (PC) were sufficient to explain 83% of the variance of the data figure , with raWe also found that WM volume, as well the PAL performance indices, made large contributions mainly in higher PCs. The WM volume dominates the PC4, whilst age makes large contributions to the PC3 and PC4. PAL reaction time makes a small positive projection diagonally across the PC space, but is largely disassociated from WM volume. Positive T2 coefficients in PC1 and PC3 are associated with negative coefficients for PAL accuracy and positive coefficients for PAL time. This means that longer T2 implies poorer pattern recognition and longer reaction times. This is of particular interest as the TBSS identified regional T2 increases to be associated with AD.We also examined whether the unsupervised PCA, having reduced the 32-dimensional data to 4 dimensions, would reveal potential differences between participant groups. Figures 4.We have found increases in the coherence lifetime of nuclear spin phase in WM, heuristically described by a mono-exponential decay time constant at long echo times (i.e. T2), to be associated with ageing and poorer performance in cognitive tasks, the increases being regionally greater in AD. We have also found that both the tract-wise median T2 and its rate of change with respect to age differs in different WM tracts.et alet alOur data also indicate, consistent with the literature, that older age is associated with lower FA in all WM tracts examined in the sampled age range  echo onwards. There is literature supporting the view that decoherence in WM is better  described by bi-exponential kinetics . Again, coverage was sacrificed for resolution. Continued improvements in MRI technology and pulse sequences may in future ameliorate this. Most major WM tracts are partially or fully covered, as well as the most of the temporal lobe and entire limbic system, cerebellum and brain stem.5.In all, it appears that T2 in WM is affected by age in later life to a similar or greater extent than common DTI parameters and may be a powerful complement to other more popular imaging techniques. WM is an appealing source of imaging biomarkers since damage and stress alter its magnetic environment in ways that are not so in GM. For this reason, continued deployment of MRI techniques which detect changes in magnetic environment in response to stress of damage, rather than volume loss occurring afterwards, is likely to be powerful in detecting and characterizing various pathologies.


There existed controversies about the association between the response to chemotherapy for osteosarcoma (OS) patients and the genetic polymorphisms in excision repair cross-complementation group (ERCC1 and ERCC2) genes. We aimed to perform a meta-analysis to comprehensively evaluate the association.We searched multiple databases for literature retrieval including the PubMED (1966 ∼ 2017), Embase (1980 ∼ 2017), and the Web of science (1945 ∼ 2017). The overall odds ratios(OR) and their corresponding 95% confidence interval (CI) were calculated for the three polymorphisms under the dominant, recessive, and allelic models.P = 0.005; recessive model: OR = 1.791, P = 0.003; allelic model: OR = 1.677, P = 0.003), and OS patients carrying C allele in rs11615 polymorphism were more likely to response to chemotherapy. In terms of ERCC2 rs1799793 polymorphism, this polymorphism was significantly associated with the response to chemotherapy for OS patients under recessive model , and patients with AG + AA genotype in rs1799793 polymorphism were more appropriate to receive chemotherapy. With respect to ERCC2 rs13181 polymorphism, this polymorphism was not correlated with the response to chemotherapy for OS patients under all three models.From six eligible articles in our study, we found that for ERCC1 rs11615 polymorphism, a significant association was detected between the chemotherapy response and the polymorphism under all three models (dominant model: OR = 2.015, Our meta-analysis suggested that among Chinese population, the rs11615 and rs1799793 polymorphisms were significantly correlated with the response to chemotherapy for patients with OS, and patients with CC or TC + CC genotypes in ERCC1 rs11615 polymorphism or AG + AA genotype in ERCC2 rs1799793 polymorphism were more suitable for chemotherapy. Osteosarcoma, the most common primary malignancy of bone, is a devastating disease due to its rapid dissemination and poor prognosis –3. ApproThe current well-established strategy for the treatment of newly diagnosed OS was the combination of neoadjuvant chemotherapy, surgical resection for metastatic OS patients, and the adjuvant chemotherapy after surgery . The neoERCC1 and ERCC2 genes are associated with the response to chemotherapy for OS. A retrospective study showed that ERCC2 rs1799793, a DNA repair polymorphism, was a predictive factor for chemotherapy response in OS patients [ERCC1 and ERCC2 genes for the response to chemotherapy for OS. Study from Yang et al. found that polymorphism of ERCC1 rs11615 did not significantly influence the response to chemotherapy in patients with OS [The nucleotide excision repair (NER) pathway, a highly powerful and sophisticated DNA damage removal pathway, has been believed to play important roles in cancer progression and response to platinum-based chemotherapy , 9. Excipatients . A studypatients . However with OS . The ERC with OS . Herein,The PubMED (1966 ∼ 2017), Embase (1980 ∼ 2017), and the Web of science(1945 ∼ 2017) were searched for study retrieval with a combination of Medical Subject Headings (MeSH) and text words relating to “ERCC1”, “ERCC2”, “osteosarcoma”, and “chemotherapy” as the search strategy. We retrieved literatures from the database inception to March 5th, 2017. The reference lists of identified articles and related reviews were examined to avoid any omission of eligible studies by the above electronic search strategy.In order to get more reliable estimations, we pre-defined strict inclusion criteria as follows: (1) all the participants were OS patients treated with chemotherapy; (2) detecting the relationship between the response to chemotherapy, and the SNPs in ERCC1 and ERCC2 genes such as rs11615, rs1799793, and rs13181 polymorphisms; (3) studies conducted on Chinese population; (4) providing available genotype data of relevant polymorphisms in ERCC1 and ERCC2 genes; (5) full-text studies published in English. Articles were eliminated if one of the following existed: (1) other SNPs rather than rs11615, rs1799793, and rs13181 polymorphisms; (2) SNPs of ERCC1 and ERCC2 genes are risk factors for OS survival; (3) literature types such as news, books, communications, letters, and reviews.The following data were collected independently from incorporated studies by two reviewers according to the mentioned inclusion and exclusion criteria: the first author, year of publication, number of patients, treatment approaches, genotyping methods, the age and gender ratio of patients, and genotyping data of ERCC1 rs11615, ERCC2 rs1799793, and ERCC2 rs13181 polymorphisms.P less than 0.05 was regarded as statistically significant. The pooled odds ratios (ORs) were calculated for dominant model, recessive model, and allelic model for ERCC1 rs11615, ERCC2 rs1799793, and ERCC2 rs13181 polymorphisms, respectively. The evaluation of response to chemotherapy was identified as the previous relevant studies [I2 index as assessment of the heterogeneity among the incorporated studies. If the I2 was less than 50%, we believed there was no significant heterogeneity and adopted the fixed-effects model to calculate the OR and its corresponding 95% CI. Otherwise, the DerSimonian and Laird (D-L) random-effects model was selected for the calculation of OR and 95% CI. Begg’s funnel plots were constructed to examine the publication bias. The noticeable asymmetry in the shape of funnel plot indicates publication bias. Egger’s tests were performed for further investigation, and the significance level was set at 0.05. The Rosenthal’s fail-safe numbers were calculated to estimate stability of the results [Nfs0.05 = (ΣZ/1.64)2−n, where Z is Z scores for the individual significance values, and n is the number of studies. A fail-safe number is often considered robust if it is greater than 5n + 10 [We followed the Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA) guidelines . In our  studies , 16, 17. 