Atypical movement was demonstrated in the majority of infants with an absent MEP from the MAH  compared to those with a present MEP  (p = 0.191). We found that age influences the ability to elicit an MEP from the MAH, and motor outcome may be related to MAH MEP absence. Assessment of MEPs in conjunction with current practice of neuroimaging and motor assessments could promote early detection and intervention in infants at risk of CP.Diagnosis of cerebral palsy (CP) after perinatal stroke is often delayed beyond infancy, a period of rapid neuromotor development with heightened potential for rehabilitation. This study sought to assess whether the presence or absence of motor evoked potentials (MEPs) elicited by transcranial magnetic stimulation (TMS) could be an early biomarker of atypical development within the first year of life. In 10 infants with perinatal stroke, motor outcome was assessed with a standardized movement assessment. Single-pulse TMS was utilized to assess presence of MEPs. Younger infants (3–6 months CA,  Perinatal stroke, occurring between 20 weeks’ gestation and the 28th day of life, often leads to cerebral palsy (CP), a diagnosis of disordered movement due to a disturbance to the developing brain in utero or infancy ,2,3. CP TMS is a form of electromagnetic non-invasive brain stimulation which can assess cortical excitability and CST organization through depolarization of corticospinal neurons . A knownThis cross-sectional pilot study was approved by the University of Minnesota’s (UMN) Institutional Review Board, Fairview Health Services, the Clinical and Translational Sciences Institute, Center for Magnetic Resonance Research (CMRR), and the UMN Discovery, Research, and Innovation Economy initiative steering committee. Infants were recruited from the University of Minnesota’s Masonic Children’s Hospital (UMMCH) neonatal intensive care unit (NICU) and the Children’s Hemiplegia and Stroke Association. Infants between 3–12 months of age corrected for prematurity, with a diagnosis of unilateral or asymmetric bilateral perinatal stroke confirmed by cranial ultrasound or magnetic resonance imaging (MRI), were included. Infants with MRI incompatible implanted medical devices, uncontrolled seizure, other neurological diagnoses, genetic disorders, or neoplasm were excluded. A total of 10 infants meeting criteria enrolled and participated. Parents provided informed consent for their infants to participate in this study. Data collection occurred at UMN and UMMCH between August 2016 and January 2018.min video of the infant naturally moving while lying supine with clearly visible upper and lower extremities. GMA videos were taken by 20 weeks CA at home by a parent or by a member of the research team in the UMMCH NICU follow-up clinic. All GMA videos were scored by the same GMA-certified pediatric physical therapist (C.P.). The BSID-III motor scale, which consisted of fine and gross motor subsets, was utilized for infants 6–12 months CA and was administered and scored by the same pediatric physical therapist (J.L.K). Gestational age (GA) and CA were determined by Academy of Pediatrics definitions, and medical record review [Age-appropriate standardized behavorial assessments were completed to examine motor outcome. Dependent on the postnatal corrected age (CA) for prematurity of the child, either the Prechtl General Movements Assessment (GMA) or Bayley Scales of Infant and Toddler Development III (BSID-III) was completed. The GMA was utlized for infants 3–5 months CA, and consisted of a 3–5 d review .As a means to guide TMS assessment, magnetic resonance imaging (MRI) sequences were collected at the CMRR at UMN per our published study protocol, which utilized procedures from the Baby Connectome Project for infant scanning techniques ,23. PartMuscle responses to TMS were recorded via surface electromyography (EMG) of the bilateral wrist flexors. The wrist flexors were selected as the optimal muscle group for EMG recording due to pilot testing results which demonstrated robust resting EMG amplitude and lack of cardiac artifact present in signal from the biceps bracii . The ski2 and 2002 unit set, Magstim Co. Ltd., Carmanthenshire, UK) placed on the scalp at a 45 degree angle posteriorlateral to the midsagittal plane. Dependent upon the postural control and preference of the infant, infants were positioned supported in their parent’s lap, or independently seated or standing. When possible, testing was completed with the infants’ upper extremities at rest through activities such as passive bottle feeding or watching a video to achieve a relaxed state. TMS was first delivered to the targeted M1 of the left hemisphere beginning at 50–60% maximum stimulator output (MSO) and then increased by 5% increments after 3–5 trials at each MSO with a pre-determined ceiling of 90% MSO ; range of TMS intensities (50–90% MSO)). If 90% MSO was attained and no potential MEP responses were identified, the surrounding cortex was assessed in anterior, posterior, medial, and lateral directions by moving the coil approximately 0.5 cm in each direction with 2–3 trials at each new location. The right hemisphere was then assessed following the same protocol ; range of TMS intensities (50–90% MSO)). Number of trials and intensities completed were dependent upon infant tolerance and time limitations.Single-pulse TMS was delivered to the targeted M1 with a 70 mm figure-of-eight coil  connected to a TMS stimulator . The raw EMG signal was baseline corrected and rectified. The area under the curve (AUC) of each MEP and pre-stimulus activity was cacluated as defined by Rossini et al. . A 10 msmin throughout the TMS assessments. A member of the research team followed up by phone with parents of participating infants 24 h after TMS assessment to identify any potential adverse events or behavioral changes. The Medical Monitor reviewed the documentation upon completion of the study for each infant, and if needed, evaluated any potential adverse events to determine optimal recommendations for care.Medical records were screened for eligibility criteria and reviewed by the research and medical teams prior to participant enrollment. The Medical Director determined final eligibility of all infants. Assessments of heart and respiratory rate, blood pressure, behavioral state, and stress responses were completed prior to commencing and after completing TMS assessments. Ear plugs were utilized to protect the infants’ hearing during TMS assessments. Assessment of behavioral state and stress responses were completed every 5 R version 3.4.1 .Data are presented as frequency counts and percentages, and descriptive statistics of mean, SD, and range. Proportions of groups were compared using Fisher’s exact tests. MEP responses from the MAH and less-affected hemisphere (LAH) were compared across groups defined by CA and GA. Motor outcome was compared between those with and without MEP responses within each hemisphere. For analysis, infants were grouped as younger infants (3–6 months CA) or older infants (7–12 months CA) and by GA as preterm (GA < 37 weeks) or term (GA ≥ 37 weeks). Infants were also categorized as having atypical or typical movement as determined by GMA or BSID-III. Motor outcomes  were compared to variables of CA (younger or older) and GA (preterm or term). All statistical analyses were completed using h post-TMS assesssment, and no indications or adverse events for Medical Monitor review [The infants’ tolerance to TMS was determined based on previously reported safety assessments, follow up calls with no adverse events reported by parents 24 r review .SD = 8 weeks, range: 22–41 weeks), and mean CA at time of TMS assessment was 7 months . The majority of infants were male (70%) and born preterm (70%) (The mean GA of participating infants was 31 weeks (rm (70%) . In 60% p = 0.048) with 4/5 younger infants (80%) and 0/5 older infants (0%) displaying MEPs from the MAH (p = 0.444). MEP presence based on GA group was not significantly different for the MAH (3/7 (43%) preterm and 1/3 (33%) term; p = 1) or LAH (6/7 (86%) preterm and 2/3 (67%) term; p = 1). In total, 4/10 infants (40%) displayed MEPs from the MAH and 8/10 infants (80%) displayed MEPs from the LAH  . A stati the MAH . No sign the LAH .n = 10). Though not statistically significant, the proportion of infants with atypical movement was higher among those without MEP response compared to those with MEP from the MAH (5/6 −MEP (83%), 1/4 +MEP (25%); p = 0.191) and the LAH (2/2 −MEP (100%), 4/8 +MEP (50%); p = 0.467) (p = 0.524). No significant difference in motor outcome was determined based on GA group comparison (p = 1) with 4/7 preterm (57%) and 2/3 term infants (67%) displaying atypical movement.In 5/8 infants with MEPs, the mean pre-stimulus AUC was lower in +MEP trials compared to –MEP trials . As dete= 0.467) . AtypicaAs a means to determine whether MEP absence may be an early biomarker of atypical CST development within the first year of life, we assessed the prevalence of atypical movement and MEP responses to TMS in 10 infants with perinatal stroke. Here we explore the results of our aims to investigate the relationship between MEP presence and (1) age and (2) motor outcome.A significant difference in MEP outcome was identified based on corrected age, with MEPs from the MAH present in 4/5 younger infants (3–6 months CA) and 0/5 older infants (7–12 months CA). Our results suggest that younger infants with perinatal stroke are more likely to display TMS elicited MEPs from the MAH. Interestingly, none of the older infants displayed presence of an MEP from stimulation of the MAH, signifying an early age-related neurophysiologic difference in infants with perinatal stroke. Our findings are synonymous with Eyre et al.’s report of TMS outcomes of absence of MEPs from stimulation of the injured hemisphere of