The incorporation of tendon graft into bone tunnel is one of the most challenging clinical issues in anterior cruciate ligament (ACL) reconstruction. As a biodegradable metal, Mg has appropriate mechanical strength and osteoinductive effects, thus may be a promising alternative to commercialized products used for graft fixation. Therefore, it was hypothesized that Mg based interference screws would promote tendon graft-bone junction healing when compared to Ti screws. Herein, we compared the effects of Mg and Ti screws on tendon graft healing in rabbits with ACL reconstruction via histological, HR-pQCT and mechanical analysis. The histological results indicated that Mg screws significantly improved the graft healing quality via promoting mineralization at the tendon graft enthesis. Besides, Mg screws significantly promoted bone formation in the peri-screw region at the early healing stage. Importantly, Mg screws exhibited excellent corrosion resistance and the degradation of Mg screws did not induce bone tunnel widening. In tensile testing, there were no significant differences in the load to failure, stress, stiffness and absorption energy between Mg and Ti groups due to the failure mode at the midsubstance. Our findings demonstrate that Mg screws can promote tendon graft healing after ACL reconstruction, implying a potential alternative to Ti screws for clinical applications. Injuries to the anterior cruciate ligament (ACL) are common for those individuals especially with high-level physical activity. Currently, 5 million new ACL ruptures are estimated to occur each year in the world367Currently, titanium (Ti) and degradable polymer based interference screws are the most commonly used tendon graft fixation devices in ACL reconstruction919et al. reported that the use of Mg fixators may favor the formation of fibrocartilage at the intra-articular aperture in rabbits after ACL reconstructionet al. also found that Mg interference screws could effectively inhibit the degeneration of the tendon graft during the remodeling phase via reducing the expression level of MMP-13, indicating that more collagen fibers in the tendon graft were preserved to connect the surrounding bone tissue for higher knee stabilityInjectable bone cements, growth factors and stem cells have been already reported as potential therapy options to promote tendon graft healing after surgery in animal models1415161718192021Therefore, it is of significance to investigate the effects of Mg implants on the tendon graft healing quality in the middle section and the alteration of bone tunnels in ACL reconstruction model. Herein, it was hypothesized that Mg based interference screw could significantly enhance the incorporation of tendon graft within a bone tunnel when compared to traditional Ti interference screws. To test the hypothesis specifically, ACL reconstruction surgery was performed in rabbits by using Mg or Ti based interference screws for histological, radiographic and mechanical testing at different time points.The serum Mg levels in testing animals were found within the normal reference range at all examined time points during the four month implantation periodAs shown in 2/μm vs. 12.1 ± 3.9 μm2/μm) at week 16 (The fibrous tissue with massive cellularity and vascularity was formed and uniformly distributed in the mid-tunnel interface between tendon graft and bone as early as 3 weeks after surgery and then increased over time within the first 12 weeks in both Ti and Mg groups . At 16 w week 16 . The res week 16 .P < 0.01), indicating an acceleration of bone remodeling after the use of Mg screws. In the following healing stages, MAR values were then reduced in both Mg and Ti groups  in bone tissue around screws in Mg group was significantly higher than that in Ti group at week 3  while the gap between Mg screws and bone was gradually refilled by newly formed bone tissue at week 12 and 16 37in situ release of degradation products from implants, the deterioration manner in the mechanical integrity of screws may well match the tendon graft healing process without fixation concerns. Only 10% loss in the apparent volume of Mg screws was observed after 16 weeks. More importantly, the degradation of Mg screws did not induce bone tunnel widening during the entire experimental period. As the low production of artifact caused by the Ti hardware on imaging did not significantly alter the bone related parameters (P = 0.496) and BMD , CT analysis was suitable for the assessment of temporal change of peri-tunnel bone quality in both Mg and Ti groups. The peri-tunnel bone mass, which is commonly reduced after ACL reconstruction in both preclinical and clinical casesAlthough Mg based screws showed favorable biological effects via rameters , includiHowever, the direct mechanical proof on graft bonding strength was not delineated as the failure sites of all the femur-tendon graft-tibia complex (FTGTC) samples in tensile testing were in the tendon graft midsubstance in both Mg and Ti groups. Actually, the primary mode of failure was widely reported at midsubstance after reconstruction in animals especially in the long-term study394142ex vivo CT scans of rabbits is about 1:4.5. Christiansen’s findings showed that the ratio of voxel size to trabecular thickness between 1:4 to 1:6 did not significantly affect BV/TV3 to approximate 820 mg HA/cm3 when the ratio of voxel size to trabecular thickness increased from 1:6 to 1:4. Therefore, BMD values may be underestimated while BV/TV may not be affected in trabecular outcomes for scans with 41 μm voxel size in our study. Although the relatively large voxel size is the limitation of HR-pQCT when applied to scan smaller structures, the ex vivo scanning model can effectively address the concerns about the inter-group variation. Currently, the smallest voxel size in the updated HR-pQCT devices can be down to 17 μm, indicating that we can get more satisfactory results in the future study if the conditions are met.However, there are several limitations in the present study. Firstly, as the metallic screws may easily cause damage to the soft-tissue grafts owing to their sharp edges, biodegradable polymer screws may be theoretically more favorable for fixation of soft-tissue grafts4546In summary, the present study suggested that the Mg based biodegradable interference screws might be an alternative to traditional inert fixators for treating tendon graft-bone junction healing in ACL reconstruction owing to the promoted mineralization at the fibrous entheses. In addition, the degradation products of Mg implants did not cause significant changes in bone tunnel size and inflammatory responses in rabbits. The biosafety and bio-efficacy experiments have been tested and guaranteed in small animals, so it is of significance to extend the research in large animals for the ultimate translation of Mg screws in clinical trials.A total of 112 ACL reconstruction procedures were performed in left knees of 6-month-old male New Zealand White rabbits by using the long digital extensor tendon autograft via transtibial technique according to our established experimental protocol approved by the Animal Ethics Committee of the Chinese University of Hong Kong (13/041-MIS-5), which was in accordance with the guidelines for the ethical treatment of animals . The surThe blood samples of rabbits in Mg group were collected at week 0 (baseline), 2, 4, 6, 12 and 16 postsurgically for serum Mg level determination by using inductively coupled plasma-mass spectroscopy .IL-1 and IL-6 levels were measured in serum of the rabbits after the use Mg and Ti screws at week 0 (baseline), 1, 2, 4 and 6 by means of ELISA (Enzyme Linked-Immuno-Sorbent Assay) method by using commercial kits .To label newly formed bone, the fluorescent staining agent calcein green (10 mg/kg) was subcutaneously injected into these animals assigned for four time points at 14, 28, 63 and 84 days postsurgically prior to xylenol orange (90 mg/kg) injection at 21, 42, 84 and 112 days after surgery, respectively. All the rabbits were then sacrificed for sample harvest at day 3 after xylenol orange injection. Mineral apposition rate (MAR) in the bone tissue for the region of interest (ROI) adjacent to the implants was calculated to compare the apposition rates at different healing stages according to the standardized definitionet al.Both decalcified and undecalcified samples were used for histological examination under a microscope . Briefly, all these harvested femora samples were fixed in 4% neutral buffer formalin for 48 h and then embedded in paraffin or methylmethacrylate (MMA) according to previously published protocols prior to tissue sectioning at mid-tunnel siteSix rabbits were anesthetized and immobilized in the scanner tube of HR-pQCT  for scanning their knees at week 0, 3, 6, 12 and 16 after operation. The fixed parameters for X ray-tube were set: 60 kV (tube voltage), 1 mA (tube current), 41 μm (an isotropic voxel size). A series of 2D tomographic gray-images (80 slices) in the mid-tunnel along the direction perpendicular to the screws were segmented for 3D reconstruction of peri-tunnel trabecular bone . In ordeThe femur-tendon graft-tibia complexes (FTGTC) were harvested and then stored in −80 °C prior to biomechanical testing after thawing at room temperature. Except for the ACL graft, the suture on the tibial side and all the soft tissue were carefully removed while interference screws were kept in femoral tunnels prior to the fixation of femur and tibia with custom-designed jigs in a uniaxial mechanical testing machine . The tensile tests were performed in the femur-ACL graft-tibia complex with the knee flexed to 90° according to previously published protocols with a preload of 1 N and a rate of 50 mm/min until failurepost hoc test was applied for statistical analysis of BV/TV, BMD, serum IL-1 and IL-6 levels in the two groups by using SPSS 17.0 software . The unpaired two-tailed Student’s t-test was used to compare the mechanical data between Mg and Ti groups. All the data were expressed as mean ± standard deviation (SD) with the significant level set as P < 0.05.The non-parametric Mann-Whitney U-test was performed for calculation of significant differences in mineralized area in graft healing interface, histological assessment scores and MAR between Mg and Ti groups while one-way ANOVA with Tukey’s How to cite this article: Wang, J. et al. Biodegradable Magnesium Screws Accelerate Fibrous Tissue Mineralization at the Tendon-Bone Insertion in Anterior Cruciate Ligament Reconstruction Model of Rabbit. Sci. Rep.7, 40369; doi: 10.1038/srep40369 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Purkinje cells (PCs) in Crus 1 represent whisker movement via linear changes in firing rate, but the circuit mechanisms underlying this coding scheme are unknown. Here we examine the role of upstream inputs to PCs—excitatory granule cells (GCs) and inhibitory molecular layer interneurons—in processing of whisking signals. Patch clamp recordings in GCs reveal that movement is accompanied by changes in mossy fibre input rate that drive membrane potential depolarisation and high-frequency bursting activity at preferred whisker angles. Although individual GCs are narrowly tuned, GC populations provide linear excitatory drive across a wide range of movement. Molecular layer interneurons exhibit bidirectional firing rate changes during whisking, similar to PCs. Together, GC populations provide downstream PCs with linear representations of volitional