Traditionally, the diagnosis of post-dural puncture headache (PDPH) relied upon the patient’s history regarding dural puncture and symptoms, such as orthostatic headache. However, such evidence may not always be reliable or specific. We report an unexpected diagnosis with spontaneous intracranial hypotension (SIH), which was confirmed upon examination of Magnetic Resonance (MR) images in a patient who was initially suspected to have PDPH because he had recently undergone a uncertain dural puncture.A 45-year-old man had undergone a thoracic epidural catheter insertion for perioperative analgesia prior to general anesthesia induction. Due to intermittent dripping of fluid while the epidural needle was being advanced, a dural puncture was suspected. The patient complained of an orthostatic headache after recovery from surgery, therefore a PDPH was suspected. MR images revealed signs of SIH: dural sinus engorgement, contrast enhancement along the neural sleeves of the left C6–7, bilateral C7-T1, T1–2, T2–3, T3–4, T4–5, and T5–6. Computed tomography-guided epidural blood patching (EBP) was performed the following day, with the patient experiencing immediate relief of the headache.The benefits of radiological imaging in this case included confirming the correct diagnosis, guiding the accurate level and proper approach of EBP, distinguishing the epidural space from the intrathecal space, and ultimately increasing the likelihood of successful EBP. Spontaneous intracranial hypotension (SIH), which is characterized by spontaneous cerebrospinal fluid (CSF) leakage, has an incidence of 5 cases per 100,000 people each year , 3. The This 45-year-old male denied having any systemic diseases or any surgical history prior to admission to our center for a thoracoscopic lung segmentectomy for cryptococcus infection. Epidural catheter insertion for perioperative analgesia was performed prior to the induction of general anesthesia without sedation. The first attempt of epidural catheter insertion was performed with a Tuohy needle using the loss of resistance to air technique at the T7–8 interspinous space. However, an intermittent fluid drip was discovered as the Tuohy needle was advanced. A dural puncture was suspected, and the procedure was then repeated through the T8–9 interspinous space. However, an intermittent fluid drip was found once again as the Tuohy needle was advanced, so the procedure was abandoned. Both general anesthesia and the surgery were performed uneventfully.The patient complained of both headache and dizziness when he sat up in the ward after the operation. The symptoms were attenuated when he was in a supine position. PDPH was suspected and hydration and analgesics were prescribed. By the fifth postoperative day, the patient was unable to either stand or walk for more than 5 min without recurrence of the headache. A neurologist was consulted, and meningitis was considered as a differential diagnosis. Brain and whole spine MR imaging were ordered for further evaluation.The MR images revealed dural sinus engorgement, contrast enhancement along the neural sleeves of the left C6–7, as well as bilateral C7-T1, T1–2, T2–3, T3–4, T4–5, and T5–6. High signal intensity stripes with a length less than the width of the thecal sac were found, and type C lesions were defined [Iatrogenic trauma of the dura at T7–8 and T8–9 was initially suspected because fluid leakage was observed while a Tuohy needle was being advanced at these levels. However, there was no evidence of CSF leakage at T7 to T9 according to the MR images. The leakage location was far from the Tuohy needle puncture levels. During the loss of resistance technique, changes of pressure in the epidural space and an induced dural tear might have explained the cause of the accident, as Thomas and Thanthulage described . Congeni2 [2, while the delivery speed was 6.7 ml/min. Goeller et al. also demonstrated marked caudal epidural space pressure elevation associated with the bolus injection of a local anesthetic [The normal dura strength varies from 0.4 to 1.1 kg/mm2 . Klumpne2 . The peaesthetic . SIH aftesthetic . In our Based on this case, distinguishing SIH from PDPH based on a patient’s subtle medical history may not be as accurate as we had thought. In this patient, the CSF dripped intermittently from the needle, rather than spurting out continuously as is the normal experience after a dural puncture by an 18-gauge Tuohy needle. Thus, if an atypical manifestation occurred due to an epidural catheterization, MR imaging (or any type of advanced imaging) would be a reasonable option to provide additional information regarding the true cause of the manifestation, including neoplastic or non-neoplastic lesions –13. The Smith first recommended non-targeted EBP at the lumbar area, as the location was less risky than the thoracic or cervical areas . HoweverThe benefits of radiological imaging in this case included confirming the correct diagnosis, helping place the EBP injection into the epidural space, and ultimately increasing the likelihood of success of EBP.


Gaucher disease (GD) is one of the most prevalent lysosomal storage diseases and is associated with hormonal and metabolic abnormalities, including nutritional status disorders, hypermetabolic state with high resting energy expenditures, peripheral insulin resistance, hypoadiponectinaemia, leptin and ghrelin impairments, hypolipidaemia, linear growth deceleration and growth hormone deficiency, delayed puberty, hypocalcaemia and vitamin D deficiency. Specific treatments for GD such as enzyme replacement therapy and substrate reduction therapy display significant effects on the metabolic profile of GD patients.Hormonal and metabolic disturbances observed in both adult and paediatric patients with Gaucher disease type 1 (GD1) are discussed in this review. The PubMed database was used to identify articles on endocrine and metabolic disorders in GD1. GD1 appears to facilitate the development of disorders of nutrition, glucose metabolism and vitamin D insufficiency. Metabolic and hormonal diseases may have a significant impact on the course of the underlying disease and patient quality of life.Conditions relating to hormones and metabolism can be wide-ranging in GD1. Obtained findings were intrinsic to GD either as a deleterious process or a compensatory response and some changes detected may represent co-morbidities. Actively seeking and diagnosing endocrine and metabolic disorders are strongly recommended in GD1 patients to optimize healthcare. GBA gene (encodes glucocerebrosidase) located on chromosome 1q21.31or PSAP gene (encodes prosaposin) located on chromosome 10q22.1, underlie GD. The progressive accumulation of glucocerebroside causes clinical manifestations of the disease D) levels are the gold standard parameter to evaluate vitamin D deficiency , 84. The0 ng/mL) , 86. A 20 ng/mL) .Mikosch et al. found a total mean of 25(OH) D level of 58.2 ± 30.3 nmol/L (23.28 ± 12.12 ng/ml) in 60 GD1 patients from England, which is clearly below the threshold of deficiency . GD1 patAccording to the “Management goals for type 1 Gaucher disease: An expert consensus document from the European working group on Gaucher disease”, no consensus was achieved for statements on vitamin D deficiency. The diagnosis and treatment of hypovitaminosis D was regarded as good clinical practice rather than a management goal for GD1 . Measurevitamin D receptor (VDR) gene polymorphisms have become popular recently in the general population and in specific groups of patients, including GD1 patients. The AA genotype of the c.1024 + 283G > A (rs1544410) gene variant in the VDR gene seems to be a risk factor for low BMD, osteoporosis and pathological fracture in GD1 patients [VDR Bsml polymorphism was associated with skeletal involvement, including osteonecrosis and/or pathological fractures, in GD1 subjects [VDR gene (ApaI aa genotype) and malignancies in GD patients [Studies on patients . VDR Bsmsubjects . Lieblicpatients . More stA number of studies have suggested a link with different metabolic changes, although the clinical significance of this is questionable. The heterogeneity of the patients’ clinical profile and the low number of subjects were among the limitations of the studies that made it challenging to confirm whether the obtained findings were intrinsic to GD as a detrimental or a compensatory process. What is more, some changes may in fact be co-morbidities. Regarding the liver, it may be dysfunctional, although the rare occurrence of liver failure is rather a consequence of conditions such as, e.g. hepatitis, autoimmune diseases or infections. Finally, it is necessary to focus on other disease processes and mechanisms when individuals with a diagnosis of GD do not follow a typical course of the disease or show characteristics, which are inconsistent with the key features of reticuloendothelial involvement.Metabolic and hormonal diseases are widespread in the world population. Gaucher disease appears to facilitate the development of some of the discussed diseases, especially disorders of nutrition and glucose metabolism. Many metabolic and hormonal diseases result in pathological changes, often with the most severe effects on the course of the underlying disease and patient’s quality of life. GD1 patients are at increased risk of peripheral insulin resistance and characteristic lipid alterations. An increased focus on detecting hormonal and metabolic disturbances, especially nutritional status disorders, insulin resistance and lipid alterations, is strongly recommended to optimize the healthcare therapy in GD1 patients. Effective ERT treatment seems to have positive effects on most metabolic and hormonal diseases, but could promote a tendency to gain weight. The existence of a casual relationship and confirmed, reasonable pathophysiological mechanism, is hard to prove because of a low incidence of GD in the population and a relatively high incidence of metabolic and hormonal disorders, such as obesity or insulin resistance, in the general population. It is necessary to conduct further longitudinal studies on larger GD1 patient groups to monitor the risk of metabolic and hormonal disorders, especially with the emergence of SRT and the transition of patients to it from ERT.


