Developing an effective safety emergency plan for coal mines is crucial to prevent and reduce accidents, as well as improve the emergency response capability. This paper identifies and analyzes potential accident hazards in the Burtai coal mine through a comprehensive and practical investigation of risk factors. Based on a mathematical model of a mine fire disaster relief algorithm, the MATLAB software is used to conduct numerical simulations of the dynamic spreading process of the fire smoke. Several escape routes are determined based on the simulation results, including the main escape route at the working face of coal mining, the main escape route at the working face of tunneling, and the main escape route against the reverse wind in the main inclined shaft, the auxiliary adit, and the main air intake roadway. The results presented in this study can provide guidance for improving fire emergency escape and rescue plans of the Burtai coal mine. Therefore, establishing an effective safety emergency plan is important for providing an appropriate response to an accident, escape, and rescue. When an accident occurs, providing a fast, orderly, and effective escape and rescue plan is necessary to ensure a rapid control of the accident and timely achieve self-rescue, mutual rescue, and disaster escape so as to minimize casualties and property losses9.The coal industry is a top high-risk industry with poor production conditions and a constantly changing operating environment. Many types of disasters and hazards such as floods, fires, gas, coal dust, and roof accidents can cause severe threats to the production safety of coal mines. Coal mine safety issues are the top priority in the national production safety11. When a major disaster occurs in a mine, it can severely hurt underground workers and even threaten their lives13. Fire, vehicle collisions, and rockfalls are the most common emergencies in mineral and metal underground mines15. In most areas of a mine, fire can affect roof stability and produce toxic gases, thus causing hypoxic conditions and making spontaneous escape and rescue operations impossible17. Mine rescue operations take a certain time regardless they are carried out by mine rescue teams or local rescue services and emergency medical services. There are time delays because the rescue team has to go into remote mines, arrange rescue operations, and navigate in complex environments to reach the accident site, which can be far underground. When a disaster happens, a large amount of toxic gases is generated, and the presence of fire and wind pressure can even accelerate gas diffusion. Therefore, workers must be evacuated rapidly from a disaster site to the safe zone. However, underground mines have complex structures with limited escape routes. These factors impose significant difficulties to escape planning and realization. Therefore, to reduce the death rate from accidents and ensure worker safety, it is critical to plan an escape route ahead23.Underground mining is the main method of coal extraction in China and many other coal-producing countries. In recent years, with a continuous increase in mining depth, the possibility of various disasters, such as gas explosions, eruptions, gas outbursts, and mine floods, has increased25. A lack of these qualifications can severely affect mining safety. At the very first moment of an emergency, understanding the current situation can significantly improve escape and rescue. A rescue route refers to an optimal route for the rescue personnel to enter the disaster area from a safe area, whereas an escape route refers to an evacuation route for the underground personnel to leave the dangerous area27.The Mine Safety Technology and Training Committee has recommended that mine workers must possess the following qualifications to ensure effective escape and rescue in the case of emergency: escape knowledge, ability to determine an evacuation route for a specific mine, and general knowledge about escape and rescue for decision making in emergencyAt present, most researches on the planning of emergency escape routes are mostly carried out with indoor buildings as the research object. The geological structure of the mine is complex and the tunnels are crisscross, so the existing indoor disaster escape route research conclusion has little application value in the coal mine. The existing research on coal mine emergency escape route planning is mostly carried out from the existing safety management regulations. Through continuous improvement of emergency management system and continuous safety training, it aims to enable underground workers to master more disaster escape and escape skills as much as possible, so as to improve the success rate of emergency disaster escape. Every mine and every emergency situation are unique, and the causes of different coal mine accidents are different. For different mines, a certain disaster may lead to different consequences. Therefore, the research on emergency disaster escape route of mine fire should be carried out for a specific mine. The research results obtained in this way are universal and valuable.In this work, we analyzed potential accidents of the Burtai coal mine and establishes a mathematical model to simulate the dynamic fire propagation process of a smoke flow. Based on the simulation results of the constructed model, optimal escape routes are determined. The results obtained from this work can provide guidance for fire emergency planning of the Burtai coal mine.2 and an average elevation of 1300 m. The Burtai coal mine has complex terrain, including many crisscrossing valleys. The geological reserve of its coal seam is 3.3 billion tons, and its recoverable reserves account for 1.85 billion tons. According to the annual mining yield estimation of 20 million tons, the service life of this mine will be 71.3 years. There are a total of ten recoverable coal seam layers in this mine. The currently mined layers are as follows: 2–2 in the first layer and 4–2 in the second layer. The coal seam dip angle is 1°–3°. The coal seam thickness is in the range of 0.87–7.32 m, with an average value of 3.78 m. The resource reserve utilization thickness is in the range of 0.87–7.24 m, with an average value of 3.67 m. The recoverable thickness in the bifurcation area is between 0.90 and 5.00 m, with an average value of 2.28 m. The recoverable thickness in the combined area is between 0.87 and 7.24 m, with an average value of 5.90 m. The thickness of the coal seam changes regularly with a tendency of thickening in the middle and thinning in the southeast and west, i.e., thickening in the combined area and thinning in the bifurcation area.The Burtai coal mine is a super-size mine located in Inner Mongolia, China, with an area of 192.8559 km3/min; the total air intake and return volumes are 15,241 m3/min and 15,482 m3/min, respectively. Inverted ventilation for the entire mine is achieved by using multiple main fans with fan reversal. The coal mining method is comprehensive mechanized coal mining along the long wall. The roof is managed by using the total straddle method. Further, tunneling is achieved by using continuous shearers, anchor diggers, and comprehensive diggers. Finally, shuttle cars and conveyors are used to transport coal.Several mining approaches have been adopted in the Burtai coal mine, including inclined shaft, adit, and vertical shaft. Also, partition extraction ventilation is used in this mine. Initially, five shafts were arranged, including the main inclined shaft, an auxiliary adit, a Sundingholuo air intake shaft, an industrial square air return inclined shaft, and a Sundingholuo air return shaft. The first three shafts induct air, and the last two shafts return air. The total air volume of the mine is 15,212 m3/t, and the absolute gas emission is 23.99 m3/min. The relative and absolute emissions of carbon dioxide are 0.41 m3/t and 11.01 m3/min, respectively. The mine has a low gas level without outburst danger related to coal and gas. The coal dust explosion indices of the 2–2 coal and the 4–2 coal are 33.82% and 36.55%, respectively, which indicates high explosive properties. The spontaneous combustion tendency grades of the 2–2 coal seam and the 4–2 coal dust fall in class II spontaneous combustion, and the ignition period is usually 1–3 months. When there is floating coal in the roadway with poor ventilation, spontaneous combustion may occur. Moreover, a coal dust explosion can be caused by a gas explosion, electrical equipment detonation, friction sparks, impact sparks, a fire caused by electric welding, and spontaneous coal combustion.According to the gas grade certification of the Burtai coal mine in 2021, the relative gas emission is 0.88 m29. For miners, it is very difficult to find a reasonable and safe emergency escape route in such a complex underground environment.Depending on the type of coal mined and the conditions of the working face, the fire may spread rapidly, intensify and burn for a long time. Because of this, the normal and safest choice is self-escape30. Therefore, it is necessary to analyze and plan the emergency escape route in advance through numerical simulation, so as to provide guarantee and support for underground workers to make reasonable choices in emergency situations.Table Based on the analysis on the potential hazard sources above and factors affecting the escape routes of Burtai coal mine, combined with the reality of Burtai coal mine, a fire emergency escape simulation algorithm for the coal mine is proposed and described as follows:t is given by:v(t) is the walking speed of a worker at a moment t in m/s; v0 denotes the normal walking speed (m/s); kt is the walking speed variation with time; k1 denotes the impact coefficient of a roadway slope, and 0 < k1 < 1 when the roadway slope is J > 0, k1 = 1 when J = 0, and k1 > 1 when J < 0; k2 represents the impact coefficient of roadway obstacles, and k2 = 1 when either there are no obstacles on a roadway or obstacles do not affect workers’ walking, and 0 < k2 < 1 when there are obstacles on a roadway and they affect workers’ walking; k3 is the impact coefficient of the roadway shape, and 0 < k3 < 1 when the roadway shape is unfavorable for walking; otherwise, k3 = 1.First, an average walking speed of a mine worker is calculated. The walking speed of a mine worker at a moment l(i) is the length of roadway i (m), and i (m/s).The time spent by a worker passing through a roadway is given by:i is obtained by:i (m/s).The time required by the smoke front to pass through a roadway G = , V is a collection of nodes, and m denotes the number of nodes, and E is the collection of branches, and n is the number of branches, and G, Gw is the polluted area, and other sub-graphs represent safe areas and are denoted as Gs28, showing the following relationship with the polluted area:In a directed graph 31. The collection of nodes from the ending node of the current branch to the pathways in a safe area is denoted by Ps and expressed as:In the escape process, workers may follow the wind direction or run against the wind direction. Therefore, a depth-first search (DFS) method based on an undirected graph should be used to determine an escape routeWhen a fire occurs, the front of the fire smoke flow may spread to a certain location The path Pesc selected from the collection of safe pathways Ps, which can be expressed as follows:Ps and x is the remaining distance of escape (m); x of the current branch f is the number of branches before branch j (s).An escape route is determined as a collection of escape pathways The best escape route is the shortest path in the collection of all feasible escape paths from the fire location to any node in the safe areas, and it is defined as follows:Based on the established mathematical model, the simulations are conducted using MATLAB software. First, the simulation calculation program was written, and then the simulation calculations were performed, as shown in Fig. A ventilation network diagram was drawn according to the overall structure of the Burtai coal mine, as shown in Fig. 32. Based on the mathematical model of the mine fire spreading process and actual ventilation network data, the dynamic calculation of mine fire spread was conducted. According to the simulation results, fire source parameters, and personnel location, the main disaster escape routes were obtained. The main steps to determine an optimal escape route by numerical simulation are shown in Fig. Based on the monthly ventilation report of the Burtai coal mine, the simulation data of the dynamic spreading process of the smoke flow in a fire were obtained, as shown in Table The ventilation network diagram of the Burtai coal mine is shown in Fig. There are three main disaster escape routes at the coal mining working face, which are as follows  comprehensive mining face → 22,103 transport chute → 2–2 auxiliary coal transport roadway → auxiliary transport adit → ground;Route 2: 22,103 (2) comprehensive mining face → 22,104 transport chute → 2–2 auxiliary coal transport roadway → auxiliary transport adit → ground;Route 3: 42,102 comprehensive mining face → 42,102 transport chute → 4–2 auxiliary coal transport roadway → 4–2 dark auxiliary coal transport adit → 2–2 auxiliary coal transport roadway → auxiliary transport adit → ground.There are six main disaster escape routes at the tunneling working face, which are as follows:Route 1: 12,101 transport chute tunneling face → 12 auxiliary coal adit → 2–2 auxiliary coal transport roadway → auxiliary transport adit → ground;Route 2: 12,101 return air chute tunneling face → 12 auxiliary coal adit → 2–2 auxiliary coal transport roadway → auxiliary transport adit → ground;Route 3: 22,201 return air chute tunneling face → central auxiliary transport roadway → 2–2 coal auxiliary transport roadway → auxiliary transport adit → ground;Route 4: 22,202 retreat chute tunneling face → central auxiliary transport roadway → 2–2 coal auxiliary transportation roadway → auxiliary adit → ground;Route 5: 42,103 transport chute tunneling face → 42,103 transport chute → 4–2 auxiliary coal transport roadway → 4–2 dark auxiliary coal transport adit → 2–2 auxiliary coal transport roadway → auxiliary transport adit → ground;Route 6: 42,104 return air chute tunneling face → 42,103 transportation chute → 4–2 auxiliary coal transport roadway → 4–2 dark auxiliary coal transport adit → 2–2 auxiliary coal transport roadway → auxiliary transport adit → ground.There are four main disaster escape routes from the fire with the reversed wind in the main inclined shaft, the auxiliary transport adit, and the main air intake roadway, which are as follows:Route 1: staff in the 2–2 coal and panel 1 → 2–2 coal return air roadway → industrial square return air inclined shaft → ground;Route 2: staff in the 4–2 coal → 4–2 coal return air roadway → 4–2 dark coal return air inclined shaft → 2–2 coal return air roadway → industrial square return air inclined shaft → ground;Route 3: staff in the 2–2 coal and panel 2 → central auxiliary transport roadway → 32# connected roadway grid bypass → central return air roadway to wait for rescue;Route 4: staff at the bottom of the air intake vertical shaft → central auxiliary transport roadway → 32# connected roadway grid bypass → central return air roadway to wait for rescue.Potential accidents and hidden danger of the coal mine are identified based on the geographical location, geological conditions, and actual production situations of the Burtai coal mine.A mathematical model of a mine fire rescue algorithm is constructed for the hidden danger and potential accidents. This model is used to conduct numerical simulation on the dynamic spreading process of the fire smoke flow in the Burtai coal mine.Several escape routes are determined based on the simulation results, including the main escape route at the coal mining working face, the main escape route at the tunneling working face, and the main escape route against the reverse wind in the main inclined shaft, auxiliary adit, and the main air intake roadway.Based on a self-established mathematical model of a mine fire disaster relief algorithm, numerical simulations of a dynamic spreading process of the fire smoke are conducted, and the main disaster escape route in the Burtai coal mine is determined. The main conclusions of this study can be drawn as follows:In future work, the main disaster escape routes in all mining areas of the Burtai coal mine could be determined, and technical support for improving the emergency rescue plan of the coal mine could be provided.


