We propose and numerically demonstrate two novel terahertz absorbers made up of periodic single- and double-layer decussate graphene ribbon arrays. The simulated results show that the proposed absorbers have narrowband near-unity terahertz absorption with ultra-wide frequency reconfiguration and angular stability. By tuning the Fermi level of graphene ribbons, the over 90% absorbance peak frequency of the absorber with single-layer graphene structure can be flexibly adjusted from 6.85 to 9.85 THz for both the transverse magnetic (TM) and transverse electric (TE) polarizations. This absorber with single-layer graphene demonstrates excellent angular stability with the absorbance peaks of the reconfigurable absorption bands remaining over 99.8% in a wide angle of incidence ranging from 0 to 70°. The tuning frequency can be significantly enhanced by using the absorber with double-layer graphene structure from 5.50 to 11.28 THz and 5.62 to 10.65 THz, approaching two octaves under TM and TE polarizations, respectively. The absorbance peaks of the reconfigurable absorption band of this absorber for both polarizations maintain over 70%, even at a large angle of incidence up to 70°. Furthermore, an analytical fitting model is also proposed to accurately predict the absorbance peak frequencies for this variety of absorbers. Benefitting from these attractive properties, the proposed absorber may have great potential applications in tunable terahertz trapping, detecting, sensing, and various terahertz optoelectronic devices. Terahertz waves generally refer to electromagnetic waves with a frequency of 0.1–10 THz. Terahertz technology has broad applications in terahertz communication, spectroscopy, detecting, sensing, and imaging ,3,4. THzfH − fL)/fL × 100%. Li et al. demonstrated a tunable mid-infrared narrowband absorber using parallel double-layer graphene ribbons with an RFTR of 18.7% from 22.73 to 27 THz and a wide angular stability of 60° [Graphene is a monatomic carbon layer two-dimensional material that has attracted a great deal of attention in the past decade because of its unique electrical performance, low intrinsic loss, and photonic properties . Since gy of 60° . He et ay of 60° . Zhang ey of 60° . An enhay of 60° ,27. DespTo address this issue, we propose a new variety of frequency-reconfigurable narrowband near-unity terahertz absorbers with significantly enhanced RFTR and angular stability using single/double-layer decussate graphene ribbon arrays (S/DLDGRAs). To demonstrate the properties and mechanisms of the absorbers, we present the geometric configurations and study their performance, including terahertz absorbance spectra, effective impedance characteristics, field distributions, frequency reconfiguration, and angular stability of the proposed absorbers. It was found that by tuning graphene Fermi level, the RFTR for the over 90% absorbance peak frequency of the proposed absorber with SLDGRA reached 43.8%, ranging from 6.85 to 9.85 THz for both TM and TE polarizations under normal incidence. The RFRT for the over 90% absorbance peak frequency of the absorber with DLDGRA could be further extended to 105.1% (89.5%), ranging from 5.50 (5.62) to 11.28 (10.65) THz, approaching two octaves for the TM (TE) polarization under normal incidence. The absorbance peaks of the different absorption bands for both absorbers maintained over 70% in a wide range of angle of incidence from 0° to 70°. Furthermore, we propose a simple analytical fitting model to accurately predict the reconfigurable absorbance frequencies of the absorbers. Finally, we systematically draw the conclusion of the whole work.2 dielectric layer, and a gold (Au) reflection layer from the top to the bottom. The absorber with DLDGRAs can be interpreted as the transformation of the absorber with a SLDGRA by separating two graphene ribbon arrays into different layers with a distance of h1 while keeping the identical thickness of the dielectric layer. Here, we display the top views of the unit cells of the proposed absorbers with S/DLGRAs in σg = σintra + σinter (Unit: S) [σintra and interband contribution interσ are given by:ω is the radian frequency, μc is the chemical potential or Fermi level, T0 is the temperature, Γ is the phenomenological scattering rate, Γ = 2τ−1, τ is the relaxation time, e is the charge of an electron, ξ is energy, ћ is the reduced Plank’s constant, and kB is Boltzmann’s constant. The relative dielectric constant of ZrO2 was set as εr = 4.41 [Lx and Ly are the periods of the unit cell, and Wx and Wy are the widths of the graphene ribbons in x and y directions, respectively. The thickness of the upper and lower ZrO2 spacers, the Au reflecting layer, and the total thickness of the absorbers were set as h1, h2, h3, and h = h1 + h2 + h3, respectively. In this study, we assumed the parameters as Lx = Ly = 4 µm, Wx = Wy = 0.9 μm, h1 = 1 μm, h2 = 4 μm, h3 = 1 μm, T0 = 300 K, τ = 0.6 ps [g =Z 1g/σ without thickness [A = 1−T–R, where reflectance R = |S11|2, transmission T = |S21|2, and S denotes the scattering parameter, respectively [μc of graphene can be easily tuned from 0 to 1.0 eV via chemical or electrostatic doping [The schematic of the proposed frequency-reconfigurable wide-angle terahertz absorbers with S/DLDGRAs is illustrated in Unit: S) ,29, wherr = 4.41 ,31 and tr = 4.41 . In the = 0.6 ps , respecthickness ,35 and tectively . In addic doping ,37, makiIn this section, we first study terahertz absorbance spectra, frequency reconfiguration and angular stability of the proposed absorbers with SLDGRA. Then, to demonstrate a wider frequency reconfiguration, we present and investigate the properties of the absorbers with DLDGRA. Furthermore, we also proposed a simple analytical fitting model to accurately predict the reconfigurable absorbance peak frequencies of this variety of absorbers.A, transmission T, and reflectance R for both TE and TM polarizations with μc = 0.3 eV under normal terahertz incidence (θ = 0°). It is clear that excellent narrow-band terahertz absorption was achieved with the absorbance peak approaching 100% at 6.85 THz. Because of the axisymmetric unit cell, as shown in Z, respectively. We observed that for upper green point, Re(Z) = 1.0004, and for the lower green point, Im(Z) = −0.00114, at the absorbance peak frequency of 6.85 THz, implying the impedance of the absorber matched the free space well. Therefore, since the extremely low reflectance resulting from smooth impedance matching together with zero transmission enabled by the thick gold layer, the near-unity absorbance of the absorber was achieved. E|, and magnetic field distributions, |H|, for the TM and TE polarizations along the cut-plane on the top graphene layer at 6.85 THz. The localized surface plasmon polaritons (LSPPs) were excited and strongly confined on the graphene ribbons, leading to ultra-high terahertz absorption. As expected from the symmetric configuration and polarization-independent property, the |E| and |H| for TM polarization were exactly 90° shifted from that of the TE polarization. Therefore, this absorber with a SLDGRA showed a clear independence of polarization, which has a significant advantage over the polarization-sensitive absorber with single graphene ribbon array. First, we investigated the terahertz absorption performance of the proposed absorber with a SLDGRA under normal incidence. The identical geometric parameters are shown in μc and angle of incidence θ on the absorbance spectra. μc. It was found that changing the μc of the graphene ribbon had a significant influence on the absorption frequency band. As μc increased from 0.2 to 0.8 eV, the absorbance peak frequency band red-shifted accordingly from 5.58 to 11.32 THz. Especially, good frequency reconfiguration characteristics were clearly observed. As μc increased from 0.3 to 0.6 eV, the over 90% absorbance peak frequency varied from 6.85 to 9.85 THz, demonstrating a wide absolute frequency tuning range of 3 THz with the RFTR of 43.8%, which was larger than the recently reported works [μc of 0.3 and 0.6 eV, for the first and the last absorption bands with over 90% peak absorbance, respectively. The results showed that the absorber also possessed an excellent angular stability for narrowband terahertz absorption characteristics. Owing to the wide-angle localized resonance of SPPs in the SLDGRA structure, the peak absorbance remained over 99.8%, even at a large oblique incidence angle of 70° for both cases. This absorber with wide frequency reconfiguration, absolute polarization independence, and excellent angular stability may have significant potential applications in the terahertz regime.To demonstrate the properties of wide frequency reconfiguration and angular stability, we studied the dependence of Fermi level ed works ,25,26,272 separation (h1) of the double-layer decussate graphene ribbon arrays was set as 1 µm while the rest of the geometric parameters of this absorber were fixed to be the same as the absorber with a SLDGRA. The simulated absorbance spectra for TM and TE polarizations with different μc at normal terahertz incidence are displayed in μc. For TM polarizations, the reconfigurable over 90% absorbance peak frequency significantly switched from 5.50 to 11.28 THz, corresponding an enhanced RFTR of 105.1%, as the μc increased from 0.2 to 0.8 eV. For TE polarization, the reconfigurable over 90% absorbance peak frequency switched from 5.62 to 10.65 THz, corresponding to a RFTR of 89.5%, as the μc varied from 0.3 to 1.0 eV. Compared with the RFTR of the absorber with a SLDGRA, ultra-wide frequency reconfiguration with 2× more RFTR was achieved in the absorber with DLDGRAs. To further enhance the absorbance peak frequency tuning range, we propose the absorber with DLDGRAs, as shown in μc = 0.3 eV under normal incidence. For TM polarization, |E| distributions of the LSPPs at the absorbance peak frequency of 6.81 THz along upper and lower graphene plane are shown in E| were mainly confined around upper-layer graphene ribbon edges. Therefore, the terahertz absorption for the TM polarization was mainly caused by LSPPs located at the upper-layer graphene ribbons. Similarly, the electric distributions for the TE polarization at the absorbance peak frequency of 5.62 THz along upper and lower graphene