It is proposed that the addition of graphene into the base grease effectively enhanced the tribological properties and thermal conductivity.During mechanical processes, violent friction and wear between the friction contact surfaces not only causes wear to mechanical components, reducing the instrument life, but also causes friction heat, reducing the working efficiency of machines during operation. The addition of graphene-reinforced grease to the mechanical friction surface can effectively reduce the friction coefficient and improve the thermal conductivity. In this work, the tribological properties and thermal conductivity of base grease with graphene were investigated systematically. The tribological results showed that the grease with 2 wt% graphene had the best tribological properties among all these greases. The wear scar diameter and average friction coefficient of graphene grease with 2 wt% graphene reached 0.43 mm and 0.10 , respectively. In addition, the average friction coefficient and wear scar diameter increased proportionally with the increasing load and frequency. The thermal conductivity of the grease with 4 wt% graphene reached 0.28 W (m K) The addition of graphene-reinforced grease to the mechanical friction surface can effectively reduce the friction coefficient and accelerate heat transportation. Grease and lubricating oil with some nanoparticles can ameliorate the tribological performance more effectively compared with grease and lubricating oil without nanoparticles.3 However, there is a disadvantage in that the nanoparticles gather together and settle down, as time goes by, when the nanoparticles are dispersed in oil. Compared with oil, there is no trouble in grease. Owing to its high viscosity, grease with nanoparticles retains its effectiveness. It can effectively enhance the anti-wear properties and decrease friction on metal–metal contact surfaces. As a lubrication additive dispersed in grease, many nanoparticles are available, such as inorganic nanoparticles, carbon nanoparticles and other 2D structural materials.4–6Increasing industrialization has provided peace and well-being, but it causes much energy waste. A portion of this energy waste is produced by friction and wear in mechanical systems. Many researchers have tried to control friction and wear by lubricating rubbing surfaces; one of the most effective ways is adding grease or oil to lubricate the surface to reduce the friction coefficient, which not only effectively decreases energy waste and improves the working efficiency of the machine, but also reduces damage to the equipment. Both grease and lubricating oil are superior lubricating materials.2 hybrid orbitals, is a carbon material with a two-dimensional structure, which has attracted lots of attention owing to its exceptional electric, thermal, physical and mechanical properties.7 Graphene can be validly applied in various fields, such as solar cells, supercapacitors, sensors, thermal conductivity composites and tribological materials.8–12 When adding graphene into lubricating materials, such as grease, the lubricity of the materials can be enhanced effectively.13 Besides, compared with the base lubricating material, the thermal conductivity of the composite is also improved.14Graphene, with a carbon atom possessing sp15. The use of graphene as an additive in base grease to enhance the tribology had been widely reported. Zhang16 added graphene prepared by physical stripping method to lithium-based grease, and studied the tribological properties and critical load pressures for grease with different graphene contents (0–2 wt%). Wu17 studied a composite nanomaterial composed of a bubble structure composed of molybdenum disulphide/redox graphene (MoS2/RGO) and found that adding it to grease greatly improved its wear resistance. Singh18 compared the tribological and rheological properties of grease with different nanoparticles added . Wang19 added graphene–copper nanoparticles (GN/Cu) prepared by reduction to grease, then studied the tribological properties of the GN/Cu grease. Kamel20 studied the effects of different concentrations of graphene on the tribological properties of calcium-based greases.As an additive, graphene can validly improve the anti-wear and reduce the friction lubrication performance of grease, owing to its favourable mechanical properties and extremely smooth atomic surface. As a result of graphene's extremely small particle size and large surface area, the oxidation resistance of grease with graphene is greatly enhanced21–23 The relevant interpretation and wear mechanisms of graphene have been studied and the relevant published work can be used for Graphene has been reported to improve the tribological properties and thermal conductivity of grease or composites in many relevant recently published studies. Actually, many researchers have found that graphene has excellent tribological properties and thermal conduction performance and they are now paying more attention to single fields, like tribological properties or thermal conductivity. However, these researchers have not combined the tribological properties with the thermal conductivity of grease.via the four-ball test. We also investigated the load-carrying and frequency-carrying capacity of graphene-reinforced grease to complete the tribological properties. The critical value of oil film rupture and the complete failure of oil were also studied carefully. At the same time, the thermal transportation properties of graphene-reinforced grease were studied and verified by infrared thermography.In this work, the graphene dispersion in grease could enhance the tribological properties and thermal transportation. Graphene could be completely dispersed in grease owing to the “shear-thinning” phenomena. The friction and wear resistance were measured Graphene was purchased from Ningbo Morsh Tech. Co., Ltd. (China). The base grease was purchased from Shaoxing G&N Lubricate Materials Co., Ltd. (China).Each graphene grease sample was prepared by adding graphene into the base grease at 25 °C and then mixing using a mixing machine (Flacktek SpeedMixer DAC 150 FVZ-K) at 3500 rpm for 15 min. 490 g of base grease and 10 g of graphene was divided into five parts . Owing to the “shear-thinning” phenomena, the viscosity of the base grease decreased a lot at the high shear rate (3500 rpm) and the graphene could be dispersed in grease effectively at this frequency. Moreover, the high shear stress makes it impossible for the graphene to agglomerate together. Thus, we obtained graphene-reinforced grease with superior even dispersion.via the four-ball test , as shown in Fig. S1.via a series of calculations. The infrared thermal images were captured with a Ti400 infrared camera .The graphene was investigated by a scanning electron microscope  operating at an accelerating voltage of 10 kV. X-ray photoelectron spectroscopy (XPS) was carried out with an AXIS Ultra DLD spectrometer . Atomic force micrographs (AFM) were obtained with a Dimension 3000 scanning probe microscope  in tapping mode. The Raman spectra were recorded using a Reflex Raman system  with a laser wavelength of 532 nm. The viscosity of the graphene-reinforced grease with different graphene concentrations was determined using a rotary rheometer  at 25 °C. The friction and anti-wear properties were performed  PBM data was replaced with SVG by xgml2pxml:<glyph-data id="z.dbd" format="PBM" resolution="300" x-size="8" y-size="10" xml:space="preserve">00000000000000000000000000000000111111110000000011111111000000000000000000000000</glyph-data><svg xmlns="http://www.w3.org/2000/svg" version="1.0" width="13.200000pt" height="16.000000pt" viewBox="0 0 13.200000 16.000000" preserveAspectRatio="xMidYMid meet"><metadata>Created by potrace 1.16, written by Peter Selinger 2001-2019</metadata><g transform="translate scale" fill="currentColor" stroke="none"><path d="M0 440 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z M0 280 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z"/></g></svg>C, C–C, and C–O, respectively. AFM is a valid technique for judging the thickness of graphene. The AFM image and the corresponding height profile of the graphene, from which the thickness was found to be approximately 6.5 nm, are shown in 28 The D band at ∼1330 cm−1 and the G band at ∼1580 cm−1 are composed of sp3 hybridized carbon and sp2 bonded carbon atoms, respectively.29The morphology and structure of the graphene were characterized by using SEM/AFM and XPS and Raman spectra, respectively, as shown in ID/IG) demonstrates the quality of the graphene nanosheets.30In addition, the ratio of the D band and the G band , the viscosity and shear stress of graphene grease changed a lot at 0.1 r s−1. Meanwhile, the viscosity increased slowly at the shear rate of 60 r s−1, which was the shear rate we used for blending the graphene grease we prepared. There was no obvious change at the rate of 1000 r s−1, as shown in As a non-Newtonian fluid, the viscosity of the grease will be weakened at higher shear rates, which is called “shear-thinning” phenomena.33 The variation of the friction coefficient as a function of time when lubricated by base grease with different levels of graphene ranging from 0 to 4 wt% is shown in The tribological properties of the base grease and the graphene-reinforced grease, such as friction coefficient and wear, are shown in 34 we could get the friction state. For the four-ball test with all of the balls the same size, the Hertzian formula could be simplified as follows:N is the positive pressure on the contact friction surface under the four-ball test, μ is the friction factor of the ball friction surface, R is the radius of the balls, and E is 2.085 × 105. The Hertzian contact diameter was calculated to be 0.30 mm under 400 N load, which is 31% lower than that of 2 wt% graphene grease, which means the lubrication state belongs to boundary lubrication. The average diameters of the wear scars were measured by the four-ball test from four repeated experiments, as shown in By calculating the Hertzian contact diameters,35 of graphene-reinforced grease was investigated as shown in The maximum non-seizure load (PB), related to the protective film on the friction surface,36–38 It was found that the thermal conductivity of the graphene-reinforced greases increased with increasing graphene content. The thermal conductivity of the base grease was 0.18 W (m K)−1 at 25 °C. When the graphene concentration was increased to 4 wt%, the thermal conductivity increased to 0.28 W (m K)−1, as shown in Many researchers have found that the thermal conductivity of composites can be improved with the addition of graphene into the composites.