To investigate the effect of fetal sex on fibroids’ growth during pregnancy according to the hCG serum levelsObservational study conducted from January 2007 to December 2016 on women with ultrasound identification of uterine fibroids who had a pregnancy within 1 year from diagnosis. The fibroids diameter was determined during the pre-pregnancy ultrasound, early first trimester (5–7 weeks), late first trimester (11–13 weeks), second trimester (19–21 weeks), and third trimester (31–33 weeks). The diameter growth was calculated in each interval between two ultrasounds. The hCG serum levels were determined both in early and late first trimester. The correlation between hCG levels and fibroid diameter was evaluated. Obstetric outcomes collected were gestational weeks at birth and the rate of cesarean section. Neonatal outcomes were birthweight and Apgar score at 1 min.p < 0.01), late first trimester , and second trimester . The hCG serum levels resulted higher in women with a female fetus: 61406 (50554-71760) mU/ml vs 46016 (37160-56744) mU/ml (p < 0.01). A positive correlation between hCG levels and fibroid diameter was found both for male and female fetuses .Eighty-seven of the included women had a male fetus, and 70 had a female fetus. A progressive increase of fibroid diameter was observed from pre-pregnancy to second trimester for both fetal sexes. In third trimester, the mean ± SD fibroid diameter of female fetuses showed a slowdown, while the mean ± SD fibroid diameter of male fetuses continued to grow. Women carrying a female fetus presented a higher fibroid diameter in early first trimester (33.5 ± 13.3 mm vs 27.4 ± 11.0 mm, Women with female fetus seem to have a higher growth of fibroids up to second trimester of pregnancy. This process may be mediated by the higher serum hCG levels found in women expecting a female fetus. Uterine fibroids are the most common benign gynecologic tumors in the reproductive years, with an incidence directly related to age –3. The pRecent evidence shows that fibroids can increase the risk of adverse obstetrical outcomes , or breech presentation) with a direct correlation with their number and size . The dimThe growth of uterine fibroids during pregnancy appears to occur more rapidly up to mid-pregnancy, with a peak in the first trimester and stabilization or initial regression in the second half of pregnancy . NumerouOne of the factors that most influences the bio-humoral condition of the first weeks of pregnancy seem to be the fetal sex and, specifically, women with female fetus may present higher values of hCG compared to male fetuses , 11.Thus, the objective of this study was to evaluate the effect of fetal sex on the growth trend of uterine fibroids and to verify the possible correlation with hCG levels.This was an observational study conducted on a cohort of child-bearing age women followed from pre-pregnancy to post-partum. The population presented in this study represents an extension of a cohort analyzed in a previously published paper by our research group .All patients presented an ultrasound diagnosis of uterine fibroids performed at the Obstetrics and Gynecologic Section, Department of Odontostomatologic and Specialized Clinical Sciences, of Università Politecnica delle Marche , from January 2007 to December 2016, with no immediate indication for medical therapy or conservative surgical treatment, and which became pregnant within 1 year from diagnosis.The first eligibility criterion was the presence of at least one fibroid with a mean diameter higher or equal to 10 mm identified before pregnancy during a transvaginal ultrasound performed for mild gynecological symptoms . All women with a spontaneous singleton pregnancy that occurred within 1 year from the initial diagnosis of uterine fibroids constituted the study population.As exclusion criteria, we considered recourse to in vitro fertilization techniques, diagnosis of multiple pregnancy, miscarriage, or ectopic pregnancy.All enrolled patients performed routine pregnancy assessments and delivered at our center. Each included patient signed an informed consent to ultrasound execution and data collection. The local ethical committee approved the execution of the present study .As background characteristics, we collected maternal age at pregnancy, BMI , number of previous pregnancies, smoking, previous oral contraceptive use, previous spontaneous miscarriage, previous cesarean delivery, and previous therapy for fibroids.All included women underwent five ultrasound examinations: the first in the pre-pregnancy period , the second between 5 and 10 completed gestational weeks (early first trimester), the third between 11 and 13 completed gestational weeks (late first trimester), the fourth between 19 and 21 completed gestational weeks (second trimester), and the fifth between 31 and 33 completed gestational weeks (third trimester). During the pregnancy ultrasounds, we also collected the obstetric parameters such as the vitality of the embryo or the fetus, the placental location, and the amount of amniotic fluid.Ultrasounds were performed with a Voluson 730 PRO  and a 3.5–5.5 MHz probe by the same senior sonographer. We defined uterine fibroids as well-defined round uterine lesions, with heterogeneous echogenicity, shadows at the edge of the lesion or internal fan-shaped shadowing, and circumferential flow around the lesion . We deteWe defined as “change in fibroid diameter” (∆D) the difference between the final mean diameter (FD) of each fibroid and the starting mean fibroid diameter (SD) in two consecutive ultrasounds (FD–SD). We defined the diameter growth rate (GRD%) of fibroids in two successive ultrasounds by the following formula: GRD% = (100*∆D/SD).Human chorionic gonadotropin serum levels (hCG) were collected for each patient both during early first trimester  and during late first trimester , with an ELISA test .We considered as obstetric outcomes the gestational age at delivery, the rate of preterm birth , and the rate of cesarean section.The following neonatal outcome was collected after birth: sex , weight, and Apgar score at 1 min.r coefficient. A multiple regression analysis was conducted considering as dependent variable fibroid diameter at early first trimester ultrasound and late first trimester ultrasound and as independent variables the maternal age, maternal BMI, number of fibroids, FIGO classification, hCG levels at 5–7 weeks, and hCG levels at 11–13 weeks. A p value < 0.05 was regarded as a statistically significant.The statistical software used was SPSS 20 . The normality of each variable was evaluated by the D’Agostino-Pearson test. Normally distributed variables  were expressed as arithmetic mean ± standard deviation, while not-normally distributed variables  were reported as median and interquartile range (IQR). Qualitative variables were expressed as numbers and percentages. The chi-square test, the Mann-Whitney test, or the one-way ANOVA test was used for variable comparison, as appropriate. Correlation between linear variables was evaluated with the Pearson’s During the study period, 10,197 childbearing age women were subjected to a gynecological ultrasound at our institution for mild gynecological symptoms . Among these, 3784 (37.1%) were diagnosed with at least one uterine fibroid with a diameter greater than 10 mm. For 2399 (63.4%) women, it was a new diagnosis, while the remaining 1385 were aware that they were affected by uterine fibromatosis. A pregnancy was obtained within 1 year from diagnosis in 213 women, and 157 of them fulfilling the eligibility criteria were considered for the final analysis. More specifically, 18 women were excluded because of spontaneous miscarriage, 10 because of multiple pregnancies, 7 for IVF treatments, and 21 did not complete all the ultrasound evaluations  patients had a male fetus, while 70 (44.6%) had a female fetus. Table p = 0.01).At the pre-pregnancy ultrasound, 119 patients (66.1%) presented a single fibroid, and 38 (33.9%) multiple fibroids; the total number of identified lesions was 180. The number, site, and location of fibroids remained the same until the second trimester, allowing the paired comparison between pre-pregnancy and first or second trimester. Table p = 0.92) (Table p < 0.01), with an increase up to late first trimester (p < 0.01), and no difference between late first and second trimester  (Table p < 0.01)  Table . The dias) Table . Women w1) Table . The mea1) Table .Table 3p = 0.08) and the late first trimester .No difference in fibroid diameter between women with or without previous therapy for fibroids emerged both during the early first trimester , while no difference emerged between the early first and late first trimester (17 (0–38)% vs 23 (0–64)%, p = 0.16) or between first and second trimester (0 (− 12–7)% vs 2 (0–12)%, p = 0.07). The diameter growth rate was different between females and males between second and third trimester (6 (4–9)% vs 6 (− 7–8)%, p = 0.02).The diameter growth rate (%) between pre-pregnancy and early first trimester was significantly higher in women with a female fetus, in comparison to a male fetus (33 (8–68)% vs 25 (5–47)%, p = 0.04). A positive correlation with the gestational weeks was found for both the fetal sexes .The median (IQR) level of hCG in male fetus was 30309 (3856–47604) mU/ml, significantly lower than the female fetus hCG level of 34403 (2432–62350) mU/ml (p = 0.06) and 11–13 weeks .The hCG levels of women with or without previous therapy for fibroids were not different: 5–7 weeks . Conversely, a significant difference was found between during late first trimester (11–13 weeks): 46016 (37160-56744) mU/ml vs 61406 (50554-71760) mU/ml, p < 0.01, when the pregnancies with female fetus reached higher levels  and female  median (IQR) levels of hCG during early first trimester (5–7 weeks)  and female fetuses   and for male fetuses was 0.68, . During the late first trimester, the Pearson’s r coefficient for females was 0.87 , and for males was 0.75 . No difference between correlation coefficients was found during the early first trimester (p = 0.94), while in late first trimester the r coefficient for females was significantly higher than males (p = 0.02).A positive correlation between hCG levels and fibroid diameter during first trimester was found both for male (01) Fig. . More spAt the multiple regression analysis, only female sex and hCG values at 5–7 weeks or 11–13 weeks were independently associated with fibroid diameter at early first trimester ultrasound and late first trimester ultrasound Table . Maternap = 0.85), rate of preterm birth  or rate of cesarean section  emerged between women with male or female fetuses. The mean ± SD neonatal weight of male fetuses was 3351 ± 504 gr, and of female was 3197 ± 552 gr (p = 0.09). The median (IQR) Apgar was 9 (9–9) for males and 9 (9–9) for female (p = 0.82).No difference in gestational age at delivery , while in female fetuses, a reduction in fibroid diameter was noted during third trimester.In the first trimester, when the difference between males and females was more evident, we noticed a significantly higher hCG serum level in patients with female fetus rather than patients with a male fetus, with a direct correlation between hCG increase and fibroid diameter.This trend of growth is in line with the literature; indeed, Benaglia et al. observed in 2014 a significant growth of fibroids in the first period of pregnancy in patients who underwent ART procedure .The significant growth rate observed in the first weeks of pregnancy seems to be related to the variation of the hCG serum levels in the same gestational period. This hypothesis is supported by laboratory data that showed the presence of hCG receptors on the fibroid cells  and fromp < 0.002) [p < 0.0001) in patients with a female fetus rather than patients with a male fetus both during the first and the second trimester on a wide population (1.1 million) of women subjected to screening [To date, it is widely documented that the values of hCG are influenced by fetal sex. Yaron et al. reported that in patients who underwent to ART procedures, from the 14th and the 20th day post-fertilization, women attending a female fetus had higher levels of hCG than women attending a male (< 0.002) . Adibi ecreening . The resThe different growth trend observed between males and females during third trimester  may not be related only to hCG serum levels. Indeed, other hormones , placental hormones (human relaxin RLNH1), and angiogenic markers  that present differences between fetal sexes may play and additional role on fibroid growth during pregnancy.Our results are strengthened by a large number of included women and by the early and close monitoring of fibroid characteristics. Moreover, the two subgroups of women with male and female fetus were comparable for all the socio-demographic and sonographic features.In our series, the discrepancy of fibroid growth between the two fetal sexes was not associated with a different incidence of adverse obstetric outcomes. This lack of association could be explained by the fact that a significant part of the patients included in the study had a single myoma of small size, under the threshold for an increased risk of adverse obstetric outcomes. Furthermore, it is important to underline that the genesis of obstetric diseases is a multifactorial event, not attributable only to the presence of uterine fibroids.A potential limitation of our study is the sonographic method of evaluation of diameter of fibroids, with the inevitably ultrasound-related degree of imprecision. We tried to overcome this limitation by using a standard technique for fibroid diagnosis and measuring. For these reasons, the determination of fibroid diameter and the evaluation of diameter increase could be affected by an inevitable risk of inaccuracy.In conclusion, uterine fibroids seem to grow rapidly both during first and second trimester of pregnancy, with faster growth in women expecting a female fetus. This difference could be explained by the direct correlation between hCG serum levels and fibroid diameter found in these patients. After second trimester, the growth continues for male fetuses until the third trimester but it stops for female fetuses.The early determination of fetal sex  could be useful in patients with fibroids and female fetus for a more accurate counseling about the risk of growth of uterine fibroids and the possible onset of adverse obstetric outcomes.These patients with a higher risk could be initiated to more intensive screening programs, with monitoring of fibroids dimensions, in order to more effectively prevent, diagnose, and handle eventual complications. However, future studies are needed to evaluate the risk of adverse obstetric outcomes related to the growth of fibroids, also in populations with multiple and larger lesions.