plane are shown in Next, it is remarkable that this absorber was no longer polarization-independent because of the non-axisymmetric DLDGRA structure. To better understand this characteristic, we depict the simulated electric field distributions for TM and TE polarizations along the cut-planes on upper and lower graphene layers with θ and operating frequency with μc of 0.2 and 0.8 eV for TM polarization in μc of 0.3 and 1.0 eV for TE polarization in Furthermore, we also investigated the angular stability of the proposed absorber with DLDGRAs. Here, for example, we present the simulated absorbance spectra as a function of incidence angle fp and μc. According to the quasi-static analysis in References [fp and μc is given by k = 0.1 denotes a dimensionless fitting constant, W is the width of graphene ribbon, εe is the effective permittivity of the background materials surrounding the graphene ribbon array, and εo is the permittivity of vacuum, respectively. Here, we first compared the fp of the absorber with a SLDGRA obtained from this analytical fitting mode and the simulated results, as shown in εe was expressed as εe = (εZro2 + 1)/2. It was found that with μc varying from 0.2 to 0.9 eV, fp increased from 5.58 to 11.97 THz. The analytical fitting results matched well with the simulation results. Then, we verified this analytical fitting mode for the absorber with the DLDGRA case. Because it is polarization-dependent, we compared the fitting and simulation results separately for TM and TE polarizations, as shown in εe was also approximately expressed as εe = (εZro2 + 1)/2. With the same values of εe for the absorber with DLDGRAs under TM polarization and the absorber with SLDGRA, the same analytical predictions of fp are shown in both εe should be calculated by εe = εZro2 instead. Both analytical prediction and the simulation results clearly showed that fp increased from 5.50 (4.60) to 11.97 (10.09) THz for TM (TE) polarization with μc varying from 0.2 to 0.9 eV with good agreement. Therefore, this analytical fitting mode can be used for the efficient prediction of the reconfigurable absorbance peak frequencies of this variety of absorbers.To predict the reconfigurable frequencies of the proposed absorbers, we further studied the relationship between the absorbance peak frequency ferences ,40, a fiμc from 0.3 to 0.6 eV. For the absorber with DLDGRAs, the absorbance peak frequencies of each reconfigurable bands could be significantly extended from 5.50 to 11.31 THz and 5.60 to 10.61 THz under TM and TE polarizations, respectively, approaching two octaves. Good angular stability of these absorbers was achieved, with the absorbance peaks of the reconfigurable absorbance bands maintained over 70% in a wide angle of incidence ranging from 0° to 70° for both polarizations. We proposed an analytical fitting model to predict the absorbers’ reconfigurable absorbance peak frequencies. Our results demonstrated that the proposed frequency-reconfigurable absorbers offer significant potential to realize novel reconfigurable optoelectronic devices, which may have great applications in the emerging terahertz regime.We systematically demonstrated a new variety of frequency-reconfigurable absorbers using S/DLDGRAs. The absorbers had excellent properties of narrowband near-unity terahertz absorbance, ultra-wide frequency reconfiguration, and angular stability. For the absorber with a SLDGRA, the over 90% absorbance peak frequency could be flexibly adjusted from 6.85 to 9.85 THz for both polarizations under normal incidence by tuning


Integrating graphene with other nanomaterials has created a variety of graphene nanocomposites with extraordinary chemical, optical, mechanical, and electrical properties. These composites could combine the characteristics of their components to achieve structural stability and multifunctionality with proper design of the interfacial interactions. The field of graphene nanocomposites has developed rapidly over the past decade, with many applications in various fields, such as chemical/biomedical sensing, tissue engineering, and bioimaging.−1, respectively. Moreover, this sensor achieved a temperature sensitivity of 2.33 nm/5 °C. These findings can expand the range of use for a nanoscale real-time temperature sensor with resistance to electromagnetic interference. Another project fabricated a temperature sensor consisting of two metal layers and two ethanol-sealed elliptical resonators, connected to a straight waveguide using two rectangular tubes [2 for light detection (405 to 1310 nm) [This Special Issue contains six research articles and two reviews in total, covering structure-proprieties of graphene and its applications in sensing organic solvents, light and DNA, as well as in tissue engineering. Among these, four articles reported graphene nanocomposites for chemical/biomedical sensing applications. One article reported fabrication of graphene-like ZnO/graphene oxide (GO) nanosheets for acetone vapor sensing . The senIn one of the remaining two research articles, the authors fabricated chitosan/graphene oxide (CS/GO) composite scaffolds for bone regeneration . The othOne of the two review articles investigates the application of graphene nanocomposites for DNA detection. This review comprehensively summarizes recent trends in DNA detection using graphene and graphene-related nanomaterials including graphene nanopore (GNP), graphene nanoribbon (GNR), GO, reduced graphene oxide (rGO), and graphene-nanoparticle (G-NP) hybrid materials . It alsohttps://www.mdpi.com/journal/molecules/special_issues/graphene_nanocomposites


Since one source of intracellular Ca2+ is N-methyl-d-aspartate receptors (NMDARs) related and the function of NMDARs can be regulated by neuroinflammation, we therefore hypothesized that dysregulation of BDNF/TrkB signaling mediated by NMDAR/Ca2+/calpain might be involved in the pathogenesis of POCD.Postoperative cognitive decline (POCD) is a recognized clinical phenomenon characterized by cognitive impairments in patients following anesthesia and surgery, yet its underlying mechanism remains unclear. Brain-derived neurotrophic factor (BDNF) plays an important role in neuronal plasticity, learning, and memory via activation of TrkB-full length (TrkB-FL) receptors. It has been reported that an abnormal truncation of TrkB mediated by calpain results in dysregulation of BDNF/TrkB signaling and is associated with cognitive impairments in several neurodegenerative disorders. Calpains are CaIn the present study, 16-month-old C57BL/6 mice were subjected to exploratory laparotomy with isoflurane anesthesia to establish the POCD animal model. For the interventional study, mice were treated with either NMDAR antagonist memantine or calpain inhibitor MDL-28170. Behavioral tests were performed by open field, Y maze, and fear conditioning tests from 5 to 8 days post-surgery. The levels of Iba-1, GFAP, interleukin-1β (IL-1β), IL-6, tumor necrosis factor-α (TNF-α), NMDARs, calpain, BDNF, TrkB, bax, bcl-2, caspase-3, and dendritic spine density were determined in the hippocampus.Anesthesia and surgery-induced neuroinflammation overactivated NMDARs and then triggered overactivation of calpain, which subsequently led to the truncation of TrkB-FL, BDNF/TrkB signaling dysregulation, dendritic spine loss, and cell apoptosis, contributing to cognitive impairments in aging mice. These abnormities were prevented by memantine or MDL-28170 treatment.Collectively, our study supports the notion that NMDAR/Ca2+/calpain is mechanistically involved in anesthesia and surgery-induced BDNF/TrkB signaling disruption and cognitive impairments in aging mice, which provides one possible therapeutic target for POCD. Postoperative cognitive decline (POCD) is a recognized clinical phenomenon characterized by cognitive impairments in patients after anesthesia and surgery, especially in the elderly . POCD reBrain-derived neurotrophic factor (BDNF) is a neurotrophin widely expressed in the central nervous system, which plays a critical role in neuronal survival and differentiation, and synaptic plasticity through activation of its full-length receptor (TrkB-FL) , 5. Dysr2+-dependent cysteine proteases that play a physiologic role by the cleavage of several substrates, including the neurotrophin receptor TrkB [2+ concentrations and one source of intracellular Ca2+ is NMDARs related. Importantly, one recent study showed that amyloid-β peptide (Aβ) induced the overactivation of NMDARs and calpain, and then triggered the formation of a truncated isoform (TrkB-T′) and an intracellular domain (ICD) fragment, and ultimately disrupted BDNF/TrkB signaling, which can be prevented by a NMDAR antagonist memantine [Calpains are intracellular Cator TrkB , cytosketor TrkB . A calpator TrkB  and epiltor TrkB . The ovetor TrkB  and alsotor TrkB . Calpainemantine . HoweverInflammation has been proved to be a potential source of reactive oxygen species for inducing NMDARs hypofunction and nonsteroidal anti-inflammatory drugs (NSAIDs) can improve impaired NMDAR-dependent synaptic plasticity and age-related cognitive dysfunction . In addiOne hundred and forty-four 16-month-old male C57BL/6 mice (26–36 g) were obtained from the Animal Center of Southeast University, Nanjing, China. Animals were housed in groups of 3–5 individuals per cage in a standard condition with access to food and water ad libitum in a colony room kept at 19–22 °C and 40–60% humidity, under a 12-h light/dark cycle (light from 07:00 to 19:00). The experiments began after all the animals acclimated to the environment for 2 weeks. The study protocol was approved by the Ethics Committee of Zhongda Hospital, Medical School, Southeast University, and all procedures were performed in accordance with the approved guidelines. The schematic timeline of the experimental procedure is summarized in Fig. Exploratory laparotomy was performed under isoflurane anesthesia described as our previous studies , 23. MicThe NMDAR antagonist memantine   was intrA white opaque plastic chamber  was used as the open-field arena. The open-field test was performed at 5 days post-surgery to assess the exploratory locomotor activity. Each mouse was placed in the center of the arena and left to explore it for 5 min while activity was automatically