5n + 10 .Based on our search strategy, we retrieved 56 literatures from PUBMED, 86 from Embase, and 43 from the Web of science. Twenty-five duplicated articles were removed, leaving 130 literatures for further assessment. After screening the titles, 54 literatures were eliminated. Then the remaining 76 articles were estimated for eligibility according to our inclusion and exclusion criteria. Finally six eligible articles , 20, 21 P = 0.005, Fig. P = 0.003, Fig. P = 0.003, Fig. Five studies including 1019 OS patients were incorporated to evaluate the association of the rs11615 polymorphism and the response to chemotherapy. The results could be found in Table I2were lower than 50% for all the three models, so the fixed-effects model was used to achieve the OR and 95% CI. The ORs for dominant model (AA + AC versus CC), recessive model (AA versus AC + CC), and allelic model (A versus C) were 1.058 (95% CI: 0.793–1.516, P = 0.757, Fig. P = 0.355, Fig. P = 0.392, Fig. All eligible studies together pooled 1133 OS patients involved in the association of the response to chemotherapy and the rs13181polymorphism. Table P = 0.06, Fig. P = 0.036, Fig. I2 was 57.60%, so the random effects model was applied for yielding the OR and 95% CI. The OR for G versus A was 1.328 (95% CI: 0.943–1.87, Fig. P was higher than 0.05, which implied that no significant association was observed under allelic model.There were five eligible studies for the analysis of association between the rs1799793 polymorphism and the response to chemotherapy for OS patients. The results were displayed in Table P in both Begg’s and Egger’s test were higher than 0.05, which further provided evidence of no publication bias in our study. Rosenthal’s fail-save number suggested that the results in allelic model of rs11615 groups and all three rs1799793 groups were comparatively reliable. However, the publication bias could not be ignored in dominant model and recessive model of rs11615 groups, and all three rs13181 groups (Table We observed no obvious asymmetry in the shape of funnel plots Figs. , 6 and 7ps Table .Fig. 5FuIn the current study, we performed a meta-analysis to assess the association of the response to chemotherapy for OS patients and the rs11615, rs1799793 and rs13181 polymorphisms. Our results showed that for ERCC1rs11615, OS patients carrying CC or TC + CC genotypes were more likely to respond to the chemotherapy, and patients with C allele in rs11615 polymorphism were more appropriate to receive chemotherapy. With respect to ERCC2 rs1799793, the response rate to chemotherapy in patients with AG + AA genotype was significantly higher than that in patients carrying GG genotype, and patients carrying AG + AA genotype in rs1799793 polymorphism were more suitable for chemotherapy, while for ERCC2 rs13181, the polymorphism had a null effect on the response to chemotherapy for OS patients.Despite there is only 3 cases of OS per 1,000,000 individuals, OS is the most primary malignant of bone tumor worldwide occupying approximately 56% of all bone tumors , 23. It ERCC1 rs11615 polymorphism might influence the clinical outcomes and response to chemotherapy for patients with OS, and patients with CC genotype in ERCC1 rs11615 polymorphism had better response to chemotherapy [ERCC1 rs11615 polymorphism for patients with OS could be an underlying candidate predictor in clinical chemotherapy treatment.ERCC1, located at 19q13, encodes a rate-limiting enzyme in NER pathway, which can repair chemical drug-induced DNA damage , 27. Theotherapy  which waERCC2 gene was likely to influence the chemotherapy response, and OS patients with AA genotype in ERCC2 rs1799793 were likely to have better chemotherapy response, whereas the rs13181 polymorphism was not associated with the chemotherapy response [ERCC2 gene was also not correlated with the response to chemotherapy for patients with OS. However, when enlarging the sample size by pooling all related data together in our meta-analysis, we detected that OS patients with AG + AA genotype of rs1799793 polymorphism were more likely to have good chemotherapy response which suggested AG + AA genotype could be a potential predictor for OS patients in clinical diagnosis and chemotherapeutic treatment. What we found were significant supplements to the molecular mechanism research of OS and will greatly benefit the OS patients in the future.ERCC2, located at 9q13.3, encodes a DNA helicase which causes repair of single-strand DNA injury . ERCC2 gDuring the study retrieval, we found nine papers regarding the relationship between the chemotherapy response and the rs11615, rs1799793, and rs13181 polymorphisms in ERCC1 and ERCC2 genes for OS patients. Considering the different genetic and nationality backgrounds, studies conducted on Spanish , SloveniTo our knowledge, the present study is the first meta-analysis to explore the correlation of the chemotherapy response and the rs11615, rs1799793, and rs13181 polymorphisms in ERCC1 and ERCC2 genes for OS patients among Chinese population. However, there are some limitations in the current study. Firstly, as we mentioned above, there is only one article assessing the association between the response to chemotherapy and the polymorphisms of rs11615, rs1799793, and rs13181 for OS patients for three different nationalities including Spanish, Slovenian, and Italian, respectively. Although, we performed our meta-analysis on Chinese population with six eligible studies incorporated, with more studies becoming available for other nationalities, the overall meta-analysis and subgroup analysis stratified by nationalities would be conducted. Secondly, the genotyping methods in the included studies were not exactly the same, which might cause bias in our meta-analysis. Additionally, unpublished articles were not considered in our study.The current meta-analysis suggested that for OS patients among Chinese population, the rs11615 and rs1799793 polymorphisms were significantly associated with the chemotherapy response, and patients with CC or TC + CC genotypes in ERCC1 rs11615 polymorphism or AG + AA genotype in ERCC2 rs1799793 polymorphism were more likely to have good response to chemotherapy. These SNPs may be candidate pharmacogenomic factors capable of indentifying OS patients who are more appropriate to receive chemotherapy.