Noroviruses are major causative agents of nonbacterial acute gastroenteritis in humans. Ten genogroups of noroviruses have been identified to date, among which genogroup I (GI) and genogroup II (GII) noroviruses are major pathogens for humans. GI and GII noroviruses are further classified into nine and 27 genotypes, respectively. Noroviruses are well known to bind to histo‐blood group antigens (HBGAs). Many studies have revealed that virus‐like particles (VLPs) from different genotypes exhibit distinct patterns of HBGA binding, but the assay conditions used in these studies were not identical. To enable comparison of the binding to HBGA of nine GI genotypes, I purified VLPs from insect cells and analysed their HBGA‐binding profiles. Although each genotype exhibited a distinct pattern of HBGA binding, Lewis b antigen was commonly recognized by all of the genogroup I strains, suggesting that this antigen plays a critical role in the pathogenesis of noroviruses. Histo‐blood group antigens (HBGAs) are thought to be cellular attachment factors for human noroviruses. Using virus‐like particles from the genogroup I noroviruses, their HBGA‐binding profiles were investigated. Herein, I discovered that Lewis b antigen is a common ligand for all of the genogroup I strains, suggesting its critical role in the infection event. BSAbovine serum albuminD‐PBSDulbecco's phosphate‐buffered salineFUT2the α1,2‐fucosyltransferase gene known as the secretor geneGalgalactoseGalNAcN‐acetylgalactosamineGIgenogroup IGIIgenogroup IIHBGAhisto‐blood group antigenHSAhuman serum albuminLNDFHN‐difucohexaoselacto‐LNFPN‐fucopentaoselacto‐VLPvirus‐like particleNoroviruses that belong to the family Caliciviridae are major causative agents of nonbacterial acute gastroenteritis in humans . Ten genT = 3 icosahedral particle [T = 1 icosahedral symmetry [A single norovirus virion is composed of 180 molecules of the VP1 capsid protein, and 90 dimers of VP1 proteins self‐assemble to form a particle . Since tparticle . Normallparticle , 7. The Noroviruses are well known to bind to histo‐blood group antigens (HBGAs) . EspeciaAs summarized in Table m sodium citrate buffer (pH 4.2) was used in place of distilled water. After preparation, the integrity of VLPs was evaluated by transmission electron microscopy with uranyl acetate used as a stain  were transfected with the constructed pORB plasmids together with Sapphire Baculovirus DNA (Allele Biotechnology) to obtain recombinant baculoviruses, which were then used for VLP production in Trichoplusia ni cells (Oxford Expression Technologies). Typically, VLPs were collected from culture media and separated by isopycnic CsCl density gradient centrifugation. VLPs sedimented by ultracentrifugation were resuspended in distilled water unless otherwise stated and stored at 4 °C. For the preparation of the GI.4 Chiba VLPs, 20 main Fig. .The rabbit antisera reacted with the GI.1, GI.2, GI.3, GI.4 or GI.6 strains were laboratory stocks. The rabbit anti‐GI.9 VLP antiserum was prepared by Scrum, Inc.  . For theThe HBGA binding assay was performed as described previously . The bovkaleida graph software , I evaluated whether the data of the HBGA binding assay for VLPs , the Michaelis–Menten equation, Eqn  could be collected from culture media of the T. ni insect cells, and these VLPs were morphologically homogeneous, showing typical 38‐nm diameter particles, as indicated in our previous reports , 22. In For the determination of the HBGA‐binding profiles of VLPs from the nine different GI genotypes mentioned above, the binding assay was performed using commercially available BSA or HSA conjugates of HBGAs. As shown in Fig. The binding patterns of each genotype are summarized in Table Helicobacter pylori recognizes the Lewis b antigen for infection [Most importantly, VLPs from all of the genotypes bind to the Lewis b antigen, suggesting that this HBGA plays a critical role in norovirus infection; this reflects the similar finding that the microaerophilic bacterium nfection . It is anfection .Regarding the GI.1 Norwalk , 28, GI.in vitro biding assay, neither the Miyagi VLP nor the TCH‐060 VLP could bind to Lewis a and x antigens. No binding was observed although the VLPs reacted with HBGAs at 4 °C (data not shown), as was observed for the TCH‐060 VLPs [in vitro HBGA binding assay may not have precisely revealed all of the characteristics of VLPs. There is no doubting that the TCH‐060 VP1 protein were complexed with Lewis a and x antigens [In contrast, there was a marked difference among the GI.7 strains. Shanker et al.  reported060 VLPs . VP1 pro060 VLPs  of HBGA 060 VLPs  are cons060 VLPs , there iantigens . ConsideIt is also notable that the GI.6 VLPs bind to a wide variety of HBGAs Fig.  and thatAlthough there is some variation in the binding profiles, binding to Lewis antigens is likely a common characteristic of the GI noroviruses . The resThe following question thus arises: what is the role of HBGAs including the Lewis b antigen in the process of norovirus infection? For many years, HBGAs have been assumed to be receptors for noroviruses. Since HBGAs actually affect humans' susceptibility to some genotypes of noroviruses , 11, 12,The author declares no conflict of interest.YS designed and performed experiments, analyzed data and wrote the manuscript.Fig. S1. Transmission electron microscopic images of VLPs prepared from insect cell culture. VLPs were prepared as described under the Materials and Methods in the main text and stained with uranyl acetate for taking images. The bars indicate 200 nm, except for panel F, in which it indicates 100 nm.Click here for additional data file.Fig. S2. Symbolic representation of the HBGAs used for the binding assay.Click here for additional data file.Table S1. The parameters for the binding between VLPs and HBGAs.Click here for additional data file.