recorded by a video tracking system.The Y maze is a symmetrical three-arm maze  and is used to evaluate the spontaneous alternation performance at 6 days post-surgery. Each mouse was placed in the center of the Y maze and could explore freely throughout the three different arms of the maze during an 8-min session. The sequence and total number of arms entered were recorded. Arm entry was complete when the hind paws of the mouse had been completely placed in the arm. The alteration was determined from successive consecutive entries to the three different arms on overlapping triads in which all arms were represented. For example, a sequence of entries to the three arms ABC, ACBABACABA, would generate four “successful” alternations, ACB, CBA, BAC, and CAB. Percentage alternation is the number of triads containing entries into all three arms divided by the maximum possible alternations  × 100. The re-entry into the same arm was not counted for analysis .Mice were trained for fear conditioning at 7 days post-surgery. Each mouse was placed into a conditioning chamber  and allowed to explore freely for 3 min. Then a 30-s tone  was delivered followed by a 2-s foot shock (0.7 mA). After that, the mouse stayed in the chamber for another 30 s and then returned to the home cage. Contextual fear conditioning (a hippocampus-dependent task) was assessed 24 h after training. For the contextual fear conditioning, each mouse was placed back into the same chamber in which they were explored for 5 min without tone or foot shock and scored for the freezing behavior. Freezing behavior was defined as the absence of all visible movement except for respiration. Given that no difference was observed in the auditory-cued fear test (a hippocampus-independent task) between control and surgery group in our previous studies , 23, theThe entire dissected hippocampus was harvested and subjected to Western blot analysis. The samples were lysed as described previously , 23. Pro.; Sigma, St Louise, MO, USA) and transcardially perfused with saline, followed by 4% paraformaldehyde (PFA) in phosphate-buffered saline . Brains were removed and postfixed in 4% PFA for 2 h and dehydrated in 30% sucrose at 4 °C overnight and then embedded in O.C.T. compound. The brains were cut coronally into 10-μm-thick sections from bregma − 1.70 to − 2.30 by a freezing microtome and mounted on glass slides. The sections were blocked with 1% bovine serum albumin (BSA) for 1 h at room temperature. And then the sections were incubated with primary antibodies: rabbit anti-Iba1  and rabbit anti-GFAP  in 1% BSA at 4 °C overnight. Sections were washed with PBS three times and incubated with goat anti-rabbit IgG-FITC  and goat anti-rabbit IgG-Cy3  for 1 h at room temperature. After washing out the secondary antibodies, sections were incubated with 4′,6-diamidino-2-pheny-lindole (DAPI) for nuclear staining. Fluorescent images were captured by a confocal microscope . Six sections of the hippocampus per mouse were analyzed by ImageJ  for immunofluorescence analysis. Three non-overlapping fields of each section in the hippocampal Cornu Ammonis 1 (CA1) area was randomly acquired by a counting frame size of 0.4 mm2. Positively stained areas were defined that the number of pixels per image with intensity in which was above a predetermined threshold level. The immunoreactivity of a protein was quantified by percentage area with positive staining to the total area of the imaged field. All quantitative analyses were performed by an experimenter blinded to the group of each sample.Mice were deeply anesthetized with 2% sodium pentobarbital in saline . Data are presented as mean ± S.E.M. Differences between groups were assessed with one-way ANOVA followed by post hoc Tukey multiple comparisons. The dendritic intersections were analyzed by repeated-measures ANOVA followed by post hoc Tukey multiple comparisons. A significant difference was considered as F = 16.34, p < 0.0001; Fig. F = 20.06, p < 0.0001; Fig. We performed immunostaining by using antibodies of Iba1 and GFAP on day 1 post-surgery, respectively. Compared with control + vehicle (con + veh) group, the intensity of Iba1 [F3, 0 = 16.34F3, 0 = 16.34F = 14.55, p < 0.0001; Fig. F = 19.50, p < 0.0001; Fig. F = 16.02, p < 0.0001; Fig. F = 7.250, p = 0.0018; Fig. F = 0.6109, p = 0.6158; Fig. F = 10.13, p = 0.0003; Fig. F = 8.462, p = 0.0008; Fig. Next, we detected the levels of proinflammatory cytokines on days 1 and 8 post-surgery. On day 1 post-surgery, the IL-1β [F, 20 = 14F, 20 = 14F, 20 = 14F, 20 = 14F, 20 = 14F = 234.5, p < 0.0001; Fig. F = 4.599, p = 0.0132; Fig. F = 4.854, p = 0.0107; Fig. To evaluate whether NMDAR inhibition by memantine could affect anesthesia and surgery-induced activation of calpain, we detected αII-spectrin levels and the formation of calpain-specific spectrin breakdown products (SBDPs). αII-spectrin is a major substrate for calpain and caspase-3 proteases. The activation of calpain could result in the cleavage of αII-spectrin and then produce breakdown products with distinct molecular weights. The expected molecular weight of αII-spectrin is 250 kDa, whereas the SBDP is 150 kDa. Our data showed that the levels of SBDPs were significantly increased, which was accompanied by decreased levels of αII-spectrin in the sur + veh group, suggesting the activation of calpain. However, this increase was ameliorated by memantine treatment [F = 64.15, p < 0.0001; Fig. F = 4.064, p = 0.0062; Fig. F = 4.958, p = 0.0020; Fig. F = 4.325, p = 0.0044; Fig. To determine whether TrkB is truncated by calpain activation, we administrated MDL-28170, an inhibitor of calpain. The levels of SBDPs were significantly increased and the levels of αII-spectrin were decreased after anesthesia and surgery, which were reversed by memantine or MDL-28170 treatment [F = 8.311, p < 0.0001; Fig. F = 3.739, p = 0.0095; Fig. F = 5.399, p = 0.0012; Fig. F = 4.754, p = 0.0026; Fig. F = 19.98, p < 0.0001; Fig. F = 17.00, p < 0.0001; Fig. The presence of calpain-cleaved fragments occurs early in neural cell pathology and may be indicative of necrotic and excitotoxic neuronal injury and death. As reported previously, cell apoptosis plays an important role in POCD. In the current study, we showed that the levels of bax [F = 0.1977, p = 0.9609; Fig. F = 0.009950, p > 0.9999; Fig. F = 3.949, p = 0.0072; Fig. We used Sholl analysis to assess dendritic branching and spine density in the CA1 region in the hippocampus. There was no significant difference in the total number of dendritic intersections [F = 0.07516, p = 0.9957; Fig. F = 0.05177, p = 0.9982; Fig. F = 0.1552, p = 0.9776; Fig. F = 6.052, p = 0.0002; Fig. F = 5.489, p = 0.0004; Fig. The open field was used to evaluate the locomotor activity and exploratory behavior. During the 5-min test session, there was no significant difference in the total distance [In the present study, we showed that anesthesia and surgery-induced neuroinflammation overactivated NMDARs, and the abnormal activation of NMDARs triggered the overactivation of calpain, which subsequently led to the truncation of TrkB-FL, BDNF/TrkB signaling dysregulation, dendritic spine loss, and cell apoptosis, contributing to cognitive impairments in aging mice. Of note, NMDARs antagonist memantine or calpain inhibitor MDL-28170 attenuated these abnormalities, suggesting tackling abnormal activation of NMDARs or truncation of TrkB-FL may be a therapeutic strategy for POCD Fig. .Fig. 10Patients suffering from cognitive impairments after anesthesia and surgery have been recognized for more than 60 years. Recently, a multi-specialty working group recommended “perioperative neurocognitive disorders” (PND) as an overarching term for cognitive impairments diagnosed in the preoperative or postoperative period –33. PND Although various mechanisms have been proposed to be involved in the development of POCD, neuroinflammation is believed to play an initial and central role. In the present study, we showed that IL-1β and IL-6 levels were significantly increased and lasted for 8 days post-surgery. Our results were consistent with the previous studies , 22, 34,There is accumulating evidence suggesting a strong correlation between neuroinflammation and NMDAR dysfunction, eventually resulting in deficits of synaptic plasticity and cognitive impairments. It has been showed that microglia is activated initially and become a major cellular source of a variety of proinflammatory cytokines in a rat model of chronic neuroinflammation, which has negative effect on long-term potentiation requiring NMDAR activation . In addi2+/calpain is mechanistically involved in neuroprotective effects of memantine in anesthesia and surgery-induced cognitive impairments in aging mice. In a rat model of postoperative pain-induced cognitive impairments, memantine treatment could improve memory deficits without any effect on NMDAR expression [We previously demonstrated that NMDARs calcium/calmodulin-dependent kinase II pathway was involved in the pathogenesis of POCD . Therefopression . Interespression . Thus, tBDNF plays an important role in neuronal plasticity, learning, and memory through the activation of TrkB-FL receptors , 5. It hTrkB is expressed as a full-length, catalytically active isoform (TrkB-FL), as well as several alternatively spliced truncated isoforms lacking the intracellular kinase domain, including TrkB.T1, TrkB.T2, and TrkB.T-Shc , 52. BotThere are some limitations in this study. Firstly, we mainly observed relatively short-term cognitive performance by Y maze and fear conditioning tests after anesthesia and surgery. Using Morris water maze to measure spatial cognition are needed in our future studies. Secondly, memantine is a partial antagonist of NMDAR receptor that block the pathological activation of NMDARs, while not interfering with normal synaptic transmission . ClinicaIn summary, our study demonstrated that neuroinflammation overactivated NMDARs, at least in part, plays a key role in overactivation of calpain, cleavage of TrkB-FL receptor, BDNF/TrkB signaling dysfunction, dendritic spine loss, cell apoptosis, and consequent cognitive impairments. Hence, identifying viable therapeutic strategies to tackle abnormal activation of NMDARs or calpain may provide effective interventions for POCD.


