Current technology development ensures a variety of advanced materials and options for reinforcing concrete structures. However, the absence of a uniform testing methodology complicates the quantification and comparative analysis of the mechanical performance of the composite systems. The repeated mechanical loads further complicate the issue. This research extends the recently developed residual stiffness assessment concept to the repeated loading case. It provides an engineer with a simplified testing layout and analytical model to quantify the residual flexural stiffness of standardized laboratory specimens subjected to repeated cycling loads. This model explicitly relates the particular moment and curvature values, requiring neither iterative calculations nor the load history. Thus, this feature allows residual stiffness quantification under repeated loading conditions, including complete reloading of the beam samples imitating the structural strengthening procedure; the proposed technique is equally efficient in quantifying the residual stiffness of the beam samples with any combinations of fiber-reinforced polymer (FRP) reinforcements, i.e., embedded bars, near-surface-mounted strips, and externally bonded sheets. This study employs 12 flexural elements with various reinforcement and loading layouts to illustrate the proposed methodology’s efficiency in quantifying the residual strength of the tension concrete, which estimates the efficiency of the reinforcement system. The explicit quantifying of the residual resistance of the FRP reinforcement systems under repeated load cycles describes the essential novelty of this work. Technology development provides various materials for reinforcing and strengthening concrete structures ,2. FiberVarious FRP materials, i.e., embedded bars, near-surface mounted (NSM) strips, and externally bonded reinforcement (EBR) sheets, exhibit diverse mechanical performances when reinforcing composite systems. The literature has extensively documented these performances . StudiesConcrete structures often experience cyclic rather than static loading in practical engineering applications. Li et al.  investigAn alternative approach to studying the flexural behavior of RC specimens under various loading conditions is using the acoustic emission method. Mat Saliah and Md Nor  extensivThe absence of a standardized methodology for quantifying reinforcement efficiency in composite systems subjected to repeated loads motivated this study. Several studies have examined the behavior of RC beams with composite reinforcement under repeated loading. For instance, Fathuldeen and Qissab  investigUnlike the structural analysis design, the proposed procedure focuses on the mechanical performance of composite materials, particularly reinforcement systems. As described in the previous section, recent achievements in engineering, developing advanced fibers and reinforcement materials , and other reinforcement solutions caused problems in setting an efficient solution for a particular situation. Furthermore, the combination of fibers and continuous reinforcement  in hybrid systems e.g., ,11) comp comp11])As mentioned in Therefore, Gribniak et al. ,22 develAt the same time, the comparative analysis of the equivalent stress–strain relationships of alternative reinforcement systems can estimate the improvement of the reference solution. For instance, Gribniak and Sokolov  evaluateJakubovskis et al.  transforThis study adapts the proposed testing layout and the simplified analytical model to analyze the residual flexural stiffness of the laboratory specimens subjected to repeated mechanical loads. It employs the concept of the equivalent residual stresses acting on concrete in tension to measure the structural performance of alternative composite reinforcement systems and their variation with the load cycles of various intensities. This analytical model explicitly relates the bending moment and curvature values, quantifying the equivalent stresses acting in the concrete under the assumption of the rectangular stress distribution. This solution requires neither iterative calculations nor the load history definition.At the same time, the simplified analytical model  does notThe explicit nature of the analytical expressions, which do not require any additional modifications regarding the previous publications ,22,29, erd is the effective depth; rA is the cross-section area; rE is the modulus of elasticity; n is the number of the different reinforcement parts; iE, iA, and id are the modulus of elasticity, cross-section area, and effective depth of the i-th reinforcement component.The model  considerThe strain distribution follows the Euler–Bernoulli hypothesis b. NumeroThe smeared crack model describes the stress–strain behavior of the tension concrete b,c. VariIdealized elastic material laws define the mechanical behavior of the reinforcement and the compressed concrete. This modeling approach substantially simplifies the mathematical expressions and ensures a straightforward solution.The rectangular distribution of the tensile stresses in concrete defines the equivalent stress σt* d. This cThe proposed analytical model uses the following assumptions:Fr and Fc are the internal forces acting on the tensile reinforcement and the compressed concrete; rd is the efficient depth of the transformed reinforcement (Equation 1); h and cy are the height and gravity center coordinate of the cross-section in rd > h condition is acceptable).The latter two assumptions allow quantifying the stiffness of the beams subjected to repeated loads with the solution expressed in terms of the equivalent tensile stresses. The following equilibrium equations of internal forces and bending moments for the centroid of the equivalent stress diagram d define cy for the particular curvature κ and bending moment Mext:cE is the modulus of elasticity of concrete; h and b are the height and width of the cross-section in κ is the average curvature of the pure-bending zone subjected to the external bending moment Mext; Equation 1 describes the remaining parameters.The above equation system relates the internal forces and stresses acting on the cross-section, employing the strain compatibility condition b. Referecy ≤ h] condition describes the neutral axis position:The above polynomial has three roots, and the [0 < t stress d and avet stress b:(5)σt*=Mext, average curvature in the pure-bending zone κ, cross-section geometry  detectors have a 2456 × 2085 pixel resolution and operate at 12.2 frames per second. The lighting equipment Arri ensures the quality and accuracy of the digital images.The verification capability of the deformation monitoring results defined the second condition for developing the testing setup shown in h and b represent the height and width of the cross-section in d, A, and E are the reinforcement parameters ; tf describes the tensile strength of the reinforcement, and the subscripts “1” and “2” correspond to the transformed reinforcement components in Equation 1. cf’) on the testing day; the column “Age” specifies the testing age. The column “cf’” specifies the average value and standard deviation determined for four identical cylinders; the remaining parameters of the concrete necessary for the analysis  were determined using the average compressive strength values  loads the test samples with a 0.4 mm/min velocity; Dion 7 software ensures the loading system control and data acquisition process. The load cycles are formed in a semi-automatic manner with manual load reduction to the minimum cycle boundary. The data logger Almemo 5690-2 collects the load cell and LVDT outputs every second.Mser. These load cycling numbers were set arbitrarily in this study to avoid measurement errors and provide several data points for averaging.This study focuses on the stiffness decrease under repeated mechanical loads. Still, the analytical model  does notMmax (exceeding Mser by 15%). Subsequently, the operator interrupts the loading process when it reaches the upper boundary Mmax, manually reducing the load to the minimum cycle boundary Mmin (corresponding to approximately 85% of Mser). After that, the bending moment rises again under computer control to the maximum cycle load Mmax, continuing the loading cycles. The complete unloading finalizes the first loading stage, resulting in the residual deformation expressed as curvature rκ1. Typically, this loading process is repeated three times (with five load cycles in each repetition) with the complete unloading of the beam samples, investigating the residual deformation trends. Each subsequent loading stage starts at the same speed as the previous loading round. However, the curvature analysis accounts for the residual deformations from all past loading stages , shifting the curvature diagrams as shown in Mult, Mser) and the cycle boundaries (Mmin and Mmax).The sign “≡” relates the loading conditions to the particular beam sample. For instance, the symbol “≡S1” refers to the loading condition of the S1-EBR beam, determining the exact service moment , the theoretical moment, corresponding to the steel yielding, limits the ultimate cycle load formulas .The target loadings of the elements, combining steel and NSM reinforcements, were set to represent the loading conditions of alternative test samples  for comparison purposes.During the physical tests, the loading process starts automatically at 0.4 mm/min until the maximum load of the cycle Gribniak et al.  proposedCrackstop M Ultra ∅0.022 × 13 mm and 4.2 kg of Durus EasyFinish ∅0.7 × 40 mm) were used in the concrete to avoid a sudden failure of the shear zone. All the beams were produced in steel forms and were unmolded 2–3 days after the casting. After that, all samples were stored in laboratory conditions at an average of 73% humidity and 20 °C before the testing day or forming external FRP reinforcement systems.The illustrative experimental program employs laboratory-mixed concrete, using the same mix design as the previous studies ,22,29. TMapeWrap C UNI-AX sheets with the dry fabric’s 100 × 0.164 mm equivalent thickness were attached to the most tensioned surface of the beam samples. These sheets were placed along the beam and attached to the concrete surface using a two-component MapeWrap 31 epoxy resin  bars. The GFRP reinforcement system employs R1, R2, R3, etc.) captured with the DIC system. However, these cracking results could only qualitatively illustrate the composite performance of the reinforcement system in involving the tension concrete resisting the load. Thus, the number of cracks (in the post-cracking stage) could reveal the tension-stiffening effect [g effect . Remarkag effect .L1–L9 LVDT devices and the surface deformations captured by DIC and LVDTs. This instrumentation allows cross-verifying the test measurements, which is vital during the cyclic tests because of a certain inertness of the LVDT devices under reversed load and DIC sensitivity to the cameras’ movements [l equal to 600 mm, as iu is the displacement obtained by the iL device (i = 1…9).Gribniak et al.  also demovements ,50. Gribovements  describeRemarkably, complete reloading followed all load cycling sets to measure the residual deformation of the beam samples. The latter values are vital for the residual stiffness analysis—the origin of each consequent diagram  coincidenρ ratio  subjected to 3.710 kNm load. This outcome demonstrates the efficiency of the strengthening solution.The residual curvature analysis of the results from nρ ratio  and loadnρ ratio . In addinρ value, i.e., 2.45% vs. 1.22% (nρ ratio (1.18%). This allows the authors to hypothesize that the embedded GFRP bars cannot ensure sufficient resistance to repeated loads because of high deformability, corrupting the bond