OXA influenced ~28% of dLGN neurons recorded in vivo by either excitation or suppression of neuronal firing. OXA-responsive neurons did not show any spatial distribution nor represent a coherent group of dLGN cells, and responded to OXA similarly across the light–dark cycle. Interestingly, some OXA-responsive neurons worked in a cortical state-dependent manner, especially during the dark phase, and ‘preferred’ cortical activation over slow-wave activity induced by urethane. The corresponding patch clamp study confirmed these results by showing that < 20% of dLGN neurons were excited by OXA under both light regimes. The results suggest that OXA is involved in the development of the visual system rather than in visual processes and further implicate OXA in the mediation of circadian and arousal-related activity.Orexins  are hypothalamic peptides playing crucial roles in arousal, feeding, social and reward-related behaviours. A recent study on juvenile rats suggested their involvement in vision modulation due to their direct action on dorsal lateral geniculate (dLGN) neurons. The present study aimed to verify whether a similar action of OXA can be observed in adulthood. Thus,  Derived from a common precursor prepro-orexin and synthetized exclusively in the lateral hypothalamus (LH), orexins bind to two G protein-coupled receptors: OX1 selective for OXA and non-selective OX2, binding both peptides with similar affinity2. Orexins exert their multiple effects by means of extensive projections throughout the brain7.Orexin A and B (OXA and OXB), also known as hypocretin 1 and 2, are hypothalamic neuropeptides11, orexins follow a circadian pattern of expression and action. Significant daily variation has been found for the level of prepro-orexin mRNA and OXA immunoreactivity in the hypothalamus14, OXA in the cerebrospinal fluid15, as well as the number and activity of orexin neurons in rodents17. Increased levels of peptide production, expression and associated neuronal activity have been observed during the dark phase of the photoperiod, which in nocturnal rodents is a time of prolonged wakefulness and motor activity16. Indeed, orexin neurons increase their firing rate during active wakefulness, decrease the frequency of discharges when animals approach sleep and become silent during slow-wave and REM sleep19. Considered as a neuroexcitatory agent23, OXA has also been found to inhibit neuronal activity25. Interestingly, day/night differences have been reported for this mechanism of action in the SCN26.Engaged in the regulation of rhythmically occurring processes  and controlled by a master circadian pacemaker the suprachiasmatic nucleus (SCN)in vitro study showed that OXA exerts multiple excitatory actions on neurons in the dorsal lateral geniculate nucleus (dLGN), a thalamic relay for vision27. Therefore, this sensory modality has been suggested to be modulated by arousal and a circadian factor, both of which are related to orexins27. Certainly, visual processing in the dLGN differs according to vigilance state29, and the neuronal activity of the dLGN has recently been reported to be influenced by the general state of the brain under different types of anaesthesia31.Recently, an in vivo and in vitro. Considering the diurnal variation in the actions of orexins, as well as orexinergic fibre density in the dLGN27, the peptide was applied in both phases of the photoperiod. Additionally, the light responsiveness, relationship with the general brain state, regularity of the discharge pattern and presence of infra-slow oscillatory activity of dLGN neurons were characterised.The present study aimed to investigate the putative action of OXA on neuronal activity in the dLGN in adult Wistar rats patch clamp study on juvenile Wistar rats suggested orexin involvement in vision modulation due to the direct, excitatory action of orexins on dLGN neurons. Moreover, immunohistochemical staining showed sparse, circadially modulated orexinergic innervation in the dLGN27. We aimed to verify whether similar action of OXA can be observed in adulthood and, if so, whether it is influenced by the time of day. Therefore, we compared sensitivity of dLGN neurons to OXA application during in vivo and in vitro recordings under two light regimes: light and dark  and 117 under scotopic conditions during the dark phase . The recorded neurons were evenly distributed across the entire dLGN, as shown in Fig. In total, the activity of 235 dLGN neurons was recorded The effect of 200 µM OXA infusion on the spontaneous firing of dLGN neurons was first tested during the light phase. In total, 118 neurons were subjected to that experimental protocol; however, due to observed changes in the cortical state influencing the spontaneous firing rate of dLGN neurons during or just after OXA infusion Fig. , only 10Importantly, 13 out of 27 OXA-responsive neurons were tested under both cortical states, and five different response types were observed: activation (n = 5) or suppression (n = 1) under both phases, activation (n = 3) or suppression (n = 3) only during cortical activation and activation (n = 1) only under SWA state, suggesting that OXA at least to some extent works in a state-dependent manner and that alterations in the ECoG may influence dLGN neuron physiology, as the responses were often observed only during one cortical phase.All neurons responsive to OXA (either activated or supressed) were characterised as non-regular with a mean CV of 1.20 ± 0.08 and 1.02 ± 0.04 during the SWA and cortical activation state, respectively, and their CV did not differ from the mean CV of neurons not responsive to OXA . Moreover, the mean CV was not changed during the response to OXA , OFF (exhibited a rebound activation after the stimulus termination) and sustained (maintained elevated firing during the stimulus presentation). The light-induced responses were assessed based on the peristimulus time histograms (bin size = 0.1 s) and 6 OXA-responsive neurons were characterised as transient ON, 4 as transient OFF and 1 as sustained. Among OXA-activated neurons were transient ON, transient OFF, sustained and non-responsive types, while only 1 out of 7 OXA-supressed neurons was light responsive (transient OFF). Also, neurons responding to OXA application only during one cortical state could be characterised as transient ON, OFF or not light-responsive. The above analysis suggests that OXA-responsive dLGN neurons represent different neuronal populations, and only some of them (46%) are directly engaged in vision formation.Electrophysiological characteristics of OXA-responsive dLGN neurons showed that 19 out of 23 tested neurons were modulated by the cortical state , 12 out of 26 tested were sensitive to light stimulation  and 8 exhibited both of these properties. Moreover, infra-slow oscillatory neurons (n = 3) were among these cells. Among OXA-responsive neurons receiving retinal input, all three known types36. Thus, we decided to verify whether a light background impacts the ability of dLGN cells to respond to OXA infusion. To this end, electrophysiological recordings were performed during the light phase under a photopic and scotopic lighting background. In total, 22 neurons were subjected to that protocol, and the response of each of them to OXA was tested four times . OXA infusion statistically (≥3 SD) influenced the activity of 8 dLGN neurons, and both activation (n = 4) and suppression (n = 4) of firing were observed. Among OXA-responsive cells, 3 responded under all four conditions tested (2 cells were activated and 1 suppressed), and 5 responded only during the cortical activation state (2 cells were activated and 3 were suppressed). Importantly, there were no changes in cells responsiveness to OXA between background lighting conditions, meaning that if cells responded to the drug under scotopic conditions, they also responded under photopic conditions. In this way, we ruled out the possibility that a high firing rate of dLGN neurons under photopic conditions makes neurons insusceptible to OXA. Moreover, these observations confirm the above result suggesting that OXA may work in a state-dependent manner in the dLGN. Due to the small sample size, no statistical test could be performed; however, stronger suppression of firing was observed under a photopic background, most likely due to the higher spontaneous firing rate .The dLGN receives strong excitatory input from retinal ganglion cells, which significantly influences its spontaneous activity27. Thus, the effect of 200 µM OXA pressure-driven infusion on the spontaneous firing of dLGN neurons was tested during the dark phase. In total, 117 cells were subjected to that experimental protocol; however, only 97 were further analysed. OXA infusion significantly influenced the spontaneous firing rate of 28% of tested dLGN neurons, and both activation (n = 14) and suppression (n = 14) of activity were observed  and received retinal innervation (17/28 from which 11 were characterised during SWA phase and exhibited transient ON (n = 1), OFF (n = 3) and sustained (n = 6) responses). Moreover, 11 of them expressed both of these properties. Similarly like in the light phase, responses to light could not be linked to any particular type of OXA-responding neurons, as transient ON, OFF and sustained responses were observed in activated and supressed neurons.in vivo. Interestingly, significantly more neurons were influenced by OXA during cortical activation state in the dark phase than in the light phase .Finally, we compared the electrophysiological properties of OXA-responsive dLGN neurons recorded during the light and dark periods. There were no differences in the magnitude of the response  and the mean time of the response between OXA-activated neurons .In total, 50 neurons were recorded (25 neurons per phase). In both phases, responses to OXA application were observed, but were much more rare (8/50) than in juvenile ratset al.27 were due to developmental changes in orexinergic innervation of the dLGN, an additional immunohistochemical experiment was performed. Using the same technique as Chrobok et al.27 we showed that the dLGN of juvenile rats is sparsely innervated by orexinergic fibres  in ~28% of dLGN neurons recorded 38, and some inconsistencies were found. Reports by Bayer et al.23 and Govindaiah & Cox38 did not find any orexin-responsive neurons in the dLGN; however, they performed an overview of orexin action on different thalamic neurons. The total number of tested neurons in the dLGN in both studies was relatively low thus, orexin-responsive neurons may have been omitted from the recorded populations. On the other hand, Chrobok et al.27 focused on orexin action on the rat dLGN by performing a comprehensive study on Wistar rats. The authors showed that ~75% of dLGN neurons are excited by orexins regardless of light regime and with the involvement of voltage-dependent calcium influx and closure of GIRK channels. Moreover, their immunohistochemical study revealed