However, there are disadvantages to using S1P clinically as an EG restorative therapy. A potential alternative is the S1PR agonist fingolimod (FTY720). The aim of this study was to assess whether FTY720 prevents EG shedding in injured cultured human umbilical vein endothelial cells.Shedding of the endothelial glycocalyx (EG) is associated with poor outcomes in a range of conditions including sepsis. Fresh frozen plasma (FFP) restores the damaged EG to baseline thickness, however the mechanism for this effect is unknown, and some components of FFP have adverse effects unrelated to the EG. There is some limited evidence that sphingosine-1-phosphate (S1P) within FFP restores the EG by activating the endothelial cell S1P receptor 1  by exposure to adrenaline, TNF-α and Hp < 0.001), consistent with EG shedding. Syndecan-4 and thrombomodulin mRNA expression was increased (p < 0.001) and decreased (p < 0.05), respectively, by the injury. Syndecan-4 shedding was not affected by treatment with FTY720, whereas FFP attenuated syndecan-4 shedding back to baseline levels in the injured cells and this was unaffected by W146. Neither treatment affected syndecan-4 or thrombomodulin mRNA expression.The injury resulted in a 2.1-fold increase in syndecan-4 (1 and upregulation of SDC-4 production. Further studies to examine whether FTY720 or another S1PR agonist might have EG-protective effects under different conditions are warranted, as are investigations seeking the mechanism of EG protection conferred by FFP in this experimental model.FTY720 did not prevent syndecan-4 shedding from the EG in the HUVEC model of endothelial injury, suggesting that activation of S1PR does not prevent EG damage. FFP prevented syndecan-4 shedding from the EG via a mechanism that was independent of S1PR The endothelial glycocalyx (EG) is a 0.2–5 μm layer of glycoproteins, proteoglycans, glycosoaminoglycans and plasma proteins that lines the vascular endothelium . It is aDamage to the EG causes intraluminal shedding of its components. This occurs in critical illness including trauma and sepsis and is correlated with poor outcomes . While i1–5) [1-3 are expressed on endothelial cells [1. Activation of S1PR1 on endothelial cells by S1P in vitro inhibits matrix-metalloproteinases (MMPs), preventing EG shedding [Sphingosine 1-phosphate (S1P) regulates the cardiovascular, immune, nervous and endothelial systems via the five S1P-specific G protein-coupled receptors (S1PR1–5) . Of thesal cells . A potenshedding , and in shedding . FFP conshedding , and theshedding .2, which has an opposing effect to S1PR1. Activation of S1PR1 promotes vascular integrity, whereas S1PR2 is critical for regulating the endothelial response to inflammatory stimuli causing increases in vascular permeability, and a pro-adhesive and procoagulant phenotype [1 and S1PR2 expression on the endothelium determines the phenotypic response to an inflammatory stimulus [1, or blockade of S1PR2, will result in a greater endothelial and EG-protective effect than non-selective activation of S1PR1 and S1PR2.There are some disadvantages to using S1P clinically as an EG restorative therapy. Firstly, S1P is not approved for therapeutic use and hence translation into practice would take considerably longer than an approved agent due to the necessary regulatory processes. Secondly, because S1P is a non-selective agonist it also activates endothelially expressed S1PRhenotype . The balstimulus . Potenti1 but approximately tenfold less affinity for S1PR3 and very little for S1PR2 [2-adrenoceptor agonists [A potential candidate drug that addresses both these issues is FTY720 (fingolimod), an S1PR agonist with high affinity for S1PRor S1PR2 , 14. FTYor S1PR2 , and hasor S1PR2 . The maiagonists , 16. In agonists , 17–20. Human umbilical vein endothelial cells (HUVECs) were purchased from Lonza . Cells were seeded onto fibronectin-coated 75-cm2 flasks using complete media  and grown to confluence in a humidified, 5% CO2 incubator at 37 °C. Cells were lifted from the flasks using 0.25% trypsin/EDTA solution  . Media wCells were assigned to one of six groups: no injury or treatment (control group), injury only, injury treated with FTY720 with and without W146, and injury treated with FFP with and without W146.As FTY720 is a pro-drug requiring phosphorylation to its active form by EC , cells a1 antagonist W146  was prepared in 100 mM sodium hydroxide  and stored at – 80 °C. Cells assigned to W146 groups were exposed to 10 A 5 mM solution of the S1PR2O2 for 4 h in a 5% CO2 incubator at 37 °C. Cells assigned to the FFP groups were exposed to 25% FFP . All cells were incubated with 0.2 U/mL heparin  to prevent fibrin formation given the use of FFP.Cells assigned to the injury groups were then exposed to 1.0 nM adrenaline , 10 ng/mL TNF-α , and 100 g and stored at − 80 °C for later analysis. Cells were stored in RNALater  at 4 °C for 24 h then at − 20 °C prior to qPCR analysis. The experimental protocol is illustrated in Fig. After 4-h exposure to injury and treatment conditions, supernatant was removed, centrifuged for 5 min at 10,000×Syndecan-1 (SDC-1), syndecan-4 (SDC-4), and thrombomodulin (TM) are components of the EG and were measured in HUVEC supernatant using commercially available enzyme-linked immunosorbent assay (ELISA) kits according to manufacturer instructions .Isolation of mRNA was performed using an Isolate II RNA mini kit  and reverse transcription was then performed with a SensiFAST cDNA synthesis kit . Gene expression levels were measured by quantitative PCR using a SensiMix SYBR No-Rox kit  and a Rotor-Gene Q thermal cycler  according to the manufacturer’s protocol. Primers for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), SDC-4, TM, and glypican-1 (GP-1) were as described by Liu et al.  and Yangp < 0.05. All data are expressed as mean ± SEM for n = 5 experiments. As we had a small sample size, normality was assessed using the Shapiro–Wilk test and for all data this did not demonstrate evidence of non-normality (p > 0.05). Visual inspection of the QQ plots was also consistent with normally distributed data. The Brown–Forsythe test for all data was consistent with approximately equal variances between groups (p > 0.05). We therefore chose to use a parametric test. An analysis of variance was performed with the Tukey’s multiple comparisons test.Statistical analysis was performed with GraphPad Prism 9 . Statistical significance was inferred at p < 0.01). Pre-exposure to FTY720 did not reduce the amount of SDC-4 shed into the supernatant. SDC-4 levels returned to uninjured baseline levels in the FFP treated groups. The inhibitory effect of FFP was not reversed by the addition of W146. The concentration of SDC-4 in the FFP sample (no cells) was 58 ng/mL.Cell injury caused a 2.1-fold increase in the amount of SDC-4 in HUVEC supernatants  that is used clinically and well above the threshold known to improve vascular permeability, but it is possible this is too low to achieve EG protection. Clinically, maximal lymphopenia is seen at trough levels of 10 ng/mL, and optimal efficacy for immunosuppression in transplantation is around 50 ng/mL . The natr FTY720 , 14. Thial cells .1 and, to a lesser extent with tenfold less affinity, S1PR3 selectivity [1 is thought to mediate most of the effects of S1P on the endothelium including S1P’s EG-protective effect and has a functional antagonistic relationship to S1PR2 [3 on the endothelium, but it does appear to have a similar function to S1PR2 [1-3 by S1P but not from activation of S1PR1 and S1PR3 by FTY720.A less likely but possible reason that FTY720 does not prevent EG shedding is because of its S1PRectivity , 14. S1Pto S1PR2 . Less is2O2) at concentrations that have previously been demonstrated to induce EG shedding in vitro [The conditions associated with EG shedding are diverse, and include ischaemia and reperfusion, sepsis, trauma, atherosclerosis, and diabetes . These cin vitro , 32 and in vitro , 33.Exposure to these agents for 4 h induced EG shedding as evidenced by significantly higher SDC-4 levels in the supernatant of the injury group compared to non-injured controls. SDC-4 is one of the four sub-types of trans-membrane syndecans that form the main structural elements of the EG. Most clinical studies have measured SDC-1 as a marker of EG shedding as the predominant syndecan shed into the blood of critically unwell patients is SDC-1 and -3, and SDC-4 and SDC-2 levels are not significantly elevated compared to healthy controls . SDC-1 c1 [1 receptor to prevent MMP-mediated shedding of EG components. However, it is still possible that FTY720 has a differential effect on SDC-1 and SDC-4 shedding through an unknown mechanism. The effect of FTY720 on the shedding of SDC-1 and other EG components should be assessed in a flow cultured model to investigate this further.Given these differences, the clinical relevance of a statically cultured HUVEC model of EG injury and treatment is unclear and may be a limitation of this study. However, although not well characterised, SDC-1 and SDC-4 appear to have similar, but not identical, roles in regulating vascular endothelial function , 37, 41.1 . Therefo1 [1 did not alter the efficacy of FFP, suggesting that the EG-protective properties of FFP are not mediated by this receptor as has been suggested by previous studies where S1P-depleted FFP regained its EG-protective properties with S1P supplementation, as measured by SDC-1 shedding [1 in the mechanism of FFPs EG-protective effect, and then whether specifically targeting this pathway will achieve the benefits of FFP while avoiding its adverse effects.The results of this study are consistent with the well-established EG-protective effects of FFP . The mec1 . In thisshedding . It may 1 or by upregulation of SDC-4 production. The search for the mechanism(s) of FFPs protective effect is ongoing. Furthermore, it should be noted that while EG shedding is associated with poor outcomes in critical illness, there is no evidence yet that protection and repair of the EG improves clinical outcomes. The identification of a targeted EG therapy will allow this hypothesis to be tested.In conclusion, this study suggests that FTY720 does not prevent EG shedding in injured cultured HUVECs. The previously reported attenuation in endothelial cell hyperpermeability by FTY720 , 17–20 m


Heart failure with preserved ejection fraction (HFpEF) is a multi-organ disorder that represents about 50% of total heart failure (HF) cases and is the most common form of HF in the elderly. Because of its increasing prevalence caused by the aging population, high mortality and morbidity, and very limited therapeutic options, HFpEF is considered as one of the greatest unmet medical needs in cardiovascular medicine. Despite its complex pathophysiology, numerous preclinical models have been established in rodents and in large animals to study HFpEF pathophysiology. Although age and sex differences are well described in HFpEF population, there are knowledge gaps in sex- and age-specific differences in established preclinical models. In this review, we summarize various strategies that have been used to develop HFpEF models and discuss the knowledge gaps in sex and age differences in HFpEF. Heart failure (HF) is a leading cause of death worldwide and its prevalence is estimated to be 1%–2% of the adult population in developed countries, rising to ≥10% among people over 70 years of age . HF is aHFpEF consists of about 50% of total HF cases , and itsExtensive clinical and preclinical studies have resulted in the development of efficient therapies for HFrEF . HoweverHFpEF is recognized to be a multi-systemic disorder with broad phenotypic heterogeneity . PrognosAnimal models of HFpEF should mimic the phenotypes and symptoms observed in patients with HFpEF. Diastolic dysfunction as well as concentric hypertrophy and fibrosis are the main cardiac characteristics for HFpEF. Patients with HFpEF also develop systemic derangements such as exercise intolerance and pulmonary congestion/edema. Different strategies for induction of HFpEF risk factors, such as aging, hypertension, and metabolic stress, have been used alone or in combination to reproduce diastolic dysfunction and other phenotypes of HFpEF in animal models.The aging heart shows phenotypic changes such as cardiac hypertrophy, diastolic dysfunction, worsened