with concrete. The following equivalent stiffness analysis checks this hypothesis.Increasing the sample numbers will help identify more meaningful tendencies. However, s. 1.22% , which cThe moment–curvature diagrams in The schematic in In addition to the tripled residual curvatures , Figure nρ ratio (1.83%) among the test samples (except for the hybrid reinforcement systems). This outcome proclaims the inefficiency of the typical steel reinforcement for resisting cycling load, requiring a further unpractical nρ ratio increase. Furthermore, it reveals the need to renew structural design principles and tailor the materials’ mechanical properties for construction [A sharp inclination of the trend line of the equivalent stresses of the S12-S sample (with only steel reinforcement) highlights another bond-degrading mechanism characteristic of embedded bars. This stiffness decrease results from the cover cracking and the corresponding reduction in the concrete efficiency—only the concrete between the bar reinforcement and compressive zone b contribtruction . This anIn this context, the CFRP reinforcement systems demonstrate outstanding performance under the cycling load. The equivalent stresses in the S1 to S3 EBR samples reach the 1.86 MPa value and do not decrease below 0.70 MPa under the 3.71 kNm service load cycling; the NSM systems demonstrate the 1.16 MPa stress and preserve the minimum 0.64 MPa stresses, though these values correspond to the 2.22 kNm bending moment. The S10-S/NSM hybrid system improves the latter values correspondingly to 1.65 MPa and 0.87 MPa with the same tendency of stress decay as the S5-NSM beam. Under the increased load cycles, the S11-S/NSM proves a further increase in mechanical performance: the maximum and minimum equivalent stresses are equal to 2.11 MPa and 1.15 MPa.The S12-S/NSM beam represents the strengthening situation of the S12-S element. At the load stages “2” and “3”, the S12-S/NSM sample demonstrates very similar equivalent stresses to the S11-S/NSM beam . HoweverThe results of i describes the values corresponding to successive loading stages; the negative ratio corresponds to the average stress reduction because of the load repetitions.Here, the subscript In addition to the beam S12-S discussed above, e moment  in combie moment h.Comparing the stiffness decay tendency  of the SThe proposed testing procedure is suitable for quantifying the residual stiffness decrease under repeated mechanical load, including the complete load removal between the loading cycles. The quantification employs the equivalent stresses acting in the concrete in tension under the assumption of the rectangular stress distribution. This simplified model approximates the tensile stresses with sufficient accuracy—the average approximation error (regarding the “exact” solution) does not exceed 7%. On the other hand, the “exact” inverse analysis reported in the literature is inapplicable to the specimens under cycling loads, which makes the proposed methodology irreplaceable for this study’s purpose.This study reveals a limited ability of glass fiber-reinforced polymer (GFRP) bars to ensure the bonding performance under repeated loads. The concrete deformations exceed the bonding limit, making the reinforcement inefficient because of a relatively low modulus of elasticity (60 GPa) of the GFRP bars. Moreover, the bonding problem becomes apparent only for repeated loads—the previous tests did not identify the bond deterioration problems of GFRP bars under monotonic load for the same reinforcement configurations. On the other hand, this drawback is untypical for alternative reinforcement schemes considered in this study, proving the viability of the proposed analysis methodology.The carbon fiber (CF) reinforced materials demonstrate outstanding mechanical performance under repeated loads. The externally bonded reinforcement (EBR) system ensures the equivalent stresses, which do not decrease below 0.70 MPa; the near-surface mounted (NSM) system preserves the minimum 0.64 MPa stresses. The hybrid reinforcement system, combining steel bars and NSM CFRP strips, improves this value to 0.87 MPa. Under the increased load cycles, the hybrid reinforcement demonstrates a further increase in mechanical performance—the equivalent stresses exceed 1.15 MPa, exceeding 50% of the tensile resistance of the concrete.The NSM reinforcement system efficiently strengthened the beam sample with steel reinforcement bars tested until 85% of the theoretical load-bearing capacity. The load repetitions did not affect the residual resistance of the strengthened specimen, preserving the averaged equivalent stresses at an approximately 1.3 MPa level. Further studies should reveal the hybrid reinforcement system’s efficient layout and steel-to-CFRP ratio.This manuscript proposes composite reinforcement systems’ residual stiffness analysis procedure under repeated mechanical loads. The experimental program demonstrates the effectiveness of the proposed methodology, analyzing the bending test results of 12 beam samples with various reinforcement types. The following essential conclusions result from this work:


Well-documented scientific evidence indicates that mobile health  apps can improve the quality of life, relieve symptoms, and restore health for patients. In addition to improving patients’ health outcomes, mHealth apps reduce health care use and the cost burdens associated with disease management. Currently, patients and health care providers have a wide variety of choices among commercially available mHealth apps. However, due to the high resource costs and low user adoption of mHealth apps, the cost-benefit relationship remains controversial. When compared to traditional expert-driven approaches, applying human-centered design (HCD) may result in more useable, acceptable, and effective mHealth apps. In this paper, we summarize current HCD practices in mHealth development studies and make recommendations to improve the sustainability of mHealth. These recommendations include consideration of factors regarding culture norms, iterative evaluations on HCD practice, use of novelty in mHealth app, and consideration of privacy and reliability across the entire HCD process. Additionally, we suggest a sociotechnical lens toward HCD practices to promote the sustainability of mHealth apps. Future research should consider standardizing the HCD practice to help mHealth researchers and developers avoid barriers associated with inadequate HCD practices. Mobile health  is defined by the World Health Organization  as the pWhen compared to traditional design methods, incorporating HCD into research methods resulted in more usable, acceptable, and effective health care interventions ,21. NumeThe Medical Research Council  recommenCurrently, there are no standardized HCD processes, and HCD practices are reported inconsistently in mHealth studies ,32,34. HNot surprisingly, mHealth studies used traditional methods that have been widely used in health care research involving patients and HPs. Most HCD studies in mHealth development relied excessively on traditional interviews and focus groups. Both techniques are well suited to pose direct questions but have a limited ability to elicit tacit knowledge . PlayingA review of the HCD practice literature specific to mHealth app development indicates five constraints associated with the long-term efficacy of mHealth apps: (1) limited coherence, (2) lack of considerations for long-term viability, (3) undefined measurement, (4) long development time, and (5) publication bias.Coherence is the degree of consistency when HCD practice is enforced when developing a mHealth app. The incorporation of HCD into the development of mHealth apps necessitates the use of other theories, but they have not yet been developed methodically ,45,46. TAnother major concern is the lack of long-term viability of mHealth apps developed through HCD practice partially due to a different interpretation of outcomes between the design and the public health perspective . For insChallenges exist in defining and then selecting measures of success in design outcomes. During the HCD evaluation phase, randomized controlled trials (RCTs) and usability testing may not be sufficient to validate the success of a mHealth app as there are often multiple indirect outcomes that cannot be easily quantified and aggregated  . AdditioNew forms of technology are constantly emerging, and mobile technology is evolving quickly. Meanwhile, valid and reliable mHealth development is always time-consuming, especially the development process with users involved. Therefore, designed mHealth apps should be released in the future, which means they exist outside of the understanding of technology and user paradigms at the time of the HCD process. This can be problematic. Potential solutions for the development team include speculative design, which involves imagining life in the future to create a better-fitting design . HoweverPositive results are more likely to be reported or published, implying publication bias ; and thiWe reviewed the current HCD practices in mHealth studies and identified interconnected gaps that indicate a lack of comprehensive HCD practices. These gaps in reported HCD practice of mHealth development lead to poor adoption rates and a lack of long-term sustainability. Here, we provide 5 recommendations for future mHealth studies with the goal of developing sustainable mHealth apps using HCD. The recommendations aimed to address the identified gaps and explore the potential of HCD in addressing prevalent challenges with mHealth sustainability. This viewpoint attempts to encourage a discussion toward evidence-informed or evidence-based HCD practice in mHealth development, which can then help address existing gaps in science.Because mHealth design solutions from HCD practice are typically localized, lifestyle-focused, and are part of a system solution, “culture norms” must be considered . In suppEvaluation of the HCD process and participant experience is just as critical as evaluation of the design solution. However, it is largely unexplored . When buNovelty is an important aspect to address since lost interest is one of the reasons why the majority of users do not use such apps frequently and may even stop using them for a short time . GamificVarious studies have raised concerns about the quality of mHealth apps, especially the credibility of the medical information in the apps, because some mHealth app development may be driven by developers without professional medical expertise . One of Aside from information reliability, data privacy and security are also of concern. In a cross-sectional analysis of 36 top-ranked apps for depression and smoking cessation available in public app stores, 29 transmitted data to Facebook or Google, but only 12 accurately disclosed this in a privacy policy . MoreoveEmery and Trist  coined tIn mHealth apps have been widely developed and have proven successful, in some instances, in improving patient outcomes and quality of life. However, low adoption and sustainability are known challenges for mHealth apps. In this paper, we reviewed and synthesized the current HCD literature focused on mHealth app development and identified the gaps in current mHealth studies. We offer 5 recommendations for future HCD practice to develop sustainable mHealth apps, which can potentially address the gaps mentioned and contribute to the sustainability of mHealth apps. In particular, a new HCD process with a sociotechnical lens was proposed, which illustrates the integration of HCD phases with an STS in mind. Future research should consider standardizing the HCD practices to help mHealth researchers and developers avoid barriers associated with inadequate HCD practices.