−1 at 25 °C, which is an increase of 55.6% compared with the base grease. It was proposed that the addition of graphene into base grease could not only enhance the tribological properties but also increase the thermal transportation performance.In summary, the tribological behavior and thermal conductivity of base grease reinforced with graphene were studied. The lubrication and anti-wear properties of the base grease were ameliorated with the addition of graphene, indicating that these graphene-reinforced greases show better tribological properties than those of the base grease. Compared with the base grease, the friction coefficient and anti-wear properties of the graphene-reinforced grease were reduced by 15.3% and 14% under 400 N load and 20 Hz frequency, respectively. We investigated the effects of load and frequency on the lubrication and anti-wear properties of the greases with different concentrations of graphene and found that the friction coefficient increased with increasing load but not with increasing frequency. Meanwhile, the thermal conductivity of the graphene-reinforced grease increased with increasing graphene concentration. The thermal conductivity of the grease with 4 wt% graphene was 0.28 W (m K)There are no conflicts to declare.RA-009-C9RA09201C-s001


At the neuromuscular junction (NMJ), motor neurons and myocytes maintain a bidirectional communication that guarantees adequate functionality. Thus, motor neurons’ firing pattern, which is influenced by retrograde muscle-derived neurotrophic factors, modulates myocyte contractibility. Myocytes can be fast-twitch fibers and become easily fatigued or slow-twitch fibers and resistant to fatigue. Extraocular muscles (EOM) show mixed properties that guarantee fast contraction speed and resistance to fatigue and the degeneration caused by Amyotrophic lateral sclerosis (ALS) disease. The TrkB signaling is an activity-dependent pathway implicated in the NMJ well-functioning. Therefore, it could mediate the differences between fast and slow myocytes’ resistance to fatigue. The present study elucidates a specific protein expression profile concerning the TrkB signaling that correlates with higher resistance to fatigue and better neuroprotective capacity through time. The results unveil that Extra-ocular muscles (EOM) express lower levels of NT-4 that extend TrkB signaling, differential PKC expression, and a higher abundance of phosphorylated synaptic proteins that correlate with continuous neurotransmission requirements. Furthermore, common molecular features between EOM and slow soleus muscles including higher neurotrophic consumption and classic and novel PKC isoforms balance correlate with better preservation of these two muscles in ALS. Altogether, higher resistance of Soleus and EOM to fatigue and ALS seems to be associated with specific protein levels concerning the TrkB neurotrophic signaling. Motor neurons (MNs) interact with myocytes through the neuromuscular junction (NMJ) to guarantee their adequate functionality in a feedback loop mode through the neuromotor and neurotrophic control mechanisms. In brief, MNs regulate the properties of the myocytes they innervate through their firing activity pattern, determined by their intrinsic properties and by the inputs that they receive. In their turn, myocytes influence MNs through the retrograde activity-dependent secretion of neurotrophic factors .One MN innervates several myocytes to form a motor unit (MU). At their time, several MU innervate all the myocytes in one muscle to generate a complete functional contraction. Consequently, fast and slow skeletal muscles are differently innervated and combine their intrinsic characteristics to organize the functionality of a whole limb. MNs are named fast-twitch fatigable (FF), fast-twitch fatigue-resistant (FR), and slow-twitch fatigue-resistant (S) in accordance with the properties of the myocytes they innervate . In theiAmong the muscles used in the present study, Extensor Digitorum Longus (EDL) and Tibialis Anterior (TA) are fast extensor and flexor muscles, respectively, mainly composed of fast-twitching fibers. On the other hand, Soleus (SOL) is composed of slow-twitching fibers . Type I The Brain-derived neurotrophic factor (BDNF)/Tropomyosin receptor kinase B (TrkB) signaling is a retrograde neurotrophic signaling tightly related to NMJ stability and neurotransmission. In brief, nerve-induced muscle contraction regulates the pathway to retrogradely modulate the activity of presynaptic protein kinases PKC and PKA on SNARE-SM targets , 2019, din vivo in SOD1-G93A mice  were kept in the animal facility under standard conditions: constant temperature (22 ± 2°C), relative humidity (50 ± 10%), a 12-h light/dark schedule and unrestricted access to food and water. Experiments were performed under the approval of the Ethics Committee of Animal Experimentation of the Universitat Rovira i Virgili, in accordance with the European Union Directive 2010/63/EU guidelines for the humane treatment of laboratory animals. Muscles were obtained from six animals from the colony (n = 6) to be used as biological replicates.Adult male Sprague Dawley rats ]. Insoluble materials were removed with two centrifugations at 4°C: 1,000 g for 10 min and 15,000 g for 20 min. The final supernatant contained the whole-cell fraction lysate. Protein concentrations were determined by DC protein assay .Animals were euthanized and immediately afterward Extensor Digitorum Longs (EDL), Tibialis Anterior (TA), Soleus (SOL), and the four rectus EOM muscles were dissected from their insertions and frozen in liquid nitrogen. Homogenization was performed with an overhead stirrer  in ice-cold lysis buffer [in mM: NaCl 150, Tris–HCl 50 (pH 7.4), EDTA 1, NaF 50, PMSF 1, NaThe western blotting procedure was performed as previously described . Proteinpost-hoc test . The criterion for statistical significance was: *p < 0.05, **p < 0.01, and ***p < 0.001.All values are represented as mean ± standard deviation (SEM) within each muscle. Also, each circle represents the value of one animal, normalized in relation to EDL muscle, whose value was transformed to 1, to visualize proportional data distribution. The statistical significance of the differences between the experimental groups was evaluated under a non-parametric Kruskal–Wallis test followed by Dunn’s NTR, (ii) the coupled serine-threonine kinases C (PKC isoforms) and their priming kinase phosphoinositide-dependent kinase 1 (PDK1) and the different subunits of the cAMP-dependent kinase (PKA), and (iii) PKC and PKA targets related with ACh release (Munc18-1 and SNAP-25).To determine the expression of the presynaptic TrkB neurotrophic signaling pathway that regulates synaptic vesicles exocytosis at the NMJ, we analyzed the entire TrkB pathway at the synapses of EDL, TA, SOL, and EOM muscles in its three molecular levels: (i) the neurotrophic factors BDNF and NT-4 and their receptors TrkB full length (TrkB.FL) and truncated (TrkB.T) and p75NTR expression is proportional to muscular fatigue resistance, being less expressed in EDL muscles and upregulated in SOL and EOM. On the other hand, TrkB is more expressed and phosphorylated in the fast, fatigable EDL and TA than in the fatigue resistant muscles SOL and EOM in healthy young adult Sprague Dawley rats. However, results reveal that TrkB.FL is proportionally more phosphorylated in SOL and EOM muscles, as the proportion between phosphorylated and total TrkB.FL detected (pTrkB.FL/TrkB.FL ratio) increases in these muscles. Furthermore, the proportion between full and truncated TrkB detected (TrkB.FL/TrkB.T) ratio is kept through all the studied muscles.Understanding neurotrophic factor consumption requires the analysis of the receptors that trigger their signaling. Thus, Next, we studied three relevant PKC isoforms involved in neurotransmission at the NMJ that are activated following the downstream TrkB signaling , 2019. AOn the contrary, novel nPKCε abundance is higher in SOL and EOM than in EDL and TA but it is less phosphorylated . FinallySince PKC and PKA are closely related to regulating ACh release at the NMJ , we alsoBoth PKC and PKA phosphorylate targets are keys in the exocytosis of ACh synaptic vesicles. Munc18-1 is an essential, neuron-specific protein involved in neurotransmitter release , specifiSNAP-25 is a component of the SNARE core complex that is essential for functional neurotransmission . Thus, iSkeletal muscles differ from one specie to the other, with activity patterns, disease conditions, and aging, among others and despite sharing functional characteristics. In consequence, skeletal muscle tissue is very plastic and its small molecular variations can induce significative differences despite being in the same generic group . Until now, it was unknown whether the TrkB signaling is differently expressed in fast and slow limb muscles and in EOM, which have specific functional characteristics, resistance to fatigue, and different vulnerability to ALS neurodegenerative disease . Here, wNT-4 is an activity-dependent neurotrophic factor that mediates NMJ growth and remodeling . PreviouConcerning BDNF, results show a differential consumption of mBDNF among the different studied muscles, which is higher in fatigue-resistant muscles, where TrkB isoforms expression is lower. TrkB can follow the degradative pathway after binding to its ligand  and actiThus, fatigue-resistant muscles present