myocardial performance, and preserved systolic function at rest but impaired while performing exercise . The oveConsistent with the observations in humans, studies on C57BL/6 mice  showed that aging results in left ventricular and left atrial hypertrophy, diastolic dysfunction, worsened myocardial performance, and a modest decline in systolic function . In addiSenescence-accelerated mouse (SAM) is a model of accelerated aging. Untreated male senescence-prone (SAMP8) mice develop diastolic dysfunction at 6 months of age , whereasAlthough aging animal models and patients with HFpEF share some common features such as cardiac hypertrophy, diastolic dysfunction, and exercise intolerance, aging alone does not result in HF in most animal models . HoweverHypertension is one of the major comorbidities contributing to the development of HFpEF. Because of its structural and functional consequences on the cardiovascular system, hypertensive stress is widely used in different animal models to study HFpEF.Dahl salt-sensitive (SS) rat model is a commonly used HFpEF model . TreatmeDeoxycorticosterone acetate (DOCA)–salt hypertension leads to hypervolemia due to an impairment of renal sodium handling where the kidneys reabsorb more water and sodium . When yoSpontaneously hypertensive rat (SHR) is an inbred rat strain that has a predisposition to the spontaneous development of hypertension . At arouAngiotensin II plays a major role in cardiac homeostasis and regulates such processes as afterload, preload, cardiac hypertrophy, and fibrosis . Young mWhile animal models of hypertension display cardiac features of HFpEF at an early stage, extracardiac features of HFpEF are not well represented and some hypertensive models progress to HFrEF at a later stage . TherefoObesity and diabetes are two of the major comorbidities in HFpEF . PatientLepr gene, which encodes the leptin receptor and that leads to the worsen affinity of leptin to its receptor  develop insulin resistance and dysfunctional leptin receptors due to a mutation in receptor . This mureceptor . Howeverreceptor .Ob/ob mouse is a leptin-deficient model in C57BL/6J background that spontaneously develops obesity and type 2 diabetes. Concentric hypertrophy, diastolic dysfunction, and preserved LVEF have been observed in ob/ob female and male mice . HoweverDb/db leptin receptor-deficient mouse model is characterized by a point mutation in the gene that encodes the leptin receptor in C57BL/6J or C57BL/6KsJ background. This mutation leads to obesity and results in type 2 diabetes . At 6 moThese models of metabolic stress have marked obesity with (ob/ob and db/db mice) or without diabetes (Zucker rats) and develop cardiac features of HFpEF in both sexes. Sex differences in cardiac HFpEF features have been observed, but age differences have not been determined in these models.Early attempts on the development of HFpEF animal models mainly focused on the use of single perturbations  to induce the clinical features of HFpEF. However, because HFpEF is a systemic disorder with multiple comorbidities, these single-hit models typically do not mimic certain symptoms of patients with HFpEF. Very recently, researchers established several novel models that utilize perturbations on two or more risk factors of HFpEF to recapitulate major clinical features of this complex syndrome .Obese ZSF1 rat is one of the first models that combine more than one perturbation to induce HFpEF phenotype in rodents. This model established by crossing female Zucker diabetic rat and male spontaneously hypertensive HF rat develops both cardiac and extracardiac HFpEF phenotype . Nguyen l-arginine methyl ester (l-NAME), respectively. After 15-week treatment, two-hit stress–treated mice display preserved EF and significant diastolic dysfunction accompanied by extracardiac HFpEF phenotypes including pulmonary congestion and exercise intolerance (l-NAME-only– and HFD-only–treated mice, two-hit stress–treated mice exhibit more severe HFpEF phenotypes and developed impaired cardiomyocyte contraction and relaxation kinetics (Recently, olerance . This moolerance . Comparekinetics . Using tkinetics .l-NAME and HFD would present exacerbated HFpEF progression, including the development of significant HFpEF phenotypes in old females. This two-hit model was later used to demonstrate the roles of NAD metabolism and Xbp1s-FoxO1–regulated lipid accumulation in the pathogenesis of HFpEF (L-NAME and HFD on female mice resulted in significantly attenuated phenotype as compared to male counterparts . Female of HFpEF .A recent study by Withaar et al.  combinedA more recent study by The aforementioned two- and multi-hit strategies generate novel animal models that display typical cardiac and extracardiac features of human HFpEF patients. Sex differences in HFpEF phenotypes were observed in two of these models , and advThe aforementioned murine and rat models have some limitations due to their sizes and different cardiac structure and function as compared to large animals and humans . Large aIn recent years, multi-hit swine HFpEF models were also established. Using streptozotocin injection, HFD and renal artery embolization, Despite sharing more similarities in their cardiovascular physiology with humans, large animal preclinical models have their limitations. Primary limitation of large animal preclinical models is the high general cost compared to rodent models . This ofMost of the pharmacological and device therapies for HFpEF investigated in completed clinical trials have been shown to be ineffective. So far, only the recent EMPEROR-Preserved clinical trial showed that empagliflozin reduced the combined risk of cardiovascular death or hospitalization for HF in patients with HFpEF . It is iIt is highly unlikely that a single animal model will fully recapitulate the phenotypes of the heterogenous HFpEF population. For example, a limitation of HFpEF models induced by hypertension is the fact that, in the majority of patients with HFpEF, it continues to display HFpEF symptoms even when their blood pressure is under control . Some moMany preclinical HFpEF studies used animals of one sex or mixed sex, limiting the interpretation of sex-specific differences in HFpEF pathogenic mechanisms. Because of the sex disparities in HFpEF, it is important to include both sexes in HFpEF research and analyze the outcomes separately to determine any potential sex-specific mechanisms. While it is well established that the prevalence of HFpEF sharply increases with age , the maj


As a near-physiological pacing innovation, left bundle branch area pacing (LBBAP) has drawn much attention recently. This study was aimed to investigate the electrophysiological characteristics, unipolar/bipolar pacing parameters and mid- to long-term effects and safety of three different pacing methods and identify possible predictors of adverse left ventricular remodeling.Ninety-two patients were divided into the LBBAP group, right ventricular septal pacing (RVSP) group and right ventricular apical pacing (RVAP) group. Baseline information, electrophysiological, pacing and echocardiographic parameters were collected.p < 0.01). The stimulus to left ventricular activation time (Sti-LVAT) was the shortest in the LBBAP group, followed by the RVSP and RVAP groups . LBBAP had a significantly lower tip impedance during the procedure and 3-month follow up as compared to RVSP and RVAP (p < 0.001). Higher bipolar captured thresholds were observed in LBBAP during the procedure (p < 0.001). Compared to the baseline values, there was a greater reduction in left ventricular end-diastolic dimension (LVEDD) in the LBBAP group (p = 0.046) and a significant enlargement in LVEDD in the RVAP group (p = 0.008). Multiple regression analysis revealed that the Sti-LVAT was a significant predictor of LVEDD at 12 months post-procedure. At the 24-h post-procedure, significant elevations were observed in the cTnI levels in LBBAP (p < 0.001) and RVSP (p < 0.05). More transient RBB injury was observed in LBBAP. But no significant difference was found in cardiac composite endpoints among three groups (p > 0.05).The three pacing methods were performed with a similar high success rate. The paced QRSd was significantly different among the LBBAP, RVSP and RVAP groups (105.93 ± 15.85 ms vs. 143.63 ± 14.71 ms vs. 155.39 ± 14.17 ms, LBBAP demonstrated a stable captured threshold, a low tip impedance and a high R-wave amplitude during the 12-month follow-up. Left ventricular remodeling was improved at 12 months post-procedure through LBBAP. The Sti-LVAT was a significant predictor of left ventricular remodeling. LBBAP demonstrated its feasibility, effectiveness, safety and some beneficial electrophysiological characteristics during this mid- to long-term follow-up, which should be confirmed by further studies. Electrical pacing is an important and effective therapy for the patients with bradycardia and cardiac conduction dysfunction. Traditional right ventricular apical pacing (RVAP) causes left ventricular electrical and mechanical dyssynchrony, resulting in atrial fibrillation or heart failure (HF) , 2. AlteCurrently, His bundle pacing (HBP) is accepted as a real physiological pacing technology for its feasibility and clinical benefits , 6. HoweThis was a retrospective cohort study of patients with indications for pacemaker implantation treatment of symptomatic bradycardia according to the established guidelines . All con4̴-6 on the surface 12-lead electrocardiogram (ECG) showed the shortest and most constant left ventricular activation time (LVAT)  by the transventricular-septal method as previously described . In our The 5076 pacing lead (Medtronic Inc. USA) was implanted at the mid-RV septum as previously described , 14. LikThe 4574 pacing lead (Medtronic Inc. USA) was routinely implanted at the RV apex in a standard fashion.Considering the intrinsic bundle branch block and atrioventricular (AV) conduction time, AV delay programming was individualized as previously described .Here, LVAT indicates the rapidity of left ventricular free wall activation. The short LVAT (< 80 ms) suggested capture of the conduction system. We defined the Sti-LVAT of the LBBAP or RVSP as the duration from the stimulus artifact to the peak of the R wave and the Sti-LVAT of the RVAP as the duration from the stimulus artifact to the trough of the R wave in lead V5/6 Fig. A, B, C.The clinical baseline characteristics were collected. Echocardiography was performed by two experienced echocardiologists. The electrophysiological and echocardiographic parameter data were collected, including the paced QRS duration (QRSd), paced QRS axis, Sti-LVAT, left ventricular end-diastolic dimension (LVEDD), left atrial diameter (LAD) and left ventricular ejection fraction (LVEF). The unipolar (tip) and bipolar pacing parameters were measured during the procedure. Patients were followed at 1 week, 1 month, 3 months and 12 months post-procedure. The mid-term follow-up was defined as 3–6 months and the long-term follow-up as more than 6 months. The echocardiographic parameters and pacing parameters were recorded at outpatient visit. The levels of cardiac troponin I (cTnI) and plasma N-terminal pro-B type natriuretic peptide (NT-pro-BNP) were recorded and evaluated at baseline and each follow-up.p value < 0.05 was considered significant.Data were analyzed using SPSS 22.0 . Continuous data are reported as the means ± standard deviations (SDs) or median (interquartile ranges). Categorical data are expressed as numbers and percentages and were compared by χ2 test or Fisher exact test. An analysis of variance (ANOVA) and Tukey’s post hoc test were performed for multiple comparisons. Repeated-measures ANOVA was applied to compare the mean values of variables before the procedure, during implantation, and at each follow-up. A linear regression analysis was done in all cases. Stepwise multiple regression analysis was used to determine which variable provided the best estimate for heart function. A two-sided A total of 142 patients underwent the first pacemaker implantation from August 2017 to November 2020. After the exclusion of 22 patients who were lost to follow-up, 5 patients more than 89 years of age, 9 patients with LVEF ≤ 35%, 11 patients with MI and 3 patients with HCM, 92 consecutive patients were enrolled in the study. In the LBBAP group, LBBAP was successfully