Inspiratory flow limitation (IFL) is a critical symptom of sleep breathing disorders. A characteristic flattened flow-time curve indicates the presence of highest resistance flow limitation. This study involved investigating a real-time algorithm for detecting IFL during sleep. Three categories of inspiratory flow shape were collected from previous studies for use as a development set. Of these, 16 cases were labeled as non-IFL and 78 as IFL which were further categorized into minor level (20 cases) and severe level (58 cases) of obstruction. In this study, algorithms using polynomial functions were proposed for extracting the features of IFL. Methods using first- to third-order polynomial approximations were applied to calculate the fitting curve to obtain the mean absolute error. The proposed algorithm is described by the weighted third-order (w.3rd-order) polynomial function. For validation, a total of 1,093 inspiratory breaths were acquired as a test set. The accuracy levels of the classifications produced by the presented feature detection methods were analyzed, and the performance levels were compared using a misclassification cobweb. According to the results, the algorithm using the w.3rd-order polynomial approximation achieved an accuracy of 94.14% for IFL classification. We concluded that this algorithm achieved effective automatic IFL detection during sleep. Respiratory event identification is necessary for diagnosis and treatment of sleep-disordered breathing (SDB), including obstructive sleep apnea/hypopnea syndrome (OSAHS), upper airway resistance syndrome (UARS), and snoring. These three conditions comprise the entire SDB spectrum, ranging from the most severe to the mildest , 2. The Patients with UARS are characterized by upper airway collapse (apnea-hypopnea index ≤ 5 events per hour) or more than 30% of total sleep time with inspiratory flow limitation (IFL) during sleep. In recent years, IFL events have been recognized as a vital diagnostic tool for mild SDB , 9. AccoFor automatic noninvasive assessment of IFL and related changes in UARS or OSA, the recognition of both airflow and intrathoracic pressure changes is required; this has been described in previous studies , 14. VarThis study investigated a real-time feature extraction algorithm for detecting IFL during sleep. We propose IFL detection methods involving weighted polynomial functions for IFL feature extraction. For comparative study, both the flatness index (FI) method and polynomial approximation were used for IFL detection. Vital insights into the ability of the proposed methods to automatically detect IFL were thereby obtained.To evaluate the potential feature extraction metrics (or indicators) of IFL and to determine the best cut points for each metric, a development data set was obtained from previous studies , 20 ClusThirteen volunteers with OSAHS were recruited from the community. These volunteers underwent full nocturnal polysomnography  recordings using EEG, ECG, EOG, EMG, and Spo2 derived at a sample frequency of 256 Hz at the sleep center of Taipei Veterans General Hospital in Taipei, Taiwan. The study was approved by the Institutional Review Board of Taipei Veterans General Hospital, Taiwan (2012-11-001AC). Sleep stages were manually scored by experts using Rechtschaffen and Kales criteria. Apneas and other respiratory events were scored by applying standard criteria . The res strongly negative, (2) balanced, and (3) strongly positive. The aim was to not exclude the influence of subjective judgment. Each physician was asked to judge the inspiratory flow following visual assessment and to note the IFL degree as a certainty value. The percentage of agreement was 89.3% between both physicians. The test set created with classical non-IFL criteria consisted of 637 breaths and that with the IFL criteria consisted of 456 breaths (IFL Level 1 (363) and IFL Level 2 (93)), resulting in a total of 1,093 breaths being obtained by strong positive certainty. The distribution is illustrated in Respiratory event-related arousal (RERA) is believed to be the characteristic event of OSA. Our test set was obtained from 13 OSA subjects either with or without RERA while sleeping. Their breathing cycles were analyzed as follows. Polysomnography was applied for prescoring to detect each subject's breathing—for 150 seconds prior to RERA or a nonrespiratory-event, and for 150 seconds subsequently, thereby obtaining a section containing non-IFL and IFL breathing cycles. A set of 2565 respiratory data was obtained from the 72 sections collected during sleeping. Subsequently, 1093 respiratory data items were selected from the set through subjective visual detection. Processing of visual detection was conducted as follows. For the separate processing of the non-IFL and IFL data, two physicians with more than 3 years of clinical experience assigned levels of 1–3 in accordance with their respective experience. We used three levels of certainty in our interviews, namely, (1)fs is the inspiratory flow signal, M is the mean of the inspiratory flow, and D is the number of breaths. In this study, the value of D is 1 because the flow-time curve relates only to a single breath. Flatness is determined by the absolute value of variance between 25% and 75% for the inspiratory flow from the average of all values in the same period. FI detects IFL at 50% of the middle area of an inspiratory signal. A normal inspiratory flow-time curve is rounded or quasi-sinusoidal. For this reason, it is easy to determine whether an inspiratory breath is normal or limited. Examples of FI detection are shown in A patented FI process  was usedTo achieve rapid convergence and efficiency, the Levenberg-Marquardt (L-M) method was achieved for obtaining least-squares coefficient solutions. The polynomial models were used to fit the inspiratory flow data and to calculate the residues as feature extraction. Using the residuals, a receiver operator curve was produced to determine the threshold of non-IFL, IFL Level 1, and IFL Level 2 classifications.Polynomial Approximation. The inspiratory flow signal may be in compliance with a k-order linear equation  and [IFL1 → NIFL]. The reasons for this may be that (1) the FI method and first-order polynomial approximation applying 50%–60% standard deviation of the IFL middle waveform as a characteristic value and (2) using the second-order polynomial approximation can only indicate a turning point. It may describe a non-IFL waveform but can describe only the trend in the IFL waveform. That is, it cannot act as effective data of flow-limited breath for improving the classification. The second-order polynomial approximation is characterized by only one deflection. In other words, it does not approximate as closely as the first-order approximation (see Previous studies by Aittokallio et al. , 20 haveIn conclusion, the third-order algorithm demonstrated high performance for IFL detection. The w.3rd-order polynomial function significantly improved the population and was successfully applied for classification of severe IFL. This comparative study provides vital insights into the efficacy of automatic detection of IFL for reducing mean airway pressure, improving subjective sleepiness, and ensuring adherence to treatment in patients with mild SDB.


Caenorhabditis elegans, changes in environmental osmolarity are commonplace. It is known that the nematode acutely avoids shocks of extremely high osmolarity. Here, we show that C. elegans also generates gradually increased aversion of mild upshifts in environmental osmolarity. Different from an acute avoidance of osmotic shocks that depends on the function of a transient receptor potential vanilloid channel, the slow aversion to osmotic upshifts requires the cGMP-gated sensory channel subunit TAX-2. TAX-2 acts in several sensory neurons that are exposed to body fluid to generate the aversive response through a motor network that underlies navigation. Osmotic upshifts activate the body cavity sensory neuron URX, which is known to induce aversion upon activation. Together, our results characterize the molecular and cellular mechanisms underlying a novel sensorimotor response to osmotic stimuli and reveal that C. elegans engages different behaviors and the underlying mechanisms to regulate responses to extracellular osmolarity.Environmental osmolarity presents a common type of sensory stimulus to animals. While behavioral responses to osmotic changes are important for maintaining a stable intracellular osmolarity, the underlying mechanisms are not fully understood. In the natural habitat of  Caenorhabditis elegans responds to the upshifts of extracellular osmolarity by gradually increasing reorienting movements, and the aversive response requires a conserved cGMP-gated channel subunit TAX-2. TAX-2 acts in several neurons that are exposed to the body fluid to regulate the response to osmotic upshifts. This behavior uses different channels and sensory neurons than those previously characterized in acute avoidance of osmotic shocks, which is mediated by a transient receptor potential vanilloid channel in a sensory neuron open to the outside. Thus, our results reveal a novel sensorimotor response to extracellular osmolarity and the underlying mechanisms.Extracellular osmolarity presents an ecological factor that is essential for the survival of all organisms. However, how animals respond to environmental osmolarity is not fully understood. We show that A stable intracellular osmolarity is essential for survival. Both hyperosmotic and hypoosmotic conditions, when the extracellular osmolarity is higher and lower than the intracellular osmolarity, respectively, alter the water content, the volume, and the ionic concentration of a cell. These perturbations often change the activity of the intracellular proteins and nucleotides, resulting in detrimental effects on the physiology and the function of a cell . Many orThe physiologic response to hyperosmolarity has been well studied in yeast, where osmotic upshifts induce the biosynthesis of glycerol that functions as an osmolyte to increase intracellular osmolarity . In contCaenorhabditis elegans encodes several homologs of the mammalian TRPV channels, one of which is OSM-9, which acts in a polymodal sensory neuron ASH to detect strong osmotic shocks that induce acute avoidance. Expression of the mammalian TRPV4 in ASH substitutes for the function of osm-9 in directing avoidance of osmotic shocks  channels have been characterized for their osmosensory functions. TRPV1 is expressed in the central osmosensory neurons and the Trpv1 knock-out mice lose the response to hyperosmolarity in these neurons . TRPV4 ic shocks .C. elegans provides an opportunity to investigate this question. The nematode is often found in rotten organic materials, a heterogeneous