Modelling in anaerobic digestion will play a crucial role as a tool for smart monitoring and supervision of the process performance and stability. By far, the Anaerobic Digestion Model No. 1 (ADM1) has been the most recognized and exploited model to represent this process. This study aims to propose simple extensions for the ADM1 model to tackle some overlooked operational and metabolic aspects. Extensions for the discontinuous feeding process, the reduction of the active working volume, the transport of the soluble compound from the bulk to the cell interior, and biomass acclimation are presented in this study. The model extensions are included by a change in the mass balance of the process in batch and continuous operation, the incorporation of a transfer equation governed by the gradient between the extra- and intra- cellular concentration, and a saturation-type function where the time has an explicit influence on the kinetic parameters, respectively. By adding minimal complexity to the existing ADM1, the incorporation of these phenomena may help to understand some underlying process issues that remain unexplained by the current model structure, broadening the scope of the model for control and monitoring industrial applications. Anaerobic digestion (AD) is expected to play a crucial role in tackling climate change and assisting in the transition toward a circular economy . ModelliRegardless of the model complexity, modelling, as such, is an abstraction of the reality, since several assumptions, considerations, and simplifications are included. In fact, even the ADM1 model can be considered as a parametric gray-box approach . These aThis study aims to analyze some aspects that have not been considered in AD modelling and to evaluate their incorporation into the ADM1 model in a simple way. Two operational aspects, namely the feeding strategy and the working volume reduction, and two metabolic aspects, the extra- intra- cellular transport and the adaptation, are tackled with new extensions of the ADM1 model proposed in this study. We aim at broadening the scope of the model by adding minimal complexity to the existing common model base.Four model extensions are proposed, two regarding operational aspects and two regarding metabolic aspects. Before describing the model equations, we provide theoretical background and the rationale behind the extensions that are proposed in the present study. For modelling purposes, a typical assumption is that the digester is continuously fed, which does not occur in reality. A continuous feeding rate is suggested to avoid process imbalances in digester operation . Most fuAlong with the substrate, particularly with semi-solid waste, such as sludge or manure, inorganic materials, such as sand and grit, will enter the digester tank and accumulate due to sedimentation at the bottom or in dead zones where the agitation system is less effective. This buildup will lead to the need for maintenance and the cleanup of the tank. This should be somehow monitored to anticipate the moment it will need to be carried out . FurtherThe ADM1 model considers that any change in substrate composition will have an immediate impact on the growth rate of the microorganisms. Specifically, any change of soluble substrate will have an immediate influence on the biomass kinetics. This is clearly seen by looking at the expression of the growth rates which are based on the Monod kinetic. Of course, the instantaneous effect can be partially smoothed depending on the values of the affinity constant and the maximum substrate consumption rate. Likewise, the same instantaneous effect will take place for substrate inhibition. It is known that the cell membrane is impermeable to water-soluble compounds, such as glucose, amino acids, and LCFA, which means that a transport mechanism takes place inside the cell, and it has its own kinetics. Once inside the cell, these molecules enter their catabolic pathways. The transport will need time that, compared to the traditional modelling, will lead to a certain delay of the biomass response. This delay has been studied in other microorganisms such as microalgae . The impMicrobial adaptation has been a widely studied topic in AD because the capacity of microorganisms to adapt will determine the robustness of the process performance. Adaptation can be related to a change in the abundance of specific microbial species , population shift or change in dominance, or a mutation that results from changes in the activity of an enzyme that will provide a selective advantage by allowing growth under new conditions ,25. Partc) is set to zero so that the particulate material will enter the hydrolysis reaction directly partitioned as carbohydrates, proteins, and lipids. The hydrolysis and