sparse orexinergic innervation of the dLGN with maximal fibre density during the dark period. The discrepancies between the high number of orexin-responsive neurons and the sparse orexinergic innervation of the dLGN were explained by a possible engagement of volume transmission. Interestingly, however, we hypothesised that this difference may be due to developmental changes, as they used juvenile (14–21 days old) and adult (5–6 weeks old) rats for electrophysiology and immunohistochemistry, respectively. Rodent retina and the visual system are developed at the time of eye-opening (P12–P14); however, the functionality of the retina and specificity of the retinogeniculate synapse take approximately three weeks spanning eye opening to display the activity and connectivity typical of adult animals43. In fact, the present study confirms this hypothesis by showing that a relatively lower proportion of dLGN neurons are sensitive to OXA in in vitro (~20%) and in vivo (~28%) preparations in adult rats. Notably, we used the same technique (patch clamp recordings) as Chrobok et al.27 to determine the effects of OXA on dLGN neurons in adult rats; therefore, the observed difference between the results is not due to technical issues. Thus, the next obvious question is whether the dLGN of juvenile rats is densely innervated by orexinergic fibres. Surprisingly, our immunohistochemical data showed sparse orexinergic innervation of the dLGN in immature rats at ZT 1–2. Moreover, the orexinergic innervation did not follow any spatial pattern explaining random distribution of OXA-responsive neurons in the dLGN in our in vivo study. In the work of Chrobok et al.27 the density of orexinergic fibres was slightly higher in the anterior direction, however we did not observed such pattern. The quantitative analysis showed that in the present work the mean area fraction of orexinergic fibres is a bit higher than in a previous work on adult animals. There are few explanations for that. First of all, confocal laser parameters during scanning are adjusted to the observed immunoreactivity, so some differences could appear there. Secondly, different parameters could be used in Bernsen’s adaptive thresholding method. Importantly, all previous studies examining orexinergic innervation of the brain showed dense orexinergic fibres in a wide range of brain areas but no or very low density in the dLGN46. The possible explanation for the discrepancies between sparse orexinergic innervation (the present data) and high sensitivity to OXs27 in the juvenile rat dLGN might be due to developmental changes in orexin receptors. In fact, remarkable differences in the mRNA levels of prepro-orexin and orexin receptors occur during the first weeks of the postnatal period. Moreover, developmental changes are specific for brain regions. The mRNA level of prepro-orexin in the LH48, OXR1 in the ventromedial hypothalamic area and OXR2 in the paraventricular nucleus47 increases until the third postnatal week and then stabilises to further decrease with age. The hypothalamic level of mRNA for OXR1 and OXR2 has been shown to decrease with brain maturity, similar to the cortical level of OXR250. Moreover, OXR1 mRNA was present in the infant cerebellum but not detectable at all in maturity49. Similarly, expression of OXR2 mRNA in hypoglossal motoneurons peaks around P20 and then strongly decreases51. This difference in expression across development is not an unusual situation, as different peptide receptors, including those for vasopressin52 and somatostatin53, which are also present in the visual system54, have been found to be more strongly expressed in the developing than in the adult brain. As mentioned before, the dLGN changes remarkably during the first few weeks of the postnatal period, including changes in total RNA and DNA contents, which stabilises around P4055. Taking these rationales into account, we suggest that orexins may strongly influence dLGN postnatal development. Indeed, such a role for orexins in different brain regions has been suggested previously56. Alternatively, the low number of OXA-responsive neurons in the dLGN of adult animals may be connected to concentration-dependent activity of OXA58. In the present work concentrations of 200 nm and 200 µm were used for patch clamp and in vivo studies, respectively. Even though, similar results were acquired for both preparations, there is still a possibility that higher concentrations would have more broaden responses. On the other hand, generally OXs concentration in nanomolar and micromolar range was used for the in vitro61 and in vivo64 studies, respectively.The effects of orexins on dLGN activity were studied before in slice preparation by three different groupsin vivo recordings under urethane anaesthesia regardless of light regime. Importantly, urethane has already been proven to be a good anaesthetic for experiments investigating diurnal variations in electrophysiological recordings in the SCN67 and medial habenula68. Additionally, urethane induces cyclic fluctuations in cortical and/or brainstem activity mimicking sleep episodes: slow-wave and desynchronization states; thus, urethane anaesthesia was used previously as a model of sleep (but not sleep-wake cycle)73. OXs are generally considered excitatory in both in vivo and in vitro preparations. Excitation of spontaneous neuronal activity by OXs has been observed under urethane anaesthesia in the hippocampus62, globus pallidus74, substantia nigra pars compacta63, dorsal raphe (DRN)75 and laterodorsal tegmental nucleus (LTD)64. Moreover, increases in neuronal activity in vitro upon OX application have been showed in numerous brain structures, such as the locus coeruleus77, tuberomammillary nucleus78, DRN79, intergeniculate leaflet80 and paraventricular nucleus of the thalamus61. However, an inhibitory action of OXA is not rare. OXA mainly suppresses neuronal firing in the SCN82 with different mechanisms across the light-dark cycle26. Moreover, OXA inhibits the activity of neurons expressing melanin-concentrating hormone in the LH by acting on local GABAergic interneurons83 and OX1R mRNA expression was found on the putative GABAergic interneurons in the LTD and pedunculopontine tegmental nuclei84. Additionally, hippocampal pyramidal neurons have been showed to be suppressed during in vivo recordings upon cerebral ventricle OXA infusions by indirect activation of local inhibitory interneurons85. In line with these studies, our data show that OXA application in vivo can have a suppressive outcome on spike firing. Because we did not observe hyperpolarisation of dLGN neurons during patch clamp recordings, we speculate that the suppressive effect may be due to activation of local GABAergic interneurons. In fact, interneurons in the dLGN make inhibitory connections with relay cells86. In our in vivo recordings, OXA-suppressed neurons constituted 7% and 14% of all neurons recorded during light and dark phase, respectively. Distinction regarding a neuronal type: thalamo-cortical vs local interneurons in in vivo approach was not made, as even though general criteria exist for such analysis, it has not been published before specifically for the rat dLGN cells. Patch clamp recordings would enabled such discrimination and experiment focusing on dLGN interneurons would help in interpretation of our data, however considering low percentage of interneurons in the dLGN (~10%), low response rate to OXA in the structure of adult rats and challenging methodological aspects regarding patch clamp recordings in adult animals, such experiment would not seem to draw any conclusions. Importantly, local OXA infusion was not limited to the recorded neuron; thus, in some cases, we may have also observed transsynaptically mediated effects.The present work shows that dLGN neurons may be excited or suppressed by OXA during 86, such a wide distribution of OXA-responsive neurons is not surprising. More importantly, these results extend those of Chrobok et al.27 by showing that OXA influences not only neurons contributing to direct vision formation but also dLGN neurons receiving signals from non-retinal inputs (for review see86). The very strong modulatory innervation of the dLGN derives from the brainstem cholinergic nuclei that control network states during arousal, sleep and wakefulness88. Interestingly, the present data show that some OXA-responsive dLGN neurons work in a state-dependent manner under urethane anaesthesia. These OXA-responsive neurons were more often found during the dark period, and they ‘preferred’ cortical activation than SWA state. Orexinergic neurons promote arousal and are more active during wakefulness in the dark period and become silent during sleep19. During naturally occurring slow-wave sleep, thalamo-cortical dLGN neurons are mostly hyperpolarized and generate lower firing rates, whereas during REM sleep and waking states, they are depolarised and fire more often90. Moreover, neurons switch between bursting and tonic firing modes, with the latter prevailing during slow-wave sleep93. These differences reflecting the state-dependence of dLGN neuron physiology have been examined in detail in terms of their light responsiveness97 and with little attention to the state-modulatory influences of pharmacological agent application98. Iontophoretical administration of acetylcholine to single dLGN cells caused excitation of firing only during waking and REM sleep89, whereas adrenergic receptor agonists worked mainly during slow-wave sleep98. On the other hand, OXA was showed to work in a state-dependent manner on the serotoninergic DRN neurons causing excitation only during naturally occurring sleep75. Our results coincide well with these studies, as we report that state-dependent OXA-responsive dLGN neurons responded during the cortical activation state and were more often observed during the dark period. These neurons were mostly modulated by the ECoG and were characterised by a higher firing rate during cortical activation than during SWA state. We hypothesise that state-dependent OXA-responsive dLGN neurons may be engaged in mediating arousal-related activity, however taking into consideration the heterogeneous nature of the dLGN and small number of such neurons; it may be difficult to test.OXA-responsive dLGN neurons could not be linked to any specific population of dLGN neurons. Some of them  were sensitive to light stimulation and were thus most likely directly engaged in vision formation. The majority were modulated by the general brain state according to the simultaneously recorded ECoG under urethane anaesthesia. Moreover, OXA-responsive neurons did not express the same firing pattern, as some of them were classified as infra-slow oscillatory during the light phase. Considering the large heterogeneity of dLGN neuronsin vivo and in vitro in adult rats compared to previously published data on juvenile rats27 and sparse orexinergic innervation of the nucleus in both studies, we suggest that