The Brighton Collaboration criteria have standardized the clinical and laboratory‐supported diagnosis of Guillain–Barré syndrome (GBS) and Miller Fisher syndrome (MFS) in a way that is applicable in many parts of the world with variable resources. The caveat within the criteria, “absence of an identified alternative diagnosis for weakness” makes GBS a diagnosis of exclusion. Accurate diagnosis of GBS requires a good understanding of an updated, locally contextualised list of mimics, and features that distinguish them from GBS. Magnetic resonance imaging (MRI) brain showed restricted diffusion in both caudate heads, and T2 hyperintensity in the periaqueductal area and bilateral dorsomedial thalamus. Cerebrospinal fluid (CSF) analysis was normal. A history of chronic alcoholism was elicited. She was diagnosed as Wernicke's encephalopathy with beriberi neuropathy. She recovered fully with intravenous thiamine treatment.1.2A 54‐year‐old man had pain, numbness, and weakness in limbs over 2 days. He had asymmetrical flaccid weakness in limbs with reduced reflexes and patchy sensory loss. He progressed to symmetrical weakness in all limbs. CSF was normal. NCS within a few days of symptoms showed multiple motor conduction blocks and reduced sensory potentials. He was diagnosed with GBS. He did not improve with intravenous immunoglobulin (IVIg). NCS at the third week showed inexcitable nerves. The asymmetric painful weakness raised the possibility of mononeuritis multiplex. Further investigations revealed a diagnosis of vasculitic neuropathy associated with cryoglobulinemia. His weakness gradually improved with immunosuppressant drugs.1.3A 25‐year‐old man presented with abdominal pain, tea‐colored urine, altered mental state, and seizure. He was treated with intravenous phenytoin. Two days later, he developed flaccid tetraparesis and ophthalmoplegia. MRI brain showed mildly enhancing T2 and diffusion‐weighted imaging (DWI) hyperintensities in bilateral caudate nucleus heads, periaqueductal gray matter, mammillary bodies, superior and inferior colliculi, bilateral hypoglossal and vestibular nuclei. CSF was normal. NCS showed length‐dependent axonal sensorimotor polyneuropathy. His urine ketones were raised as he was not fed on account of the abdominal pain. He was treated with intravenous thiamine for Wernicke's encephalopathy with improvement in ophthalmoplegia and MRI brain findings. However, he continued to have flaccid weakness, hyporeflexia, autonomic dysfunction, and required mechanical ventilation. GBS overlapping with MFS was considered and he was treated with IVIg. He did not improve. He was finally diagnosed with acute intermittent porphyria (AIP) when the urine test was positive for porphobilinogen. All medications that can worsen AIP were withdrawn. Patient eventually improved to premorbid status. We believe the MRI changes were due to concomitant Wernicke's encephalopathy.1.4A 35‐year‐old man was started on insulin therapy for newly diagnosed diabetes mellitus. HbA1c decreased from 12.8% to 5.9% over 3 months. He developed maculopathy, central retinal vein occlusion, orthostatic hypotension, supraventricular tachycardia, hypoglycemia spells, and recurrent syncope. Four days after an episode of Group B Salmonella gastroenteritis, he developed flaccid paraparesis, areflexia, retention of urine, and distal painful sensory loss. He was diagnosed as GBS. He was treated with IVIg. NCS showed axonal‐demyelinating polyneuropathy. CSF showed elevated white blood cell (WBC) count (90 cells/μl) and raised protein (1.27 g/L) with normal glucose. The diagnosis was revised to treatment‐induced neuropathy of diabetes mellitus because of persistent and progressive small fiber neuropathy and dysautonomia many weeks later. He gradually improved with optimization of blood sugar and neuropathic pain medications.1.5A 57‐year‐old man with stage four diffuse large B‐cell lymphoma, treated with eight cycles of rituximab, cyclophosphamide, doxorubicin, vincristine, prednisolone (RCHOP) therapy over 5 months, presented with acute‐onset right facial weakness, bulbar palsy, flaccid weakness in lower limbs with areflexia, and distal sensory loss in all limbs. He was diagnosed as GBS and treated with IVIg. NCS showed mild length‐dependent axonal sensorimotor polyneuropathy. CSF showed raised WBC count (79 cells/μl), elevated protein (2.99 g/L), and reduced glucose (2.1 mmol/L). CSF cytology showed B‐lymphoma cells. His diagnosis was revised to diffuse large B‐cell lymphoma with leptomeningeal metastasis. He was transferred to a cancer center for treatment of lymphoma.1.6A 28‐year‐old man presented with acute‐onset flaccid tetraparesis with hyporeflexia. NCS showed prolonged F‐wave latencies and nonuniform slowing of motor nerves consistent with GBS. CSF showed raised WBC count (10 cells/μl), mildly elevated protein (0.53 g/L), and normal glucose. HIV serology was positive. He recovered fully after IVIg treatment. In the next 1 year, he had few more episodes of weakness that resolved with IVIg and eventually resolved with highly active antiretroviral treatment. His final diagnosis was chronic inflammatory demyelinating polyneuropathy related to HIV seroconversion.1.7A 51‐year‐old man presented with difficulty walking for 2 weeks that was preceded by upper respiratory tract infection. He had symmetrical tetraparesis and reduction in reflexes. Cranial nerves were normal. CSF showed raised WBC count (9 cells/μl), raised protein (>3.00 g/L) and normal glucose. NCS showed demyelinating polyneuropathy. He was diagnosed with GBS, and IVIg was administered. A month later, he was readmitted with progressive tetraparesis, areflexia, and distal sensory loss. His diagnosis was revised to acute‐onset chronic inflammatory demyelinating polyneuropathy (A‐CIDP). His condition eventually improved with corticosteroid and mycophenolate mofetil.1.8A 53‐year‐old woman presented with vertiginous giddiness and unsteady gait for 2 weeks. She was treated with steroid prior to coming to our center. Clinical examination showed complex ophthalmoparesis, and bilateral lower motor neuron facial weakness. There was mild left upper limb dysmetria and she could not tandem walk. She had normal power, reflexes, and sensation. She was diagnosed with GBS overlapping with MFS. NCS showed only absent bilateral blink reflex. CSF showed elevated protein (0.60 g/L), no pleocytosis, and normal glucose. However, MRI brain showed nonenhancing signal abnormalities in the facial colliculi region of pons, periventricular white matter, and subcortical white matter. Final diagnosis was clinically isolated syndrome of brain stem demyelination. Three months later, she only had mild residual left facial weakness.1.9A 37‐year‐old man with systemic lupus erythematosus (SLE), presented with acute‐onset of severe loss of sensation associated with areflexia in all limbs and inability to walk. He was diagnosed with GBS and treated with IVIg. CSF showed normal WBC count (3 cells/μl) and raised protein (1.01 g/L). NCS within 1‐week from the onset was normal but subsequent study in the second week detected nonlength‐dependent reduction in sensory potentials. His mobility improved over few weeks but continued to have sensory loss and persistent postural hypotension. Anti‐Ro (SSA) was weakly positive. He was finally diagnosed as subacute sensory neuronopathy from underlying Sjogrens‐SLE. With escalation of immunosuppression, he gradually improved but he remains disabled by severe sensory ataxia and orthostatic hypotension.1.10A 19‐year‐old man undergoing military recruitment developed acute sequential weakness and numbness of his limbs during a field military camp, where he had to sleep and assume various prone postures on hard grounds for prolonged periods. He had symmetric flaccid weakness and patchy sensory loss. Cranial nerves were normal. NCS showed multiple conduction blocks, prolonged distal motor latencies and F‐wave latencies, and reduced sensory potentials. CSF analysis was normal. He was diagnosed with GBS and treated with IVIg. With the additional finding of thickened nerves, hereditary