infants with unilateral perinatal stroke after 12 months of age . AsymmetA relationship between gestational age and MEP presence was not determined, with preterm and term infants displaying no significant differences in MEP outcomes. No known prior TMS studies have investigated the relationship of gestational age and MEP presence in infants with perinatal stroke. However, one study in older children with CP determined preterm or term birth did not appear to influence MEP outcomes or motor performance . In contA strong trend of atypical movement in 83% of infants with absence of MEPs from the MAH compared to 25% with present MEPs suggests a relationship between cortical excitability and motor outcome may exist in infants with perinatal stroke at risk of CP. Eyre et al. reported similar findings in infants with unilateral perinatal stroke, with absence of MEPs from the injured hemisphere being associated with failure to display a grasp by two years of age . Though No consistent trends were demonstrated regarding amount of pre-stimulus activity and MEP presence in infants. The mean pre-stimulus AUC was lower in +MEP trials compared to −MEP trials in 5/8 infants with MEPs, contradictory to reports that higher pre-stimulus muscle activity facilitates MEP elicitation in older children and adults . MEPs weAbsence of MEPs from the MAH is a demonstrated biomarker of atypical CST organization in older children with CP ,15,16,17Myelination and synaptogenesis begin in the last trimester and continue throughout childhood as the brain continues to mature, with the first three years of life being an especially prolific period of neural development . The forIdentification of a potential sensitive period for experience-dependent plasticity of the CST could inform optimal timing for intervention. Given our findings that infants 6 months of age or less were more likely to demonstrate presence of MEPs elicited from the MAH, rehabilitation with the first 6 months of life may have the greatest impact on optimal CST organization. In a rodent stroke model, rehabilitation interventions harnessing experience-dependent plasticity have been shown to increase synaptic density in layer V pyramidal neurons in the motor cortex through training in an upper limb reaching task . The incThough a trend of MEP absence was identified in infants with atypical movement, our small sample size and the heterogeneity of our sample limits our ability to determine whether statistically significant differences exist. The heterogeneous diagnosis of perinatal stroke affects a variety of brain areas and results in variable clinical presentations. As such, it is possible that lesion location and the unilateral or bilateral nature of the lesion may influence TMS and motor outcomes. The assessment of MEPs with established protocols, such as resting motor threshold, is not feasible in infants as they cannot remain consistently at rest or complete repeated contractions at a constant force, resulting in deviation from typical TMS protocols and MEP analysis. Our protocol utilized concurrent activities to encourage relaxation of the upper extremities, such as bottle feeding or watching a video, to attempt to facilitate consistent resting muscle activity during TMS testing. MEPs were analyzed with a requirement of an amplitude greater than 120% of the variance of pre-stimulus activity to overcome variability in fluctuating levels of muscle activity. Latencies for TMS elicited MEPs are yet unknown in infants, thus we included MEPs occurring within 15–50 ms after the TMS stimulus given consistent patterns of post-stimulus EMG activity in our data. Infants may display different neural conduction velocities from older children and adults due to still developing myelination ,37. In tAn analysis of CP diagnosis outcome in our full sample of 10 infants was not possible at this time due to variable length of follow-up and inconsistent identification of diagnosis (definition and timing). Physicians commonly wait to provide a diagnosis of CP to ensure abnormal motor findings are not transient and watch to determine whether they will resolve . As suchAbsence of TMS elicited MEPs from the more-affected hemisphere was found to be related to corrected age and may be associated with atypical movement in infants with perinatal stroke at risk of cerebral palsy. TMS assessments could promote early identification of biomarkers of atypical corticospinal tract development within the first year of life. Longitudinal TMS assessments of infants with perinatal stroke could inform whether MEPs from the more-affected hemisphere are present early in infancy and lost later in development. Further investigations could identify a sensitive period of corticospinal tract development and guide optimal timing for intervention to maximize motor outcomes after perinatal stroke.


We find ERG mediates the late phase of spike-frequency adaptation in pyramidal cells and is recruited later than both SK and M currents. Attenuation of ERG during coincident depolarization and ACh release leads to reduced late phase spike-frequency adaptation and persistent firing. In neuronal ensembles, attenuating ERG enhanced signal-to-noise ratios and reduced signal correlation, suggesting that these two hallmarks of cholinergic function in vivo may result from modulation of intrinsic properties.Most neurons do not simply convert inputs into firing rates. Instead, moment-to-moment firing rates reflect interactions between synaptic inputs and intrinsic currents. Few studies investigated how intrinsic currents function together to modulate output discharges and which of the currents attenuated by synthetic cholinergic ligands are actually modulated by endogenous acetylcholine (ACh). In this study we optogenetically stimulated cholinergic fibers in rat neocortex and find that ACh enhances excitability by reducing Ether-à-go-go Related Gene (ERG) K Understanding how modulatory systems function to govern neural circuits is a fundamental question in neuroscience. Abundant work  has demoThe specific cellular mechanisms that enable ACh to have such wide-ranging and important functional effects remain unresolved despite extensive efforts to define targets of mAChRs modulation. Previous work has identified large set of outward and inward currents that could explain the increase in excitability traditionally found with cholinergic stimulation . However+ currents that appear to govern SFA in neocortical pyramidal cells . Endogenous ACh released by optogenetic stimulation modulates neuronal discharges by selectively attenuating one of these currents (ERG). Acetylcholine acting through mAChRs enhanced intrinsic excitability by selectively reducing the late phase of SFA normally mediated by ERG. The ACh-triggered changes in excitability and SFA were occluded by three different ERG channel antagonists. Examining consequences of ERG modulation by ACh in virtual ensembles, we find an enhancement in SNR and reduction in signal correlation among neurons similar to that observed in vivo following cholinergic stimulation. These results suggest that changes in population neuronal activity associated with common behavioral phenomenon such as attention  did not modulate the membrane potential over the 2 s window we typically used to assay excitability changes or directly trigger discharges . We founThe increase in neuronal excitability we find with paired ACh release was repeatable across multiple trials with no modulation of excitability observed in interleaved control trials without ChR2 stimulation . The excUnexpectedly, the increase in neuronal excitability elicited by ACh was not uniform during the response to the depolarizing step. Instead, release of endogenous ACh selectively advanced the timing of APs triggered after ~ 500 ms from the step onset . The incIn these initial experiments, ACh was released by light trains that began 2 s before the depolarizing step and ended 2 s following step offset. In seven experiments, we systemically varied the timing of the light pulses relative to the depolarizing step . The onlThe delayed increase in excitability we find with ChR2 stimulation appeared to reflect both an increase in average spike frequency shown in  and a chReleasing ACh via light stimulation of ChR2 altered SFA kinetics starting approximately 800 ms into the step response , left. Iak steps , inset. quencies .The delayed increase in excitability in response to moderate duration (2 s) steps suggests that release of endogenous ACh with ChR2 should selectively modulate responses to long duration steps while leaving responses to short duration stimuli unaffected. We confirmed that prediction by testing responses to 100 ms step stimuli and found no increase in the number of APs evoked with stimulation . In the same 5 cells, pairing light stimulation with longer duration steps (750–2000 ms) increased the number of APs directly demonstrating the specificity of ChR2 modulation to long-duration responses in these experiments .Two different mechanisms can explain the pronounced delay before ChR2 stimulation increased the spiking rate. This delay could reflect either the time required to initiate muscarinic receptor signaling pathways  since our results thus far suggest endogenous ACh modulates a current with slow kinetics; we wanted to avoid activating mAChRs while the target current was activating rapidly early in the step response. As shown in the example trace in Finally, we confirmed using conventional early/late ISI ratio measurements that under all three timing conditions ChR2 reduced SFA and promoted near tonic firing . These r+ channel is likely a potential candidate as we  without affecting the initial current response assayed over the first 100 ms  relate to the time scale we find for modulation of SFA by endogenous ACh.While our findings showed that ERG antagonists