environment that presents osmotic stimuli in various ways. It is known that C. elegans acutely avoids high osmolarity by generating reversals immediately on a direct contact. This response is mediated by a homolog of the mammalian TRPV channels in the sensory neuron ASH that is open to the outside I, PR671 tax-2(p671)I, am1 osr-1(rm1)I, BE93 dpy-2(e8)II, ZC2533 tax-2(p691)I; yxEx1291, ZC2547 tax-2(p671)I; yxEx1315, ZC2691 tax-2(p691)I; yxEx1325, ZC2407 yxEx1257, ZC2408 yxEx1258, ZC2406 yxEx1256, ZC2393 yxEx1248, ZC1552 yxEx778, CX7102 lin-15(n765)X; qaIs2241X.All strains were cultivated under the standard conditions . The strtax-2 genomic region contained the tax-2 coding region, 2.3 kb 5' sequence and 400 bp 3' sequence. Two PCR fragments with an overlapping sequence of 500 bp were generated to cover the entire tax-2 genomic sequence and were injected together into the tax-2(p691) animals at 10 ng/µl each. A PCR product that was generated from a fusion PCR from the above two overlapping fragments and contained the same genomic region was injected into the tax-2(p671) animals at 5 ng/µl. The primers used in these experiment are as follows: forward primer, CTTGAGCCTCTCGACCCATACC; reverse primer, CACATACTGAGCCTGCACTG; and the additional primers for the overlapping regions, GATCTGACAATCGTCGCCAGTG, and TGGGCTACTTCGAGCATTTCTG.The rescuing Pgcy-36::tax-2 construct or the Pstr-3::tax-2 construct, a PCR product of the gcy-36 promoter  or the str-3 promoter  was cloned into the Invitrogen pCR 8 TOPO TA vector to make an entry clone. The PCR product of tax-2 cDNA  was introduced into a destination vector derived from pPD95.77. LR recombination (Invitrogen) between the Pgcy-36 entry clone or the Pstr-3 entry clone and the tax-2 cDNA destination clone were used to generate Pgcy-36::tax-2 or Pstr-3::tax-2, respectively. Each of the constructs was injected into the tax-2(p691) mutant animals at 5 ng/µl to generate transgenic lines.To generate the C. elegans responds to NaCl; therefore, to control for the behavioral response to NaCl, all of the solutions used in the study had the same concentration of NaCl at 50 mm. Specific osmolarity was generated with glycerol, NaCl, or sorbitol. Glycerol was used when the solute was not specifically described. The final osmolarity of each solution was calculated by adding up the osmolarity contributed from each solute in the solution. We did not consider the effects on osmolarity that were generated by potential interactions among solutes, which were usually small (see the chart below). The osmolarity from each solute is the total number of elements the solute contributes when fully dissolved in water. Assuming that NaCl is fully ionized at the concentrations used, 1 mm NaCl contributes to 2 mOsm, and 1 mm glycerol or sorbitol contributes to 1 mOsm. The calculation was validated by measuring the osmolarity of each solution with an osmometer (Vapro Vapor Pressure Osmometer 5520), as shown in the following chart.The osmolarity of the standard nematode growth medium (NGM) was measured to be 158 mOsm.C. elegans generated continuous body thrashes that were interrupted by reversals and big body bends that resembled the shape of Ω. We categorized both reversals and big body bends as turns. Specifically, to identify turns, the MATLAB code fitted the image of a worm in each frame into an ellipse and calculated the eccentricity of the ellipse. The eccentricity was smaller during turns, because the shape of the worm was more circular. A turn was defined to occur when the eccentricity was smaller than a threshold value, which was previously determined with manually detected turns. We measured the swimming speed by quantifying the number of body thrashes per minute. We counted the curving of a worm body toward either side as one thrash. We also measured the volume of the four droplets before and after the assay for multiple assays and found no significant difference . Wild-type control and comparative genotypes were tested sequentially on the same day in the same experiment.The droplet assay was performed essentially as described with modifications . In eachWorms were cultured on regular NGM plates (∼150 mOsm). To measure the dehydration caused by different osmotic conditions, a worm was first transferred onto an agar plate that had the same osmolarity as the cultivating plate to take pictures with an Eclipse TE2000U microscope with a CoolSNAP EZ CCD camera (Nikon). The worm was then soaked in a 200 or 400 mOsm solution for 8 min and returned to the original agar plate to take another set of pictures. Three pictures were taken for each worm under each condition. The outline of each worm was manually traced, and the area of the worm body was measured with the NIS-element software (Nikon) in pixels. To minimize the subjective bias during the measurement, the picture files were randomly ordered and renamed using a MATLAB code. The measurement was performed with the researcher blind to the conditions.NGM plates with osmolarity of 400 mOsm were made by adding sorbitol to the mixture of the regular NGM plates, which had osmolarity of 150 mOsm. To adapt animals to the hyperosmotic conditions, wild-type worms were cultivated on the 400 mOsm NGM plates for one generation before testing. During the assay, adapted adults were soaked for 10 min in a 400 mOsm solution balanced by glycerol and then transferred to droplets of the 200 or 400 mOsm solution to record behavior. Control animals were cultivated on regular NGM plates, soaked in the regular NGM buffer for 10 min, and tested in droplets of the 200 and 400 mOsm solution in parallel.Fbase) to calculate the change in GCaMP6 signal (ΔF) for every fame for the first 120 s recording for each animal [ΔF = (F − Fbase)/Fbase * 100%]. The bleaching effect was corrected by the built-in function “Bleach Correction” of Fiji  by generating reversals within a few seconds of a direct encounter, and the avoidance requires the C. elegans homolog of the mammalian TRPV channels in the sensory neuron ASH. In contrast, on direct contact C. elegans does not generate a similar reversing response to hyperosmotic stimuli ranging from 200 mOsm to 1 Osm , respectively, of the area that their bodies occupied on a flat image after swimming in a glycerol solution of 200 or 400 mOsm for 8 min . In addition, we measured how fast the worms thrashed their bodies in the 150 and 400 mOsm solutions  and found that they thrashed significantly less in the 400 mOsm glycerol solution  and dpy-2(e8) mutant animals. Previous studies have shown that a prolonged exposure to hyperosmotic conditions (1 Osm) decreases the body volume and reduces mobility in C. elegans due to the loss of water. Both the osr-1(rm1) and dpy-2(e8) mutants harbored mutations that generated resistance to the water loss induced by hyperosmotic conditions (osr-1(rm1) mutants nor the dpy-2(e8) mutants increased their turning rates in response to the osmotic upshift from 150 to 400 mOsm (osr-1(rm1) mutant animals generate normal avoidance to nose touch or the repellent 1-octanol (dpy-2(e8) mutant animals normally avoided  gentle touches to the anterior part of the body  in the polymodal sensory neurons ASH  and the che-1(p679) mutants, which were defective in the development and differentiation of the sensory neurons AFD and ASE, respectively (twk-18 in the sensory neuron ADF [Psrh-142::twk-18(gf)] (twk-18(gf) hyperpolarizes and inhibits the activity of the expressing neurons ] . The exp neurons . None of neurons . These rtax-2-expressing sensory neurons that regulate the aversive response to the osmotic upshift, we expressed the wild-type tax-2 cDNA selectively in a few sensory neurons in the tax-2(p691) mutants and examined the potential rescuing effects. Because we found that exposure to the osmotic upshift from 150 to 400 mOsm for a few minutes generated a significant shrinkage of the worm body, we hypothesized that osmotic upshifts might alter the osmolarity of the body fluid. Thus, we expressed tax-2 in AQR, PQR, and URX (Pgcy-36::tax-2), which comprised a set of sensory neurons that were exposed to the body cavity. We found that the expression of tax-2 in AQR, PQR, and URX fully rescued the defect of the tax-2(p691) mutant animals in responding to the osmotic upshift from 150 to 400 mOsm  had no detectable rescuing effect in the aversive behavioral response  (twk-18(gf) in either AIY alone [Pttx-3::twk-18(gf)] or primarily in AIB [Pinx-1::twk-18(gf)] generated no obvious phenotype in the aversive response evoked by the osmotic upshift (Pttx-3::twk-18(gf); Pinx-1::twk-18(gf)] significantly reduced the increase in the turning rate in response to the osmotic shift from 150 to 400 mOsm. In addition, expressing twk-18 in AIB, AIY, and AIZ together  similarly suppressed the aversive behavior, but expressing twk-18 mainly in AIB and AIZ [Podr-2b(3a)::twk-18(gf)] was not sufficient to generate a detectable defect   to inhibk-18(gf)  and exam upshift . In conte defect . Togetheglr-1 promoter to block presynaptic release from several command interneurons and motor neurons  via the OSM-9 TRPV channel, which acts in the polymodal sensory neuron ASH, which is open to the outside (C. elegans also generates an aversive behavioral response when it is continuously exposed to the upshift of osmolarity for a couple minutes. The locomotory response evoked by the osmotic upshift is characterized by a gradual increase in the frequency of turns and reversals. In addition, this slow behavioral response is regulated by the function of a cGMP-gated channel subunit TAX-2 in a set of sensory neurons, AQR, PQR, and URX, that are exposed to the body fluid. Osmotic upshifts activate URX, which is known to generate aversive behavioral responses on activation. The aversive response to the osmotic upshift requires a motor circuit that underlies navigational locomotion. Together, our findings characterize a new type of aversive behavioral response that C. elegans uses to cope with an environmental stress, osmotic upshifts; and to identify the underlying molecular, cellular, and circuit mechanisms.Under the standard laboratory conditions, the nematode  outside . Here, wC. elegans, resulting in death (C. elegans increases the production of glycerol, which increases the intracellular osmolarity and prevents water loss (Escherichia coli (On changes in the extracellular osmolarity, animals use both homeostatic responses and behavioral strategies to alleviate the osmotic stress. It is known that a hyperosmotic condition can dehydrate in death . During ter loss . Here, whia coli . Meanwhihia coli . The comtax-4, which also encodes a cyclic nucleotide-gated channel subunit and regulates many sensory responses to chemicals, is dispensable for the aversive response to the osmotic upshifts (The gradual increase in the turning rate in response to the osmotic upshift requires the cGMP-gated channel subunit TAX-2, which is known to regulate chemosensory responses downstream of the G-protein and cGMP signaling pathways . Howeverupshifts . The difupshifts . These rC. elegans prefers a range of the ambient oxygen concentrations and that the AQR, PQR, and URX neurons contribute to the regulation of the behavioral preference. The oxygen response of URX is modulated by the fat storage of the animal (gcy-35 or gcy-36 or several other guanylyl cyclases that are known to express in AQR, PQR, or URX do not disrupt the behavioral response to the osmotic upshift. While URX is able to respond to both an increase in the environmental oxygen concentration and an increase in the environmental osmolarity, the neuronal response, measured by genetically encoded calcium reporters, that is evoked by the osmotic upshift appears to be much milder than that evoked by the oxygen upshift (We also show that TAX-2 acts in a set of sensory neurons, AQR, PQR, and URX, to regulate the increased turning rate in response to the osmotic upshift. The AQR, PQR, and URX neurons are exposed to the body cavity . These ae animal , and URXe animal . These fe animal ; however upshift . The dif upshift . Therefo