the disintegration constant were set at 0.5 d−1. The latter remains active only for the transformation of the biomass that has gone through decay. The rest of the parameters were set equal to the values presented by Rosen and Jeppsson [The base for the model extensions is the ADM1 developed by Batstone et al.  togetherJeppsson .The feeding strategy extension does not entail a modification of the model itself, but instead implies that the algorithm that solves the equations is divided in two operation modes working in a cycle: a batch reactor when no feeding is occurring and a continuous reactor when the feeding is taking place. The final conditions of a batch period correspond to the initial conditions of a continuous one, and vice versa. The mass balance for a substrate The working volume reduction comprises a modification of the mass balance for each state variable since the volume is no longer constant, so the classic mass balance of the chemostat needs to change. Because there is still an inlet and exit of media, the resultant balance is a mix between the balance for a fed-batch reactor and a continuous one. The general mass balance for a substrate 3 d−1 units. This parameter depends on the substrate characteristics, the mixing, and the pumping system, just like other apparent parameters such as the So, the question is, how does the working volume diminish over time? Here, we assume that the volume reduction is linear as a function of time (modelled by Equations (5) and (6), in its integral and differential form, respectively), with the slope , where only one new parameter is added to the model, the deposition rate alpha .The expression that represents the rate of transfer between the bulk and inside the cell, given by Equation (9) for the case of sugar consumers, is similar to the one used to model liquid-gas transfer. This means that the phenomenon is governed by the gradient of the soluble monomer concentration between the exterior and the interior and a transport coefficient.In the case of the adaptation model extension, an explicit time-dependent function was added to the model. Considering that the adaptation process usually requires the microorganisms to develop a new enzymatic machinery, and as a first approach to the issue, the hydrolytic constant for the particulate carbohydrates was selected to be modified as the biomass adapts. Using Equation (10), the hydrolytic constant is now expressed as a function of time by a saturation-type equation.® and the solver ode15s was used to solve the ODE system. A virtual digester of 330 m3 total volume (300 m3 working volume) operating at a hydraulic retention time (HRT) of 30 d (continuous flow of 10 m3 d−1) at mesophilic conditions (35 °C) was considered. The inlet characteristics of the substrate and the assessment conditions for each model extension are presented in The model and the extension were implemented and simulated in Matlab 20212 and HCO3. Thus, more (or less) CO2 is dissolved in the liquid phase , causing a change in the CO2 and CH4 concentrationThe biogas production, the biogas composition, and the pH of the digestate for 18 days of operation for each of the assessed feeding strategies are shown in As observed, the methane content in general is below than the average expected values (60–65%), which is explained by the fact that, for this study, the original values of the ADM1 were used. According to some studies, the value of some coefficients related to the methane formation for carbon need to be adjusted higher ,33.The pH is less affected by the feeding strategy than the biogas flow, which is expected since the variables measured in the liquid media are normally more robust due to the influence of the working volume and the HRT of the process. Nevertheless, some pH variations are observed, but they do not reach values that could be considered dangerous for the digester operation since they remain close to neutrality (depending on the lower and upper limits for pH inhibition). 3 d−1 of the accumulation rate of inert material, the impact on biogas production is low. Indeed, at this rate, the biogas decreases by 5% due to the loss of working volume. At a rate of 0.6 m3 d−1, the biogas production decreases 16% compared to the values when no working volume is lost . At higher accumulation rate values, the reactor collapses with a sharp drop in the biogas production and pH associated with the washout of the methanogenic biomass. It is interesting to note that the effect of the working volume has, for a long period, a steady negative effect on the biogas production that steadily decreases due to the reduction of the biomass activity. However, the collapse of the system could occur in a matter of a few days. In this case, the system will need to be