achieved in 30 patients (90.91%) and the remaining 3 patients received RVSP instead due to an inability to screw the lead deep enough to reach the left bundle branch area. In the RVSP group, RVSP was successfully performed in 21 patients (95.45%), and the remaining 1 patient received RVAP due to the high pacing threshold in the RV septum. In the RVAP group, RVAP was successfully performed in 37 patients (100%). The study flowchart is presented in Fig. p = 0.021). The clinical baseline characteristics are summarized in Table At baseline, the LBBAP group had more patients characterized by the LBBB pattern than the RVSP group and the RVAP group . Compared with intrinsic QRSd, the paced QRSd was significantly narrower in the LBBAP group , and significantly wider in the RVSP group  and the RVAP group . In the LBBAP group, left bundle branch (LBB) potential was recorded in 21 (70.0%) patients, and the current of injury (COI) was recorded in 7 (23.3%) patients. The incidence of selective LBBP in LBBAP was 53.3%. The Sti-LVAT among the three groups was significantly different . Compared to the RVSP group, tip captured thresholds of the ventricular lead in the LBBAP group were significantly increased . LBBAP had significantly high bipolar captured thresholds, followed by those in RVAP and RVSP (p < 0.001). In contrast, LBBAP had significantly low tip impedance, followed by those in RVSP and RVAP (p < 0.001). Intraprocedural parameters among the three groups are summarized in Table In the LBBAP group, the paced ECG morphology was characterized by an RBBB pattern and a narrow QRS complex. In the RVSP group and RVAP group, the paced ECG morphology showed wide QRS and LBBB patterns. The paced QRSd was significantly different among the LBBAP, RVSP and RVAP groups . On the contrary, a significant enlargement in LVEDD was observed at 12 months post-procedure in the RVAP group when compared with baseline , 1 week post-procedure  and 3 months post-procedure    among the three groups at baseline Table , 1 week 4) Table .Table 3Ep < 0.001) . However, the difference was not significant in ΔLVEDD between RVSP and RVAP (p = 0.181).Encouragingly, there was a significant difference in changes in LVEDD (ΔLVEDD) from baseline to 12-month follow-up over time among the three groups   ng/ml vs. 0.014  ng/ml, p = 0.014], and remained stable in the RVAP group  .In comparison to baseline, the cTnI levels at 24 h post-procedure significantly increased in the LBBAP group  , which was maintained at 12 months post-procedure when compared with the RVAP group (p = 0.049). However, the difference was not significant in the tip impedance between the RVSP and RVAP groups or in tip/bipolar captured thresholds, R-wave amplitude and bipolar impedance among the three groups during the 12-month follow-up (p > 0.05). There was no significant difference in the average percentage of ventricular pacing among the three groups during the 12-month follow-up (p > 0.05). The pacing parameters during the follow-up period are summarized in Table At 1 week and 3 months post-procedure, the LBBAP group had a significantly lower tip impedance than the RVSP and RVAP groups , 3 months post-procedure , and at 12 months post-procedure   , NT-pro-BNP preprocedure  and QRSd preprocedure . Stepwise multiple regression analysis, using the LVEDD at 12 months post-procedure as the dependent variable, and age, Sti-LVAT, NT-pro-BNP and QRSd preprocedure as independent variables in a predictive model, showed that only Sti-LVAT  was a significant predictor of the LVEDD . Another case in LBBAP suffering from transient fever 3-day post-procedure was treated with antibiotic treatment for three days. No serious complications, including coronary artery injury, septal perforation and pocket infection, occurred during 12 months follow-up period. During 12 months follow-up, a case with diabetes in RVSP suffered from unstable angina and received percutaneous coronary intervention (PCI) in right coronary artery. Five patients in RVAP and one in RVSP were hospitalized with heart failure during the follow-up. No cardiovascular related death was observed in the three groups.A case with CHD in RVAP suffered from pocket hematoma 2-day post-procedure and received intermittent compression hemostasia. More transient RBB injuries were observed in LBBAP than and in RVSP and RVAP during the procedure  LBBAP was feasible with the same high success rate as RVSP and RVAP. (ii) LBBAP, characterized by an RBBB pattern, yielded the narrowest QRSd and the shortest Sti-LVAT among the three pacing methods and maintained physiological LV activation. (iii) Tip impedance, rather than bipolar impedance, was significantly lower in the LBBAP group than in the RVSP and RVAP groups during the 3-month post-procedure. LBBAP demonstrated stable pacing parameters during the 12-month follow-up regardless of lead polarity. (iiii) A significant reduction in LVEDD and NT-pro-BNP levels was achieved in the LBBAP group during the follow-up. (iiiii) The Sti-LVAT was a significant predictor of the left ventricular size at 12 months post-procedure.Recently, LBBAP, as a near-physiological pacing innovation, demonstrated its feasibility in some previous observational studies , 17, witp < 0.001) [p < 0.01), suggesting LBBAP produces a better ventricular electrical synchronization after procedure. The QRSd in LBBAP was the shortest in comparison with that in RVSP and RVAP , which is similar to the finding of Chen K et al. [QRSd is one of the most important indicator of correction of electromechanical dyssynchrony. Chen et al. reported the paced QRSd was 111.85 ± 10.77 ms in the LBB pacing group and 160.15 ± 15.04 ms in the RVP group (< 0.001) . The mag< 0.001) . HoweverK et al. . They fop < 0.001), which depended on whether the LB-Purkinje conduction system was captured. The Sti-LVAT was longer in RVSP and RVAP because activation propagation of the endomyocardium was slower than that of the natural LB-Purkinje conduction system. Thus, the Sti-LVAT may also be used as an important indicator of evaluating ventricular electromechanical dyssynchrony.LVAT, another important electrophysiological index, indicates the rapidity of left ventricular lateral precordial myocardium depolarization. A constantly short Sti-LVAT was an essential criterion of the capture of the LBB during LBBAP . PreviouP < 0.001) and reduced LVEDD  by LBBAP after 1-year follow-up [p = 0.046). Meanwhile, a significant difference in ΔLVEDD from baseline to 12-month follow-up over time was noted among the three groups  . The significant change in LVEDD over time among the three groups could demonstrate the efficacy of LBBBAP.The nonphysiological ventricular activation pattern, such as in RVSP and RVAP, results in electromechanical dyssynchrony and adverse clinical outcomes , 2. Howeollow-up . In thisEncouragingly, although the LBBAP group included more patients with LBBB and slightly higher NT-pro-BNP at baseline, we observed a significant reduction in the LVEDD and NT-pro-BNP values during LBBAP at 12 months post-procedure, suggesting that this more rapid ventricular activation improves LV function and reverses ventricular remodeling. Lan S et al. reported the patients with wide baseline QRS (> 120 ms) were observed to have significantly improved LVEF and reduced LVEDD , which wIn this study, we found that the Sti-LVAT was correlated with the LVEDD at 12 months post-procedure. Moreover, stepwise multiple regression analysis showed that the Sti-LVAT was an independent predictor of the LVEDD at 12 months post-procedure. All these findings indicated that the short Sti-LVAT during LBBAP represents near-physiological ventricular activation pattern, which translates into rapid ventricular activation and electromechanical synchronization, reverses ventricular remodeling. On the contrary, a wide Sti-LVAT means a prolonged ventricular activation time and a loss of physiological ventricular activation patterns, which might result in impaired left ventricular function.Recently, more concerns have been focused on the pacing parameters of LBBAP. Previous studies by Chen et al.  and ChenIn accordance with the previous study , a mild This was a single-center study with small sample sizes. The lack of randomization might lead to heterogeneous population and bias estimation on the results. The case crossover and the lack of the propensity score matching method might have resulted in bias estimation. In our study, the percentage of ventricular pacing was reduced due to the inclusion of the patients with SSS, which might have affected the final results. The comparisons between the patients with a higher percentage of ventricular pacing were more convincing. A total of 15 LBBB patients were included in our study. Subgroup analysis was not performed due to a small sample size. The 12-month follow-up was relatively short, and the long-term effects of electrophysiological and pacing parameters of LBBAP remain unclear at present. Prospective, randomized, long-term, large-scale and multicentered studies are required to determine the feasibility and effectiveness of LBBAP.LBBAP was feasible with the same high success rate as RVSP and RVAP. LBBAP demonstrated a stable and low captured threshold and unipolar pacing impedance and a high R-wave amplitude during the 12-month follow-up. Significantly improved LV remodeling at 12 months post-procedure was achieved through LBBAP. The Sti-LVAT was a significant predictor of LVEDD at 12 months post-procedure. LBBAP demonstrated its feasibility, effectiveness, safety and some beneficial electrophysiological characteristics during this mid- to long-term follow-up, and these results should be confirmed by further studies.


M. jannaschii Fsr (MjFsr) and ANME-Fsr belong to two phylogenetically distinct groups, FsrI and FsrII, respectively. MjFsrI reduces sulfite to sulfide with reduced F420 (F420H2), protecting methyl coenzyme M reductase (Mcr), an essential enzyme for methanogens, from sulfite inhibition. However, the function of FsrIIs in ANME, which also rely on Mcr and live in sulfidic environments, is unknown. We have determined the catalytic properties of FsrII from a member of ANME-2c. Since ANME remain to be isolated, we expressed ANME2c-FsrII in a closely related methanogen, Methanosarcina acetivorans. Purified recombinant FsrII contained siroheme, indicating that the methanogen, which lacks a native sulfite reductase, produced this coenzyme. Unexpectedly, FsrII could not reduce sulfite or thiosulfate with F420H2. Instead, it acted as an F420H2-dependent nitrite reductase (FNiR) with physiologically relevant mK values . From kinetic, thermodynamic, and structural analyses, we hypothesize that in FNiR, F420H2-derived electrons are delivered at the oxyanion reduction site at a redox potential that is suitable for reducing nitrite  but not sulfite . These findings and the known nitrite sensitivity of Mcr suggest that FNiR may protect nondenitrifying ANME from nitrite toxicity. Remarkably, by reorganizing the reductant processing system, Fsr transforms two analogous oxyanions in two distinct archaeal lineages with different physiologies and ecologies.Anaerobic methanotrophic archaea (ANME), which oxidize methane in marine sediments through syntrophic associations with sulfate-reducing bacteria, carry homologs of coenzyme FIMPORTANCE Coenzyme F420-dependent sulfite reductase (Fsr) protects methanogenic archaea inhabiting deep-sea hydrothermal vents from the inactivation of methyl coenzyme M reductase (Mcr), one of their essential energy production enzymes. Anaerobic methanotrophic archaea (ANME) that oxidize methane and rely on Mcr, carry Fsr homologs that form a distinct clade. We show that a member of this clade from ANME-2c functions as F420-dependent nitrite reductase (FNiR) and lacks Fsr activity. This specialization arose from a distinct feature of the reductant processing system and not the substrate recognition element. We hypothesize FNiR may protect ANME Mcr from inactivation by nitrite. This is an example of functional specialization within a protein family that is induced by changes in electron transfer modules to fit an ecological need. Halobacteriota (2–7–420-dependent sulfite reductase (Fsr) , converting this potent inhibitor of methyl coenzyme M reductase, an essential enzyme for the methanogens, to sulfide, an essential sulfur source for