occlude the increase in neuronal excitability triggered by endogenous ACh, previous work using bath application of cholinergic receptor agonists such as CCh have identified other Kdulation  that likdulation  in contedulation  and othedulation . Presuma+ channels, we first used a conductance-based model of pyramidal cells to ask if our cumulative spike count assay was sensitive enough to detect changes in SFA kinetics. This initial computational approach also reveals how changing K+ current dynamics affects experimental measures of SFA such as cumulative spike count assays. In + conductance whose kinetics was predefined  and which followed either the time course of the current step stimulus (‘continuous K’) or increased linearly during the step (‘Linearly-increasing K’). The maximal conductance of both types of K+ currents was set to reduce the total number of APs evoked during the step by an equivalent amount (~6 APs). Cumulative spike count plots revealed different SFA kinetics generated by each type of K+ conductance with the + current was introduced . The delayed increase in SFA with the linearly-increasing K+ current was not because of a specific step amplitude used in these simulations. Linearly-increasing K+ currents generated delayed SFA increases across a large range of stimulus amplitudes .Before conducting tests with pharmacological agents that block different K+ current in the simulations show in 2+ buffering in these experiments when assaying changes in SFA with XE991 and Terf. However, we found similar delayed SFA modulation with Terf when SK currents were not attenuated  before the blockers we applied. Reducing SK currents using NS8593 triggered a rapid increase in excitability with a + conductance  and the M current (green). Among these two K+ subtypes initially recruited, our results suggest that SK begins to slow firing before the M current because only SK modulators could influence the first spike time. Later in the response the influence of SK diminishes while SFA mediated by M current is maintained. Finally, a third K+ subtype slowly develops  that further slows firing. In response to physiological (5–15 Hz) firing rates, ERG-mediated SFA begins after approximately 500 ms. While all three K+ currents that contribute to SFA can be modulated by artificial mAChR agonists, our results indicate that endogenous ACh selectively attenuates the ERG mediated component. Because of this specificity, cholinergic stimulation is able to selectively modulate long-lasting responses while leaving responses to brief stimuli unaltered  of bath CCh, we found similar results. All three structurally-diverse ERG antagonists tested  abolished persistent firing in ChR2  and then repeated that process within virtual ensembles composed of the entire ChR2 data set. In each pairwise test, we computed the correlation (Pearson’s R) between the spike counts in corresponding time bins within the step response in both cells . While iAcross 24 light-stimulated ACh release experiments, we found significantly less temporal signal correlation in responses acquired during light stimulation than in control conditions. This reduction in temporal signal correlations is reflected by the majority of experiments showing less correlation when ACh is released than in control (step-only) conditions in the plot shown in + current was reduced . While previous reports have studied the isolated contributions from these currents, especially SK  channels in mediating at least partially AHP and SFA , little is known about how these different currents function together to effect adaptation in response to physiological discharges.The intrinsic currents underlying SFA have been studied extensively in many cell types with most reports focusing on SK-mediated AHP and the M current. The Cat of APs  but is pt of APs . Multipl and SFA . While Shelation . Previou current  and ERG  current . While t+ currents contribute to SFA by assaying afterhyperpolarizations following different duration depolarizing steps. Crill and colleagues (2+ -activated K+ channels (BK) channels like contribute to the fastest AHP  that were subthreshold for AP generation. We confirmed that prediction by demonstrating that SK inhibitors reduced the AHP following brief subthreshold depolarizations. Finally, bifurcations in cumulative SFA plots revealed with SK inhibitors occurred early in the step response and before the bifurcation time generated by M and ERG blockers. We deduced the order that leak K+ currents contribute to SFA by comparing bifurcation time following pharmacological blockade of either the M current or ERG (the latter tested under two different Ca2+ buffering conditions). These results suggest that following the initial phase of SK-mediated adaptation, the M current and then ERG further slow firing. In addition to different onset latencies, the K+ currents that mediate SFA appear to have different kinetics with SK currents reducing soon after their maximal functional effect while M currents remained constant and the adaptation mediated by ERG currents increased with time.Our results implicated SK and two leak K2+ current  responses by selectively regulating one constituent current. All three K+ currents we find contribute to SFA in L5 neocortical pyramidal cells  have been reported to be attenuated following mAChR activation , and was recorded 2–4 weeks later (P35-P50). Rats aged P14-P21 were used in all other experiments not involving ChR2 optogenetics. All experiments were performed under guidelines approved by the Case Western Reserve University Animal Care and Use Committee.Sprague-Dawley rats of either sex  were deeply anesthetized with 5% xylazine/10% ketamine . Under aseptic conditions, the skull was exposed and small holes with ~ 1 mm in diameter were drilled bilaterally. Stereotaxic coordinates of NB are : AP −1.4, ML 2.5 ~ 3.5, DV 6.0  c.f. . ChR2 wa2PO4, 6.2 MgSO4, 26 NaHCO3, 10 dextrose, and 1 CaCl2, equilibrated with 95% O2/5% CO2. Horizontal slices (300 Sprague-Dawley rats were anesthetized with ketamine and decapitated. The brain was then dissected and transferred into ice-cold (~0°C) artificial CSF (ACSF) composed of the following (in mM): 124 NaCl, 2.54 KCl, 1.23 NaHescribed . Slices 2PO4, 1.2 MgSO4, 26 NaHCO3, 10 dextrose, 2.5 CaCl2, equilibrated with 95% O2/5% CO2. Whole-cell recordings were made using an Axopatch 1D Amplifier (Molecular Devices/Molecular Dynamics). Patch-clamp recording electrodes with resistances of 3–8 MΩ were pulled from 1.5 mm outer diameter thin wall borosilicate glass tubing (WPI), using a micropipette puller . The pipettes contained the following (in mM): 140 K-methylsulfate , 4 NaCl, 10 HEPES, 0.2 EGTA, 4 MgATP, 0.3 Na3GTP, and 10 phosphocreatine. In some experiments, 10 mM bis(2-aminophenoxy)ethane-N,N,N’,N’-tetra-acetic acid (BAPTA) was substituted for EGTA in the internal solution to enhance Ca2+ buffering. Individual neurons were visualized under infrared differential interference contrast (IR-DIC) video microscopy . Recordings were low-pass filtered at 5 kHz  and acquired at 10 kHz using a simultaneously sampling 16-bit data acquisition system  operated by custom software written in VB.NET on a Windows-based PC . Membrane potentials related to current-clamp recordings in the text and illustrations were not corrected for the liquid junction potential. Membrane potentials related to voltage clamp recordings were adjusted for the the liquid junction potential to enable comparisons with prior ERG biophysical studies. We calculated the correction factor to be + 10.3 mV based on the method outlined in Intracellular recordings were performed in a submerged chamber maintained at 30°C and perfused continuously (~2 ml/min) with ACSF containing the following (in mM): 124 NaCl, 3 KCl, 1.23 NaHWe recorded from L5 pyramidal cells that generated ‘regular spiking’ in response to 2 s duration depolarizing currents, as defined by previous neocortical studies . For 61 + currents since extracellular TTX would limit ChR2-stimulated ACh release. We used a modified ACSF with higher K+ concentration  was obtained from Alomone Labs and dissolved directly in ACSF .+ channel blockers and green traces and bars represent experiments using bath application of CCh.473 nm LED light (custom built) was used to stimulate axon fibers projected from NB in neocortical slices through the epifluorescence pathway. LED illumination focused directly on the recorded neuron through 63x Zeiss water objective (NA 0.9) with no restriction through the field diaphragm. Brief (2 ms) light pulses were controlled by the TTL output of the ITC-18 to stimulate ChR2. Throughout the illustrations, orange traces denote experiments that included ChR2 stimulation while blue traces and bars represent experiments using KHodgkin-Huxley (HH) model of neurons was built based on + currents, To introduce variability in spike timing, noisy conductance term equation , denoted+ currents were added onto the HH model with + current, and + current conductance with maximum amplitude of 1. ‘No K’ denotes no artificial K+ current was added to the HH model. ‘Linear K’ denotes linearly increasing artificial K+ current, with slope + current during the entire period of the stimulus.Artificial K+ currents, we calculated the metric that we termed ‘Slope’  was calculated by the inverse of the coefficient of variance . Methods used to estimate signal correlation were based on approach used by  In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by Richard Aldrich as the Senior Editor, a Reviewing Editor, and three reviewers. The following individuals involved in review of your submission have agreed to reveal their identity: Lorna Role (Reviewer #1).Thank you for submitting your article "Selective attenuation of ERG by endogenous acetylcholine reduces spike-frequency adaptation and network