neuropathy with liability to pressure palsy (HNPP) was considered. Further enquiry revealed a possible family history of HNPP. One copy (deletion) of the PMP22 gene region on chromosome 17 was demonstrated. We cannot exclude the possibility of GBS occurring in a patient with HNPP; the absence of raised CSF protein in second week of illness points against it  amplitudes that increased after short (10 s) exercise test and decreased during 3‐Hertz repetitive nerve stimulation test. He was diagnosed with Lambert–Eaton myasthenic syndrome (LEMS). Voltage‐gated calcium channel (VGCC) antibody was present. Computed tomography (CT) of the thorax showed left lung suprahilar mass which on biopsy revealed small cell carcinoma. He was treated with plasma exchange and 3,4‐diaminopyridine. He also received chemotherapy and radiotherapy for small cell lung carcinoma. His weakness improved after 6 months.2We have listed the conditions that were mistaken for GBS in a tertiary care hospital of a developed country. We would like to highlight the following general practical points that are useful in distinguishing them from GBS.Unless Bickerstaff encephalitis is in consideration, altered mental state should not be present in GBS. The temporal evolution of neurological deficits and severe deficits that remain without significant recovery , or presentation of relapsing pattern  should prompt a review of diagnosis. Sural sparing pattern of sensory abnormality, defined as a greater decrease in median and or ulnar sensory nerve action potential (SNAP) compared to that of the sural SNAP, was present in only one patient with GBS mimic (patient 13). He had sensory neuronopathy and sural sparing pattern was present in the NCS performed on Day 8. This corroborates the observation of Derksen et al.  that surCytoalbuminologic dissociation in spinal fluid examined about 1 week after symptoms is useful in the diagnosis of GBS (Wong et al., The spectrum of disorders that resembles GBS evolves with changes in the practice of medicine, patient demographics, and disease epidemiology. Local setting should also be considered. For instance, we expect acute beriberi neuropathy to be less frequent in countries where alcohol consumption is low for cultural–religious reasons. In our center, improvements in access to laboratory investigations and neuroimaging have removed previously common differential diagnoses of GBS such as acute spinal shock and hypokalemic periodic paralysis.In conclusion, accurate diagnosis of GBS requires a good understanding of an updated, locally contextualised list of mimics, and features that distinguish them from GBS.


Acropora muricata, Pocillopora damicornis and Isopora palifera) at three distinct locations  along Australia’s Great Barrier Reef (GBR). Annual growth rates of all species were highest at Lizard Island and declined with increasing latitude, corresponding with differences in temperature. Within locations, however, seasonal variation in growth did not directly correlate with temperature. Between October 2012 and October 2014, the highest growth of A. muricata was in the 2013–14 summer at Lizard Island, which was unusually cool and ~0.5 °C less than the long-term summer average temperature. At locations where temperatures reached or exceeded the long-term summer maxima, coral growth during summer periods was equal to, if not lower than, winter periods. This study shows that temperature has a significant influence on spatiotemporal patterns of branching coral growth, and high summer temperatures in the northern GBR may already be constraining coral growth and reef resilience.Coral growth is an important component of reef health and resilience. However, few studies have investigated temporal and/or spatial variation in growth of branching corals, which are important contributors to the structure and function of reef habitats. This study assessed growth  of three branching coral species ( The value and productivity of coral reef ecosystems are strongly linked to the condition of coral assemblages, and especially the structural complexity and habitat diversity provided by coral-rich habitats5. Accordingly, widespread declines in coral cover and structural complexity, as reported in a number of locations7 directly effect local diversity and productivity9, thereby undermining the ecological and economic value of reef ecosystems. Sustained and ongoing declines in abundance of corals are largely attributed to increasing incidence of whole coral mortality caused by acute disturbances7. However, acute disturbances  as well as chronic disturbances  can have important demographic consequences for reef building corals11, further contributing to declines in live coral cover.Coral reefs are important ecosystems, providing invaluable goods and services to tropical nations12. However, at large latitudinal scales, aragonite saturation state, temperature, and light control the geographic distribution of corals, as they are critical factors for photosynthesis (energy acquisition) and calcification13. Beyond the latitudinal limit of reef formation (~30°), aragonite saturation is generally too low (Ωarag < 3) to enable reef accretion and formation14. At lower latitudes coral growth generally increases in accordance with increasing temperature, carbonate saturation and light intensity12. Across 14 degrees of latitude on Australia’s Great Barrier Reef (GBR) (10 °S to 24 °S), an increase of 1 °C in annual average sea surface temperature (SST) was correlated with an increase in calcification rates by 0.3 g cm−2 year−1 for massive Porites15.Coral growth rates are known to be strongly linked to environmental conditions. Individual coral growth is regulated by a wide range of both biotic and abiotic factors17. Most notably, corals may bleach when the local temperatures exceed typical summer maxima by 1 °C18. However, even before corals bleach sustained increases in local temperatures may lead to declines in growth or performance. For example, the optimum temperature for coral calcification is typically 1–3 °C below the local summer maximum20, such that ocean warming will constrain coral growth by reducing the time that environmental conditions are conducive to maximum rates of calcification12. Ocean warming is purported to be key factor contributing to observed declines in growth of massive corals across many reef locations .Despite the generally positive relationship between coral growth and temperature, ocean warming is of great concern for scleractinian coralsarag) on coral reefs and how this will effect demography of scleractinain corals is very limited25. Increases in atmospheric carbon dioxide (CO2) from 280 ppm in the pre-industrial period to present day concentrations of 401 ppm26 have resulted in a decrease in the surface ocean pH from a global average of 8.21 to 8.10, as well as corresponding shifts in aragonite saturation27. As the aragonite saturation declines, the carbonate saturation state of the internal calcifying fluid declines and the rate of calcification decreases28. Albright et al.29 used alkalinity enrichment to demonstrate that net community calcification increases significantly when ocean chemistry is restored to conditions expected to have occurred in pre-industrial times.While the effects of ocean warming on coral growth are relatively well understood, current knowledge regarding projected changes in aragonite saturation (ΩAcropora muricata (cf. A. formosa), Pocillopora damicornis and Isopora palifera) at three distinct locations along the Great Barrier Reef, Australia  of three branching corals  were recovered and healthy, and were used in analyses of coral growth. Of those excluded, 20% had died since staining but were still intact, while the remainder were not found. For P. damicornis, 90% (108/120) of the colonies stained were found healthy and used in this study. Of the 12 colonies not included, 7 died, 4 were not found and 1 did not stain well. For Isopora palifera, 90% (27/30) of stained colonies were found healthy and used in the study, while the fate of the other 10% is unknown (possibly lost