anabolism in this hyperthermophilic methanogen  –18. Fsr al vents . Geneticthanogen –21. All ironment , 22. It ironment , 19, 22.42− utilization in ANME-2 is likely assimilatory in nature and not coupled to methane oxidation  retrieves reducing equivalents from F420H2 and the C-terminal dissimilatory sulfite reductase (Dsr) unit (Fsr-C) utilizes them to reduce sulfite to sulfide  and is incapable of reducing sulfite with F420H2. We describe the thermodynamic and structural basis for this distinction and discuss the possible ecophysiological relevance of the enzyme.The enzyme Fsr is a two-domain protein, where the N-terminal F sulfide , 22. A pnd FsrII . With oncteriota , 14; MetMethanosarcina acetivorans strain carrying pDS701, a replicable expression vector for ANME2c-FsrII, was constructed for the study. An extract of cells of this strain that were induced with tetracycline was able to catalyze sulfite-dependent oxidation of reduced methyl viologen. This activity was absent in native M. acetivorans extracts, which lack Fsr (M. acetivorans(pDS701) under tetracycline induction. Similar to MjFsrI  value was 0.2 ± 0.01 μmol of F420H2 oxidized or 0.3 ± 0.02 μmol electrons transferred per min per mg enzyme  is a 6-electron process and that for hydroxylamine to ammonium (NH2OH to NH4+) is a 2-electron process, ANME2c-FsrII-6D reduced hydroxylamine 21 times faster than nitrite. In a reaction mixture with 0.08 μmol of F420H2 and 0.40 μmol of nitrite, ANME2c-FsrII-6D produced 0.02 ± 0.001 μmol of F420 and 0.006 ± 0.005 μmol of ammonia after 30 min of reaction; these values represent averages from three independent assays. Considering that 3 mol of F420H2 (6 electrons) would be needed to reduce 1 mol of nitrite to ammonia, the above values represent 90% recovery of reducing equivalents from F420H2 into ammonia, indicating that it was the sole product. ANME2c-FsrII-6D was not able to utilize NADH or NADPH as electron donors with nitrite or hydroxylamine as an oxidant. The second partial reaction of the nitrite reductase activity could not be assayed because nitrite chemically oxidized the electron source, reduced methyl viologen (MV·+); the first partial reaction did not involve nitrite, and the respective activity was assayed as described in the following section. The kcat values for the nitrite reductase activity with respect to F420H2 and nitrite were 19 and 18 min−1, respectively, and for the sulfite reductase activity it was 64 min−1 with respect to both HSO3- and MV·+ (−1 was recorded for the hydroxylamine reductase activity with respect to NH2OH (The lack of sulfite reduction activity with F nitrite –31. Furtg enzyme . Similarg enzyme . The appg enzyme . These kand MV·+ . A turnoto NH2OH .420H2 (the predicted native electron donor), NADH, and NADPH. ANME2c-FsrII-6D did not oxidize F420H2 with sulfite or thiosulfate as electron acceptors and similar results were obtained in assays with NADH and NADPH as reductants. These conclusions were based on activity assays both with and without 0.5 mM DTT and more than 10 independently generated purified preparations.We tested the ability of ANME2c-FsrII-6D to reduce relevant sulfur oxyanions with the reductants: F420H2 by the enzyme, we examined if ANME2c-FsrII-6D could catalyze the following two partial reactions that have been observed with MjFsrI  as the direct electron acceptor, the second partial reaction is observed with reduced methyl viologen (MV·+) as the reductant  for sulfite reduction from the second partial reaction. In assays with a fixed concentration of sulfite (1 mM) and a concentration range of 29 to 408 μM for MV·+, the apparent mK for MV·+ was determined to be 137 ± 26 μM and the value of maximum velocity (Vmax) was 1 ± 0.1 μmol of MV·+ oxidized or 1 ± 0.1 μmol electrons transferred per min per mg enzyme  supplied in the assay toward sulfide production from sulfite, which is a 6-electron reduction process. The enzyme was able to oxidize F420H2 with a mixture of methyl viologen (MV2+) and metronidazole, where the former served as the direct artificial electron acceptor for the enzyme and was reduced to MV·+, and metronidazole regenerated MV2+ from MV·+. From assays with 2.5 μM MV2+, 1.5 mM metronidazole, and a concentration range of 2 to 80 μM for F420H2, the apparent mK for F420H2 was determined to be 5 ± 1 μM and Vmax was 0.5 ± 0.02 μmol of F420H2 oxidized or 1 ± 0.04 μmol electrons transferred per min per mg enzyme  for sulfite; the standard error for the fitting suggested that the inhibition did not follow a standard model, and the mechanism underlying this departure is currently unknown.The pattern seen in the double-reciprocal or Lineweaver-Burk plots of the data collected at a fixed concentration of 40 μM for Factivity . In this420H2 oxidation and sulfite or nitrite reduction, respectively, we analyzed the features for ANME2c-FsrII-6D-N and ANME2c-FsrII-6D-C separately. From a comparison with MjFsrI, where the residues 1 to 311 and 325 to 620 represent the N- and C-terminal domains  represent two distinct domains performing two parts of the overall reactions, F domains , 22, theMjFsrI-N are homologs of the F420H2 dehydrogenase subunit F of F420H2:quinone oxidoreductase (FqoF) complex of A. fulgidus (420H2:phenazine oxidoreductase (FpoF) complex of Methanosarcina mazei  carried sequence features that are typical of a siroheme- clusters, one of which may be ligated to the siroheme, whereas DsrAs in A. fulgidus and D. vulgaris assemble only one [Fe4-S4] cluster in addition to the siroheme-[Fe4-S4] unit.In the current study, an AlphaFold2 modeling and alignment with X-ray crystallographic structures of proposed , 22 contFsrII-Cs  and 8. Titional) . Of thessubunits . In A. fresidues  and B. Tse units : (i) assditional  and D;   was used as an electron donor, the enzyme was able to reduce this oxyanion (The sulfite binding site of a DsrA subunit of a dissimilatory sulfite reductase is comprised of four positively charged residues (PDB IDs nd 2V4J) , 37. In and K213  and the and K213 . An analand K213  and 7. Sand K213  and struand K213  14). Al. Al3MM5 and K213  and 7. T sulfite . Howeveroxyanion  and E.in silico model yielded the following possibilities in terms of the nature and number of the iron-sulfur cluster: MjFsrI, 5 or 6 [Fe4-S4] clusters; ANME2c-FsrII-6D, 4 or 5 [Fe4-S4] clusters with or without an [Fe3-S4] cluster. Our chemical assays suggested that ANME2c-FsrII-6D carries 15 Fe and 15 acid-labile sulfur units which closely correspond to four [Fe4-S4] clusters. The difference between the iron-sulfur cluster contents of MjFsrI and ANME2c-FsrII-6D likely would pertain to the N-terminal domain or specifically the motif B of this unit.For the combined N-terminal and C-terminal domains the Methanosarcina acetivorans, and purified the enzyme, and characterized its biochemical properties. This is the first case where a homogenous and active form of an ANME enzyme with elaborate metallocenters was produced using a methanogen as the expression host, purified, and biochemically characterized. An ANME methyl-coenzyme M reductase, also a metalloenzyme, was previously expressed in M. acetivorans; however, the recombinant enzyme has yet to be purified to homogeneity  as the electron donor, the cell extracts did not exhibit a sulfite reductase activity (data not shown). All follow-up work with ANME2c-FsrII-6D was subsequently conducted in Methanosarcina acetivorans as the expression host, which presented several advantages over M. maripaludis for this study. Not only is M. acetivorans a genetically amenable methanogen, but this archaeon is also phylogenetically closely related to the ANME-2c, both belonging to the order Methanosarcinales  (4-S4] clusters (590) carrying a siroheme unit has been isolated from a Methanosarcina species  , and theclusters , and a r species . As menta Dsr-LP , can gen protein  and 3. TMjFsr-I which reduces sulfite to sulfide with F420H2 as a reductant , we examined the potential for ANME2c-FsrII-6D to reduce nitrite. Assays with the ANME-2c FsrII confirmed active reduction of nitrite to ammonia with F420H2 as the reductant, with a mK value for nitrite (5 μM) that is comparable to that of bona fide nitrite reductases described from other organisms (Table S1). The mK value of ANME2c-FsrII-6D for the electron donor F420H2 was also comparable to that of other F420H2-utilizing enzymes, including MjFsrI and F420H2-dependent thioredoxin reductase (DFTR) from M. jannaschii, FpoF of Methanosarcina mazei, and FqoF of Archaeoglobus fulgidus (Table S1)  , 48, 49.mK value for sulfite was 26 times higher than that of nitrite, and this property was consistent with ANME2c-FsrII-6D being a nitrite reductase. The catalytic efficiency (kcat/mK) values for nitrite and sulfite reduction could not be compared, as these two reactions had to be measured with different electron donors .A comparison of the respective kinetic constant values  revealedn donors , althougdonors 2631. Consior NH2OH . Based oor NH2OH  is distimK of the enzyme for sulfite (~130 μM) was 11-fold higher than that of MjFsrI (12 μM)  that was comparable to that of MjFsrI (21 μM)  , suggest nitrite  and G. H (21 μM)    . These s420H2 is a hydride donor, the first step of electron transfer in an Fsr involves FAD, the only available hydride acceptor in the enzyme residing in the N-terminal domain  and FsrI of M. jannaschii (MjFsrI) could deliver F420H2-derived electrons to the siroheme-[Fe4-S4] center at different redox potentials.We rationalize the above-stated hypothesis in terms of the cofactor contents and modeled structures of ANME2c-FsrII-6D and MjFsrI  and 7. Tand FqoF , 32, 48,contents  and 5, pHalobacteriota  center of F430 in Mcr in methanogens, rendering this essential enzyme inactive , a close methanotrophic relative found in terrestrial environments that is capable of directly oxidizing methane with nitrate reduction (nrfAH) using menaquinol as the electron donor , a role that is notably distinct from group I Fsr. In the hyperthermophilic methanogen M. jannaschii, FsrI catalyzes F420 coupled sulfite reduction and may also serve as a nitrite reductase. The FsrII enzyme is hypothesized to restrict the use of sulfite via fine-tuned control of the redox potential of the electrons delivered at its oxyanion reduction site. If the prediction about the physiological role of ANME-2c FsrII is proven to be correct, it would show that both FsrI and FsrII protect the same enzyme, methyl coenzyme M reductase (Mcr), from inactivation by two structurally similar oxyanions, sulfite and nitrite, respectively, in archaea from different habitats involved in methane production and consumption. Our study also showed that subtle changes in the predicted protein structure likely alters the properties of the redox centers, contributing to functional diversification in the Fsr protein family. These insights, along with the available heterologous expression system for an ANME-FsrII (this study) and MjFsrI  containing 7.5 ppm of H2S at a pressure of 105 Pa. The jar was left inside the anaerobic chamber , which contained a mixture of N2, CO2, and H2 (76:20:4 [vol/vol/vol]), and maintained at 37°C. To select M. acetivorans strains harboring pDS701, an M. acetivorans–Escherichia coli shuttle vector, puromycin was added to the solid or liquid medium at a final concentration of 2 or 1 μg/mL, respectively.in WWM60  was growin WWM60 . For groM. acetivorans using a liposome-mediated transformation method, and the transformant was selected on HS-TMA solid medium containing puromycin (M. acetivorans(pDS701) was induced by addition of tetracycline to the liquid culture to a final concentration of 100 μg/mL, and the induced cells were examined for the expression of the heterologous protein via SDS-PAGE analysis of cell extracts.The coding sequence for FsrII was previously PCR amplified from the metagenomic DNA extracted from an ANME-2-dominated methane seep sediment and cloned into the plasmid pCR4-TOPO . For theuromycin . The exp2 and H2 (96:4 [vol/vol]) and maintained at room temperature (~25°C). For the centrifugation of higher-volume materials, a Nalgene centrifuge bottle  was filled and sealed inside the chamber, then centrifuged outside, and taken back into the chamber for the collection of the supernatant and pellet. All column-chromatographic steps were performed employing gravity flow. Followings are the details of