correlation" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.The reviewers find that the manuscript describes a timely and interesting set of results regarding the mechanisms by which synaptically released ACh increases the excitability of L5 pyramidal neurons. The authors show that optogenetic stimulation of ACh fibers increases firing frequency, and using pharmacological approaches they argue that the target of ACh is hERG channels. There have been relatively few electrophysiological staudies of the role of hERG in the CNS despite its widespread distribution, and this paper suggests that these channels may be an important target. The authors speculate that acetylcholine and depolarization-dependent attenuation of ERG channels may account for acetylcholine's hypothesized effects on persistent activity, working memory, and signal-to-noise ratio.Essential revisions:The reviewers raise a number of concerns that must be adequately addressed before the paper can be accepted. Some of the required revisions will likely require further experimentation.1) The identification of hERG as the key target of ACh is not air-tight. Experiments with bath applied ACh or agonists have shown previously that muscarinic stimulation activates calcium-dependent non-specific cation conductances as well as blocking M-channels. Here the hERG current in these cells is not isolated in voltage-clamp recordings that could support the conclusion that hERG is present in these cells and acting late to modulate spike-frequency adaptation . It will be critical for the authors to demonstrate that synaptically released ACh specifically blocks the hErg mediated current in voltage-clamp, providing direct evidence for (1) the modulation of the hErg current by ACh and (2) the time course of ACh-induced block of Erg currents, which currently the authors only indirectly inferred.2) Terfenedine may not be the best choice as an identifier of hERG currents as its primary target is H1 receptors. The use of the more specific compound, E4031, is a useful addition, but some of these experiments have an unacceptably low n . The n's must be increased for this essential experiment.3) In some figures, data from experiments with reasonable n values are presented only as bar graphs. The preferred presentation is to show a full scatter plot of all of the data because it's useful for readers to see individual data points to understand variability more clearly than standard error alone.+ conductance ("linear K") to get a time-dependent SFA effect proves anything; and (d) why adding stochastic noise directly to the membrane potential is better than adding stochastic noise to the *conductances* - since real background fluctuations in vivo are mediated by conductance fluctuations.4) The modeling adds only modestly to the present work. Its contribution would be greater if the authors could explain: (a) why a one-compartment model of ventral cochlear nucleus neurons helps us understand layer 5 pyramidal neurons with elaborate dendrites; (b) how a model that does not contain BK, SK, or M channels deepens our understanding of SFA; (c) in what way putting in by hand a time-dependent KThe modeling should be strengthened considerably or simply eliminated. Essential revisions:The reviewers raise a number of concerns that must be adequately addressed before the paper can be accepted. Some of the required revisions will likely require further experimentation.1) The identification of hERG as the key target of ACh is not air-tight. Experiments with bath applied ACh or agonists have shown previously that muscarinic stimulation activates calcium-dependent non-specific cation conductances as well as blocking M-channels. Here the hERG current in these cells is not isolated in voltage-clamp recordings that could support the conclusion that hERG is present in these cells and acting late to modulate spike-frequency adaptation . It will be critical for the authors to demonstrate that synaptically released ACh specifically blocks the hErg mediated current in voltage-clamp, providing direct evidence for (1) the modulation of the hErg current by ACh and (2) the time course of ACh-induced block of Erg currents, which currently the authors only indirectly inferred.We conducted three additional sets of experiments to address this point. First, we tested two additional ERG blockers (E-4031 and ErgToxin) using the same current-clamp protocol presented in the R0 version. Both E-4031 and ErgToxin1 blocked persistent firing, as we reported previously for terfenadine. While terfenadine has actions on other targets besides ERG, ErgToxin1 and E-4031 are highly specific for ERG channels. These current-clamp experiments also set up the new voltage-clamp studies employing E-4031 that the reviewers requested. We also used this battery of three ERG blockers in our previous JN paper on ERG modulation of persistent firing by bath carbachol and used terfenadine to determine ERG current kinetics and modulation by ACh under voltage-clamp conditions.Second, we performed voltage-clamp experiments to define the properties of K current modulated by ChR2 stimulation . These recordings demonstrated that ACh release reduced an outward current with very slow kinetics that appeared to initiate after ~0.5 seconds of depolarization. Both the late onset and slow kinetics of the ACh-sensitive current fit well with properties we inferred from our analysis of how spike-frequency adaptation changed with ChR2 stimulated ACh release and, in separate experiments, when ERG current was attenuated by terfenadine. We also show that the ERG blocker suggested by the reviewers, E-4031, greatly reduced the ACh-sensitive slow outward current, providing additional evidence supporting our hypothesis that ERG is a target of ACh modulation.Finally, we conducted additional current-clamp experiments testing whether blockade of other K currents could interfere with persistent firing. Consistent with our previous results, we now report that SK attenuation  could not block persistent firing triggered by coincident ACh release and intracellular depolarization. Together, these studies provide additional evidence for ERG as a target of ACh modulation and also define time course of the underlying outward current, as requested by the reviewers.2) Terfenedine may not be the best choice as an identifier of hERG currents as its primary target is H1 receptors. The use of the more specific compound, E4031, is a useful addition, but some of these experiments have an unacceptably low n . The n's must be increased for this essential experiment.As outlined in response #1 above, we conducted additional experiments using E-4031, the antagonist suggested by the reviewers, as well as the ERG-specific peptide blocker ErgToxin1 that we used in previous paper on ERG in JN. We also clarified in the R1 text that we had previously tested fexofandine, another H1 receptor blocker that is structurally similar to terfenadine but does not affect ERG channels. We reported in our JN paper last year that it did not affect persistent activity even when used at 3x times the concentration used in our terfenadine experiments. We also increased the Ns of the SK blocker experiment that was flagged to 3 3) In some figures, data from experiments with reasonable n values are presented only as bar graphs. The preferred presentation is to show a full scatter plot of all of the data because it's useful for readers to see individual data points to understand variability more clearly than standard error alone.We have reformatted most of the figure panels that were bar graphs to include raw data points. In most of the light/no-light comparisons we have also included connecting lines to allow the reader to see corresponding data points in each experiment. We also retained some bar graphs without including individual data points when the means represented a very large number of trials or when the goal of the illustration was to illustrate how similar multiple mean values were rather than to make a claim about statistically significant differences between means.+ conductance ("linear K") to get a time-dependent SFA effect proves anything; and (d) why adding stochastic noise directly to the membrane potential is better than adding stochastic noise to the *conductances* – since real background fluctuations in vivo are mediated by conductance fluctuations.4) The modeling adds only modestly to the present work. Its contribution would be greater if the authors could explain: (a) why a one-compartment model of ventral cochlear nucleus neurons helps us understand layer 5 pyramidal neurons with elaborate dendrites; (b) how a model that does not contain BK, SK, or M channels deepens our understanding of SFA; (c) in what way putting in by hand a time-dependent KThe modeling should be strengthened considerably or simply eliminated.We included the modelings results in the original manuscript for two different reasons. First, to provide easily understood reference data sets to illustrate the analysis methods we developed to define modulation in spike-frequency adaptation (the cumulative spike count panels and the slope methods for estimated the kinetics of the ACh-modulated current). We prefer to retain these panels in the main illustrations as they provide helpful illustration of the analysis work flow that would be difficult to replace using real biological data. The second application of the model was to demonstrate that we could replicate the network ensemble changes that we find occur with ACh release. We agree with the reviewers that the unusual nature of the model implementation we used  limits the additional impact of these simulations without including more work supporting this approach. We therefore moved the network simulation results that were in Figure 8G,H to Figure 10—figure supplement 1 and deemphasized the conclusions from modeling work.