stopped and reinoculated completely with all the operational and waste management consequences that a situation such as this entails. By having a well-established model, this accumulation rate could be estimated from the biogas production and could be used to estimate the need for a clean-out and digester maintenance.The biogas production, composition, and the pH behavior in the digester, for a year of operation, at different accumulation rates of solid material (α), are shown in −1, the system operates without a limitation given by this transport process. This can be related to a system well adapted to the substrate, where the transport of monomers to the interior of the cell is a consolidated mechanism. Under these conditions, the concentration inside the cell is similar to the one in the bulk. Thus, similar biogas production values are obtained. At lower values of the transport coefficient, −1), the measured variables begin to be limited by this extra- and intracellular transport process, and the system reaches a steady-state with lower biogas production accompanied by a modified abundance of the microbial species given by the steady-state substrate concentrations. Similar results were obtained for a culture of Escherichia Coli in a chemostat [−1, the biogas is mostly produced by no methanogenic biomass. Thus, it is composed mainly of CO2, whereas at values of 0.01 d−1, the biogas drops to almost zero, with a minimum generation of biogas made up of H2 and CO2. As the transport coefficient values decrease, the pH values tend to level off to lower values which are explained by the low generation of VFAs due to the transport restrictions. The fact that no extra- or intra- cellular transport is normally considered can lead to an overestimation of the digester performance as well as an anticipation of the system’s response since the delay related to this transport is not accounted for. This delay depends on the types of compounds that need to enter the cell. In this study, the same transport coefficient value was considered for all the monomers, which is probably not very accurate in a real case. For instance, the light required in a microalgae culture has a very fast transport kinetic, and thus a minimum delay is observed [The response of the biogas production flow, the biogas composition, and the pH of the digestate regarding the proposed extra- and intracellular transport kinetics extension is shown in hemostat . Lower vobserved , whereasobserved ,34,35. Iobserved .The extra- and intracellular transport can cover some aspects concerning the biomass adaptation response, but it still considers a fixed set of parameters. In this case, the adaptation extension of the model includes the dynamic evolution of the hydrolytic coefficient over time. In the incorporated function for this extension, the values of 3 d−1 are obtained. This behavior can be explained by a higher accumulation of VFAs as the time goes on and the adaptation process moves along, and it comes as a response to the previous period where the biogas values are notoriously below the base case where the process is strongly limited by the hydrolysis reaction. The fact that this extension affects the hydrolysis reaction leads to the methane content having the opposite behavior, as when adK increases, the methane content also increases. This effect upon the hydrolysis reaction has a more important influence on CO2 production, which makes the biogas composition to behave this way. With In general, the pH does not seem to be significantly affected in the representation of the adaptation process. It remains between recommended values to operate digesters.The simulated response of a digester when a new (co)substrate is added on day 100th is presented in Making the kinetic parameters or stoichiometric coefficients become variables of the model has been evaluated in a handful of studies. Overall, most of these studies have focused on the stoichiometry of the ADM1 representing the anaerobic digestion process. The catabolic yield for acetate and butyrate were defined as a function of the hydrogen concentration and the pH in the digester by Rodríguez et al. , althougAs one may think, these theoretical approaches presented here need to be validated with experimental results, particularly with the extensions where new parameters were incorporated. For the working volume reduction extension, the operation in a (semi) continuous mode of a sludge digester at the same HRT and OLR for long period of time can be used to calibrate the accumulation rate parameter. Under those conditions, one can expect that the only variable that can change the reactor performance is the reduction of working volume due to the accumulation of inert material. In the case of the extra- intra- cellular