a sustained activity of the TrkB.FL signaling pathway correlated with their stability through disease and supported by a higher TrkB.FL phosphorylation ratio in SOL and EOM. Thus, the higher TrkB.FL phosphorylation ratio observed in SOL and EOM seems to be related to the maintenance of neurotransmission. Indeed, several studies related TrkB signaling with mitochondrial function maintenance and promotion . TherefoNTR (NTR (NTR-mediated plasticity mechanisms to refine and optimize mature adult NMJ connections (NTR–/– mice showed impaired structural complexity correlated with altered synaptic function and increased susceptibility to fatigue (NTR expression in fast plantaris muscles of ALS mice (NTR in the limb muscles of these animals (NTR levels are proportional to fatigue resistance. Indeed, p75NTR is upregulated in EOM myogenic progenitor cells, in relation to higher proliferative and fusion rates and better regenerative properties (NTR seems to be related to selective muscle resistance.On the other hand, the neurotrophic factor receptor p75NTR  is widelNTR , where iNTR . ParticuNTR (NTR . Similarnections . Indeed, fatigue . These rALS mice  while itALS mice . Further animals . In accooperties . Thus, pAs a result of TrkB.FL-mediated PLCγ1 activation, mature PKCs are anchored to the membrane . PKC matOnce PKCs are phosphorylated by PDK1, they can sense intracellular second messengers and bind to the membrane for substrate phosphorylation . AfterwaOn the other hand, novel nPKC isoforms are more active in slow fibers  in correcAMP-dependent kinase regulatory isoforms have a specific tissue and subcellular distribution . RIα andPKCs phosphorylate several proteins that are essential for the exocytotic mechanism. On the one hand, Munc18-1, which favors membrane fusion when associated with the SNARE complex , dependsThe present results show that Munc18-1 and pMunc18-1 levels are higher in EOM than in EDL, TA, and SOL. However, despite PKC activity differences, the Munc18-1 phosphorylation ratio is maintained through the four muscles. In SOD1-G93A mice, the amount of Munc18-1 able to interact with the SNARE complex is reduced due to over-phosphorylation  and in rOn the other hand, SNAP-25, a member of the exocytotic SNARE complex, is phosphorylated by PKC at S187 in response to synaptic activity and elevated intracellular calcium levels, which induces its translocation to the membrane. At the NMJ, nPKCε mediates this process especially during synaptic activity, while muscle contraction exerts negative feedback to return S187 phosphorylation to basal levels . In partOn the other hand, SNAP-25 T138 is phosphorylated by PKA . This phThe TrkB signaling is crucial for the correct bidirectional communication between the MN and the muscle fibers that they innervate , 2019. TThe raw data supporting the conclusions of this article will be made available by the authors, upon reasonable request.The animal study was reviewed and approved by Ethics Committee of Animal Experimentation of the Universitat Rovira i Virgili.LJ-B: data collection and quantitative analysis and statistics. VC-M: graphical abstract design. LJ-B, JT, ML, and NG: literature search. LJ-B, JT, and ML: manuscript preparation. JT and ML: conception and design. All authors data interpretation, read, and approved the final manuscript.


Lactococcus lactis spp. cremoris has been associated with promising immunomodulatory results in preclinical trials. The aim of this study was to investigate the pharmacodynamic (PD) effects of three monoclonal microbial formulations of L. lactis spp. cremoris (EDP1066) on the immune response to keyhole limpet hemocyanin (KLH). Potential effects on the gut microbiota were also investigated.https://trialsearch.who.int). Eighty-one healthy subjects  were randomized to 28 days of enteric-coated capsules at five doses (n = 13) , freeze-dried powder at one dose (n = 12)  or five doses (n = 12), minitablets at one dose (n = 12) or five doses (n = 12), or placebo (n = 20) prior to KLH immunization. Antibody responses and circulating regulatory T cells (Tregs) were measured after KLH immunization, and skin responses were evaluated after a KLH rechallenge by laser speckle contrast imaging and multispectral imaging. Ex vivo lymphocyte (phytohemagglutinin) and monocyte (lipopolysaccharide (LPS)) cytokine release assays were explored in the minitablet-treated groups only. The prevalence of L. lactis spp. cremoris in the gastrointestinal tract and the impact on the fecal microbiota were assessed by qPCR and 16S rRNA sequencing, respectively.The trial was registered on Netherlands Trial Register  from placebo: −44.2%, 95% confidence interval (CI) −65.3% to −10.3%), interleukin (IL)-1β , and IL-6 . The fecal presence of L. lactis spp. cremoris increased during treatment by all EDP1066 formulations and normalized 5 days after the last dose. Microbiome α-diversity did not change by the treatments compared to placebo.Repeated-measures analysis of covariances revealed no significant treatment effects on the antibody responses to KLH, number of Tregs, or KLH skin rechallenge outcomes. L. lactis spp. cremoris in minitablet formulation impacted ex vivo whole blood LPS cytokine response. The clinical impact of these effects awaits further investigations.The EDP1066 formulations did not affect the immune response to KLH immunization in healthy individuals. However, exposure to trialsearch.who.int, trial ID NL7519. Over the past decades, evidence has emerged for an interplay between the systemic immune system and the intestinal microbiome –3. The eLactococcus lactis spp. cremoris identified from powders used in dairy product manufacturing. Preclinical data of EDP1066 on both in vitro immune cell cultures and in vivo murine immune challenge and disease models show promising results; however, these data are not currently available in the public domain. In separate independent research, L. lactis spp. cremoris restored T-cell impairment in aged mice , providing essential information on proof-of-pharmacology during early-phase drug development (Keyhole limpet hemocyanin (KLH) is a metalloprotein derived from the hemolymph of the marine mollusk, elopment –34. KLH elopment  and sincelopment , 36–41.ex vivo immunomodulatory activity of EDP1066 was explored by whole blood stimulation with the Toll-like receptor 4 ligand lipopolysaccharide (LPS) and phytohemagglutinin (PHA) for monocyte and lymphocyte stimulation, respectively. Finally, we aimed at assessing the impact of EDP1066 on the fecal microbiota, next to routine safety and tolerability assessments.The primary aim of the present study was to characterize the pharmacodynamic (PD) effects of EDP1066 on the systemic immune response to an intramuscular immunization with KLH and secondary to evaluate the effects on a subsequent KLH skin rechallenge. Because the exposure sites within the GI tract for ingested microbes may depend on the formulation and therefore be important for the immunomodulatory effect , we alsohttps://trialsearch.who.int).The independent Medical Ethics Committee “Medisch Ethische Toetsingscommissie van de Stichting Beoordeling Ethiek Biomedisch Onderzoek”  approved the study prior to any clinical study activity. All subjects provided written informed consent before participation. The trial was registered on the Netherlands Trial Register, currently available for consultation at the International Clinical Trial Registry Platform  are widely used in drug product manufacturing, and none of the excipients were expected to elicit immune system modulation.All EDP1066 and placebo formulations were manufactured and provided by Evelo Biosciences Inc. . The doses tested were based on the results of a separate first-in-man study . The hig11 total cells per capsule, administered orally at a dose of 10 capsules daily (5× Capsules). Two other groups received EDP1066 as free freeze-dried powder with an achlorhydria regimen administered orally at a dose of 3.0 * 1011 (1× Powder) and 1.5 * 1012 (5× Powder) total cells daily. The achlorhydria regimen consisted of omeprazole 40 mg and aluminum hydroxide/magnesium hydroxide 200/400 mg administration 3 h prior to each EDP1066 dose. Both drugs increase the gastric pH  and 10 (5× Minitablets) capsules daily. Participants were dosed once daily for 28 consecutive days. Compliance was confirmed by the supervised administration of the study treatment during the in-clinic period. Administration at home was recorded by an electronic diary. Intramuscular KLH immunization was performed in the left deltoid muscle after the completion of the third administration of EDP1066/placebo. KLH immunization was administered as 0.1 mg of Immucothel® adsorbed in 0.9 mg of aluminum hydroxide (Alhydrogel®) into 0.5 ml of NaCl 0.9% as previously described (®) and saline in 0.1 ml of NaCl 0.9% intradermally in the left and right ventral forearms, respectively, 23 days after KLH immunization. The skin challenge response was quantified prior to and 2 days after intradermal KLH administration. These are similar intervals between assessments as in our previous studies, which also detail the methodology , Leiden, the Netherlands, based on the principles of the Declaration of Helsinki. An overview of the study design is shown in stric pH –46 and wescribed . All subhodology , 47, 48.g for 10 min with a temperature of 2°C–8°C, and the serum was aliquoted. The aliquots were stored at a temperature of −40°C until shipment and analysis. Samples were assessed by quantitative enzyme-linked immunosorbent assay (ELISA) for anti-KLH IgM and IgG as previously described ) ) . For the®, Miravex, Dublin, Ireland) as previously described  , and erythema quantification was performed with multispectral imaging  or 2 ng/ml of LPS  for 24 h at 37°C, 5% CO2. After 24 h, the supernatant was collected, and cytokines were measured using qualified ELISA-based assays by Ardena Bioanalytical Laboratory. Interferon gamma (IFN-γ) and IL-2 were measured in the PHA-stimulated samples; tumor necrosis factor (TNF), IFN-γ, IL-1β, IL-6, and IL-8 were