We investigated whether short term high fructose intake may induce early hepatic dysfunction in rats and to test whether allopurinol treatment may have beneficial effects. Twenty male Sprague-Dawley rats received 20% fructose in drinking water  and 10 control rats received tap water. After 14 days, the hepatic response to an acute fructose load was evaluated, and in fasted animals, respirometry studies in freshly isolated mitochondria were performed. In fasting rats, we did not find differences in systemic or hepatic uric acid and triglyceride concentrations among the groups, but mitochondrial respiratory control rate was significantly decreased by high fructose feeding and correlated with a reduced expression of Complex I, as well as decreased aconitase-2 activity. On the other hand, in fructose fed rats, an acute fructose load increased systemic and hepatic uric acid, triglycerides and oxidative stress. Fructose feeding was also associated with fructokinase and xanthine oxidase overexpression and increased liver de novo lipogenesis program (fatty acid synthase (FAS) and cell death-inducing DFFA-like effector C (CIDEC) overexpression, ATP citrate lyase (ACL) and acetyl coA carboxylase (ACC) overactivity and decreased AMP-activated protein kinase (AMPk) and endothelial nitric oxide synthase (eNOS) activation). Allopurinol treatment prevented hepatic and systemic alterations. These data suggest that early treatment with xanthine oxidase inhibitors might provide a therapeutic advantage by delaying or even halting the progression of non-alcoholic fatty liver disease (NAFLD). The role of high fructose intake (HFI) is well recognized in the development of lipids abnormalities and metabolic syndrome in laboratory animals and in humans . A well-Hepatic steatosis is the earliest manifestation of metabolic syndrome in the liver, and it is followed by the development of non-alcoholic steatohepatitis  In additn = 5) were used to evaluate the response of an acute fructose load by the liver (2 g/kg/BW). To test the effects of high glucose intake and its liver handling after and acute load 4 rats were given glucose 20% for 14 days and received an acute load of glucose (2 g/kg/BW). In the remaining fasted animals (n = 5 each group), liver tissue was excised to perform respirometry studies in freshly isolated mitochondria and to measure the intrahepatic and plasma concentrations of uric acid and triglycerides.All the studies were approved by the Internal Animal Care and Use Committee (Permit No INC/CICUAL/PIL/002/2019) and were conducted in agreement with the National Institutes of Health guide for the care and use of laboratory animals . We studied male Sprague-Dawley rats obtained from the Instituto Nacional de Cardiología Ignacio Chávez vivarium. Rats  were allocated in individual acrylic cages in the following groups: 10 rats received 20% fructose in drinking water plus vehicle (water) by gavage (F20% group), 10 rats received 20% fructose in drinking water plus allopurinol (5 mg/day in 1 mL of water) by gavage (F20% + AP group), and 10 rats received tap water and were included as controls. All groups were followed for 14 days. The dose of fructose was chosen as it has been reported that the highest consumers of added sugars in the USA had a fructose intake of 20–25% of caloric intake  and protein content was determined by the Bradford method. Thirty micrograms of protein were loaded per lane for SDS/PAGE (10% w/v) separation and then transferred to PVDF membranes . Membranes were incubated overnight at 4 °C, with the following primary antibodies: OXPHOS , Aconitase-2  Bax  Bcl2  ATP citrate lyase  phospho T447 + S451 ATP citrate lyase , Acetyl CoA carboxilase , Fatty acid synthase  KHK  Xanthine oxidase  CIDEC  SREBP1 , PCNA  AMPKα  phosphor AMPKα ,1:2000 dilution. Danvers, MA, USA) eNOS  phosphor Ser1177 eNOS  and visualized by using a horseradish peroxidase (HRP) secondary antibody  and the ECL Clarity . Immunoblots were analyzed with Image Studio Lite 5.2 software .Soluble fraction of hepatic tissue was extracted for 30 min on ice bath in MAP kinase buffer [in mmol/l 25 HEPES , 150 NaCl , 4 EDTA, 25 NaF , and 1 Nap < 0.05. Post hoc analysis was performed using the Bonferroni test.The data were analyzed using Prism 8 . Results are presented as mean ± SD and were analyzed by one-way ANOVA. The significance was set at Water intake was 35 ± 2.8 mL/d in normal rats. Fructose in drinking water increased significantly the mean water intake to 58 ± 5 mL/d in F20% and 53 ± 10 mL/d in F20% + AP groups . In bothAs there were no changes in systemic neither in hepatic uric acid and triglycerides concentrations after 14 days of increased fructose intake, we evaluated the effect of an acute oral dose of fructose in fasted rats. After 2.5 h of fructose load, we observed a sharp increment in ADP/ATP ratio caused by an ATP decrement and ADP increment in the liver of rats that received 20% fructose beverage during 2 weeks A. ATP deTo study whether there were mitochondrial changes induced by fructose intake, we evaluated the whole respiratory chain in isolated mitochondria of fasted rats. Oxygen consumption was stimulated by feeding Complex 1 with malate/glutamate and Complex 2 with succinate/rotenone . SignifiAs Bcl2 and Bax proteins play important roles in regulating mitochondrial function and its ratio is associated with caspase-independent apoptosis, we evaluated their expression. A twenty percent fructose beverage increased the Bax/Bcl2 ratio, and allopurinol precluded this effect C.Fructose intake has been associated with the overexpression of KHK (30). In this study, we tested whether fructose intake can also upregulate XO expression. Fructose induced the overexpression of both enzymes; allopurinol concomitant treatment partially prevented this effect A,B.To assess the lipogenic response to an acute load of oral fructose, we evaluated the following enzymes and proteins implicated in the de novo hepatic lipogenesis p-ATP citrate lyase/ATP citrate lyase ratio (p-ACL/ACL acetyl CoA carboxylase (ACC), fatty acid synthase (FAS), cell death-inducing dFF45-like effector C (CIDEC) and the nuclear translocation of the sterol regulatory element-binding protein 1 (SREBP1c) . FructosActivation of the AMPk has been demonstrated to attenuate liver steatosis, therefore we evaluated the effect of allopurinol in this parameter. High fructose feeding reduced the activation of AMPk, noted by a reduction in phospho (p)AMPk/AMPk ratio compared to control. Allopurinol rescued the activation of AMPk A. FructoHigh fructose diets, especially when the fructose is given in liquids, are well-known to cause obesity and metabolic syndrome, but typically it takes several months. Indeed, short-term feeding of fructose (< 4 weeks) is often associated with minimal metabolic findings, leading some investigators to propose that fructose is benign . HoweverOne potential explanation is the fact that normally the gut has low expression of Glut5, the fructose transporter, and with exposure to fructose, these transporters can be induced ,27. FrucHere, we evaluated the effect of fructose on metabolic parameters in rats that had been ‘primed’ with two weeks of fructose provided in the water. During these two weeks, the fructose fed fats showed no difference in a variety of metabolic parameters  compared to control rats. Nevertheless, when rats were exposed to an acute load of fructose, they showed dramatic changes in metabolism driven by alterations in ATP and mitochondrial function, and of interest, most of these mechanisms were mediated in part by the generation of uric acid in the nucleotide degradation pathway, as shown by the effects of allopurinol.Fructose given as a 20% solution in drinking water did not induce an increment in body weight in this short-term study (14 days) compared to control rats receiving tap water. Nevertheless, allopurinol treatment induced a mild but significant weight loss despite equivalent food and water intake compared to F20% of animals. Allopurinol has been rarely associated with acute liver injury, mainly in older patients with concomitant kidney damage, as kidney is more sensitive to allopurinol toxic effects. Thus, we believe that the weight loss observed in F20%+Allopurinol rats was not related to renal toxicity by allopurinol since proteinuria and creatinine clearance were comparable among the studied groups, suggesting that allopurinol was well tolerated. Thus, the effect of allopurinol might be related to lower feed efficiency, e.g., less weight gain per calorie ingested. Such an allopurinol effect on body weight has also been observed in overweight humans on a high fructose diet .Previous studies have shown that the hepatic alterations induced by fructose may take eight weeks or longer to develop in rodents depending on the dose . TherefoSince hepatic mitochondrial dysfunction is one of the earliest manifestations of liver steatosis, fructose as well as uric acid is associated with mitochondrial oxidative stress , we measPreviously it was shown that high fructose diet induces apoptosis of the hepatic tissue , we confOne of the prolipogenic effects of fructose is related to its ability to stimulate the activity and expression of the enzymes associated with fatty acid synthesis . In thisAdditional protective effects of allopurinol were that maintained the activation state of AMPk and eNOS, as inactivation of both enzymes has been associated with liver steatosis ,39. In cIn summary, short-term fructose exposure induces early manifestations of systemic and hepatic alterations only noticed when an acute fructose load is administered and ratify the importance of increased uric acid synthesis secondary to fructose metabolism by fructokinase in these deleterious effects. These data also suggest that an early treatment with xanthine oxidase inhibitors might provide therapeutic advantage by delaying or even halting the progression of non-alcoholic fatty liver disease.