movement, while inhibitory networks invert these signals. The exquisite sensitivity of neurons at each processing stage enables faithful propagation of kinematic representations through the cerebellum. Cerebellar Purkinje cells (PCs) linearly encode whisker position but the precise circuit mechanisms that generate these signals are not well understood. Here the authors use patch clamp recordings to show that selective tuning of granule cell inputs and bidirectional tuning of interneuron inputs are required to generate the kinematic representations in PCs. For tactile sensation, rodents rhythmically sweep their whiskers back and forth to scan the proximal surrounding. Such active whisking enables animals to explore, identify and discriminate objects with impressive degrees of sensitivity and capability2. This behaviour has served as a well-defined paradigm to study active sensory processing and has yielded many insights into the neuronal circuit basis of sensorimotor control10. The cerebellum is strongly implicated in sensorimotor processing11, and recent studies in the vibrissae regions of the rodent cerebellum have highlighted its functional role in the control of voluntary whisker movement8 and in sensorimotor learning tasks12. However, the precise role(s) performed by this structure during voluntary whisking is poorly understood and the implications for cerebellar function remain unclear.Animals actively probe and interact with the world by moving to acquire sensory information. Self-motion has sensory consequences that enable the nervous system to guide and adjust future movement, with sensorimotor brain circuits constantly engaged to optimise this process16. In lobule Crus 1, the majority of PCs encode whisker set point through linear bidirectional changes in simple spike firing rate17. Such remarkable linear encoding of a single kinematic parameter requires precise integration of both excitatory (PF) and inhibitory (MLI) inputs that together provide whisking-related signals to the dendrites of PCs. However, the functional contribution of PFs and MLIs to the generation of PC movement signals is not known.The organisation of the cerebellar cortex is relatively simple and is comprised of a densely packed input layer, the granule cell layer (GCL), which provides excitatory drive via parallel fibres (PFs) to Purkinje cell (PC) dendrites and molecular layer interneurons (MLIs). PCs integrate excitatory and inhibitory synaptic inputs from PFs and MLIs in order to shape spike output for the entire cerebellar cortexBecause granule cells (GCs) transform mossy fibre (MF) input into excitatory PF drive to both MLIs and PCs, it is essential to determine how these cells encode whisker movement prior to processing at subsequent stages of the cerebellar circuit. GCs are the smallest and most abundant neurons in the brain. They receive only a small number of MF inputs (<7), suggesting that GCs may individually encode movement more selectively than PCs. However, their small size and high packing density has precluded measurement of their activity during whisking.20, whereas MLIs exert potent feedforward and lateral inhibition to regulate the firing rate and spatiotemporal dynamics of PC simple spiking23. As a result of this organisation, close examination of the inhibitory network is also required to obtain a complete understanding of information flow through the cerebellar cortex.GABAergic inhibitory interneurons (INs) substantially influence information transmission at multiple stages in the cerebellar cortex. Golgi cells (GoCs) provide feedforward and feedback inhibition that mediates the excitability and gain of GCs in the input layer17. Unlike downstream PCs, whisker movement is associated solely with increased GC activity in the cerebellar input layer. Patch clamp recordings of the activity of INs reveal that reciprocal firing patterns of PCs are generated via the di-synaptic GC-IN-PC inhibitory pathway24. Our results demonstrate that processing of whisker movement signals occurs sequentially at successive stages of the cerebellar circuit in order to generate precise bidirectional estimates of whisker position in PCs.In this study, we reveal the circuit mechanisms that govern bidirectional linear encoding of whisker movement and establish how information about whisker motion is conveyed by excitatory and inhibitory inputs to PCs. We have used patch clamp recordings of the activity of GCs to investigate the representation of movement in these cells. Approximately one-third of PCs exhibit spike rate reductions during free whisking17, but the origin of these decreases in activity is unclear. A reduction in net excitatory drive to PCs could result from reduced activity within upstream populations of MFs and PFs, or alternatively via inhibitory operations within the cerebellum. To examine the underlying mechanism, we examined GC and IN activity during epochs of voluntary whisking.Around 1 out of 3 of movement-responsive PCs exhibit reduced firing rates during free whiskingn = 32, N = 22 mice) and cell-attached  patch clamp recordings from GCs in the vibrissal areas of the cerebellar cortex (lobule Crus 1) of awake mice. To correlate GC activity with whisker movement, we simultaneously tracked spontaneous whisking via a high-speed camera  and fast membrane time constant . These measurements are in good agreement with the data obtained from the lateral hemispheres of anaesthetised rodents30. The mean resting membrane potential of GCs was −63.8 ± 1.0 mV (n = 32). In both WC and CA recordings, GCs exhibited low baseline firing rates , bursting patterns of spike output31 as reflected in high coefficient of variation of interspike interval , and short half-width of action potentials . Although the mean firing rate of CA recordings was significantly different from that of WC recording , this was likely due to our inability to identify silent GCs in CA mode. Accordingly, the whole-cell data revealed a substantial fraction of GCs (n = 26/32) that remained silent in the absence of whisker movement , we observed significant positive correlations between whisker position and membrane potential  prior to the onset of whisking  of GCs lagged movement, and overall there was a broad distribution of latencies between GC depolarisation and movement ; (2) increased amplitude of individual EPSC events and/or (3) elevated sensitivity to synaptic input mediated by enhanced glutamate spillover from neighbouring synapses34. To understand the relationship between GC depolarisation and whisker movement, we performed voltage-clamp recordings to measure EPSCs25. GCs were clamped at −70 mV  and EPSCs were recorded during bouts of quiescence and whisking  enhanced frequency of MF input(EPSCs);  increase17. We examined the relationship between EPSC rate and: (1) raw whisker position, (2) the slowly varying parameter, set point  int Fig. . In 11 rn = 5), we were able to perform consecutive voltage- and current-clamp recordings, and directly compare movement-related changes in EPSC rate and membrane potential. We observed a close correspondence in both the profile . Unlike brief sensory-evoked bursting in anaesthetised rodents30, movement-related bursts were longer lasting, containing on average 31.5 ± 17.9 spikes with an inter-burst interval of 2.1 ± 0.8 s. Accordingly, the mean CV of ISI was extremely high for GCs .To determine how an increased rate of MF EPSCs influences GC output during whisking, we measured GC firing patterns Fig. . In apprn = 9 out of 12 cells; P < 0.05, Kolmogorov–Smirnov test comparing distribution of angle at all times to its distribution at times of action potential occurrence for each recording), and also phase  tuning to kinematic features in WC recordings Fig. . GC outpR2 = 0.32, protraction: R2 = 0.64, P < 0.05, ANOVA, Fig. To determine how GC populations respond during changes in whisker set point, the tuning curves of individual GCs were normalised with respect to the range of movement observed during individual recordings (see ‘Methods’ section). This enabled us to average across cells for a given set point percentile and obtain the mean tuning curve for the population as a whole. Whereas individual GCs displayed sharp tuning at one or multiple set point positions Fig. , the mea35 configurations . To test this prediction, we determined the relationship between IN instantaneous firing rate and whisker position  between relative changes in firing rate and mean whisker position over a certain range , meaning INs exhibited larger changes in firing rate in order to encode the same degree of movement, and indicating that these cells may be more susceptible to rate saturation . To test the fidelity of movement encoding by INs, transfer functions were computed from individual spike trains and corresponding whisker positions . Using this approach, it was partially possible to predict the dynamics of whisking trajectory in real time , and mechanisms such as short-term plasticity  do not influence the overall profile of EPSC amplitude between these two conditions. The observed increase in the rate of synaptic input during whisking resembles dramatic increases in excitability observed in lobule V GCs when mice are running41. However, individual GCs in Crus 1 receive rate-modulated input that confers kinematic tuning to specific features of whisking behaviour.GCs are small in size and vast in number, providing the sole source of excitatory drive within the cerebellar cortex, but little is known about the properties of these neurons in awake, behaving animals. In the absence of movement, GCs display low baseline firing rates Fig. . During 30, and the timing of GC output18, and further work is required to confirm these proposals. However, our results show that MF input rate governs the subthreshold tuning of individual GCs during voluntary whisker movement.A large proportion of GCs exhibit tuning to positional changes, in particular with respect to the slowly varying kinematic parameter, set point, but also to whisker phase. The rate of MF input (EPSCs) varies with respect to whisker angle/phase, and we observed a direct correspondence between the tuning of MF input rate, membrane potential and action potential output in individual GCs Figs. e and 4i.GC membrane potential tuning is typically rather broad Figs. , but the43, and that efferent rather than re-afferent drive dominates cerebellar activity during free whisking  are activated during whisking, implying a shift from sparse to a dense mode of activation during such behaviour32. However, the selectivity of individual GCs to whisker position may ensure that overall low firing rates are preserved on a moment-by-moment basis. Measures of population sparseness will ultimately require simultaneous monitoring of many GCs47 during whisker movement (or monitoring of PF activity48) to examine the fraction and identification of active GCs in Crus 1.These measurements from the input layer support the notion that whisker input to the cerebellar cortex is dense and widespreading Fig. . Classic51. The congruent arrangement of somatosensory and motor input to lobule Crus 1 means that individual GCs might integrate convergent sensory- and motor-related signals about the whisker8. Integration of afferent inputs from functionally distinct origins, e.g., ex-afferent sensory input during touch, in addition to efferent motor copy relating to other aspects of movement, such as locomotion, might be required to promote firing in the approximately