P = 0.015). Multivariate analysis identified sPD-L1 and the neutrophil-to-lymphocyte ratio as independent prognostic factors for OS. During chemotherapy, more patients achieved complete response (CR)/partial response (PR) as their best response when sPD-L1 was decreased at the first response assessment (P = 0.038). In the patients who achieved CR/PR as their best response, sPD-L1 was significantly higher at the time of disease progression than at the first response assessment (P = 0.025). In conclusion, the sPD-L1 level at diagnosis exhibits a prognostic value in pancreatic cancer. Furthermore, sPD-L1 dynamics correlate with disease course and could be used to understand various changes in the tumor microenvironment during chemotherapy.In pancreatic cancer, acquiring a sufficient amount of tumor tissue is an obstacle. The soluble form of PD-L1 (sPD-L1) may have immunosuppressive activity. Here, we evaluated the prognostic implications of sPD-L1 in unresectable pancreatic cancer. We prospectively enrolled 60 patients treated with first-line FOLFIRINOX chemotherapy. We collected blood samples at diagnosis, first response assessment and disease progression. Serum sPD-L1 levels were measured using enzyme-linked immunosorbent assays. The median sPD-L1 level was 1.7 ng/mL . Patients with low sPD-L1 level (<4.6 ng/mL) at diagnosis showed better overall survival (OS) than those with high sPD-L1 level ( In particular, PD-L1 is the most promising immune checkpoint molecule used as a potential target for immunotherapy.With recent advances in immuno-oncology, many biomarkers of immune checkpoints have received increasing attention due to their roles as prognostic factors and therapeutic targets. Biomarkers such as cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) ligands, interleukin-10, transforming growth factor β (TGF-β), and programmed death-ligand 1 (PD-L1) are well-known molecules involved in escape from host immune surveillance and are associated with cancer cell proliferation1. Tumor cells are then able to escape immune recognition and the host immune response. In various cancers, such as lung cancer, breast cancer, and renal cell carcinoma, overexpression of PD-L1 in tumor cells results in aggressive behavior and is associated with a poor prognosis5. Additionally, PD-L1 expression can predict treatment response in anti-PD-1/PD-L1 agent-treated patients6. Therefore, an evaluation of PD-L1 expression could predict patient prognosis and treatment response in certain types of cancer.PD-L1, a B7 superfamily member, is expressed in tumor cells and binds to programmed death-1 (PD-1) on T cells. After the PD-L1/PD-1 interaction occurs, inhibitory signals are transmitted to T cells, thereby suppressing T cell proliferation and reducing cytokine secretion8. Therefore, the use of soluble markers could help in understanding the dynamics of the interaction between host immune responses and tumor cells or the tumor microenvironment.A biopsy is needed to evaluate PD-L1 expression in tumor tissue and the tumor microenvironment, which is the primary limitation of this method. However, soluble serum biomarkers have been regarded as promising surrogates because they can reflect the tumor status and predict survival outcomes through a minimally invasive modality9. In addition, a few studies have recently reported the dynamics of sPD-L1 in cancer patients during chemotherapy treatment8.The soluble form of PD-L1 (sPD-L1) has been reported to have prognostic value in various cancers, including lymphoma, gastric cancer, and lung cancer12. However, the results of an early trial of a monotherapy checkpoint blockade approach were disappointing and suggested that evaluating the immune checkpoint alone may be insufficient to explain the poor prognosis of pancreatic cancer13. Therefore, the role of PD-L1/PD-1 in pancreatic cancer requires further investigation, especially because in many cases, it is difficult to acquire a sufficient amount of tumor tissue to evaluate PD-L1 expression, and the value of sPD-L1 has not yet been studied.Despite previous study results, in pancreatic cancer, the prognostic role of the PD-L1/PD-1 signaling axis remains uncertain. Some studies confirmed the prognostic importance of the PD-L1/PD-1 signaling axis in pancreatic cancerThe aims of this study were to measure serum sPD-L1 levels in unresectable pancreatic cancer patients who were treated with palliative first-line chemotherapy and to evaluate the prognostic role of sPD-L1 as well as its dynamics during chemotherapy.2 on day 1), irinotecan (180 mg/m2 on day 1), leucovorin (400 mg/m2 on day 1), fluorouracil , and a continuous infusion of 5-FU . Clinical data were collected by reviewing medical records that included demographic information and the results of laboratory exams, including total bilirubin, albumin, and cancer antigen 19-9 (CA 19-9) levels and blood cell counts . The neutrophil-to-lymphocyte ratio (NLR) and the platelet-to-lymphocyte ratio (PLR) were calculated by dividing the neutrophil or platelet count, respectively, by the lymphocyte count. The results of a laboratory test performed at the time of diagnosis with unresectable pancreatic cancer were used in the analysis. We evaluated the disease state during FOLFIRINOX chemotherapy by computed tomography examination every three chemotherapy cycles. Response assessment was performed according to the Response Evaluation Criteria in Solid Tumors (RECIST) criteria version 1.114. The best overall response rate (ORR) was defined as the proportion of patients who achieved a complete response (CR) or partial response (PR) as their overall response during chemotherapy15.We prospectively recruited pathologically confirmed unresectable pancreatic cancer patients who were treated with FOLFIRINOX as their first-line palliative chemotherapy regimen. Between 2013 and 2015, 60 patients who provided informed consent for the biomarker analysis study at Seoul National University Hospital were enrolled; all were ethnically Korean. FOLFIRINOX chemotherapy consisted of oxaliplatin  according to the manufacturer’s instructions16. Each sample was analyzed in duplicate.We prospectively collected blood samples from patients at the time of diagnosis (prechemotherapy), at the first response assessment , and at the time of disease progression. Serum sPD-L1 levels were measured using an enzyme-linked immunosorbent assay t-test or one-way ANOVA, as appropriate. Progression-free survival (PFS) and overall survival (OS) were estimated using the Kaplan-Meier method. PFS was defined as the time from the date of initiation of FOLFIRINOX chemotherapy to the date of disease progression. OS was defined as the time from the date of initiation of FOLFIRINOX chemotherapy to the date of either death or last follow-up. A receiver operating characteristics (ROC) curve was used to determine the cut-off values of the NLR, the PLR, and sPD-L1 levels to best predict survival. The cut-off values used for albumin, total bilirubin, and CA 19-9 levels were the corresponding normal values. Clinical variables with univariate P-values < 0.2 were considered for inclusion in multivariate analyses, which were performed using the logistic regression model or Cox proportional hazard model, as appropriate. All statistical tests were two-sided, and a statistically significant difference was defined as P < 0.05. Statistical analyses were performed using IBM SPSS version 23.0 .Fisher’s exact test or Pearson’s chi-squared test was used to compare categorical variables. The comparison of continuous variables was performed using an independent or paired This study was approved by the institutional review board at Seoul National University Hospital  and was conducted in accordance with the guidelines of the Declaration of Helsinki for biomedical research. Informed consent was obtained from all participants.The mean and median values of the sPD-L1 level at initial diagnosis (prechemotherapy) (n = 60) were 2.2 and 1.7 ng/mL , respectively. ROC curve analysis was used to determine the cut-off value of sPD-L1, and the cut-off value of 4.6 ng/mL achieved the highest combination of sensitivity and specificity for the prediction of OS.P > 0.05).Baseline characteristics of the patients are summarized in Table Patient responses to FOLFIRINOX chemotherapy during the follow-up period were as follows: 2/60 (3.3%) patients achieved a CR; 20/60 (33.3%) patients achieved a PR; 30/60 (50%) patients achieved stable disease (SD); and 8/60 (13.3%) patients exhibited progressive disease (PD). Twenty-two (36.7%) patients achieved a CR or PR as their overall response during chemotherapy.The median follow-up duration of the 60 patients was 11.4 months , and the median PFS and OS were 6.5  and 10.3  months, respectively.P = 0.061) and high sPD-L1 level  showed a trend toward worse PFS.In a univariate analysis for PFS, older age (≥60 years), a low sPD-L1 level (<4.6 ng/mL), a low NLR (<1.83), and a low PLR (<109.6) were associated with prolonged PFS Table ; Fig. 1AP = 0.015) and low NLR  were independent prognostic factors for prolonged OS.In a univariate analysis for OS, the prognostic factors indicative of improved OS were older age (≥60 years), a low sPD-L1 level (<4.6 ng/mL), and a low NLR <1.83) .In patients diagnosed with metastatic pancreatic cancer, only a low sPD-L1 level was an independent factor for prolonged OS in univariate and multivariate analyses  were obtained from 25 patients.P = 1.000; Table vs. 9/35 [25.7%] for younger age, P = 0.037; HR, 4.858, 95% CI, 1.344–17.563, P = 0.016) and a decreased sPD-L1 level at the first response assessment time point (postchemotherapy) compared with the initial diagnosis time point (prechemotherapy)  were predictive factors for the achievement of a best overall response (CR + PR) during FOLFIRINOX chemotherapy.When we evaluated the role of sPD-L1 in predicting treatment response during FOLFIRINOX chemotherapy, the sPD-L1 levels at diagnosis (prechemotherapy) could not predict the best ORR in a univariate analysis  and decreased sPD-L1 after chemotherapy  were predictive factors for the achievement of a best overall response.Similar results were observed in patients with metastatic pancreatic cancer. Older age  during FOLFIRINOX chemotherapy, sPD-L1 levels were decreased at the first response assessment time point compared to the time of diagnosis. However, this change was not statistically significant  and the first response assessment time point showed better responses to FOLFIRINOX than those whose sPD-L1 levels increased after chemotherapy.18. Although recent palliative chemotherapies have improved the median OS of metastatic pancreatic cancer patients up to 8.5–11.1 months, the efficacy of current treatment options is still very limited20. In the era of immunotherapy, large randomized clinical trials have reported promising outcomes in patients with various cancers, such as lung cancer, melanoma, and renal cell carcinoma23. However, in pancreatic cancer, clinical trials of anti-PD-L1 monotherapies have shown disappointing outcomes24. The reason for this failure could be partly explained by the nonimmunogenic tumor microenvironment of pancreatic cancer25. The efficacy of immune checkpoint blockade has been related to the expression of PD-L1 by tumor cells and PD-1 by activated T cells. However, a previous study revealed that pancreatic cancer had low expression of both PD-1 and PD-L126. In addition, although patients have high levels of PD-L1 expression, highly immunosuppressive microenvironment elements, such as regulatory T cells, myeloid-derived suppressor cells and tumor-associated macrophages, might affect the unsuccessful treatment response to immunotherapy25.Pancreatic cancer is a highly lethal malignancy worldwide27. However, several studies have reported a prognostic role for PD-L1. Tessier-Cloutier et al. demonstrated that increased PD-L1 expression (>10%) detected by immunohistochemistry was associated with poor disease-specific survival in resected pancreatic cancer patients 11. Yamaki et al. revealed that PD-L1-positive patients showed worse OS than PD-L1-negative patients who underwent surgical resection 12. However, in an analysis using sPD-L1, Kruger et al. reported that sPD-L1 did not predict adverse outcomes in patients with advanced pancreatic cancer . They found that the levels of sPD-L1 were increased in patients with elevated C-reactive protein levels (P < 0.001), suggesting that sPD-L1 could be a marker of systemic inflammation in advanced pancreatic cancer28.Due to its nonimmunogenic nature, pancreatic cancer has not yet been evaluated in a way that fully elucidates the prognostic role of PD-L1. It has been suggested that an immune checkpoint might not entirely represent the histopathological hallmarks of this malignancyIn this study, we also demonstrated the dynamics of sPD-L1 during homogenous FOLFIRINOX chemotherapy. sPD-L1 levels at the first response assessment time points were lower than those at the time of diagnosis. sPD-L1 levels were also higher at the disease progression time point than at the first response assessment time point. Although this study did not achieve statistical significance due to the small sample size, the observed changes in the sPD-L1 levels according to disease status were clinically important. In patients who achieved a CR or PR as their overall response, the sPDL1 levels were significantly increased at the disease progression time point compared to the first response assessment time point. Therefore, dynamic changes in sPD-L1 levels during chemotherapy correlated with disease progression.30.Although inflammatory markers such as the NLR and PLR did not correlate with sPD-L1 levels in our study, elevated NLR levels were associated with poor OS. This finding was in accordance with the results of previous studies that showed a relationship between increased systemic inflammation and poor outcomes in pancreatic cancerThe present study has several limitations. It was a single-center study, and the sample size was small. Therefore, further large-scale studies are needed to confirm our results. We enrolled only advanced-stage pancreatic cancer patients who were treated with FOLFIRINOX chemotherapy as a palliative first-line treatment to focus on a relatively homogeneous population. Patients who received other standard of care treatments, such as gemcitabine/nab-paclitaxel combination therapy or gemcitabine monotherapy, were excluded. A study of pancreatic cancer patients treated with these standard regimens is warranted to further support our conclusion for advanced pancreatic cancer. Despite these limitations, to the best of our knowledge, this is the first study to show the dynamics of sPD-L1 levels in unresectable pancreatic cancer patients treated with homogenous FOLFIRINOX chemotherapy. This study provides evidence that the prechemotherapy sPD-L1 level could be a prognostic factor for OS and that the dynamics of sPD-L1 levels during chemotherapy could predict treatment responses. In routine clinical practice, sPD-L1 measurement in patient blood samples could be easily incorporated. However, the standardization of this measurement should be further validated beforehand.In conclusion, pretreatment sPD-L1 levels play a significant role in predicting survival outcomes in advanced pancreatic cancer patients treated with FOLFIRINOX chemotherapy. Additionally, the dynamics of sPD-L1 levels during chemotherapy could be used to predict the best treatment response during chemotherapy.