Genetic susceptibility is likely involved in nasopharyngeal carcinoma (NPC), a cancer caused by Epstein-Barr virus (EBV) infection. Understanding of genetic factors involved in NPC and how they contribute to EBV-induced carcinogenesis is limited. We conducted whole-exome capture/sequencing among 251 individuals from 97 multiplex families from Taiwan  using SeqCap EZ Human Exome Library v3.0 and Illumina HiSeq. Aligned sequences were filtered to identify likely-to-be-functional deleterious variants that co-segregated with disease. Ingenuity Pathway analysis was performed. Circulating magnesium levels were measured in 13 individuals in 2 families with NIPAL1 mutations and in 197 sporadic NPC cases and 237 controls. We identified variants in 12 genes likely involved in cancer pathogenesis, viral infection or immune responses to infection. These included genes postulated to be involved in magnesium transport (NIPAL1), EBV cell entry (ITGB6), modulation of EBV infection , telomere biology , modulation of cAMP signaling (RAPGEF3), DNA repair , and Notch signaling . Pathway based analysis demonstrated enrichment for Notch signaling genes . Evaluation of individuals within NIPAL1 families suggested lower serum magnesium in NPC compared to unaffected members. A significant reduction in serum magnesium levels was observed among sporadic NPC cases compared to controls  and is consistent with findings demonstrating a role for magnesium channeling in T-cell responses to EBV. We identified novel genes associated with NPC that point to new areas of inquiry to better understand genetic factors that determine the fate of viral infections and/or otherwise predisposes to NPC. An intriguing feature of NPC is that while the infection that causes this cancer (EBV) is nearly ubiquitous worldwide (>90% adults infected) and a necessary cause for undifferentiated forms of NPC that account for over 90% of all NPC cases in high-risk regions, the cancer itself occurs much less frequently and with a geographic distribution that is strikingly uneven1. This suggests that factors other than EBV are important determinants of which individuals develop NPC. An important role for host genetic susceptibility is suggested by (1) the strong predilection of this disease for individuals of particular ethnicities , (2) migrant studies showing residual elevated incidence in second and third generation migrants from high-risk to low-risk regions, (3) observed familial clustering of NPC in endemic regions, and (4) epidemiological studies showing that a family history of NPC is associated with a 4-8 fold increased risk of disease1. The study of NPC therefore provides a unique opportunity to elucidate the interplay between oncogenic infections and host genetics in cancer etiology.Nasopharyngeal carcinoma (NPC) is an epithelial tumor caused by Epstein-Barr virus (EBV), an infection that typically occurs in childhood or adolescence and persists lifelong3. These results support decades of research demonstrating a role for HLA genes (genes located within the MHC region) and NPC, likely via differential ability of distinct HLA alleles to effectively trigger an immune response targeting EBV infection via presentation of viral antigens to cytotoxic T-cells5. However, while findings regarding the association between HLA genes and NPC have been strong and consistent, polymorphisms in HLA genes only explain a minority of NPC cases and other genetic factors are therefore likely to be involved. These additional host genetic susceptibility factors, however, have remained largely elusive.Genome-wide association studies (GWAS) of sporadic NPC have reproducibly highlighted the link between this cancer invariably caused by EBV and variation in the MHC region on chromosome 66. In addition to studies of sporadic NPC, studies of familial NPC have identified chromosomal regions linked to NPC development, but findings have differed across studies and the genes involved remain unknown7. Recent whole exome sequencing (WES) studies of NPC have identified additional genes that might be important in NPC development, including KMT2C and MST1R9. Taken together, these findings highlight the complex nature of genetic susceptibility factors linked to NPC development.One meta-analysis of four NPC GWAS followed by a replication effort recently reported a significant modest (~20% increase in risk) association between SNPs located within the CLPTM1L/TERT, MECOM, TNFRSF19, and CDKN2A/B gene regions and NPCTo further understand genetic susceptibility factors involved in NPC development and how inherited genetic factors might contribute to NPC, we conducted a large-scale NPC multiplex family study followed by whole exome sequencing (WES). WES was performed on 251 individuals from 97 informative NPC multiplex families. We report the identification of novel inherited genetic variants that segregate with NPC within multiplex families. Some variants potentially operate via biological pathways that might impact EBV infectivity and/or immune control or are modulated by oncogenic EBV infection. Other novel variants involve genes in telomere maintenance and DNA repair, confirming the likely role of these biological pathways in NPC development.Results from 251 individuals from 97 NPC families were included in this analysis. NPC cases were 43.1 years (range: 18–80) at diagnosis, on average. Obligate carriers and unaffected family members were, on average, 68.0 (range: 47–91) and 67.6 (range: 53–87) years, respectively. 69.8% of cases were male. The corresponding percentage for obligate carriers and unaffected family members was 52.4% and 36.0%.We identified potential rare variants in 608 genes and present these genes in Supplemental Table To evaluate all candidate genes identified, pathway based analysis of genes included in Supplemental Table 10.When the cBioPortal and Cosmic public databases were queried, we identified evidence for alterations in NOTCH1 (one truncating and one missense mutations) in 4% of the 56 tumors evaluated in a study conducted in Singaporeet al.8 and 56 NPC germline/normal samples from Lin et al.10. Overall, we observed three variants from Table et al. study. This included one NPC case with a missense variant in PSEN2 (p.His169Asn) and a second NPC case with the missense variant in CLPTM1L (p.Arg510Trp); this latter variant was observed only once in gnomAD with a frequency of 0.00003. Finally, we observed the missense variant in BRD2 (p.Glu762Asp) in one NPC case from the study of Dai et al.8.We evaluated the 20 germline variants in Table NPC families with at least two NPC cases/obligate carriers sequenced were examined for rare variants in two postulated NPC susceptibility genes KMT2C and MST1R. Supplemental Table 11. We therefore explored our NIPAL1 findings further by testing family members within the 2 families with NIPAL1 variants for total serum magnesium levels, and by evaluating the association between total serum magnesium levels and sporadic NPC using routinely available pre-treatment magnesium testing results from NPC cases and non-cases attending the ENT clinic at the National Taiwan University Hospital. We observed reduced, albeit not statistically significant, serum magnesium levels among the 6 NPC cases from our families with NIPAL1 variants compared to 7 unaffected individuals from these same families  , magnesium transport (NIPAL1), EBV entry into epithelial cells (ITGB6), modulation of EBV , telomere biology , modulation of cAMP signaling (RAPGEF3), or DNA repair . Pathway based analysis provided support for a role in NPC pathogenesis for additional genes in the Notch signaling pathway. Finally, direct testing for circulating magnesium levels in individuals within our families and sporadic NPC cases and controls provided support for the role of magnesium transport genes such as NIPAL1 in NPC. These findings add to the known list of candidate genetic susceptibility factors linked to familial NPC that previously included polymorphisms in HLA and selected other genes10. Notch signaling is an evolutionarily conserved pathway known to play an important role in cell differentiation, proliferation and apoptosis17. Extensive work has linked Notch and Notch signaling genes to both hematopoietic and solid tumors17. Of note, EBV, the virus linked to NPC development, encodes a functional homologue of intracellular notch (EBNA2) that has been shown to act as a constitutively activated form of Notch20. EBNA2 is known to be critical in initial EBV infection and necessary for EBV-transformation of infected B cells, suggesting an important role for modulation of Notch signaling in establishment of the lifelong viral infections linked to subsequent NPC development19. Our results suggest that inherited changes in Notch and other genes in the Notch pathway may predispose to the development of NPC and one might speculate that such a link is due to modulation of its viral cause, EBV infection.We identified rare variants potentially linked to NPC within 6 genes involved in Notch signaling, including variants in NOTCH1 and in an extracellular Notch ligand, DLL3. This is consistent with a report from a previous study of 56 sporadic NPC cases in which alterations in NOTCH1 were noted for 4% of cases evaluated21. We did not observe evidence for rare mutations in MAGT1 in the NPC families included in our study. Taken together, these findings suggest that genes that regulate magnesium transport across cells might be important determinants of NPC risk and one potential explanation for such a link is that these genes are important determinants of a host’s ability (or lack thereof) to mount an effective immune response against EBV, the virus linked to NPC.We also identified rare variants potentially linked to NPC within a magnesium transport gene, NIPAL1. We further showed evidence suggesting that circulating magnesium levels are reduced in individuals with NPC, both in the context of NPC multiplex families who carry NIPAL1 variants and of sporadic NPC. While our finding of reduced circulating magnesium levels in sporadic NPC cases compared to controls does not directly implicate NIPAL1, our findings take on added significance when considered in light of the recent discovery that null mutations in another magnesium transport gene, MAGT1, which abrogates magnesium flux in NK and CD8 T cells, predisposes to uncontrolled EBV infection and EBV-associated