two-thirds of Crus 1 GCs that are not active during whisker movement alone.Recent studies have revealed the convergence of functionally distinct afferent inputs onto single GCs across the mouse cerebellum40 in firing rate during voluntary whisking. Given that MLIs outnumber PCs by a factor of ten52 while GoCs are relatively sparse, we consider that the majority of our INs recordings are MLIs. However, we were unable to identify sub-classes on the basis of electrophysiological characteristics , placing them in a critical position to control information flow within cerebellar circuits. By making direct electrophysiological recordings from single INs in behaving mice, our experiments demonstrate INs in lobule Crus 1 show linear changes53 solely exhibit reduced firing rates  in downstream PCs24. In addition to reciprocal connectivity between interneurons, PCs collaterals also project back to neighbouring PCs and MLIs in the adult cerebellum55. This feedback projection could likewise offer an additional route for sign inversion. Together, GC-IN-PC, GC-IN-IN, GC-PC-PC and GC-PC-IN pathways may act in concert with the GC-PC connection to determine the firing rate change of PCs and INs in a bidirectional manner.GC populations represent normalised set point information via monotonic increases in firing rate, i.e., movement either in front or behind the resting position is associated with significant increases in spiking activity. In contrast, some INs, along with PCsIn contrast to GCs, INs exhibit quite distinct tuning properties with respect to whisker position. The most prevalent functional IN class consists of ‘unidirectional’ cells that exhibit altered firing rates at only forward or backward positions. This property is surprising, given that, across the entire population, upstream GCs encode both forward and backward positions via elevated firing. It is therefore possible that unidirectional INs receive excitatory inputs from specific subsets of GCs , while ‘bidirectional’ INs may also receive inhibitory input from local unidirectional INs. The circuit mechanisms underlying this organisation remain unclear, and functional imaging of IN populations may reveal whether such functional heterogeneity is spatially organised . At present, our results highlight the surprising complexity of function exhibited by cerebellar interneurons.60. However, it has remained unclear how such movement-related signals propagate through the circuitry of the cerebellar cortex. The present study reveals that linear encoding is prominent in INs upstream of PCs, and indicates that broad sampling of PF inputs underpins the linear modulation of firing rate in INs and PCs that encode set point.The role of the cerebellar cortex during sensorimotor behaviour has been widely debated. During voluntary whisking, a majority of PCs cells encode movement via linear changes in firing rate to represent salient kinematic parameters61.Our results provide a platform from which to address the crucial role of the lateral cerebellum in active sensory processing. Compared to multi-joint limb movements, whisking is a relatively simple behaviour, and we have focused on the movement with a single degree of freedom (forward and backward), though it is known that animals can also move single whiskers in three dimensions65, to fully establish the importance of the cerebellum in sensorimotor learning.Beyond the representation of whisker position, cerebellar circuits also encode other kinematic features of whisking, including phase, which is robustly represented by neurons in somatosensory neocortex. Given the prominent sensory input received by Crus 1 (from both neocortex and periphery), a necessary next step is to determine the influence of external sensory input  on the activity of cerebellar neurons during active whisking. Furthermore, it will be essential to record from different components of the cerebellar cortex during the acquisition and performance of vibrissal-based sensorimotor tasks2, 1 MgCl2 in mM) and subsequently sealant . Ipsilateral whiskers were partially trimmed with one whisker row left untouched (row C or D). At least 2 h following these procedures, habituation and recording sessions were started. Mice were carefully placed on a cylindrical treadmill and the head-post was gently loaded into fixation clamp for painless immobilisation of the head. At least 1 h of habituation was allowed for the mice to be acclimated to the recording environment. Habituated mice showed normal grooming, whisking and locomotion behaviours on the treadmill. After removal of sealant and agar, recordings were performed in the dark in a single session lasting up to 3 h17. Randomisation and blinding were not appropriate, given the nature of the experiments, and were not performed.The care and experimental manipulation of animals was performed in accordance with institutional and UK Home Office guidelines. 4- to 8-week-old C57BL/6 mice of both genders were used in this study. Animals were group housed in a 12–12 reverse light-dark cycle and all experiments were carried out during the dark phase. Prior to electrophysiology experiments, mice were anesthetised with 1–2% isoflurane under aseptic conditions, and a lightweight head-post was attached to the skull using glue Histoacryl  and acrylic dental cement . A circular chamber was built with cement over the lateral hemisphere of the cerebellum to allow subsequent access for electrophysiological recording. The chamber was then filled with a silicone-based elastomer  and sealed with a layer of nail varnish. A non-steroidal anti-inflammatory drug  was provided via intra-peritoneal administration during surgery to support recovery. Implanted mice were given 2–5 days for recovery, during which time Buprenorphine (0.8 mg/kg) jelly was provided for postoperative analgesia. On the day of the recording, anaesthesia was induced and a small cranial window (1–1.5 mm) was drilled over lobule Crus 1. The dura was removed with fine forceps and the craniotomy was covered with 1.5% low-melting point agar dissolved in cortex buffer . The data were filtered at 10 kHz, digitised at 25 kHz using an ITC-18 interface  and transferred to a computer using AxographX software (www.axograph.com). In whole-cell recordings, resting membrane potentials were recorded immediately after formation of whole-cell configuration and series resistances ranged between 20–40 MΩ. Bridge balance in current-clamp mode was applied in interneurons, but not in granule cell recordings, given their much higher input resistances in comparison to series resistances66. No current was injected unless otherwise stated. Membrane potentials were not corrected for liquid junction potentials. Neuron types in the cerebellum (GCs and INs) could be readily identified by their characteristic electrophysiological properties41.Patch clamp recordings were made from cerebellar GCs and interneurons in awake mice using a Multiclamp 700B amplifier . Recordings were made between depths of 200–1000 μm from the pia surface using borosilicate glass pipettes (6–8 MΩ) filled with internal solution containing (in mM): 135 K-gluconate, 7 KCl, 10 HEPES, 10 phosphocreatine, 2 Mg-ATP, 2 Na2-ATP, and 0.5 Na67—http://whiskertracking.janelia.org—and a customised graphical user interface in MATLAB (Mathworks). Whisker azimuth angles were measured along the longitudinal axis ; protraction corresponded to increases in whisker angle. Because whiskers, especially those from the same row, move in synchrony, one of the traced whiskers was routinely used for analysis concerning whisking, as changing whisker did not affect the results68.Under infrared light illumination, whisker movements were filmed with a high-speed camera  operating at 250 fps Video acquisitions were controlled by Streampix 6 software  and externally triggered by TTL pulses generated via the ITC-18 to synchronise with electrophysiological acquisition. Whisker position was tracked offline using open-source softwareThe data are presented as mean ± SEM unless otherwise stated. All the data analysis was carried out in MATLAB (MathWorks) and Axograph. In whole-cell recordings, input resistance of GCs was measured from steady-state voltage deflections during 400 ms step current injections of –20 pA. Interneuron input resistance was calculated using 400 ms current injection of –50 pA. GC synaptic events were detected using a template-matching algorithm in Axograph X, where a representative EPSC event was selected to serve as the template in each cell and event detection was set at least three times the standard deviation of the baseline noise. All detected EPSCs were visually inspected. Cell-attached recordings were first high-pass filtered at 20 Hz. Action potentials were detected automatically using an amplitude threshold in Axograph X. Sample size estimation was not performed due to the technically challenging nature of the recordings and instead post hoc tests were performed to assess statistical significance.69. Kinematic feature phase was defined as the angle of the Hilbert transform on band-pass filtered (6–30 Hz) whisker angle. A phase of zero corresponds to maximal protraction and a phase of ±π denotes maximal retraction in a whisk cycle68.Whisking epochs were visually identified off-line. Traced whisker position was first low-pass filtered at 30 Hz using a 4-pole Butterworth filter run in forward and reverse directions, and subsequently up-sampled to 1 kHz. Kinematic feature set point was derived by low-pass filtering whisker angle at cutoff frequency 6 HzP < 0.001) was used to evaluate significant change in firing rate or EPSC rate across the population of GCs. Significant changes in membrane potential distribution between non-whisking and whisking epochs were determined using a 2-sample Kolmogorov–Smirnov test (P < 0.05). Membrane potential and the whisker position data were truncated into 1 s segments centred on individual whisking onsets (0.5 s preceding- and post-onset). To examine the temporal relationship between membrane potential depolarisation and whisker position change, normalised cross-correlations were computed for the individual data segments and averaged across segments. The time at the nearest maxima (peaks) above the upper/lower 95% confidence bounds defined the time delay between the two signals.Spikes were grouped into bursts with inter-spike interval (ISI) > 50 ms. Wilcoxon signed-rank test (P < 0.05) was used to compare the distribution of angle (position/set point) at all times with the distribution at times of EPSC/spike occurrence. The distribution of kinematic features at EPSC/spike times was normalised by the amount of time spent in individual bins to generate the tuning curve (in terms of Hz). Kuiper’s test (P < 0.05) was used to assess phase tuning. Modulation depths were calculated as the maximal rate (preferred) minus the minimal rate (least-preferred) divided by the mean in the tuning curve68. The firing rate tuning curves of individual significantly modulated GCs were normalised into percentiles in 5% increments with respect to the range of set point in individual mice. A population set point tuning curve was generated via averaging across cells for individual percentiles.To identify GC tuning to kinematic features, a two-sample Kolmogorov–Smirnov test (n = 2× (ISIn+1 − ISIn)/(ISIn+1 + ISIn). Comparisons of the spike rates were made between quiet and whisking epochs using the non-parametric Wilcoxon signed-rank test, where P < 0.05 was recognised as a significant