the purification experiment.Heterologously expressed ANME2c-FsrII-6D was purified primarily by following a previously described procedure . All steM. acetivorans(pDS701) containing recombinant ANME2c-FsrII-6D was resuspended in 30 mL of a 100 mM potassium phosphate buffer, pH 7 (buffer A), containing 0.2 mg/mL DNase I . The resulting suspension that contained the lysed cells resulting from the osmotic shock in the low-ionic-strength solution was homogenized via serial passages through 18-, 22-, and 25-gauge needles in that sequence. The lysate was centrifuged at 48,000 × g and 4°C for 20 min. The supernatant obtained from this step was fractionated on ice by precipitation with ammonium sulfate at two sequential stages, representing 30% and 60% saturations of the salt. At each stage, the suspension was centrifuged at 48,000 × g and at 4°C for 20 min. The pellet from 60% (NH4)2SO4 saturated extract was dissolved in a 1 M (NH4)2SO4 solution prepared in buffer A and fractionated over chromatography resins, each packed in a 1- by 20-cm column . The first step involved a column with 6 mL of phenyl-Sepharose resin  that was pre-equilibrated with 1 M (NH4)2SO4; all (NH4)2SO4 solutions were prepared in buffer A. After the sample was loaded onto it, the column bed was washed with the following (NH4)2SO4 solutions in buffer A at 1 M, 0.75 M, 0.5 M, 0.25 M, and 0 M in that sequence, and in each case the volume was 18 mL, except that the first was 36 mL. For each wash, fractions of 6-mL volumes were collected. Eluates from 0 M (NH4)2SO4 wash contained Fsr activity and were pooled. This pool was loaded onto a column packed with 6 mL of QAE-Sephadex  that was pre-equilibrated with buffer A. The flowthrough from the column contained Fsr activity, and the respective fractions were pooled and then loaded onto a column with 4 mL F420-Sepharose resin that was pre-equilibrated with buffer A; F420-Sepharose was prepared as described previously . Initial results were filtered with a 1% false discovery rate (FDR) threshold at the peptide level estimated by the target–decoy approach . A 100-μL aliquot of this filtered extract was resolved on a 4.6- by 250-mm Vydac analytical HPLC C18 column  by using a previously described method  as the reductant and following methods described previously , 500 μM sodium sulfite or nitrite, and 12 μg purified ANME2c-FsrII-6D. Reduced F420 (F420H2) was generated via chemical reduction of F420 dissolved in water with NaBH4  to MV·+ (reduced methyl viologen), and metronidazole continuously removes MV·+, which is a colored product of the reaction, by chemically oxidizing it to colorless MV2+ , and the progress of the reaction was monitored at 560 nm ; MV·+ w chamber .420H2 as the electron donor was also used to test the ability of ANME2c-FsrII-6D to reduce thiosulfate and hydroxylamine, and here, these electron acceptors were used at a final concentration of 500 μM in place of sulfite and nitrite. Similarly, the ability of the enzyme to use NADH and NADPH in place of F420H2 as electron donors was examined with sulfite and nitrite as electron acceptors, and in each case, the concentration of the reduced coenzyme was 50 μM and the progress of the reaction was followed at 340 nm. For the kinetic studies, the concentrations of the relevant substrates were varied. Coenzyme F420 was purified from Methanothermobacter marburgensis 2(SO4)2, was used as the standard.The iron and acid-labile sulfur contents of ANME2c-FsrII-6D were estimated via bathophenanthroline and methylene blue methods, respectively –81. In e–2 and H2 (96:4 [vol/vol]), and these employed a micro-gas diffusion cell made up of a 5.6-mL, 75- by 10-mm borosilicate glass Kimax tube  with an insert of a 30- by 3.5-mm capillary tube   and brought outside the anaerobic chamber, and 100 μL of 0.1% (wt/vol) N,N-dimethyl-p-phenylenediamine in 20% (vol/vol) H2SO4 was added to it with a syringe. Then, the rubber stopper of the tube was removed, and 50 μL of 1% (wt/vol) FeCl3 in 4% (vol/vol) H2SO4 was added to the assay mixture. After incubation under air at 25°C for 15 min, the absorbance of the resultant solution was read at 670 nm. Sodium sulfide was used as the standard. From the values of Fe and sulfide (moles per microgram of protein) derived from the above-described assays, and considering that the theoretical subunit mass of the protein is 69.10 kDa, the values for the Fe and acid-labile sulfur contents of ANME2c-FsrII-6D were calculated.For the estimation of the acid-labile sulfur content, the first few steps occurred inside an anaerobic chamber containing a mixture of Nlle, NJ) , 82. TheThe ammonia produced in an enzymatic reaction mixture was captured and assayed following the above-described protocol for the determination of acid-labile sulfur content, except that for the ammonia assay, the capillary was filled with 1 M HCl, the release of ammonia gas was initiated by the addition of 1 M NaOH to the reaction mixture that was placed in the annular space, and the ammonia concentration in the solution retrieved from the capillary was estimated via a glutamate dehydrogenase-based assay employing a kit .Protein concentration was estimated via Bradford assay  using thv = Vmax [S]/(mK + [S])  by using Solver in Microsoft Excel (v = Vmax [S]/{mK (1+[I]/iK) + [S]} (where [I] is inhibitor concentration and iK is the inhibition constant), using an R statistical package . The predicted structures were visualized using PyMOL . Docking of prosthetic groups into the modeled 3D structures of MjFsrI and ANME2c-FsrII-6D were performed as a structural alignment by employing the align command line in PyMOL; for the N-terminal half (Fsr-N), the alignment was performed with the Methanothermobacter marburgensis F420-reducing [NiFe]-hydrogenase subunit B , and for the C-terminal half (Fsr-C), it was performed with the Archaeoglobus fulgidus dissimilatory sulfite reductase subunit A .Multiple-sequence alignment of protein sequences was performed using MUSCLE  with def setting  run on t setting  (https:/


SAUNA  mice are commonly used in HFpEF pre-clinical studies and demonstrate cardiac, lung, kidney, and white adipose tissue impairments. However, the SkM  has not been described in this preclinical HFpEF model. We sought to characterize the soleus skeletal muscle in the HFpEF SAUNA mice and investigate its translational potential.Skeletal muscle (SkM) phenotypic switching is associated with exercise intolerance in heart failure with preserved ejection fraction (HFpEF). Patients with HFpEF have decreased type-1 oxidative fibers and mitochondrial dysfunction, indicative of impaired oxidative capacity. The d-aldosterone or saline (Sham) infusion by osmotic pump implantation, and 1% NaCl drinking water was given for 4 weeks. Mice were euthanized, and the oxidative-predominant soleus muscle was collected. We examined fiber composition, fiber cross-sectional area, capillary density, and fibrosis. Molecular analyses were also performed. To investigate the clinical relevance of this model, the oxidative-predominant, vastus lateralis muscle from patients with HFpEF was biopsied and examined for molecular changes in mitochondrial oxidative phosphorylation, vasculature, fibrosis, and inflammation.HFpEF was induced in mice by uninephrectomy, soleus muscle of HFpEF mice compared to Sham. Expression of targets of interest such as a reduction in mitochondrial oxidative-phosphorylation genes, increased VEGF-α and an elevated inflammatory response was also seen. The histological and molecular changes in HFpEF mice are consistent and comparable with changes seen in the oxidative-predominant SkM of patients with HFpEF.Histological analyses demonstrated a reduction in the abundance of oxidative fibers, type-2A fiber atrophy, decreased capillary density, and increased fibrotic area in the SAUNA model recapitulates the SkM phenotypic switching seen in HFpEF patients. This model is suitable and relevant to study SkM phenotypic switching in HFpEF.The HFpEF  HFpEF aherapies .Exercise intolerance and limitations in aerobic capacity are central features of HFpEF , 9. PrioMYH) 7 and are enriched with mitochondria primarily utilizing oxidative metabolism to generate ATP (MYH1) and type-2B glycolytic fibers (MYH4), have less mitochondrial content and predominantly use glycolysis for metabolism. Finally, the type-2A oxidative fibers are also considered “fast twitch” (MYH2), but they utilize oxidative metabolism and have intermediate contractile properties between type-1 and type-2X/type-2B. Human SkM has three major fiber types , while in mouse SkM, there are four major types   oxidative fibers express myosin heavy chain  , 34. Botxidative –36  mouse model is characterized by exercise intolerance and pulmonary congestion in addition to cardiac  (soleus muscle) of this model has not been characterized. Therefore, we sought to investigate the soleus muscle phenotype in HFpEF mice and establish the clinical relevance of these findings in human HFpEF by utilizing SkM biopsies from the oxidative-predominant vastus lateralis muscle and biomarkers obtained from patients with HFpEF.We, and others, previously demonstrated that the HFpEF ed LVEF) –40 and eed LVEF) , 41–44. All data that support the findings of this study, including methods and study materials, are available from the corresponding authors upon reasonable request.An expanded Materials and Methods section is available in the vastus lateralis muscle biopsies and human serum samples, respectively. All subjects provided written informed consent prior to entry into the study.The Institutional Animal Care and Use Committee at Boston University School of Medicine approved all procedures related to the handling and surgery of the mice. The University of Pennsylvania and Boston University Medical Campus Institutional Review Boards approved the human studies, including human vastus lateralis muscle was performed in HFpEF patients and control subjects without a history of HFpEF. The HFpEF patients had New York Heart Association (NYHA) class II/III symptoms, an LVEF ≥ 50%, and were on stable medical therapy for at least one month. See inclusion and exclusion criteria for the human HFpEF patients in the Using the suction-modified Bergstrom technique , percutaSAUNA model. Mice were anesthetized with 80 mg/kg ketamine and 8 mg/kg xylazine intraperitoneally. All mice underwent uninephrectomy and then received either a continuous infusion of 0.9% normal saline (Sham) or d-aldosterone  for 4-weeks via osmotic minipumps . All mice were also given 1.0% sodium chloride drinking water. At 4-weeks, mice were sacrificed and the soleus muscle  was colHeart rate (HR), systolic and diastolic blood pressures (BP) of HFpEF mice and Sham were measured at the end of the 4-weeks using a non-invasive tail-cuff BP-2000 blood pressure analyzer (Visitech Systems).2.To examine the fiber composition, immunofluorescence analysis of myosin heavy chain (MHC) isoforms was performed by probing the mice muscle sections with primary antibodies against MHCI , MHCIIa , MHCIIx (type-2X fast twitch/glycolytic fiber) and dystrophin (SkM-specific marker). Unstained fibers were identified as type-2B fast twitch/glycolytic fiber and fibers co-expressing MHCI and MHCIIa were identified as type-1/2A hybrid fiber.  