extension, the addition of a specific soluble substrate in a pulse manner can be carried out while observing the system response in terms of biogas flow and pH. This way the transport coefficient can be estimated depending on the observed delay between the system’s disturbance and the system’s reaction to it. For the adaptation extension, an experiment where a sudden change of the type of substrate used, without changing the total OLR and the HRT, could be used to estimate the adaptation coefficient.Four new extensions to the ADM1 model have been proposed to cover two operational aspects, namely the feeding strategy and reduction of the working volume as a result of solid material accumulation, and two metabolic aspects, namely the transport of monomers from the bulk to the cell and the biomass adaptation to a substrate given the time of exposure. The feeding strategy can influence the curve of biogas production and, consequently, the quality sensors used to measure it, and it has an effect on the downstream process for biogas purification and storage. The average daily production is not affected as long as inhibitory levels of some intermediate variables are not exceeded. The reduction of the working volume of the reactor depends on the rate of material accumulation, which can be estimated from the biogas production data. This phenomenon leads to a reduction of the activity of the microorganism and eventually to a total collapse of the anaerobic digester. The extra- and intracellular transport kinetics, regarding the soluble compounds, considered in the model application allows us to account for the delay in the response of the system compared to a conventional instantaneous model kinetic. Variable kinetic parameters as a function of the time of exposure can be considered to obtain a more realistic picture of the adaptation that takes place during, e.g., the start-up of the digester or the addition or replacement of the substrate fed into the reactors.


Background: Knee osteoarthritis (KOA), a chronic degenerative disease, is mainly characterized by destruction of articular cartilage and inflammatory reactions. At present, there is a lack of economical and effective clinical treatment. Zhuifeng Tougu (ZFTG) capsules have been clinically approved for treatment of OA as they relieve joint pain and inflammatory manifestations. However, the mechanism of ZFTG in KOA remains unknown.Purpose: This study aimed to investigate the effect of ZFTG on the TLR4/MyD88/NF-κB signaling pathway and its therapeutic effect on rabbits with KOA.Study design:In vivo, we established a rabbit KOA model using the modified Videman method. In vitro, we treated chondrocytes with IL-1β to induce a pro-inflammatory phenotype and then intervened with different concentrations of ZFTG. Levels of IL-1β, IL-6, TNF-α, and IFN-γ were assessed with histological observations and ELISA data. The effect of ZFTG on the viability of chondrocytes was detected using a Cell Counting Kit-8 and flow cytometry. The protein and mRNA expressions of TLR2, TLR4, MyD88, and NF-κB were detected using Western blot and RT-qPCR and immunofluorescence observation of NF-κB p65 protein expression, respectively, to investigate the mechanism of ZFTG in inhibiting inflammatory injury of rabbit articular chondrocytes and alleviating cartilage degeneration.Results: The TLR4/MyD88/NF-κB signaling pathway in rabbits with KOA was inhibited, and the levels of IL-1β, IL-6, TNF-α, and IFN-γ in blood and cell were significantly downregulated, consistent with histological results. Both the protein and mRNA expressions of TLR2, TLR4, MyD88, NF-κB, and NF-κB p65 proteins in that nucleus decreased in the ZFTG groups. Moreover, ZFTG promotes the survival of chondrocytes and inhibits the apoptosis of inflammatory chondrocytes.Conclusion: ZFTG alleviates the degeneration of rabbit knee joint cartilage, inhibits the apoptosis of inflammatory chondrocytes, and promotes the survival of chondrocytes. The underlying mechanism may be inhibition of the TLR4/MyD88/NF-kB signaling pathway and secretion of inflammatory factors. Knee osteoarthritis (KOA) is a chronic osteoarticular disease with a higher incidence during middle age, especially in the joints, and it is associated with the spine, knee, and hip joints. KOA is characterized by degeneration of articular cartilage, subchondral bone sclerosis, and osteophyte formation . OA is aEarly joint pain is the first symptom. The degree of pain becomes increasingly serious with increase in joint activity, affecting the quality of life of patients and eventually causing disability . TherefoModern medicine recognizes that KOA is a disease of the whole joint, including lesions of various tissues . HoweverUnlike the acquired immune system, the innate