L-Methionine (Met) is an essential alpha-amino acid playing a key role in several metabolic pathways. Rare inherited metabolic diseases such as mutations affecting the MARS1 gene encoding methionine tRNA synthetase (MetRS) can cause severe lung and liver disease before the age of two years. Oral Met therapy has been shown to restore MetRS activity and improve clinical health in children. As a sulfur-containing compound, Met has a strongly unpleasant odor and taste. The objective of this study was to develop an optimized pediatric pharmaceutical formulation of Met powder, to be reconstituted with water, to obtain a stable oral suspension. Organoleptic characteristics and physicochemical stability of the powdered Met formulation and suspension were evaluated at three storage temperatures. Met quantification was assessed by a stability-indicating chromatographic method as well as microbial stability. The use of a specific fruit flavor  with sweeteners  was considered acceptable. No drug loss, pH changes, microbiological growth, or visual changes were observed at 23 ± 2 °C and 4 ± 2 °C with the powder formulation for 92 days, and the reconstituted suspension for at least 45 days. The developed formulation facilitates the preparation, administration, the dose adjustment and palatability of Met treatment in children. MARS1 gene. This methionyl-tRNA allows the initiation of translation, by appropriate positioning at the initiation AUG codon of the mRNA, a 40S ribosomal subunit containing the initiator methionyl-tRNA and a 60S ribosomal subunit binding to form the 80S complex to start initiation and the transition to elongation [L-methionine (Met) is a vital sulfur-containing α-amino acid playing a key role in mammalian metabolism such as methyl donor reactions, as well as diverse cellular functions such as mRNA translation and protein synthesis . Althougongation ,4.MARS1 have been described such as the Charcot-Marie-Tooth disease type 2, a group of peripheral nervous hereditary disorders involving sensory and motor peripheral nerves dysfunctions [MARS1 [Diseases caused by variants of unctions . Anothers [MARS1 . This sps [MARS1 . This muMARS 1 gene has been described as being severe and usually manifesting in early childhood. It included dyspnea, cough, digital clubbing and severe respiratory distress often leading in death in childhood due to lung fibrosis, reaching 59% with half of them occurring before the age of two years [The particular type of PAP caused by mutations in the wo years . Therapewo years . Indeed,wo years ,7. Dailywo years .To our knowledge, there is currently no validated pediatric pharmaceutical form of Met for the oral route. Given the young age of patients, we aimed to develop a pharmaceutical liquid form of Met. A liquid dosage form facilitates administration to young children and adjustment of the dose, and improves the acceptability of the treatment ,9. BeingPharmaceutical grade Met pure powder was obtained from Fagron , sodium benzoate and xanthan gum pharmaceutical pure powders were obtained from Cooper . Pharmaceutical sodium carmellose, sodium citrate, and citric acid were supplied by Caelo . Sucralose was provided by Inresa pharmaceutical . Strawberry, orange, litchi, and candy floss flavors were purchased from Scrapcooking . Acetonitrile and orthophosphoric acid were of analytical grade, and purchased from VWR chemicals . Distilled sterile water was purchased from Aguettant .® software . A Polaris® C18 column  maintained at 30 °C using a column heater was used. The mobile phase consisted of acetonitrile and water (0.5/99.5 v/v) adjusted to pH 2.1 with orthophosporic acid. The column temperature, the mobile phase, the flow rate, the injection volume and the detection for detection and quantification were set at 30 ± 2 °C, 1.0 mL/min, 30 µL and 205 nm, respectively.Met assay was performed by a high-performance liquid chromatography (HPLC) system  with HPG-3200SD quaternary pump and WPS-3000TSL autosampler, coupled to a Dionex MWD-3000 diode array detector (DAD). HPLC system data acquisition  was carried out using the Chromeleon2) were calculated to study linearity. Accuracy was determined using quality controls, and expressed as the percentage of recovery determined by the following equation: The method was validated according to the guidelines by assessing the linearity, accuracy, specificity, and precision . Two ston = 3). The inter-days analysis was performed by calculating the RSD on each quality control recorded over three days (n = 9). Additionally, repeatability was estimated by recording 10 times a 200 µg/mL Met solution and calculating the RSD. Precision of the method was studied using quality controls recorded in triplicate on three independent days. The intra-days analysis was performed by calculating the relative standard deviation (RSD) of calculated compared to theoretical concentration on each quality control recorded in triplicate the same day (The limit of detection (LOD) and limit of quantification (LOQ) for Met were evaluated based on response standard deviation and calibration curve slope based on the following equations: LOD = The selectivity of the method was ensured so that no chromatographic peak of an excipient or degradation product coincided with that for Met. Chromatograms of Met in the presence of all formula excipients were visually inspected to detect changes in shape of the Met peak as well as by the analysis of the Met peak UV spectra recorded by DAD (200–300 nm).2O2) stress conditions for 72 h (H72) at 70 °C, and photolytic stress for 32 h (H32) with samples taken and analyzed at various time points. To perform the forced degradation study, a stock solution of Met with excipients  equipped with a xenon lamp and a daylight-Q filter (Q-Lab), which simulates natural sunlight (wavelength from 300 to 800 nm) operating in window mode and complying with ICH Q1B recommendations. The light beam obtained simulate CIE Standard Illuminant D65 (Q-Lab), with irradiance set at a constant intensity constant (1.5 W/m2 420 nm). The temperature was controlled and set at 23 ± 2 °C.Then, four stress conditions were applied on the Met formula described in ents see  was prepA powder mixture ready to be reconstituted with water has been formulated to obtain a suspension of Met at 200 mg/mL suitable for an oral administration in children. It was composed of a mix of pharmaceutical grade Met, and selected pharmaceutical grade anhydrous excipients such as preservative (sodium benzoate), viscosifier and thickener , pH buffer (sodium citrate and citric acid), sweetener , and aroma .m/v) were tested by the same taste scoring methodology.To make the taste and smell of the preparation more attractive, a fruit flavoring has been added in a selected amount: orange, cotton candy, lychee and strawberry. Five adults rated the Met formulas containing the selected fruit flavor on a subjective scale from 1 to 5, 5 being the best score for taste. Different amounts of sucralose (0.05–0.2% w/v) was performed to assess particle distribution and agglomeration. The uniformity of Met content was determined using two methods. First, after 10 s of manual bottle shaking, samples were taken (in triplicate) at three sampling levels: at the bottom, middle, and top of the suspension. These samples (n = 3) were analyzed for Met content by HPLC assay. Secondly, suspension samples  were withdrawn with an oral syringe  after manual bottle shaking for 10 s, and at different times after water reconstitution of the bulk powder: 1 h, 5 h, and 3 days. All samples were analyzed for Met content using the HPLC assay.Visual inspection of Met suspensions containing various amounts of xanthan gum  according to the manufacturer’s instructions. Briefly, the cup is immersed in the suspension at 25 °C until it is filled, and the time needed to leave the surface of the liquid until the first break point in flow is monitored. The kinetic viscosity is then calculated and transformed by mass volume correction to obtain absolute viscosity.The final optimized composition of the Met formulation was detailed in To prepare one container at 200 mg/mL Met powder oral formulation, ingredients are exactly weighed as described in Met stability studies were performed with the Met formula shown in Powder samples of each preparation were analyzed on days 0, 3, 7, 14, 21, 30, 45, 60, 75 and 90. After shaking, 200 mg of each sample were diluted into a 1000 mL volumetric flask with distilled water and mixed for 10 min before being analyzed by HPLC.Following water addition and shaking , aliquots of suspension from each preparation were obtained on days 0, 3, 7, 14, 21, 30, and 45. After shaking, a sample was collected and diluted to 1/1000th in water before being analyzed by HPLC.® Expert Pro-ISM pH sensor (Mettler-Toledo). A visual inspection was carried out by the examination of each sample under daylight and in front of a white background, under which aspect, color and odor of each sample were noted and after shaking for 30 s for Met suspension study. Potential hydrogen (pH) was measured with digital SevenEasy S20 pH meter  equipped with a calibrated InLabA microbiological stability study was performed  to evaluate the antimicrobial preservation efficacy of the reconstituted Met formulation. The test was performed as described by the European Pharmacopoeia v. 8.0 for non-sterile products. This test consists of voluntarily contaminating the Met suspension, using a calibrated inoculum of selected microorganisms and counting them after each selected time . Six strains of microorganisms were used, namely three fungi  and three bacteria . A neutralizing solution was used to ensure that any preservative effect of the formulation was neutralized for microbial counting. Microorganisms were counted in tryptic soy and Sabouraud agar culture media for bacteria and fungal culture, respectively. For each strain, one mL containing appropriate number of colony-forming units (CFU) per mL was added to nine mL of the neutralizing agent, for testing in the absence of the product. One mL of the previous suspension was added into Petri dishes for each medium. This procedure was also employed to the Met formula. CFU number per dish was done after an incubation of five days maximum at 30–38 °C for Trypticase medium, and three days at 20–25 °C for Sabouraud. The method was judged validated when CFU number in one mL of inoculated sample was at least 50% of that obtained in the control . At each time point, the logarithmic reduction in the number of viable microorganisms relative to the value obtained for the inoculum was calculated. This logarithmic reduction was then compared to the values as recommended (see European Pharmacopoeia chapter 5.1.3).After one year of Met suspension use, a questionnaire was given to the parents of 7 children . To evalData analysis was performed using Excel 365  and Prism . Descriptive statistics for continuous variables were expressed as mean ± standard deviation (SD) unless otherwise specified.2 = 0.994). The Met retention time was ~6.5 min. Selectivity was assessed (no peak deformation or change in UV spectra in the presence of excipients) and no matrix effect was demonstrated. As shown in n = 10). In addition, the accuracy profile was within the acceptability limits of 10% . Of the fruit flavors tested, the 5% strawberry flavor was found to be satisfactory in masking the Met odor (m/v). Below 0.1% of sucralose content, the preparation was not sufficiently sweet, while above 0.2% it was judged overly sweet.The quantities of some excipients were optimized to obtain a better acceptability in terms of odor, taste and uniformity of the content. Regarding odor and taste, a range of fruit flavors were tested in proportions from 1% to 6% (Met odor . To imprm/v). A visual inspection of the water reconstituted Met suspension containing xanthan gum at 0.3% showed a homogeneous particle distribution after manual shaking without powder agglomerate/cake over the 45 days of the study.The amount of xanthan gum used was studied from 0.1% to 0.5%. The preparation was judged to be overly thick and improper to withdraw it with oral dispenser with an amount of xanthan gum higher than 0.3%  for its antimicrobial properties at pH below 5, and citrate was added to the formulation for a target pH of 4.5 [n = 3). The density of the suspension having been measured at 1.02 ± 0.03 g/mL (n = 3) the absolute viscosity of the Met suspension corresponds to 425.1 mPa.s.Regarding the other excipients, sodium benzoate was added at a regular concentration of 0.3%  [Rare ILLD diseases characterized by double homozygous Ala393Thr/Ser576Leu mutations on To this end, we developed a 200 mg/mL Met bulk powder. An oral Met suspension is easily obtained after water reconstitution by caregivers or family. A stability indicating analytical assay was validated for measuring Met to ensure the stability validation of pharmaceutical Met formulation. The Met assay developed fulfills analytical and ICH requirements , and was shown specific for Met stability assays as no interference peaks were detected as demonstrated by the forced Met degradation studies performed with the formula containing excipients as detailed in Acceptability of drugs is crucial for children due to inherent characteristics of this population including swallowing difficulties and strong taste sensitivity ,19,20. AIn terms of palatability, Met is a sulfur amino acid with a strong and unpleasant smell and taste . OptionsCritical product quality attributes to be considered for oral suspensions include physicochemical characteristics of the solution such as viscosity, potential for foaming, air entrapment, sedimentation, and sticking of the suspended active substance to the primary container and to the measuring device. Where particles sedimentation cannot be avoided, easy re-suspension with moderate shaking is required to reduce the risk of dosing errors due to inhomogeneous distribution of the active substance. The risks of under- and overdosing to the child because of inadequate shaking should be considered. In view of the relatively low water solubility of Met (~56 mg/mL) , an oralThe physico-chemical stability study was demonstrated for 92 days at room temperature for the unreconstituted Met bulk powder formulation, and for at least 45 days at refrigerated or ~22 °C storage for the Met suspension. The preliminary results obtained from the stress degradation studies confirmed the sensitivity of Met for oxidation. The need for an antioxidant  was tested during the accelerated degradation test, without noticeable improvement . The phyA survey was given to the parents to evaluate acceptability of the Met formulation. It showed that all parents were satisfied with this Met suspension. Indeed, the survey indicated that the taste and smell of Met were improved. The taste and odor masking of Met could probably have been modified by the use of other strategies such as cyclodextrin or liposomal complexes. However, these strategies do not dispense with the addition of a flavoring which, in children, seems to be mandatory for a better oral acceptance, especially if the treatment is of long duration. Moreover, the choice was made to develop a formulation that could be more easily transposed to other pharmacy departments and without making the formulation too attractive (candy type), as the risks of overdose in Met are not well known .This study allowed the validation of an original formulation of Met in the form of an oral powder to be reconstituted. It was optimized in terms of odor and taste by using ingredients such as sucralose and strawberry flavors. Adequate dispersibility of Met in the suspension was ensured by the addition of thickeners. The stability study showed 92-day stability at 4, 23, or 40 °C for the bulk powder. Once reconstituted with water, microbiological and physicochemical studies confirm the stability of the suspension for at least one month. This pharmaceutical form appeared to be more suitable in terms of treatment administration and patient acceptability for the pediatric population, as shown by the survey conducted after at least one year of treatment. Indeed, it facilitates the gradual adaptation of dosages over time and the administration of the treatment by the parents to the children.


