Inflammation plays an important role in the pathogenesis of intervertebral disc (IVD) degeneration. The proinflammatory cytokine tumor necrosis factor alpha (TNF‐α) has shown markedly higher expression in degenerated human disc tissue compared with healthy controls. Anti‐inflammatory treatment targeting TNF‐α has shown to alleviate discogenic pain in patients with low back pain. Therefore, in vitro and ex vivo inflammatory models utilizing TNF‐α provide relevant experimental conditions for drug development in disc degeneration research.The current method article addressed several specific questions related to the model establishment.(a) The effects of bovine and human recombinant TNF‐α on bovine nucleus pulposus (NP) cells were compared. (b) The required dose for an inflammatory IVD organ culture model with intradiscal TNF‐α injection was studied. (c) The effect of TNF‐α blocking at different stages of inflammation was evaluated.3 disc volume, as indicated by increased nitric oxide, glycosaminoglycan, interleukin 6 (IL‐6), and interleukin 8 (IL‐8) release in culture media, and upregulation of MMP3, ADAMTS4, IL‐8, IL‐6, and cyclooxygenase (COX)‐2 expression in NP tissue. However, results in human NP cells showed that the time point of anti‐inflammatory treatment was crucial to achieve significant effects. Furthermore, anticatabolic therapy in conjunction with TNF‐α inhibition would be required to slow down the pathologic cascade of disc degeneration.Outcomes revealed that bovine and human recombinant TNF‐α induced equivalent inflammatory effects in bovine NP cells. A bovine whole IVD inflammatory model was established by intradiscal injection of 100 ng TNF‐α/ cm This article describes the methods for establishment of proinflammatory intervertebral disc cell and organ culture models induced by TNF‐a. Compared with the small animals like mouse, rat and rabbit, the IVDs from large animals such as sheep, dog and cow are more similar to human. They show comparable size and loss of notochordal cells in early adulthood as human IVD.Does TNF‐α from bovine and human have the same proinflammatory effect on bovine NP cells?What is the optimal dose of TNF‐α when utilized within an IVD inflammation organ culture model induced by TNF‐α intradiscal injection?Does TNF‐α inhibition at different stages of inflammation have equal anti‐inflammatory and/or regenerative effects on NP cells?In the present study, we sought to establish TNF‐α induced in vitro and ex vivo IVD inflammation models, which would represent preclinical testing systems for screening of anti‐inflammatory drugs for disc degeneration treatment. Specifically, the following questions were addressed within this study:22.1Alpha Minimum Essential Medium (αMEM) has shown an advantage compared with Dulbecco's Minimum Essential Medium (DMEM) in terms of numbers and quality of cells acquired in mesenchymal stem cells isolation and expansion.2.24Cl, 10 M KHCO3, and 0.1 mM EDTA in Milli‐Q water) to remove the red blood cells. The chopped tissue was digested with 0.2% w/v Pronase  in αMEM  for 1 hr, then digested with 65 U/mL collagenase type II  in αMEM / 10% fetal bovine serum  in a spinner flask for 12 to 14 hours at 37°C. The digested cell suspension was filtered through a 100 μm cell strainer to obtain a single‐cell suspension. NP cells were expanded in αMEM supplemented with 10% FBS and 100 U/mL penicillin and 100 mg/mL streptomycin , incubated at a hypoxic condition of 2% O2 at 37°C. Culture medium was changed twice a week. Passage 2 and 3 NP cells were used in the current study.Human NP cells were isolated from traumatic IVDs  with ethical approval . General consent was obtained from all patients before surgery. All studies were performed in accordance with the ethical standards as laid down in the 1964 Declaration of Helsinki and its later amendments or comparable ethical standards. The IVDs were classified as mildly degenerated by MRI (Pfirrmann grade 2‐3). Bovine NP cells were isolated from caudal intervertebral discs of 6 to 12‐month‐old calves from local abattoirs immediately after death. NP cell isolation was performed as described previously.2.32 in 12‐well plates with DMEM medium (containing 4.5 g/L glucose) supplemented with 10% FBS. After cell attachment (24 hours after cell seeding), the medium was exchanged to serum‐free experimental medium  with or without inflammatory inducers 10 ng/mL human recombinant TNF‐α  or 10 ng/mL bovine recombinant TNF‐α . After another 72 hours of culture, the cell monolayer was lysed and RNA was isolated for gene expression analysis.Bovine NP cells were seeded at a concentration of 60 000/cm2.42. One day after seeding, cells were treated with 10 ng/mL (low dose) or 50 ng/mL (high dose) TNF‐α in serum‐free experimental medium as described above for bovine NP cells TNF‐α experiments. The samples were collected at three timepoints, 6, 24, and 48 hours after treatment, for gene expression analysis.Human NP cells were seeded into a six well‐plate at a cell density of 30 000/cmTo investigate the effect of TNF‐α blocking with the TNF‐α inhibitor Etanercept , NP cells were seeded as described above and cultured for 24 hours to allow for cell attachment. Hereafter, cells were divided into 4 different groups: (1) iNP—cells were treated with 10 ng/mL TNF‐α for 48 hours, (2) iNP‐Eta—cells were treated with 10 ng/mL TNF‐α and immediately after 1 μg/mL Etanercept was added for 48 hours, (3) iNP‐24 hours‐Eta—cells were treated with 10 ng/mL TNF‐α, 24 hours after 1 μg/mL Etanercept was added, and (4) iNP‐24 hours‐FM—cells were treated with 10 ng/mL TNF‐α, 24 hours after replaced to fresh medium without TNF‐α. Cells treated with serum‐free culture medium as described above served as negative control. All the cells were harvested for gene expression analysis at 72 hours after seeding. The concentration of Etanercept used here was selected according to previous studies, showing that Etanercept at 0.01, 0.1 and 1 μg/mL induced less than 8% cell death in TNF‐α transfected Jurkat cells, and in human NP cells and AF cells cultured with Etanercept at 100, 250, 500, 1000, and 2000 μg/mL, cell proliferation was only suppressed with Etanercept at 500 μg/mL or higher.2.52 × π × disc height. The surfaces of EPs were cleaned using a Pulsavac Wound Debridement Irrigation System  with Ringer's buffer to remove the cutting debris and blood clots. After prewashing in PBS with 10% P/S, IVDs were cultured in 6‐well plates with 7.5 mL IVD culture medium, DMEM supplemented with 1% P/S, 50 mg/mL Primocin , 2% FBS, 50 μg/mL ascorbate 2 phosphate, 1% ITS+, 1% NEAA, at 37°C, 5% CO2.Bovine caudal IVDs were collected from fresh sacrificed 6 to 12‐month‐old calves from local slaughterhouses. Disc dissection was performed as described previously.2.63 of disc volume, was firstly injected into the disc, 30 minutes after 20 μL Etanercept, containing 10 μg Etanercept per 100 ng TNF‐α, was injected into the disc. TNF‐α: 40 μL of TNF‐α, containing 100 ng TNF‐α/cm3 of disc volume, was injected into disc 30 minutes after 20 μL PBS was injected. PBS: 40 and 20 μL PBS was injected into disc sequentially. The injection was performed using a 30‐gauge insulin needle, after the first dynamic loading on day 1. The intradiscal injection dose of Etanercept was kept at the same ratio of TNFα to Etanercept as in vitro, which is 1:100. IVDs were cultured with daily dynamic loading and free swelling recovery overnight, the disc size of IVDs was measured before and after loading.IVDs having a diameter of 1.5 to 2.0 cm were selected for the current study. IVDs were cultured free swelling during the night. Dynamic loading was performed, at 0.02 to 0.2 MPa, 0.2 Hz for 2 hours per day within a bioreactor.2.7RNA samples were collected from monolayer NP cells by adding 0.5 mL TRI reagent  with 2.5 μL polyacryl carrier (Molecular Research Centre Inc) per well. RNA isolation was performed according to the manufacturer's specifications. RNA isolation from NP tissues was performed as described before.−ΔΔCT method, with RPLP0 as an endogenous control. The RPLP0 showed similar Ct value with different treatments, indicating TNFα and Etanercept did not show an influence on the house keeping gene expression.SuperScript VILO cDNA Synthesis Kit (Invitrogen) was used for cDNA synthesis with 400 ng RNA per sample. The quantitative real‐time polymerase chain reaction (qRT‐PCR) was conducted on QuantStudio6 PCR System (Applied Biosystems). The primers and probes used in qRT‐PCR for human and bovine samples are shown in Table 2.8IL‐6 and IL‐8 content in bovine IVD organ culture media were measured with enzyme‐linked immunosorbent assay (ELISA) kits . Capture antibody: anti‐bovine IL‐6 polyclonal antibody , anti‐bovine IL‐8 polyclonal antibody . Detection antibody: Biotinylated‐anti‐bovine IL‐6 , Biotinylated‐anti‐bovine IL‐8 . Experiments were performed according to the manufacturer's protocol. The results of the ELISA were presented as the original concentration in the media without normalization.2.92−), using the Griess Reagent Kit . The NO concentrations in the media are presented in the results section without normalization.The amount of Glycosaminoglycans (GAGs) released in IVDs culture media was measured by using the 1,9‐dimethylmethylene blue dye method.2.10t‐test was used to determine differences between two groups; One‐way ANOVA was used to determine differences between three or more groups. For the not normally distributed data, Mann‐Whitney U test was used to determine differences between two groups; Kruskal‐Wallis test was used to determine differences between three or more groups. P < .05 was considered statistically significant.Statistical analyses were performed using the GraphPad Prism 7 software . D'Agostino‐Pearson omnibus normality test was used to define whether the data were normally distributed. For data that were normally distributed, unpaired 33.1Bovine NP cells were treated with 10 ng/mL bovine or human recombinant TNF‐α. Catabolic gene expression as well as proinflammatory mediators are illustrated in Figure 3.2Human NP cells were treated with human recombinant TNF‐α at a concentration of 10 or 50 ng/mL. Samples were collected at three time points, 6, 24, and 48 hours, for gene expression analysis. As shown in Figure To investigate the anti‐inflammatory treatment with TNF‐α blocking at different time points, NP cells were treated with TNF‐α 10 ng/mL for 48 hours (iNP), TNF‐α immediately followed by 1 μg/mL Etanercept for 48 hours (iNP‐Eta), TNF‐α for 24 hours followed by 1 μg/mL Etanercept for 24 hours (iNP‐24 hours‐Eta), and finally TNF‐α for 24 hours then replacing to fresh basal medium without TNF‐α for 24 hours (iNP‐24 hours‐FM). NP cells treated with basal medium served as control . Results . Etanercept was injected 30 minutes after TNF‐α injection, at a ratio of Etanercept(w): TNF‐α(w) = 100:1 as anti‐inflammation positive control (TNF‐α + Etanercept). Discs were injected with the same volume of PBS as negative control (PBS). Discs were cultured with daily physiological loading and culture media after overnight free swelling were collected daily for NO, IL‐6, IL‐8, and GAG measurement. NP tissue was collected at two time points, at 1 (day 2) or 4 days (day 5) after TNF‐α injection. As shown in Figure Consequently, intradiscal injection was performed with TNF‐α at 100 ng/cmdays day  after TN4Anti‐inflammatory therapy has been considered as a promising approach to delay the IVD degeneration and relieve discogenic pain. TNF‐α, as a pro‐inflammatory factor, has been reported to be associated with IVD degeneration and discogenic pain.In the current study, several specific questions related to the inflammatory model were investigated. Firstly, due to the scarce access to human IVD tissue and especially to healthy samples, bovine IVD cells and bovine caudal whole IVDs have been widely used in spine research. While using TNF‐α for inflammation induction of bovine disc cells or organs, one question which has not been well addressed is whether human and bovine recombinant TNF‐α imply the same effect on bovine disc cells, or whether the TNF‐α receptors on bovine disc cells can also transmit the signaling from human recombinant TNF‐α. Our results showed both bovine and human recombinant TNF‐α can equally induce inflammation in bovine NP cells in vitro Figure . These r3 disc volume. This has effectively induced inflammation in bovine NP tissue, as shown by increased NO, GAG, IL‐6, and IL‐8 release in culture media, and upregulated MMP3, ADAMTS4, IL‐8, IL‐6, and COX2 expression in NP tissue on both day 2 and day 5. These results showed that TNF‐α intradiscal injection at the adjusted dose increased expression of catabolic enzymes and inflammatory mediators in the whole IVD organ culture system, which is consistent with previous NP tissue and cell culture studies.Secondly, the required dose for an inflammatory IVD organ culture model based on intradiscal TNF‐α injection is unclear. Takahashi et al reported that the concentration of TNFa in herniated disc tissue is a dozens of pgs per 100 mg tissue.Our result showed that TNF‐α downregulated gene expression of type II collagen in cell culture, but not in whole organ culture. In contrast, Seguin et al showed that TNF‐α decreased expression of type II collagen in NP tissue culture.Annular puncture may induce disc degeneration depending on the disc size and needle size.R2 = .935, Figure Analysis of the culture medium was undertaken to investigate whether the molecule release was related to the disc volume. This was performed with IVDs cultured under physiological loading and without TNF‐α injection. The initial GAG release on day 1 from discs of different donors showed a high variation, which may result in an inundating difference between experimental groups. The day 2 GAG release was highly related with day 1, evaluated with linear regression  and NO (B) release in culture media after overnight free swelling were measured. Each bar represent the result of one disc. No statistical analyze was applied.Click here for additional data file.Figure S2 Regression analysis of the GAG, NO, IL‐6, and IL‐8 release content in the conditioned media of bovine IVDs cultured during day 1 and day 2, under physiological culture condition without TNF‐α injection.Click here for additional data file.