OXA is particularly important for the postnatal development of the visual thalamus. At present, however, its role in this process remains unclear and requires further investigation. Second, a comprehensive analysis of the electrophysiological properties of OXA-responsive dLGN neurons showed a large heterogeneity, thus extending its previously suggested role from modulation of visual processes to more global effects on dLGN functioning. OXA-responsive neurons were characterised as receiving not only visual information but also modulatory influences from the brainstem and/or cortex. Moreover, the state-dependent action of OXA, intensified during the dark period, further implicates its contribution to the mediation of circadian and arousal-related activity to the dLGN, which may result in the facilitation/attenuation of incoming information and/or information processed by the dLGN. Lastly, we emphasise the importance of circadian control of not only clock-related structures and simultaneous ECoG and spike recordings, especially in experiments on the thalamus.Three main conclusions can be driven from the present study. First, taking into consideration the relatively low number of OXA-responsive dLGN neurons recorded 99.All experimental procedures were performed in accordance with Polish Animal Protection Law, approval (196/2012) of Local Ethics Committee of Jagiellonian University in Krakow and regulations and standards of the Directive 2010/63/EU of the European Parliament and of the Council of 22 September 2010 on the protection of animals used for scientific purposes, the 3Rs law, and the ARRIVE guidelines for reporting experiments involving living animals with respect to anaesthesia and animal handlingad libitum. Zeitgeber time (ZT) 0 was designated as the time of lights on. Experiments were performed during two different light regimes: light phase (ZT 1–11) and dark phase (ZT 13–23). Regardless of the experimental protocol (in vivo or in vitro study), animals were removed from their home cages during the early portion of the light (ZT 1–2) or dark (ZT 13–14) phase. For in vivo experiments, rats were directly anaesthetised with an i.p. injection of urethane . The dark phase group received the injection in a darkened room, and immediately after their eyes were covered with black foil. The surgery and electrophysiological recordings were performed in a darkened room with the light intensity at the animal’s eyes < 0.1 µW/cm2 through the experimental sessions. The rig was covered with a light-impermeable material. Electrophysiological experiments during the light phase were conducted in the same experimental room with room lights on (~100 µW/cm2). The electrophysiological recordings spanned the middle portion of the light (ZT 3–10) and dark (ZT 15–22) phases. The experimental design is shown in Fig. in vivo, 13 for in vitro and 4 for immunohistochemistry experiments).All experiments, except for the immunohistochemistry study, were conducted on adult, male Wistar rats bred in the animal facility of the Institute of Zoology and Biomedical Research at Jagiellonian University in Krakow. Animals were housed under a 12:12-h light:dark cycle at a temperature of 22–23 °C and 60% humidity with food and water available bregma100. The dura was punctured, and the exposed brain tissue was covered with a drop of mineral oil  to prevent drying. The recordings were taken at a depth of 4.0–5.5 mm from the first contact of the electrode with the cortical surface. Borosilicate glass micropipettes (resistance: 5–10 MΩ) pulled on a horizontal puller  and filled with 4% Chicago Sky Blue in 2% NaCl  were used to acquire neuronal signals. The recorded single-unit (sometimes multi-unit) activity was amplified 10,000 × , filtered between 300 Hz and 3 kHz and digitised at 20 kHz using an Axon Instruments CyberAmp 380 , Micro mkII interface and Spike2 software . Data were stored on a computer, and all further analyses were performed off-line.Adult Wistar rats weighing 270–400 g (2–3 months old) were used. Following urethane injection and prior to the surgery, each animal was checked for withdrawal and ocular reflexes to assess the level of anaesthesia . The physiological state of the animal was continuously monitored by electrocardiogram and body temperature readouts. The animals’ head was secured in a stereotaxic apparatus  and an initial incision was made to expose the scull surface. Skin and all soft tissues were removed to reveal sutures and stereotaxic points. A craniotomy was performed above the dLGN, 4–5 mm posterior and 3.3–4.3 mm lateral to www.molex.com, cat no. TSP100170) glued to the Tygon tube and connected to a 5-µL Hamilton syringe . Next, the fused silica tube was glued 200–300 µm above the tip of the recording electrode under visual microscopic control. The same system was used 3 to 4 times and was tested for leaks and washed out with sterilized water each time. The system was pre-loaded with 200 µM OXA diluted in saline or saline and 200 nL of the fluid was infused. In control experiments , 200 nL of saline was slowly infused during on-line monitoring cell activity to confirm outflow, signal stability and lack of cell response. There were no significant differences between the mean firing rate or CV before and after saline infusion  and connected to CED Micro mkII interface and Spike2 software .103) was held open, and the pupil was dilated by a topical application of atropine solution . The cornea was moisturized with mineral oil to prevent drying. A light-emitting diode  was placed in front of the eye and connected to a Master-8 stimulator . Regardless of the experimental group (light or dark phase), all animals were presented with 5-s light pulses with 25-s intervals, repeated 6–8 times. The intensity of the light stimulus differed, as it was presented against photopic and scotopic backgrounds, and was 220 and 100 µW/cm2 for the light and dark group, respectively. The light intensity was measured with a GENTEC P-LINK .The majority of recorded cells were tested for light responsiveness. Light stimulations were mostly presented during cortical activation phase (it is longer and more stable under urethane anaesthesia). Thus, the eye contralateral to the craniotomy  was ejected for 15 min from the recording electrode containing ChSB  and theta (~4 Hz), respectively.Simultaneously recorded alternating changes in ECoG signal: cortical SWA and activation states were identified based on the work of Clement 31. Briefly, a cross-correlation coefficient (CC) analysis was performed on delta power band derived from ECoG signals containing both activated and SWA phases  and simultaneously recorded FR of the dLGN neurons. Neurons were characterised as significantly correlated with the ECoG when their maximal CC values exceeded confidence limits representing a 2-fold increase in their respective standard errors. Based on maximal CC values, dLGN neurons were classified into SWA-ON and activation-ON  calculated as a ratio of the SD of the ISI to its mean. The Young’s regularity criterion was used105.The infra-slow oscillatory activity was identified by applying fast Fourier transform and autocorrelation analysis to ~900-s-long intervals, as shown before in detailet al.62. The baseline firing rate was averaged for at least 50 s (max 200 s when examining infra-slow oscillatory pattern of activity) before OXA application with the condition that the recording had to be obtained under one cortical phase. To assess the beginning and end of the response to OXA, the firing rate was averaged for 10-s bins with a 1-s sliding window starting from 10 s before OXA infusion . An increase or decrease in firing rate was considered significant if it exceeded or fell below 3 standard deviations (SDs). The SD was calculated by averaging the firing rate of sequential time bins (10 s) within 50 s before OXA application.To characterise dLGN neurons as OXA-responsive, we used a similar method as Chen Statistical comparisons were based on the results of two-way ANOVA followed by Tukey’s test, paired/unpaired t tests, Mann–Whitney, Kruskal–Wallis, Wilcoxon tests, Fisher’s and Chi-square exact tests performed in Prism, version 5 . Significance was set at p < 0.05. Data are presented as the mean ± SEM.59. In short, adult male Wistar rats (5–6 weeks old) were anaesthetised with isoflurane  and decapitated between ZT 1–2 or ZT 13–14. Next, the brain was quickly removed and immersed in ice-cold, oxygenated  cutting artificial cerebrospinal fluid (ACSF), composed of (in mM) 185 sucrose, 25 NaHCO3, 3 KCl, 1.2 NaH2PO4, 2 CaCl2, 10 MgCl2 and 10 glucose. Then, the block of brain tissue containing the thalamus was placed on the cold plate of the vibroslicer . Coronal slices, 250 µm in thickness, including the dLGN, were cut and transferred to the pre-incubation chamber filled with recording ACSF containing (in mM) 123 NaCl, 25 NaHCO3, 3 KCl, 1.2 NaH2PO4, 2 CaCl2, 2 MgCl2 and 10 glucose. Slices were kept in this solution for 30 min at 32 °C, followed by 60 min at room temperature before placement in the recording chamber (21 °C–24 °C).To obtain brain slices containing the dLGN, we used a previously described procedureRecording electrodes were placed in the dLGN under visual microscopic control . Recordings were performed in whole-cell configuration obtained by applying negative pressure from an Ez-gSEAL100B Pressure Controller . The recorded signal was amplified by a SC 05LX amplifier , low-pass filtered at 3 kHz and digitized at 20 kHz. Spike2 and Signal  software were used for the recordings. A liquid junction potential of approximately −13 mV was added to the measured membrane potential. Typical recordings lasted ~1000 s.2, 4 Na2ATP, 0.4 Na3GTP, 1 EGTA and 0.05% biocytin . Rectangular current pulses  were applied to monitor the membrane resistance during each recording. Neurons for which the membrane resistance changed by more than 15% during the recording or that had a membrane potential more positive than −35 mV  were excluded from further analysis.Experiments in current clamp mode (holding current = 0 pA) were conducted at room temperature (21 °C–24 °C). Patch pipettes pulled on a horizontal puller  were filled with normal intrapipette solution containing (in mM) 125 potassium gluconate, 20 KCl, 10 HEPES, 2 MgClmax). Δmax is described as the highest point of the examined value reached during the response.All data were analysed using MATLAB  and GraphPad Prism 6  software. Changes in the firing rate and membrane potential were considered to be significant if they differed from baseline by more than three standard deviations (SDs). Data are presented as the mean ± SEM or maximal change of membrane potential ± SEM  was freshly prepared from 200 µM stocks , and 200 µM or 200 nM was used for the TM . Slices were then scanned on an A1-Si Nikon Inc. (Japan) confocal laser scanning system built on an inverted Nikon Ti-E microscope (Japan). Image processing was performed with the use of ImageJ2 software . Quantitative analysis of the orexinergic fibres was performed using similar protocol to Chrobok et al.27 and the whole area of the dLGN was scanned. The results are presented as area fraction (%) ± SEM .Four male juvenile Wistar rats (14–21 days old) were deeply anaesthetised with pentobarbital  and sacrificed (ZT 1–2) by transcardial perfusion with PBS followed by 4% PFA. Brains were quickly removed and immersed in 4% PFA overnight. Next, blocks of tissue containing the dLGN were cut into 100- µm-thick slices on a vibroslicer  and rinsed twice in PBS. Then, slices were transferred to solution containing 0.6% Triton X-100  and 10% NDS  at room temperature. After 2 h, slices were rinsed in fresh PBS and incubated with goat OXA antisera  in PBS, 2% NDS and 0.3% Triton X-100 overnight at 4 °C. Next, slices were washed twice in PBS and incubated with secondary anti-goat AlexaFluor 488-conjugated antibodies  for 24 h. Finally, slices were rinsed in PBS and mounted on glass slides in FluoroshieldSupplementary Information