Biosurfactants are attracting much interest due to their potential application as therapeutic agents in the medical and cosmetic field. Previous studies have demonstrated that biosurfactant such as sophorolipid (SL) exhibits immunomodulatory effects. In this article, we found the potential of sophorolipid for inhibiting histamine-induced itch and preliminarily explored its molecular basis. First, behavioral tests indicated that SL can remit the histamine-induced scratching behaviors of mice. Second, SL can suppress the the calcium influx induced by histamine, HTMT and VUF8430 in HaCaT cells. RT-PCR analysis showed that the histamine-induced upregulation of mRNA levels of phospholipase Cγ1, 1,4,5-trisphosphate receptor (IP3R), and protein kinase Cα can be inhibted by SL, suggesting that SL may impede the PLC/IP3R signaling pathway activated by histamine. In further tests, the capsaicin-induced calcium influx can also be inhibited by SL. The immunofluorescence and molecular docking analysis indicated that SL acts as an inhibitor of transient receptor potential vanilloid-1 (TRPV1) activation to decrease calcium influx against stimuli. In summary, these results revealed that SL may inhibit histamine-induced itch by decreasing PLC/IP3R signaling pathway activation and modulating TRPV1 activity. This paper indicates that SL can be a useful treatment for histamine-dependent itch. Mast cells that produce histamine are usually located in the upper layer of the dermis of healthy skin, where the host organism is confronted with extraneous bacteria and antigens. Upon stimulation, mast cells will secrete histamine as a response4. Unmyelinated C-fibres, which are not responsive to mechanical stimuli, can be used to treat histamine-induced pruritus or sensitivity. Additionally, although itch-sensing neurons possess a repertoire of receptors that allows for a degree of direct irritants detection, itch is often initiated by epidermal and immune cells, and passed to sensory afferents through endogenous itch mediators5. Researches over the past decade have shown that itchy sensation is not purely a neuronal phenomenon but a complex involvement of skin, immune, and neural processes to complete this enormous task7. Histamine also has the capacity to stimulate human keratinocytes through histamine receptors, resulting in the upregulation of inflammatory cytokine, anti-microbial peptide and matrix metalloproteinase (MMPs), which can lead to inflammatory skin diseases9. Studies have indicated that H1 and H4 histamine receptors are strongly associated with allergic conditions, skin inflammation and itching10. Besides, H1 and H4 receptor antagonists are able to lessen the scratching behavior of those suffering from atopic dermatitis (AD)12. Recent research has suggested that the use of next-generation anti-histamines that target both H1 and H4 receptors may be an effective treatment for skin irritation and itch14.Itch (pruritus), a disagreeable sensation of the skin, is classified into two distinct types: histamine-dependent and histamine-independent. It is widely acknowledged that histamine is an endogenous molecule responsible for eliciting itch, acute inflammation, and other forms of rapid hypersensitivity reactions+ ions, capsaicin, and heat, which has been suggested to participate in itchy sensations as well as the transmission process of pain15. Research has revealed that TRPV1 is expressed not only in the central nervous system and sensory neurons but also in human epidermal cells17. An immunofluorescence study reported in 2001 was used on primary keratinocytes from healthy individuals to detect the presence of TRPV1 for the first time. Subsequently, the primary keratinocytes is identified as the source of TRPV118. Also, another research using in-vivo mice model only expressing epidermal TRPV1 gene has shown that the specific and selective activation of TRPV1 on keratinocytes is sufficient to induce pain and itch19. Studies conducted on cultured human epidermal keratinocytes (HaCaT) have demonstrated that the influx of calcium, mediated by TRPV1, can inhibite proliferation and induce apoptosis20. TRPV1 is also a key component in initiating physiological reactions following the activation of histamine receptors15. The activation of TRPV1 has been linked to the calcium-dependent production and release of signaling molecules such as PLC, IP3, diacylglycerol (DAG), and PKC22. It has been suggested that TRPV1 inhibitors can impede the increase of MMPs and interleukin 1β, 2, 4 caused by UV exposure, which are linked to inflammation23. Therefore, the activation of TRPV1 channel on keratinocytes is similar to that in dorsal root ganglion (DRG) and can be utilized as a target to gauge the intensity of itching.The transient receptor potential vanilloid-1 (TRPV1) channel, a type of non-specific cation channel, is activated when exposed to H25. Our previous investigation has demonstrated that SL is effective in suppressing the calcium influx in RAW264.7 cells stimulated with LPS25. Therefore, it can be proposed that SL may have the capacity to inhibit histamine-induced calcium influx and be further applied as a treatment for skin deseases, such as pruritus. This study aims to determine whether SL has the potential to inhibit histamine-induced itch and affect the mechanics of histamine-induced intracellular calcium elevation in keratinocytes. We anticipate that these results will provide a new perspective for the application of SL in the medical and cosmetic field.Recently, there has been a surge in the search for biodegradable, natural and environmentally friendly ingredients for the treatment of pruritus. Previous studies have shown that Sophorolipid has the ability to alleviate inflammation, which is attributed to the suppression of the NF-κB pathway and the consequent reduction in the abundance of cyclooxygenase-2 and inducible nitric oxide synthase25. Histamine, loratadine, JNJ7777120 and capsaicin were obtained from Aladdin Biochemical Technology Co. Ltd. . HTMT and VUF8430 were purchased from Kuma Biochemical Technology Co. Ltd. . Fluo-4 AM, rabbit polyclonal TRPV1 antibody, Cy3-labeled goat anti-Rabbit IgG antibody and Hanks' Balanced Salt Solution (HBSS) were obtained from Beyotime Biotechnology Co. Ltd. .A non-pathogenic strain of Candida bombicola  was used to produce SL through a fermentation process, as described in a prior study2 at 37 °C. The DMEM medium was supplemented with 10% (v/v) fetal bovine serum (FBS) . The cells were maintained by replacing the culture medium with a new complete medium every 2–4 days.Human epidermal keratinocytes (HaCaT) were sourced from the BeNa Culture Collection . The cells were cultivated in a modified version of Dulbecco's Eagle medium   at 37 °C in a humidified atmosphere with 5% COFemale mice of the C57BL/6 J inbred strain, aged 6–8 weeks, were procured from Shiyanjia Lab . Mice were housed in a temperature-controlled (22 ± 2 °C) and humidity-controlled (40–70%) animal room under a 12-h light/dark cycle, with free access to food and water. The study was performed in accordance with relevant guidelines and regulations of the Institutional Animal Care and Use Committee of the Jiangnan University. All experimental protocols were approved by the International Association for the Study of Pain. We followed the guidelines of ARRIVE .2) was depilated with electric hair clippers 24 h before starting the experiments. Mice were placed in a box (294 mm × 190 mm × 130 mm) for approximately 30 min for acclimatisation before each experiment. All drugs were injected intradermally with a 30G needle to the de-haired area behind the ear. Immediately after the injections, mice were recorded on video for 30 min and the number of scratch bouts counted in 5-min as a bin with an observer blinded to treatment. One scratch bout was defined as a lifting of the hind limb towards the injection site. The animals were divided into eight groups: four of them were used as control  drug groups; the rest of them including histamine, and different concentrations of SL plus histamine were used to observe scratching behavior. In the inhibited experiments, SL  was administered by intradermal injection 30 min before the subcutaneous injection of histamine . All drugs were injected in a volume of 100 μL.The back area of mice ear  staining was used to visualize epidermal thickness and inflammatory cells infiltration. A light microscope was employed to view sections at a magnification of 400×.For TRPV1 immunostaining, the skin segments of mice were first incubated with a primary rabbit polyclonal TRPV1 antibody (dilution factor: 1:100) overnight at 4–8 °C and then washed with PBS to remove the primary antibody. The sections were then incubated with the secondary Cy3-labeled goat anti-Rabbit IgG antibody for 1 h at room temperature. The nuclei were counterstained by 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) staining. All the sections were examined immediately and photographed with a laser confocal microscope .2+]i was quantified using the fluorescent probe Fluo-4 AM, a technique that has been reported in other study previously25. HaCaT cells were incubated in HBSS supplemented with Fluo-4 AM at a concentration of 5 μM and CaCl2 at 1.2 mM at 37 °C for a period of 30 min. Following Fluo-4 AM loading, the cells were washed three times with HBSS before being exposed to various samples