measured in the LPS-stimulated samples.The percentage of circulating Tregs was evaluated by flow cytometry. Venous blood was collected in sodium heparin tubes by venipuncture at the time points indicated in L. lactis spp. cremoris strain. The fecal samples were analyzed using a qPCR with a lower limit of quantification of 5.0 copies/5 ng DNA. For gut microbiome analyses, extracted DNA was prepared for Illumina sequencing via PCR amplification of the variable region 4 of the bacterial 16S rRNA gene. After PCR purification using AMPure XP beads , sample-specific barcodes using Illumina Nextera XT Index kit  were appended to the PCR products during a second PCR. The PCR products were purified for a second time, and lastly, the PCR products were equimolarly pooled and sequenced on the Illumina MiSeq platform using the MiSeq v3 sequencing kit.Fecal concentrations of EDP1066 for stool persistence and prevalence and the gut microbiome were measured by Diversigen Inc.  using validated bioanalytical assay methods. In short, fecal microbial DNA was extracted based on the Zymo Research  fecal DNA extraction methodology. EDP1066-specific primers and probes had been developed to enable the detection of the Safety and tolerability were monitored by physical examination, assessment of vital signs, laboratory parameters , and ECG data from 12-lead ECGs at regular intervals. Subjects were monitored continuously for adverse events (AEs). Participants were also asked to daily complete the Bristol Stool Scale (BSS) and questions regarding defecation patterns using an electronic diary app in order to obtain insight into the participants’ stool patterns at the time of fecal sample collection.ex vivo monocyte cytokine release assays required log transformation. The general treatment effect and specific contrasts were reported as the estimated difference (ED) with a 95% confidence interval (CI) and graphically as ED with 95% CI, as least squares mean (LSM) with 95% CI, or as mean with SD. Fecal EDP1066 concentration was reported graphically as median with range. Fecal microbiome endpoints were analyzed using Python  by Diversigen Inc. Read count and relative abundance tables were calculated at the genus level and retrieved using custom Python scripts and the One Codex Python library, an in-house curated database of bacterial marker genes including 16S ribosomal RNA. The relative abundances of all microorganisms at the genus level were calculated to present the occurrence of the Lactococcus genus relative to all microbial DNA in the samples. Diversity trend analysis was performed using the Shannon diversity index. The Shannon diversity index was calculated for all samples using the One Codex Python library. Results were aggregated and plotted using custom Python scripts. To determine whether some genera were more or less abundant in placebo vs. EDP1066 treated individuals, read count tables were fed to ANCOM, a statistical framework for the analysis of microbiomes. Fecal microbiome diversity was reported graphically as median with an interquartile range.The sample size was based on previously performed power calculations on KLH challenge endpoints . In ordeThe study was conducted between February 2019 and January 2020. Ninety-five subjects were enrolled in the study of which 81 were treated Figure 1No statistically significant treatment or formulation effects were observed on the humoral KLH challenge outcomes. Observations closest to the desired treatment effect were lower anti-KLH IgG Figure 3There were no consistent EDP1066-dependent changes in the percentage of circulating Tregs over all groups, though Tregs were significantly increased in subjects treated with 5× Powder  was comparable among time points and treatment groups, albeit some changes could be observed on individual levels for a subset of the participants (data not shown). Overall microbiome diversity seemed to be slightly lower in EDP1066-treated samples; however, many of these differences probably occured due to the small sample size when calculations were performed for individual groups. When Shannon diversity indices were aggregated across all the groups, the mean Shannon diversity was very stable between time points and treatment groups  with no distinction between EDP1066 and placebo treatment (Table S1). One subject was withdrawn from further treatment after the second EDP1066 dose due to a possible hypersensitivity reaction to EDP1066 consisting of a mild burning sensation and itch of the throat lasting approximately 6 h. No abnormalities were found upon physical examination and additional vital sign measurements. Due to the mild and limited nature of the AEs, no further diagnostics were conducted. No earlier hypersensitivity AEs after EDP1066 administration had been reported. The subject also did not report any allergies to cheese or other dairy products. Allergic reactions to excipients used in the 5× Capsule formulation  have been reported before; however, these are very rare –51. Plac12 total cells, five times the expected therapeutic dose, did not result in consistent significant effects on KLH challenge responses and LPS- and PHA-driven cytokine release in whole blood cultures. We demonstrated that L. lactis spp. cremoris was detected in the fecal samples and increased during the 28-day treatment period for all EDP1066 formulations tested. However, the fecal levels returned to baseline levels 12 days after the end of treatment, indicating no prolonged persistence. Overall, EDP1066 was considered safe and well-tolerated. To the best of our knowledge, the current trial is the first to investigate the effects of orally administered L. lactis spp. cremoris in high doses on systemic immune responses and the gut microbiome.In this study, we showed that daily EDP1066 treatment in encapsulated, powdered, and minitablet formulations and daily doses up to 1.5 * 10+ T cells were significantly increased in subjects treated with 5× Powder compared to placebo, it should be noted that these percentages remained within the general range of Tregs in the CD4 population as reported in the literature (5%–10%) (in vitro human dendritic cell (DC) cultures, without significant induction of pro-inflammatory cytokines (unpublished data), and EDP1066 significantly reduced KLH- and ovalbumin-induced ear inflammation in mice and improved intestinal pathology and weight loss in an acute dextran sulfate sodium-induced colitis mouse model (unpublished data). Also in contrast to our results, other preclinical trials reported that L. lactis spp. cremoris restored T-cell impairment in aged mice  , 53. Theged mice  and thatse model . Probiotse model , 25. Mulse model –58. Anotse model . Single ex vivo in both the 1× and the 5× Minitablets groups, reaching statistical significance for IL-1β, IL-6, and TNF-α in the 1× Minitablets group, which may indicate innate immune system inhibition . Duodenal EDP1066 exposure was hypothesized to be important for the immunomodulatory effect, as immune cell subsets are found at the highest concentrations in the duodenum and jejunum, particularly the CD103+CD11b+ DCs, which are thought to play distinct roles in intestinal immune homeostasis  and as minitablets in non-coated capsules. The minitablets in non-coated capsule formulation were expected to achieve the highest concentration of relatively intact EDP1066 bacteria in the duodenum. Non-coated capsules were used to ease the intake of relatively large numbers of enteric-coated minitablets and to preserve the blinding. Based on L. lactis spp. cremoris is used by the dairy industry, it is likely that participants have developed at least some intestinal tolerance to this microbe (in vitro prediction of the release criteria of the enteric-coated capsules and minitablets may underestimate the time to release, suggesting that the true release of EDP1066 was in the distal small intestine or colon rather than in the proximal small intestine.The current human trial did not confirm previous findings from preclinical trials that oral administration of EDP1066 had immunomodulatory effects as measured on antibody response to KLH immunization or skin immune responses to KLH re-challenge. There are several potential explanations for the suboptimal translation of EDP1066 activity between mice and humans. Firstly, it was impossible to do conventional allometric scaling between mice and humans. Other than for most medicinal products, the exposure to EDP1066 was considered to remain restricted to the GI tract. This was hypothesized to be sufficient, since the mechanism of action of EDP1066 only requires local interaction with cells of the GI mucosa, driving subsequent systemic effects. Under these conditions, assumptions of traditional allometric scaling may not hold true. For this reason, relative GI mucosal surface area and stool mass are key parameters for allometric scaling. The relative GI mucosal surface area has been estimated as a function of body mass to the ¾ power . As the  microbe , possiblIn conclusion, oral EDP1066 treatment for healthy volunteers did not consistently result in significant immune modulation. Future clinical studies should build onto the insights obtained in this study and further investigate formulation versus local release and dose–effect relationships, which will ultimately be beneficial not only for EDP1066 but also for the field of therapeutic human commensals in general.The datasets for this article are not publicly available due to concerns regarding participant/patient anonymity. Requests to access the datasets should be directed to the corresponding author.The studies involving human participants were reviewed and approved by Medisch Ethische Toetsingscommissie van de Stichting Beoordeling Ethiek Biomedisch Onderzoek. The patients/participants provided their written informed consent to participate in this study.All authors wrote the manuscript. AI, DM, MM, PG, and MS designed the research. MM, PG, and MS performed the research. HG and EK analyzed the data. All authors approved the final version of the manuscript.