Discovered in 1991 in a screen for genes involved in spindle pole body duplication, the monopolar spindle 1 (Mps1) kinase has since claimed a central role in processes that ensure error-free chromosome segregation. As a result, Mps1 kinase activity has become an attractive candidate for pharmaceutical companies in the search for compounds that target essential cellular processes to eliminate, for example, tumour cells or pathogens. Research in recent decades has offered many insights into the molecular function of Mps1 and its regulation. In this review, we integrate the latest knowledge regarding the regulation of Mps1 activity and its spatio-temporal distribution, highlight gaps in our understanding of these processes and propose future research avenues to address them. D. D58]. D8.a), and microtubules compete with NTE–NDC80 interactions in vitro [figure 3b). Another potential consequence of this is that MPS1 molecules, now unable to cluster on kinetochores, cannot be efficiently reactivated once they become inactive. MPS1's ability to re-bind may also depend on Aurora B activity, and metaphase tension states may thus further ensure that re-binding is prevented. Molecular insights into how Aurora B promotes MPS1 localization will determine whether this is the case.In order for anaphase to initiate, SAC silencing needs to occur once all chromosomes have formed stable attachments to microtubules of the mitotic spindle ,116. As figure 3in vitro ,15. In aDrosophila cells, by directly inactivating the kinase [b). Cytoplasmic PP1 activity can further assist this process.Residual low levels of Mps1 remain on metaphase kinetochores ,117, yete kinase . Aurora e kinase ,125. A d9.trans-autophosphorylation, resulting in rapid activation of a substantial pool of MPS1. This pool is now proficient in eliciting a strong SAC signal around the time of nuclear envelope breakdown (a). Activation of MPS1 now causes the MR–NDC80C interaction to diminish, increasing its turnover rate and decreasing its steady-state kinetochore levels (b). As more and more kinetochores become attached, this signal is further dampened. In parallel, biorientation of chromosomes reduces Aurora B activity near kinetochores, promoting PP1 binding. PP1 then dephosphorylates Mps1 substrates and potentially directly inactivates any residual Mps1 molecules at or near kinetochores, essentially quenching the SAC signal emitted from these bioriented chromosomes (b). Relocalization of MPS1 to attached kinetochores at this stage is severely hampered by the microtubules, further ensuring that inactivated MPS1 can no longer be reactivated. When a sufficiently large pool of MPS1 is inactivated, all SAC activity is quenched and APC/C activity is initiated.Aside from a possible small pool of active MPS1 prior to mitosis, the majority of MPS1 molecules are inactive. When kinetochores are assembled, MPS1 is recruited to the NDC80 complexes via its NTE and MR regions E. This creakdown a. Activae levels F. This ae levels b. As moromosomes b. Reloca10.We have outlined current knowledge on how localization and activity of the chief executive officer of the SAC, the kinase Mps1, is regulated. Understanding its role in the protection of genome stability is far from complete, however, and several key lacunas need to be filled.Though Mps1 activity peaks in mitosis, Mps1-dependent formation of MCC complexes already occurs in interphase. Very little is known about how and where this pool of Mps1 becomes activated. Answers will be greatly assisted by the development of a live biosensor assay, similar to those that clarified essential features of Aurora B activity dynamics . Upon enCompetition with microtubules ensures that Mps1 kinetochore levels become greatly reduced once stable attachments have been formed and this is a major SAC silencing mechanism. Once Mps1 is displaced from kinetochores, how exactly does inactivation take place and what are the dynamics of it? Nevertheless, a fraction of Mps1 remains bound to kinetochores even in the presence of stable attachments . This beMuch progress has been made in recent years with regard to understanding the principles that underlie faithful chromosome segregation and normal mitotic progression. The importance of attachment error correction and the SAC in these processes and the involvement of Mps1 are undisputed. Elucidating the finer points of Mps1 regulation in the coming years will provide crucial insights into how genomic stability is ensured.


The advent of personalized cancer treatment resulted in the shift from the administration of cytotoxic drugs with broad activity spectrum to a targeted tumor-specific therapy. Aligned to this development, the focus of this study revolved around the application of our novel and patented microtube array membrane (MTAM) in the US National Cancer Institute (NCI) developed an HFA (hollow fiber assay) assay; hereinafter known as MTAM/HFA. Electrospun poly-L-lactic acid (PLLA) MTAM was sterilized and loaded with cell lines/patient derived tumor cells (PDTC) and subcutaneously implanted into the backs of BALB/C mice. Anticancer drugs were administered at the respective time points and the respective MTAMs were retrieved and the viability tumor cells within were quantified with the MTT assay. Results revealed that the MTAMs were excellent culture substrate for various cancer cell lines and PDTCs (patient derived tumor cells). Compared to traditional HFA systems that utilize traditional hollow fibers, MTAM/HFA revealed superior drug sensitivity for a wide range of anticancer drug classes. Additionally, the duration for each test was <14 days; all this while capable of producing similar trend outcome to the current gold-standard xenograft models. These benefits were observed in both the in vitro and in vivo stages, making it a highly practical phenotypic-based solution that could potentially be applied in personalized medicine. The development of anticancer drug research are often plagued with poor translatability between the developmental phases and the clinical phases . This isPatient-derived xenografts (PDXs), established from tumor tissue directly engrafted into immune-deficient mice, are one possible solution. There is increasing evidence that suggest PDXs consistently conserve biological features of the parental malignancies , while maintaining the complex interaction between implanted tumor cells and its microenvironments ,5,6,7,8.About 20 years ago, Dr. Hollingshead, M. et al. at the National Cancer Institute (NCI)/NIH, USA, proposed a unique in vivo hollow fiber assay (HFA) that can dramatically reduce the screening time required, the number of animals and the quantity of required candidate compound ,17. ThisRecently, we developed a new class of hollow fibers known as the microtube array membrane (MTAM), which consisted of ultra-thin, homogenously porous, one-to-one connected individual fibers ,21,22. TIn this study, we strive to demonstrate the MTAM/HFA in screening of several types of cancer cell lines against several types of anticancer drugs in both in vitro and in vivo settings. In addition, we also will demonstrate the use of MTAM/HFA in the determination of the drug sensitivity of patient’s tumor cells, which could potentially increase the accuracy and sensitivity of anticancer drug screening within a clinically practical time frame.2O) and air dried for 24 h.Preparation of poly-L-lactic acid (PLLA) microtube array membranes (MTAM) with different tube wall porosity has been reported previously ,10. BrieHuman bone chondrosarcoma, fibroblast-like (SW1353) cell line, fibro sarcoma (HT1080) cell line, human lung adenocarcinoma (A549), colon cancer cell line (HCT116) and glioblastoma multiform (GBM) were used and maintained in conditional medium. The cell culture and all assays in this study were performed in DMEM/F-12, RPMI, and HDMEM  supplemented with 10% FBS , 50 U/mL penicillin, and 50 mg/mL streptomycin  at 37 °C in a 5% carbon dioxide atmosphere.2O and placed in a container containing ddH2O for ultra violet (UV) light sterilization. After sterilization, the fibers were flushed under sterile conditions. SW1353, HT1080, and A549 cells (2 × 105 cells/10 μL) were drawn into a 0.5 cm × 1.5 cm PLLA/30 MTAM via capillary action; and into CellMax fibers measuring 1 cm. The ends of the respective fibers were sealed by pliers at 0.5 cm intervals several times. The cell containing fibers were then cultured in six-well plates filled with complete growth culture medium.The respective fibers were washed three times using ddH5 cells/10 μL, and the ends of the fibers were sealed with a plier. Next, these fibers containing cells were maintained in six-well plates in the complete growth culture medium. The corresponding anticancer drugs were administered accordingly and incubated in 37 °C in a 5% CO2 atmosphere. At a predetermined time, fibers were cut into pieces then analyzed by the BCA protein assay kit (Pierce). The absorbance at 562 nm was measured by ELISA . All experiments were performed three times with 12 replicated wells for each sample and control per assay.The respective cells that were cultured within six-well plates were siphoned into the respective fibers at a density of 2 × 105 cells/10 μL) was siphoned into a 0.5 cm × 1.5 cm PLLA/30 MTAM and CellMax fibers measuring 1 cm via capillary action. Next, the ends of the fibers were sealed by plier at 0.5 cm intervals several times until the ends of the fibers were not be cocked. After preparation, the samples were transferred to culture dishes containing complete medium and incubated overnight at 37 °C in a 5% CO2 atmosphere prior to implantation into mice. Anesthesia  was induced into the respective mice by via inhalation. For subcutaneous (s.c.) implants, a small skin incision measuring 1–2 cm was made at the nape on the back. Samples were inserted with a caudal through the subcutaneous tissues. The skin incision was closed with a skin staple. After 24 h of cell proliferation, the