for stimulation. The spectrofluorometer  was used to assess the fluorescence of Fluo-4-Ca2+ at excitation at 488 nm and emission in the range of 512–520 nm. Data was documented at intervals of 10 s.The influx of intracellular calcium concentration i influx to varying levels. SL still inhibited histamine-induced [Ca2+]i influx when H1 or H4 receptor blocker was present. In addition, we investigated whether the inhibitory action of SL in the presence of H1 or H4 receptor blocker can be blocked in a dose related manner. As shown in Fig. 2+]i elevation. Furthermore, we explored if SL can suppress the excessive intracellular calcium increased by specific histamine receptor agonists. As shown in Fig. In normal human epidermal keratinocytes, studies have demonstrated that the use of histamine receptor blockers can suppress the histamine-induced calcium ion influx28 IP3 binds with the specific IP3 receptor (IP3R) and is implicated in the regulation of calcium ion flow. It is well known that IP3 and DAG are capable of triggering the activation of the pertinent PKC, a kind of signaling molecule which is also closely related to the transportation of calcium ions22. Both extracellular and intracellular sources of calcium ions are essential for initiating and maintaining the reactions. As shown in Fig. PLC has a crucial role in the signaling pathways of histamine-induced itch networks. Upon activation of H1/H4 receptors, PLC is stimulated, resulting in the generation of IP3 and DAG2+]i influx in HaCaT cells. After pre-treatment with various concentrations of SL, we could observe an obvious decrease in fluorescence intensity of [Ca2+]i; however, the inhibitory effect of SL was not dose-dependent. To better understand the inhibitory effect of SL, we found applying SL alone does not stimulate intracellular calcium flow significantly compared to the Vehicle group (1% DMSO). This implies that SL may potentially modulate the activity of the TRPV1 channel and then reduce the [Ca2+]i influx triggered by capsaicin.To explore the impact of SL on the following downstream channels of histamine-induced fluctuation, we investigated the TRPV1 ion channel, a widely known pathway for calcium ion flow. Capsaicin was selected to be the activator of TRPV1 channel. As shown in Fig. 29, TRPV1 can be detected in the cytoplasm and surrounding the nucleus of HaCaT by immunofluorescence. To further clarify the antagonistic effect of SL against TRPV1 activation, we used immunocytochemistry detection to mark the presense of TRPV1 protein in HaCaT cells. As shown in Fig. According to previous articleTo examine SL’s action on histamine induced TRPV1 activation, we also conducted immunohistochemistry test of TRPV1 staining on mice skin epidermis after the mice were euthanized. As shown in Fig. The results of immunocytochemistry and immunohistochemistry indicated that SL can modulate TRPV1 activity against capsaicin and histamine.Finally, a molecular docking assay was used to determine whether SL has a direct interaction with TRPV1. The diacetylated lactonic SL ligand was docked with TRPV1 protein complex and the calculated binding free energy is -7.4 kcal/mol. As shown in Fig. 8. In medical clinics, anti-histamines are the most widely used medications to relieve itch30. A multitude of studies have demonstrated that natural plant extracts, such as osthole27, quercetin13 and red ginseng extract14 have the ability to suppress histamine-induced itch. This is the first investigation to suggest that SL, a kind of biosurfactant, can have an antipruritic action in C57BL/6 J mice against histamine, as well as alleviate further skin harm caused by scratching.In recent years, there has been an increase in researches on the treatment of pruritus, an uncomfortable sensation on the skin. Suppression of itching is indispensable because it not only alleviates the unpleasant sensation but also help prevent further scratching, which is generally a sign of several common diseases, such as thyroid disease and atopic dermatitis. It is widely accepted that histamine is the main stimulus of allergic and itching reactions28. The increase in intracellular calcium resulting from this phenomenon can lead to upregulated levels of of pro-inflammatory cytokines and may even induce apoptosis. It has been proved in previous studies that an overabundance of intracellular calcium can result in certain negative pharmacological effects associated with the receptor, the most common of which is pruritus32. The results of this article shown in Fig. Histamine is identified when it binds to histamine receptors, initiating the downstream signaling pathway and subsequently activating a number of intracellular signaling molecules, such as the adenylate cyclase and PLC pathways33. Among these receptors, histamine H1 and H4 receptors are essential components in the transmit of histamine-associated itch signals between epidermal cells and peripheral sensory nerve endings34. Previous studies have demonstrated that neither histamine H1 nor H4 receptor blocker can block histamine-induced scratching behavior completely. Only when both histamine H1 and H4 receptor antagonists are associated with the transmission process of the itch signaling to the central nervous system can the histamine-induced scratching behavior be nearly suppressed34. Also, Itch caused by specific receptor activators cannot be inhibited by other receptor blockers. For example, thiopamine, a H3/H4 receptor antagonist, has been found to be ineffective in reducing scratching behavior caused by HTMT35. Extensive research has been conducted in recent years to elucidate the effects of H1 and H4 receptors on histamine-induced calcium influx both in vitro and in vivo models11. It has been demonstrated by various researchers that in human epidermal keratinocytes, histamine receptor blockers can also play an inhibitory role in histamine-induced calcium ion influx32. To ascertain if the inhibitory effect of SL is associated with histamine receptors, we conducted experiments to evaluate if SL can further inhibit histamine-induced calcium ion influx when histamine H1 or H4 receptor blockers are present. The observations in this study suggested that SL can affect the action of histamine H1 and H4 receptor. In addition, the increasing intracellular calcium induced by specific activation of H1 or H4 receptors can be reduced by different concentrations of SL. It is conceivable that SL may not act as a direct selective agent of H1 or H4 receptors, but instead, SL plays a partial role via the interaction with H1 and H4 receptors to inhibit the following signaling transduction.Histamine action is widely recognized as being mediated by four distinct receptors, namely H1–H421. IP3 attaches to a particular IP3 receptor (IP3R) and is involved in the regulation of calcium ion movement. It has been reported that IP3 and DAG can stimulate the corresponding PKC, which is also linked with the conveyance of calcium ions22. We employed an RT-PCR analysis to examine the effect of SL on gene expressions that can be stimulated by histamine. Our research showed that histamine significantly elevated the mRNA levels of PLCγ1, IP3R, and PKCα and SL can reduce their expression to a certain degree.After histamine binded with its specific receptors, the subsequent PLC, IP3, and DAG signaling elements are activated, resulting in the opening of corresponding calcium ion channels. Then this allows for the increase of calcium influx, which will lead to the generation of action potentials and neuro-sensitivity28. Research evidence has indicated that TRPV1 is involved in the calcium response activated by histamine and the conduction of itch signals when the H1 or H4 receptor is stimulated15. We hypothesized that SL inhibits histamine-induced itching by modulating the activity of TRPV1, which was validated by demonstrating that SL was able to reduce the rise of [Ca2+]i caused by capsaicin (Fig. TRPV1 has been well-acknowledged as a vital ion channel during the downstream signaling process of histamine stimulationcin Fig.  and 7. M14. Therefore, for a comprehensive understanding of SL's antipruritic effect, further examination on chloroquine-induced itch is required.Although our article found the antipruritic effect of SL with keratinocyte and mice models, there are several limitations in the current study. For example, the result of 0.25% SL in scratching behavior is not consistent with the skin tissue change, which needs additional verification to explain the phenomenon. In addition, the mechanism of itching is complex and we need to investigate more molecular pathways and establish different models such as sensory neurons to confirm the function of SL. Apart from histamine-dependent itch, there is also the presence of itch that is not related to histamine. Chloroquine, an anti-malaria drug, has proved to induce histamine-independent pruritus as a side effectThis is the first research to indicate that SL, a kind of biosurfactant, is capable of alleviating histamine-induced itching by suppressing the activiation of the PLC/IP3R pathway and modulating TRPV1 ion channel activity in HaCaT cells, thus lowering the intracellular calcium level. This investigation suggests that SL may be a potential addition to medical and personal care products as it has an antipruritic effect and can help in relieving skin diseases.Supplementary Information.