due to poor tag retention).Staining of branching corals to monitor growth is a common method, however some loss of stained branches and colonies is inevitable. Moderate levels of background coral mortality and injuries are common for fast growing and typically short-lived, branching corals−1) (mean ± SE) compared to Davies Reef (3.81 ± 0.42 cm 6-month−1) and Heron Island (2.49 ± 0.08 cm 6-month−1). Linear extension rates measured at Lizard Island were significantly greater than Heron Island  and greatest (6.56 ± 0.43 cm 6-months−1) linear extension rates were in the 2012–13 summer and 2013–14 summer, respectively  decreased along the north-south latitudinal gradient along the GBR and were largely reflective of variation in linear extension  was 40% less than Lizard Island (3.70 ± 0.4 g cm−2) and significantly varied  and Heron Island , compared to Davies/Trunk Reefs (0.91 ± 0.11 cm 6-month−1) and Heron Island (0.69 ± 0.09 cm 6-month−1). There were significant differences in linear extension between Lizard and Heron Island , but not Heron , significantly greater than Davies/Trunk (1.07 ± 0.07 g cm−2) and Heron Island (0.84 ± 0.06 g cm−2)  Fig. . Along t−2) Fig. . CalculaI. palifera was 1.49 ± 0.03 cm yr−1. Linear extension was greatest at Lizard Island (1.64 ± 0.06 cm yr−1) but was not significantly different to growth rates recorded at Davies Reef (1.51 ± 0.06 cm yr−1) or Heron Island (1.33 ± 0.06 cm yr−1)  compared to the Lizard Island and Davies Reef  compared to Davies Reef and Heron Island  and monthly average SST  among reefs. Throughout the course of the study (Oct 2012–Nov 2014), the average SST at Lizard Island was 26.8 °C (ranging from 23.2 to 30.6 °C), compared to 26.1 °C (22.5–30.3 °C) at Davies Reef and 24.1 °C (17.4–28.6 °C) at Heron Island. Summer temperatures in both 2012–13 and 2013–14 were lower than the long-term monthly averages for all locations, ranging from ~0.5 °C cooler than expected for Lizard Island to 1.0 °C cooler than expected for Heron Island  and calcification (g cm−2 6-month−1) were significantly related to SST  and month  during the study was greatest at Heron Island (539 ± 27 μmol s−1 m−2), compared to Lizard Island (375 ± 30 μmol s−1 m−2) and Davies Reef (371 ± 22 μmol s−1 m−2). When examining the relationship between calcification, density and linear extension rates of A. muricata and P. damicornis with light intensity (PAR), for all growth variables, there was no significant relationship  among sectors, there was a significant variation among sectors  averaged 3.3 ± 0.0334. Diel patterns in aragonite saturation determined for Lizard Island  were on average highest just before dawn , coinciding with a period of extensive rainfall at Lizard Island  were inverse to those of pCO2 and CT but with the highest values at dusk and lowest at dawn  at Lizard Island averaged 3.3 ± 0.2 Table . ComparaA. muricata . As well, linear extension rates varied among locations in approximate accordance with spatial variation in annual average SST . Therefore, the relationship between SST and latitude focused solely on data from the area of interest, the Indo west-Pacific. There was a significant negative relationship of decreased linear extension with increasing latitude . Linear extension increased with SST to an optimal value at 26.7 °C . °C Fig. , howeverA. muricata, P. damicornis and I. palifera) were highest at Lizard Island, in the northern GBR (14.7 °S), and declined at higher latitude. Spatial variation in coral growth was largely consistent with variation in observed temperatures; average annual SST at the two most northern reefs were similar , however sea temperature at Heron Island was ~2 °C less (24.1 °C). Growth rates of A. muricata increased by 1.9 cm yr−1 for every 1 °C increase in average annual temperature across the three study locations. For P. damicornis, linear extension increased 0.3 cm yr−1 per 1 °C among locations. Despite contrasting morphologies, the rate of increase for P. damicornis was the same as that determined for massive morphologies of Porites (0.31 cm yr−1 per 1 °C) on the GBR15. Growth rates of Isopora increased by 0.11 cm yr−1 per 1 °C among the three study locations.This study showed that growth rates of three common coral species  is similar to that determined at Lizard Island in Oct–Nov 2008 and 2009 38 The average for the central sector at Davies Reef was slightly higher (Ωarag = 3.7)33 than the other sectors and may reflect the variance that can be observed due to factors outlined above. Importantly, variation among locations in growth rates of the three corals species considered in this study  was more consistent with differences in local temperature, rather than light or aragonite saturation.In addition to temperature, another key environmental driver of coral reef calcification is aragonite saturationarag = 3.3) at Lizard Island and Heron Island, despite 8.76° latitudinal separation and 2 °C difference in SST. It seems despite lower temperatures and concomitant greater solubility of calcium carbonate at Heron Island, the benthic community at Heron Island can buffer the reef carbonate saturation state39 and maintain an average aragonite similar to the lower latitude site of Lizard Island. However the variability in available carbonate ions (Ωarag) at Heron Island  is almost double that of Lizard Island . The reef flat of Heron Island is characterised by a greater seawater residency time that can result in large fluctuations in carbonate chemistry40. Therefore, the locally adapted corals of Heron Island at the southern GBR may be able to withstand changes in seawater chemistry, as they are naturally subjected to large intra-annual variation in temperature and pH40. However, periods of anomalous temperature and pH exposure do not coincide at Heron Island; the greatest diel temperature range was recorded in austral spring (October) and largest diel variability in pH occurred in the fall (June)40. It is likely that the effects of increasing temperature on coral growth will be exacerbated with declining pH41 but temperature-induced increases in coral metabolism42 may counteract any negative response such that the synergistic affects remain unclear. Understanding how these stressors synergistically affect coral growth and survival will be imperative for predicting persistence of the reef community.Mean within-reef aragonite saturation states were comparable  for both Isopora palifera as this is the first study to publish growth characteristics on this species. The only other record of linear extension for Isopora sp. (I. cuneata) was at a subtropical location in Australia, Lord Howe Island, that averaged 1.96 ± 0.07 cm yr−1 from 2010–1143. This is a greater growth rate than reported for the genus in this study on the GBR (1.43–1.64 cm yr−1) but comparison between the two studies is flawed as the species and methodologies between the two studies differed. The colonies from Lord Howe Island were not sliced to the dominant axis of growth and linear growth may have been overestimated; In this study, initial investigation revealed the linear extension determined externally from the stain line to column tip without sectioning was overestimated by an average of 0.35 ± 0.1 cm . As a result, in this study all columns of I. palifera were sectioned to determine linear extension.A meta-analyses was not completed on 12. At the low latitude site Lizard Island, the lowest growth rate of A. muricata was in the 2012–13 summer  from what is often experienced would be more favorable for growth. This trend of constrained coral growth in summer months has also been reported in Western Australia44 and in the Persian Gulf45. At the higher latitude site of Heron Island, positive temperature anomalies in the winter months may be leading to higher growth rates, unrestricted by cooler temperatures. Similarly, P. damicornis did not display expected seasonal trends in growth rates , whereas increasing temperatures may increasingly constrain coral growth  at lower latitudes. Continued monitoring of coral growth rates in combination with environmental conditions, such as temperature