difference70. Overall firing rates during whisking and non-whisking were calculated by averaging the spike rates of all epochs comprising the two respective conditions. To generate peri-event time-histograms, spike trains were aligned by the onsets of whisking bouts and averaged across trials. Corresponding whisking epochs were aligned at the onset and averaged to reveal the mean whisker movement within bouts.Spike rates were calculated across all whisking and non-whisking epochs in the recording sweeps as the total number of spikes divided by the duration of an epoch. CV2 of ISI was calculated using CV2P-value smaller than 0.05 justified linear modulation of firing rate by whisker position.To determine instantaneous firing rates, a 100 ms rectangular window function was slid along IN spike train with 1 ms step. All linear regression fits for IN instantaneous firing rate and average whisker position was performed using the Basic Fitting GUI in MATLAB. Analysis of Variance (ANOVA) was used to determine whether variation in whisker position arises among different instantaneous firing rate groups of a given IN, and a http://chronux.org/) applied in MATLAB was used to generate transfer functions via multi-taper estimation described in detail elsewhere68. Briefly, using 10–20 s of training data set with both quiet and free whisking epochs, the transfer function H(f) was calculated from the Fourier transform of the spike train S(f) as a sequence of Dirac delta functions, and the Fourier transform of the whisker position θ(f) viaS(f)test to reconstruct the Fourier transform of the whisker trajectory θreconstruct(f) byThe relationship between the IN firing pattern and whisker position was modelled by a linear transfer function, which was calculated in order to decode whisking trajectory from single neuron spike trains. Open-source Chronux Software (All the relevant data are available from the authors.Supplementary InformationPeer Review File


Natural killer (NK) cells play a major role in anti-viral immunity as first line defense during hepatitis B infection, particularly in untreated patients whose T cells functions are profoundly impaired. Cytokine interferon (IFN)-γ and tumor necrosis factor (TNF)-α produced by NK cells are important anti-viral factors. However, there is lack of a quantifiable model to evaluate cytokine responses by NK cells. In this study, almost half of the patients (47.9%) beyond treatment criteria had high cytokine activity, although it was lower than those recommended for antiviral therapy (78.2%). Moreover, we developed a model that low levels of HBsAg, HBcAb, and albumin and high fibrosis values predicted strong antiviral cytokine production by NK cells. Based on the cut-off score (0.361) obtained from the multivariable model, patients with 67%, 8%, 92%, and 74% in immune-active (IA), immune-tolerant (IT), immune-inactive (IC), and grey zone (GZ), respectively, showed active antiviral cytokines produced by NK cells. These results suggest that those who possess activated cytokine responses beyond the current treatment criteria may have potential implications for the timing of antiviral therapy to achieve better virus control. For those affected, lifelong infection carries the risk of developing cirrhosis, hepatocellular carcinoma (HCC), and liver failure. Efforts to understand the immunopathogenesis of CHB focus on the intricate relationship between the virus and the host. The natural course of this relationship progresses predictably through the following four recognized sequential phases: immune tolerant (IT); immune active (IA); inactive carrier (IC); and hepatitis B e antigen (HBeAg)-negative immune reactivation2. Although this phase-focused view of CHB allows for the design of rational therapeutic practices, the distinctions among the phases of infection are not absolute3.Despite the availability of a prophylactic vaccine, chronic hepatitis B (CHB) affects nearly 350 million people worldwide4. However, an area of ongoing debate in CHB is the management of certain subgroups of patients beyond this treatment threshold, including patients with ALT < 2 x ULN, those in the immune-tolerant stage, patients with intermittently elevated ALT that can escape typical clinical surveillance, and those in the so-called “inactive carrier stage”5. There are no formal treatment recommendations for these patients, although a liver biopsy would be considered for some of them. These patients are believed to either lack an HBV immune response or have a favourable prognosis and, therefore, are not considered treatment candidates8. Indeed, HBV-specific T-cell reactivity is weak either due to T-cell exhaustion or viral escape14. Nevertheless, this does not fully explain why the immune system is sometimes able to either control infection or induce liver injury15. Moreover, some studies challenge the dogma of defective HBV-specific T cells in younger immune-tolerant patients. Up to 37% of CHB patients with normal ALT already have significant necrotic inflammation, fibrosis and even cirrhosis on liver biopsy18.Current treatment guidelines recommend the presence of active liver inflammation on histology or elevated serum levels of alanine aminotransferase (ALT) of more than 2 upper limit of normal (ULN) level plus elevated HBV DNA above 2000 IU/mL (HBeAg negative) or 20,000 IU/mL (HBeAg positive) as criteria for initiating antiviral therapy21. NK cells produce interferon (IFN)-γ and potent cytotoxic effector cells to mediate immunity against HBV infection22. Based on their expression of CD56, NK cells are classified into NKdim and NKbright subsets. NKbright cells are primary cytokine producers, while NKdim cells mainly perform cytotoxic functions in the periphery27. NK cells are also reported to boost the efficacy of PEGylated interferon-α in CHB treatment, which correlated with peak virological responses28.Natural killer (NK) cells are a major component of the innate immune system and perform crucial functions in defence against viral infection and cancer30. Other studies suggested that NK cells can exhibit activated phenotypes that are associated with viral clearance32. Recent findings indicated their immunomodulatory function, suggesting regulatory roles for NK cells in other immune cells33. However, these studies have limitations: (1) There are no detailed comprehensive comparisons of cytokine functions in NK cells between CHB patients who are strongly recommended for initiation of routine antiviral therapy and those who are beyond the treatment criteria according to current HBV guidelines from international associations; (2) there are inadequate data to evaluate the correlation between NK-cell driven antiviral cytokines and clinical-virological parameters; and (3) there is lack of a quantifiable model to calculate cytokine responses by NK cells in CHB patients.A systematic review and multiple studies have identifies the phenotype and function of NK cells in CHB patients. Several studies have found that impaired IFN-γ from NK cells in CHB contributes to viral persistenceIn our current study, we, therefore, performed a comprehensive investigation of NK cell frequency, phenotype, and cytokine production in a large cohort of well-characterized CHB patients. We displayed an overall picture of NK cell production of antiviral cytokines in CHB patients who are beyond the treatment regimen criteria (CAN) compared to those who satisfy the recommendations (CA) for antiviral therapy. The relevance of cytokine expression and a broader repertoire of clinical-virological variables were clarified. A mathematical model with unique specificity and sensitivity was generated to evaluate the innate immunity. This report provided a feasible and quantifiable predictor to identify the efficiency of NK cell antiviral activity in CHB patients.Consecutive adult patients with CHB infection observed in the dedicated viral hepatitis clinic of the Third Affiliated Hospital of Sun Yat-sen University between July 2015 and July 2016 were recruited. Patients who received antiviral treatment  within the previous 6 months; patients with human immunodeficiency virus (HIV), hepatitis C virus, or hepatitis D virus coinfection; and patients with end-stage liver insufficiency, autoimmune disorders, immunosuppressive treatment, cirrhosis, and malignancies were excluded. The study was approved by the Institutional Review Board of the Sun Yat-sen University. Informed consent has been obtained from each patient involving in this study. All methods were performed in accordance with the relevant guidelines and regulations.4. Blood was also obtained from age-matched non-HBV infected healthy controls (n = 16). Information on the demographics , body mass index (BMI), HBV markers , hepatic panel , HBV genotypes, and liver stiffness measurement  is listed in Table Of the 226 individuals eligible for participation in this study, 33 were excluded because of missing values, leaving 193 patients available for analysis. The classification and denomination of the patients with CHB in this work were based exclusively on serologic and biochemical parameters in accordance with published international treatment guidelines as follows: (1) inactive carriers (IC): normal ALT level, HBeAg antibody (anti-HBeAg) positive, and low HBV DNA level; (2) chronic active hepatitis with recommendation for initiation of antiviral therapy (CA): elevated ALT level, HBeAg positive or negative; and (3) CHB infection without a strong recommendation to receive antiviral therapy (CAN), including patients during the immune-tolerant phase and those who are in the “grey zones”, meaning that ALT and HBV DNA levels are not in the same traditionally characterized phases34. Isolated PBMCs were stained for surface markers, fixed, permeabilized with IntraPreReagent , and further stained with antibodies directed against intracellular markers. Leukocytes were stimulated with Leukocyte Activation Cocktail  at 37 °C for 4 h prior to the intracellular staining using Pharmingen’s staining protocol. Anti-human mAbs against CD3-PE-CF594, CD56-FITC, IFN-γ-PE, and TNF-α-PE with corresponding isotype-matched controls were purchased from BD Biosciences . Data were acquired on a Gallios instrument  and analysed with FlowJo software .Peripheral blood mononuclear cells (PBMCs) were isolated from fresh blood samples using Ficoll density gradients as described previouslyUpon recruitment to the study, serum was tested for HBsAg, anti-HBsAg, anti-HBeAg, HBeAg, and HBcAb using commercial kits . HBV genotype was performed by direct sequencing. Quantitative HBsAg was measured by the Elecsys HBsAg II Quant reagent kits  according to the manufacturer’s instructions. HBcAb levels were quantified with a chemiluminescence immunoassay . Serum HBV DNA level was measured by Roche COBAS Ampliprep/COBAS TaqMan HBV Test v2.0 (dynamic range from 20 to 1.7E + 08 IU/mL), . Fibrosis levels were defined by liver stiffness measurements .2 test for categorical variables. We explored the association between two continuous variables using a linear regression model and Pearson correlation. The association between the continuous and categorical variable was used the Kruskall-Wallis test. For the cluster analysis, we used the K-Means method to separate the sample into two clusters based on the levels of anti-viral cytokines on total NK cells, NKdim cells and NKbright cells. A multivariate logistic regression model was employed to select the combination of clinical variables associated with the two