The chimera modules divided the cases into three classes that respectively recapitulated the traits of mesenchymal epithelial reverse transition (MErT), epithelial mesenchymal transition (EMT), and hybrid/partial EMT. This indicates a close correlation between the reprogramming governed by EMT regulators and DSRCT biology, which was further confirmed by miRNA-21 and is consistent with the broad morphological spectrum of DSRCTs. Starting from the miRNA-200/ZEB1 axis, we also found that DSRCTs carry a signature of immunological ignorance that is not responsive to PD-L1 blockade. Evidence that the up-regulation of miRNA-200 and E-cadherin, and quite a high level of miRNA-21 expression segregate with the MErT supports the idea that, in addition to the hybrid/partial state, MErT is also enriched in stemness: the androgen-positive cases, whose stemness traits were confirmed by stem cell arrays, all fell into these two classes. Our findings also confirmed that tumoral cell PDGFRA expression correlates with desmoplasia, and demonstrated the co-expression of PDGFRA and ISLR/Meflin, another marker of pluripotency. Despite the limited number of cases, these findings provide unexpectedly relevant information concerning the pathogenesis of DSRCTs, and prove the validity of miRNA-based chimera circuit modelling in the clinico-pathological setting.To gain new insights into desmoplastic small round cell tumors (DSRCTs) by means of gene expression profiling (GEP). Formalin-fixed, paraffin-embedded surgical specimens obtained from seven pretreated DSRCT patients were interrogated using GEP complemented by immunohistochemistry, a cancer stem cell array, and miRNA  EWS-WT1 translocation and are therefore included with simple sarcomas in the molecular sarcoma classification [EWS-WT1 chimera leads to the transcription of a number of the target genes involved in proliferation, survival and invasion, including PDGFRA [MET and PIK3CA mutations have been identified [in situ hybridisation (ISH) analyses provided a comprehensive picture of the pathogenetic alterations characterising DSRCTs, which can be summarised in the following four main cues. First, a high degree of plasticity due to the metastable state of hybrid/partial EMT tumor cells that at one hand favours tumor stemness and local spreading, and on the other hand is consistent with the broad morphological spectrum of the disease. Second, a PD-L1 immunoscore of both tumor cells and host immunocomponent that fits with the “non-inflamed” tumor type. Third, an unexpected enrichment in the stemness of AR-positive cases that may explain the limited effectiveness of this target treatment in DSRCTs. Fourth, a reinterpretation of the role of PDGFRA which, together with ISLR/Meflin (a new player closely related to pluripotency), is confirmed as contributing to desmoplasia.DSRCTs are rare, ominous, small round cell sarcomas mainly affecting young men that bear the g PDGFRA . Furtherg PDGFRA  that mayentified , and theentified , and theentified  and SMARentified , thus suentified . The conentified  and, on entified . This hyentified  or “chimentified , which sISLR/Meflin, a gene that was significantly differentially expressed in cluster 1  , and cleISLR/Meflin and PDGFRA, we used PDGFRA immunostaining and, in the absence of a commercially available Meflin antibody working on formalin fixed paraffin embedded (FFPE) tissue, carried out ISH against PDGFRA and ISLR/Meflin using a PDGFRA-positive gastrointestinal stromal tumor (GIST) as an in-built control  showed a low level of PDGFRA expression and ISH revealed the expression of reported , but thee Figure . AltogetIn this study, GEP was used as a starting point to identify candidate oncogenic pathways in DSRCTs, after which we used IHC, ISH and CSC array analyses in an attempt to contextualise and, when possible, provide a picture of the most significant processes dictated by the molecular analysis. The results demonstrated the high degree of plasticity/flexibility of DSRCT cells orchestrated by the EMT/MErT process, which explains the broad histological spectrum of the tumors. The relevance of EMT/MErT in this tumor is underlined by its dual epigenetic regulation via miRNA200/miRNA-34 and miRNA-21: the first is mainly involved in governing the switch, and the second endows pluripotency. Current thinking concerning “partial EMT” suggests that the metastable status of tumoral cells  is the key to the apparent paradox that the local progression of a primary tumor is facilitated by the acquisition of mesenchymal traits, whereas colonisation in metastatic sites requires rapid reversion to an epithelial phenotype . The proin vitro analysis and IHC and ISH investigations of histological sections [Using chimeric modules consisting of TFs (ZEB1 and SLUG) and miRNAs (miRNA-200 and miRNA-34), our DSRCTs could be divided into three groups that respectively recapitulated the MErT, hybrid/partial EMT and EMT phenotypes : a commercial probe KIT (Vysis EWSR1 Break Apart FISH Probe Kit) for EWSR1 break apart and an in house labeled BAC probe mix  for WT1 break apart. The antibodies and experimental conditions used for IHC staining in this investigation are detailed in Supplementary Methods.The study was approved by the Independent Ethics Committee of the Fondazione IRCCS Istituto Nazionale dei Tumori di Milano (INT-MI). All of the patients whose biological samples were included in the study gave their signed consent to donate the tissue remaining after the diagnostic procedure had been completed to INT-MI. We interrogated the database of the Department of Pathology at our Institution and identified seven primary DSRCTs surgically removed after combined chemotherapy between 2000 and 2016. The clinical data (including follow-up data when available) were obtained from the patients’ records and are summarised in http://linus.nci.nih.gov/ was used for the differential expression analysis.The gene expression analysis was made using FFPE material obtained by direct dissection from ten 7 μm methylene blue-stained sections of non-necrotic, contamination-free normal tissue representative of tumoral areas. RNA was isolated using the Qiagen RNeasy FFPE kit  in accordance with the manufacturer's instructions. The gene expression profiles were generated using Illumina HumanHT12_v4 WGDASL  , which allows the expression profiling of 29,377 probes from degraded RNA. Reverse transcription, oligo annealing, ligation, amplification, labelling, probe purification, hybridisation, and chip washing procedures were carried out in accordance with the manufacturer's instructions. The Illumina IScan Reader was used to scan the arrays, and Illumina BeadScan software was used to acquire the images and recover the primary data, which were then normalised using BeadStudio software and the quantile method. The data have been deposited in the Gene Expression Omnibus repository (Accession No. GSE90904). The DSRCTs underwent unsupervised tumor subtype identification using k-means clustering and 1Pearson correlations as the distance matrix following the procedures implemented in the R package ConsensusClusterPlus  on the bmiR-21 (# 38102-15), miR-34a (# 38487-15) and miR-200c (# 38536-15); the negative control was the Scramble miR probe (# 99004-15) .MiRNA ISH was performed as previously described . FFPE tiISLR/Meflin and pdgfra mRNA expression was studied by means of ISH using the target probes ISLR  and the RNAscope 2.5 High-Definition detection kit (brown)  in accordance with the manufacturer's instructions (see Supplementary Methods for details).2 first strand kit . The RNA obtained from a representative FFPE glioblastoma sample obtained from an additional patient was included as a reference/control. After retro-transcription, the cDNA was pre-amplified using the human cancer stem cell RT2 PreAMP pathway primer mix  in accordance with the manufacturer's instructions. The pre-amplified cDNA was then used as a template for the human cancer stem cell RT2 profiler PCR array . The arrays were amplified using an ABI 7900 HT fast real-time PCR with RT2 SYBR Green ROX qPCR as the mastermix . All of the raw data  The expression profile of the glioblastoma was compared with of each DSRCT. After appropriate corrections for preamplified samples, all of the analyses passed the quality control criteria. The data were normalised by using a housekeeping pool . The fold-changes were calculated using the 2 −ΔΔ Ct method with a value of 2 as a fold regulation cut-off value. The fold-changes are expressed as Log10.RNA was extracted as above described from one MErT, two hybrid/partial EMT and one EMT case, and 1 μg was retro-transcribed using the RT