lymphomas22. In the context of EBV and NPC pathogenesis, integrins are of relevance because studies suggest that they are important for EBV anchoring/fusion to epithelial cells, thus facilitating its entry into and infection of epithelial cells24. Given the role of integrins in EBV infection of epithelial cells, variants in genes that code for these proteins could modulate efficiency of viral-cell interactions that lead to infection of epithelial cells and yield epithelial cells exposed to EBV virions (such as those at the nasopharynx) either more or less susceptible to EBV infection and therefore more or less susceptible to the development of EBV-associated epithelial cancers such as NPC. Future studies aimed at understanding the effect of variants in ITGB6 and other integrin-coding genes on EBV infectivity could shed light on the specific mechanism(s) that explain our current findings.Rare variants within the ITGB6 gene that codes for an integrin were also identified in our NPC families. Integrins are transmembrane proteins expressed in epithelial cells that are important for cell-to-cell and cell–to–extracellular matrix communications28. Interestingly, LMP1 has been shown to be expressed frequently in NPC tumors29.Additional genes coding for protein with which EBV interacts and that we found to contain rare variants that segregate with disease in our NPC families include BCL2L12 and NEDD4L. These genes code for protein products that are known to interact with LMP1, an EBV protein that functions as a viral oncogene via interactions with BCL26. Our findings therefore support a role for telomere maintenance and its regulation and DNA repair in NPC pathogenesis.We also identified rare variants within genes that have previously been shown to be involved in processes important for tumorigenesis (independent of EBV infection). These include genes involved in DNA repair  and telomere biology . With respect to the latter, it is noteworthy that a recent meta-analysis of NPC GWAS and replication study implicated polymorphisms in CLPTM1L/TERT region with NPC9. Although not definitive, KMT2C and MST1R were proposed as candidate susceptibility genes for NPC. We, therefore, reviewed the families in the current study with at least two NPC cases/obligate carriers sequenced for rare variants in these two candidate genes. Although multiple rare variants were observed, only one variant in MST1R fully co-segregated with NPC in a family with >2 NPC cases/obligate carriers sequenced. This observed missense variant produced a protein change (p.G356D) that was not predicted to be deleterious in any in silico program reviewed including SIFT, PolyPhen, FATHMM, Mutation Taster, Mutation Assessor, Provean, LRT, or CADD. In addition, this variant was observed in East Asian non-cancer subjects from ExAC with a relatively high frequency of 0.0017 suggesting that it is likely not disease-related. The MST1R variant reported by Dai et al.8 that was most strongly associated with NPC (c.G917A:p.R306H) was not observed in any of the 97 NPC families in the current study. In summary, there was little additional supporting evidence for either proposed NPC candidate gene.After completion of our analyses, two WES studies of NPC cases and controls were identified based on published worket al.8 and 56 NPC germline/normal samples from Lin et al.10 and evaluated the 20 germline variants reported in Table We searched for publicly available NPC WES datasets to further investigate variants identified in the multiplex NPC families. We conducted variant calling (see methods) for two WES datasets that included 39 early-age-of-onset NPC samples from Dai Our study is not without limitations. One limitation of exome sequencing studies such as ours, is the limited ability to define precise mechanisms for our findings. While our findings, particularly those for genes involved in EBV infection, response and life cycle are suggestive, future laboratory studies will be required to further elucidate their biological functions and relationship to tumorigenesis for EBV-associated cancers. Another limitation of our work is the fact that all variants identified occurred in a small number of families, only a subset of which were observed in all cases/obligate carriers within the families, suggesting that they are unlikely to explain a large fraction of NPC. Nonetheless, they point to important genes potentially involved in NPC development that might suggest avenues for future investigation aimed at identifying additional, more common changes (genetic or epigenetic) in these genes/pathways that might explain a larger fraction of NPC cases. Finally, by design, we targeted the identification of rare genetic alterations with a dominant mode of inheritance. Therefore, alterations that operate in a non-dominant fashion  might have been missed by our study.Strengths of our study include the fact that it is embedded within the largest study of NPC multiplex families conducted to date, that a sizeable number of individuals were sequenced within these families, and that the sampling frame for identifying our multiplex families relied on a long standing nationwide cancer registry maximizing the representativeness of the NPC multiplex families evaluated. Our ability to follow-up findings for NIPAL1 by demonstrating reductions in magnesium levels in familial and sporadic NPC is also a strength of our work.Results from this study implicate variation in numerous genes involved in Notch signaling, magnesium transport, EBV entry into epithelial cells, modulation of EBV infection, telomere biology, and DNA repair in the development of NPC. Our findings with respect to magnesium transport genes, which may be important for activation of T-cells in response to EBV infection, are of particular note, given our independent finding of reduced magnesium levels among NPC cases. Our findings pave the way for future work to better understand genetic factors that are important in response to viral infections and/or otherwise predispose to virus-associated cancers.33. Briefly, 20,450 NPC cases diagnosed between 1980 and 2003 were identified through the National Cancer Registry, 10 tertiary care hospitals and select outpatient clinics that treat NPC. As in other NPC endemic areas, over 90% of these cases are expected to be of histological types linked to EBV infection1. We successfully screened 10,178 (49.8%) cases for a family history of NPC. Ultimately, 358 NPC multiplex families were identified and recruited into our study. From these families, a total of 3,216 individuals  were included in the parent study, consisting of NPC cases, their parents, up to 5 siblings, and for deceased NPC cases, their spouse and up to 3 children. When it was necessary to link affected individuals in our families, additional relatives were recruited. Blood (in acid citrate dextrose buffer vacutainers) from which DNA was extracted using a salt precipitation method was obtained from participants and stored at −80 °C. Subjects provided informed consent and the protocol was reviewed/approved by human subjects review committees in Taiwan  and the United States . All research was performed in accordance with relevant guidelines and regulations.Participants were part of the previously described cancer registry based NPC Multiplex Family Study in TaiwanWe performed WES on 97 families deemed to be most informative for study by virtue of having 3 + NPC cases (N = 35) or 2 affected individuals where at least 2 NPC cases or obligate carriers had biological material available (N = 62). In total, WES was performed on 205 affected individuals, 21 obligate carriers , and 25 unaffected family members (21 blood relatives and 4 relatives through marriage) . Pools of captured DNA underwent paired-end sequencing using an Illumina HiSeq according to Illumina-provided protocols for 2 × 100-bp paired-end sequencing. Each exome was sequenced to high depth to achieve a minimum threshold of 80% of coding sequence covered by at least 15 reads, based on the UCSC hg19 ‘known gene’ transcripts. The exome data analyzed in this paper are archived in the CGR exome build Ensemble_New_Annotation dated 2014-01-09.Whole-exome capture/sequencing was performed at the NCI Cancer Genomics Research Laboratory (CGR) as previously described36. Briefly, sequencing reads were trimmed by Trimmomatic (v0.32)37 to remove short read pairs with at least one end <36 bp. Reads were aligned to the hg19 reference genome using Novoalign software (v3.00.05) (http://www.novocraft.com). The alignments were refined by removing duplicate reads in Picard software, MarkDuplicate module (v1.126) (http://picard.sourceforge.net/), short fragments (<200 bp), and sequence pairs not in complementary direction when mapped to reference. Sites with indels were realigned locally by Genome Analysis Toolkit38 (GATK v3.1), the RealignerTargetCreator and IndelRealigner modules.Details of the bioinformatic pipeline for variant alignment/calling have been publishedhttp://bcb.io/2013/02/06/an-automated-ensemble-method-for-combining-and-evaluating-genomic-variants-from-multiple-callers/) was used to integrate the results from the three callers. Support Vector Machine learning algorithm was used to identify an optimal decision boundary to produce a more balanced decision between false and true positives.Variant discovery and genotype calling were performed globally among all subjects using UnifiedGenotyper and HaplotypeCaller modules from GATK and the FreeBayes variant caller (v9.9.2). An Ensemble variant calling pipeline (v0.2.2 http://evs.gs.washington.edu/EVS/), dbNSFP39: database of human nonsynonymous SNPs and function predictions (https://sites.google.com/site/jpopgen/dbNSFP), Combined Annotation Dependent Depletion (CADD) scores (cadd.gs.washington.edu), the Molecular Signatures Database (MSigDB) (http://www.broadinstitute.org/gsea/msigdb/index.jsp), the National Center for Biotechnology Information (NCBI) Clinically Relevant Sequence Variations (ClinVar) and Single Nucleotide Polymorphism database (dbSNP) databases40 build 137, the 1000 Genomes Project41, the Exome Aggregation Consortium (ExAC) database (http://exac.broadinstitute.org/), and the Human Gene Mutation Database (HGMD)42.Variants were annotated via a custom CGR in-house script based on public data including Ensembl, refGene, and UCSC KnownGene databases, the dataset from University