immune mechanism is mainly involved in tissue repair, wound healing, apoptosis, and cell debris removal, serving as the initial line of defense against infection. The immune cells of the innate immune mechanism mainly include macrophages, granulocytes, mast cells, and dendritic cells. Fast-acting innate immune cells play an important role in inducing inflammation . Among tThe TLR family comprises ten types of transmembrane protein receptors in humans (TLR 1–10). TLR 1, 2, 4, 5, 6, and 10 are located on the cell surface, which mostly rely on the myeloid differentiation factor 88 (MyD88) and cytoplasmic adaptor, further activating the inflammatory response with the nuclear factor kappa B (NF-κB). The other portion of TLRs, including TLR 3, 7, 8, and 9, is located intracellularly and can directly activate IRFs across MyD88 to induce inflammatory responses. Within this, the TLR4 receptor is the only two-way TLR that triggers internalization into the intracellular-activated IRF pathway after activation of MyD88. TLR10, the newly discovered TLR in humans, is also the only known anti-inflammatory molecule in the TLR family . Its mecThe mechanism to slow down the occurrence and development of OA is the current research focus. As a national treasure of the Chinese nation, traditional Chinese medicine (TCM) plays an irreplaceable role in maintaining human health. Zhuifeng Tougu (ZFTG) capsules, a clinical drug approved for OA, are composed of classic famous prescriptions, including Xiaohuoluo pills and the Linggui Zhugan and Jiuwei Qianghuo decoctions, which effectively relieve joint pain and inflammation in patients with OA. It can expel wind, eliminate dampness, dredge meridians and collaterals, dispel cold, and relieve pain. The composition and dosage in the automatic production line of ZFTG are illustrated in ZFTG was produced and provided by Hinye Pharmaceutical Co., Ltd.  and produced according to the automatic production line displayed in Approximately 2g of ZFTG was weighed accurately and placed in a conical flask with a stopper before adding 50 ml of pure water for ultrasonic treatment  for 30 min. Then, the capsules were removed, centrifuged  for 10 min, and the supernatant was filtered, followed by continuous filtration.A proper amount of paeoniflorin reference standard was accurately weighed and mixed with methanol to prepare a solution containing 20 μg per 1 ml.Altogether, we used a UPLC H-Class system from Waters Corporation , MSA3-6P-OCE-DM millionth electronic balance from Sartorius Co., Ltd. , MS105DU from Mettler-Toledo Group , and KQ250-DB from Kun Shan Ultrasonic Instruments Co., Ltd. .−1; column temperature: 40°C; injection volume: 1 μl; detection wavelength: 230 nm; many theoretical plates: not less than 5000 according to the paeoniflorin peak; separation degree with other peaks: greater than 1.0. All components were detected within 60 min. The gradient elution process is illustrated in Chromatographic column: Waters HSS T3 column ; mobile phase: A was acetonitrile, B was 0.1% formic aqueous acid solution; flow rate: 0.5 ml minPaeonia veitchii Lynch ; peak 4 belonging to Gentiana macrophylla Pall ; peak 5 belonging to Aconitum kusnezoffii Reichb ; peak 8 belonging to Notopterygium incisum Ting. ex H. T. Chang ; peak 9 belonging to Saposhnikovia divaricata (Turcz.) Schischk ; peak 10 belonging to Ligusticum chuanxiong Hort ; and peak 11 belonging to Glycyrrhiza uralensis Fisch .According to the chromatographic conditions, six batches of ZFTG were prepared as test solutions for UPLC analysis  using paeoniflorin as the reference substance  purchased from the National Institutes for Food and Drug Control . The chromatograms were recorded for 60 min, and the UPLC fingerprints of six batches of ZFTG were obtained . The resad libitum access to normal water and a standard diet, the rabbits were randomly separated into two groups: the control (BC) (n = 9)and KOA (KOA) (n = 33). To generate a model of OA, we chose the modified Videman method ]. All rabbits were kept in individual cages in the Animal Center Laboratory of Hunan University of Chinese Medicine [establishment license number: SYXK]. The feeding temperature was between 24 and 26°C, and the humidity was between 50% and 70%. After 2 weeks of acclimatization with n method  and the n = 6) received saline gavage (10 ml/kg body weight). The gavage dose of ZFTG and GS in rabbits was equivalent to that used in patients.Six weeks after modeling, three rabbits were randomly selected from the BC or KOA groups for KOA model validation, according to the random number table method. After model validation, the KOA model rabbits were randomly divided into five groups: model control ; high-dose ; medium-dose ; low-dose ; or positive control groups , with a total of 30 