Cervical degenerative disease is one of the most common spinal disorders worldwide, especially in older people. Anterior cervical corpectomy and fusion (ACCF) is a useful method for the surgical treatment of multi-level cervical degenerative disease. Anterior cervical disc replacement (ACDR) is considered as an alternative surgical method. However, both methods have drawbacks, particularly the neck motion decrease observed after arthrodesis, and arthroplasty should only be performed on patients presenting with cervical disc disease but without any vertebral body disease. Therefore, we designed a non-fusion cervical joint system, namely an artificial cervical vertebra and intervertebral complex (ACVC), to provide a novel treatment for multi-level cervical degenerative disease. To enhance the long-term stability of ACVC, we applied a hydroxyapatite (HA) biocoating on the surface of the artificial joint. Thirty-two goats were randomly divided into four groups: a sham control group, an ACVC group, an ACVC-HA group, and an ACCF group (titanium and plate fixation group). We performed the prosthesis implantation in our previously established goat model. We compared the clinical, radiological, biomechanical, and histological outcomes among these four different groups for 24 weeks post surgery. The goats successfully tolerated the entire experimental procedure. The kinematics data for the ACVC and ACVC-HA groups were similar. The range of motion (ROM) in adjacent level increased after ACCF but was not altered after ACVC or ACVC-HA implantation. Compared with the control group, no significant difference was found in ROM and neutral zone (NZ) in flexion-extension or lateral bending for the ACVC and ACVC-HA groups, whereas the ROM and NZ in rotation were significantly greater. Compared with the ACCF group, the ROM and NZ significantly increased in all directions. Overall, stiffness was significantly decreased in the ACVC and ACVC-HA groups compared with the control group and the ACCF group. Similar results were found after a fatigue test of 5,000 repetitions of axial rotation. The histological results showed more new bone formation and better bone implant contact in the ACVC-HA group than the ACVC group. Goat is an excellent animal model for cervical spine biomechanical study. Compared with the intact state and the ACCF group, ACVC could provide immediate stability and preserve segmental movement after discectomy and corpectomy. Besides, HA biocoating provide a better bone ingrowth, which is essential for long-term stability. In conclusion, ACVC-HA brings new insight to treat cervical degenerative disease. Cervical degenerative disease is a prevalent problem in our aging population. It can be in the form of herniated discs and spondylosis in the cervical spine associated with axial neck and/or radicular arm pain and neurological symptoms. Surgical and nonsurgical treatments have been used to halt or reverse the disease process. Posterior approachesas well as anterior techniques, such as anterior cervical discectomy and fusion (ACDF) and anterior cervical corpectomy and fusion (ACCF), are the most commonly used approaches.Because of its high rate of clinical success (relief of symptoms and favorable outcomes),anterior cervical discectomy and fusionhas been considered by many orthopedist and neurosurgeons as the standard surgical treatment for cervical myelopathy or radiculopathy, secondary to cervical degenerative disease and refractory to a conservative treatment. The antArtificial cervical disc replacement (ACDR) has developed and is now increasingly used as an alternative to ACCF. The first clinical trial involving spinal arthroplasty occurred between 1962 and 1970 and implied the implantation of Fernstrom ball in 207 cases. Nowadays, some clinical trials are currently testing various artificial cervical disc devices while some have been approved by the United States Food and Drug Administration. Arthroplasty seems to be as safe as ACCF and is becoming a substitute for fusion. Cepoiu-Martin et al. performeFor multi-level cervical degeneration disease, there is currently no method to rebuild cervical stability and preserve the motion function after decompression. There is no role for the indiscriminate use of ACDR. ACDR should simply not be considered as superior or as a replacement of fusion. Besides, if there is a vertebral body disease, such as a primary tumor, metastatic cancer, tuberculosis, vertebral body deformity, fusion is still the most commonly used surgical technique. To address these issues, we developed a novel artificial cervical vertebra and intervertebral complex (ACVC) that can provide immediate stability after anterior cervical corpectomy and maintain the motion of the surgery segment. Theoretically, this motion-preserving joint system may prevent adjacent segment degeneration.10(PO4)6(OH)2; HA) has been reported to have a high biocompatibility, a low degradation rate, and the ability to increase the efficiency of bone-implant integration in order to improve osteointegration of implants because of its similar composition to bone tissue and its ability tocreate chemical bonding withbones[2 and HA. This suggests that artificial joints pretreated with an HA coating may induce strong bonding with the bone via the natural growth of new HA. For this reason, we applied HA biocoatings to the surface of the ACVCs to determine whether the HA biocoating would increase the implant’s interaction with bone and improve its long-term stability.Moreover, long-term stability of prostheses is necessary for orthopedic implants. For uncemented prostheses, many parameters could affect long-term stability, including the implant design, surface texture, material biocompatibility, the presence of a bioactive coating, and the biomechanical properties of the surrounding bone. Various biocoatings have been proposed as effective methods for improving the bone-implant interface stability. In recent decades, hydroxyapatite  from the Animal center of Xi'an Jiaotong University Second Affiliated Hospital were randomly divided into 4groups. This included: a control group (Group A), an ACVC group (Group B), an ACVC-HA group (Group C), and an ACCF group (Group D). In the control group, each goat received a sham anterior cervical operation that fully exposed the anterior vertebral surface without disturbing the bony, disc and ligamentous structures. In the ACVC and ACVC-HA groups, ACVC and ACVC-HA were separately implanted in the goats after decompression. In the ACCF group (decompression and fusion group), each animal received an anterior cervical decompression and fusion, using a titanium mesh cage and an anterior plate. To exclude cervical spine abnormalities, radiological images were obtained using a C-arm fluoroscopic imager before surgery.All the internal instruments were made by professional engineers in Northwest Nonferrous Metal Research Institute (China) and were composed of titanium alloy (Ti6Al4V). The ACVC  with 12-hours dark-light cycle. Each goat was given access to food and water 2 through 8-mm diameter endotracheal tube. Then the surgeries were performed in supine position by a transverse skin incision at the C3 level from the right side[The surgical procedures and postoperative animal care have been well documented in our previous study. Brieflyight side. The antSurgery time, blood loss, recovery time, and time before eating were recorded. Surgery time was the period from skin incision to skin suture. Blood loss was estimated using a vacuum extractor and blood gauze. Recovery time was the period from the end of surgery until the goat could stand by itself. Time before eating was the period from the completion of surgery until the goat began to eat by itself. Food and water intake was limited during the first 24 hours after surgery to reduce the risk of intestinal tympanites. A normal diet was established two days after surgery. Cefazolin (25 mg/kg) was administered twice a day for two days after surgery. Goat care was ensured by trained animal care staff. Eating habits, ambulatory activities, health status, and neurological functions were monitored daily for the first 4 weeks, twice weekly for Weeks 5 to 12, and weekly from the 12th to 24th week.Post-surgery pain was the major suffering during the post-surgery recovery. Analgesia or anesthesia was used to relieve the post-surgery pain. NSAIDS  and opioids (such as meperidine and morphine) were used to relieve the post-surgery pain if necessary. Soft food (such as cabbages) was provided within the first week after surgery.Anteroposterior and lateral radiographic films were taken under general anesthesia at weeks 1, 6, 12, and 24 after surgery. Computed tomography (CT) analyses were performed to determine the extent of bony fusion and the position of the prosthesis at weeks 6, 12 and 24 post-surgery. All animals were observed for 24 weeks before euthanasia with an overdose of pentobarbital (200 mg/kg).Criteria for early euthanasia during the experiment for humane endpoints: weight loss of 20–25%, inappetance up to 5–7 days, severely weakness, moribund state, infection fails to respond to antibiotic therapy, and severe organ system dysfunction non-responsive to treatment.The biomechanical test protocol has been described previously . To inveintact − ROMinstrumented)/ROMintact, and SI NZ = (NZintact − NZinstrumented)/NZintact.To determine the stability of the specimens in the different groups, the C6 and C7 segments were removed. A multidirectional flexibility test was used in a non-destructive manner for the C1-C5 segments. The end vertebrae of the specimens  were transfixed with perpendicular pins, to enhance the fixation with mounting jigs. The C1 to C5 vertebrae were then mounted in fast-drying epoxy resin  Limited, HK). A fatigue test comprised of 5,000 repetitions of axial rotation  was used during the kinematic test. A similar biomechanical test was performed. The kinematic behavior of each specimen was compared by examining the range of motion (ROM) and the neutral zone (NZ) of the C2-C4 segments of the intact and instrumented goat spines from the fifth loading cycle before and after the fatigue test. Throughout the biomechanical testing, special care was taken throughout the tests to keep the specimens moist using a 0.9% saline solution. A stability index ROM (SI ROM) and a stability index NZ (SI NZ) were introduced to quantify the stability provided by the implantation to the spine. These indices were similar to those described by Zhang et al. . The SI After the biomechanical testing, each sample was fixed in cold Karnovsky fixative containing 4% paraformaldehyde and 5% glutaraldehyde for two weeks. The specimens were dehydrated in an alcohol series and embedded in polymethyl-methacrylate. Transverse sections of 100 μm thickness were carried out with a Leica SP1600 sawing microtome . The specimens were then ground to the desired thickness. Masson’s trichrome staining and Van Gieson staining were used for transmitted light microscopy.All results were analyzed using SPSS Version 13.0 . The data regarding surgery characteristics, ROM, NZ, and stiffness were analyzed using are repeated-measure analysis of variance (ANOVA). Post hoc tests were performed using Bonferroni correction for multiple comparisons. The level of statistical significance was set at P<0.05.All surgeries were successful. Surgery time, blood loss, recovery time, and time before eating are shown in The goats tolerated the artificial joints very well during the experiment. All goats survived until 24 weeks after surgery. No abnormal neurological reflexes or neurological complications were found. One goat in the ACVC group and two goats in the ACCF group developed wound infections 8 to 12 days