animals were treated with the corresponding anticancer drugs. The respective fibers were retrieved at the respective predetermined time points. A general clinical observation  was made twice daily by a veterinarian.All animal studies were approved by the animal research ethics committee (LAC-2016-0207). Thirty Male BALB/c mice at 6–7 weeks of age were purchased from BioLASCO Taiwan Co., Ltd.  and were free of known pathogens at the time of use. All procedures were performed in compliance with the guidelines of the Institutional Animal Care and Use Committee, which are based on the guidelines of the Association for Assessment and Accreditation of Laboratory Animal Care including facility (protocol number LAc-2U14-U193). Mice were housed in the TMU Laboratory Animal Center  in a conventional environment at constant temperature (20 °C ± 3 °C) and humidity (50% ± 20%). To prepare samples, a sterile field was established in a biological safety cabinet. Immediately prior to cell siphoning, the respective fibers were individually rinsed with condition media. The cell suspension , heck and neck cancer (IRB: N201704053) and breast cancer (IRB: N201604012) whom were diagnosed at Taipei Medical University Hospital (TMUH). Surgical specimens and core needle biopsy were taken before any treatment. All patients signed an informed consent for the usage of their tumor samples for research.Responses to therapy were then converted to clinical outcomes (based on RECIST) from changes in tumor volume over the course of treatment.Data are presented as mean values ± SD. Statistical significance was analyzed by two-tailed Student’s t-test. Values of P < 0.05 (n = 5) were considered significant.The PLLA MTAM appeared to be an off-white thin film to the naked eye. Conversely, when observed under the SEM , the unique microstructures consisted of one-to-one, individually connected submicron scale hollow fibers. The surface of the respective MTAM consisted of homogenously distributed pores of about 200 nm. In terms of the dimensions of the individual lumens, the height × width measured 60 μm × 40 μm, and a lumen wall thickness of 2.2 ± 0.4 μm a. The PL5 cells/10 μL were defined as the optimal seeding density for in vitro and in vivo growth within hollow fibers over 4 days , or surgical excisions (colon cancer). The cell viabilities from these two groups were detected by MTT assay. The results demonstrated that patient derived tumor cells had better proliferation rate in MTAM a. Then, The nanoporous PLLA MTAM possessed self-supporting structure can be easily handled and manipulated into most the configurations as desired throughout the experimental procedures. In regards to the cell loading procedure, the process primarily employed the capillary force that existed in the respective lumens, thereby siphoning the droplet of cell culture media up into these lumens. Additionally, due to the transparent wall of the PLLA MTAM, the tumor cells that were siphoned into the PLLA MTAM were clearly visible. It was observed that the PLLA MTAMs were not as mechanically sound as CellMax fibers; nevertheless, this did not hamper the use of the PLLA MTAMs throughout the duration of the experiment as the PLLA MTAMs were sufficiently strong. For ease of tracking, the PLLA MTAM can be colored through the addition of food grade dye to the polymer solution prior to electrospinning. The addition provided a high contrast between the PLLA MTAM and the surrounding soft tissue; a highly beneficial characteristic for ease of identification of the PLLA MTAM.Results indicated that the MTAM with the diameter size between 50 and 60 μm performed the best in terms of cell survivability. Potentially, at this diameter, oxygen was allowed to freely permeate deep into the respective PLLA MTAM thereby allowing tumor cells in the PLLA MTAM to survive better since oxygen appeared to be one of the most important nutrient for cells proliferation . An altePrior to in vitro and in vivo toxicity test, the IC 50  was conducted and the IC 50 was around 15 mM. Based on the results in High failure rates are often associated with the lack of reliable preclinical models to predict the effects these newly developed drugs in a setting resembling the complex nature of the tumor environment. One method to overcome this problem is to utilize a patient-derived xenograft model (PDX) . The graIntraperitoneal hollow fiber activity was also found to be a better predictor of xenograft activity when compared to subcutaneous hollow fiber activity or intraperitoneal plus subcutaneous activity combined . CompareAdditionally, several other limitations were observed in the use of PDX as a standard modality in modeling human cancers. Namely, the loss of tumor microenvironment and immune-response , selectiThe in vitro and in vivo data gathered in this study demonstrated that MTAM is a superior culture substrate for cancer cells when compared to traditional HFs, as well as being a significantly more sensitive and reliable system for anticancer drug screening. The MTAM/HFA system conferred significant benefits in terms of the cost, rapidness, reliability and the reduction in the overall number of required animal used in a study; while maintaining comparable trend outcome compared to the current gold standard-traditional xenograft model. Clinically, we also demonstrated the potential of MTAM/HFA-PDTC, which demonstrated its practicality, ease of use and reliability. In short, the range of benefits of the MTAM/HFA system made it a highly practical phenotypic-based solution that could potentially be applied in applications such as anticancer drug screening for personalized medicine and as a pre-xenograft model the development of anticancer drugs.


The interpolated bathymetry was tested for accuracy against independent depth data provided by Fisheries and Oceans Canada . Quantitative terrain attributes were generated to better understand seascape characteristics at multiple spatial scales, including slope, rugosity, aspect, and bathymetric position index. Landform classification was carried out using the geomorphons algorithm and a novel method for the identification of previously unmapped tributary canyons at the continental shelf edge are also presented to illustrate some of many potential benefits of crowd-sourced regional seafloor mapping.Seafloor mapping can offer important insights for marine management, spatial planning, and research in marine geology, ecology, and oceanography. Here, we present a method for generating regional bathymetry and geomorphometry maps from crowd-sourced depth soundings (Olex AS) for a small fraction of the cost of multibeam data collection over the same area. Empirical Bayesian Kriging was used to generate a continuous bathymetric surface from incomplete and, in some areas, sparse Olex coverage on the Newfoundland and Labrador shelves of eastern Canada. The result is a 75m bathymetric grid that provides over 100x finer spatial resolution than previously available for the majority of the 672,900 km Marie Tharp and Dr. Bruce Heezen used early single-beam echosounders to produce the first continuous, three dimensional visualization of the North Atlantic seafloor in 1957 . Twenty Study of the benthic environment often involves study of the patterns and processes that shape the seabed itself (i.e. geomorphology). The relationship between a species or a community and their environment is fundamental to the concept of habitat –9. For mLophelia pertusa using 50, 100, 250, 500, and 1000 m bathymetric grids and associated terrain attributes, and found that accurate predictions of this habitat type required bathymetric data finer than a 250m grid. Development of 100m grid regional bathymetry for the Terre Adélie and George V continental margin in Antarctica has also shown to greatly improve geomorphological interpretation over existing bathymetric datasets (GEBCO and ETOPO1), including new information on the extent and complexity of inner-shelf valleys [The most widely used seafloor dataset is the General Bathymetric Chart of the Oceans (GEBCO) . GEBCO b valleys . Many ot valleys , oceanog valleys , geohaza valleys , 28, and valleys  have bee valleys . However valleys . It is e valleys .Alternative approaches to bathymetric data collection can help meet current information needs. Our study focuses on the potential benefits of crowd-sourced seafloor mapping, a relatively recent field of study with the capacity to provide large amounts of data at minimal cost . The IntWith a sufficiently large participating community, crowd-sourcing is a very efficient way to collect large amounts of data. Furthermore, repeated sampling by different actors helps reduce overall error rate and size –35. At sOur study area is part of a passive continental margin  and is b2 and tested the accuracy of the resulting bathymetry against independent depth data provided by the department of Fisheries and Oceans Canada. The interpolated bathymetry we have produced is a 75 m grid; over 100 times finer than existing GEBCO data which is the best bathymetry for most of this region. In this paper, we describe the geostatistical methods used to generate continuous bathymetry. We also provide a few examples of how crowd-sourced bathymetry can contribute to an improved understanding of the region through the quantification of terrain attributes, classification of bedforms, and identification of previously unmapped tributary canyons. This work opens the door to various applications in marine ecology, conservation, resource management, marine geology, and oceanography.We interpolated Olex data over an area of approximately 673,000 kmOlex AS provides a commercial charting system that allows fishing vessels to collect and share bathymetric data . The equOnce collected and provided to Olex AS, bathymetric data are corrected based on predicted tides and local chart datum reference levels. Variables like sound velocity, echosounder installation depth, and vessel heave/pitch/roll are rarely associated with the provided depth soundings. Instead, transducer depth correction and a sound velocity coefficient are calculated based on comparison to the rest of the crowd-sourced dataset. If new data contributions provide soundings within a 5.6m cell that is already populated, the shallowest depth value is retained. A simple linear interpolation is also applied to the Olex data to produce the 45m grid used in this study, however each cell contains at least one real sounding from the underlying 5.6m grid. Olex reports vertical accuracy of approximately 0.3m, based on comparisons of their processed data to independent bathymetry. For this study, processed Olex bathymetry data for the Newfoundland and Labrador waters of Canada’s east coast were made available as XYZ points.2 in the study area was calculated using the Point Density tool in ArcGIS Pro 2.0. Areas with fewer than 100 data points per 10 km2 were excluded from further processing as this level of sampling was insufficient to