The prevalence of obstructive sleep apnea (OSA) has greatly increased in recent years. Recent data suggest that severe and moderate forms of OSA affect between 6 and 17% of adults in the general population. Many papers are reporting the significantly increased prevalence of OSA in patients suffering from fibrotic diseases, including idiopathic pulmonary fibrosis (IPF). Therefore, we performed a systematic review and meta-analysis regarding the dependency between IPF and OSA. Due to the lack of papers focusing on IPF among OSA patients, we focused on the prevalence of OSA among IPF patients. In the search strategy, a total of 684 abstracts were identified, 496 after the removal of duplicates. After the screening of titles and abstracts, 31 studies were qualified for further full-text analysis for eligibility criteria. The final analysis was performed on 614 IPF patients from 18 studies, which met inclusion criteria. There were 469 (76.38%) IPF patients with OSA and 145 (23.62%) without. The mean age varied from 60.9 ± 8.1 up to 70.3 ± 7.9. The obtained prevalence was 76.4 (95% CI: 72.9–79.7) and 75.7 (95% CI: 70.1–80.9) for fixed and random effects, respectively. The median prevalence of OSA among non-IPF patients for all the ethnics groups included in this study was 16,4% (IQR: 3.4%–26.8%). The study provides strong evidence for the increased prevalence of OSA in IPF patients when comparing with the general OSA prevalence. VEGF), erythropoietin, and aldolase-A [SERPINE1, TIMP1, and VEGF. Additionally, HIF1α signaling pathway was inhibited with nintedanib (tyrosine kinase receptor antagonist that suppress collagen formation and is used to treat IPF), what led to the blockage of pro-fibrotic processes in the extracellular matrix [Obstructive sleep apnea (OSA) is one of the most common sleep disorders in the world, with an estimated prevalence between 6–17% of adults in the general population in its moderate or severe forms . OSA is dolase-A ,12,13,14r matrix . At the We conducted a systematic review in accordance with Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA).Studies included in this review met the following criteria: (1) adult participants of either sex, (2) mean/median age of participants > 60 years old, (3) participants with a diagnosis of OSA based on the apnea–hypopnea index (AHI) > 5 or ≥5, (4) participants were evaluated for sleep-disordered breathing by objective method–in laboratory PSG and/or with at home PSG, and/or polygraphy (5) participants were evaluated for IPF, (6) the study was reported in English , (7) croThe age criterium for study inclusion is results from the great prevalence of IPF in older patients with a median age at presentation around 66 years .PubMed.gov (accessed on 4 October 2021)), “PubMed Central” (PMC), “Embase” (on Ovid), and “Scopus” (on Scopus.com (accessed on 4 October 2021)). Paper extraction from databases was performed on October 4th, 2021. The detailed search strategy is included in The systematic literature search was performed with the use of online databases including: “PubMed”  as well as data pertaining to source characteristics of included studies .The Freeman–Tukey transformation was employed to calculate the weighted summary proportion in both the fixed and random effects models. Moreover, we conducted tests assessing heterogeneity and the publication bias (Egger’s test and Begg’s test juxtaposed with funnel plot). All calculations were made with the usage of MedCalc Statistical Software version 19.1.2 .The final analysis was performed on 614 IPF patients from 18 studies. There were 469 (76.38%) IPF patients with OSA and 145 (23.62%) without. The mean age varied from 60.9 ± 8.1 in the study by Pihtili et al.  to 70.3 2 of 57.7% (95% CI: 28.6%–74.9%) and p-value of 0.001. As the heterogeneity was significantly observed, the random effects model was the preferred model. The publication bias was assessed using the funnel plot accompanied by both Egger’s test and Begg’s test  for the first one and Kendall’s Tau of -0.066 and p-value of 0.701 for the second one  needed to be determined by standard PSG examination or home PSG examination.OSA is one of the most common sleep respiratory diseases. Its prevalence estimation ranges from 6–17% in the general adult population [IPF is a relatively rare pulmonary condition correlated with a considerable number of comorbidities such as pulmonary hypertension, pulmonary embolism, lung cancer, gastroesophageal reflux, and cardiovascular comorbidities ,58. AnotIPF is one subtype of ILD. Even though there are a number of studies providing evidence that OSA is correlated with ILD ,63,64, wApart from the complex and not fully discovered pathogenesis of OSA in patients with IPF, these conditions are linked with each other through other common comorbidities, which makes IPF management even more challenging and complex. Pulmonary hypertension is one of the most important and severe manifestations of IPF . IPF patThe significant prevalence of OSA in IPF patients raises a question about the impact of OSA treatment on the course of IPF. Continuous positive airway pressure (CPAP) therapy is the most effective treatment for OSA. There is an ongoing discussion about the effectiveness, safety, and acceptance of CPAP therapy in IPF patients . AlthougAnother important problem with CPAP compliance in the group of IPF patients is oxygen supplementation. In the course of IPF, alveolar hypoxia progresses and eventually oxygen therapy is required . ConceivOur study concentrates on the assessment of OSA prevalence among IPF patients, although there are emerging studies providing evidence about the impact of OSA on interstitial lung injury, possibly leading to the development of IPF. The results of research regarding the correlation between OSA and lung injury markers suggest that OSA patients have elevated plasma KL-6 levels  which may suggest that alveolar wall permeability is increased in OSA . AdditioThe main formal limitation of this review is the lack of registration in any prospective registry, e.g., PROSPERO, which may raise doubts about the validity of this paper, even though we performed the literature search, data extraction, and statistical analysis as described earlier in the section methods and results. We prepared our systematic review in accordance with the PRISMA protocol and good practice for statistical analysis. Another limitation of our study is the lack of longitudinal data on IPF–OSA patients. The controls in our study include inconsistent data on OSA prevalence in the general population ethnically matched with the study population.Despite the limitation, our review is one of the first systematic approaches to establish the high prevalence of OSA in IPF patients. The results of this research may suggest that IPF–OSA patients as a separate clinical group might benefit from a more personalized diagnostic process and treatment.In conclusion, this review is a summary of numerous literature reports regarding the relationship between OSA and IPF. The study provides strong evidence for the increased prevalence of OSA in IPF patients. Yet, it is important to highlight that no precise data about the nature and reasons for this relationship were available. Further research in this field can lead to a better understanding of described correlation whereby facilitation of complex diagnostic and treatment processes.


Camsap3-cKO, which intended to eliminate CAMSAP3 in limited organs including the inner ear, olfactory bulb, and kidneys. Immunofluorescent staining of vestibular organs demonstrated that CAMSAP3 proteins were significantly reduced in Camsap3-cKO mice and that aged Camsap3-cKO mice had significantly shorter kinocilia than their wildtype littermates. Transmission electron microscopy showed that aged Camsap3-cKO mice were in fact missing that the central MT pair in kinocilia more often than their wildtype counterparts. In the examination of behavior, wildtype and Camsap3-cKO mice performed equally well on a swim assessment, right-reflex test, and evaluation of balance on a rotarod. However, Camsap3-cKO mice showed slightly altered gaits including reduced maximal rate of change of paw area and a smaller paw area in contact with the surface. Although Camsap3-cKO mice had no differences in olfaction from their wildtype counterparts, Camsap3-cKO mice did have kidney dysfunction that deteriorated their health. Thus, CAMSAP3 is important for establishing and/or maintaining the normal structure of kinocilia and kidney function but is not essential for normal olfaction. Our data supports our hypothesis that CAMSAP3 is critical for construction of the central MT pair in kinocilia, and that the central MT pair may be important for building long and stable axonemes in these kinocilia. Whether shorter kinocilia might lead to abnormal vestibular function and altered gaits in older Camsap3-cKO mice requires further investigation.Kinocilia are exceptionally long primary sensory cilia located on vestibular hair cells, which are essential for transmitting key signals that contribute to mammalian balance and overall vestibular system function. Kinocilia have a “9+2” microtubule (MT) configuration with nine doublet MTs surrounding two central singlet MTs. This is uncommon as most mammalian primary sensory cilia have a “9+0” configuration, in which the central MT pair is absent. It has yet to be determined what the function of the central MT pair is in kinocilia. Calmodulin-regulated spectrin-associated protein 3 (CAMSAP3) regulates the minus end of MTs and is essential for forming the central MT pair in motile cilia, which have the “9+2” configuration. To explore the role of the central MT pair in kinocilia, we created a conditional knockout model (cKO),  Cilia, found on cell surfaces, are microtubule (MT)-based organelles that play essential roles for cell development, proliferation, differentiation, migration, signal transduction, etc. The structure, length, and function of cilia must be tightly regulated because their dysfunction is associated with numerous diseases collectively called ciliopathy disorders . Based oVestibular kinocilia, located on hair cells of the vestibular organ, are primary sensory cilia present throughout the murine lifespan. Compared to other primary sensory cilia, vestibular kinocilia are exceptionally long, some being 3–4 times longer than adjacent microvilli called stereocilia . The actCamsap3tm 1a/tm1a mice, the global knockdown (KD) of CAMSAP3 expression is associated with impaired ciliary motion, leading to phenotypes of Primary Ciliary Dyskinesia (PCD), which includes hydrocephalus, subfertility, and impaired mucociliary clearance. Dysfunctional mucociliary clearance leads to hyposmia, anosmia, rhinosinusitis, and otitis media. We occasionally noticed Camsap3tm 1a/tm1a mice (Camsap3-KD for short) with a head tilt to one side. Therefore, we suspect that CAMSAP3 is also needed to construct the central MT pair in kinocilia on vestibular hair cells and predict that the central MT pair in vestibular kinocilia is essential to establish the long and stable cilia required for vestibular function.Calmodulin-regulated spectrin-associated protein 3 (CAMSAP3), also called Marshalin , is a MTCamsap3 gene from a mouse genome leads to global Camsap3-KO mice dying prematurely  for tissue specific cKO by using Cre-loxP system.CAMSAP3 is expressed ubiquitously in mice. The global KD and global knockout (KO) CAMSAP3 in mice could potentially affect all cells that express CAMSAP3. Indeed, removal of maturely . Even thocessing  without ing loss . To inveCamsap3-cKO would allow us to examine CAMSAP3’s role in vestibular hair cells without influence from other health issues. TgPax2-Cre mouse line is widely used to establish cKO mouse models for inner ear (IRES-Cre was inserted into Pax2 (paired box gene 2), a transcription factor that is crucial in the developmental and proliferation of multiple cells and organs. Cre expression was detected at E8.5 in the otic placode in the Pax2-Cre mouse line (Camsap3-cKO by crossing TgPax2-Cre mice with Camsap3tm 1c/tm1c (fl/fl), aiming to eliminate CAMSAP3 expression in Cre-expressed tissues, which include most of the cells in the inner ear, midbrain, cerebellum, the olfactory bulb, and kidneys Wtsi (referred to as Camsap3-KD) mouse model was obtained from the Wellcome Trust Sanger Institute. The original Camsap3-KD line on the C57B6N background was re-derived on FVB murine backgrounds to increase their viability 36 , a strain of pseudo-wildtype (WT) mice. This WT tm 1cCamsap3 floxed line was further crossed with the TgCrePax2 mouse line, Tg(Pax2-Cre)1Akg/Mmnc (MMRRC stock number 10569)  Camsap3-cKO for short. All animals were maintained by heterotypic breeding, and the genotypes were determined by detecting the inserted cassettes in mouse tail samples performed by Transnetyx .The iability . As showstrategy . A targeocessing  without -FLPe)36  to deletr 10569)  to creatCre expression was limited in a few tissues including the inner ear, olfactory bulb, and kidneys of TgCrePax2 mice, the exon 7 of camsap3 was expected to be fully removed from these tissues of Camsap3-cKO mice. To verify the exon 7 exclusion, genomic DNA was purified from the inner ear of WT and Camsap3-cKO mice using the QIAamp DNA FFPE Tissue Kit (Qiagen). PCR was used to verify the exon 7 exclusion using the PCR Master Mix  with forward primer L1L2-BactP-MD-F (GCTGGCGCCG GAACC) and reverse primer C3Floxed5-R (TTGGCCTGGGGAACATGAC). Conditions for the thermal cycler were: step 1–95°C, 2 min; step 2–95°C, 30 s; step 3–55°C, 30 s, step 4–72°C, 90 s; repeat steps 2–4, 35 × ; step 5–72°C, 10 min. Expected sizes were 1,307 base pairs (bp) for WT, and 199 bp for Camsap3-cKO mice. Both male and female mice were used in all experiments.Since Camsap3-cKO littermates were placed in a 5-gallon bucket filled with room temperature water and were required to swim for 3 min. Behavior was observed, and any abnormal behavior was recorded. The swim test protocol was a modified version of a protocol described previously (WT mice and their eviously .Camsap3-cKO littermates were placed in a clear plastic box (1′′× 2′′× 5′′). The box was quickly flipped upside down and the time it took each mouse to flip themselves back upright was recorded.We modified a protocol described previously  to assesCamsap3-cKO and their WT littermates. Following the Camsap3-cKO mice and 10 WT littermates completed eight trials, with the first three trials functioning as an acclimation and familiarization period. The final five trials were recorded for analysis.The rotarod test was created by Camsap3-cKO littermates were placed on the treadmill one at a time and were required to run. The mice were then evaluated at treadmill speeds of 10, 17, and 24 cm/s. The DigiGait assay was run by Northwestern Behavioral Phenotyping Core. The DigiGait Imaging System software allowed us to examine 52 different aspects of each mouse’s gait  consists of a motorized transparent treadmill belt sitting above a digital camera which records the ventral view . WT micee’s gait . The anaCamsap3-cKO littermates for immunofluorescence as previously described (RRID: AB_2556548), goat anti-mouse IgG2b Alexa 647 at 1:500 (Thermo Fisher Scientific AB_143165), Hoechst at 1:1000 , and Phalloidin-Alexa 568 at 1:2000 , or Phalloidin-Alexa 647 at 1:400 (Thermo Fisher Scientific A22287) for 2.5 h at room temperature. The stained samples were subsequently mounted in Fluoromount Aqueous Mounting Medium . When mounting the whole mount utricle samples, a 0.12 mm deep Secure-Seal spacer  was placed between the slide and the cover glass in order to avoid destruction of the vestibular kinocilia on hair cells. Immunostained samples were imaged using a Nikon C2 or A1R+ confocal microscope using objects of a plan Apo VC 20×/0.75 DIC N2 (Nikon) and a plan Apo 60×/1.4 oil (Nikon). All images were taken as 12-bit, which allows for 0–4,095 range. For vestibular kinocilia length measurement, Z-stacks images were captured from the apical surface of utricle hair cells to the tip of vestibular kinocilia . To identify regions of interest 1 μm thick sections were collected, stained with Toluidine Blue O and examined by light microscopy. 70 nm tissue sections were collected on 200 mesh copper grids and stained with uranyl acetate and Reynolds lead citrate, and examined using a FEI Tecnai G2 Spirit Transmission Electron Microscope. The number of MTs in kinocilium was counted and sorted by structure: “9+2,” “9+0,” “8+2,” or “8+0.” The first number indicates the numbers of MT doublets outlying, and the second number implies the amount of MTs in the center.Utricles from WT mice and their Camsap3-cKO littermates were food restricted but provided water ad libitum for 24 h prior to testing. Mice were given a piece of a Nutter Butter cookie during this day to familiarize the mice with its scent. Testing was performed during daylight, starting at 9 AM. The base of a Sterilite translucent storage box (cage) [47 cm (L) × 37.8 cm (W) × 28.3 cm (H)] was filled with bedding to a depth of 3 cm. Each mouse was placed in an acclimation bin which was identical to the testing bin in every way, with the exception of no food being present, and were given 5 min to adjust to the enclosure. Mice were then transferred individually to the testing cage, which had ∼ 0.5 g of Nutter Butter cookie hidden in the center of the cage under the bedding. As soon as the mouse entered the testing bin a stopwatch was started and the latency to find (LTF) the cookie was recorded. Following the experiment, mice were returned to their home cages and given their full allotment of food.The Buried Food Test was used to evaluate olfaction in mice . WT miceCamsap3-KD, were born with smaller bodies along with sub-infertility, hydrocephalus, anosmia or hyposmia, sinusitis, respiratory distress, otitis media, and hearing loss , the expected short fragments (199 bps) were detected, but not in mice without the Pax2-Cre allele (Pax2-Cre negative) or original homozygous Camsap3tm 1c mice. We noted that the long PCR band, 1307 bp, was also detected in Camsap3-cKO mice with low intensity. As we did not perform cardiac perfusion before extracting genomic DNA from the inner ear, we suspected that cells from the circular bloodstream in the inner ear could contribute to the long PCR bands as the genomic DNA from these cells were not affected by Pax2-Cre expression. It is also possible that the exon 7 of Camsap3 was not completely removed from all cells in the inner ear. To verify whether Camsap3 was expressed in the vestibular hair cells, we performed immunofluorescence using vestibular organs collected from Camsap3-cKO and their WT littermates, with the ages of postnatally day (P) 23 to 260. In WT, CAMSAP3 expression was found in the vestibular organ including the hair cells in cristae . In sum, we created a cKO model that significantly reduced CAMSAP3 proteins in the vestibular system.As previously reported, the global KD mice, ing loss . In cont kidneys . To test cristae , utricle cristae , and sacCamsap3-cKO mice  and their WT littermates  around 1-year old (P296-P384) were examined using immunofluorescent staining of anti-acetylated tubulin and phalloidin, which labeled vestibular kinocilia (green) and stereocilia , respectively .To investigate CAMSAP3’s role in establishing kinocilia on the vestibular hair cells, utricles collected from ectively . We meassistency . From easap3-cKO . An unpade novo formation of the central MT pair in a “9+2” configuration observed in the axons of motile cilia  at the center of axonemes. This is demonstrated in a transverse section of WT vestibular kinocilia (Camsap3-cKO image in Camsap3-cKO mice showed similar structure. The majority of Camsap3-cKO vestibular kinocilia (55%) have the “9+0” configuration. In addition, irregular configurations, “8+2” and “8+0,” were found in both WT (12%) and Camsap3-cKO (21%). Distribution of the MT configurations between WT and KO was statistically significant different as analyzed using a Kolmogorov-Smirnov distribution comparison test (p = 0.0003). Our data suggests that vestibular kinocilia of Camsap3-cKO were more likely missing the central MT pair than