after surgery. These wound infections were cured by applying antibiotics and changing bandages daily. One goat in the ACVC-HA group and another goat in the ACCF group suffered respiratory infections and recovered by using antibiotics.No bone fracture, joint dislocation, or screw loosening were found during the experimental period. To investigate the cervical spine ROM separately, we used the three-dimensional laser optoelectronic camera system . There wThe biomechanical data of the C2-C4 segment recorded before the fatigue test  and 4 coThere was no significant NZ difference in flexion-extension and lateral bending among control, ACVC and ACVC-HA groups. In rotation, NZ was significantly greater for ACVC and ACVC-HA groups than for the control group. For the ACCF group, NZ was significantly decreased in all directions. The stiffness was significantly decreased in all directions for the ACVC and ACVC-HA groups, while it was increased for the ACCF group. We found similar results for the ROM, NZ, and stiffness in the ACVC and ACVC-HA groups. Similar C2-C4 biomechanical data were found after the 5,000repetitions of the axial rotation fatigue test .The SI ROM and SI NZ are reported in Histology sections of the goat cervical spines showed no inflammatory response 24 weeks after surgery, as well as no significant metallic wear debris at the implant-bone interface. Newly formed bone was found around the vertebral body component of the ACVC and the self-drilling fixation screws.In the sections located at the ACVC vertebral body component level , we founThe magnified images showed that bone formation around the implant surfaces was fragmentary and that a large portion of the bone tissue was separated from the implant surface by soft tissue in the ACVC group . In contACCF and ACDF have been considered to represent the standard surgical treatments of cervical myelopathy or radiculopathy with degenerative disc disease that are refractory to conservative treatment. Because of its high rate of clinical success in relieving symptoms, ACCF is usually chosen to treat multi-level cervical degeneration disease. The surThe two main advantages of this metal-on-metal unconstrained artificial cervical joint system are its ability to allow physiological motions and its potential to reduce stress at the bone-implant interface. This articulation allows anterior-posterior translation, constrained flexion-extension, and unconstrained rotation, which theoretically mimics the normal cervical physiological motion. To eliminate the risk of dislocation, it is important to have the articulation closed and the posterior longitudinal ligament and surrounding soft tissues aid in providing stability.Besides, we designed several structures to enhance the long-term stability, such as the HA biocoating, the rough and grit-blasted endplate component, and the hollow vertebral body component. Autologous grafting is the gold criteria for fusion in various types of ACCF techniques because it has a high fusion rate compared with discectomy alone or implanted cages. However, the main shortcoming of autologous bone graft is the harvesting of bone, primarily from the iliac crest, which may results in short- and long-term morbidities. These include increased blood loss, infection, donor site pain, hematoma, and peripheral nerve injury. CompareThe selection of biomaterials for the artificial joint system depend on the good mechanical properties (high strength and low modulus closer to bone), biocompatibility, superior corrosion and wear resistance, and osseointegration. The reasons we chose Ti6Al4V were because of its adequate mechanical properties, good biocompatibility, valuable imaging characteristics, and low elasticity modulus. Besides, there was no acute neural orsystemic histopathologic response to the Ti6Al4V included in a 6-month in vivo animal model. But lonWe designed this ACVC implant to preserve motion in the degenerative segment of the cervical spine after the anterior cervical discectomy and subtotal corpectomy. To test the ACVC biomechanical properties, we compared its kinematic data with those of the intact and ACCF states.First of all, we investigated the adjacent segmental movement. Our data confirmed that once the cervical vertebrae (C2-C4) were fused, the adjacent segment (C4-C5) would compensate for the decreased ROM. Finally, the total motion of the cervical spine (C2-C6) was still less than that of the control group. In contrast, by using this novel motion-preserving cervical joint system, namely ACVC or ACVC-HA, the movement of C2-C4 could be preserved and C4-C5 would not compensate for the altered ROM in flexion-extension and lateral bending. Moreover, rotation increased at the C2-C4 level after ACVC and ACVC-HA implantations, which resulted in a decreased ROM for the C4-C5 and C5-C6 segments. Park et al. reportedCompared with the intact state (control group), no difference was observed for both ACVC and ACVC-HA groups in the ROM and NZ during flexion-extension and lateral bending. During rotation, the ROM and NZ were increased. The stiffness was decreased in all directions. The fold change of stiffness decrease in rotation (0.10) was much greater than that in flexion-extension (0.48) and lateral bending (0.38). Compared with the ACCF group, the ROM and NZ were significantly increased and the stiffness decreased in all directions. These biomechanical results indicated that thanks to the ball-in-trough articulation structure design, the ACVC could preserve the three-dimensional motion of cervical spine. Compared with the intact state, the fold change of ROM decrease in the ACCF group was much greater in flexion-extension (0.09) than in lateral bending (0.23) and rotation (0.71). The fold change of NZ decrease was also much greater in flexion-extension (0.25) than in lateral bending (0.27) and rotation (0.42). The fold change of increase in stiffness was much greater in lateral bending (×19.46) than in flexion-extension (×11.43) and rotation (×7.29). The cylinder titanium cage combined with the anterior plate successfully provided strong stability in all directions. A positive, null, or negative stability index value indicates that the spinal construct was respectively, more stable than, equally as stable as, or less stable than the intact spine. Therefore, a higher stability index value indicates a greater immediate stability of the spinal construct . In the All of the above results indicate that, compared with the intact state and the ACCF group, the ACVC (or ACVC-HA) successfully restored the flexion-extension and lateral bending abilities after discectomy and subtotal corpectomy. The absence of any biomechanical difference before and after the fatigue test for every group indicate that the fixation screw could provide primary stability.The histological results indicated that the amount of new bone formed was much greater in the ACVC-HA than the ACVC group. Furthermore, the bone-implant contact was much higher in the ACVC-HA than the ACVC group. This higher bone-implant contact indicate that the HA biocoating offered great osteoconductivity.In the sections located at the screw level, we found fibrous tissue around the screws in the ACVC group and newly formed bone in the ACVC-HA group. This may have occurred because there were less micro-motion and osteolysis after implantation in the ACVC-HA than the ACVC group. The HA coating formed a strong interface between the bone and screws, providing strong initial stability in the early stage after implantation. The MAO-treated, HA-coated screws significantly improved the bone reaction to implant and increased the fixation stability. Thus, they served as an effective method to decrease screw-loosening by providing both mechanical interlocking and biochemical support at the bone-implant interface.The histological results suggest that the HA coating may encourage bony ingrowth to provide a morphological fixation of the implant to the bone, which may improve the interlocking between the implant and the bone. The bony ingrowth into the implant is accelerated by the bioactivity and the osteoconductive property of the HA coating. The HA coating on the titanium alloy implant increased the osteoconductivity and inhibited soft tissue infiltration, thus offering a mechanical interlocking attachment for short- and long-term fixation. Furthermore, the HA coating immobilizes osteoblastic cells and facilitate vascularization in bone tissue at the early stage after implantation. During the later stage, peri-implant bone formation was stimulated by the calcium ions degraded by the HA coating .Our histological results are in agreement with the findings of other studies that have used calcium-phosphate ceramics for various applications. Lim et al. performeSeveral limitations are inherent to this study. First of all, this is an in vivo animal study. The length and size of cervical vertebrae in goat is different from those of humans. Besides, in humans, the most common symptomatic levels are C5/6 and C6/7. Agenetic and environmental etiology may likely explain this difference. In our study, we choose the C2-C4 level as the surgery level, because of the biomechanical properties of C2-C4 in goat, which is similar to those of humans. Secondly, the follow-up duration is limited. In animals, it may be appropriate to follow an in vivo study up to only 24 weeks. However, it is difficult to comment on the function and longevity of a device designed to remain intact and preserve motion after only 24 weeks in humans. That is why we included a fatigue protocol in the biomechanical test, though the number of fatigue cycles was limited as well. Thirdly, we are planning to conduct further experiment aiming at improving the durability and stabilization capacity of our ACVC for clinical application, such as applying biocoating on the surface of ACVC to improve the bone-implant integration for long-term stability. Fourthly, the low wear resistance of Ti6Al4V was the main shortcoming of this joint system, which may result in implant loosening and wear debris. Thus we are planning to develop a metal-on-UHMWPE articulation to solve this problem.We have successfully performed the in vivo implantation of ACVC, ACVC-HA and titanium cage in goats. Goat has been proven to been excellent animal model for cervical spine biomechanical studies, especially for the artificial joints and biomedical materials. The biomechanical results indicate that, compared with intact state and ACCF, ACVC and ACVC-HA provide stability and preserve segmental movement for at least 6 months after discetcomy and corpectomy. The histological results demonstrate that hydroxyapatite biocoating provide better bone ingrowth, which is essential for long-term stability. Also, ACVC-HA brings a new insight to treat cervical degenerative disease.