Though idiopathic pulmonary fibrosis (IPF) is characterized by single-organ involvement, many comorbid conditions occur within other organ systems. Patients with IPF may present during evolution different complications and comorbidities that influence the prognosis and modify the natural course of their disease. In this chapter, we highlight common comorbid conditions encountered in IPF, discuss disease-specific diagnostic modalities, and review the current treatment data for several key comorbidities. The diagnosis and treatment of these comorbidities is a challenge for the pulmonologist specialized in interstitial lung diseases (ILDs). We will focus on pulmonary emphysema, lung cancer, gastroesophageal reflux, pulmonary hypertension, obstructive sleep apnea (sleep disorders), and acute exacerbation of IPF. Idiopathic pulmonary fibrosis (IPF) is a progressive, chronic, and unpredictable disease with few useful treatments and a poor survival.Although the appearance of new treatments, such as nintedanib and pirfenidone, has given new hope to these patients, its management is complex and is influenced by the coexistence of several comorbidities. The presence of these comorbidities may decrease patients’ survival and quality of life and might accelerate the progression of the disease. Some comorbidities might coexist with IPF because of a common risk factor for both diseases  or, in other cases, they could be a consequence of IPF itself . There is increasing evidence that the early diagnosis and treatment of comorbidities is as important as the treatment of idiopathic pulmonary fibrosis itself.Smoking is a common risk factor for both emphysema and pulmonary fibrosis ,2,3. NotThe syndrome of combined pulmonary fibrosis and emphysema (CPFE) has been proposed as an important phenotype of pulmonary fibrosis and is defined by the presence of emphysema and parenchymal pulmonary fibrosis in the same patient . In thisIt has been suggested that cigarette smoke is the main causal factor, given the fact that a history of smoking is constant in all the published cohorts . SeveralKaolinite or aluminium silicate, for example, is an inorganic substance with industrial use which is present in tobacco smoke and has been isolated in the alveolar macrophages of smokers with pulmonary fibrosis and emphysema ,3,5.Usually, individuals with CPFE tend to be males with an extensive smoking history and increased oxygen requirement ,2,3,4. The coexistence of emphysema and fibrosis determines a characteristic functional profile that contrasts with the degree of dyspnea manifested by these patients. Pulmonary mechanics are altered by emphysema and pulmonary fibrosis. In emphysema, elastic forces are reduced, and both lung compliance and pulmonary volumes are increased. Pulmonary fibrosis leads to increased lung elastic recoil, decreased compliance, and reduced lung volumes . The sim1), and the total pulmonary capacity (TLC) are usually within normal values or only slightly decreased, while the diffusing capacity of carbon monoxide (DLCO) is disproportionately reduced , the forced expiratory volume in the first second (FEV reduced  6]. The. The1), These physiologic hallmarks of CPFE likely reflect the opposing impact of parenchymal fibrosis and parenchymal destruction on airflow and lung volumes, along with their additive impact on gas exchange. High-resolution computed tomography (HRCT) is part of the routine diagnostic evaluation of all patients with suspected IPF , and rouThe recognition of CPFE has potential management implications. Some studies suggest that CPFE is associated with reduced survival , but othRegarding therapy, nintedanib and pirfenidone seem to have a similar effect in CPFE patients as that produced in subjects with IPF alone. The ASCEND (Pirfenidone) and INPULSIS (Nintedanib) studies recruited patients with emphysema. However, the whole spectrum of severity of emphysema was not represented in those studies, because those patients who presented an obstructive ventilatory pattern or a very severe reduction of DLCO on lung function testing were excluded. It is unclear whether patients with IPF and CPFE can benefit from treatments with inhaled drugs, such as long-acting beta-agonists, long-acting muscarinic antagonists, and inhaled corticosteroids ,6,7,8. WGastroesophageal reflux (GER) is another common comorbidity in patients with IPF ,10,11, aIf GER is severe, it may imply pulmonary aspiration of gastric fluid, which may have a role in the natural history of IPF, as it might be a trigger of alveolar damage.Esophageal pH monitoring remains the gold standard for diagnosis of acid GER, with a reported sensitivity and specificity over 80% ,15. ReceThe survival of IPF patients might be reduced if GER is left untreated. Medications to treat GER are a predictor of better survival in subjects with IPF. Reflux has also been related to exacerbations in patients with pulmonary fibrosis. It has been demonstrated that bronchoalveolar lavage (BAL) pepsin levels are raised in 30% of the cases with acute exacerbation of IPF (AE-IPF) . These fOn the other hand, it has been found that the presence of reflux occurs more frequently when pulmonary fibrosis is asymmetric, with one lung more affected than the other, and this finding suggests a relationship between these diseases .In patients in whom both diseases occur, reflux is often silent and associated with preserved esophageal function but with slow clearance of gastric acid, and it is usually present in the supine position. There is a need for further investigation of the association between GER and IPF and the effect of anti-acid therapy in patients with this disease. Although the effectiveness of anti-reflux surgery is controversial, and, thus, this treatment should not be performed routinely, it should be considered for selected IPF patients.Compared to the general population, individuals with IPF have a nearly five-fold increased risk of developing lung cancer, with 3–22% of cases affected, and an estimated incidence of 11 cases per 100,000 person-year ,20,21.The strong link between IPF and cigarette smoking  likely eSome studies show that squamous cell carcinoma predominate over adenocarcinoma , while aThe survival among those patients with IPF and comorbid lung cancer is poor ,23, and While surgical resection of early-stage lung cancer may be curative, IPF severity and disease evolution must be taken into consideration, because of the increased risk of postoperative morbidity and mortality. These patients have a higher mortality associated with the surgical treatment, usually due to the development of an acute exacerbation (5–15%), which leads to a short-term mortality of approximately 50%. Exacerbations may also occur after treatment with radiation therapy or chemotherapy  . The strRecent studies suggest that the anti-proliferative effects of pirfenidone and nintedanib may synergize with concurrent chemotherapeutic oncologic agents, but additional research is needed to confirm this hypothesis ,23,24,252  ≥25 mmHg, a pulmonary artery wedge pressure ≤15 mmHg, and elevated pulmonary vascular resistance >3 wood units with a cardiac index <2.5 L/min/m2  26]. An. An2 , which estimates the right ventricular systolic pressure (RVSP) by the systolic tricuspid regurgitation velocity at Doppler as a surrogate for mPAP, is the first and most widely used test to search for PH . Althoug 80 mmHg . When th 80 mmHg . Unfortu 80 mmHg . Therefoclinicaltrials.gov NCT02951429, NCT02802345) that are currently enrolling participants. Nowadays, there is not an effective specific therapy for PH associated with IPF. Furthermore, several trials have demonstrated that pulmonary vasodilators might increase the v/q mismatch, which could result in a worsening of the hypoxemia and disease progression in IPF patients.In addition to enhance the non-invasive detection of PH, there is a need to evaluate new treatments that might improve either the hemodynamic or the clinical parameters in patients with IPF-PH. Therapies used to treat group 1 pulmonary arterial hypertension, such as endothelin receptor antagonists, phosphodiesterase-5 inhibitors, guanylate cyclase stimulators, or prostacyclin analogues, have not been effective to treat the underlying PH in the setting of IPF. Raghu et al. reported negative results for ambrisentan, an endothelin receptor type-A selective antagonist, for the treatment of IPF in patients with RHC-proven PH . The triWith the approval of pirfenidone and nintedanib, a new scenario is open for the role of these drugs in PH associated with IPF. A tyrosine kinase inhibitor as imatinib has been reported to improve exercise capacity and hemodynamics, but serious adverse events led to a high rate of study discontinuation in the active treatment arm, precluding definitive conclusions. The antiproliferative and anti-inflammatory activity of pirfenidone could theoretically be beneficial in the treatment of PH associated to IPF, but presently there is no definitive data that support this hypothesis. Thus, the possible beneficial role of nintedanib in PH due to IPF remains an open area of investigation . Although hypoxemia correction confers a survival advantage in patients with COPD, the role of long-term oxygen therapy in PH-IPF has not been clearly established. Moreover, it has been suggested that long-term oxygen therapy for this disease does not result in either improvement of PAP or reversal of pulmonary vascular remodeling. Nevertheless, the only therapeutic approaches to PH in the setting of IPF currently recommended by the European Society of Cardiology/Eureopean Respiratory Society (ESC/ERS) guidelines are to correct hypoxia with supplemental oxygen therapy, to treat the underlying interstitial lung disease, and to refer the appropriate candidate for lung transplantation . PulmonaIn conclusion, research in PH-IPF can be considered an emerging field. Further prospective studies are required to determine the best methods to detect PH in patients with IPF. In addition, and more importantly, current strategies for treating PH in IPF are far from perfect, and this fact should stimulate investigators to explore whether PH-IPF patients might benefit from novel therapies.In healthy people, sleep is a state of restoration which covers approximately one-third of human life. Overall across studies, sleep is reported to be markedly disturbed in patients with IPF . In fact1 and FVC percentages. These findings suggest that obese IPF patients with decreased pulmonary function might have an increased risk for OSA, especially during REM sleep [Recent studies have revealed that moderate-to-severe obstructive sleep apnea (OSA) syndrome, defined by an apnea–hypopnea index (AHI) > 15 events per hour, is frequent in patients with IPF . MoreoveEM sleep . MermigkEM sleep . HoweverEM sleep . InvestiEM sleep .2) had greatest sleep-related desaturation, especially during REM sleep. Thus, nocturnal hypoxemia seems to be common between patients with IPF , and this also has a negative impact on the quality of life and daytime function [Anyway, most studies have agreed on the finding of disturbance of sleep architecture. The abnormal sleep macroarchitecture in patients with IPF is characterized by abnormal sleep stage distribution with reduced slow-wave and REM sleep, increased