WT vestibular kinocilia.To further verify the role of CAMSAP3 in formation of the central MT pair in vestibular kinocilia, the MT structure differences between WT and sing TEM . We examsing TEM  from 8-mCamsap3-cKO mice just as their WT littermates. In addition, the shape and orientation of stereocilia on vestibular hair cells from Camsap3-cKO were quantitatively similar to those on WT mice despite the vestibular kinocilia from Camsap3-cKO mice were shorter and more likely to miss the central MT pair , and the average male Camsap3-cKO mouse weighed 26.49 g ± 4.904 (n = 13). For female mice older than 8 months, the average WT mouse weighed 30.56 g ± 4.687 (n = 8), and the average female Camsap3-cKO mouse weighed 22.38 g ± 1.727 (n = 7). WT mice older than 8-month were significantly larger than the Camsap3-cKO mice regardless their sex  mice, ages ranging from P22 to P388, we found that there were no notable differences between the Camsap3-cKO and WT mice regarding their ability to swim. The right-reflex test assesses whether the mice can re-right themselves when flipped upside down. The same group of 26 WT and 27 Camsap3-cKO mice completed the right-reflex test. We found no difference in performance between the WT and Camsap3-cKO mice as each of the mice was able to flip themselves back to upright immediately (within 1 s). Subsequently, we analyzed the balance ability of Camsap3-cKO mice by rotarod apparatus. On the Rotarod, we assessed 12 WT  and 8 Camsap3-cKO  mice, ages P37–P153, and found that there was no significant difference in TTF between the two groups (data not shown).Since CAMSAP3 was essential to build or maintain normal structure of vestibular kinocilia , 3, we flex test . The swiCamsap3-cKO mice was also examined using the DigiGait Imaging System  is the maximal paw area captured by the camera and corresponds with the time of “peak stance”  and 58.9% (hind limbs) of the paw area WT mice used. Maximum dA/dt (cm2/s) is the maximal rate of change of paw area in contact with the treadmill belt during the braking phase. At a low treadmill speed (10 cm/s), there was no substantial difference between the WT and Camsap3-cKO mice in their dA/dt for both fore and hind lambs. However, as the treadmill speeds increased, the difference between the WT and Camsap3-cKO mice became more apparent. At a high treadmill speed (24 cm/s), the maximum dA/dt decreased in Camsap3-cKO mice’s hind limbs (−10.5%), while WT mice had increased maximum dA/dt (+10.0%). Similar to paw area, maximum dA/dt in Camsap3-cKO mice were always slower than their WT littermates independent of walking speed. These differences exist despite there being no difference in the body length, width  than their WT littermates  and Camsap3-cKO mice  from 8 litters of mice, ages P55–P313. Mice were placed in a large container filled with bedding, and their latency to find (LTF) a buried Nutter Butter cookie was recorded. As shown in Camsap3-cKO mice regardless their sex. We then examined CAMSAP3 expression in the olfactory bulb. Olfactory bulbs from P180 WT and Camsap3-cKO littermates were collected and stained with anti-Camsap3. As shown in Camsap3-cKO mice, suggesting that CAMSAP3 has a minimal role in olfactory bulbs. We also compared CAMSAP3 expression in olfactory sensory neurons, where CAMSAP3 was abundantly expressed  in vestibular kinocilia than their WT littermates. Although a very small portion of irregular MT configuration is commonly found in WT cilia, an increased proportion of irregular MT configurations in cilia was often associated with damage or degeneration of cilia  of Camsap3 transgenic mice using video-oculography. Video-oculography quantifies eye movement data to evaluate vestibular function, and could highlight vestibular issues that we were not able to capture in this study.Balance disequilibrium is a significant contributor to falls in the elderly. The most common cause of balance dysfunction is due to abnormal hair cells from the vestibular sensory epithelia of the vestibular organ. Because of malfunction of kidneys in Camsap3-cKO mice could directly lead to vestibular function requires further investigation.In summary, our data suggest that CAMSAP3 is critical for construction of the central MT pair in vestibular kinocilia, which may be essential for building long and stable axonemes in these kinocilia on vestibular hair cells. Whether abnormal vestibular kinocilia of The original contributions presented in this study are included in the article/The animal study was reviewed and approved by the Institutional Animal Care and Use Committees of Northwestern University.JZ designed and performed some of the experiments and analyzed the data. JO’D performed some of the experiments and analyzed some of the data. JO’D and JZ wrote the manuscript. Both authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Background: Idiopathic pulmonary fibrosis (IPF) is the interstitial lung disease with the highest incidence and mortality. The lack of specific markers results in limited treatment methods for IPF patients. Numerous prognostic signatures represented effective indexes in predicting the survival of patients in various diseases; however, little is investigated on their application in IPF.Methods: This study attempted to explore the clinical markers suitable for IPF by constructing a prognostic signature from the perspective of 7-methylguanosine (m7G). An m7G-related prognostic signature (m7GPS) was established based on the discovery cohort with the LASSO algorithm and was verified by internal and external validation cohorts. The area under the curve (AUC) values were utilized to assess the accuracy of m7GPS in predicting the prognosis of IPF patients and the ability of m7GPS in screening IPF patients. Kaplan-Meier curves and Cox regression analyses were used to identify the relationship of m7GPS with the prognosis of IPF individuals. Enrichment analyses, CIBERSORT algorithm, and weighted gene co-expression network analysis were applied to explore the underlying mechanisms and correlation of m7GPS in IPF.Results: The two m7G regulatory genes can divide IPF into subtypes 1 and 2, and subtype 2 demonstrated a poor prognosis for IPF patients (p < 0.05). For the first time in this field, the m7GPS was constructed. m7GPS made it feasible to predict the 1–5 years survival status of IPF patients (AUC = 0.730–0.971), and it was an independent prognostic risk factor for IPF patients . The conspicuous ability of m7GPS to screen IPF patients from the healthy was also revealed by an AUC value of 0.960. The roles of m7GPS in IPF may link to inflammation, immune response, and immune cell levels. Seven genes  were identified as hub genes of m7GPS in IPF. Three drugs  were considered sensitive to patients with high m7GPS risk scores.Conclusion: This study developed a novel m7GPS, which is a reliable indicator for predicting the survival status of IPF patients and is identified as an effective marker for prognosis and screening of IPF patients. Idiopathic pulmonary fibrosis (IPF) is the most common interstitial lung disease with unknown etiology, mainly characterized by chronic and progressive pulmonary fibrosis. In the United States and Europe, the annual incidence of IPF is 5–20 per 100,000 . Lung trRNA methylations are common epigenetic modifications in eukaryotes, including N1-methyladenosine (m1A), N6-methyladenosine (m6A), 5-methylcytosine (m5C), and 7-methylguanosine (m7G). The application of RNA methylation-related prognostic signatures for promoting the individualized treatment of human diseases has received considerable critical attention. For example, a prognostic signature constructed based on m6A-modified long noncoding RNAs was identified as a risk factor and predictor of the prognosis of patients with low-grade gliomas . An m1A-For the first time in this field, this study established an m7G-related prognostic signature (m7GPS) for IPF based on the discovery cohort and verified it by the internal and external validation cohorts. Further, the study also explored the prognostic value and potential molecular mechanisms of m7G regulatory genes (m7GRGs) in IPF, which may promote the understanding of m7GRGs in IPF and reveal clinical markers for predicting the prognosis of patients with IPF.2 (x + 1). The two cohorts included multiple bronchoalveolar lavage samples obtained from IPF patients (n = 176) and normal healthy individuals (n = 20) (n of Freiburg-Siena = 112) and healthy (n of Freiburg-Siena = 20) groups, the SMOTE (synthetic minority oversampling technique) algorithm was used to balance the sample size. This process was completed by the “DMwR” package for R (v4.1.0), and the target samples of both the IPF group and the healthy group (for the new Freiburg-Siena cohort) were set to 120. The new data produced based on SMOTE were used to explore the expression difference and identification effect of m7GRGs between the IPF and healthy groups.Three IPF-related cohorts and the corresponding clinical data were collected from the Gene Expression Omnibus database, containing the Freiburg, Siena, and Leuven cohorts, all of which belong to the series GSE70866. Given that data of the Freiburg cohort and Siena cohort were detected with the same platform GPL14550, they were combined as the Freiburg-Siena cohort for further analysis. The gene expression levels of Freiburg-Siena and Leuven cohorts were normalized with log(n = 20) . ConsideBUD23, METTL1, and RNMT, were collected from the literature survey , the average method was used to identify various m7G subtypes related to m7GRGs, with clustering by the k-means algorithm and describing the distance metric by euclidean. The k values were set as 2–20, and the optimal k value was determined by the cumulative distribution function plot. The identification of m7G subtypes was performed by utilizing the “ConsensuClusterPlus” package  > 1 and adjusted p-value < 0.05, and the step was finished with the help of “limma” package , IPF patients from the Freiburg-Siena cohort were randomly classified into the discovery cohort (70%) and internal validation cohort (30%). The