This study aimed to evaluate the effects of ischemia-reperfusion injury (IRI) on the risk of hepatocellular carcinoma (HCC) recurrence after liver transplantation. Data of 195 patients were retrospectively analysed. Post-reperfusion aspartate (AST), alanine transaminase, and lactate dehydrogenase (LDH) levels were the primary measures of IRI. Tumour recurrence was the primary endpoint. Post-reperfusion AST was a continuous risk factor for tumour recurrence in patients within Milan criteria (p = 0.035), with an optimal cut-off of 1896 U/L. Recurrence-free survival of patients within Milan criteria and post-reperfusion AST of <1896 and ≥1896 U/L was 96.6% and 71.9% at 5 and 3.7 years, respectively (p = 0.006). Additionally, post-reperfusion AST and LDH exceeding the upper quartile significantly increased the risk of HCC recurrence in patients within Milan criteria  and to a lesser extent, in patients within Up-to-7 criteria . No other significant IRI effects were found in patients beyond the Up-to-7 criteria and in analyses stratified for independent risk factors for recurrence: tumour number and differentiation, alpha-fetoprotein, and microvascular invasion. Thus, IRI exerts major negative effects on the risk of HCC recurrence after liver transplantation in patients within standard and extended criteria. The Milan criteria defined transplant eligibility for HCC patients for more than two decades; however, the limits are now being expanded according to morphological and biological tumour features6. Nevertheless, discussion on widening the pool of potential candidates is controversial owing to a major and relatively constant shortage of deceased donors. Further expansion of the selection criteria will inevitably lead to increased waiting times for both HCC and non-HCC populations. In HCC patients, markedly prolonged times on the waiting list are characterised by more common dropouts, possibly leading to the development of more aggressive tumours7. Owing to increased rates of listing and privileged positions of HCC patients under the current allocation policies, a higher number of HCC transplant candidates may have even more detrimental effects on non-HCC patients’ waiting times and pre-transplant mortality9. Because widening the donor pool with living donors and high-risk or extended criteria deceased donors is a common strategy, it appears to have major relevance, particularly for HCC patients.Hepatocellular carcinoma (HCC) remains one of the most common indications for liver transplantation11. Changes in hepatic microenvironment caused by IRI promote seeding and the development of metastases, whereas IRI-induced proinflammatory response, release of growth factors, mobilization of progenitor cells, and transformation of cancer cells to more aggressive phenotypes may potentiate the formation and growth of metastases at both local and remote sites16. Because grafts procured from high-risk deceased donors and to a lesser extent, partial grafts procured from living donors may be more susceptible to IRI, the use of these grafts may increase the risk of post-transplant HCC recurrence. This hypothesis was subject to numerous studies with inconsistent results. Although transplantations of grafts procured from living donors or high-risk grafts procured from deceased donors after cardiac death or those who were older and had hepatic steatosis or other risk factors were reported to have adverse effects on outcomes after liver transplantations for HCC in several studies, the results of available studies are not completely consistent23. However, recent reports found that prolonged ischemic times, directly related to the magnitude of IRI, increased the risk of post-transplant HCC recurrence25. Nevertheless, data on the direct effect of the magnitude of IRI on the risk of HCC recurrence after deceased donor liver transplantation are limited. Therefore, this study aimed to evaluate the association between the degree of graft IRI as indicated by post-reperfusion transaminase and lactate dehydrogenase (LDH) levels and the risk of post-transplant HCC recurrence after deceased donor liver transplantation with respect to patients’ initial risk profile.Experimental studies demonstrate the increased risk of cancer recurrence associated with ischemia-reperfusion injury (IRI)This was a retrospective observational study. In total, 250 liver transplantations were performed for HCC patients between January 2001 and June 2016 at the Department of General, Transplant and Liver Surgery . Patients with fibrolamellar HCCs and those with combined HCC/cholangiocarcinoma were not included. After exclusion of 55 patients with missing measurements of transaminase levels 2 h after reperfusion, the final study cohort comprised 195 liver transplant recipients. The study protocol was approved by the institutional review board of the Medical University of Warsaw. Informed consents were not obtained from the patients due to the retrospective nature of the study, which is in line with institutional review board and national regulations. All methods were performed in accordance with the relevant guidelines and regulations. No organs were procured from prisoners.27.The degree of IRI was represented by three variables, namely, serum alanine transaminase (ALT), serum aspartate transaminase (AST), and serum LDH levels; each was assessed from a blood sample obtained 2 h after portal reperfusion. These variables were the primary factors of interest. Peak serum bilirubin concentration, international normalised ratio (INR), and gamma-glutamyl transpeptidase (GGTP) activity over the first 7 post-transplant days were additionally analysed as variables associated with IRI. The duration of cold and warm ischemia was defined as the time from clamping of the donor aorta until the removal of the graft from the preservation solution and that from the removal of the graft from the cold preservation solution until portal reperfusion, respectively. The sum of cold and warm ischemic times formed the total ischemic time. All grafts were procured from donors after brain death. Tumour recurrence over the 5-year post-transplant observation period was the primary end-point. Recurrence-free survival was calculated from the date of transplantation until tumour recurrence and censored at the date of last available follow-up, death for non-HCC related causes or 5 years post-transplantation (whichever occurred first). Details on the surgical technique, perioperative care, immunosuppression, and follow-up protocol are provided elsewhereFirst, post-reperfusion ALT, AST, and LDH levels were assessed for their potential effect on the risk of post-transplant tumour recurrence in all patients. Other independent predictors of recurrence were also assessed, including peak post-transplant bilirubin concentration, INR, and GGTP activity. Furthermore, the analyses were adjusted for their potential confounding effects in bivariable analyses. Subgroup analyses were subsequently performed to determine the potential differences in associations between IRI degree and the risk of HCC recurrence according to patients’ initial risk profile, based on fulfilment of selection criteria and established independent predictors of recurrence.Continuous and categorical variables are given as medians (interquartile ranges) and numbers (percentages). The Kaplan-Meier method was used for survival calculations, and log-rank test was used for intergroup comparisons. A Cox proportional hazards regression analysis was performed to evaluate the associations between particular factors and the risk of recurrence. A multivariable model was created using forward stepwise method with p thresholds of 0.05 and 0.150 for inclusion and exclusion of variables, respectively. An additional series of bivariable analyses were performed to adjust the effects of IRI to potential confounding effects of independent risk factors for tumour recurrence. Spearman correlation coefficients were calculated to evaluate the associations between ischemic times and donor age and post-reperfusion laboratory measurements. Post-reperfusion AST, ALT, and LDH levels; peak post-transplant bilirubin concentration; and peak post-transplant GGTP activity were transformed to their natural logarithms prior to their analyses as continuous variables. Additionally, they were assessed as categorical factors using the upper quartile for division. Receiver operating characteristic (ROC) curves were constructed to determine the optimal cut-offs of continuous factors in predicting recurrence. Hazard ratios (HRs) and c-statistics were presented with 95% confidence intervals (95% CIs). Significance threshold was set to two-tailed p values of 0.05. Analyses were computed in STATISTICA version 13  software. The datasets generated and/or analysed during the current study are available from the corresponding author on reasonable request.The characteristics of the 195 patients are shown in Table The median follow-up period was 37.5 months. A total of 27 patients developed HCC recurrence with recurrence-free survival rates of 90.8%, 83.4%, and 81.0% at 1, 3, and 5 years, respectively. Univariable analyses revealed that post-reperfusion AST (p = 0.521), ALT (p = 0.773), and LDH (p = 0.575) levels and peak 7-day post-transplant bilirubin concentration (p = 0.592), INR (p = 0.553), and GGTP activity (p = 0.534) were not significantly associated with recurrence in all patients (Table For further analyses, the patients were divided into subgroups based on the fulfilment of selection criteria and independent predictors of recurrence. Cut-offs for tumour number of ≥3 and alpha-fetoprotein concentration of ≥48.3 ng/ml were derived from the corresponding ROC curves. As a continuous variable, post-reperfusion AST significantly influenced the risk of HCC recurrence only in patients within the Milan criteria (p = 0.035, Table In patients within the Milan criteria, recurrence-free survival at 1, 3, and 5 years was 98.8%, 96.6%, and 96.6%, respectively, when post-reperfusion AST level was <1896 U/L as opposed to 86.2%, 86.2%, and 71.9% at 1, 3, and 3.7 years, respectively, when post-reperfusion AST level was ≥1896 U/L (p = 0.006, Fig. 16. Our study results demonstrate a major negative effect of IRI on the risk of post-transplant HCC recurrence, although limited to patients with low tumour burden.In the era of donor shortage and increasing utilization of high-risk grafts to partly ameliorate its negative effects, the problem of potential association between the degree of IRI and the risk of HCC recurrence after liver transplantation is of utmost importance. According to the available results of experimental studies, hepatic IRI, universally present in the setting of liver transplantation, increases the risk of metastasis formation both within the ischemic and remote sites through changes in the local microenvironment, induction of inflammatory response, induction of metastatic potential of circulating cancer cells, and systemic release of pro-tumourigenic cytokines5. Nevertheless, a major significant negative effect of post-reperfusion AST and LDH levels was observed for patients within the Milan criteria, which still determine the majority of liver transplant recipients28. Similar findings, although of remarkably lesser extent, were found for patients within the Up-to-7 criteria, whereas the magnitude of IRI did not influence the risk of recurrence in patients beyond the extended criteria. This indicates that the clinical relevance of IRI is limited to generally low-risk populations and diminishes with increasing tumour burden. This appears to be particularly importantly because it demonstrates the possibility of using high-risk grafts to expand the donor pool for high-risk HCC candidates in the context of discussion on widening the boundaries of existing selection criteria29. Notably, the safe use of extended criteria allografts preferentially for patients with advanced