Virus-like particles (VLPs) have emerged as a powerful scaffold for antigen presentation and delivery strategies. Compared to single protein-based therapeutics, quality assessment requires a higher degree of refinement due to the structure of VLPs and their similar properties to extracellular vesicles (EVs). Advances in the field of nanotechnology with single particle and high-resolution analysis techniques provide appealing approaches to VLP characterization. In this study, six different biophysical methods have been assessed for the characterization of HIV-1-based VLPs produced in mammalian and insect cell platforms. Sample preparation and equipment set-up were optimized for the six strategies evaluated. Electron Microscopy (EM) disclosed the presence of several types of EVs within VLP preparations and cryogenic transmission electron microscopy (cryo-TEM) resulted in the best technique to resolve the VLP ultrastructure. The use of super-resolution fluorescence microscopy (SRFM), nanoparticle tracking analysis (NTA) and flow virometry enabled the high throughput quantification of VLPs. Interestingly, differences in the determination of nanoparticle concentration were observed between techniques. Moreover, NTA and flow virometry allowed the quantification of both EVs and VLPs within the same experiment while analyzing particle size distribution (PSD), simultaneously. These results provide new insights into the use of different analytical tools to monitor the production of nanoparticle-based biologicals and their associated contaminants. The presence of EVs in conditioned supernatants could also be observed by SEM-Alcian Blue staining  for HEK 293 and 146 ± 42 nm (n = 188) for Sf9 cells f9 cells , indicatf9 cells E and thef9 cells F. ODVs pf9 cells G 7]. Ot. Otn = 5f9 cells E. Differf9 cells I,J. Gag n = 371) was obtained in HEK 293 samples, whereas an average of 261 ± 104 nm (n = 794) was measured for Sf9 supernatants  in order to calculate the mean nanoparticle diameter , indicating that the swarm effect could not be the main reason behind these results (SRFM, NTA and flow virometry were compared for the quantification of nanoparticles in HEK 293 and Sf9 samples . Compare results . On the Analysis of the different nanoparticle populations in each platform revealed that at least 50% of the total particles produced did not correspond to VLPs . The posThe selection of an adequate analytical method is essential in VLP characterization processes. Among the six methodologies studied in this work, cryo-TEM was shown as the best method to resolve nanoparticle structures while maintaining its native conformation. This technique allowed for a detailed characterization of the different EV and BV subpopulations co-produced with VLPs in HEK293 and Sf9 productions. Alternatively, the high-throughput analysis of VLPs and their differentiation from other contaminant particles was achieved by flow virometry, SRFM and NTA. Among them, flow virometry showed to be the fastest method for PSD analysis while also allowing to simultaneously quantify different nanoparticle subpopulations. Nonetheless, further improvements of these methods are required since different quantification results are observed between techniques.


ZFS-1 obese rats showed features of HFpEF and atrial cardiomyopathy in vivo: increased left ventricular (LV) mass, E/e’ and LA size and preserved LV ejection fraction. In vitro, LA cardiomyocytes exhibited more mitochondrial-fission (MitoTracker) and ROS-production (H2DCF). In wildtype (WT), pro-inflammatory TNF-α impaired cellular Ca2+ homeostasis, while anti-inflammatory IL-10 had no notable effect . In HFpEF, TNF-α had no effect on Ca2+ homeostasis associated with decreased TNF-α receptor expression (western blot). In addition, IL-10 substantially improved Ca2+ release and reuptake, while IL-10 receptor-1 expression was unaltered. Oxidative stress in metabolic syndrome mediated LA cardiomyopathy was increased and anti-inflammatory treatment positively affected dysfunctional Ca2+ homeostasis. Our data indicates, that patients with HFpEF-related LA dysfunction might profit from IL-10 targeted therapy, which should be further explored in preclinical trials.Metabolic syndrome-mediated heart failure with preserved ejection fraction (HFpEF) is commonly accompanied by left atrial (LA) cardiomyopathy, significantly affecting morbidity and mortality. We evaluate the role of reactive oxygen species (ROS) and intrinsic inflammation  related to dysfunctional Ca In heart failure (HF) with preserved ejection fraction (HFpEF), patients suffer from typical HF symptoms , while presenting normal left ventricular ejection fraction (>50%). HFpEF is increasingly prevalent and currently accounts for approximately half of all HF patients . While pThe prevalence of co-morbidities which are marked by endothelial dysfunction and systemic inflammation, such as obesity, diabetes mellitus type 2, arterial hypertension, dyslipidemia and renal dysfunction, is strikingly high amongst HFpEF patients . Mechani2+ handling in metabolic HFpEF-related left atrial cardiomyopathy  during electric pacing are reported as ΔF/F0. Tau of decay  is reported as a mesuare of Ca2+ reuptake. For early Ca2+ release (ER) site analysis: F50 was defined as 50% of the CaT amplitude and the corresponding time-to-F50 (TF50) calculated as an indicator of ER. Scan lines along the longitudinal axis were grouped into 1 µm intervals, indicating active couplons [Left atrial cardiomyocytes were incubated with Tyrode solution in the presence or absence of TNF-α (10 nM) or IL-10 (10 nM) at 37 °C for 30 min. Sub-sequently, cells were loaded with Fluo-4 for another 30 min at 37 °C (in the continuous presence of the respective cytokine). The cells were then washed twice with Tyrode solution and transferred to an LSM 800 laser scanning microscope. Experiments were conducted at 37 °C at steady state following approximately 1 min of electric stimulation at 1 Hz. Cacouplons ,25. ER wLeft atrial tissue was homogenized in lysis buffer (composition (in mM): 20 Tris-HCl, 137 NaCl, 20 NaF, 1 sodium orthovandate, 1 sodium pyrophosphate, 50 β-glycerophosphate, 10 EDTA, 1 EGTA, 1 PMSF, 10% glycerol, 1% NP 40, 4 µg/mL aprotinin 4 µg/mL pepstatin A, 4 µg/mL leupeptin. Lysed tissue was run on 16.5% Tricine gels and transferred to a nitrocellulose membrance for 120 min. Total protein was stained with Ponceau S. Blocking of non-specific binding was performed with 5% dried milk in Tris-buffered salin with 0.1% Tween-20. Membranes were probed with anti-TNF-α receptor 1/2, anti-IL-10 receptor 1/2  and anti-GAPDH  for 12 h at 4 °C. Secondary antibodies were either anti-mouse IgG  or anti-rabbit IgG . The signal was recorded with an Odyssey CLx System  and quantified with the LI-COR Image Studio software .p-values are reported in the figure legend. A p-value below 0.05 was assumed to be of statistical significance.Results are shown as mean ± standard error. Statistical tests and Left ventricular function was assessed in vivo with echocardiography A and lefTo investigate the contribution of mitochondrial function to the pathology of HFpEF-related atrial cardiomyopathy, left atrial cardiomyocytes were isolated and mitochondria examined in vitro. Structural analysis of mitochondria revealed a reduced perimeter to area ratio in HFpEF, indicating enhanced mitochondrial fission A,B. The 2+ cycling in HFpEF, CaT of left atrial cardiomyocytes were recorded after exposure to TNF-α and its anti-inflammatory counterpart IL-10  sites was conducted to evaluate the origin of improved Ca2+ release  [Abnormal Caks of HF ,27. Dysftabolism . In turnizations ), or viazations) . In thiszations)  and ROS 2+ handing of left atrial cardiomyocytes in HFpEF may be impaired associated with an upregulation of inflammatory pathways  pathway activation by upregulating extracellular signal-regulated kinase 1/2 (ERK 1/2) mitogen-activated protein kinase (MAPK) phosphorylation [2+ removal and CaT amplitudes, IL-10 also facilitated a notable acceleration and synchronization of Ca2+ release kinetics  or agonizing IL-10 . Large clinicals trials evaluating TNF-α inhibitors in HF  , Anti-TNn humans . However2+ handling. Anti-inflammatory treatment with IL-10 facilitates an attenuation of Ca2+ signaling of left atrial cardiomyocytes in HFpEF. IL-10 substitution might pose a viable treatment strategy for atrial dysfunction  in the setting of metabolic syndrome-related HFpEF and its efficacy should be further evaluated in preclinical trials.Cardiomyocytes in metabolic HFpEF-related atrial cardiomyopathy exhibit mitochondrial fission, increased oxidative stress and dysfunctional Ca