Leuven cohort was identified as the external validation cohort.The gene expression data of the Freiburg-Siena cohort was used to select differentially expressed genes (DEGs) in various m7G subtypes by the threshold of the absolute value of log package . Based ok-fold cross) were set by default  Cox regression and Random Forest. Considering that LASSO Cox regression was more common in this field , it was  default .Using time-dependent receiver operating characteristic (ROC) curves  producedA nomogram was constructed based on prognosis-related factors identified from univariate Cox analysis, where the survival probabilities of IPF patients can be predicted. Calibration curves were utilized to detect the accuracy of the nomogram in predicting the survival condition of patients. Decision curve analysis (DCA)  was applDifferential expression genes (DENGs) between the high-risk group and low-risk group were identified for exploring the potential molecular functions and KEGG (Kyoto Encyclopedia of Genes and Genomes)  signalin2 was set as 0.85 and identified the minimum soft threshold; 3) the correlations between genes in each module and m7GPS risk score were evaluated by gene significance and module membership; 4) the genes of the essential module were output for selecting hub genes. Subsequently, hub genes of m7GPS were identified in Cytoscape (v3.9.0) based on the “degree” algorithm. The same steps were also utilized for internal and external validation cohorts.The weighted gene co-expression network analysis (WGCNA) determined candidate hub genes of m7GPS. In detail, the median absolute deviation (MAD) of all genes in the discovery cohort was calculated, and genes with a MAD value of more than one were screened for performing WGCNA. The steps of WGCNA were as follows: 1) the top 5,000 genes with the highest MAD values were divided into various modules related to the m7GPS risk score using the “WGCNA” package ; 2) The The “oncoPredict” package  was applThe expression difference of m7GRGs between IPF and control groups was identified by Wilcoxon rank-sum test. The test was also utilized to evaluate clinical characteristics—ages, genders, GAP (gender-age-physiologic) stages, and subtypes—between high and low risk score IPF groups. All the analyses in this study were carried out in R (v4.1.0) and Cytoscape (v3.9.0). The design of this study is shown in n = 240), the expression level of METTL1 in the IPF group was higher than that in the normal group (p < 0.05); in contrast, the expression of RNMT between the two groups was not statistically different (p = 0.42)  by the unsupervised clustering algorithm  . IPF patlgorithm . Subtypelgorithm . The anap > 0.05; Based on the Freiburg-Siena cohort, there were 1,579 DEGs between the various subtypes (subtype 2 versus subtype 1), including 1,490 upregulated expression genes and 89 downregulated expression genes . Using uCCL2, CCL7, HS3ST1, MRVI1, TM4SF1, and TPST1—was constructed (METTL1 and RNMT) can be seen in Based on the discovery cohort and the LASSO Cox regression algorithm, the m7GPS containing six candidate genes—structed . The coestructed , and thestructed , suggestBy analyzing the discovery cohort, m7GPS can highly accurately predict the 1–5 years survival status of IPF patients  were related to the poor prognosis of IPF individuals  and risk score of m7GPS demonstrated conspicuous accuracy in distinguishing IPF patients and healthy individuals  of the m7GPS (based on m7G subtypes) . To explThe DENGs between the high-risk and low-risk groups (classified based on the median risk scores in the cohort) were determined by discovery cohort data, and they were used to perform the molecular functions and KEGG analyses. The same calculations were performed for the internal and external validation cohorts. The results showed that 13 functions were screened simultaneously from three cohorts , such asThe data analysis results of the three cohorts showed that IPF patients with high m7GPS risk scores had increasing levels of activated mast cells . In addi2 , the soft threshold was set as 12 to construct a scale-free network  and the subtypes based on them represented essential roles in IPF. According to previous reports, METTL1 overexpression was associated with poor prognosis in patients with intrahepatic cholangiocarcinoma, and they also found the carcinogenic role of METTL1-mediated m7G tRNA modification in promoting intrahepatic cholangiocarcinoma. METTL1-mediated m7G tRNA modification regulated the expression of target genes such as EGFR and EFEMP1, thereby accelerating bladder cancer progression. Our study identified upregulated METTL1 in BLA cells of IPF patients, and increasing levels of METTL1 indicated a poor prognosis of IPF; to our knowledge, the prognostic value of overexpressed METTL1 in IPF has not been reported before, indicating the novelty of this finding. The other m7GRG, RNMT, was considered to promote the proliferation of tumor cells by RNMT in BLA cells of IPF patients and healthy persons and the significant prognostic value of this gene in IPF. Even so, the combination of METTL1 and RNMT expression levels can classify IPF into two molecular subtypes (subtype 1 and subtype 2). The prognosis of patients in subtype 2 was more unfavorable than those in subtype 1, indicating the importance of m7G subtypes.The two m7GRGs  is also worthy of further investigation.Collectively, this study constructed a novel m7GPS composed of six genes. m7GPS is a reliable indicator for predicting the survival probability of IPF patients and is identified as an effective marker for prognosis and screening of IPF patients. The underlying mechanisms of m7GPS in IPF may relate to inflammation and immune response. Several drugs potentially sensitive to patients with high m7GPS risk scores were also investigated. In a word, m7GPS may serve as a novel marker for IPF.


These results suggest that schematic cognitive models of body dissatisfaction and eating disorders body may also be applicable to men, however more research is needed.Over the past 30 years, attentional bias for body shape and weight content has been implicated in the precipitation and maintenance of body dissatisfaction and eating disorders. Although the existence of this bias toward body stimuli is well-established in female populations, it is comparatively understudied in men. This review aimed to examine the nature of this visual attentional bias toward male bodies in male samples across a range of different attentional paradigms, including eye-tracking, dot-probe, and the visual search task. Results were heterogenous, finding some evidence that men with higher body dissatisfaction and eating disorder symptoms demonstrated an attentional bias toward desirable bodies of  Body dissatisfaction can be defined as a negative subjective evaluation of one’s body as a whole or relating to specific aspects of one’s body such as body size, shape, muscularity/muscle tone, and weight Grogan, . Over thCooper , 2005 suVitousek and Hollon’s  cognitivWilliamson et al.  built upAttentional bias can be defined as the tendency of one’s perception to be affected by recurring, salient thoughts  stimuli and body dissatisfaction in women has been examined in many studies through a variety of paradigms, however very few have dealt with men. This may be problematic as body weight and shape concerns, and indeed psychopathology of male eating disorders may differ from females  examining the evidence for attentional biases toward desirable male bodies of others and (2) toward undesirable features of one’s own body across a range of different paradigms for measuring attention. Results are summarized in Table  examininBias toward desirable body features of others. Nine studies were identified showing that men who are dissatisfied with their bodies tend to display attentional biases toward desirable body features of others. Using eye tracking, Stephen and colleagues , and (2) dissatisfied men tend to have attentional biases toward perceived negative or nonideal features of their own body. However, there are at least four studies which provide evidence counter to this proposed attentional model. So, what does this mean for schematic cognitive models of body dissatisfaction and eating disorders? Effectively, these results suggest some evidence that existing cognitive models . A further example is seen in that typical measures of attention such as the dot probe task are unable to differentiate between attentional engagement and attentional disengagement biases, and as such are measuring different facets of attentional bias , a relatively low number compared with the magnitude of research that has been conducted on their female counterparts and hardly large enough to conclusively generalize the nature of these biases to the male population. Additionally, many of these participants were nonclinical and/or university students (approximately 65%). Of the remaining 35% of participants, 108 (18%) were Chinese gym attendees (Jin et al., A further limitation of the existing examination of attentional bias in men is the sheer lack of empirical data. All existing studies identified for this review, of which there were only 11, returned a combined sample of 618 men (A final limitation of the extant literature is that to date, there are no studies investigating whether body-related attentional biases play a causal role in body image and eating disturbances in men. Causality could sufficiently be examined through the use of ABM paradigms (Renwick et al., Based on these limitations, several recommendations for future studies are proposed. First, future studies should determine the ideal paradigm for best examining attentional bias toward body stimuli. Although on face-value eye-tracking may present as the most comprehensive measure of attentional patterns, the field would benefit from an experimental comparison of the sensitivity for detecting attentional bias across the four paradigms. Notably, paradigms that provide measures of attentional engagement and attentional disengagement, such as the ARDPEI, should be utilized (Dondzilo et al.,