stress and within reef carbonate chemistry, will be essential to better appreciate likely impacts of climate change on corals and reef ecosystems.In conclusion, growth rates of three dominant coral species varied spatially along the GBR, largely in conjunction with differences in average annual temperatures. Based on limited temporal sampling of carbonate chemistry, within-reef aragonite saturation was comparable among these locations, suggesting that reef ecosystems do have considerable capacity to buffer latitudinal gradients in oceanic aragonite saturation. There was also no consistent trend in variation in solar radiation among reefs relative to observed variation in growth rates, though this may impose increased constraints on coral growth at higher latitudesAcropora muricata (cf. A. formosa), Pocillopora damicornis, and Isopora palifera) that were common and abundant at widely separated locations along the length of the GBR. Acropora muricata is a staghorn coral that often dominates in shallow water lagoons, forming large mono-specific thickets. P. damicornis is an abundant, ubiquitous species and the recent amendment of the Pocillopora complex taxonomy52 was used to aid identification of P. damicornis. I. palifera is abundant in high wave energy areas forming submassive clumps that contain columns and ridges53.Coral growth  was quantified for three coral species  at each reef, while also selecting areas with high abundance of each of the specific study species. In the central sector, P. damicornis was poorly represented at the first reef visited (Davies Reef) and therefore, additional sampling was undertaken at another nearby reef, Trunk Reef , winter 2013 (Apr 2013–Oct 2013), summer 2013–2014 (Oct/Nov 2013-Mar/Apr 2014), winter 2014 (Apr/May 2014–Oct-Nov 2014). Individual branches were enclosed in a 1 L plastic bag in situ and secured with an elastic band. Alizarin red was injected with a needle under the elastic band into the bag. Ten colonies of A. muricata, with three branches per colony were stained at each site, totalling 20 colonies and 60 branches per reef  in October 2012. In April 2013, the stained colonies were collected and another set stained. This cycle of staining continued for two-years totalling 720 branches.The sampling regime for A. muricata. Linear extension (cm) was measured with Vernier calipers, recording the minimum distance from the stain line to tip of the apical polyp. Branches that had died prior to final collection were excluded from analysis. In addition, 0.02% (16/720) of branches had switched to zooxanthellae free (non-growing) tips through the course of the study that can result from interior conditions of the colony being less suitable for growth or branches growing too closely together55 and were excluded from analysis. To determine bulk skeletal density of the branching species A. muricata, corals were cut along the stain line with a geological saw using a 2 mm saw blade. The dry weight (g) of the cut branch tips was recorded. Branches were dipped in paraffin wax and the total enclosed volume was determined using water displacement technique56. Skeletal bulk density (g cm−3) was then determined by dividing the dry weight by the enclosed volume. Calcification (g cm−2) rates were calculated by multiplying the linear extension by the density15. However, these calcification rates are only of the apical newly grown branch and are not a measure of whole colony calcification.Growth measurements were determined for each branch of P. damicornis encompassed one year, separated into two 6-month periods at each reef: summer 2013–2014 (Oct/Nov 2013–Mar/Apr 2014) and winter 2014 (Apr/May 2014–Oct/Nov 2014). 20 colonies per location (10 at each site) were stained. Colonies (max 20 cm diameter) were removed from the substrate and put into a 70 L clear plastic bag in situ with Alizarin dye released into the bag. For P. damicornis, the number of coral branches measured with callipers ranged from 10–20 . Branch measurements for P. damicornis were randomly sampled along the longest axis of growth (often the most upward projecting). Measured branches were sectioned with a saw and the bulk density and calcification was determined as described for A. muricata. In addition, areas of partial mortality due to algal overgrowth or smothering due to sediment were excluded. Calculated rates of density and calcification were averaged among branches and determined at the colony level.The sampling regime for I. palifera were determined for an entire year (Oct/Nov 2013–Oct/Nov 2014) at one site per location . At each location, 10 colonies were stained  with 2–3 columns per colony. Columns of I. palifera were enclosed in a 4 L clear plastic bag, sealed at the base with an elastic band and the dye injected under the elastic band with a needle. To determine the linear extension of I. palifera, stained coral columns were sliced at 4.4 mm thickness until the maximum vertical axis of growth was determined and then photographed with a reference scale. Linear extensions were determined for each column by taking three measurements along the main vertical growth axis from the stain line to the end of the column on the digital image in Image J Fiji57. Density and calcification rates of I. palifera were determined from the digitised images of x-radiography adapted from Carricart-Ganivet & Barnes58 and detailed methodologies can be found in Supplementary Materials.Growth rates of 59 was used to fit linear mixed-effects (lme) models using the statistical program R60. Separate analyses were conducted for each coral species . For all models the fixed effect was location and as a random effect sampling period which was nested in site and then colony. Model selection was informed by Akaike information criterion (AIC) which measures the relatively quality of the model for the data set. The best models were selected based on lowest AIC, then fit by restricted maximum likelihood  in linear extension, calcification, and density of corals, the package nlme−1), density (g cm−3) and calcification (g cm−2 yr−1) were determined averaging the 6-month means (x) for A. muricata and P. damicornis and standardizing for variation in sample size (N) between study periods using equation Annual rates of linear extension cm yr−, densitywhich was then converted to an annual rate.http://www.aims.gov.au/docs/data/data.html, accessed March 6, 2015) and details of determining monthly averages found in Supplementary Materials. For each location, the long-term (2001–2011) average monthly SST was determined for comparison to the study period (2012–2014). Two-way ANOVA was used to determine significant variation between location SST/light and monthly time period. The relationship of A. muricata and P. damicornis density, linear extension and calcification rates were compared to the sampling period average SST and light for each reef and sampling period using linear regression (lm). As I. palifera was only investigated for one year at 3 sites, a statistical relationship cannot be reliably established due to limited sample size.Sea surface temperature (SST) and light (PAR) throughout the study was determined from the Integrated Marine Observing System . Satellite SST data of higher-resolution, both spatially  and temporally (weekly), were also derived for the period 1985–2012 from the Pathfinder v5.2 night-only, 4 km-daily SST dataset , with gaps filled following Heron et al.61. These data were recently used by Heron et al.62 to examine recent temperature trends and thermal stress patterns. However, as these data did not overlap the time periods of interest, 4 km-monthly composites were calculated and used to bias adjust the spatially and temporally lower resolution ERSST data to ensure relevance to the study. Trends in bias-adjusted ERSST were calculated annually from 1965 to 2014. In addition, accumulated thermal stress was calculated from ERSST using the Degree Heating Months (DHM) metric. Values were converted to Degree Heating Weeks (DHW32) for ease of comparison with established ecologically-relevant thresholds for significant bleaching and mortality of 4 and 8 °C-weeks32. At each reef, bias-adjusted ERSST during the course of the study (2012–2014) was compared to the long-term average (1965–2000) using a Student’s t-test.Historic sea surface temperature trends were determined from the Extended Reconstructed Sea Surface Temperature (ERSST) dataset  for the dates of each study where provided; when sampling dates were not included, the year prior to publication was used. Study depths ranged from 0.5–15 m for A. muricata and 2–7 m for P. damicornis but depth was not reported in all the studies and could not be included in the analysis . For P. damicornis, the relationship of linear extension with latitude required a square root transformation to normalize the residuals.Linear extension is the metric most often reported in the literature for quantifying growth of branching corals60. All linear models were tested graphically to meet the assumptions of linear models .Statistical analysis was completed in RThe datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.Supplementary Materials