identified clusters. Stepwise variable selection with AIC criteria was used to select the important clinical variables. The logistic regression coefficients were used to generate nomograms. Discrimination was evaluated by analysing the area under the receiver operating characteristic curve (ROC curve). We used the Youden Index to generate the optimum cutoff for the clinical variables. The nomogram was created using the rms package of R software. All the other statistical tests were performed using R software version 3.2.2. Statistical significance was set to 0.05. We used Bonferroni correction to correct for the multiple comparisons.We compared two patient groups using the Mann-Whitney test for continuous variables and the χdim subset tended to be lower in CHB patients than in healthy subjects, while the lowest proportion was observed in the CA group, even though both patient groups failed to show statistically significant differences from healthy subjects. Instead, the NKbright subpopulation was increased in CA compared with CAN patients (p = 0.0004) and healthy subjects  or beyond (CAN) current treatment guidelines, respectively. The frequency of the circulating NK cells and the NK581 Fig. . This innts Fig. .Figure 1p = 0.0023) or 33.2 for TNF-α (p < 0.0001)). Similar results were observed in the proportions of NK cell subsets producing IFN-γ and TNF-α between the two distinct patient groups  compared with the CAN cohort  and low (Low group) antiviral immunity within the groups of CA and CAN based on the combined expression of the two cytokines Fig. . Almost ers Fig. . Finallyers Fig. .Taken together, these results show that a certain number of CHB patients who are beyond the current treatment guidelines still produce antiviral cytokines.bright cells correlated with higher HBV DNA levels, which were limited to the CA group   , fibrosis , or virological  indicators. We found higher numbers of NKups Fig. , 2.Figur2 = 0.0500, p = 0.0294; r2 = 0.0913, p = 0.0072; and r2 = 0.1193; p = 0.0021; for groups of total NK, NKdim, and NKbright cells, respectively)  Fig. . Howeverly) Fig. .Figure 4To further evaluate the impact of the clinical-virological factors on the frequency and cytokine-producing function of the NK cell subsets, a multivariate logistic regression model, extended to 14 variables readily available in clinical practice, was used to predict the functional response of NK-cells in CHB. Stepwise variable selection with AIC criteria was used to select the important clinical variables. Four clinical parameters  were selected in the final model Table . We thenGiven the dynamic nature of CHB, the current CHB cohort plus an additional 19 patients who were in the inactive CHB phase (IC) were assessed by the above model to predict their innate cytokine functions. Therefore, the 193 patients were categorized into 4 phases as follows: IT phase (n = 13), IA phase (n = 103), IC phase (n = 19), and grey zone . Patients in each phase were segregated into 2 groups with either active or inactive NK cell cytokine function based on the individual estimates derived from the nomogram. Table Overall, the current predictive nomogram revealed distinct NK cell cytokine function in CHB patients in different disease stages and clearly separated individuals with high innate cytokine activity from those with low activity although they might be in the same phase of CHB infection.In the present study, we performed detailed and comprehensive analyses to determine the innate immunity of patients with CHB who meet the stringent inclusion (CA) or exclusion (CAN) criteria of the treatment guidelines. We showed the following: (1) Nearly half of the CAN patients preserved similar levels of cytokine expression by NK cells as those in the CA group, although the average level was lower than in the CA group; (2) patients with higher baseline levels of HBcAb, HBsAg, and ALB, and lower levels of fibrosis had depressed NK-cell-driven IFN-γ and TNF-α levels; and (3) a statistically predictive nomogram was created based on the Cox regression model tailored to individual patients to identify antiviral cytokines produced by NK cells in CHB.35. Analysis of cytokine production by circulating NK cells indicated that NK cells from CA patients with CHB infection displayed, on average, a superior ability to produce IFN-γ and TNF-α compared to those of CAN patients and healthy individuals. Nevertheless, 47.9% of CAN patients can be categorized into the group of high NK immunity of cytokine production by K-means analysis. This group of patients with high levels of antiviral cytokines seemed to be neither in an immune tolerant or an inactive state. Instead, they are more likely similar to the immune activate status and may benefit from antiviral therapy. This finding is consistent with the results from children and young adults during immunologic tolerance, which show higher antiviral immunity than healthy controls3. Therefore, ALT or HBV DNA levels are inadequate to reflect the presence/absence of an antiviral NK-cell response. Moreover, an active virus-specific immune response was detected in the liver in the absence of elevated ALT levels in both patients and animal models5.The data presented in this study do not support the hypothesis that naïve patients have defective NK-cell IFN-γ productionWe pooled the two patient groups to develop a model for the prediction of the NK-produced cytokine response to HBV. Although the model is based on data from patients enrolled with predefined inclusion and exclusion criteria, the generalizability of our results is probably good because of the large sample size and detailed, comprehensive analysis. With the strong association between several key parameters  and the extent of NK-cell cytokine activity in the model, we then generated a nomogram that can be used to calculate the predicted likelihood of an NK-cell-produced cytokine response in an individual patient. With this model, we were able to categorize the patient populations in each clinical phase into two groups of high ( > = 0.361 NK score) or low NK cytokine function (<0.361 NK score). We observed that 45.31% of patients who were not recommended to receive treatment had high levels of antiviral cytokines produced by NK cells. CHB has been traditionally characterized into four phases, including the IT, IA (HBeAg positive), IA (HBeAg negative), and IC phases, reflecting the dynamic relationship between viral replication and evolution and the host immune response. However, some patients are not included in the above characteristic phases but are in a “grey zone (GZ)”. The data presented here showed that approximately 7.69%, 66.99%, 26.32%, and 44.83% of the patients in the IT, IA (HBeAg positive and negative), IC, and GZ phases, respectively, had high levels of NK-cell-specific cytokines. It should be noted that almost half of the CHB patients in the GZ group tended to have similar levels of cytokine production by NK cells as those for whom treatment is recommended. They possess the ability to produce NK-cell driven IFN-γ and TNF-α to counteract HBV infection. These observations illustrate the complexity of defining the host immune response to the virus.37. HBcAb is considered to be accompanied by low levels of HBV DNA and a depressed immune reaction in the liver38. Similar to these results, our data implied that HBcAb is a strong indicator of NK cell-mediated immune quiescence. In addition to HBcAb, HBsAg and ALB were also identified as predictors of a lower antiviral response from NK-cell-produced cytokines. HBsAg is expressed by cccDNA-derived mRNA and released as a part of an infectious particle or non-infectious subvirion particles, the latter being produced independently of HBV replication39. Both in vitro and vivo studies showed that HBV proteins dysregulate cytokines, such as interleukin (IL)-10 or IL-18, which mediate cell signalling, to interfere with IFN-γ production by NK cells40. Thus, it is not surprising that patients with higher quantities of HBsAg express less antiviral cytokine by NK cells. However, higher fibrosis values were associated with increased cytokine expression by NK cells in the current patient population. It is well-known that hepatic stellate cells (HSCs) play a fundamental role in the development and pathogenesis of liver fibrosis43. The activation of HSCs and the transdifferentiation to myofibroblasts represent crucial paths towards fibrosis44. During the development of fibrosis, IFN-γ can be activated to exert its antifibrotic effect against the accumulation of HSCs in the liver45. Therefore, we proposed that the relationship between IFN-γ and fibrosis might be less significant in patients with advanced fibrosis or cirrhosis.In the current multivariable regression model, we found inverse associations between the levels of HBcAb, HBsAg, and ALB with cytokine production by NK-cells, while the fibrosis value had a significant positive association with cytokine expression. HBcAb is a serological marker, representing either the history or presence of HBV infection. The expression of HBcAb only is the most characteristic feature of occult HBV infection along with inactive immunity46. It is widely assumed that waiting to initiate antiviral therapy until the occurrence of clinically active liver disease is an adequate standard of care. However, symptoms often are not apparent until a patient has terminal liver damage. One of the important reasons for not including other HBV-infected individuals beyond the current guidelines is the lack of immune activity or the presence of immunological tolerance in these patients. The viral burden is well-tolerated in these patients; thus, there is no indication for treatment. Rather, treatment is ill-advised because it may promote drug resistance47. The data presented in this study can identify HBV-infected individuals with high or low antiviral cytokine expression by NK cells, particularly HBV-specific T cells which are usually exhausted during native CHB infection. Higher IFN-γand TNF-α produced by NK cells has been demonstrated correlated to viral inhibition and HBsAg clearance51. In addition to direct antiviral effect, these cytokines could further regulate adaptive immune responses by activating T and B cells53. Some studies also uncovered that NK cells may play a detrimental role in HBV chronicity thorough killing HBV specific CD8+ cells. Such negative immunomodulatory functions were closely related to TNF-related apoptosis-inducing ligand (TRAIL) expressed on NK cells. Although TRAIL expression was correlated to the detrimental effect of NK cell against HBV-specific T cells, it could be reduced by antiviral therapy especially in those NUC treated patients after HBsAg clearance. The improved HBV-specific T cell functions were also observed at the meantime33. Collectively, patients could benefit from viral load reduction for increasing antiviral cytokine production and decreasing detrimental function of NK cells. Thus, we arbitrarily assumed that the proportion of patients in the CAN group with higher NK-cell cytokine function possibly can be the candidates of an earlier treatment intervention before irreversible liver damage occurs.Since antiviral therapy for the treatment of CHB delays or prevents cirrhosis and reduces the incidence of HCC, its benefits are obvious. The questions of when to start treatment and flexibility of treatment initiation threshold have become more important and more controversialThe strengths of this study include the following: (1) its novel information regarding the behaviour of NK cells in CHB patients regardless of whether they meet the treatment criteria. The patient population was reclassified into high or low activity of NK-cell cytokine production by K-means analysis. The demonstration that a certain number of subjects from the CAN group can produce high levels of NK-cell specific cytokines is direct evidence that these patients cannot be in a state of defective immunology. This observation illustrates the complexity of defining disease activity in CHB and the limitations of disease classification based on serology or biochemical markers alone. (2) The model could potentially be applied clinically to stratify CHB patients by their levels of efficient cytokines produced by NK cells. Those with high antiviral responses would be considered for protocols involving more frequent surveillance intervals and possibly antiviral strategies, while those with low responses would be triaged to less frequent surveillance protocols. However, our practical model is preliminary and we should include more patients to study in the future. And questions that remain unanswered by our study and merit future inquiry are as follows: (1) How does the cytokine activity in NK-cells change during CHB? A prospective longitudinal assessment would be desirable to identify the potential impact of disease fluctuations on cytokine composition and effector function. (2) Can our clinical prediction model of NK-cell specific cytokine be validated in a large cohort of NUC-treated patients? (3) Is the quantity of cytokines produced by intrahepatic NK cells correlated with or is regulated independently by peripheral NK-cell producing cytokines? (4) Ideally, large confirmatory groups would have been used for an external validation. An ongoing clinical trial may allow for further validation of the model in the near future.We have shown that almost half of the patients not meeting the treatment guidelines had high levels of NK-cell cytokine production, contrary to the current opinion of having either immune insufficiency or less liver injury. Moreover, IFN-γ and TNF-α can be incorporated into a clinical prediction model that can be used to stratify the relative antiviral response due to NK-cell cytokine production in CHB patients. Our immunological data provide a new argument to suggest that patients who preserve a high immune response to viral antigens may be suitable treatment candidates although they do not meet current treatment criteria.Supplementary material


MITF promoter. Our data give new insight into the complex regulation of MITF, a key regulator of melanoma biology, and support previous findings that link metabolic disorders such as hyperglycemia and diabetes with increased melanoma risk.It is well know that cancer cells have adopted an altered metabolism and that glucose is a major source of energy for these cells. In melanoma, enhanced glucose usage is favoured through the hyper-activated MAPK pathway, which suppresses OXPHOS and stimulates glycolysis. However, it has not been addressed how glucose availability impacts on melanoma specific signaling pathways that drive melanoma cell proliferation. Here we show that melanoma cells are dependent on high glucose levels for efficient growth. Thereby, glucose metabolism controls the expression of the melanoma fate transcription factor MITF, a master regulator of melanoma cell survival and proliferation, invasion and therapy resistance. Restriction of glucose availability to physiological concentrations induces the production of reactive oxygen species (ROS). Increased ROS levels lead to the up-regulation of AFT4, which in turn suppresses MITF expression by competing with CREB, an otherwise potent inducer of the  Cutaneous melanoma is a type of skin cancer that originates from melanocytes, specialised cells residing at the basement membrane of the epidermis. The main role of melanocytes is to protect the skin from the damaging effects of UV radiation through the synthesis and secretion of melanin. At the genetic level melanomas are characterised by the presence of activating mutations in genes regulating the RAS/MAPK pathway. Between 40–50% of human melanomas harbour activating mutations in BRAF, while approximately 15–20% of melanomas possess mutations in the NRAS gene [Throughout progression, the viability of melanoma cells depends on their capacity to adapt to new and challenging environments through a diverse range of survival mechanisms such as autophagy, endoplasmic reticulum stress or metabolic reprogramming. Cancer cells are subject to the Warburg effect, by which cells metabolize elevated amounts of glucose through a high (and inefficient) glycolytic rate at the expense of increased lactate production and lower oxidative phosphorylation (OXPHOS) . The Warwww.diabetes.co.uk). On the other hand, in diabetic patients fasting levels of glucose are not correctly regulated and concentrations of glucose can reach and surpass 2 mg/ml (11 mM). Notably metabolic related conditions such as diabetes, obesity and hyperglycemia have been linked to higher risk of a variety of cancers  but also to melanoma [The need for high levels of glucose has initiated several clinical trials testing drugs that target glucose metabolism and has been extensively applied for cancer imaging –8. In thmelanoma –11.Despite evidence for the crucial role played by glucose as a fuel source for cancer cells and the links between high blood glucose levels and melanoma incidence, little is known about how glucose availability impacts on specific drivers of melanoma progression. Therefore we studied the effect of glucose availability on melanoma cell growth with the aim to gain insight into the relevant signalling mechanisms affected by the dependence of melanoma cells on glucose.In order to assess how glucose levels impact on melanoma cell proliferation 501mel (BRAFV600E), A375 (BRAFV600E) and WM266-4 (BRAFV600D) melanoma cells were cultured in the presence of high levels of glucose  or 5 mM of glucose . Cell proliferation was analysed by determining the relative cell number compared to day 0 every 24 hours for 3 days Figure . We obseIn view of these results we decided to characterize the signalling mechanism by which glucose stimulates melanoma proliferation.Due to the central role played by the BRAF/MAPK pathway in melanoma proliferation and cell cycle progression we first assessed if glucose restriction could affect ERK activation. However, glucose deprivation did not inhibit the activation of the MAPK pathway, and even led to an increase in phospho-ERK in A375 and WM266-4 cells Figure , which cTo assess whether there was a causal link between glucose-mediated MITF expression and glucose-dependent growth in melanoma cells, we ectopically expressed MITF from the cytomegalovirus (CMV) promoter , which wThus, in summary, in melanoma cells, it appears that an altered glucose metabolism has seized control over the expression of MITF, thereby rendering these cells dependent on glucose to drive proliferation.MITF promoter fragment containing most of the relevant regulatory sites  and physiological (5 mM) glucose levels  –26. FurtWe first tested if restriction of glucose availability in melanoma cells increases ROS production. Indeed, culturing A375, WM266-4 or 501mel cells with 5 mM glucose significantly induced ROS levels Figure . In thisAccordingly, under 5 mM glucose NAC restored MITF expression and blocked ATF4 up-regulation in a dose dependent manner Figure . ImportaThe functional consequence of this mechanism was seen when melanoma cell growth was assessed under glucose restriction conditions, whereby ROS scavenging using NAC significantly restored cell numbers at low glucose concentrations in all three melanoma cell lines Figure Together, our data strongly suggest that glucose deprivation suppresses MITF expression through ROS induced ATF4 up-regulation, which in turn results in reduced melanoma cell proliferation Figure .The deregulated metabolism that cancer cells have adopted makes these cells dependent on the supply of metabolic substrates. In this study we have attempted to gain insight into the dependence of melanoma cells on glucose availability. We demonstrate that melanoma cells have become dependent on glucose for their growth, which is fuelled by high glucose concentrations, whereas glucose withdrawal i.e. restricting glucose to physiological concentrations reduces melanoma cell proliferation. Although the role of glucose transporters as markers of melanoma progression and the effect of BRAF inhibition on melanoma cell metabolism have been studied , 30–35, MITF promoter. Under physiological conditions the CRE site is bound by CREB, which is one of the strongest transcriptional inducers of the MITF promoter [MITF promoter and contributes to the basal expression of MITF. On the other hand, we show that ATF4, when expressed, is able to reduce the amount of CREB binding to the MITF promoter. Thereby it appears to act as both, competitor of CREB and transcriptional repressor.We demonstrate that limiting the levels of glucose availability results in the expression of the transcription factor ATF4, which is a known mediator of stress pathways, including hypoxia/anoxia, nutrient deprivation and endoplasmic reticulum stress , 36, 37.promoter . While dpromoter , we findInterestingly glucose restriction seems to induce further hyper-activation of the MAPK pathway while reducing melanoma cell proliferation. While this seems contradictory in the first place, the explanation lies in the fact that in melanoma cells MITF is a crucial stimulator of cell cycle progression downstream of ERK . This meIt is well known that in order to promote tumour formation cancer cells under oxygen stress produce pro-angiogenic factors such as VEGF to promote the formation of newly formed vessels and provide the nascent tumour with oxygen and nutrients from the blood. Previous reports have suggested that the pro-angiogenic response of melanoma to low levels of oxygen depends at least in part on HIF1α mediated down-regulation of MITF . In thisin vivo settings if in melanoma glucose deprivation might contribute to the angiogenic and pro-metastatic responses triggered by low oxygen availability and whether ATF4 complements HIF1α's role in promoting the formation of new blood vessels to irrigate and feed the tumour.In the future it will be interesting to investigate in We identified a role for ROS in the regulation of ATF4, and in line with previous reports, we show that glucose deprivation induced the production of reactive oxygen species (ROS) , 41. ROSSimilarly complex is the role of elevated ROS in melanoma progression . IncreasCould glucose-dependent ROS production in melanoma cells explain reports correlating hyperglycemia and obesity with increased melanoma risk –11? In lMelanoma cell lines were cultured in Dulbecco's Modified Eagle's Medium (DMEM) (SIGMA) supplemented with 0.5% penicillin and streptomycin (SIGMA) and 10% bovine calf serum . Cells were grown at 37°C in a 5% CO2 environment. Cell lines had been authenticated by short tandem repeat allele profiling 6 months prior to the study. Cell stocks were expanded and vials kept in liquid nitrogen. New aliquots were thawed every 5–7 weeks. Human melanocytes were from Cascade Biologics and where grown according to manufacturer´s guidelines. Medium with varying concentrations of glucose was obtained by mixing DMEM with 4.5 g/l (25 mM) or no glucose in the