rabbits (six rabbits in each group). The control group  and western blotting (WB) analyses. Individuals were euthanized according to the IAEC animal experimentation guidelines. All experimental protocols were approved by the Committee of Ethics on Animal Experiments at the Hunan University of Chinese Medicine (LLBH-202007070001).The validation method included comparing the functional activities and general observation of knee cartilage to assess cartilage damage. After the general observation, frozen sections of the knees (5 mm thick) were collected and observed under a microscope using a modified 100 and 400 OA Research Society International (OARSI) scoring system . The OARAll collected sections were dewaxed with dimethylbenzene, soaked in graded ethyl alcohol, and washed with distilled water. Parts of the sections were stained with hematoxylin and eosin (H&E), dehydrated with gradient alcohol (95%–100%), and sealed. Urea and trypsin antigen were used to repair other parts of the sections, which were subsequently treated with primary and secondary antibodies, and then DBA was added for 5 min to develop color and observed with a microscope. Other sections were immersed in EDTA buffer (pH 9.0), heated, and then cooled to room temperature. Before adding the primary and secondary antibodies, the sample was first cleaned with 0.01M PBS (pH 7.4–7.6), submerged in 75% alcohol, Sudan black dye solution was added, and then stained with DAPI working solution at 37°C until sealed. The H&E staining solution and immunohistochemistry reagents and kits were purchased from Wellbio (K435960 and K484350) and Beijing ZSGB company (600D54 and 600W23), respectively. The immunofluorescence reagents were purchased from ProteinTech (SA00013-2). The stained cartilages were observed and imaged using a light microscope . Immunohistochemical sections were examined for IL-1β, IL-6, TNF-α, and IFN-γ. Immunofluorescence sections were used to observe the expression of NF-κB p65.Primary chondrocytes were obtained from rabbit knee cartilage. Chondrocytes were maintained in 10% Dulbecco’s modified Eagle’s medium (DMEM). After digesting the cells with trypsin, adherent cells were collected and subcultured. Chondrocytes from passage 3 (P3) were used for further analysis. The P3 generation of chondrocytes was randomly divided into nine groups and then treated with different concentrations of ZFTG  to select the best concentration.The chondrocytes were modeled with 10 ng/ml IL-1β and grown on glass slides fixed with 4% paraformaldehyde. Inactivated endogenous enzymes were identified with toluidine blue and collagen type II  for immunocytochemical staining under microscopic observation. The P3 generation of chondrocytes was randomly divided into five groups. In addition to the control group, the other groups were treated with IL-1β and then treated with different concentrations of ZFTG . The selection of these concentrations was based on the screening results of the previous drug concentration.4 cells/well, followed by treatment with IL-1β and various ZFTG concentrations at the indicated dosages for 24 h. After cell intervention for 24 h, 20 μl CCK-8 solution was added into each well and incubated for 4 h in a 5% CO2 incubator at 37°C. The optical density (OD) values at 450 nm were measured using a microplate reader .The Cell Counting Kit-8 (CCK-8) was used to assess the cytotoxicity of various concentrations of ZFTG against the P3 generation of chondrocyte rabbits. Cells were seeded in 96-well plates at a density of 10The apoptosis of P3 chondrocytes treated with IL-1β (10 ng/ml) and various ZFTG was measured using flow cytometry. Cells were digested with 0.25% trypsin, which contained 0.02% EDTA, before collecting the cell suspension. The cells were collected after the suspension was centrifuged for 5 min (1500 rpm), washed with PBS, and mixed with Annexin V-FITC and propidium iodide. Annexin V was subjected to fluorescein FITC labeling of apoptotic chondrocytes using the Annexin V-FITC apoptosis detection kit .In vivo and in vitro experiments of the cartilage tissue weighing approximately 0.025 g were carried out, and chondrocytes were precooled, washed with ice and PBS, and crushed with 300 µl RIPA lysate in a biological sample homogenizer, and the complete protein was extracted. The protein supernatant was mixed with loading buffer, boiled in water for 5 min, and placed in an ice box for medium-speed cooling. BCA detection  was used to carry out protein quantification, and electrophoresis was carried out for 130 min according to the results. After blocking with 5% non-fat-dried milk at room temperature for 1 h, membranes were incubated with primary antibodies overnight at 4°C, and then incubated with horseradish peroxide-conjugated secondary antibodies at room temperature for 90 min. The ECL reagent  was incubated with the membrane for 1 min, and the exposure was performed in the chemiluminescence imaging system .