Central diabetes insipidus is characterised by arginine vasopressin deficiency. Oxytocin is structurally related to vasopressin and is synthesised in the same hypothalamic nuclei, thus we hypothesised that patients with acquired central diabetes insipidus and anterior hypopituitarism would display an oxytocin deficiency. Moreover, psychological research has demonstrated that oxytocin influences social and emotional behaviours, particularly empathic behaviour. We therefore further hypothesised that central diabetes insipidus patients would perform worse on empathy-related tasks, compared to age-matched and gender-matched clinical control  and healthy control groups.n = 20, 8 males; clinical control: n = 15, 6 males; healthy control: n = 20, 7 males) provided two saliva samples which were analysed for oxytocin and completed two empathy tasks.Fifty-six participants  had significantly lower oxytocin concentrations compared to healthy control participants. Hypopituitary patients also performed significantly worse on both the reading the mind in the eyes task and the facial expression recognition task compared to healthy control participants. Regression analyses further revealed that central diabetes insipidus patients’ oxytocin concentrations significantly predicted their performance on easy items of the reading the mind in the eyes task.Hypopituitarism may therefore be associated with reduced oxytocin concentrations and impaired empathic ability. While further studies are needed to replicate these findings, our data suggest that oxytocin replacement may offer a therapeutic approach to improve psychological well-being in patients with hypopituitarism. Central diabetes insipidus (CDI) is an endocrine disorder with an estimated prevalence of 1 in 25000 . It seemOT is known to initiate uterine contractions during parturition and to initiate the milk let-down reflex during lactation. There is currently no recognised endocrine phenotype associated with OT deficiency. However, psychological research has shown that OT plays an important role in a range of social behaviours including increasing an individual’s ability to identify emotional expressions , 5, incrThe only study (to our knowledge) to investigate the presence of an OT deficiency in a patient group found that patients with childhood-onset craniopharyngiomas who had grade 1 lesions to the hypothalamus as a result of surgical resection had significantly lower OT concentrations compared to those with grade 2 lesions and those with no hypothalamic involvement . The samThe present study is a logical extension of this research: to investigate whether patients with acquired CDI would present with lower salivary OT concentrations, compared to a clinical and a healthy control (HC) group, and if so, whether this would have an effect on their emotion recognition skills.Fifty-five adults were recruited from the Endocrinology clinic at the University Hospital Wales into one of three groups: the CDI group, the clinical control (CC) group, and the HC group. The diagnosis of CDI and anterior hypopituitarism (HP) was established as per routine clinical practice by the water deprivation test, and a combination of basal and dynamic tests of anterior pituitary function, respectively. Inclusion criteria included documented biochemical evidence of anterior HP and CDI, if present. Participants were also required to receive full hormone replacement therapy for at least 3 months prior to study entry, while those with a history of functioning pituitary adenomas  were included only if in biochemical remission. CC patients were recruited using the same inclusion criteria, but were excluded if there was a history of partial or transient CDI. All participants in the CDI group had acquired CDI as a result of transsphenoidal pituitary surgery. Participants were excluded from the study if they were under 18 years of age, pregnant or breastfeeding, or if they were unable to comply fully with the protocol and study instructions. Participants were also excluded if they presented with any concomitant condition which could compromise the study objectives and/or preclude the protocol-defined procedures . Finally, both the CC and HC participants were matched by age and gender to CDI patients; a one-way analysis of variance (ANOVA) revealed that there was no significant difference in age between the three groups, and there was a similar gender distribution between groups (see Table The study protocol was approved by Cardiff University (study sponsor), the Research and Development Office at the Cardiff and Vale University Health Board and by the Cambridge Central Research Ethics Committee (15/CMC/6297). All participants provided written informed consent, were fully debriefed at the end of the study, and financially compensated for their participation.The RMET is a pre-existing validated measure of cognitive empathy created by Baron-Cohen et al. . ParticiA modified version of the FER task  was usedα = .682; fantasy: α = .722; personal distress: α = .767; perspective taking: α = .814).The Interpersonal Reactivity Index (IRI)  is a welα = .752; mind reading: α = .702; restricted and repetitive behaviour: α = .429; imagination: α = .629; attention to detail: α = .706).The Autism Quotient short version  is an aα = .876; father: α = .890).The Relationship Structure Questionnaire (ECR-RS)  is a preWe chose to measure OT via saliva because (a) previous research has demonstrated saliva is a valid medium for measuring OT , 15, 22,Participants were instructed to abstain from alcohol for 24 h and caffeine for an hour prior to testing. Participants were only allowed to drink water during the study and if any food had been consumed before the start of a session, participants were asked to rinse their mouths thoroughly before any saliva samples were taken. All testing was carried out between 09:00 and 12:00 in order to control for any hormonal circadian rhythms.On arrival, participants’ height and weight were measured in order that their body mass index (BMI) could be calculated. After a brief period (approximately 10 min) of acclimatisation to the testing facility, participants were asked to provide their first saliva sample before completing either the RMET or FER task. Given the similarity of these tasks, the order in which they were presented was counterbalanced to control for any learning effects that might arise from the first task. Participants then completed the personality measure questionnaires before providing their second saliva sample, approximately 30 min later. After this, participants completed a behavioural task that will not be discussed here, before completing either the RMET or FER task according to their counterbalance condition. Participants were fully debriefed before leaving.An analysis of covariance (ANCOVA) was conducted to assess the effect of group on participants’ OT concentrations, while controlling for age, medication and BMI. A priori power calculations were based on our recent study in which salivary OT concentrations were assessed