tumours was already reported30. Conversely, none of the subgroup analyses performed in high-risk patients, including those beyond particular selection criteria, with ≥3 tumours, alpha-fetoprotein concentration ≥48.3 ng/mL, or with tumours either poorly differentiated or with microvascular invasion, revealed a significant effect of IRI on the risk of HCC recurrence. Therefore, while these findings point toward the possibility of the utilization of grafts more prone to IRI for high-risk HCC patients, they also indicate limited clinical relevance of reducing IRI in these patients.Importantly, initial analyses performed in all patients failed to reveal any significant associations between post-reperfusion AST, ALT, and LDH levels and HCC recurrence risk, irrespective whether the factors were analysed as continuous or categorical variables. However, the study cohort comprised patients with a wide range of tumour burden due to a liberal selection policy utilised in the authors’ department before establishment of precise criteria31. However, the degree of IRI is driven by the interplay of several donor risk factors, of which a single component may not necessarily be an adequate measure of IRI32. In the present study, the laboratory measures of graft ischemia were significantly, yet poorly correlated to graft ischemic times, which in fact is consistent with the results presented by other authors25. This may partly explain the inconsistent results of studies on the effect of duration of graft ischemia and particular donor factors on HCC recurrence risk, as these may not always accurately reflect the magnitude of IRI31.In contrast to the use of post-reperfusion transaminases and LDH levels as surrogates of IRI degree in the present study, previous studies focused on the negative effects of prolonged graft ischemia or donor characteristics25. These populations were characterised by 18F-fluorodeoxyglucose tumour avidness on pre-transplant positron emission tomography and vascular invasion, both of which are known surrogates of biological aggressiveness. Although positron emission tomography data were not available, categorization of patients based on pre-transplant alpha-fetoprotein concentration and tumour differentiation, which are important markers of tumour biology, did not reveal any significant effects of IRI and neither did the analyses stratified for microvascular invasion. The reason for this discrepancy is unclear, although it may be related to a wider spectrum of tumour burden in patients included in the present study. Of note, post-operative peak transaminases did not emerge as risk factors for HCC recurrence in these previous reports. However, we chose post-reperfusion AST, ALT, and LDH levels routinely assessed in our department and not peak levels over the postoperative period in order to minimise the effect of events other than IRI on these parameters.In contrast to the significant effects of IRI limited to low-risk patients found in the present study, two previous analyses specifically aimed at the effect of ischemic times on tumour recurrence revealed the presence of significant associations particularly in high-risk HCC patients33. The protective effects of ischemic preconditioning with respect to the development of metastases were also reported in a recent experimental study14. The use of machine perfusion devices has also been shown to decrease the magnitude of IRI and recently even enabled the development of a strategy to practically eliminate its negative consequences36. Although the present study does not provide any evidence for the effects of these measures in liver transplantation for HCC, it provides a rationale for prospective trials aimed at addressing this issue.The results of the present study point toward the importance of strategies aimed to decrease IRI particularly for patients within the standard selection criteria. A single retrospective study revealed decreased magnitude of IRI, as illustrated by low transaminase levels and decreased risk of HCC recurrence in patients receiving prostaglandin E1 analog alprostadil in the early period after liver transplantation23. Although subject to additional warm ischemia and thus potentially increased magnitude of IRI, their use in HCC patients may be confounded by other factors, including but not limited to, non-random allocation and differences in other donor characteristics. Furthermore, the duration of warm ischemia was not identified as a significant predictor of HCC recurrence. Finally, the main findings of our study are based on the results of univariable subgroup analyses. Therefore, the findings may be confounded by the effects of other risk factors for tumour recurrence. Although there was no particular policy at the authors’ department for the allocation of high-risk grafts to higher-risk HCC patients, the results may also be confounded by non-random allocation of grafts more prone to IRI to patients within the Milan or Up-to-7 criteria, yet at higher initial recurrence risk.This study had several limitations besides those inherent to its retrospective nature. Donor characteristics other than a baseline variable of age were neither analysed for associations with post-reperfusion transaminase and LDH levels nor as predictors of tumour recurrence. However, such analyses were beyond the scope of this study, specifically aimed at the effect of IRI on post-transplant HCC recurrence rather than on its determinants. Because all recipients received grafts from donors after brain death, this study did not directly address the issue of using grafts from donors after cardiac death for HCC patients, which was recently shown not to increase the risk of post-transplant recurrenceIn conclusion, the magnitude of IRI is strongly associated with the risk of tumour recurrence in patients within the Milan criteria and to a lesser extent, in patients within the extended criteria. Available measures to decrease IRI should be evaluated as a method to prevent HCC recurrence after liver transplantation, specifically in patients with low tumour burden.


Owing to the alkyl-chain interdigitation, nanoparticles very close to each other result and an interaction among their surface plasmon resonances may take place. Surface plasmon interaction causes a splitting of the absorption band whose characteristics depend on the aggregate shape. Since shape-less aggregates are generated, a multiple-splitting of the silver surface plasmon absorption band is observed, which causes a broad absorption spreading on the whole visible spectral region. Amorphous polystyrene containing interdigitated silver nanoparticles has a dark-brown or black coloration, depending on the nanoparticle numerical density, but since the inter-particle distance slightly increases at melting point of interdigitation crystallites a reversible termochromic effect is observed at this special temperature. In particular, the material coloration changes from dark-brown to yellow which is the coloration produced by the surface plasmon absorption of isolated silver nanoparticles. This reversible thermochromism can be finely controlled by modifying the structure of thiolate groups, and precisely, the strength of interactions acting inside the interdigitation crystallites.Technologically useful reversible thermochromic materials can be prepared using very simple polymer-embedded nanostructures. In particular, silver nanoparticles capped by long-chain alkyl-thiolate molecules (i.e., Ag The properties of metals and semiconductors mainly depend on their electronic configuration, which results significantly modified when they are reduced to a nanoscopic scale. Consequently, these solids may show anomalous physical behaviors as their size approaches to a few nanometers ,2,3,4,5,The mesoscopic regime extends over a dimensional range which is depending on the type of phenomenon under consideration, and it may range from 1 nm to 100 nm for certain phenomena, or be very close to only one nanometers for others. For example, to observe luminescence in metallic phases a size of a few nanometers is required  ,7,8, whiNanoscopic solids can be used in an embedded form and polymers represent a very convenient type of matrix. Usually, these nanostructured materials are named nanocomposites because they are morphologically similar to the discontinuous polymeric composites ,15,16,17A conveniently selected polymer containing nanoscopic metal or semiconductor particles may show some of the physical characteristics of the hosted phase. A functional material whose properties are based on some anomalous physical behavior of matter at mesoscopic regime represent a ‘mesoscopic device’ . Example2 may diffuse into the polymer matrix, modifying the chemical nature of the metal surface and therefore affecting significantly the functional properties of the nanoparticle system. Actually, also noble-metal nanoparticles can be chemically modified at surface owing to the enhanced chemical reactivity of nanoscopic metals. Usually, a chemical passivation treatment of the metal nanoparticle surface is the only way to achieve stable functional properties for these systems. Transition metal nanoparticles are high electrophilic species owing to the presence of empty d- and f-orbitals of the surface atoms, and therefore they may absorb nucleophilic ligand molecules on their surface, leaving to nanoparticle compounds. For example, strong ligand molecules like phosphines  can be stably bonded to the metal nanoparticle surface leaving to air-stable nanoparticle compounds.Therefore, metal and semiconductor nanoparticles can be used as building-blocks of a mesoscopic device. Polymer-embedding prevents naked-nanoparticles from aggregation and self-assembly, however these nanoparticles are quite unstable also in a polymer-embedded form because of surface oxidation and/or contamination phenomena. Small molecules like oxygen, water, SOHowever, thiolate-capping is a very common chemical passivation treatment for noble-metal nanoparticles, like for example the thiolate-capped gold nanoparticles, named thioaurites . Thioaurn, Ptn, Pdn, etc.) may give thiol-derivatized nanoparticles that are much more stable than the starting structures [nH2n+1-SH), containing a number of carbon atoms higher than 10 have an important physical characteristic, consisting in the co-crystallization of alkyl chains present on the surface of neighbor nanoparticles by interdigitation , two different topologies are possible for such systems: (i) nanoparticles may form a contact-free dispersion when capped by short-chain thiolate, and (ii) nanoparticles may leave to small coagulation aggregates when coated by long-chain thiolate. In the first case, nanoparticles are perfectly distributed inside the embedding dielectric matrix and an uniform inter-particle distance (much larger than the nanoparticle diameter) characterizes this system. In the latter, nanoparticles form small aggregates uniformly dispersed inside the amorphous polymeric matrix, and inside these aggregates, nanoparticles touch each other but their surfaces are separated by the thin thiolate-capping mono-layers and therefore their surface appear located at a distance of approximately twice the thickness of thiolate coating. In particular, this type of nanoparticle aggregates are named ‘coagulation aggregates’ because they are different from the ‘coalescence aggregates’, where the nanoparticle metallic cores result sintered together . When thIn the case of nanoparticles capped by thiolate molecules and organized in form of aggregated structures, because of thiolate chain interdigitation, the optical properties of the resulting polymer-embedded systems are strongly dependent on the temperature. In fact, the optical properties result significantly modified at melting point of crystallites produced by the interdigitated chains, showing a step-discontinuity closed to this special point.Therefore, optical devices