The maximum rate of sulfite reductase for corrosive relevant collection strains and SRB strains isolated from heating systems were increased by 3 to 10 times. A completely different picture was found for the intestinal SRB Vmax in the strains Desulfovibrio piger Vib-7 (0.67 µmol/min × mg−1 protein) and Desulfomicrobium orale Rod-9 (0.45 µmol/min × mg−1 protein). The determinant in the cluster distribution of SRB strains is the activity of the terminal enzyme of dissimilatory sulfate reduction—sulfite reductase, but not APS reductase. The data obtained from the activity of sulfate reduction enzymes indicated the adaptive plasticity of SRB strains that is manifested in the change in enzymatic activity.A comparative study of the kinetic characteristics  of key enzymes of assimilatory sulfate reduction (APS reductase and dissimilatory sulfite reductase) in cell-free extracts of sulphate-reducing bacteria (SRB) from various biotopes was performed. The material for the study represented different strains of SRB from various ecotopes. Microbiological (isolation and cultivation), biochemical (free cell extract preparation) and chemical (enzyme activity determination) methods served in defining kinetic characteristics of SRB enzymes. The determined affinity data for substrates  were 10 times higher for SRB strains isolated from environmental (soil) ecotopes than for strains from the human intestine. The maximum rate of APS reductase reached 0.282–0.862 µmol/min×mg Clostridia, assimilating sulfates [Sulfate-reducing bacteria (SRB) are known to be the main producers of the biogenic hydrogen sulfide in the biosphere. They can be found in a number of natural ecotopes and in human-caused and industrial systems, such as sea sludge deposits, hydrothermal springs, freshwaters, soils, anaerobic muds, as well as wastewaters, oil and gas fields, on the surfaces of metal constructions of gas and oil communications, in oil tankers, thermal systems, sewers, reinforced concrete and metal installations, etc. ,5,6,7,8.sulfates ,17.The fundamental metabolic feature of SRB is the hydrogen sulfide production as the result of dissimilatory sulfate reduction, i.e., sulfidogenesis ,19,20. Bsat, EC 2.7.7.4), and the following reaction is the reduction of adenosine-5′-phosphosulfate (APS) to adenosine-monophosphate (AMP) and sulfite by adenylylsulfate reductase , and subsequent reduction to hydrogen sulfide by dissimilatory sulfite reductases  [The sulfate-reduction pathway of SRB is catalyzed by three key enzymes: ATP sulfurylase, APS reductase, and sulfite reductase. The initial stage is the activation of sulfate by ATP sulfurylase enzyme (.8.99.3) . Two ele.8.99.3) ,29. AdenDesulfovibrio genus [Archaeoglobus fulgidus, Archaeoglobus profundus, Pyrobaculum islandicum [Salmonella enterica [Clostridium pasteurianum [DSR enzymes of various types occur in all organisms capable of reducing sulfite/sulfate during anaerobic respiration in all the SRB genera, especially of the io genus ,30,31,32landicum ,34,35, wenterica  and Closeurianum , but theeurianum .p < 0.05) different rates of enzymatic reaction, catalyzed by ATP sulfurylase in the bacterial strains, isolated from various environmental ecotopes. Based on cluster analysis, the parameters of physiological and biochemical characteristics of SRB from different ecotopes were divided into three clusters that corresponded to the location of their isolation . The sulfate reduction inhibition effects of the initial stage in SRB isolated from intestinal strains have also been shown. The process can be recognized as the unusual adaptation of SRB to the intestinal environment [In our previous study, we characterized the initial step of the dissimilatory sulfate-reduction pathway. The enzymatic parameters of ATP sulfurylase in cell-free extracts of SRB isolated from various ecotopes such as soils, corrosion products and human large intestine were determined in the research. The obtained results indicate significantly  in cell-free extracts of SRB from various ecotopes. The results in the experiment conduction provide more information about the aggressiveness of SRB in different environments.Desulfovibrio desulfuricans DSM642 and Desulfovibrio vulgaris DSM644 (GenBank: AF418179.1) were isolated from corrosion products and soil near a gas-main and obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) (Braunschweig Germany). Desulfovibrio sp. 10 (UCM B-11503) (GenBank: KC886400) were isolated from corrosion products of steel construction of DniproHES and were obtained from the Ukrainian Collection of Microorganisms at D.K. Zabolotny Institute of Microbiology and Virology of NAS of Ukraine , as well as corrosion strains: Desulfovibrio sp. TC2 (UCM B-11504), Desulfotomaculum sp. TC3 (UCM B-11505) and Desulfomicrobium sp. TC4 (UCM B-11506) were isolated from city heat system construction’s corrosion products . The intestinal SRB strains Desulfovibrio piger Vib-7 (GenBank: KT881309.1) and Desulfomicrobium orale Rod-9 (GenBank: MF939896.1) were isolated from the human large intestine (human feces) and obtained from at the Laboratory of Anaerobic Microorganisms of the Department of Experimental Biology at Masaryk University .Sulfate-reducing bacteria (SRB) were isolated from various ecotopes, including soil, corrosion products and human feces. Isolation, purification and identification were previously described ,41,42 an−1 was used in this modified Postgate C medium [−1 solution of NaOH was used. The redox and anaerobic conditions were controlled by resazurin sodium . Low redox potential (Eh = −100 ...−200 mV) for anaerobic condition was achieved by the addition of 2% ascorbic acid or 2% solution of sodium sulfide (1 mL L−1 of cultivation media). The tubes were filled with media, which were inoculated with SRB cultures (5% v/v), then closed by rubber plug to provide anaerobic conditions. The corrosive and soil SRB were cultivated at +28 °C for 7 days, while the intestinal bacteria were grown at +37 °C for 3 days.Cultivation of SRB was performed in liquid modified Postgate C medium . The higC medium . To adju2 and turbidimetric determination of it in the form of BaSO4. For the suspension stabilization, glycerol was used. Suspension of 40 mg L−1 BaCl2 has been prepared in 0.12 mol L−1 HCl. The resulting solution was mixed with glycerol in a 1:1 ratio. To the 1 mL of sample supernatant after centrifugation at 5000× g at 23 °C  was added 10 mL of prepared BaCl2:glycerol solution (in 1:1 ratio) and carefully stirred. The mixture was allowed to stand for 10 min and right after that the absorbance was measured at 520 nm . As a control, the measurement was repeated with the same method using a cultivation medium. The calibration curve has been constructed with the same process. Calibration solutions have been prepared in distilled water at concentrations of 2, 4, 8, 16, 24, 32, 40, and 48 µmol L−1 of sodium sulfate [The content of sulfate in the medium was determined by the turbidimetric method right after the seeding and after 24 h of cultivation. Its essence lies in the precipitation of sulfate ions with the BaCl sulfate .−1 aqueous solution of zinc acetate. Right after this, 2 mL of a 0.75 g mL−1 of p-amino dimethylaniline in solution of sulfuric acid (2 mol L−1) was added. The mixture was left to stand for 5 min at room temperature. After that, 0.5 mL of 12 g L−1 solution of ferric chloride dissolved in 15 mmol L−1 sulfuric acid was added. After standing for another 5 min at room temperature, the mixture was centrifuged at 5000× g at 23 °C . The absorbance of the centrifuged supernatant was determined to measure sulfide ions at a wavelength of 665 nm by spectrophotometer . As a control, the measurement was repeated in the same method using a cultivation medium. The calibration curve was constructed with the same process. Calibration solutions were prepared in distilled water at concentrations of a 12.5, 25, 50, and 100 µmol L−1 of sodium sulfide [Production of hydrogen sulfide by SRB has been measured right after inoculating and after 24 h of cultivation spectrophotometrically. A 1 mL sample was added to 10 mL of a 5 g L sulfide .−1 EDTA, 50 mmol L−1 potassium phosphate buffer, pH = 7.5) was added to centrifuged sediment cells to bind and depose heavy metal ions. After this procedure, the suspended bacterial cells containing 0.096–0.927 mg of protein × mL−1 were obtained. The cells were homogenized using the ultrasonic homogenizer  at 20 kHz for 5 min at 0 °C. The soluble fractions were placed into centrifugal tubes and cell-free extracts were separated from the cell fragments by centrifugation for 30 min at 14,000× g and at 4 °C . Supernatant was then used as cell-free extracts. The protein concentration in the cell-free extracts was determined by the Bradford method (1976) [Cell-free extracts were prepared from the bacterial cell gained from the exponential phase of growth . The bacteria were grown anaerobically in modified Postgate C liquid medium . The cold (1976) .3Fe(CN)6 (4 µmol), Na2SO3 , and cell-free extract (50 µL). The incubation temperature was 27 °C  and the reaction was started by the addition of sulfite-EDTA solution. Ferricyanide reduction was spectrophotometrically  measured at 420 nm. The enzyme can work in both directions [The activity of adenosine-5′-phosphosulfate reductase was measured by the AMP-dependent reduction of ferricyanide in the presence of sulfite. In a total of 3 mL of reaction mixture was contained Tris-HCl buffer , AMP (10 µmol), Narections .32− was reduced [2SO3 , NADH (0.2 mmol L−1), cell-free extract (50 µL) in total of 0.5 mL minus 27 µL of 1.2 mol L−1 HCl by which the reaction was started. The incubation temperature was 27 °C  and absorbance was read at 340 nm per minute for 10 min from the spectrophotometer .Sulfite reductase was assayed by the principles of Warburg’s effect based on the measurement of oxidation of NADH at 340 nm while SO reduced . The rea0 (linear accumulation of product (P) in time). The kinetic parameters that characterized APS reductase, and sulfite reductase such as Michaelis constant  and maximum rate of these reactions (Vmax) were determined by the Lineweaver-Burk plots [32−) was constructed in the coordinates (1/V on 1/S), where S is the concentration of the reagent (SO32−), and V is the rate of enzymatic oxidation of SO32− at a concentration of SO32− for APS reductase, reduction of SO32− at a concentration of SO32− for sulfite reductase, respectively  and Statistica 13 (http://www.statsoft.com/) software programs. Cluster analysis was performed by the single linkage method with the calculation of the Euclidean distances. Using the experimental data, the basic statistical parameters  were calculated. The research results were treated by methods of variation statistics using Student’s t-test. The significance of the calculated indicators of the line was tested by Fisher’s F-test. The approximation was accurate when p ≤ 0.05 [Statistical calculations of the results were carried out using the MS Office (2010), Origin 8.0 , for all the SRB strains. Conversely, the production of sulfide ions for all SRB strains was increased by 26 to 52% (v/v). Desulfotomaculum sp. TC3 strain consumed the highest amount of SO42− (88% (v/v)) in comparison with other genera. Furthermore, the high consumption activity was shown by Desulfovibrio sp. TC2 (84% (v/v)), Desulfovibrio sp. 10 (84% (v/v)). The lowest consumption of sulfate ions was observed for the intestinal strains of D. orale Rod-9 and D. piger Vib-7; the reduction of sulfate ions in the culture medium was within the range: 62% (v/v) and 64% (v/v), respectively.It was shown that in cultural media, the concentration of sulfate-ions decreased intensively by 62 to 88% (Desulfovibrio sp. TC2 (52%), Desulfotomaculum sp. TC3. The lowest percentage (26% (v/v)) of sulfate was consumed by D. vulgaris DSM644. The increase in the production of sulfide ions (26 to 34%) by the intestinal strains of Rod-9 and Vib-7 was similar to that of D. vulgaris DSM644 and Desulfovibrio sp. 10 (26 to 30%).Among the tested strains, the maximum production of sulfide ions was revealed by bacteria isolated from the same ecotope—city heat system—It has to be emphasized that the production of sulfide ions did not correlate with the consumption of sulfate ions. The process of sulfate reduction may reflect the functional differences between SRB strains isolated from different habitats. Therefore, the kinetic parameters of key enzymes, including APS reductase and sulfite reductase, were subsequently analyzed.Activated sulfate as APS is further reduced to sulfite by APS reductase and least to hydrogen sulfide by sulfite reductase ,28. ThesD. piger Vib-7 (5.666 nkat), D. vulgaris DSM644 (3.900 nkat) and Desulfotomaculum sp. TC3 (3500 nkat) isolated from different ecotopes. However, according to the activity of the final step of the dissimilatory sulfate reduction enzyme (sulfite reductase), the highest activity is shown in the cell-free extracts of Desulfovibrio sp. 10 (8600 nkat), D. vulgaris DSM644 (7050 nkat) collection strains, and Desulfotomaculum sp. TC3 (8383 nkat) and Desulfovibrio sp. TC2 (6000 nkat) isolated from city heat networks. The lowest activity of sulfite reductase was found in intestinal strains of SRB, 0.466 and 0.533 nkat, which is 16.1 and 18.4 times lower, respectively, according to the value of the activity of Desulfovibrio sp. 10.The specific activity data for both enzymes were different for SRB strains from different ecotopes. Activity values for APS reductase and sulfite reductase were in the range of 0.113–0.340 and 0.028–0.516 U/mL (0.8333–5.666 and 0.466–8.600 nkat), respectively. The highest activity of APS reductase was detected in cell-free extracts of the following strains: −1 for APS reductase and 3.53 and 46.73 mmol L−1 for sulfite reductase. This parameter differs in representatives of even one genus, Desulfovibrio. By affinity for the substrate for the enzyme APS reductase, the highest affinity was shown in the intestinal strains such as D. piger Vib-7 (4.33 mmol L−1), D. orale Rod-9 (3.57 mmol L−1), and SRB Desulfovibrio sp. 10 (2.42 mmol L−1). For the intestinal D. piger Vib-7 strain, it is shown that the high specific activity of APS reductase (5.666 ± 0.483 nkat) coincides with the affinity for the substrate, expressed by the Michaelis constant. Although D. vulgaris DSM644 had the higher specific activity of APS reductase, the value of KmAPS (0.99 mmol L−1) was the lowest KmAPS (0.99 mM) among the studied strains. Rod-9 has the lowest specific activity of APS reductase, but a significant affinity to APS substrate (KmAPS = 3.57 mmol L−1). The specific activity of APS reductase for SRB strains isolated from different ecotopes did not match the affinity for the substrate.The affinity rows were shown to estimate the substrate specificity, evidenced by the value of the Michaelis constant . It is vDesulfovibrio sp. 10 (46.73 mmol L−1), Desulfotomaculum sp. TC3 (46.17 mmol L−1) and D. vulgaris DSM644 (31.67 mmol L−1). It is characteristic that the smallest specific activity of sulfite reductase was detected in intestinal strains of SRB (0.466–0.533 nkat) and the least affinity for the substrate was determined according to the data of the Michaelis constant (3.53–3.86 mmol L−1). Therefore, the specific sulfite reductase activity for all tested SRB strains isolated from different ecotopes practically coincided with the affinity for the substrate.The data of activity of the enzyme in the final step of the sulfate reduction process corresponded to the calculated Michaelis constant values, and the affinity for the substrate (sulfite) was the highest in 0) and maximum (limiting) Vmax rates of enzymatic reactions were determined  and D. piger Vib-7 (0.675 µmol/min × mg−1 protein). The maximum rate of APS reductase reached 0.282–0.862 µmol/min × mg−1 of protein, which is only 10 to 28% higher than similar initial values.Important indicators of the reaction kinetics involved in the processes of dissimilatory sulfate reduction are the rates of product conversion reactions. The initial . It is significantly higher by 2.81–4.61 times than the minimum initial velocity of 0.049 µmol/min×mg−1 protein for DVI-10 strain. The maximum rate of sulfite reductase reached values of 0.23–0.516 µmol/min×mg−1 protein (for corrosion-active collection strains and SRB strains isolated from heating systems). The rate increased by 2.98–10.32 times. However, a completely different picture was found for intestinal SRB, where the Vmax in strains for D. piger Vib-7 (0.067 µmol/min × mg−1 protein) and D. orale Rod-9 (0.045 µmol/min×mg−1 protein) was decreased significantly by 5.24 and 3.06 times, respectively.For sulfite reductase, VTo analyze the affinity of SRB strains according to the kinetic parameters of two enzymes of dissimilatory sulfate reduction, a cluster analysis was performed. Principal component analysis (PCA) was used to separate SRB strains  according to their parameters of APS and sulfite reductases separately and together .The following strains, TC3, TC4, DVI-10, DSM642, VIB-7 and TC2, formed one cluster by the parameters of APS reductase. DSM644 and ROD-9 were separated from this cluster. Conversely, no differences were observed between SRB strains in sulfite reductase parameters. Based on the activity of both enzymes (APS and sulfite reductase), VIB-7 and ROD-9 were separated from the rest of the SRB.The sulfate reduction enzymes are located in the cytoplasm and peripheral plasma. The sulfate ions can be transported into the cells simultaneously with protons and some sulfate-reducing bacteria, absorbing sulfate from the flow of sodium ions . This hiDesulfovibrio and Desulfotomaculum genera, and in some oxidizing sulfite to sulfate Thiobacillus spp. [−1) of this enzyme was assayed by Huisingh et al. (1974) [Desulfovibrio sp. strain isolated from sheep rumen and described. Similar activity was also observed in a crude cell-free extract of D. desulfuricans (0.08 U × mg of protein−1) [D. vulgaris was determined to be 0.19 U × mg of protein−1 in both fractions after DEAE-Sephadex column chromatography [−1) of this enzyme was detected in supernatants of crude extracts from Desulfovibrio spp. [−1 were in the range of activities mentioned above. Intestinal SRB isolated from the large intestine of rodents (rats and mice) show differences in enzymatic activity for sulfite reductase in each sample taken (0.317 ± 0.037 to 0.702 ± 0.023 U × mg of protein−1), even in the similar group of SRB strains. The maximum rate of sulfite reductase in intestinal strains (Vib-7 and Rod-9) was lower than the initial rate in the corrosion-active SRB strains, in which the maximum rate was expected to be higher than the initial one   for a Deotein−1) . Our obtotein−1) ,52. The tography . On the rio spp. . The spe one see .−1 or soluble sulfate-ion 0.0283 moles kg−1 water) [Generally, a reduction of sulfate became a dominant biological process in the water environment (oceans and deposits), resulting in sulfidic anoxic conditions from 2.5 to 0.6 billion years ago ,55. Curr1 water)  differs 1 water)  may be a1 water) ,61.Desulfovibrio spp. strains, through the regulation of nitrate and nitrite oxidation, induction by nitrate and repression by sulfate is possible  [n = 25), a fragment of the dissimilatory sulfite reductase genes (dsrAB) that have recently been proposed to have undergone multiple lateral gene transfers [dsrAB and apsA genes are thermophilic [The sulfite reductases belong to a family of proteins that also include the assimilatory sulfite and nitrite reductases ,62. Thespressed) . It is kpressed) . Extensipressed) ,65,66,67ransfers  was alsomophilic . We can Salmonella enterica [Clostridium pasteurianum [Bilophila wadsworthia RZATAU and is involved in energy conservation by reducing sulfite to sulfide, during the degradation of taurine as an electron acceptor [CysK coded O-acetyl-serine(thiol)lyase, which is an enzyme catalyzing the reaction of inorganic sulfide with O-acetyl-serine to form the S-containing amino acid l-cysteine [In view of the fact that enterica  and Closeurianum  utilize acceptor . This enacceptor ,70. To ccysteine . Mutant cysteine . Therefocysteine ,72.The cluster analysis data confirm changes in the adaptation of SRB strains to different existence conditions, and indicate that the terminal enzyme (sulfite reductase) is crucial in the adaptation of bacterial cells to environmental conditions, namely changes in their specific activity.A comparative study of the kinetic characteristics  of dissimilatory sulfate reduction key enzymes (in particular APS reductase and dissimilatory sulfite reductase) was carried out. Determined affinity data for substrates indicate a high affinity for sulfite by SRB strains isolated from natural ecotopes, mainly in the soil environment. The maximum rate of sulfite reductase in intestinal strains (Vib-7 and Rod-9) is lower than the initial rate, and also than the initial one in the corrosive relevant SRB strains. The determinant in the cluster distribution of SRB strains is the activity of the terminal enzyme of dissimilatory sulfate reduction—sulfite reductase, not APS reductase. This difference could be caused by the presence of sulfite-reducing bacteria in environments  in which one is selective for intestinal SRB but, without isolation, the contamination of sulfite-reducing bacteria can be expected.