in 1.3% saturated picric acid , and followed by 0.1% Fast Green/0.04% Sirius red  in 1.3% saturated picric acid . The fibsoleus muscle and human vastus lateralis muscle according to manufacturer’s instructions. After reverse transcription, quantitative polymerase chain reaction (qPCR) was performed using a ViiA7 PCR system (Applied Biosystem). Details of mouse-specific and human-specific primers are provided in the Total RNA was extracted from mice soleus muscle in mice and human vastus lateralis muscle using ice-cold RIPA buffer, subjected to sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE), and transferred onto a polyvinylidene difluoride (PVDF) membrane prior incubation with specific antibodies. Details of primary and secondary antibodies used are provided in the Proteins were extracted from Circulating levels of total vascular endothelial growth factor-alpha (VEGF-α) were measured from serum samples from a second cohort of chronic ambulatory patients from the Boston Medical Center (BMC) HFpEF cohort and control subjects. See inclusion and exclusion criteria in the t test or the Mann-Whitney U test, as dictated by the distribution. Data was expressed as mean ± standard error of the mean (SEM) for mice, mean ± standard deviation (SD) or median (interquartile range) for humans. A P-value < 0.05 was considered as statistically significant. For details of the power calculations for mice and human HFpEF cohorts, see the All statistical analyses were performed using GraphPad Prism software. Normal distribution of data was verified by D’Agostino & Pearson omnibus normality test. Differences between the two groups were tested by unpaired SAUNA mice demonstrated moderate hypertension with an elevated systolic BP  and increased diastolic BP  vs. Sham. HFpEF mice had increased lung congestion as quantified by an increased wet-to-dry lung ratio . Cardiac hypertrophy, measured as an increased heart weight-to-tibia length ratio, was seen in HFpEF mice vs. Sham  and gastrocnemius muscle  vs. Sham; Notably, HFpEF mice showed calf muscle atrophy characterized by a significant reduction in the weight of both soleus muscle in HFpEF mice showed a significant reduction in the abundance of the oxidative-type fibers  were resoleus muscle in HFpEF mice as compared to Sham (vastus lateralis muscle was seen (soleus and gastrocnemius muscle of HFpEF mice demonstrated atrophy, which is also consistent with human findings reported by Haykowsky et al. (In addition, the abundance of type-2B glycolytic fibers was increased 3.4-fold in the  to Sham . Althougwas seen . Type-2Xwas seen , 56. In y et al. . In thatin vivo and in vitro overexpression of HIF-1α gene in electrically-stimulated SkM of rats for 14 days and in C2C12 myotubes caused MHC isoforms changes and a switch from a slow-to-fast fiber type, suggesting that HIF-1α or a lack of oxygen itself may trigger muscle changes. Moreover, HIF-1 has been shown to impair mitochondrial biogenesis and cellular respiration in other models (Impaired arterial delivery of blood to exercising muscle has been demonstrated in HFpEF patients, with decreased vasodilatory reserve and reduced blood flow responses to isolated muscle during exercise –60. We sr models . This wasoleus muscle of HFpEF mice. KDR is predominantly expressed in the vascular endothelial cells and mediates signaling to promote angiogenesis (both human SkM biopsies and serum samples from HFpEF patients. These results are consistent with HFpEF mice and the PROMIS-HFpEF human study where elevated circulating VEGF-α levels were seen in a large cohort of chronic HFpEF patients (In addition to impaired vasodilation discussed above, a reduction in capillary density is seen with SkM phenotypic switching in HFpEF patients . In the ogenesis , 64. Theogenesis ] or KDR patients . Althougpatients , VEGF-α patients . Thus, cvastus lateralis muscle from chronic, stable HFrEF patients (2 (A common theme to the many comorbidities seen in HFpEF is also inflammation , 67, 68.patients , 70 and ients (2 . The latients (2 , 73.Col1a1 and Ctgf gene expression. Although, no difference in these genes were seen in the human HFpEF SkM biopsy samples, FSP-1 protein expression, an indicator of the myofibroblast population, was increased in the SkM of both HFpEF mice and HFpEF patients. While the origin and role of myofibroblasts in SkM is unknown, studies show that myofibroblasts differentiate from fibro/adipogenic progenitors (SkM fibrosis is observed in aging , 75 and genitors , 48. Addgenitors , suggestFinally, it has also been suggested that central mechanisms may play a pathophysiological role in SkM dysfunction in HF. For example, HF-related myopathy has been associated with increased activity of the ergoreflex which contributes to increased dyspnea with exertion, tachycardia, and the sensation of breathlessness via autonomic pathway , 79. Intsoleus muscle was studied in HFpEF mice as its oxidative nature is comparable to the oxidative-predominant human vastus lateralis muscle (Ctgf were seen in the soleus muscle of HFpEF mice, however, we did not observe changes in capillary density (soleus muscle was studied. It is also possible that this difference may also be reflective of the disease stage. The human HFpEF patients in our study were a stable, ambulatory cohort where > 80% of patients were NYHA Class II (SAUNA mice in this study represent more advanced HF disease as they demonstrate lung congestion, decrease exercise capacity, and elevated circulating natriuretic peptides (In our study, we investigated the oxidative-predominant SkM and thus s muscle . Additio density  nor chanClass II . Our cohClass II . In contpeptides , 41–44, peptides . Finallypeptides  with regIn conclusion, SkM phenotypic switching is seen in mice with HFpEF and is comparable to the SkM phenotype seen in patients with HFpEF . Our finThe original contributions presented in this study are included in the article/The studies involving human participants were reviewed and approved by the University of Pennsylvania and Boston University Medical Campus Institutional Review Boards approved the human studies. The patients/participants provided their written informed consent to participate in this study. The animal study was reviewed and approved by the Institutional Animal Care and Use Committee at Boston University Medical Campus.vastus lateralis muscle biopsy samples, and relevant clinical data. FS provided blood samples and relevant clinical data on HFpEF patients. MV-M and ES performed the surgeries. LW performed the physiological measurement. ES and LW performed the molecular analysis. All authors contributed to manuscript revision, read, and approved the submitted version.ES, MV-M, and FS conceptualized the study, interpreted the data, wrote the first draft of the manuscript. PZ and JC provided human


Roseomonas mucosa, generated significant clinical improvement in AD lasting beyond active treatment via lipid-mediated modulation of tumor necrosis factor (TNF) signaling. To assess the potential preclinical benefit of R. mucosa in psoriasis we assessed for direct effects on surface TNF signaling in cell cultures and identified direct effects on the TNF axis. We also identified preclinical efficacy of R. mucosa treatment in the imiquimod mouse model of psoriasis. Finally, we expanded our previous environmental assessment for psoriasis to include more traditional markers of air quality and found a strong association between disease rates and ambient carbon monoxide (CO), nitrogen dioxide (NO2), and particulate matter (PM). At the current stage this work is speculative but does support consideration of further preclinical models and/or clinical assessments to evaluate any potential for therapeutic benefit through microbial manipulation and/or environmental mitigation.The cutaneous microbiome is increasingly recognized as a contributor to skin diseases like atopic dermatitis (AD) and psoriasis. Although traditionally AD and psoriasis have been viewed as having opposing immunologic findings, recent evidence suggests an overlap in ceramide-family lipid production in the protection against symptoms. We recently identified that specific environmental pollutants may drive dysbiosis through direct suppression of ceramide-family lipids produced by health-associated skin bacteria in atopic dermatitis (AD). We further demonstrated that one such bacteria,  Although the literature is less robust for the skin microbiome in psoriasis as it is for related gut dysbiosis or dysbiosis in diseases like atopic dermatitis (AD), both microbial signatures and therapeutic targeting have been described –3. We rereatment , 5. Thesreatment . For AD,reatment .R. mucosa treatment could benefit psoriasis through impacts on TNFR2 signaling and/or production of therapeutic lipids.However, unlike psoriasis , 8, TNF in vitro and models of psoriasis in vivo. Furthermore, given the associations between industrialization and psoriasis, we aimed to perform an untargeted assessment of Environmental Protection Agency (EPA) monitored pollutants versus the rate of clinical visits for psoriasis by US zip codes. Herein we describe that R. mucosa directly influences surface TNF, TNFR2, and TNF alpha converting enzyme  in cell culture models and influenced soluble TNFR2 in patients with AD. R. mucosa improved outcomes in the imiquimod (IMQ) model of murine psoriasis, a model known to be dependent on TNFR signaling balance . The cells were allowed to settle for 1 hour. RmHV1 were added at MOI 10, and cells were incubated at 37°C and 5% CO2 for 12 hours. Cells were fixed in 4% PFA, blocked with 10% normal goat serum, and labeled with antibodies for TNFR2 , TACE/ADAM17 , or TNF  overnight at 4°C. Cells were washed and secondary antibodies  were added and incubated for 1 hour at room temperature. Cells were washed and DAPI was added as counterstain (ThermoFisher). Cells were imaged in 4 different areas of well using a Cytation5, and image analysis was performed by Gen5 software as follows: each cell was identified based on nuclear stain, and fluorescence intensity was measured for each cell in image and was divided by total number of cells to obtain the mean fluorescence intensity (MFI) value for each image.Primary Neonatal keratinocytes  were seeded at 1.5x102.2Roseomonas in Atopic Dermatitis, Phase I/II; BACTERiAD I/II). Enrollment criteria, treatment formulation, and clinical scoring were all as previously described  per manufacture instructions and as previously described .2.45 CFU of R. mucosa in 10mcL of 10% sucrose was applied daily for 3 days. Ear thickness was monitored, and ears collected for histology analysis at day 11. Ear mounting to slides and H&E staining was performed by Historserv . Images of slides were collected as previously described . All mice were age and sex matched within each experiment. Imiquimod modeling of psoriasis was performed as previously described . Briefly2.52, particulate matter of 2.5 microns or less (PM2.5), PM10, and SO2 , and TNFR2. After 12 hours of co-culture, the expression for each of these markers was significantly reduced by exposure to R. mucosa.  significantly reduced the resultant swelling  1β, IL-17, IL-23, and CXCL1 . In vitr2. A previous study reported that subacute exposure to pollutants often found in vehicle exhaust, including CO, NOx, PM2.5, PM10, and benzene, are associated with psoriasis flares (2 (We used multiple models to query for psoriasis-associated pollutants. CO was ranked most important by our penalized regression models, though it may only be a marker of vehicle exhaust, urbanicity, or often-associated pollutants like NOs flares . Select s flares  and psors flares . Althougs flares , increass flares , 25. Iso2 may be nonspecific associations with population density rather than a biologic mechanism of disease. Therefore, directed investigation will be needed to test if these reported findings are relevant in psoriasis. For example, patient registries containing both location and severity could be compared in a cross-sectional and/or longitudinal study against measured CO levels in the surrounding geography. If our findings can be validated, the clinical implications include the potential for improving outcomes in and/or prevention of psoriasis via microbial manipulation and targeted environmental mitigation. In conclusion, our findings suggest that while AD and psoriasis represent distinct pathologies, they may share a potential for future therapeutic benefit through microbial manipulation of the TNF axis and/or through environmental mitigations.Thus, similar to our clinical modelling, our assessment of psoriasis-associated toxins is limited by its current speculative stage. The literature suggests possible mechanisms for CO induced inflammation through feedback loops with the host enzyme heme oxygenase 1, which is elevated in the plaques and serum of psoriasis patients and associated with metabolic syndrome –28. AddiThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by IRB of the National Institutes of Health. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin. The animal study was reviewed and approved by Animal Care Board for National Institutes of Health.PG performed all studies involving cell culture staining and wrote the manuscript. JZ performed the assessments for pollutants associated with psoriasis. IM performed serum and murine studies and wrote the manuscript. All authors contributed to the article and approved the submitted version.