Understanding the contribution of epigenetic mechanisms to cancer initiation became paramount since we know that most healthy tissues carry mutations, but only a minority undergo malignant transformation. We developed methodologies which allow locus-specific epigenetic editing of the DNA sequence, using a dCas9-effector fusion in primary human hematopoietic stem and progenitor cells (HSPCs) to investigate the impact of cancer-specific DNA methylation (DNAm) changes on cell physiology. We uncover principles of DNAm maintenance and the impact on mature cells as HSPCs differentiate in vitro and in vivo. CDKN2B (p15) promoter and ARF (p14) using dCas9-3A3L and observed DNAm spreading beyond the gRNA location. We find that despite a transient delivery system, DNAm is maintained during myeloid differentiation in vitro, and hypermethylation of the p15 promoter reduces gene expression. In vivo, edited human HSPCs can engraft the bone marrow of mice and targeted DNAm is maintained in HSPCs long term. Moreover, epigenetic changes are conserved and inherited in both myeloid and lymphoid lineages. Although the proportion of myeloid (CD33+) and lymphoid (CD19+) cells is unaffected, monocyte (CD14+) populations decreased and granulocytes (CD66b+) increased in mice engrafted with p15 hypermethylated HSPCs. Monocytes derived from p15 hypermethylated HSPCs appear to be activated and show increased inflammatory transcriptional programs. We believe these findings have clinical relevance since we found p15 promoter methylation in the peripheral blood of patients with clonal hematopoiesis. Our study shows DNAm can be targeted and maintained in human HSPCs and demonstrated functional relevance of aberrant DNAm on the p15 locus. As such, other aging-associated aberrant DNAm may impact hematopoiesis in vivo.Aging is associated with an abnormal increase in DNA methylation (DNAm) in human gene promoters, including in bone marrow stem cells. DNAm patterns are further perturbed in hematological malignancies such as acute myeloid leukemia but the physiological significance of such epigenetic changes is unknown. Using epigenetic editing of human stem/progenitor cells (HSPCs), we show that p15 methylation affects hematopoiesis in vivo. We edited the  The development of CRISPR/Cas9 for gene editing has revolutionized our ability to manipulate the genome –3. CRISP48CDKN2A) using dCas9 3A3L  in myelpatients –25, but + HSPCs from umbilical cord blood. From bulk and single-colony analyses, we show that in the absence of the editing tool DNAm is maintained at p14 and p15 as myeloid cells differentiate, with some CpGs remaining up to 99% methylated. We have combined epigenetic and genetic editing to demonstrate the feasibility of creating multiple aberrant hits within the same CD34+ cell. The edited human HSPCs can engraft murine bone marrow and recapitulate hematopoiesis with effects on differentiation in immune cell populations, which may lead to an inflammatory environment in vivo. p15 methylation is heritable in both myeloid and lymphoid lineages, further demonstrating the usability of this method for generating disease-relevant mouse models of human hematopoietic malignancy. Our study shows CRISPR/dCas9 DNAm editing in primary human HSPCs is possible and gives insights into the maintenance of de novo methylation during hematopoiesis in vitro and in vivo.In this study, we have optimized the delivery system for dCas9 3A3L to multiplex DNAm editing in human CD34SI Appendix, Fig. S1A). Human CD34+ cells were isolated from umbilical cord blood and cultured in serum-free conditions with cytokines Flt-L (fms-related tyrosine kinase 3 ligand), SCF (stem cell factor), and TPO (thrombopoietin) for 24 h (ARF), p15 (CDKN2B) and p16 (CDKN2A) gene promoters . After nucleofection, CD34+ cells were cultured with cytokines for a further 24 h before seeding into semisolid methylcellulose containing cytokines promoting myeloid differentiation in a colony-forming unit (CFU) assay. After 2 wk, colonies were counted and harvested for molecular analysis.To establish a method for targeting DNAm in HSPCs, we used the dCas9 3A3L and 3A3L-mut plasmids from our previous work , and othfor 24 h  before nromoters B. NucleoSI Appendix, Fig. S1C). In contrast to our previous work using these gRNAs . We checked publicly available chromatin immunoprecipitation data . We reason there are other structural or chromatin signals that preclude hypermethylation of p16 in HSPCs.We pooled colonies from the CFU-assay and using targeted bisulfite sequencing (bs-seq) found targeted DNAm across the p15 promoter  when using gRNAs targeting p15 alone and when using gRNAs to both p14 and p15 , indicating that different cytokine cocktails and culture conditions did not affect methylation maintenance. Finally, we found that increasing the total picomols of dCas9 3A3L mRNA and gRNAs nucleofected resulted in more hypermethylation at the p15 promoter after the CFU assay . We checked for successful epigenetic editing 2 d after nucleofecting CD34+ cells with dCas9 3A3L mRNA and gRNAs followed by culturing in serum-free conditions. We found that at 2 d postnucleofection a small percentage (<5%) of cells had evidence of DNAm at the p15 promoter when cells were nucleofected with a single gRNA targeting p15 . We speculate that this is most likely because the differentiated and dead cells in the population are not successfully edited.We also cultured cells in serum-free conditions for 14 d with SCF, Flt3-L, and TPO after DNAm targeting and found similar levels of methylation compared to the CFU assay . Gene expression of p16 and p14 was not significantly different between groups . This indicates that DNAm is maintained and the impact on gene expression persists at p15 as myeloid progenitor cells differentiate. We counted the total number of colonies formed to quantify whether targeting DNAm caused skewing of CD34+ cell differentiation, but there were no significant differences  all residing within CDKN2B and the same significantly hypermethylated CpGs comparing dCas9 3A3L to the nontargeting control . This showed there were no statistically significant off-target methylation effects maintained in the colonies.We analyzed genome-wide DNAm after the CFU assay using the Illumina Infinium MethylationEPIC (850k) array. In this experiment, we included an additional control group of dCas9 3A3L and a nontargeting gRNA, as well as dCas9 3A3L-mut, as active dCas9 3A3L has been shown to cause off-target hypermethylation . We founSI Appendix, Fig. S3 A and B). To assess whether this was dependent on the gRNA position, we designed a second gRNA for the p15 promoter , indicating that targeting different regions of the CpG island can change the efficiency of methylation deposition and spreading.The pattern of DNAm we observed across the p15 promoter was highly reproducible across different experiments  colonies or CFU granulocyte–monocyte (CFU-GM) as these are the largest colonies  and give robust targeted bs-seq data.The CFU assay offers a unique advantage compared to liquid culture as one can isolate individual myeloid colonies that have differentiated from a single CD34+ cells for 48 h and 72 h in serum-free conditions before seeding into the CFU assay and found 7 out of 10 colonies with similar levels of methylation maintenance after 14 d .As expected, there was little CpG methylation in the control group C. After We next wanted to know whether the pattern of hypermethylation at p15 had clinical relevance, as p15 is frequently methylated in myeloid neoplasm and AML –25. PrevSI Appendix, Fig. S5A). Although an unpaired t test with Welch’s correction showed no significant difference between the mean of the control vs. pre-AML groups, the F test to compare variances showed a significant difference; suggesting more variation in methylation at p15 in the pre-AML samples compared to control. To increase the power of this test, we analyzed a further 96 CHIP patient samples for p15 methylation , but found no significant difference in the mean methylation; however, the variance was again significantly different between groups. Taken together, these data indicate that p15 hypermethylation is detectable in the peripheral blood of some patients with CHIP, and pre-AML patients have increased variability of methylation at this promoter.To extend our clinical investigations, we also asked whether p15 hypermethylation is detectable in the peripheral blood of patients with clonal hematopoiesis of indeterminate potential (CHIP), with an identified known mutation. We selected samples from the European Prospective Investigation into Cancer (EPIC) cohort with known CHIP mutations in a previously published study . By perf+ cell . We found an A base insertion was the most frequent editing event using this gRNA . This insertion causes loss of function of TET2 protein in HSPCs . Targeted bs-seq analysis showed that TET2 editing alone does not result in hypermethylation at p15 ; and p15 was successfully methylated in the bulk double-edited colonies . Using targeted sequencing, we found that leaving 24 h between Cas9 targeting TET2 and dCas9 3A3L targeting p15 resulted in a small percentage of on-target mutations at the p15 promoter, increasing the time to 48 h between the editing steps prevented this on-target, erroneous editing .Next, to extend the disease modeling potential of this method, we established a protocol to create two sequential hits: genetic and epigenetic editing in the same CD34+ cell B. Our ai+ cell ], and hyin HSPCs  . We analyzed ten BFU-E colonies and found that one colony had both a homozygous TET2 mutation and over 40% average p15/p14 hypermethylation . Colony 1 was not edited at TET2, p15 or p14  and reconstitute the human hematopoietic system with no major differences in the proportions of myeloid (CD33+), lymphoid (CD19+), and progenitor (CD34+) cells (+) and increase in granulocytes (CD66b+) output from p15-methylated HSCs (+ population there were significantly fewer multipotent progenitors (MPP) and multilymphoid progenitor (MLP) cells as well as more granulocyte/monocyte progenitor (GMP) cells in the bone marrow of mice engrafted with p15-methylated HSCs  and the pattern of methylation across the p15 promoter was consistent with our in vitro data. We also seeded CD34+/CD38− cells harvested from the bone marrow into a CFU assay and saw similar numbers of colonies .Finally, to investigate the impact of p15 hypermethylation on normal hematopoiesis in vivo, we injected p15 methylated human hematopoietic stem cells (HSCs) into immunodeficient mice. We targeted methylation to p15 using dCas9 3A3L or used a nontargeting gRNA as the control in HSCs and injected them into NSG mice 48 h later. After 19 wk, the bone marrow was harvested in order to analyze the reconstitution potential and lineage output from p15-methylated HSCs. We found that p15-methylated HSCs were able to engraft (+) cells A and B. ted HSCs C. Furtheted HSCs D. StrikiSI Appendix, Fig. S9 A and B), particularly genes involved in antigen presentation (HLA genes) and proinflammatory cytokines  . Overall, the transcriptional analysis reveals that innate immune cells which have inherited p15 methylation from the HSCs in vivo could cause an exacerbated inflammatory environment.Monocytes and granulocytes harvested from engrafted mice were analyzed using RNA-seq. Both cell types show dysregulated transcriptional programs related to effector immune functions when derived from p15-HSPCs. In the monocytes, a significant proportion of the genes up-regulated are involved in mediating the activation of the immune response  F. Distin1B, TNF) , 31 appe1B, TNF) G, sugges+ cells, which will be useful for distinguishing the contributing role of aberrant events in initiating myeloid cancer. Finally, we have shown that p15 hypermethylated human HSPCs can reconstitute the bone marrow of mice and methylation was maintained for a least 19 wk, affecting the proportions of some progenitor cells, monocytes, and granulocytes in the mouse bone marrow as well as resulting in monocytes, which may create an inflammatory environment. This indicates that p15 hypermethylation may disrupt normal hematopoiesis over time, a finding that warrants further investigation based on our discovery of p15 methylation in CHIP patient peripheral blood.In conclusion, we have established a method for targeting DNAm using dCas9 3A3L in human HSPCs and shown that myeloid cells maintain methylation at p14 and p15 during differentiation, repressing the p15 gene. Individual colony analysis revealed some CpGs retained up to 99% methylation, indicating that this can be a persistent change in myeloid cells. We have also demonstrated the feasibility of dual (epi)genetic editing in CD34+, myeloid, and lymphoid lineages (up to 76% at some CpGs). p15 methylated stem cells successfully recapitulated normal hematopoiesis; however, after 19 wk, there were differences in the proportions of monocytes and granulocytes as well as some progenitor cell fractions. This finding implies that de novo promoter hypermethylation events in HSCs can be maintained permanently and may have an impact on hematopoiesis in vivo.Using an optimized form of CRISPR/dCas9 3A3L, we demonstrate that HSPCs can be edited to induce disease-specific DNAm signatures, and that this ectopic DNAm is maintained as cells differentiate into subsequent progeny in vitro and in vivo. Moreover, we show that hypermethylation of p15, as seen in myeloid disease, derails differentiation of HSPCs in vivo. Edited DNAm was also maintained at p14 and p15 gene promoters as myeloid progenitor cells differentiated in vitro, reducing p15 gene expression, without affecting colony-forming potential. Colonies had up to 99% methylation at some CpGs, suggesting de novo DNAm was permanently inherited during myeloid differentiation. P15 methylated stem cells can engraft the mouse bone marrow, and methylation is maintained in CD34The DNAm maintenance we found at p15 in vitro occurred in both the CFU assay and serum-free liquid culture. In the bone marrow of humanized mice, we showed that hypermethylated p15 is maintained in differentiated granulocytes, monocytes, and B cells, demonstrating both lymphoid and myeloid lineages inherit this DNAm from HSCs. This finding may be clinically relevant since we detected p15 hypermethylation in the peripheral blood of patients with clonal hematopoiesis, a finding in line with recent evidence of increased epigenetic age in CHIP patients , suggestA frequent problem when targeting de novo DNAm has been the potential for off-target effects, which can be substantial . Surpris33It is well established that deletion or silencing of the three crucial cell cycle regulators targeted in this study, located in close proximity in the genome  is common in many types of cancers . Specifi3842In our study, we saw no effect in vitro after targeting p15 methylation; however, after 19 wk of engraftment, we analyzed the mouse bone marrow and found differences in hematopoiesis with changes to the number of some progenitor fractions plus decreased monocytes and increased granulocytes. Monocyte gene expression changes indicate they are “preactivated” and may contribute to an increased inflammatory environment in vivo. This indicates that the biological impact of p15 hypermethylation on HSC function and differentiation, may only be revealed as cells age in the bone marrow, a clinically relevant finding given our detection of p15 hypermethylation in the peripheral blood of patients with clonal hematopoiesis; a predominantly aging-associated phenomenon with a risk of progressing to AML , 46. FutIt is likely that extrinsic factors in the bone marrow microenvironment could influence and further exacerbate the inflammatory response from p15-hypermethylated immune cells, which parallels recent work showing that HSCs with mutations in disease-relevant genes such as TET2 or DNMT3A have enhanced survival when exposed to infection or inflammation , 20. Fur+ cell using CRISPR/Cas9. Assessing how the combination of TET2 editing and p15 methylation impacts hematopoiesis in vivo would be an exciting future direction for this work. Furthermore, this method can be used to create different combinations of genetic and epigenetic edits in the same HSPC to test the functional impact on hematopoiesis, which will be a highly relevant model system in helping understand the functional impact of genetic and epigenetic changes that are being uncovered in CHIP patients.Finally, we demonstrated in vitro the technical feasibility of dual epigenetic and genetic editing in the same CD34In summary, we have adapted CRISPR/dCas9 delivery to target DNAm in HSPCs and have shown this is maintained at the p15 promoter during hematopoiesis in vitro and in vivo. Further investigation into the impact of p15 methylation in HSCs over time in vivo may reveal insights into why p15 is frequently hypermethylated in myeloid disease, unlike other cancers. By using CRISPR tools we can begin to separate the role that DNAm and gene repression plays from aberrant genetic mutations, to better understand processes that underlie cancer development.+ cells were enriched using the EasySep CD34+ Selection Kit II (StemCell Technologies). CD34+ cells were cultured in StemSpan SFEM II (StemCell Technologies) supplemented with human SCF (100 ng/µL), human FLT3 ligand (100 ng/µL) and human TPO  for 24 h before nucleofection using a 4D-Nucleofector system (Lonza). For in vivo engraftment experiments, we used the EasySep Human Progenitor Cell Enrichment Kit (Stem Cell Technologies) to deplete for lineage marker positive cells and HSPCs were isolated as described in detail previously (+CD38-) were seeded in StemSpanSFEM (Stem Cell Technologies) supplemented with 100 ng/mL rhFLT-3L, 100 ng/mL rhSCF and 100 ng/mL rhTPO. After 48 h cells were collected for CRISPR editing.Umbilical cord blood was obtained from full term donors at the Royal London Hospital (UK). The study was approved by the East London Ethical Research committee (REC: 06/Q0604/110) and all participants provided informed consent. Mononuclear cells were isolated by density centrifugation using Ficoll-Paque . For in vitro experiments CD34eviously . After s10.22025/2019.10.105.00004) has received funding from the Medical Research Council (MR/N003284/1 MC-UU_12015/1 and MC_UU_00006/1) and Cancer Research UK (C864/A14136). The genetics work in the EPIC-Norfolk study was funded by the Medical Research Council (MC_PC_13048). We are grateful to all the participants who have been part of the project and to the many members of the study teams at the University of Cambridge who have enabled this research. In total, 129 samples were selected based on the highest frequency of CHIP mutations from previously published data  using Benchling Biology Software (https://www.benchling.com/molecular-biology/) and modified synthetic single gRNAs (sgRNAs) were ordered commercially (Synthego).For DNAm targeting, the dCas9 construct was fused to the catalytic domain of DNMT3A  and portion of DNMT3L (dCas9 3A3L and dCas9 3A3L-mut) as described previously . The eff+ cells were nucleofected using a 4D-Nucleofector system (Lonza) in 20 µL of nucleofector solution using a nucleocuvette strip following the manufacturer’s instructions. Before nucleofection, 1.2 µg dCas9 3A3L or dCas9 3A3L-mut were combined with sgRNAs targeting p14, p15, and p16 to give a final concentration of 180 pmol of total sgRNA. After nucleofection, 80 µL StemSpan II was added to the nucleocuvette before transferring the CD34+ cells to a plate containing more StemSpan II media supplemented with cytokines as described above. For in vivo engraftment experiments, CD34+CD38− human cells were electroporated using the Neon system (Thermo fisher) with 1 µg dCas9 3A3L and 150 pmol p15 gRNA or a nontargeting gRNA as described.For DNAm targeting experiments, 50,000 CD34+ cells were combined with 6 µg Cas9 RNP (Integrated DNA Technologies), 150 pmol final concentration of sgRNA targeting TET2 and nucleofector solution to a final volume of 20 µL before nucleofection using the 4D Nucleofector. Cells were transferred to StemSpan II for 24 h before a second nucleofection with the dCas9 3A3L RNA and sgRNAs targeting p14 and p15 as described above.For dual genetic and epigenetic editing, CD34+ cells which had undergone DNAm targeting alone or dual genetic and epigenetic editing were cultured in StemSpan II supplemented with cytokines for 24 h before seeding into the CFU assay. CD34+ cells were counted, and 600 cells were seeded per plate into MethoCult Classic (StemCell Technologies) after DNAm editing, or 1,200 cells per plate after dual editing. Colonies were grown in an incubator for 14 d before counting and harvesting either bulk or single colonies. For single-colony harvest, individual colonies were picked using an EVOS light microscope (ThermoFisher Scientific) and deposited directly into a PCR tube for analysis.CD34DNA was extracted from bulk colonies using a PureLink Genomic DNA Mini Kit (ThermoFisher Scientific), and 250 ng was used for bisulfite conversion (Zymo) and eluted in 10 µL. For single-colony analysis, DNA was extracted by digesting cells using proteinase K (ThermoFisher Scientific) at 55 °C for 30 min and RNAse A for 5 min at RT before purification using AmpureXP beads (Beckman Coulter) and eluting DNA in 15 µL of PCR grade water. Half this volume was used for bisulfite conversion.2, 0.025 units/µL HotStarTaq, 200 µM of each deoxynucleoside triphosphate (dNTP), 0.20 μM forward and reverse primers]. The TET2 region to be sequenced was amplified using HiFi HotStart Taq (KAPA) and 10 ng of genomic DNA added to a mastermix . Primer sequences are listed in SI Appendix, Table S2.Regions to be sequenced in the p14, p15, and p16 promoters were amplified for 38 cycles using HotStarTaq (Qiagen) with bisulfite converted DNA added to a mastermix  in R usSE222181 .SI Appendix, Table S2) before qPCR to assess gene expression. Gene expression was normalized to RPS14 and data analyzed using the Pffaffl method (RNA was extracted from samples using Direct-zol (Zymo) including an in column DNAse I treatment, before cDNA conversion (ThermoFisher Scientific). cDNA was mixed with SYBR green (BioRad) and primers . Reads were trimmed using Trimgalore v0.6.5 and mapped using hisat2.2.1 to the hg38/GRCh38 genome assembly. Gene counts were generated using featurecounts v2.0.3 . DESeq2 ene sets  were useene sets . Raw reaSE222181 .W-41J Prkdcscid Il2rgtm1) were originally obtained from The Jackson Laboratory. Mice were bred in isolators with aseptic standard operating procedures in the Biological Research Facility of The Francis Crick Institute. Once weaned, mice were kept in ventilated cages. All animal experiments were performed under the U.K Home Office project license (70/8904) in accordance with The Francis Crick Institute animal ethics committee guidance. NSG-S cKitw41/w41 mice aged between 8 and 12 wk were used. Forty eight hours after the CRISPR editing with dCas9 3A3L, and p15 gRNA or a nontargeting gRNA as described, cells were engrafted into mice via intravenous injection. After 19 wk, mice were killed, the bone marrow harvested and cell populations analyzed via flow cytometry  and Appendix 01 (PDF)Click here for additional data file.