In our previous studies, we reported that myeloid differentiation protein 1 (MD1) serves as a negative regulator in several cardiovascular diseases. However, the role of MD1 in heart failure with preserved ejection fraction (HFpEF) and the underlying mechanisms of its action remain unclear. Eight‐week‐old MD1‐knockout (MD1‐KO) and wild‐type (WT) mice served as models of HFpEF induced by uninephrectomy, continuous saline or d‐aldosterone infusion and a 1.0% sodium chloride treatment in drinking water for 4 weeks to investigate the effect of MD1 on HFpEF in vivo. H9C2 cells were treated with aldosterone to evaluate the role of MD1 KO in vitro. MD1 expression was down‐regulated in the HFpEF mice; HFpEF significantly increased the levels of intracellular reactive oxygen species (ROS) and promoted autophagy; and in the MD1‐KO mice, the HFpEF‐induced intracellular ROS and autophagy effects were significantly exacerbated. Moreover, MD1 loss activated the p38‐MAPK pathway both in vivo and in vitro. Aldosterone‐mediated cardiomyocyte autophagy was significantly inhibited in cells pre‐treated with the ROS scavenger N‐acetylcysteine (NAC) or p38 inhibitor SB203580. Furthermore, inhibition with the autophagy inhibitor 3‐methyladenine (3‐MA) offset the aggravating effect of aldosterone‐induced autophagy in the MD1‐KO mice and cells both in vivo and in vitro. Our results validate a critical role of MD1 in the pathogenesis of HFpEF. MD1 deletion exaggerates cardiomyocyte autophagy in HFpEF via the activation of the ROS‐mediated MAPK signalling pathway. Microtubule‐associated protein light chain‐3 (LC3) is a major component of autophagosomes. The cytoplasmic form LC3I is converted to LC3II in autophagosomes. Therefore, LC3 was identified as the main specific autophagy indicator.Increasing evidence suggests that overproduced reactive oxygen species (ROS) regulate autophagy.Myeloid differentiation protein 1 (MD1) is a secreted glycoprotein that forms a complex with radioprotective 105 (RP105).22.1Aldosterone was purchased from Sigma‐Aldrich . Anti‐MD1 , anti‐SOD1 (1:3000), anti‐SOD2 (1:2000) and anti‐Nrf2 (1:500) were purchased from Abcam . Anti‐P38 (1:3000), anti‐p‐P38 (1:1000), anti‐P62 (1:2000), anti‐LC3  and anti‐HO‐1(1:1000) were purchased from CST . Dihydroethidium (DHE) was purchased from Invitrogen . N‐acetylcysteine (NAC), SB203580 and 3‐Methyladenine (3‐MA) were obtained from MCE . The mini‐osmotic pump was purchased from Alzet .2.2All animal care and experimental procedures were according to the guidance of Guidelines for the Care and Use of Laboratory Animals published by the United States National Institutes of Health  and were approved by the Animal Care and Use Committee of Renmin Hospital of Wuhan University. Male C57BL/6 MD1‐KO mice were purchased from the Japan RIKEN Bio Resource Centre Mouse (BRC), and Western blot validated the knockout efficiency in MD1‐KO mice Figure . The WT 2.3After the mice were anaesthetized using isoflurane (1.5%), echocardiography was performed by a MyLab 30CV ultrasound  with a 10‐MHz linear array ultrasound transducer. M‐mode images of the left ventricle at the papillary muscle level were recorded and then left ventricular ejection fraction (EF) and the fractional shortening (FS) were measured.2.4The mice were anaesthetized with pentobarbital sodium  at 4 weeks after HFpEF and then hearts were harvested. The tissues were fixed in 10% formalin, embedded in paraffin and sectioned at 5 μm. Left ventricular sections were stained with haematoxylin and eosin (H&E) for histopathology. Cryosections of fresh heart samples were stained with DHE (10 μM) for 30 min at 37°C to detect ROS production. Pictures were taken with an OLYMPUS DX51 fluorescence microscope .2.52. Then, H9C2 cells were seeded onto 6‐well in DMEM with 10% FBS for 48 hour. After that, the cells were starved for 16 hour and then were pre‐treated with siMD1or siRNA for 24 hour and then treat aldosterone (1 μM) or PBS for 18 hour.H9C2 cells were obtained from the Cell Bank of the Chinese Academy of Sciences  and were cultured in DMEM, containing 10% foetal bovine serum  and 100 U/mL penicillin, at 37°C in a humidified atmosphere with 5% CO2.6siRNAs targeting MD1 were generated by RiboBio . The cells were transfected with siMD1 or a scrambled RNA (siRNA) using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Three siRNA were generated, and the one resulted in the most down‐regulation of MD1 protein level was used for the further study.2.7Paraffin sections were also used for immunofluorescence, and slides were incubated with primary antibodies against LC3 . Slides were washed and incubated with fluorescence‐labelled secondary antibodies. The results were blindly calculated/section. After being washed 3 times with PBS, the H9C2 cells were visualized in a blinded manner under an Olympus IX53 fluorescence microscope.2.8The total proteins were extracted from the frozen heart tissues or iced cell lysates by RIPA agent . Nuclear protein extracts were isolated using commercial kits. The proteins were separated by 10% SDS‐PAGE and transferred onto PVDF membranes. Then, the membranes were blocked with 5% non‐fat milk at room temperature and incubated with the primary antibodies including antibodies against MD1, SOD‐1, SOD‐2, Nrf2, HO‐1, p‐P38, P38, P62, LC3 and GAPDH, at 4°C overnight. After incubating with the secondary antibodies at 37°C for 1 hour, the PVDF membrane were scanned and analysed by Odyssey Infrared Imaging System . The total protein levels were normalized to GAPDH.Total RNA was prepared using TRIzol reagent (Invitrogen) from heart samples or cell lysates. Two micrograms of RNA was reverse‐transcribed into cDNA with random primers using a Transcriptor First Strand cDNA Synthesis Kit . Real‐time PCR was performed using LightCycler 480 SYBR Green 1 Master Mix . The PCR conditions were as follows: 95°C for 10 minutes, followed by 40 cycles of 5°C for 10 seconds, 60°C for 10 seconds and 72°C for 20 seconds and a final step at 72°C for 10 minutes. GAPDH was used as an internal reference. All primer details are provided in Table 2.9TEM for autophagy analysis was performed according to standard operating procedures. For morphological TEM, hearts from saline or d‐aldosterone treated mice were fixed in 2.5% glutaraldehyde (Sigma) in phosphate buffer overnight at 4°C. After sample preparation, 90‐100 nm thick sections were mounted onto a 200 mesh copper grid  and imaged with an FEI Tecnai 12 120 kV transmission electron microscope equipped with an AMT XR80C 8 megapixel CCD camera.2.10t test. The differences were analysed with one‐way analysis of variance (ANOVA) with Tukey post hoc analysis. P < .05 was considered as statistically significant.All data in the tables and figures are expressed as mean ± SEM and were analysed using GraphPad Prism 8.0 software. The differences between two groups were performed with unpaired Student's 33.1To investigate whether MD1 is involved in aldosterone‐induced HFpEF, we first examined MD1 expression in an aldosterone‐induced HFpEF model. As shown in Figure 3.2Anp, Bnp and β‐Mhc  plays a major pathophysiological role in HFpEF.c Figure , moleculc Figure ‐H. The pc Figure , as indic Figure ‐K, M. Moc Figure . These o3.3Overactivated autophagy was reported to play an important role in HFpEF.3.4Reactive oxygen species (ROS) have been identified as signalling molecules in various pathways that regulate cell autophagy through several mechanisms.3.5In our previous study, we demonstrated that MD1 deficiency exacerbates cardiac remodelling by activating the MAPK pathway3.6The above results showed that knocking out MD1 may suppress HFpEF‐induced autophagy. We speculated that the inhibition of autophagy may offset the exaggerated effects of MD1 knock down by siMD1 in H9C2 cells. Consistent with our expectations, pre‐treatment with an inhibitor of autophagy, 3‐MA, significantly ameliorated Aldo‐induced autophagy. Western blot analysis showed that 3‐MA decreased the LC3II/LC3I ratio and increased P62 protein levels both in the Aldo‐treated cells and Aldo‐treated siMD1‐transfected cells Figure . These r3.7To confirm the role of autophagy in HFpEF, we treated mice with 3‐MA, an inhibitor of autophagy. We first investigated the role of 3‐MA in the acquisition of the HFpEF phenotype. As expected, the 3‐MA treatment offset the HFpEF phenotype and ameliorated the exacerbated effect of MD1‐KO on the HFpEF phenotype, as shown by the decreased HW/BW ratio, EDPVR‐k, Tau index and LW/BW ratio Figure . Then, w4In this study, we uncovered evidence that MD1 acts as a novel negative regulator of HFpEF‐induced autophagy. The expression of MD1 was down‐regulated in the HFpEF mice. HFpEF‐induced autophagy was aggravated in the MD1‐KO mice via the activation of ROS/MAPK signalling cascades. Inhibiting ROS production by NAC, inhibiting the activation of the MAPK signalling pathway by SB203580 and inhibiting cardiomyocyte autophagy by 3‐MA abolished the deleterious role of MD1 KO in HFpEF‐induced autophagy  can induce hypophosphorylation of titin, leading to increased myocardial passive stiffness.In this work, we also assessed the effects of the ROS/MAPK signalling pathway on the exacerbated role of MD1 KO in HFpEF‐induced autophagy. On the one hand, in our previous work, we reported that MD1 KO aggravated cardiac hypertrophy by activating the MAPK signalling pathway.5In conclusion, the present study indicates that MD1 may serve as a negative regulator in the pathogenesis of HFpEF. MD1 deficiency exacerbates autophagy induced by HFpEF via activation of ROS/MAPK signalling pathway. Thus, modulating MD1‐ROS/MAPK axis may provide a promising therapeutic approach for HFpEF.The authors disclose no conflict of interest.Hong Jie Yang: Conceptualization ; Data curation ; Investigation ; Project administration ; Writing‐original draft . Bin Kong: Project administration ; Resources ; Supervision . Wei Shuai: Formal analysis ; Investigation ; Software . Jingjing Zhang: Investigation ; Methodology ; Software ; Visualization . He Huang: Funding acquisition ; Supervision ; Validation ; Writing‐review & editing .