−ΔΔCt method  according to the manufacturer’s instructions. Total mRNA was used as a template for reverse transcription of cDNA  following the manufacturer’s protocol. Reverse transcription products were retained for PCR reactions and fluorescence quantitative PCR reactions. Each sample was analyzed in triplicate, and expressions of TLR2, TLR4, MyD88, and NF-κB were normalized to the expression level of β-actin. The 2t method  was usedp < 0.05 was considered statistically significant. To reduce the uncertainty and contingency of the data, we reported the mean difference between groups and the upper and lower limits of the 95% confidence interval.All data were expressed as the mean ± standard deviation. Data from two groups were compared using the Mann–Whitney test. A comparison among three or more groups was performed using one-way analysis of variance (ANOVA). SPSS for Windows (version 26) was used to analyze the data. The graphs were plotted using GraphPad Prism (version 8). A p = 0.0058). All the abovementioned results indicate that the modeling in the KOA group was successful.According to the experimental design, some rabbits were treated with modified Videman modeling at 3 months. To verify the success of the modified Videman modeling, we randomly selected three experimental rabbits from the BC and KOA groups for model verification 6 weeks later . In the After successful modeling, the rabbits in each group were treated intragastrically according to the experimental plan. Six weeks after dosing, all rabbits were killed, and the left knee joint was collected for anatomical  and histMacroscopically, we found that the knee joints of the MC group were more worn than those of the BC group and manifested as grayer, thinner, and lusterless cartilage. Because the cartilage in the bearing area of the articular surface was damaged, the articular surface was unsmooth and even dented downward, forming ulcers. Moreover, osteophytes were formed. The cartilage of the four medication groups  was better than that of the MC. In the ZFTG groups, especially the HD, the cartilage tissue was less worn, and the functional performance was better. The articular cartilage in the HD group was yellowish, more lustrous, and thicker than that in the MC, and a slight abrasion was observed in the mid-posterior region of the medial tibial plateau without significant pitting or osteophyte formation . The H&Ep = 0.028), IL-6 , TNF-α , and IFN-γ ] increased significantly than that in the BC group. The MC group presented inflammatory manifestations.To explore how ZFTG relieves joint injury, we conducted a series of tests on the blood and cartilage samples of experimental rabbits according to the previous investigation . To verip = 0.024), IL-6 , TNF-α , and IFN-γ  of the HD group had lower expression levels than those in the MC group, but the LD and MD groups had no statistical significance compared to the MC group. The abovementioned results indicated that a high-dose ZFTG could significantly inhibit inflammation by reducing IL-1β, IL-6, TNF-α, and IFN-γ in the serum of rabbits with OA. Immunohistochemical results of articular cartilage sections are demonstrated in (p < 0.001), IL-6 , TNF-α , and IFN-γ ] were significantly higher than that in the normal group. The results demonstrated that the model group had obvious inflammatory manifestations in the articular cartilage, consistent with the serum test results.The expression levels of related factors in the ZFTG groups were lower than those in the MC, and the resultant trends were consistent with those in the PC group. Among these, IL-1β , IL-6 , TNF-α , and IFN-γ  of the HD group were significantly lower than those in the MC group. Based on the results of the abovementioned blood and cartilage tests, we found that ZFTG can reduce the expression of inflammatory factors in the body and cartilage, thereby relieving the inflammatory response of KOA. This was also confirmed by the anatomical observation of the knee joint and the results of the H&E-stained section.However, the expression levels of inflammatory factors in ZFTG groups decreased, which had the same trend as the results of the positive drug group. IL-1β , MyD88 , and NF-κB ]. The test results in the ZFTG groups were consistent with those in the PC group and were lower than those in the MC group. With the increase in ZFTG concentration, the corresponding expressions of TLR4, MyD88, and NF-κB proteins decreased gradually. The protein expressions of TLR4 , MyD88 , and NF-κB  in the HD group were significantly different from those in the MC group. The protein expression of