in male undergraduates , in whomThree ANOVAs were carried out to assess whether there were any group differences in each of the self-reported personality measures .An ANCOVA was carried out to investigate any differences in total RMET scores between groups, while controlling for age and gender. To investigate the relationship between RMET performance on easy and difficult items and OT concentrations, three multiple regression models were run in order to investigate group differences.A mixed ANCOVA was carried out to assess the influence of different facial expressions, expression intensity, and group on participants’ accuracy during the FER. This analysis was repeated using participants’ OT concentrations as a covariate to identify whether OT concentrations were responsible for any observed group effects. Finally, a mixed ANOVA was carried out to assess whether there was an effect of any propensity of participants in a given group to over-report emotions during the FER, subsequently termed ‘error bias’.All reported analyses control for age and gender as covariates, although age and gender were not significant covariates in any of these analyses.Table t(31) = −1.65, p = .109, and ranged from 2 to 72 years of age; the mean age at which patients underwent surgery was 36 years of age.The age at which patients underwent surgery did not differ significantly between clinical groups, F = 3.729, p = .031, Finally, a two-way ANOVA revealed a significant difference in BMI between groups, F = 2.567, p = .086, A 3  × 2  mixed ANCOVA revealed a trend towards a significant main effect of group on OT concentrations, Normality analysis revealed an outlier in the HP group which was removed, and also that the data did not meet the assumption of normality. A log transformation was carried out, after which the transformed values met the assumption of normality. All statistical analyses reported below were carried out on the transformed data. For ease of interpretation, untransformed means and standard errors are reported.F = 4.922, p = .031, F = .193, p = .662, F = .082, p = .776, There was a significant main effect of group, M = 2.195, SE = .111) and CDI  patients had significantly lower trait empathy scores compared to HC  participants, F = 3.639, p = .033, A 4  × 3  mixed ANOVA revealed that CC  had significantly lower AQ-S scores compared to HC  participants, F = 6.269, p = .004, M = 2.710, SE = .090) and HP patients or HC participants.A 4  × 3  mixed ANOVA revealed that CC patients  × 3  mixed ANOVA revealed no main effects or interaction.F = 5.557, p = .007, A one-way ANCOVA was carried out to investigate the effect of group (CDI vs. CC vs. HC) on total RMET scores. CDI and CC patients had significantly lower RMET scores compared to HC participants Fig. , but theRegression models conducted within each participant group revealed that OT response predicted CDI participants’ performance on easy items  had significantly lower scores than HC participants , but there was no difference between CDI patients  and CC or HC participants, F = 5.680, p = .006, A mixed 4  × 4   × 3  ANOVA revealed that CC patients  correctly identified significantly fewer fearful expressions compared to HC participants , F = 4.331, p = .018, F = 11.491, p < .001, F = 6.308, p = .004, Simple effects analyses were carried out to assess the effect of group on recognition of expressions varying in emotion and intensity. Group had no effect on recognition of happy, sad, or angry expressions, but CC patients  and CC  patients had larger error biases compared to HC  participants, F = 3.962, p = .025, F = 4.002, p = .025,  = .140, and anger bias, F = 5.559, p = .007, M = 5.429, SE = .745) compared to HC participants , and both CDI  and CC  patients had a greater anger bias compared to HC participants .Finally, a 4   × 3  mixed ANOVA revealed that CDI . The results are consistent with these hypotheses: HP patients had significantly lower OT concentrations compared to HC participants, and also scored significantly lower on the RMET and FER compared to HC participants. However, the prediction that CDI patients would have significantly lower OT concentrations than CC patients was not supported. This may reflect an influence of anterior pituitary hormones on OT secretion or an unintentional disruption to hypothalamic-pituitary neurones responsible for OT synthesis during surgery, an explanation that was supported in previous research . BecauseDespite HP patients presenting with lower OT concentrations, their concentrations were not especially low. By definition, patients with CDI had acquired sufficient disruption to the hypothalamic–pituitary neurones to impair AVP release; it could therefore be reasonably anticipated that they would exhibit an equivalent and significant decrease in OT release. Although these results may potentially be explained as a result of the similarity and potential interaction between AVP and OT systems , 26, or HP patients also presented with a significant empathy deficit. Initial analyses of self-report measures found that HP patients had significantly lower trait empathy compared to HC participants. This was supported by experimental data showing that CDI patients scored significantly lower on the RMET compared to HC participants, and participants’ OT concentrations significantly predicted their performance on the easy items of the RMET: the lower their OT concentrations, the lower their score. On the FER task, CC patients had significantly lower overall scores than HC participants, which was driven by a specific difficultly in identifying fearful facial expressions. The scores of CDI patients tended to fall between the CC and HC scores. Both CDI and CC patients were poorer at identifying high intensity expressions (compared to HC participants), which one would expect to be easier to identify. Both clinical groups also demonstrated a greater error bias, compared to HC participants; CC patients demonstrated a fear bias, in that CC patients tended to over report fear when presented with other emotions, and both CC and CDI patients had an anger bias. However, participants’ OT concentrations were not related to their FER performance.These results support findings reported by Hoffmann et al.  showing In the present study, a further novel finding was that CC patients were significantly worse at recognising fearful facial expressions and while showing also a tendency to report that other facial expressions depicted fear (when they did not) is especially noteworthy. Previous research has demonstrated that clinical groups who over-report negative (in particular fearful) expressions may be at greater risk of developing mood disorders , 29. TheThere are some limitations of the study that should be acknowledged. We sought to recruit 20 participants for each group; however, we were only able to recruit 15 CC patients who were appropriately age- and gender-matched. Consequently, the results pertaining to CC patients need to be treated with some caution. A future study should therefore aim to achieve a larger sample size to confirm the generalisability of the present results. Such a study may also wish to match participants on intellectual ability to control for this variable. In addition, although we were specifically interested in the effects of OT, we acknowledge that hormones do not act in isolation and it may be the case that other hormones/neurotransmitters could explain additional variance between our groups. Importantly, a strength of the study is the age range of participants (22–74). This, in combination with the fact that age was not a significant covariate, suggests that age does not moderate any effects of group or OT on cognitive empathy. Finally, it is important to note that we did not administer intranasal OT, and thus cannot draw inferences about causality.In conclusion, the present study found that HP patients had significantly lower OT concentrations than HC participants, and also had significant deficits in cognitive empathy. Further studies are needed to replicate both these and previous findings , in ordeSupplementary Material