The enhancement patterns of dorsal epidural vein (DVCE), periradicular vein (PVCE) and intraradicular vein (IRCE) were assessed qualitatively. The quantification of IRCE was acquired by the ratio (%)  of signal intensities between the cauda equina  and psoas muscle . Receiver-operator characteristic curves were plotted to obtain imaginary cutoff values for the prediction of symptomatic appearance or operation decision. All levels were classified into seven groups on the basis of pain distribution and the presence of IRCE. PVCE was significantly related to high incidences of symptoms, unilaterality and operation. DVCE and IRCE were connected with high incidences of symptoms, bilaterality and operation. IRCE was also related to high visual analogue scale (VAS), small DCSA and high enhancement parameters. The order of the group was concordant with the degree of enhancement parameters (p = 0.000). Cutoff values of enhancement parameters for prediction were as follows: symptoms (147/123/140/121), bilaterality (165/139/157/137) and operation (164/139/159/138). Enhancement patterns and parameters could help in stratification, grading and decision-making at the surgical level.Fat-suppressed T1-weighted magnetic resonance images (MRIs) enhanced with gadolinium can evaluate the internal vertebral venous plexus and cauda equina. This study compared such findings with clinical situations and discusses whether these are helpful for symptomatic grading and selection at the surgical level in patients with lumbar central stenosis. A total of 263 patients (337 levels < 75 mm Neurogenic intermittent claudication (NIC) in lumbar stenosis can be explained by the venous congestion theory. Kobayashi et al. explained that the pathogenesis of NIC was composed of intrathecal consecutive events such as mechanical circumferential compression, the occlusion of subarachnoid space, venous congestion, nerve injury, intraradicular edema, conduction disturbance and ectopic discharge .However, extrathecal events should also be considered. The internal vertebral venous plexus (IVVP) in the lumbar spine is located between the spinal bony canal and dura mater . It commFat-suppressed T1-weighted magnetic resonance images (MRIs) enhanced with gadolinium (Gd) could show the dilation of microvessels, which is accompanied by inflammatory reactions. This technique could be useful to delineate the venous structure in cases of venous hypertension. Gd could leak through the endothelial fenestration of dilated vessels. The enhanced tissue in the lumbar spinal canal is primarily a venous structure comprising IVVP. The venous plexus can be divided into anterior  and posterior  IVVP ,3 Figur. The enhMarch 2011 and January 2020, 1815 consecutive patients with pain in the midline or lateralized back, buttock and leg underwent Gd-enhanced T1-weighted MRI with fat suppression, including the coronal, sagittal and axial planes in addition to conventional T1, T2-weighted imaging. The present study is under way to examine the relationships between pain distribution and the enhancement of outer annulus, periannular IVVP and facet capsule. All MR images were obtained with the same platform and equipment  in the same hospital to avoid scanner variations among different vendors and to assess the qualitative measurement of enhancement by using a 16-channel phased-array sensitivity-encoding (SENSE) body coil. Turbo-spine echo (TSE), an applied pulse sequence, was selected on the conventional image without enhancement. Patients were placed in a supine position. To localize most stenotic lesions, images on axial planes were obtained in parallel with each disc space. In contrast-enhanced MRI, a SPIR  sequence was used. Images were obtained in a regular sequence on the axial (repetition time (TR): 400 ms, echo time (TE): 7.4 ms, matrix scan: 256, slice thickness: 4 mm, slice gap: 0.4 mm, field of view (FOV): 220 mm, flip angle (FA): 90°, total scan duration: 3 min 8.8 s), coronal  and sagittal  planes. For the elimination of effect on the time-dependent changes showing contrast enhancement in venous structures within a spinal canal, T1-weighted images were acquired 30 s after intravenous administration of gadolinium-based contrast agents at a dose of 0.1 mmol/kilogram of body weight ; gadoterate meglumine, Uniray®, Dongkook Lifescience, Seoul, Korea (2015–2020))In a prospective, observational study conducted between 2 of the dural cross sectional area (DCSA), relative criteria <100 mm2) and lateral stenosis, which is further divided into lateral recess stenosis and foraminal stenosis [2, which can initiate a neurological dysfunction. Among cases with DCSA <75 mm2, 69 foraminal stenoses, 50 extraforaminal stenoses, five synovial cysts, four epidural lipomatosis and six indeterminate diagnoses were excluded. Cases (n = 32) with ipsilateral yellow ligament hypertrophy with DCSA >75 mm2 were excluded to rule out the confounding effect of local mechanical and inflammatory responses. Cases (n = 215) with bilateral back pain, confirmed by the medial branch block or facet capsule injection, were also excluded irrespective of DCSA. Finally, 263 patients  were included in this study. For a control group with age and sex matching, 15 asymptomatic patients  without any stenosis (DCSA = 133.5 ± 40.7 mm2 for 30 L3–4 + L4–5 levels) were recruited. This retrospective analysis of a prospective observational study was approved by the institutional review board at the hospital (File number: 2020-05-001) and informed consent was obtained from all patients.Cases with lumbar stenosis were selected among the original 1815 patients. Lumbar stenosis is composed of central  among intrathecal rootlets. In some patients, the large radiculomedullary veins showed strikingly higher signal intensities (>315) than those of other rootlets. These high signals were excluded in the subsequent measurements. The measurement of WR-IRCE and MS-IRCE on the L3–4 and L4–5 levels, respectively, were also performed in the asymptomatic control group to find a difference between such levels. The signal intensity measurement of the psoas muscle was performed within manually defined ROIs (Psoas1), which were delineated along the margin of the unilateral psoas muscle on the same enhanced axial images. In addition, circular ROIs  were designated within the medial portion of the psoas muscle corresponding to a mid to posterior disc space to avoid field inhomogeneity  of signal intensities between the cauda equina and psoas muscle on the same axial enhanced image. The signal intensity of rootlets was measured within the manually defined region of interests (ROIs) along the boundary of the white signal by a digitizer, Maroview software . To define an exact margin, intrathecal rootlets were marked on the same T2-weighted axial image. The adjustment of the gray scale (window level) was necessary for the discrimination between the rootlets and cerebrospinal fluid. Manual ROI analysis was performed by superimposing previously acquired ROI on the enhanced fat-suppressed T1-weighted image . Two metogeneity . Using tAll patients were classified as groups with bilateral buttock or leg pain, unilateral buttock or leg pain, midsacral or lumbosacral junction pain and no symptoms. The author assumed that pain distribution could have a connection with the neuroanatomical location where a venous congestion exists. The bilateral buttock and leg symptom—including neurogenic intermittent claudication—favors the severe venous congestion or edema of intrathecal rootlets, which could lead to the development of a higher IRCE value. Bilateral leg pain could be related to harsher conditions than unilateral leg pain. Pain in the midsacral or lumbosacral junction area could have originated from a less congestive status  than pain in the leg. Cases with a unilateral leg pain seem to have a localized periradicular venous congestion along the proximal traversing root  rather than the congestion of intrathecal rootlets. This pathomechanism could be concordant with a typical lateral recess stenosis. Lower IRCE values could be expected in patients with unilateral leg symptoms. However, it is rarely possible that a patient with high IRCE value, which means the congestion of other intrathecal rootlets, has unilateral, but no bilateral leg pain. In terms of the location of venous congestion, it indicated that the unilateral proximal process of dorsal root ganglion was compressed mechanically or congested enough to develop a pain, but other intrathecal rootles were insufficiently compressed mechanically or congestive. This situation could be defined as an advanced state compared to unilateral leg pain with low IRCE. In contrast to symptomatic patients, it was highly anticipated that asymptomatic patients with central stenosis would have less congestion and lower IRCE value. In addition, patients of control group were expected to have the least IRCE values compared with asymptomatic patients with central stenosis.The presence of visible IRCE is presumed to be necessary in the grouping criteria for an in-depth segmentation of symptomatic patients based on enhanced T1-weighted images with fat suppression. Therefore, the enrolled patients were graded according to the combination of pain distribution and the presence of IRCE. All levels were classified into seven groups ((Group 1): IRCE(+)/bilateral, (Group 2): IRCE(+)/unilateral, (Group 3): IRCE(−)/bilateral, (Group 4): IRCE(−)/midsacral, (Group 5): IRCE(−)/unilateral, (Group 6): IRCE(−)/no Sx, (Group 7): control). The group numbering indicates the order of predictable severity in cases with intrathecal intraradicular venous congestion or edema. In particular, only patients with an effect after an interlaminar epidural steroid injection and without an effect after medial branch block were included in Group 4.The presence of degenerative spondylolisthesis (SPL), redundant nerve root (RNR) and nerve root sedimentation sign (NRSS) were checked to compare these with the enhancement ratio to predict the presence or severity of symptoms. Spondylolisthesis could have a relation to a severe enhancement pattern, which is likely to be related to dynamic compression. RNR refers to thickened, tortuous and elongated roots of the cauda equina above the level of a long-standing, severe extradural compression . The defAmong patients without improvement after medication such as nonsteroidal anti-inflammatory drugs (NSAIDs) and gabapentin, epidural steroid injections were performed to evaluate clinical responses. Interlaminar approaches in patients with DVCE or IRCE and transforaminal approaches  in patients with PVCE were preferred according to the enhancement sites and concordant with symptomatic nature. Symptomatic patients received follow-up care for at least two months after injections. Clinical responses were assessed as poor (0–24%), fair (25–49%), good (50–74%) and excellent (75–100%) based on the reduced difference between the visual analog scale (VAS) and the preprocedural VAS, measured at two weeks (visit 1) or one month (visit 2) after injection. Because pain interventions show favorable outcomes only in short-term intervals, all follow-ups were performed within one month. The decision to operate was entirely dependent on the patients’ willingness after two months. The operation rate at the final follow-up was assessed to determine the severity of symptoms among groups. A favorable outcome was defined as good or excellent responses.Continuous variables, including age, DCSA, VAS back, VAS leg, MS-IRCE, WR-IRCE, Psoas1, Psoas2, MS/P1, WR/P1, MS/P2 and WR/P2, were evaluated. A single rater (YJJ) with more than 17 years of expertise in the spine assessed all MR images twice. An interval of at least one month was used between repeated measurements (the day of MRI and follow-up after one month). Categorical variables were sex, stenotic level, presence of symptoms, laterality of symptoms , patterns of enhancement (PVCE/DVCE/IRCE), treatment modalities (operation/block/medication), presence of SPL/RNR/NRSS and outcomes (excellent/good/fair/poor).IBM SPSS Statistics for Windows version 24  was used for all statistical analyses. The average pairwise Cohen’s kappa test was used to determine intraobserver reliability. The Landis