appropriate proportions. N-acetyl cysteine (NAC), CoCl2, dichloroacetic acid (DCA), fasentin (FAS) and hydrogen peroxide were from SIGMA.Cells were plated in 6-wel plates and the next day was DMEM 1%FCS with varying concentrations of glucose was added. The relative number of cells compared to day 0 was assessed every 24 h over a period of 3 days by fixing and staining as described in .To assess apoptosis induction and proliferation, 5000 cells were seeded per well in BD Falcon black tissue culture treated 96 well plates (SLS).After 24 h media replaced to fresh DMEM containing 25 mM or 5 mM glucose and the IncuCyte Kinetic Caspase-3/7 Apoptosis Assay Reagent (Essen BioScience) was added. Apoptotic cells can be detected by a fluorescence signal if caspase 3/7 have been activated. Cells were imaged using an IncuCyte ZOOM (Essen BioScience) under 20× lense and kept at 37°C in a humidified 5% CO2 incubator. Phase contrast and fluorescence images with two to four images per well were taken every 20 min and IncuCyte ZOOM software was utilised in real-time to measure confluency, as a proxy for proliferation, and apoptosis, respectively. IncuCyte ZOOM data and timing data were imported into Prism 6 (GraphPad) for statistical analysis and presentation.Cell lysates were prepared using RIPA buffer and analysed as described in . Primary100000 cells per well were seeded in 6-well plates and cultured under 25 mM or 5 mM concentrations of glucose. After 3 days, cells were harvested, washed and fixed in 80% ice-cold ethanol in PBS and kept at −20C. Cells were then washed in PBS and incubated in a solution containing PBS, RNase A  and Propidium Iodide  at 37°C for 1 hour. In order to assess the percentage of cells in each phase of the cell cycle DNA content analysis was performed using FACS Calibur (Becton Dickinson).Cells were labelled with 10 μM 5-ethynyl-2-deoxyuridine  for 4 h before they were formalin fixed and processed following the manufacturer's instructions. Stained cells were imaged using a BDpathway 855 Bioimager and analysed to determine the relative percentage of cells positive for EdU, indicative of the proportion of cells at the DNA-synthesis phase (S-phase) of the cell cycle.The sequence of the siRNA control or Scramble (SC) was: AAUAUAAUCACUAUCAGGUGC- siRNA against ATF4 were from GE Healthcare, Amersham. with references:ATF4 SMARTpool L-042737-01-0005ATF4 single J-005125-10 (A1)ATF4 single J-005125-13 (A4)To downregulate the expression of ATF4, 200.000 cells were seeded in 6-well plates and 24 h later the medium was replaced with serum-free DMEM. Then 200 μl of mix containing 6 μl of Lipofectamine (Thermofisher) and a final concentration of 40nM siRNA, were added drop wise to the cells and left to incubate at 37C, 5% CO2 for 6 h. Medium was then replaced with 10% FCS DMEM and cells were further cultured for 48 h before ATF4 and MITF expression was assessed.MITF promoter (−2293 to +120) and the various truncated promoters were cloned into pGL2 (Promega), and the CRE mutant −1.1 kb promoter construct was generated by PCR directed mutagenesis as described [Approximately 2.3 kb of the escribed . The seqChromatin immunoprecipitation assays, using control IgG (Santa Cruz Biotechnology) or antibodies specific for CREB , and ATF4  were performed as described previously . Primers for the M-MITF promoter specific to the region of CREB binding (−420 bp to −134 bp from ATG) were GCAGTTATTCGGCCATTGGA and GGAAGCCCTACGAGTTTGGT.®, NorthStar Scientific) according to manufacturer's instruction.Cells were cultured as indicated and then loaded with 10 μM carboxylated-H2DCFDA (Thermofisher) for 1 h. Levels of oxidised (fluorescent) intracellular dye were measured using a spectrophotometer (BIO-TEKTo assess the energetic state of melanoma cells the Cell-Titer Glo Luminiscent Cell Viability Assay (Promega) was used. According to manufacturing instructions cells seeded in black walled 96-well plates were cultured under 25 or 5 mM glucose for 48 h. Equal volumes of Cell Titer Glow reagent were added to each well and plates were placed in an orbital shaker for 10min before luminescence reading were recorded using a Synergy H1M spectrophotometer.® H1, spectrophotometer (NorthStar Scientific). This assay is based in the capacity of oxygen to quench the Extracellular O2 consumption reagent. Increased oxygen consumption is recorded as increases in fluorescence emission.As a measurement of mitochondrial function we utilized the Extracellular O2 consumption assay ab197243 from ABCAM. Cells were cultured in opaque transparent bottom- black 96-well plates under 25 mM or 5 mM glucose for 48 h, then Extracellular O2 consumption reagent was added to each well and samples were excited at 380 nm, fluorescent emission at 650nm was detected using a BIO-TEKInvasion assays analysing melanoma cell invasion into collagen  matrices were performed as previously described . In ordet-test and One-way ANOVA with Dunnett's Multiple Comparison Test performed using GraphPad Prism version 4.00 for Mac OS, GraphPad Software, San Diego California USA, www.graphpad.com.Unless indicated otherwise, data are from assays performed in triplicate, with error bars to represent standard deviations or errors from the mean. Statistics used were: predominately Student


We show that normalisation of target gene expression to that for endogenous control genes sufficiently corrects for the variable level of fragmented RNA. Recovery of target gene values was achieved in samples comprising as much as 50 percent fragmented RNA, indicating the suitability and appropriate limitation of such data treatment when applied to samples obtained from inherently necrotic tissues.Subtle molecular differences indicate the heterogeneity present in a number of disease settings. Digital-PCR (dPCR) platforms achieve the necessary levels of sensitivity and accuracy over standard quantitative RT-PCR (qPCR) that promote their use for such situations, detecting low abundance transcript and subtle changes from gene expression. An underlying requisite is good quality RNA, principally dictated by appropriate tissue handling and RNA extraction. Here we consider the application of dPCR to measures of gene expression in pathological tissues with inherent necrosis, focusing on rheumatoid subcutaneous nodules. Variable RNA fragmentation is a feature of RNA from such tissues. Increased presence of transcript fragmentation is reflected in a proportionate decrease in Agilent DV These advantages are particularly relevant for low-abundant targets1. However, there is currently little information available specifically concerning the rationale and efficacy of reference gene normalisation to correct for variable quality of input RNA in RT-dPCR. Such variation is more likely when sampling from disease-associated or tumour tissues that incorporate inherent necrosis or where significant areas are affected by mechanisms leading to cell death6. Under such conditions, the degradation of cellular RNA can be up-regulated by specific signalling pathways9, or as a consequence of therapeutic drug response10.Recent studies have shown that reverse transcription dPCR (RT-dPCR) technology can overcome the adverse impact of a range of background sample contaminants and PCR inhibitors. This enables the generation of reliable data that, in some cases, may not require normalisation to reference genes13. Studies that caution the use of degraded RNA in downstream applications highlight the characteristically unstable nature of mRNA and its susceptibility to RNA hydrolases (RNases), particularly during the acquisition, handling and storage of tissue samples. Transcript-specific degradation has also been described as a consequence of delayed post-excision or post-mortem RNA preservation13. However, the integral quality of resource tissue, particularly of valuable human pathological samples, provides an equally important barrier to molecular analysis. The underlying challenge researchers face when working with pathological, often rare, study samples is the acquisition of quality, biologically meaningful data from an adequate number of samples4.The pitfalls of using low quality mRNA in quantitative molecular analyses are well-established14, a granulomatous lesion generally associated with more severe rheumatoid arthritis. A feature of rheumatoid nodules is the “natural” presence of areas of necrotic tissue, accompanied by varying degrees of cellular apoptosis, both of which are an inherent part of the inflammatory process15. RNA from these areas is unavoidably extracted and consequently present as a variable component of nodule tissue RNA used for downstream studies. Fragmented RNA of at least 100 nucleotides in length contributes to the total RNA concentration measurement prior to RT-dPCR16, although because many transcripts may not be sufficiently intact for complete reverse transcription, correction is required to ensure that the final data reflects gene expression in functional, non-necrotic areas of the tissue.A focus of our investigations into chronic inflammation has concerned pathogenic mechanisms within the rheumatoid subcutaneous noduleHere we consider the application of nanofluidic partitioning chip-based dPCR to quantitative gene expression in tissues complicated by necrosis. We sought to utilise this technique to exploit the increased detection sensitivity and precision capabilities attributed to dPCR, when compared to qPCR, while maintaining an existing, well-validated, sample processing workflow. Results show that measures of the expression of appropriate endogenous control genes, like GAPDH and HPRT1, are proportionate to the level of mRNA of quality sufficient to allow reliable reverse-transcription. We also sought to establish suitable inclusion criteria for samples according to the accuracy of normalisation. This approach limits the undesirable impact on target gene expression values that can arise when working with tissues containing inherent necrosis, while still maintaining the high level of detection sensitivity of the digital technology.17.Retrospective histological analysis showed that all nodule tissues used for RNA extraction contained palisading monocyte/macrophages, focused on a central necrotic area (data not shown). This is a characteristic feature of granulomatous rheumatoid nodules, that we have previously described200 metric, a measure of the percentage of RNA transcripts >200 nucleotides in length. We compared the DV200 metrics measured in nodule RNA samples with those in synovial membrane RNA, finding greater variability in RNA fragmentation in nodules . Both tissue types undergo similar handling suggesting that the quality of nodule RNA reflects in vivo cellular processes characteristic of these pathological, necrotic lesions.RNA quality analysis revealed that RNA purified from nodule tissues was variably fragmented between samples, despite consistent and best practice for sample handling and RNA preservation Fig. . The extles Fig. . Consequp < 0.0001) between combined expression levels for these two endogenous control genes and the DV200 metric  and with the proportion of intact starting RNA   Fig. . Interes14) Fig.  legend.Fper se. Therefore, irrespective of quality, nodule RNA is not expected to contain transcript that might significantly influence measures of PRTN3 expression. We measured PRTN3 expression in the spiked samples using RT-dPCR. Predictably, when assessed per unit of starting RNA, the absolute copies of PRTN3 expression reduced as the proportion