based on some nanoparticle property can be classified as passive and active devices. In a passive optical device, the functional properties are not depending on external stimuli like temperature, pressure, etc. On the contrary, the functional properties of an active optical device are influenced by the effect of external stimuli like the temperature. For example, an amorphous polymer containing aggregates of thiolate-capped metal nanoparticles represents an example of active device. In fact, if the aggregates have an average size comparable with light wavelength a scattering phenomenon takes place in the film which results opaque. However, at high temperatures  the aggregates dissolves and diffuse into the polymer matrix, leading to a contact-free topology with large inter-particle distance. This material is transparent to the visible light.The surface plasmon resonance of a metal nanoparticle system is very sensitive to the inter-particle distance , and tecWhen a polymeric dispersion of aggregated silver particles transforms to a contact-free particle dispersion by the effect of a temperature increase, the system coloration switches from brown to yellow at melting point of crystallites produced by the interdigitation of thiolate-capping layers. Therefore, a variety of reversible thermochromic plastics can be simply produced by dispersing Ag nanoparticles capped by long-chain thiolates into a polymer. The color switching temperature depends on the melting point of interdigitated crystallites, since at this special temperature the inter-particle distance changes as a consequence of the expansion associated with melting process. Being the color change based on a thermodynamic transition , the thermochromism is completely reversible, quite prompt and without hysteresis.3(CH2)n- or HO-(CH2)11-) are chemical compounds not commercially available, and therefore they were obtained by chemical synthesis [3, Aldrich, 99.9%) in acetonitrile  in a dropwise manner at room temperature, under stirring (stoichiometric amounts of reagents were used). An exchange reaction (methatesis) between thiol proton and silver atom took place, according to the following scheme:Thermochromic films made of thiolate-capped silver nanoparticles embedded into amorphous polystyrene have been prepared starting from the corresponding silver-thiolate precursors whose characteristics are given in ynthesis ,23. In pA white microcrystalline precipitate was immediately produced. The solution was stirred for 24 h at room temperature, then silver(I)-thiolates was separated by vacuum-filtration, washed several times with acetone, and stored in a dry atmosphere until use.w = 230,000 g·mol-1) dissolved in chloroform. All samples were prepared using a 1:10 AgSR/PS weight ratio. The resulting AgSR/polystyrene blends were white colored and opalescent for the presence of AgSR micro-crystals. Then, the blends were isothermally annealed at 200 °C for 3 min using a hot stage for optical microscopy (Mettler). During the AgSR thermolysis stage, a fine dispersion of thiolate-capped silver nanoparticles aggregates in polystyrene were produced [12H25/polystyrene blends is shown in Homogeneous AgSR/polymer blends were obtained by slowly drying (for two days at room temperature) a mixture of AgSR and polystyrene  to bright-yellow. To produce very dark films the presence of a large amount of aggregates of thiolate-capped silver nanoparticles was required. The formation of these aggregates can be favored by causing the presence of strong concentration gradients inside the precursor silver-thiolate/polymer blend. Such silver-thiolate concentration gradients can be simply generated into the thiolate/polymer blend by a fast evaporation of the solvent (chloroform) during the film casting stage. In this case, the solution was simply put under the air flux of a fume hood, which continuously removed the solvent vapors from the liquid phase surface, allowing a fast system drying.2S,  was present in very low amount.In order to understand the crystalline structure of the silver nanoparticles, X-ray diffraction  was carried out to investigate the atomic structure of small nanoparticles. The typical XRD pattern of the as-prepared nanocomposites is shown in ±0.1 °C, the commercial Peltier device was modified by increasing the limit temperature of the hot-stage and including a water refrigeration system. In addition, to completely remove the contribution of polystyrene absorption from the optical spectra, which is quite relevant in the UV region, pure amorphous polystyrene films with a thickness exactly equal to the analyzed thermochromic films were placed in the reference sampling area and heated at the same temperature like the thermochromic samples.The temperature dependence of optical properties of the obtained nanocomposite materials, was determined by measuring the UV-visible absorption spectra of films isothermally heated at growing temperature values. This spectra were measured using a PerkinElmer Lambda 850 spectrometer equipped with a Peltier apparatus . In order to analyze the optical spectra in the temperature range 0–200 °C with an accuracy of The thermal characteristics of nanocomposite samples  were evaluated by dynamic tests performed by a Differential Scanning Calorimeter , calibrated by indium standard. Samples of 7 mg were sealed into hermetic aluminum pans and the DSC tests were done under a purging atmosphere of nitrogen gas from 70 °C to 180 °C at a rate of 10 °C/min for the different thermochromic samples.The nanocomposite samples in form of films showed a well-visible thermochromic effect, characterized by a prompt and reversible color change from dark-brown (or even black) to bright-yellow. To determine the color transition point, the thermochromic films were dipped into ethylene glycol (a non-solvent liquid for polystyrene) and the temperature increased from room temperature to 200 °C, at a controlled rate of 1 °C/min. The transition point was simply determined by eyes. The color transition temperatures found for nanocomposites based on silver nanoparticles capped by different types of thiolate molecules are given in The thermochromic effect was accurately investigated by spectroscopic characterization (UV-Vis absorption spectroscopy) of nanocomposite films at different temperatures. To understand the spectral change in The appearance of longitudinal oscillations is a well-known effect predicted in the framework of the Mie theory for aggregated nanoparticle structures . While, The color change should be related to a variation of the inter-particle distance at a special temperature and in particular above this temperature, nanoparticles do not anymore interact among themselves since they are located a higher distance and a substantial reduction of the longitudinal oscillations follows see b. TherefOptical spectra recorded for different times at temperatures above the color transition point have shown that these nanocomposite films are characterized by a very stable optical absorption during the time.2)11SAg in The optical behavior of nanocomposite films prepared by thermal decomposition of silver hexadecyl-thiolate see , silver In order to evidence the correlation between the color change and the melting of the interdigitated thiolate-capping crystallites, thermochromic films were analyzed by Differential Scanning Calorimetry (DSC). All thermograms included a first-order transition corresponding to the polystyrene glass transition temperature (Tg) at ca. 89 °C, followed by an endothermic peak. The extension of endo-thermal peaks and the corresponding enthalpy values are given in The endo-thermal signals visible in the DSC thermograms have been attributed to the melting of the crystallites generated by interdigitation of thiolate chains present on the surface of neighbor nanoparticles. As visible, the color transition points are located exactly in the temperature range of crystallite melting estimated by DSC, consequently the color switching should be related to the melting of interdigitated thiolate-capping crystallites and precisely it should be caused by the little change of the inter-particle distance caused by such melting. On the other hand, the presence of a melting point in the DSC thermogram confirms the presence of thiolate-capped silver nanoparticles inside the nanocomposite sample.2-) present into the organic chain, and this implies that very similar color switching temperatures can be obtained with these samples.The obtained DSC results clearly suggest that the degree and the amount of interdigitation for simple normal alkyl-thiolate chains is only lightly affected by the number of methylene groups 11SAg shows two different endothermic peaks. The small peak at lower temperature corresponded to the melting point of -(CH2)n- present in the organic capping layer, whereas the second peak at higher temperature can be attributed to the collapse of the interdigitation between the ends of thiolate chain on neighbor nanonanoparticles. In this case, color switching temperature results much higher for the presence of -OH groups that produced stronger interaction in the interdigitation regions. Finally, the temperatures of color switching did not significantly change with the alkyl-thiolate length, but a significant change in the thiolate structure  was able to affect the interdigitation crystals melting point.The DSC-thermogram of a sample obtained by thermal decomposition of HO to the hosted silver nanoparticle aggregates. For such a reason these devices must be exclusively based on linear polymers (thermoplastic polymers) and usually the color change is observed at a temperature higher than the polymer glass transition temperature (rubbery state). In particular, this type of thermochromism have been experimentally observed for thiolate-capped silver particles embedded into thermoplastic matrices, like polystyrene, poly(ethylene imines), poly(vinyl acetate), etc. On the contrary, as shown in To observe the thermochromic phenomenon, also the presence of long chain capping thiolates on the silver nanoparticle surface is required. Films based on amorphous polystyrene embedding silver nanoparticles capped by short-chain thiolates molecules like cyclohexyl-thiolate were transparent and showed the characteristic yellow coloration, produced by the surface plasmon resonance of insulated silver nanoparticles see . The opt2)12-S-) showed a melting point of ca. 160 °C and a well-visible color transition from brown to yellow at the same temperature.Thermochromic metal-polymer nanocomposites are technologically useful materials ,26,27, b12H25) molecules dissolved in the polymer at a temperature of ca. 200 °C. This nanocomposite material in form of film showed a prompt and completely reversible thermochromic effect, with strong chromatic variation from dark-brown or black, depending on the nanoparticles concentration, to a bright-yellow color at a temperature of 133 °C. The color switching temperature was coincident with the melting point of crystallites generated by the interdigitation of dodecyl-thiolate chains present on the metal nanoparticles surface . Such color-switching temperature only slightly changed with increasing of the alkyl-thiolate chain length (hexadecyl- and octadecyl-thiolate molecules were used). However, when a functional group, HO-, able to give stronger physical interactions (hydrogen-bridge bonds) was introduced at alkyl-chain end 11-S-), both the interdigitation crystallites melting point and the nanocomposite film color switching temperature resulted significantly modified (ca. 162 °C). Therefore, the color switching temperature of these reversible thermochromic films can be controlled by modifying of structure of the thiolate-capping molecules present on the surface of the silver nanoparticles.Dispersions of thiolate-capped silver nanoparticles in amorphous polystyrene have been produced by decomposing silver dodecyl-thiolate (AgSC