inform the interpolation. This exclusion resulted in some gaps in the final bathymetric coverage.The study area, defined by the spatial coverage of the provided Olex data, includes the continental shelf and some shelf edge throughout North Atlantic Fisheries Organization (NAFO) Sub-divisions 0B, 2GHJ, 3KLMNOPs, and 4RS, including the Flemish Cap, and a portion of the Laurentian Channel . Olex daInterpolation of the Olex data was carried out using Empirical Bayesian Kriging (EBK), a geostatistical interpolation technique that estimates the spatial relationship of the input data (defined by a semivariogram) through many iterative models that are weighted and combined using Bayes Theorem –50. ThisThe input Olex data were first divided into 129 spatial subsets of 100x100 km with 2.5% overlap to reduce processing time and non-stationarity within each subset. Each 100x100km subset was run through the EBK protocol independently . All inpThe output for each kriging window was cropped by 2.5% to remove depth estimates influenced by edge effect and to remove overlaps between analysis windows. Standard error of the interpolated values was also calculated, and all pixels with a standard error >10 m were excluded from further processing as a quality control measure. Cropped kriging windows were assembled in a mosaic with blended seam lines in ArcGIS Pro 2.0.Validation of the resulting bathymetric surface was achieved through a comparison of interpolated depth values to the GEBCO_2014 Grid, version 20150318  and analLecours et al.  demonstrThe r.geomorphons tool  was used2 cells, where n = 2, 4, 8, and 16, producing bathymetric surfaces at lower spatial resolutions. All terrain attributes were calculated for the five bathymetric surfaces to capture topographic structure of the seafloor across multiple scales. Finally, the mean value was calculated across all scales of each terrain attribute to generate a single data layer representing multiscale geomorphic structure. This method for multi-scale analysis was previously described by Dolan [Scale dependence is a fundamental problem in spatial analysis . Substraby Dolan  and was The r.geomorphons model was also applied at multiple scales through application of a variable analysis window . The flaIn addition to the terrain forms classified by r.geomorphons, a semi-automated approach was developed to identify shelf incising canyons in order to illustrate how the higher resolution bathymetry can be used to generate new information in the study region. This novel approach involves a two-step hierarchical bathymetric position index (BPI) classification. Areas identified as terrain lows through Broad scale BPI see  were man2 . The interpolated bathymetry reaches up to 647 km offshore, ranges from 1 m to 2133 m depth and extends from 60.06° W to 43.72°W and from 42.74°N to 61.95°N .Spearman’s correlation between the interpolated bathymetry and independent depth data  was both very high (0.99) and significant (p<0.001). This calculation was repeated specifically for areas where interpolated bathymetry was informed by low sounding density, as this is where bathymetric artefacts are expected to be most prevalent. Correlation was similarly high (0.95) and significant (p<0.001) when only validation data points over areas of low Olex sounding density were tested . Low sounding density is defined here by the lowest quartile —were generated at multiple spatial scales (examples shown in The majority of the Newfoundland shelf (84.5%) was classified as flat or low relief based on bathymetric standard deviation values of <3.7 m . Areas o2). Slopes are the second most prevalent features, making up 24.3% of the study area , followed by shoulders , and foot slopes . Spurs, valleys, hollows, and ridges are roughly equally prevalent in coverage . Peaks and pits are the rarest terrain types, covering 0.3% (2098 km2) and 0.4% (3115 km2) of the study area respectively.The r.geomorphons terrain classifications were generated at multiple spatial scales; the broadest scale  is discu2 of the study area, concentrated on the southern shelf break  is much higher than mean slope for the rest of the study area (0.74°) and canyon walls reach slopes of 50–65°. Most of these canyon features are too small to be identified by GEBCO bathymetry  and manyComparison of the features mapped using interpolated bathymetry to canyons mapped by Harris et al.  reveal tWhile the crowd-sourced bathymetry presented here cannot match high-resolution of hydrographic multibeam surveys, we have been able to generate a bathymetric grid that is over a hundred times finer resolution than GEBCO data for a fraction of the cost of a multibeam survey. Such data can be instrumental in supporting studies in oceanography , marine Regional biodiversity assessments and marine habitat mapping are fundamental to marine spatial planning  and ecosAlthough this study represents a >100-fold increase in bathymetric resolution for most of the study area and provides regional maps suitable for marine spatial management efforts, we do not consider seafloor mapping within this study area to be complete. Research on the impact of digital bathymetric model resolution on the prediction of marine substrates  and the Prevalent bedforms and the processes that shape local geomorphology have been previously described for this region based on seabed imagery, sediment samples, and interpretation of side-scan sonar data –74. GreaThe terrain attributes presented here provide quantified characterization of many forms of general geomorphometry which are powerful surrogates for the distribution of substrate type, benthic biodiversity  and idenThe method presented here for identification of submarine canyons is a very simple approach which classifies elongated features of low bathymetric position index that are roughly perpendicular to the shelf edge. This approach has several limitations; it relies on the quality of the training dataset, it is not fully automated, and it does not explicitly incorporate all characteristics of shelf-incising canyons, like steep walls or branching order , 79. ThiIn this paper we have described a novel method for generating continuous bathymetry from a large crowd-sourced echosounder data at a much higher spatial resolution than previously available via satellite altimetry and much larger spatial extent than available from existing MBES surveys. With very minimal thresholds applied for quality control, even areas of sparse coverage were successfully interpolated with high accuracy when tested against independent bathymetric data. The resulting interpolated bathymetry, which covers an area greater than half of the entire Atlantic Canadian shelf and provided a 480% increase in coverage when compared to the input sounding coverage for the study area, can help answer a number of scientific questions that were not possible using previously existing regional bathymetry datasets, despite the long history of bathymetric surveys in Canadian waters . The appParticular attention was given in this paper to the mapping of submarine canyons. These features are complex systems associated with high rates of ocean mixing , biologiThe crowd-sourced bathymetric data used in this paper are collected and compiled internationally wherever participating commercial vessels are active, however these data remain largely unused for the quantitative and systematic study of seafloor geomorphometry and benthic habitats. Our method offers a robust and reproducible method to make use of crowd-sourced data for applications including, but not limited to, marine conservation, resource management, and marine geology. In much of the study area, this work represents the finest resolution bathymetry data currently available, and was produced at a fraction of the cost of conventional surveys.S1 FigBPI was calculated in Benthic Terrain Modeler 2.0 at multiple scales; (a) an 8 cell inner radius and 16 cell outer radius applied to the 75m interpolated bathymetry, (b) a 25 cell inner radius and 50 cell outer radius applied to the mean interpolated bathymetry within a 300 m neighbourhood, and (c) a 100 cell inner radius and 500 cell outer radius applied to the mean interpolated bathymetry within a 1200 m neighbourhood.(PDF)Click here for additional data file.S2 FigStandard deviation within a 9 cell analysis window was calculated in Benthic Terrain Modeler 2.0 at multiple scales: (a) 75m interpolated bathymetry, (b) the mean interpolated bathymetry within a 1200m neighbourhood, and (c) the mean was taken of 5 standard deviation rasters derived from the interpolated bathymetry .(PDF)Click here for additional data file.S3 FigVRM was calculated in Benthic Terrain Modeler 2.0, for a 21 cell analysis window at multiple scales: (a) 75m interpolated bathymetry, (b) the mean interpolated bathymetry within a 1200 m neighbourhood, and (c) the mean was taken of 5 VRM rasters derived from the interpolated bathymetry .(PDF)Click here for additional data file.S4 FigSlope was calculated in Benthic Terrain Modeler 3.0 within a 9 cell analysis window at multiple scales: (a) 75m interpolated bathymetry, (b) the mean interpolated bathymetry within a 1200 m neighbourhood, and (c) the mean was taken of 5 slope rasters derived from the interpolated bathymetry .(PDF)Click here for additional data file.S5 FigStatistical aspect (i.e. slope orientation) was calculated in Benthic Terrain Modeler 3.0 for a 3x3 cell analysis window at multiple scales: (a) 75m interpolated bathymetry, (b) the mean interpolated bathymetry within a 1200 m neighbourhood, and (c) the mean was taken of 5 northerness rasters derived from the interpolated bathymetry .(PDF)Click here for additional data file.S6 FigStatistical aspect (i.e. slope orientation) was calculated in Benthic Terrain Modeler 3.0 for a 3x3 cell analysis window at multiple scales: (a) 75m interpolated bathymetry, (b) the mean interpolated bathymetry within a 1200 m neighbourhood, and (c) the mean was taken of 5 easterness rasters derived from the interpolated bathymetry .(PDF)Click here for additional data file.S1 FileTen shelf edge canyons were identified from visual assessment of the interpolated 75m bathymetric grid and measured to inform the parameters for canyon classification across the shelf edge of the entire study area.(PDF)Click here for additional data file.S2 FileThe r.geomorphons model was applied in GRASS GIS 7.4 to classify specific geomorphometry at multiple scales, based on (a) 75m interpolated bathymetry (inner search radius of 0m and an outer search radiues of 225m), (b) the mean interpolated bathymetry in a 150 m neighbourhood (inner search radius of 300m and an outer search radius of 1200m), (c) the mean interpolated bathymetry in a 1200 m neighbourhood (inner search radius of 900m and an outer search radius of 3300m), and (d) the mean interpolated bathymetry in a 1200 m neighbourhood (inner search radius of 1875m and an outer search radius of 7500m).(PDF)Click here for additional data file.