of fragmented RNA increased  in nodules sed Fig. . Normalised Fig. . HoweverT) value of ~37 by qPCR.We investigated IL27A gene expression in rheumatoid nodule tissues using dPCR, applying normalisation and appropriate corrections for RNA fragmentation. Within our nodule sample set (n = 27), absolute measures of IL27A expression ranged from 0.25 to 14.16 copies/ng RNA. As a point of reference, a sample returning absolute measures of 0.63 copies of IL27A/ng RNA using dPCR corresponded to an average cycle threshold .Based on DVles Fig. . Normalifait accompli, with potential confounders associated with available human tissue, including characteristics of any inherent pathological processes, likely to influence the quality of the tissue and yield of accurate molecular results. This study examined the characteristics of human rheumatoid subcutaneous nodules, an inflammatory lesion with inherent necrosis, associated with granulomatous inflammation. The goal was to identify those features that determine the suitability of variably necrotic tissue for downstream molecular analysis, particularly the impact on the detection of low abundance gene expression using digital PCR.Key factors in conducting research in human disease are both the availability and quality of appropriate tissue samples. This combination is often a 18. Effectively, measures of RNA integrity generate exclusion criteria that dictate the suitability of samples. The dilemma is the application of these criteria to tissues where there is inherent necrosis as part of the underlying pathological process, which is likely to impact on RNA integrity. In rheumatoid subcutaneous nodules, necrosis is a focus of the inflammatory granuloma. However, tissue necrosis is common during the development of a wide range of diseases, varying in aetiology that includes a “programmed” or “regulated” process, activated in response to death receptor ligands or cellular stress21, or through a contribution to the cytotoxicity of many chemotherapeutic agents in cancer10. In such settings, restricting molecular analysis to high quality tissues limits the available tissue and is unlikely to reflect the true pathological process in operation.The critical influence of RNA integrity on downstream molecular analysis is well recognised. Development of measures of RNA integrity from the long-time standard based on 28 S to 18 S ratio, to a predictive model of RNA integrity, the RIN, now provides a much more reliable and robust measure that subsequently correlates with downstream analysis of gene expression200 metric, indicative of the proportion of extracted RNA transcripts greater than 200 nucleotides in each sample. This metric correlated significantly with expression levels of the endogenous control genes GAPDH and HPRT1, measured by dPCR. This indicates that amongst tissue samples where RNA fragmentation has been naturally generated in-vivo, the downstream quantitation of target gene expression predominantly reflects the levels present in that portion of RNA containing the most intact transcripts.Our results show that despite nodule tissue samples being harvested and treated using optimal RNA-preservation procedures, the presence of inherent, necrotic tissue contributes considerable amounts of highly fragmented RNA. In order to gauge the extent of RNA fragmentation, we considered the DV22. Using experimentally generated mixtures of RNA and dPCR assays for proteinase 3 (PRTN3) expression, our results show that normalisation of gene expression data to endogenous control genes’ expression, also accounts for the proportion of intact transcript present amongst highly fragmented RNA. Further, the normalisation of target PRTN3 expression in THP-1 cell RNA, was effective in samples containing as much as 50% fragmented nodule RNA. However, when the proportion of fragmented RNA increased beyond 50%, and up to 75%, normalisation tended to slightly over-estimate target gene expression. Thus, when considerable RNA fragmentation is present, the expression of endogenous control genes is affected and becomes a less accurate reflection of the intact, measurable portion of RNA. Consequently, normalisation becomes less reliable. Our data suggest that implementing a 50% fragmentation threshold, circumvents any problems associated with more fragmented RNA in pathological samples.Reference gene normalisation is recommended for gene expression analysis using alternative dPCR approaches, thereby circumventing any impact from RNA sample contaminants200 metric >50% to ensure reliable normalisation, we excluded 9 of 27 samples from the final data set but subsequently revealed meaningful values for IL27A expression in those remaining. We conclude that normalisation of target gene levels to suitable endogenous controls in RT-dPCR gene expression studies is appropriate when applied to pathological samples where inherent RNA fragmentation is present. The generation of reliable, normalised data also depends on the level of fragmentation within samples, and may require the implementation of appropriate exclusion criteria. In that regard, we do anticipate similar normalisation strategies could be applied when assessing gene expression in formalin-fixed, and paraffin-embedded (FFPE) tissues, where RNA fragmentation and low RIN scores23 are an inevitable consequence of the treatment. A more likely factor limiting the assessment of gene expression within FFPE tissues is chemical modification of the RNA, fragmented or not. Consequently, adherence to the recommendations for processing and storage of FFPE tissue that is destined for gene expression analysis23 and careful selection and validation of appropriate endogenous control genes24 are additional factors for consideration.We evaluated nodule RNA samples for IL27A gene expression, comparing absolute  and normalised outcomes of dPCR. In these samples, expression of this cytokine gene was generally low by conventional qPCR. Applying the criterion of a DV25, undergoing elective surgery for nodule removal. The patient cohort comprised those of age 64 ± 9 years (mean ± SE), disease duration 21 ± 3 years, with the majority positive for both rheumatoid facter and anti-citrullinated peptide antibodies. Five patients provided multiple nodules, and 3 further patients multiple synovia, either at the same or different times. The study was approved by the New Zealand Health and Disability Multi-Regional Ethics Committee (MEC06/02/003). All patients provided informed written consent prior to tissue donation and all experiments were performed in accordance with relevant guidelines and regulations.Subcutaneous nodule tissues (n = 27) or joint synovial tissues (n = 27) were obtained from patients (n = 42) with rheumatoid arthritis (RA), as defined by 1987 American Rheumatism Association Classification criteriaNodule and synovial membrane tissues were processed immediately following surgical excision in an RNase-free environment or collected into RNAlater (Invitrogen) at 4 °C. Apportioned tissues destined for gene expression studies were snap-frozen and stored in liquid nitrogen.20 (Invitrogen) as per the manufacturer’s protocol to generate cDNA. RNA fragment and DV200 metric analysis were conducted by NZ Genomics Ltd using an Illumina Fragment analyser or Agilent BioAnalyzer and associated software.The RNeasy Mini Kit (Qiagen) was used according to manufacturers’ recommendations, with DNase digestion either on-column using the RNase-free DNase Set (Qiagen) or post-extraction with PerfeCTa DNase I, (Quantabio). Eluted RNA concentration was determined using a Qubit Fluorometer (Invitrogen) with low or high sensitivity Qubit assay kit (Molecular Probes). Up to 1000ng of total RNA was added per 20ul reaction with Superscript III Reverse Transcriptase (Invitrogen) and primed with Oligo (dT)Digital PCR (dPCR) was performed using the Quantstudio 3D Digital PCR system (Applied Biosystems) including the chip loader, ProFlex thermal cycler, and chip reader. Reactions were prepared in 15ul volumes with Quantstudio 3D Digital PCR Mastermix v2 (Applied Biosystems), Taqman gene expression assays , and appropriate sample input amounts . Reactions were loaded onto Quantstudio 3D 20 K V2 dPCR chips (Applied Biosystems) and cycled as follows: PRTN3 10 min at 96 °C, 39X , 2 min at 55 °C, held at 10 °C; all remaining assays: 10 min at 96 °C, 39X , 2 min at 60 °C, held at 10 °C. Prior to running duplex reactions, assays were validated to ensure the duplex readings were consistent with respective single-plex assay readings over a range of input cDNA concentrations (data not shown). Quantstudio 3D AnalysisSuite Cloud Software  with Poisson Plus quantification algorithm (version 4.4.10) and 95% CI was used. Imported eds files were individually assessed and fluorescence thresholds set manually to generate transcript copies/ul, then converted to copies/unit RNA.26. An average coefficient of variation (CV) of 23.3%, and M value of 0.658 for the two genes was generated.To achieve data normalisation, copies/unit RNA of the target gene were multiplied by the corresponding normalisation factor  and final data displayed as normalised copies/unit of RNA. The stable expression of the two endogenous control genes was previously assessed among equivalent nodule samples using qPCR with qbasePLUS v2.1 (Biogazelle)200 = 10%) from 4 rheumatoid nodules, then generated a series of samples containing set proportions (100–0%) of good quality THP-1 cell RNA , spiked with increasing proportions of the pooled, highly fragmented nodule RNA (0–100%). This nodule RNA contained negligible values for averaged GAPDH and HPRT1 expression (0.0017 copies/pg RNA).Available data from RNA-seq analysis identified high expression of the proteinase 3 gene (PRTN3) in unstimulated THP-1 cells . In comparison, PRTN3 expression within nodule tissues was low . This contrast in expression was used to validate the use of normalisation to account for variable fragmentation within sample RNA. We pooled naturally, highly fragmented RNA , was reverse transcribed and expression of GAPDH and HPRT1 endogenous control genes and the PRTN3 target gene measured with RT-dPCR.Samples identified as having high, medium or low IL27A expression levels by dPCR were compared for IL27A expression in routine qPCR. Ten uL reactions containing Quanta ToughMix (Quanta Biosciences), Taqman Assay (Hs00377366_m1 (VIC)), and cDNA representing 31.25ng starting RNA, were assayed in triplicate using a Roche LC480 (Roche Molecular Systems) and standard cycling and detection protocols.200 metrics between 27 nodules and 27 synovia, following a D’Agostino & Pearson test establishing normal distribution (p > 0.05). The geometric mean of the GAPDH and HPRT1 expression was correlated with the associated DV200 metric values using a two-tailed pearson correlation test with 95% CI for log-transformed nodule data (n = 27) and spiked samples (n = 5) . Absolute and normalised IL27A gene expression values were compared using a two-tailed non-parametric Mann-Whitney test, following assessment with D’Agostino & Pearson normalisty test .Graphpad Prism V7.0b (Graphpad Software) was used for statistical analyses. An unparied t test with Welch’s correction for unequal variance was used to compare the DVNodule RNAseq datasets referred to during the current study are not yet publicly available, as complete data analysis is part of on-going research. Information therein is available from the corrsponding author on reasonable request.Supplementary Figures