stage 1 sleep, multiple awakenings, decreased percentage of total sleep time, low sleep efficiency, and increased wake time after sleep onset. These patients also have an altered microarchitecture, as they present with an increased number of sleep microarousals resulting in sleep fragmentation. Therefore, these disorders in sleep architecture and the resulting poor quality of sleep impair the quality of life in patients with interstitial lung diseases (ILD) . Anotherfunction . These fThere is also some evidence that REM-associated sleep-related breathing disorders (SRBD) in IPF patients might play an important role, not only in the quality of sleep and life, but also in the mortality associated with ILD. The sleep-related desaturation in these patients might contribute to pulmonary vascular disease development, with PH as one of the possible consequences. Therefore, it might be a risk factor for increased mortality. It must be noted that, in the study by Corte et al., nocturnal oximetry was not able to distinguish those patients with ILD who desaturated because of concomitant OSA . Hence, Since SRBD in IPF patients seem to be so prevalent, even in the initial phase of IPF, and are likely to be underdiagnosed, it is important for physicians to recognize the clinical phenotype of these patients. Early detection and referral to a sleep center for evaluation is essential to promptly initiate treatment. The therapy for SRBD in patients with IPF is decided on an individual basis, although the optimization of sleep and quality of life through SRBD treatment should be recognized as a primary goal .In this sense, available tools for treating patients with IPF and underlying SRBD are oxygen therapy for REM-related SRBD and continuous positive airway pressure (CPAP) for the overlap of IPF and OSA. Oxygen administration is reasonably well tolerated and recommended for advanced forms of IPF. Nevertheless, long-term home oxygen therapy in IPF patients did not improve survival when respiratory failure had been established. Moreover, only one study was carried out to analyze the use of CPAP in patients with IPF and OSA. This investigation found that these patients had a significant improvement in quality of life and sleep, daily living activities, and survival . The stuIn conclusion, alterations in sleep architecture, breathing pattern, and desaturations in IPF patients, especially during REM sleep, as well as coexisting OSA, might have a significant negative impact on the sleep quality of these patients. Physicians should ask the patient for symptoms during the night and should maintain a high degree of clinical suspicion for the presence of possible comorbid sleep disorders. As IPF is a progressive and life-threatening disease, the role of the poor quality of sleep as a significant comorbidity must be reconsidered. The effective treatment of comorbidities in IPF patients, such as sleep disorders, should be a field of future research.Idiopathic pulmonary fibrosis is a progressive disease that can present different types of evolution overtime. Some patients may progress slowly, while others experience a rapid progression, with fast decline of the lung function. Other subjects, however, may suffer episodes of sudden deterioration. These episodes are defined as acute exacerbations (AE-IPF) .Traditionally, AE-IPF have been defined as acute, clinically significant episodes of respiratory deterioration without an identifiable cause. In recent years, the International Working Group in AE-IPF has reviewed this definition as well as its diagnostic criteria . The lasThe incidence of these episodes varies between 2% and 16% per year. Results from clinical trials of nintedanib and pirfenidone suggest that IPF therapies may help to prevent the development of AE-IPF .The aetiology of AE-IPF remains unknown. It is thought to have similarities with the aetiology of acute lung injury (ALI), so that experimental models of ALI have been used for the study of AE-IPF. AE-IPF is most likely triggered by an acute event that leads to widespread acute lung injury . There iAE-IPF is more common in patients with physiologically and functionally advanced disease . Other identified risk factors are younger age, coronary artery disease, and high body mass index .The presence of AE-IPF not only implies a high mortality during the event (up to 50%), but also a significant decrease in short-term survival after the episode. IPF patients who suffer an exacerbation have a subsequent average survival of three to four months. Mortality reaches 90% in cases in which the patient needs mechanical ventilation .Given the lack of knowledge on the pathogenesis of AE-IPF, it is understandable that there is no current effective therapy available. Treatment with corticosteroids is a recommendation based on weak-strength, very low-quality evidence. The evidence available is based on isolated case reports, and there are no controlled clinical trials that endorse the benefits of their use . The exaThe use of mechanical ventilation is also controversial because of the high mortality observed when this therapy is employed, so it is usually reserved as a life-supporting procedure for those patients who will undergo a lung transplantation. Anyway, the decision to initiate mechanical ventilation must be carefully considered on an individual basis .Several potential therapies for AE-IPF have been described over the last decade, but these studies are mostly small and uncontrolled: cyclosporine, rituximab, tacrolimus, azithromycin, and intravenous thrombomodulin are some examples .


This distinction does not imply no role for purely salience-driven, or bottom-up, attention, but the latter topic is less crucial here. The term selection history alludes to the fact that the history of selection episodes, both from the recent and more distant past, can elicit lingering selection biases that are unrelated to top-down control or to the physical salience of objects. One further claim is made in the review, namely, that signals pertaining to the current goals of the individual (top-down), the intrinsic stimulus salience (bottom-up) and selection history biases all converge onto the same priority map(s) of space, wherein the compound signal will determine within a winner-take-all architecture which particular stimulus/location will be selected first.The review article by Jan Theeuwes  mainly fIn our commentary to this interesting and stimulating review paper, we want to draw attention onto three specific points that might fuel discussion, as briefly outlined below.experience-dependent attentional control, since previous encounters with a given object and/or a given location must not have involved specific attentional operations  to exert an influence on ongoing attentional deployment. In line with this notion, some of the studies carried out over recent years to study the influence of reward on attentional control  propagation of signals from control regions to target areas responsible for visual processing of the retinal input  plastic changes in critical neural substrates.Second, we think that a neurophysiological perspective might be of help to better define the different forms of attentional control the author illustrates. Within this perspective, we propose to define: i) sm e.g., , that fosm e.g., ; ii) Topal input ; and, fiThird, we would like to make some comments regarding the schematic illustration shown in Figure 1 of the review article by Theeuwes, depicting the integration of three sources of bias within the same priority map(s) of space. Our reflection on this point is threefold. On the one hand, we remark the possibility that different sources for attentional control, or biasing signals, rather than being merged through a simple computation , as suggested by the author, might be weighted differently depending on the given context and task demands. For instance, one might conceive the possibility that, depending on the given circumstances, one or the other source will exert greater influence on the priority map, therefore being the primary determinant of attentional deployment; in other words, as recently suggested by Todd and Manaligod, a context-dependent weighting of different biasing signals for AC might be at place . On the


In other words, several TADs may belong to the same 3D community. (ii) CTCF proteins—a loop-stabilizing protein that is ascribed a big role in TAD formation—are well correlated with community borders only at one level of organization. (iii) TADs and A/B compartments are traditionally treated as two weakly related 3D structures and detected with different algorithms. With our method, we detect both by simply adjusting the resolution parameter. We therefore argue that they represent two specific levels of a continuous spectrum 3D communities, rather than seeing them as different structural entities.Several experiments show that the three dimensional (3D) organization of chromosomes affects genetic processes such as transcription and gene regulation. To better understand this connection, researchers developed the Hi-C method that is able to detect the pairwise physical contacts of all chromosomal loci. The Hi-C data show that chromosomes are composed of 3D compartments that range over a variety of scales. However, it is challenging to systematically detect these cross-scale structures. Most studies have therefore designed methods for specific scales to study foremost topologically associated domains (TADs) and A/B compartments. To go beyond this limitation, we tailor a network community detection method that finds communities in compact fractal globule polymer systems. Our method allows us to continuously scan through all scales with a single resolution parameter. We found:  Rather, across cell types and organisms, Hi-C experiments reveal that chromosomes are built up by a network of three dimensional (3D) compartments.Experiments that detect the physical contacts between DNA loci in the nucleus, such as Hi-C6)-base-pair scale, two types of coexisting structures stand out. In one, all chromosome loci belong to one of the two so-called A/B compartments, where the chromatin in one compartment is generally more open, accessible, and actively transcribed than the other. In the second type, linear subsections of the genome assemble into topological domains4, often referred to as topologically associated domains (TADs)4. Plotting Hi-C data as a heat map, TADs show up as local regions with sharp borders with more internal than external contacts. The positions of these borders are correlated with several genetic processes, such as transcription, localization of some epigenetic marks, and DNA-binding profiles of several proteins–most notably CTCF and cohesin4.At the mega . However, there are some limitations in their approach. For example, they binarize the Hi-C data (‘no connection’ or ‘connection’ with a rather arbitrarily chosen thresholds) thereby discarding contact frequency variations in the Hi-C data. Furthermore, there is no comparative study connecting their communities to biological factors or any mechanistic models. Wang et al.8 takes a step in this direction, but the method to detect the TADs itself relies on biological factor data and nontrivial threshold criteria.Some network approaches are based on community