and Koch guidelines were used to categorize the kappa value and define the strength of agreement, with values of 0.81–1.00, 0.61–0.80, 0.41–0.60, 0.21–0.40 and 0–0.20 indicating excellent, substantial, moderate, fair and slight agreements, respectively [t-tests, one-way analysis of variance (ANOVA) including Games-Howell multiple comparison test , bivariate correlation analysis and linear regression analysis to investigate the possibility of grading or stratification among groups. Additionally, statistical differences between groups were assessed. Correlations between DCSA/age/VAS and enhancement parameters were studied.Continuous variables were examined with 2 tests for the confirmation of independency, sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), accuracy, odds ratio (OR) and relative risk (RR). Incidences of these variables were evaluated and compared between groups. The correlation of PVCE/DVCE/IRCE with the presence of symptom, laterality of symptom and operation rate was investigated. The incidence of SPL was evaluated to find any relationship with visible IRCE, presence of symptom and the presence of RNR/NRSS. The influence of RNR/NRSS on DCSA, IRCE, VAS, and the presence of symptoms were also studied.Categorical variables were assessed by χReceiver-operator characteristic (ROC) curves were plotted to produce imaginary cutoff values with high sensitivity and specificity to determine the superiority of enhancement parameters in comparison with DCSA for the prediction of symptom, bilaterality of symptom, visible IRCE and operation. The most predictable parameter in terms of the area under the curve (AUC) was selected. Cutoff values were determined with appropriate sensitivity and specificity.The intraobserver reliability of DCSA, MS-IRCE, WR-IRCE, Psoas1, Psoas2, MS/P1, WR/P1, MS/P2, and WR/P2 ranged between 0.82 and 0.95 kappa, while that of DVCE, IRCE, PVCE, DS, RNR, and NRSS ranged between 0.87 and 0.97 kappa. The intraobserver agreement was excellent for all variables.p = 0.050). DCSA became significantly smaller as the group number decreased (p = 0.000). In multiple comparison tests, intergroup differences between Groups 4 and 6 were not statistically significant. Thus, DCSA was not likely to be a sufficient discriminator of various clinical manifestations. In contrast, all parameters of MS-IRCE, WR-IRCE, MS/P1, WR/P1, MS/P2, and WR/P2 increased significantly as the group number decreased (p = 0.000). F values increased in the order of MS-IRCE, WR-IRCE, WR/P1, MS/P1, WR/P2 and MS/P2. In post hoc analysis, intergroup differences were evident in the order of MS-IRCE, MS/P1, WR/P1, MS/P2, WR-IRCE and WR/P2. These results showed that the order of the group was concordant with the degree of IRCE, which indicated intraradicular venous congestion or edema. In the diagnosis of lumbar spinal stenosis, grading with enhancement parameters (MS/P1 and WR/P1) could be feasible. In comparison to DCSA, these parameters revealed the superiority of segmentation in patients with lumbar central stenosis. Demographic data on sex and level distribution were not statistically different between groups (Intergroup difference in age was only significant in Group 5 . The VASn groups . The incDCSA significantly correlated with MS-IRCE (r = −0.548), WR-IRCE (r = −0.594), MS/P1 (r = −0.501), WR/P1 (r = −0.545), MS/P2 (r = −0.486), WR/P2 (r = −0.532), VAS leg (r = −0.306) and age (r = −0.172). VAS leg had a significant correlation with MS-IRCE (r = 0.489), WR-IRCE (r = 0.491), MS/P1 (r = 0.464), WR/P1 (r = 0.470), MS/P2 (r = 0.452) and WR/P2 (r = 0.457). As such, changes in DCSA and VAS leg could be explained by corresponding changes in enhancement parameters.The PVCE(+) group had significantly higher incidences of symptoms (95.1% vs. 45.2%), unilateral (37.7% vs. 1.1%) and operation (47.0% vs. 29.4%) than those in the PVCE(-) group . The DVCDVCE had the highest sensitivity, while DVCE + PVCE + IRCE or IRCE had the highest specificity in the prediction of symptom. For unilateral pain, PVCE indicated the highest sensitivity and DVCE + PVCE had the highest specificity. For operation, DVCE had the highest sensitivity and DVCE + PVCE + IRCE had the highest specificity. In the prediction of bilateral pain, DVCE had the highest sensitivity and DVCE + PVCE + IRCE had the highest specificity.2) and higher enhancement parameters (MS-IRCE (369.8 vs. 257.1), WR-IRCE (307.3 vs. 223.2), MS/P1 (206.1 vs. 141.4), WR/P1 (171.1 vs. 122.8), MS/P2 (197.2 vs. 138.2), WR/P2 (163.7 vs. 120.0)) compared with those in the IRCE(−) group (p = 0.000).The presence of visible IRCE on sagittal or axial MR images revealed significantly higher VAS (7.3 vs. 4.5), lower DCSA (29.8 vs. 46.6 mmp = 0.023), WR-IRCE , MS/P1 , MS/P2 ) and a higher incidence of IRCE (57.1% vs. 43.8%) and DVCE (90.9% vs. 81.2%) than those of the SPL(−) group  group .p = 0.000), WR-IRCE , MS/P1 , WR/P1 , MS/P2 , WR/P2 ), higher VAS leg , lower DCSA , higher operation rate (55.0% vs. 31.4%), higher incidence of symptoms (90.3% vs. 74.6%), IRCE (73.6% vs. 26.9%) and DVCE (91.0% vs. 77.7%) than those of the RNR(−) group.RNR was significantly related to higher enhancement parameters , WR-IRCE , MS/P1 , WR/P1 , MS/P2 , WR/P2 ) and lower DCSA  compared with the NRSS(−) group. Additionally, NRSS was significantly related to a higher operation rate (51.1% vs. 11.3%) and higher incidence of IRCE (58.0% vs. 11.3%) than those of NRSS(−) group.NRSS had higher enhancement parameters , MS-IRCE (269.5), WR-IRCE (230.5), MS/P1 (146.5), WR/P1 (127.7), MS/P2 (141.5) and WR/P2 (126.1) (2), MS-IRCE (289.5), WR-IRCE (249.5), MS/P1 (162.2), WR/P1 (138.6), MS/P2 (156.8) and WR/P2 (137.3). Cutoff values for decision-making on an operation were DCSA (35.5 mm2), MS-IRCE (294.5), WR-IRCE (253.5) MS/P1 (162.2) WR/P1 (139.2) MS/P2 (157.1) and WR/P2 (139.3). Cutoff values for visible IRCE on axial or sagittal images were DCSA (34.3 mm2), MS-IRCE (301.5), WR-IRCE (266.5), MS/P1 (168.3), WR/P1 (143.6), MS/P2 (161.3) and WR/P2 (139.5).In the analysis with all predictable clinical factors, enhancement parameters had a larger area under the curve in comparison with DCSA . Cutoff  (126.1) . Cutoff Case 1. Patient  had complained of bilateral buttock and posterior leg pain for three months. Neurogenic intermittent claudication was evident after 5 min of walking. This case demonstrated the selection of symptomatic level and the reversibilty of IRCE after epidural steroid injection .Case 2. Patient  had complained of bilateral (left > right) buttock and posterior leg pain for 2 months. This case showed the reversibilty of IRCE after decompressive surgeries .Case 3. Patient  had complained of pain in bilateral lower buttock and posterolateral thigh for 2 years. The pain was aggravating for 3 months. This case indicated the sequential treatment algorithm performing interventional procedures and decompressive surgeries in the multi-level lumbar stenosis .Conventional T1- or T2-weighted imaging does not provide excellent soft-tissue contrast for regions such as the intraspinal venous plexus. Gd enhancement on T1-weighted images with fat suppression could be considered to investigate structural information and the degree of venous congestion. It is more consistently and accurately depicted after the administration of gadolinium-diethylenetriamine pentaacetic acid (DTPA), which produces a uniformly high signal intensity of the epidural venous structures outside the extradural space. However, this technique has many limitations. The gray scale also has an impact on signal intensity displayed on the images since image values are not absolute. All values present in an MR image are distributed over the dynamic gray scale . If the Several methods were applied to obtain the quantitative measurement of enhancement and to reduce all the limitations mentioned above. All images were acquired by the same protocol, platform and equipment in the same hospital. Instead of absolute measurement, a signal ratio between rootlets and psoas muscle was chosen. To avoid partial volume effects, the margin of rootlets was manually delineated on the T2-weighted axial image and subsequently, the contour was transferred into the corresponding location on the T1-weighted axial image with fat suppression. On the same image, the signal intensity of the psoas muscle was measured in the same way to prevent the effect of the dynamic gray scale. However, it was found that signal inhomogeneity, even within the psoas muscle, was changeable according to the anteroposterior direction and distance from the midline. In addition, restricted ROI as Psoas2 was attempted to compare the superiority of methodology, but showed higher signal inhomogeneity than Psoas1, depending on the sites. Nonetheless, IRCE parameters were confirmed as a severity evaluation index in the present study.Currently, there is no consensus on the specific diagnostic criteria for degenerative LSS. A reduced DCSA is a good discriminator for the presence of LSS ,13,14. HIn this study, grouping was conducted on the basis of the expected distribution of venous congestion. The group number indicated the order of severity in intraradicular venous congestion or edema. Although intergroup differences among Groups 3, 4 and 5 were not statistically significant, each group had differential enhancement parameters. This finding could be due to the small number of patients in Group 4. In a large-scale study with sufficient patients, statistical significance could be anticipated. According to results obtained in the present study, stratification or grading by MS/P1 and WR/P1 could be more useful in comparison to DCSA.Pain distribution alone could only provide limited information. Not all patients with lumbar central stenosis present with a typical symptom like neurogenic claudication. There is no correlation between the severity of stenosis and clinical complaint. The radiologic findings such as DCSA, SPL, NRSS and RNR may correspond to environmental and indirect causes for developing a root injury. They could not show the final outcome like a pathologic process that affects nerves at the root level (radiculopathy). There are certainly limits to correlation between radiologic findings and clinical complaints. As clinical symptoms are related to the neuropathological change  in the root, electrodiagnostic studies, especially electromyography (EMG), can provide confirmatory information. The primary use of EMG is to confirm nerve root dysfunction when the diagnosis is uncertain or to distinguish a radiculopathy from other lesions when the physical examination findings are unclear. Although EMG is very sensitive and specific, normal EMG results in a patient with signs and symptoms consistent with a radiculopathy do not exclude the diagnosis of radiculopathy. For example, a pure sensory lesion at the root level will have negative EMG and sensory nerve conduction study results. In this study, IRCE is also a kind of radiologic finding. Nevertheless, it has a pathologic meaning of venous hypertension which induces axonal injuries. The validity of IRCE may be considered as somewhere between DCSA and EMG. Additionally, IRCE includes changes from all sensorimotor rootlets and has also the virtue of simplicity and good visualization compared with EMG in the selection of symptomatic level in lumbar central stenosis. These advantages will be able to benefit clinicians immediately for performing pain interventions and planning a minimally invasive surgery. Considering many aspects, IRCE may be complementary to neurologic examination, DCSA and EMG results in patients with less obvious clinical complaints. So, patients with bilateral symptom were divided into IRCE(+) and IRCE(−) groups. Between Groups 1 and 3, there was no statistical difference in VAS leg (7.4 ± 1.3 vs. 7.0 ± 1.4); however, the operation rate (74% vs. 46%) was significantly different. The incidence of SPL (26% vs. 17%), RNR (65% vs. 44%) and NRSS (94% vs. 81%) was higher in Group 1 than Group 3. In addition, patients with unilateral symptom were divided into IRCE(+) and IRCE(−). Between Groups 2 and 5, there was no statistical difference in VAS leg (7.0 ± 1.1 vs. 7.0 ± 1.7), however, the operation rate (47% vs. 21%) was significantly different. The