To investigate the effect and mechanism of polydatin on bleomycin (BLM)‐induced pulmonary fibrosis in a mouse model. The lung fibrosis model was induced by BLM. The contents of TNF‐α, LPS, IL‐6 and IL‐1β in lung tissue, intestine and serum were detected by ELISA. Gut microbiota diversity was detected by 16S rDNA sequencing; R language was used to analyse species composition, α‐diversity, β‐diversity, species differences and marker species. Mice were fed drinking water mixed with four antibiotics  to build a mouse model of ABx‐induced bacterial depletion; and faecal microbiota from different groups were transplanted into BLM‐treated or untreated ABx mice. The histopathological changes and collagen I and α‐SMA expression were determined. Polydatin effectively reduced the degree of fibrosis in a BLM‐induced pulmonary fibrosis mouse model; BLM and/or polydatin affected the abundance of the dominant gut microbiota in mice. Moreover, faecal microbiota transplantation (FMT) from polydatin‐treated BLM mice effectively alleviated lung fibrosis in BLM‐treated ABx mice compared with FMT from BLM mice. Polydatin can reduce fibrosis and inflammation in a BLM‐induced mouse pulmonary fibrosis model. The alteration of gut microbiota by polydatin may be involved in the therapeutic effect. All mice were housed in specific pathogen‐free conditions in an animal facility with controlled humidity (50%–60%) and temperature (22–24°C) and under a 12 h light–dark cycle (7 AM to 7 PM) with free access to food and drink. All procedures were sanctioned by the ethics committee of Zhejiang Chinese Medical University.2.2The pulmonary fibrosis model was established by intratracheal BLM instillation in mice. Briefly, the mice were anaesthetised by intraperitoneal injection of pentobarbital (50 mg/kg). Anaesthetised mice were placed in a supine position and fixed on a sterile operating table, with the head high and the feet low and the neck skin fully exposed. Ophthalmic scissors were used to cut the neck skin approximately 2 cm along the midline after routine disinfection of the anterior neck area. The trachea of mice was fully exposed after blunt dissection, the trachea was punctured by bending the 1 mL syringe needle at an obtuse angle, the needle was inserted approximately 1.5 cm along the wall of the trachea, and the prepared BLM (5 mg/kg [5000 IU/kg] according to the body weight of micen = 6) with different tracheal instillation solutions and different follow‐up treatments: (1) Control group: intratracheal administration and intragastric administration of normal saline; (2) BLM group: intratracheal administration of BLM as above mentioned and intragastric administration of normal saline; (3) Control + polydatin group: intratracheal administration of normal saline and intragastric administration of polydatin ‐induced bacterial depletion was developed. Mice to be used for faecal transplantation were treated with Abx in advance. Mice were fed with drinking water mixed with four antibiotics  for 3 weeks following the method described previously.2.3The blood sample was collected, and the supernatant (serum) was stored at −80°C for further experiments. The faeces were collected for 16S rDNA sequencing or faecal transplantation. The left lung tissues and/or intestinal tissues were collected for pathological and ELISA analyses. The right lung was collected, and incubated at 60°C for 3–4 days to remove all moisture. Subsequently, the dry weight was measured, and the ratio of wet‐to‐dry weight was calculated.2.4Fresh faeces were collected from the control, BLM, control + polydatin and BLM + polydatin groups. Fresh faeces were collected from mice in the morning using the stress defecation method. The lower abdomen of the mice was gently pressed, and four to five complete faeces were collected from each mouse and stored in a sterilized centrifuge tube. The faeces of all mice in the target group were placed in the same centrifuge tube. The collected fresh faeces were added to sterilized saline at a ratio of 100 mg:1 mL, mixed by vigorous vortexing and centrifuged at 4°C for 3 min at 1200 rpm. Then, 200 μL supernatant was taken for enema administration in Abx‐treated mice (once per day for 2 weeks). The faecal microbiota supernatant from the control group, BLM group, control + polydatin group, and BLM + polydatin group were named control FMT, BLM‐FMT control‐Poly‐FMT, and BLM‐Poly‐FMT, respectively. The Abx‐treated mice were divided into four groups, and each group was named according to the control or BLM model and the origin of faecal microbiota to be transplanted: (1) Control + Control‐FMT; (2) BLM + BLM‐FMT; (3) Control + Control‐Poly‐FMT; and (4) BLM + BLM‐Poly‐FMT.2.5QIAamp Fast DNA Stool Mini Kit  was used for DNA extraction of faecal microbiota. Universal primers were designed using the gene fragment of the conserved region V3V4 of bacterial 16S rDNA, and PCR amplification was performed using the forward primer 341F: CTACGGGNGGCWGCAG and the reverse primer 805R: GACTACHVGGGTWTCTAATCC. The sequence of the V3V4 region was sequenced on the Illumina NovaSeq system.2.6α‐diversity is an indicator of reaction richness, diversity and evenness. α‐diversity was evaluated using QIIME2 software. The observed species, ACE, and Chao1 indices were used in this study. β diversity refers to the replacement rate of species along the environmental gradient among different communities or the dissimilarity of species composition. β‐diversity was evaluated using principal coordinate analysis (PCoA) and nonmetric multidimensional scaling (NMDS). The above analysis was completed using the R language (4.02) and QIIME2 (QIME2‐2020.2) as previously described.2.7The mice were euthanized under anaesthesia. For the haematoxylin and eosin staining, the lung tissues were quickly removed after opening the thoracic cavity, washed using precooled PBS solution, fixed in 10% neutral formalin, routinely embedded in paraffin and sectioned 48 h later. The paraffin sections were dewaxed with xylene, rehydrated with gradient concentrations of alcohol, washed with PBS and immersed in haematoxylin staining for 10 min. Stained slices were observed under an inverted microscope. For the Masson staining, paraffin‐embedded sections were deparaffinized with xylene, dehydrated with a gradient of alcohol, washed in PBS, and stained for 10 min with a 1:1 mixed Weigert's haematoxylin solution. After being differentiated for 5–15 s with acidic alcohol differentiation solution, the sections were coloured for 3 min with Masson staining solution and stained for 5–10 min with Ponceau S staining solution. The stained slices were observed under an inverted microscope.2.8The sections were routinely deparaffinized and rehydrated in gradient concentrations of alcohol. Antibody incubation was performed after antigen retrieval, blocking of endogenous peroxidase and blocking of nonspecific binding sites. The primary antibodies used were: collagen I  and α‐SMA . After overnight incubation, the subsequent steps were completed using a Pv 9000 General Purpose Two‐Step Immunohistochemical Detection Kit and DAB chromogenic solution.2.9Cells were lysed using RIPA lysis buffer  to obtain protein samples. After measuring the protein concentration with the BCA Kit (Beyotime), the corresponding volume of protein was added to the loading buffer (Beyotime), mixed well and heated in a boiling water bath for 5 min to denature the protein. Electrophoresis was carried out to separate the proteins. The membrane transfer was carried out on an ice bath with a current of 220 mA for 120 min. The membranes were rinsed for 1–2 min, put into the blocking solution for 60 min at room temperature, and incubated with primary antibodies against collagen I , α‐SMA  or GAPDH  overnight at 4°C. The membranes were washed and incubated with the secondary antibody  for 1 h at room temperature. After dripping the developer solution onto the membrane, signal detection was performed using a chemiluminescence imaging system (Bio‐Rad).2.10TNF‐α, lipopolysaccharides (LPS), IL‐6, and IL‐1β in the lung tissue, intestine and serum of mice were detected in each group according to the instructions of the ELISA kit . All operations were carried out in strict accordance with the instructions.2.11t‐test or one‐way anova, and the Tukey method was used for multiple comparisons. When p < 0.05, the difference was considered statistically significant.The experimental data obtained are expressed as the mean ± SD. GraphPad Prism 8.0 software was used for the 33.1The pathological changes and collagen deposition in mouse lung tissues were evaluated using haematoxylin and eosin and Masson staining. Figure Masson staining showed consistent results, as no collagen deposition was found in the lung tissue of the mice in the control group and the control + polydatin group, while collagen deposition was the most severe in the BLM group. Compared with the BLM group, collagen deposition was significantly reduced in the BLM + PD group. These results indicated that the BLM model was successfully established and that polydatin could effectively ameliorate BLM‐induced lung injury and collagen deposition in the mouse model. The levels of fibrotic markers, collagen I and α‐SMA, were also measured by IHC and Western blot. As shown in Figure 3.2To further evaluate the pulmonary inflammation of the mice in each group, we used ELISA to detect the levels of TNF‐α, LPS, IL‐6 and IL‐1β in the lung tissue, intestine and serum of the mice. Figure 3.3First, rarefaction curves (rank‐abundance) were used to assess whether the sequencing volume was sufficient to cover the species abundance in the sample. Figure 3.4Muribaculaceae, Lactobacillus, Lachnospiraceae, Akkermansia and Allobaculum. The main dominant species in the BLM model group were similar to those in the control group, and the abundance of Pseudomonas, Xylanophilum and Parabacteroides was relatively high in individual samples. The dominant species in the BLM + polydatin group were Muribaculaceae, Lachnospiraceae, Lactobacillus, Dubosiella, Akkermansia, Alistipes, Allobaculum and Parasutterella. Among these microflora, Akkermansia, Lactobacillus and Dubosiella are probiotics, and Lachnospiraceae is a pernicious bacterium. These results indicated that the BLM model and polydatin could alter the dominant microflora in the gut microbiota.Due to the intergroup differences in the gut microbial communities of mice in each group, a Venn diagram was used to analyse shared microflora. As shown in Figure 3.5Muribaculaceae, Lactobacillus, Akkermansia, Alloprevotella and other flora were less abundant in the BLM group. However, compared with the BLM group, the abundance of probiotics Muribaculaceae, Lactobacillus, Akkermansia, Alloprevotella and other flora in the BLM + polydatin group increased significantly  and suppresses the formation of reticular structures in lung tissue.Streptococcus pneumoniae who are receiving antibiotic therapy.Lactobacillus and Bifidobacterium supplementation in children can help improve asthma symptoms and reduce seizure frequency.The gut microbiota is acknowledged as a key innate immune system regulator that is engaged in the immunological development of the intestinal mucosa and impacts immune system development.Dubosiella, a type of probiotic, only existed in the polydatin treatment group. Dubosiella, belonging to the family Erysipelotrichaceae, was found to be significantly reduced in high‐fat diet‐ and STZ‐induced diabetic mice.Dubosiella and decrease the relative expression of activating transcription factor 4 to inhibit blood pressure in mice.Muribaculaceae, Lactobacillus, Akkermansia and Alloprevotella, were detected in all BLM mice but with altered abundance in the presence or absence of polydatin treatment. Polydatin treatment significantly increased the abundance of Muribaculaceae, Lactobacillus, Akkermansia, and Alloprevotella compared with the BLM group, indicating that polydatin could affect the composition of the mouse gut microbiota by increasing probiotic abundance. Lactobacillus was shown to protect mice against influenza virus‐induced pathology and mortality.Muribaculaceae mediated salmon peptide fraction‐induced improvement of metabolic syndrome.Akkermansia muciniphila was associated with the clinical benefit of immune checkpoint inhibitors in patients with non‐small cell lung cancer or kidney cancer.Alloprevotella was reported to be decreased in COPD patients with declining function.BLM stimulation with or without polydatin treatment caused no significant changes in gut microbiota diversity; however, the dominant species and corresponding abundance were altered. Although BLM mice with or without polydatin treatment shared the most dominant species, Complementary probiotics or FMT have been proven to restore the gut microecology and reduce inflammation in acute lung injury and pneumococcal pneumonia.In conclusion, polydatin alleviated BLM‐induced PF and altered gut microbiota species abundance. Moreover, polydatin‐induced alteration of the gut microbiota may also be involved in the therapeutic effect of polydatin in PF.Jia Yang: Conceptualization ; formal analysis ; methodology ; writing – original draft . Xiawei Shi: Data curation ; methodology ; validation ; visualization . Rundi Gao: Data curation ; methodology . Liming Fan: Investigation ; methodology . Ruilin Chen: Investigation ; visualization . Yu Cao: Investigation . Tingzhen Xu: Conceptualization ; writing – review and editing . Junchao Yang: Conceptualization ; funding acquisition ; supervision ; writing – review and editing .The authors declare no conflicts of interest in this work.