detection methods that are related to what we use here. In Cabreros 1. This goes beyond previous Louvain-like studies15 that treat the Hi-C data as a network with random connections.To overcome some of these problems, we start by acknowledging that the chromosomes have a richer 3D organization than simply TADs and A/B compartments. These are just two examples. To capture this, we develop network-based method that allows us to scan through 3D structures on all scales with a resolution parameter. In particular, our approach is based on the GenLouvain method, originally designed for network community detection. For a specific value of the resolution parameter, the method finds the optimal community structure with respect to a null model of the network that has to be specified beforehand. Based on the physics of compact polymer globules, we put forward a null model that is consistent with the average contact probabilities in real Hi-C dataet al. and Norton et al.15, treat TADs as linear contiguous sequences of chromatin. This restriction overrides the GenLouvain algorithm’s ability to find the (not necessarily contiguous) optimal community structure in the data set20. We remove this restriction in our study. Therefore, to reduce confusion, we will not use the term TAD, but rather the 3D community for the cluster of nodes that comes out of the GenLouvain algorithm since they are not necessarily linear contiguous sequences.Furthermore, most studies, such as Yan 21 to detect communities in the Hi-C maps . This algorithm offers the possibility to find communities at several scales with a single resolution parameter γ. Contrasting other methods with similar features, the spectrum of communities that we detect is not necessarily hierarchical or nested, as in e.g. Wang et al., Fraser et al., and Bianco et al.24. Instead, two different values of γ give two different collections of communities, and these do not necessarily have anything to do with each other.We use the GenLouvain algorithmQ. This function quantifies by how much more dense the connections are within the communities compared to what they would be in a particular type of network, say a random network. GenLouvain’s modularity function isAij is the number of contacts between all node pairs i and j (Aij is the adjacency matrix in standard network terminology). The resolution parameter γ controls the overall scale of communities we would like to detect, where larger values of γ correspond to smaller communities. Furthermore, i and j are connected in the real network, compared to how strongly we expect them to be given δ is the Kronecker delta that is unity only if nodes i and j belong to the same community (otherwise it is zero), and m is a normalization factor so that Q goes between −1 and +1.To find the communities, the GenLouvain method tries putting the nodes into different communities to maximize the so-called modularity function 25. For a unweighted network where Aij is either zero or one, ki and kj are the number of links for nodes i and j .One of the most popular choices for et al.1, and many papers thereafter27, the contact probability between two nodes i and j decays as a power-law with the linear distance between them, that is However, the NG null-model is too rough an approximation to find communities in Hi-C maps, because it does not obey the well-established contact patterns that we know exist in DNA, or in fact any polymer system. For example, DNA is a long polymer where nodes are arranged in a linear sequence and then folded in 3D. This sets limitations for how frequently two pieces of DNA, or nodes, can join in 3D space . As was first discovered in Lieberman-Aiden α is debatable, but we use α = 1. There are two main reasons. First, at the mega-base-pair scale of human DNA, the Hi-C data suggest that it is close to one1 . Second, in the next section we will study community detection in the fractal globule polymer . This is a compact polymer that mimics the large scale structure of human chromosomes, in particular the scaling relations of the end-to-end distance and contact frequency1. The advantage of this approach is that we have the explicit 3D coordinates for every part of the polymer (because we made it), which allows us to visualize and analyze the 3D structure of the communities that we detect. For the Hi-C map, we only have pairwise contact frequencies.Before we investigate the human Hi-C data, we wish to better understand the types of 3D structures that our community detection method picks up. To do this, we use computer-generated fractal globule polymers  model28. In a nutshell, this is a Monte Carlo method that produces a fractal globule by simulating a biased random walk on lattice where the propagation probability depends on the entire walk’s trajectory over the lattice28. This yields on-lattice space-filling polymers. To generate off-lattice fractal globules, the structure is randomized with simulated annealing where the position of a monomer is randomly displaced under the constraint of a fixed inter-monomer distance. With properly chosen parameters, the CDP method produces a fractal globule with contact frequency (probability) that decays as ~s−1 (as it should29), where s is the contour distance along the polymer. To use the notation from Tamm et al.28, we use the relation p ∝ (1 + An) with A = 104 for an unoccupied site and p to an occupied site is given by ε = 10−4. This result is consistent with the Hi-C data for the human genome at the mega-base-pair scale1. We show a realization of a CDP in Fig. As introduced in an articlei and j. The contact refers to the case where the Euclidean distance between two beads are shorter than three lattice spacings. To obtain good statistics, we first generate an on-lattice polymer, and then during the annealing stage we register all contacts in 103 random variations of the on-lattice structure. In addition, we have confirmed that different threshold values for what we consider as a contact do not qualitatively alter our results presented below. Just as in the Hi-C experiments, in the final step we normalize the contact map with the KR-norm35 so that each row and column sums to unity.From every simulated CDP polymer, we construct a contact map by counting the number of contact events between polymer beads As a reference case, we use the equilibrium globule. This is a self-avoiding polymer in a closed spherical volume. When we generate equilibrium globules, we contain it in a volume with the same diameter as the fractal globules’ radius of gyration.γ, we detect communities on various scales. On top of the contact map in Fig. γ = 0.4 (b), γ = 0.6 (c), and γ = 0.8 (d), where the boxes represent community boundaries. Based on these, we ask what the 3D structure of these communities is, and if and how they are different from the polymer as a whole.Figure γ = 0.6). In Fig. Contrasting current views on TADs, we find that 3D communities do not have to be a contiguous polymer segment. Rather, linearly distant parts of the polymer can fold in 3D to form a community. We show this in Fig. s.Furthermore, because of the above-average contact frequencies inside a community, we would like to quantify how its 3D structure differs from the fractal globule polymer as a whole. To do this, we examine the scaling relation of the end-to-end distance–the Euclidean distance between the two boundary monomers defining that (contiguous) community–with respect to the subchain length s1/3, as is expected for a space-filling curve (deviations for large-s comes from finite-size effects and insufficient statistics). For the communities, we notice that the end-to-end distances are systematically smaller than for a randomly chosen subchain. Our simulations even suggest that the scaling exponent is smaller than 1/3. Consistent properties are crosschecked in Fig. 2. For comparison, we made the same analysis for the equilibrium globule (EG). In Fig. s1/2 for s0 for N monomer ideal chain. Concurrently, the end-to-end distance normalized by the radius of gyration . We find that the community subchains in FGs tend to be more sphere-like than the FG itself, and the subchains in EGs. This leads to enhanced contact frequency between monomers within a community . The data is available in various resolutions, from 103 base pairs (1 kbp) to 106 base pairs (1 Mbp), but we used 100 kbp which is the scale where both TADs and A/B compartments can be detected2.Based on our community detection approach, we now proceed to analyze Hi-C data from human cellsγ. Similar to the simulated data, it is clear that several TADs form 3D communities. To investigate how these TADs correspond to TADs defined in other studies, we compared the border locations of each contiguous block to TAD borders defined by Rao et al.2 (the group that produced the dataset we use in this study) in Fig. γ ≈ 0.7, about 70% of the borders overlap. Moreover, several studies have shown that binding sites for the insulator protein CTCF are strongly correlated with TAD borders . We therefore check the overlap between CTCF binding sites (mapped by ENCODE36) and all borders at different γ values. We note that CTCF has the highest overlap also at γ ≈ 0.7 . Interestingly, for γ < 0.6, the chromosome consists of only two communities .We next investigate properties of the 3D communities. First, we count the number of communities and average number of TADs within each community at different γ Fig.  as well γ Fig. . The hig36 (GEO Acc. Nr: GSE88583). In Figs. γ. At small γ values where only two communities are defined, one community is clearly more active than the other. The active and less active communities are then split up into smaller communities as γ increases. Already in the original Hi-C paper by Lieberman-Aiden et al.1, they used principal component analysis on the Hi-C data to partition the chromosomes into two classes. Denoting them by A/B compartments, they found that the A compartment contains transcriptionally active chromatin, whereas the B compartment is less active.Second, we map gene activity within each community using RNA-seq data from ENCODE38 to see what types of chromatin states are enriched in different communities at different γ values  starts to split up into smaller compartments and then the less active parts of the genome (B) also start to split into smaller communities. It is clear that the genome does not split up into evenly sized sub-compartments, but rather the 3D space is dominated by a few large communities. It is striking and unprecedented that by tuning a single parameter, we detect both TADs and A/B compartments with the same algorithm. We therefore argue that TADs and A/B compartments are not two conceptually different organizational structures in the nucleus, but rather different ends of the same organizational spectrum.We took one step in this direction by looking at different types of chromatin. We use 15 chromatin states defined by ENCODE3)-base-pair sized loops to mega(106)-base-pair sized 3D structures. This pattern is consistent across organisms and cell types. To let researchers scan through the spectrum of 3D compartments, we have tailored the GenLouvain community detection method to find 3D communities in fractal globule polymer systems. Apart from verifying our method on computer-generated polymers, we have applied it to analyze human Hi-C data. First, we have found that chromatin segments belonging to the same 3D community do not have to be in next to each other along the DNA. In other words, several TADs can belong to the same 3D community. Second, we have found that CTCF proteins–a loop-stabilizing protein that is ascribed a big role in TAD formation–are only correlated well with community borders at one level of organization. It remains to find what other factors are important at higher or lower levels. Third, just by adjusting a single parameter (γ), our method picks up the two most prominent 3D compartments, TADs and A/B compartments, which are traditionally treated as two weakly related 3D structures and detected with different algorithms. Rather than seeing them as different, our work put them on an equal footing, and we argue that they represent two ends of a continuous spectrum of 3D communities of different sizes.From Hi-C experiments, it is clear that inter-phase chromosomes are built up by a network of 3D compartments on various scales–from kilo(10Supplementary Information