incidence of SPL (39% vs. 19%), RNR (74% vs. 10%) and NRSS (96% vs. 36%) were statistically higher in Group 2 than Group 5. Collectively, the pain score is not likely to reflect enhancement parameters that could indicate intraradicular pathologic changes. Most likely, there is a ceiling effect of VAS over the pathologic threshold of intraradicular venous congestion. However, the higher incidence of radiological findings  in the IRCE(+) group, compared with the IRCE(−) group, supports intergroup differences in both pathologic processes, which indicated the intraradicular congestion and prognosis and was reflected by the operation rate. Stratification should be designed meticulously under the consideration of both pathologic and symptomatic points.A gradual extension of venous congestion among enhancement patterns was expected. In this study, analyses were performed in the following manner. PVCE was symbolic of unilateral leg pain , DVCE signified the development of symptoms , PVCE + DVCE represented the development of symptoms , and IRCE denoted a bilateral symptom . The order of appearance in PVCE, DVCE, and IRCE may be concordant with the severity of venous congestion. Extrathecal events are likely to play a major role in the pathogenesis of lumbar central stenosis compared with intrathecal events.SPL showed a significant relationship with the development of enhancement patterns, but not with clinical manifestations. The absence of NRSS had the higher incidence of IRCE and operation than the NRSS(+) group. RNR was related to the higher incidence of symptom, operation, DVCE and IRCE than those of the RNR(−) group. All radiologic findings (SPL/NRSS/RNR) were significantly associated with the change of enhancement patterns. However, enhancement patterns were likely to be superior to abovementioned radiologic parameters in the prediction of clinical manifestations.The cutoff values of enhancement parameters for the prediction of clinical factors such as the development of symptom, bilateral leg pain, operation, and visible IRCE on the axial or sagittal images were estimated. These values increased sequentially in the order of the abovementioned clinical factors and could be in concordance with the severity of congestion. With these benchmarks, it is possible to predict when symptoms could be revealed; to explain whether a severe pathologic change (visible IRCE) exists; and to recommend at what of venous congestion an operation is necessary.A potential limitation of the study could be the accuracy in measurement of signal intensities. In conventional MR systems, the patient is scanned in the supine position. Venous congestion as the cause of neurogenic claudication cannot be fully evaluated. Dynamic compression during walking or repetitive exertional events cannot be examined. Nevertheless, it seems that venous enhancement patterns  can be shown at rest on the fat-suppressed contrast-enhanced T1-weighted images. This may be because static compression by circumjacent bone and soft tissue is enough to develop venous hypertension. This means that venous hypertension and neuronal damage are in an advanced stage. From the physiological point of view, upright and post-exertional MRI will be the best method to evaluate the venous congestion in incipient stage . Unfortunately, there are some compromises until now. While traditional supine scanners have a high strength magnetic field , upright and open MRI scanners have a lower field strength (0.2–0.6 T) with some compromise in image quality. Moreover, fat-suppressed contrast-enhanced T1-weighted images may have a problem relating to field inhomogeneity due to this field strength. The constant improvement in both coil technology and software will have narrowed the gap in image quality between low and higher field systems. Further studies using novel MR techniques with less field inhomogeneity are needed to reduce the variance of signal intensity. Another limitation is that the acquisition of statistical significance failed due to low case incidence among Groups 2, 3 and 4. However, intergroup differences were already confirmed between Group 2 and Group 3. Intergroup differences from group 3 to group 5 will be determined if more cases are collected in the prospective study.To conclude, with enhancement patterns and parameters, stratification and grading are feasible in patients with lumbar central stenosis. Cutoff values could help in the prediction of symptoms and decision-making at the surgical level. Collectively, such information could enable target-oriented interventional procedures or minimally invasive surgeries in patients with multilevel stenosis.


Emerging Fanconi Anemia (FA) signaling in the field of cancer research annotates the extreme importance of its center player, Fanconi Anemia complementation group D2 (FANCD2) in protecting human cells from going awry. However, a previously-unrecognized form of FANCD2, namely FANCD2-V2, is understudied. We report TRK-Fused Gene (TFG) is critical for roles played by FANCD2-V2 in early responses to DNA damage, but not for FANCD2-V1, the long-known form of FANCD2. FANCD2-V2 forms nuclear foci upon DNA damage, and both its focus appearance and disappearance are earlier than FANCD2-V1. The amino acid/aa 5-100 of TFG and the aa1437-1442 of FANCD2-V2 were identified to contribute to their interaction, which maintains the steady-state level of FANCD2-V2 protein. TFGΔaa5-100 or FANCD2-V2Δaa1437-1442-carrying cells could not show timely focus formation of FANCD2-V2 upon DNA damage and gained carcinogenicity over time. This study provides a previously-unknown key to unlock in-depth insights into maintaining genome stability, fostering translational studies on preventing, diagnosing and/or treating related diseases. Fanconi Anemia (FA) is a rare multigenic syndrome featuring clinical complications, including an early onset of aging, severe bone marrow failure and an extremely high incidence of cancer –3. To daAs a typical outcome for FA cells, the inability of FANCD2 to be monoubiquitinated or form nuclear foci appears to be a common molecular defect in response to a variety of genotoxic stresses. FANCD2 monoubiquitination or focus formation thus attracts many to study stress responses in order for an in-depth understanding of genome stability. By serendipity, we found an overlooked form of FANCD2, FANCD2-V2. Its expression is relatively higher in normal or non-malignant cells/tissues than matched malignant cells/tissues; vice versa the expression level of the long-known form of FANCD2, FANCD2-V1, is relatively lower in normal or non-malignant cells/tissues as compared to matched malignant cells/tissues . We thusTo differentiate between two versions of FANCD2 proteins in response to DNA damage, we detected the monoubiquitinated/activated FANCD2-V1 or -V2 in U2OS cells treated with ultraviolet-B (UVB) using specific antibodies raised against the carboxyl terminal unique region of FANCD2-V1 or V2 accordingly  and 1B. In looking for factors responsible for roles played by FANCD2-V2, we picked up TFG protein upon the highest number of peptides found in immunoprecipitated pellets with FANCD2-V2 specific antibodies . Indeed,Next, we began to identify which part of TFG is responsible for interacting with FANCD2-V2. As outlined in Through observing these live images, we noticed that not only FANCD2-V2’s focus duration decreased but also the focus intensity was also dramatically reduced  and 5. TKnowing the importance of FANCD2-V2 in maintaining genome stability, it is imminent to determine functional outcomes of FANCD2-V2’s timely responses to DNA damage and that of its partner TFG in maintaining its prompt response. We, therefore, detected if cell oncogenicity was affected by TFG expression and found that compromised-TFG expression promoted cell invasive capacity , consistThe accumulated studies indicate that the FA pathway has become the heart of the DNA-damage repair-signaling network, namely FA signaling or the FA signaling network –3. In thWhile studying FANCD2, we noticed a hidden form of FANCD2, FANCD2-V2. Its unique expression pattern  promptedTFG facilitates transportation from the ER to Golgi intermediate compartments (ERGIC) , 7. ThisThe U2OS and HEK293 cell lines were obtained from the shared resource center of University of Hawaii Cancer Center (purchased from ATCC). U2OS and 293T cells were maintained in DMEM medium supplemented with 10% fetal bovine serum at 37 °C in 5% CO2 (v/v). Antibodies against FANCD2-V1 or V2 were customarily made by Cocalico Biologicals, Inc. . Those recognizing FANCD2, TFG or RFP were obtained from NOVUS . The anti-vinculin and GFP antibodies were from Santa Cruz . The blue dextran, neomycin and molecular weight markers used for gel filtration were from Sigma . MG-132 was from MP Biomedicals .2 and cultured for the given period.As described previously , medium Whole cell lysates were prepared in IP lysis buffer with 1% CHAPS, and about 2mg proteins were incubated with anti-FANCD2-V2, D2-V1, TFG, GFP or Flag antibodies overnight at 4°C after pre-clearance. The incubation was continued for another 2-4h after protein A-sepharose beads  were added. The IP pellets were then washed with IP wash buffer X3 and then boiled in 1 X SDS-lysis buffer for 5 min. The supernatant was analyzed by western blotting.For immunoblotting, cells were lysed with 1 X SDS lysis buffer, or nuclear fractions were prepared with the NE-PER Nuclear and Cytoplasmic Extraction Reagents Kit  following the protocols provided by the manufacturer. Then 30-40 μg of protein lysates were separated on 10% SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were blocked with 5% non-fat milk in PBS containing 0.1% Tween-20 for 1h and probed with corresponding primary Abs and secondary Abs-conjugated with horseradish peroxidase (HRP), and detected by enhanced chemiluminescence . Vinculin or β-actin was used as a protein loading control.Gel filtration analysis was performed as described previously . Whole cCells were seeded into dishes and followed by transfection and/or treatment. Cells were fixed with 4% paraformaldehyde at a proper time period and permeabilized with 0.1% Triton X-100. After blocking with 3% goat serum for 1h at RT, these cells were incubated with anti-FANCD2-V2, V1 or TFG antibody at 4°C overnight. After washing with PBS, anti-rabbit secondary antibody conjugated with Alexa Fluor-488 or anti-mouse antibody with Alexa Fluor-568  were added and incubated for 1h at RT. Cells were washed and mounted in mounting solution with DAPI .2. Cells were then observed with 10X60 lens on a Leica SP5 Confocal Microscope. Images were acquired every 15min for 4-6h and analyzed using Leica Application Suite X.For live imaging, U2OS cells were seeded on special glass base dishes and transfected with RFP-FANCD2V1 and GFP-FANCD2V2 for 12h, then treated with UVB at 25 J/mLentiviral transduction was performed as described previously . A set oAs previously described , the ful4 cells/well were seeded to 6-well plates and cultured for 7 days. The plates were fixed and stained with crystal violet solution at day 1, 3, 5 and 7.In the cell proliferation assay, 1x104 cells/well were seeded to a 24- well chambers and cultured at 37 °C in 5% CO2 for 24h. A cotton tipped swab was used to remove non-invading cells, and the inserts were stained with crystal violet solution. Subsequently, the insert was washed with PBS and air-dried.According to the manual of BioCoat™ Matrigel Invasion Chamber , after rehydration, about 2 x10For the drug sensitivity assay, cells were plated and the next day treated with UVB and incubated with 100 ng/ml mitomycin C (MMC) for 24h. After treatment, plates were stained with crystal violet solution, then washed with PBS and air-dried.By using Image J, we measured fluorescence intensity and generated the relative red or green florescence, and r-values through Pearson’s or Mander’s colocalization coefficient.p-value <0.05 was considered as statistical significance.All values were expressed as the mean ± SD of individual samples. Samples were analyzed using Student’s t-test for two groups. A Supplementary FiguresSupplementary Video 1Supplementary Video 2Supplementary Video 3Supplementary Video 4Supplementary Video 5Supplementary Video 6