of Washington’s Exome Sequencing Project (ESP6500)  variants that had depth of <10 reads or that were in segmentally duplicated regions; 2) variants observed at a frequency >0.5% in the overall or East Asian populations in the 1000 Genomes Project, the NHLBI ESP6500 database, and ExAC database; (3) variants that were observed in >3 families in non-NPC families from our in-house NCI DCEG database of >2,000 sequenced individuals or in >50 individuals in our NPC population (based on CGR exome build Ensemble_New_Annotation dated 2014-01-09). We further applied a dominant model requiring variants to be present in all affected individuals and obligate carriers within a family to reduce the impact of phenocopies or sequencing errors. More specifically, we required that variants be observed (1) in at least 1 family with either three or more NPC cases or two NPC cases and 1 obligate carrier or (2) in at least 1 family for families with two or more NPC cases sequenced that were not siblings or parent-child combinations and for whom at least 1 unaffected individual was sequenced or (3) in at least 2 families for families with two or more NPC cases sequenced that were siblings or parent-child combinations. Within the set of identified variants, we restricted our evaluation to variants most likely to be functional by (1) excluding variants that resulted in synonymous changes , and (2) requiring that the variant was predicted to be deleterious by Combined Annotation Dependent Depletion (CADD) analysis (defined as CADD score of 15 or greater). At least one variant from each of 608 candidate genes were identified using the criteria above. These genes are listed in Supplemental Table  variants variantshttps://www.qiagenbioinformatics.com/products/ingenuity-pathway-analysis/) for the candidate genes in Supplemental Table As a second (unbiased) method for prioritizing candidate genes, we conducted a pathway analysis. Ingenuity Pathway analysis (IPA) was performed  and reports the longest high-quality stretch of each read. Only read pairs with both ends no shorter than 36 bp were used. Reads were then aligned to the hg19 reference genome using the Novoalign software (v3.00.05) (http://www.novocraft.com). Duplicate reads due to either optical or PCR artifacts were removed from further analysis using the MarkDuplicates module of the Picard software (v1.126) (http://picard.sourceforge.net/). Additionally, the analysis was restricted to only properly aligned read pairs, in the sense that the two ends of each pair must be mapped to the reference genome in complementary directions and must reflect a reasonable fragment length (300+/−100 bp). These high-quality alignments for each sample were further refined according to a local realignment strategy around known and novel sites of insertion and deletion polymorphisms using the RealignerTargetCreator and IndelRealigner modules from the Genome Analysis Toolkit (GATK v3.8.1). After two batches of BAMs were generated, SNPs and small indels joint calling on the loci presented in Table The FASTQ files of 39 early-age onset NPC and 56 NPC germline samples used by two previous studies Lin 9. Although not definitive, KMT2C and MST1R were proposed as candidate susceptibility genes for NPC. We therefore examined the Taiwanese NPC families with at least two NPC cases/obligate carriers sequenced for rare deleterious variants in these two genes to evaluate evidence for co-segregation of rare variants.To evaluate whether previously published and publicly available data on somatic alterations in NPC provide support for the primary findings reported herein, we queried the cBioPortal  and Cosmic (cancer.sanger.ac.uk/cosmic) databases to determine the extent to which alterations in the genes reported herein were noted in past studies. Also, after completion of the current analyses, two WES studies of NPC cases and controls were identified based on published work43. To independently examine the possible association between magnesium levels and sporadic NPC, we also evaluated routinely collected clinical magnesium testing results from patients in the ENT clinic at the National Taiwan University Hospital between 07/2010 and 01/2015 and compared the proportion of individuals with total serum magnesium deficiency (defined as <0.7 mmol/L or <0.78 mmol/L depending on instrumentation used) among incident NPC cases  and non-NPC controls (N = 237) tested before treatment. Diagnosis of non-NPC controls included diseases of the salivary gland (N = 154), thyroid conditions (N = 31), benign neoplasms of connective and other soft tissue (N = 13), and assorted other conditions (N = 39).Serum (500 ul) from 13 individuals  from two families with a rare NIPAL1 variant were tested for total serum magnesium  using an inductively coupled plasma atomic emission spectrometry assay as previously describedSupplemental Tables 1–4


This study aimed to develop a method to assess experienced quality of care (QoC) in nursing homes from the resident’s perspective. A narrative approach “Facilitating Care” (FC) was developed based on the INDEXQUAL framework of experienced QoC and a needs assessment. FC assesses experienced QoC by training care professionals to perform individual conversations with residents, their family and their professional caregivers (triads) in another organization than where they are employed. FC consists of three phases: 1) training, 2) data collection and registration, and 3) analysis and reporting of the results. In 2018, 16 care professionals were trained and performed 148 conversations  in 8 different nursing homes. Evaluation showed that FC teaches helpful conversation techniques and provides valuable insights into residents’ experienced QoC. Whilst the process was considered time consuming, all participants emphasized the added value of taking time for FC conversations.


RAS proteins are small GTPases that regulate signalling networks that control cellular proliferation and survival. They are frequently mutated in cancer and a commonly occurring group of developmental disorders called RASopathies. We discuss recent findings describing how RAS isoforms and different activating mutations differentially contribute to normal and disease-associated biology and the mechanisms that have been proposed to underpin this. RAS proteins are small GTPases that act as molecular switches controlling signalling pathways regulating cell proliferation and cell survival. When bound to guanosine triphosphate (GTP), RAS becomes active and signals via networks that include RAF/MEK/ERK and PI3K/AKT. Dysregulated RAS signalling is associated with oncogenesis and developmental disorders called RASopathies . ApproxiHRAS, NRAS and KRAS, the transcript from the latter can be alternatively spliced into KRAS4A and KRAS4B. They share almost complete sequence homology (90%) in their G-domain  and HRAS (9%) isoforms are less frequently mutated with melanomas accounting for half of NRAS mutant patients, while head and neck cancer and bladder cancer together account for half of HRAS mutant patients. Given that farnesyltransferase inhibitors (FTIs) only target HRAS effectively , FTIs hain vitro studies have shown that RAS isoforms appear to be differentially coupled to key effector pathways. Specifically, KRAS is a more efficient activator of RAF1 and RAC than other RAS isoforms . The. The8]. nd state .The allosteric lobe also transmits nucleotide-dependent changes in the effector lobe to specify how the G-domain and HVR interact and orientate with the membrane. Basic residues in the allosteric lobe electrostatically interact with plasma membrane phospholipids. When HRAS is GDP-bound this is mediated by R169 and K170, and when GTP binding occurs this switches to R128 and R135 . The effAs the primary mediator of membrane attachment, the HVR has a significant influence on isoform-specific RAS behaviour. All RAS isoforms are C-terminally farnesylated to provide weak membrane binding that is enhanced by a second signal comprising either palmitoylation for HRAS, NRAS and KRAS4A and/or a stretch of polylysine residues for KRAS4A and KRAS4B  10]. Th. Th10]. KRAS nanoclustering is principally driven via electrostatic interactions with anionic lipids. Phosphatidic acid (PA) and phosphatidylserine (PS), but not phosphatidylinositol-4,5-bisphosphate (PIP2), are enriched in KRAS nanoclusters ,44. DeplAnother feature of RAS organisation on the membrane that has emerged recently is that activated RAS isoforms adopt a putative dimeric state. While biophysical studies using model membranes and fully processed KRAS4B failed to detect dimerisation , cell-ba97% of Ras mutations occur at codons 12, 13 and 61, whereby single base substitutions can convert each codon into six different amino acids –53. BiocIn vivo studies point to the different oncogenic potential of individual mutations. Human melanomas are associated with codon 61 mutants, and NRASQ61R is more efficient at promoting melanoma than NRASG12D in mice [in vivo study showed that NRASQ61R/+ mice mutants displayed higher leukaemogenic activity than NRASG12D/+ [Consistent with the notion that different mutations specify differences in RAS biology, cell-based studies have shown that different signalling outputs and phenotypic responses are associated with KRAS codon 12 versus codon 13 mutations –59. For  in mice . SimilarG12V/+, LSL-KRASG12V/+, LSL-KRASG12D/+, LSL-NRASG12D/+, LSL-NRASQ61R/+) precluding comparisons of most RAS variants in a common model system. To overcome this, Winters et al. [Progress in this area has been limited by the availability of only a few genetically engineered mouse strains (Lox-Stop-Lox-(LSL-)HRASs et al.  developeIn vitro, in vivo and clinical approaches have contributed to a deep understanding of RAS biology and an appreciation of the evident complexity of the network biology and the heterogeneity of responses. Despite this, it remains difficult to pin down the answers to fundamental questions such as what are the relative contributions of the RAS isoforms to RAS biology, what makes KRAS more oncogenic than the other isoforms and how does wild-type RAS modulate oncogenic RAS signalling? While the answers are likely to be context-dependent, it is important that consensus is established around the selection of robust models and analysis pipelines that should be used in order to identify the most therapeutically tractable insights.