Liver cancer is the second leading causes of cancer-related death globally. Pyrroline-5-carboxylate reductase 1 (PYCR1) plays a critical role in metabolic profiles of tumors. Therefore, it is necessary to explore the mechanisms of PYCR1 on cell growth and survival in hepatocellular carcinoma (HCC).Protein and mRNA expression levels of PYCR1 in 140 pairs of tumor and adjacent normal liver tissues of HCC patients were analyzed by immunohistochemistry and quantitative real-time polymerase chain reaction (qRT-PCR). Expressions of PYCR1 were inhibited in BEL-7404 cells and SMMC-7721 cells using gene interference technology. The cell proliferation was detected by Celigo and MTT assay. The colony formation assay was also performed. The cell apoptosis was measured by flow cytometric assay. The effect of PYCR1 interference on tumor growth was observed by xenograft nude mice assay in vivo. The downstream pathway of PYCR1 interference was searched by microarray and bioinformatics analysis, and validated by qRT-PCR and western blot.P < 0.01). In vitro, the cell proliferation was significantly slower in shPYCR1 group than shCtrl group in BEL-7404 and SMMC-7721 cells (P < 0.001). The colony number was significantly smaller after PYCR1 interference (P < 0.01). The percentage of apoptosis cells significantly increased in shPYCR1 group (P < 0.01). In vivo, PYCR1 interference could obviously suppress tumor growth in xenograft nude mice. The volume and weight of tumors were significantly smaller via PYCR1 interference. The c-Jun N-terminal kinase (JNK) signaling pathway significantly altered, and insulin receptor substrate 1 (IRS1) were significantly down-regulated by PYCR1 interference in both mRNA and protein levels (P < 0.001).PYCR1 levels were significantly up-regulated in HCC tumor tissues than adjacent normal liver tissues in both protein and mRNA levels (PYCR1 interference could inhibit cell proliferation and promote cell apoptosis in HCC through regluting JNK/IRS1 pathway. Our study will provide a drug target for HCC therapy and a potential biomarker for its diagnosis or prognosis. Liver cancer is the second leading causes of cancer-related death globally . 90% of Growing tumors alter their metabolic profiles to meet the biosynthetic and bioenergetics demands of increased cell growth and proliferation . Many stIn this study, we aim to analyze the relationship between PYCR1 and HCC, explore the role of PYCR1 regulating c-Jun N-terminal kinase/insulin receptor substrate 1 (JNK/IRS1) pathway, and clarify the mechanisms of PYCR1 affecting HCC cell growth and survival. The achievements will probably provide us a potential drug target for therapy or a biomarker for diagnosis and prognosis.http://cancergenome.nih.gov).The tumor and adjacent normal tissues used for immunohistochemistry were collected from 90 HCC patients who underwent surgery at Shanghai Seventh People’s Hospital between 2007 and 2009. The informed consent was obtained from every study participant. The study was conducted in accordance with the Declaration of Helsinki, and the study protocol was approved by the Institutional Research Ethics Committee in 2007 (No. 2007-019). The mRNA profiles used in the study were obtained from the Cancer Genome Atlas (TCGA) database , supplemented with 10% fetal bovine serum , 100 U/ml penicillin and 0.1 mg/ml streptomycin  at 37 °C in 100% air.5/well for RNA interference. According to the results of preliminary experiment, HCC cells were infected with a certain amount of control lentivirus and shPYCR1 lentivirus. At 72 h post-infection, the expression of reporter gene GFP was observed by the fluorescent microscope. The fluorescence rate was the positive infective rate. If the infective rate was larger than 70% and the state of cells was normal, the further study could be continued.The sequences for RNA interference were 5′-TTCTCCGAACGTGTCACGT-3′ for control, and 5′- CAGTTTCTGCTCTCAGGAA-3′ for PYCR1 . The sequences were inserted into Age I and EcoR I points of GV115 vector. The shRNAs of GV-NC-GFP-shRNA and GV-PYCR1-GFP-shRNA were constructed as control lentivirus and shPYCR1 lentivirus, and positive clones were screened and identified. Subsequently, HCC cells were seeded in a 6-well plate at a density of 2 * 10Total RNA from HCC cells was extracted using TRIzol Reagent . RNA concentration was determined by NanoDrop 2000 spectrophotometer . A total of 20–100 ng RNA was reverse-transcribed using First-Strand cDNA Synthesis kits  according to the manufacture’s protocol. qRT-PCR was performed on ABI 7500 System  and used to analyze the expression levels of mRNA. Relative expression level of genes was calculated using GAPDH as the internal control. Each sample was run three times. The primer pairs used in the study are listed as follows. PYCR1 forward: 5′-GGCTGCCCACAAGATAATGGC-3′; reverse: 5′-CAATGGAGCTGATGGT GACGC-3′; GAPDH forward: 5′-TGACTTCAACAGCGACACCCA-3′; reverse: 5′-CACCCTGTTGCTGTAGCCAAA-3′.The cells were first washed with cold PBS three times, then lysed in RIPA lysis buffer  containing a complete protease inhibitor cocktail , and cell lysates were collected. Concentrations of proteins were then determined by bicinchoninic acid (BCA) protein assay kit . About 20–50 μg of proteins were separated by 10% SDS-polyacrylamide gel and then transferred to a polyvinylidene difluoride (PVDF) membrane. Subsequently, the membrane was incubated with primary antibodies against PYCR1 (1:200) , JUN (1:500) , IRS1 (1:500)  and GAPDH (1:2000)  overnight at 4 °C, and appropriate secondary antibodies (1:2000)  for 1 h at room temperature. Signals were detected by gel imaging system . The ratio of interested proteins to GAPDH was analyzed by FluorChem FC3 software. Western blot experiments were performed in triplicate.8 TU/ml) and shPYCR1 lentivirus  were seeded in 96-well plates at a density of approximately 2–5 * 103/well in quintuplicate. Cell proliferation was performed at specific times, such as 1st day, 2nd day, 3rd day, 4th day and 5th day. The fluorescence was measured to access cell counts using Celigo Imaging Cytometer . In addition, for MTT assay, 20 μl of MTT solution was added to each well. After 4 h incubation at 37 °C, the solution was removed and DMSO was added to dissolve the formazan crystals formed. The absorbance at 490 nm was measured Glomax-Multi + Detection System . At least three independent experiments were performed.HCC cells infected with control lentivirus  after 14 days, then colonies were visualized by fluorescence microscope and quantitated. At least three independent experiments were performed.Apoptosis assay was performed using FITC-Annexin V Apoptosis Detection Kit  according to the manufacture’s instruction. Briefly, the cells were collected by trypsin, washed twice with ice-cold PBS and resuspended in 1 * binding buffer. Then, 10 μl of annexin V-FITC and propodium were added into 100 μl of cell suspensions. After incubation for 15 min, the samples were analyzed using the Beckman Counter flow cytometer.6) stably expressing control lentivirus and shPYCR1 lentivirus, respectively. After the diameter of tumor was larger than 5 mm, tumor volume was determined twice per week. The tumor volume was calculated as V = (width2 * length)/2. The bioluminescence imaging was detected before sacrifice. Tumor was obtained and weighed after sacrifice. All animal studies were approved by the Ethics Committee of Shanghai Seventh People’s Hospital.A total of twenty female BALB/C nude mice  were purchased from Shanghai Lingchang Biotechnology (SCXK2013-0018). All of them were randomly separated into two groups, NC group (n = 10) and KD group (n = 10). They were subcutaneously inoculated with SMMC-7221 cells  and a core biologic pathway analysis was performed to identify molecular pathways.Total RNA was extracted from HCC cells using Trizol reagent in shCtrl group (n = 3) and shPYCR1 group (n = 3). The qualified samples were analyzed on Affymetrix GeneChip primeview human gene expression array. Differentially expressed genes were selected with a cut-off P < 0.05 was considered statistically significant. All results were confirmed by at least three independent experiments.Statistical analysis was performed by SPSS 16.0 . The data were expressed as mean ± standard deviation and analyzed using Mann–Whitney test because of abnormal distribution or heterogeneity of variance. P < 0.001, Fig. P < 0.01). Both our data and TCGA data proved that PYCR1 was over expressed in HCC tumor tissues.To find out whether PYCR1 was dysregulated in HCC patients, we determined PYCR1 protein expression levels in tumor and adjacent normal liver tissues of HCC patients by IHC. Our data revealed that PYCR1 levels were higher expressed in tumor tissues than adjacent normal ones . After sacrifice, the tumors were weighed, and obvious tumors were formed in NC group while no tumors were observed in KD group  for SAPK/JNK signaling pathway was highest , implying that PYCR1 might influence HCC cells proliferation and apoptosis by regulating JNK/IRS1 pathway.To explore the mechanisms of PYCR1 interference inhibiting cell proliferation and promoting apoptosis in HCC cells, we detected the gene expression profiling of shCtrl group and shPYCR1 group in SMMC-7721 cells. Compared with shCtrl group, 173 genes were up-regulated in shGINS2 group, and 379 genes were down-regulated. As shown in Fig. est Fig. b. Subseq+ [In this study, we investigated the role of human PYCR1, so-far a poorly studied protein. PYCR1 is one of three human PYCR isoenzymes. PYCR catalyzes the final step in the conversion of Δ1-pyrroline-5-carboxylate (P5C) to proline with concomitant oxidation of NAD(P)H to NAD(P)+ . In clinFurther investigation found that SAPK/JNK signaling pathway was significantly altered after PYCR1 interference Fig. b. C-Jun-JNK was reported to drive insulin resistance via direct phosphorylation of insulin receptor substrate (IRS) , a familMany researchers thought that amino acid concentrations were associated to insulin resistance . In the In conclusion, we report here that PYCR1 interference could inhibit cell proliferation and promote cell apoptosis in HCC, and the mechanisms might be involved in the regulation of JNK/IRS1 pathway Fig. . Our stu