NF-κB in LD and MD groups was significantly different from that in the MC group . However, there was an insignificant difference in the protein expression of TLR4 and MyD88.The expression of the TLR4/MyD88/NF-κB signaling pathway would induce differential expression of the knee joint. The protein expression is demonstrated in p < 0.001), TLR4 , MyD88 , and NF-κB ]. It was consistent with the protein expression results of cartilage proteins. Compared with the MC group, the mRNA expression level of the PC group decreased significantly. Resembling the PC group, the mRNA expression in ZFTG groups was significantly lower than that of the MC group. Similar to the positive control group, the level of serum TNF-α in ZFTG groups was significantly lower than that in the model, and the HD group demonstrated the lowest expression of mRNA . The NF-κB p65 protein was evenly expressed in the cytoplasm of the BC group, but the MC group showed significantly higher levels in the nucleus. The expression of NF-κB p65 protein in the nucleus gradually reduced following intervention with ZFTG  and lower apoptotic rate , suggesting that the intra-articular inflammation in OA patients was indeed closely related to the wear of articular cartilage. However, ZFTG could significantly affect the activity of the damaged chondrocytes and inhibit their apoptosis. The best ZFTG concentration was 400 ng/μL , which was similar to the previous experimental result.We treated the chondrocytes with ZFTG and detected cell survival . The resp < 0.001), IL-6 , and TNF-α , IL-6 , and TNF-α ]. Therefore, ZFTG had a stable anti-inflammatory effect on chondrocytes, which was consistent with the results of the previous experiment.Moreover, we detected the expression of inflammatory factors in cell supernatants. As displayed in in vivo experiment , TLR4 , and MyD88 ]. These data suggest a positive correlation between the severity of OA and expression of TLR2, TLR4, and MyD88 proteins. With the increase of drug concentration, the protein expressions of TLR2, TLR4, and MyD88 in ZFTG groups decreased gradually, and the best ZFTG concentration was 400 ng/μl . TLR2, TLR4, and MyD88 mRNA expressions in each group were consistent with the protein expression trend. The results revealed (p < 0.001), TLR4 , MyD88 ]. ZFTG could significantly reduce gene expression. TLR2, TLR4, and MyD88 mRNA expression levels were lowest in the 400 ng/μl group . Furthermore, with the decrease in drug concentration, the inhibition of ZFTG on TLR2, TLR4, and MyD88 genes weakened gradually and manifested as a gradual increase in gene level in each group.As demonstrated in revealed  that mRNThese data indicate that ZFTG can reduce chondrocyte inflammation, inhibit chondrocyte apoptosis, and repress the expression of genes and proteins of TLR2, TLR4, and MyD88.As an age-related joint degeneration, OA has been treated with general therapy , exercise plans , and medication  with existing clinical treatment strategies that aggravate the burden and risk on the gastrointestinal tract and kidneys . In addiThe occurrence and development of OA involve complex networks of inflammatory mediators. The imbalanced remodeling driven by inflammatory mediators is an important factor in inducing and promoting the development of OA . InflammThe TLRs and NF-κB signaling pathways are important for inflammatory expression and activation of chondrocytes and are highly expressed in chondrocytes stimulated by inflammation . In partRecently, the treatment of OA with TCM has been frequently reported, and its curative effect has been gradually recognized . Its effin vitro (The modified Videman method was a classic OA model for New Zealand rabbits . The patin vitro , which aIn addition, IL-1β, IL-6, and TNF-α are closely related to degeneration of articular cartilage matrix . IL-1β ain vivo experiments, we found that the expressions of IL-1β, IL-6,TNF-α, and IFN-γ in the blood and articular cartilage of the MC group were higher than those in the BC and ZFTG groups. In the in vitro experiments, the supernatant of the chondrocytes in the IL-1β group also detected high expression of IL-1β, IL-6, and TNF-α. In addition, after ZFTG intervention, the expression levels of inflammatory factors reduced significantly, similar to the results of the in vivo experiments. This not only verifies that various factors are involved in the formation of OA but also demonstrates that ZFTG has an anti-inflammatory effect. In the ZFTG groups, the concentration gradient was negatively correlated with the expression of each factor, which also provided a reference for our subsequent in vitro experiments.Using IL-1, IL-6, TNF, and other cytokines are encoded and expressed by the NF-κB classical pathway , which iIn vivo experiments found that the TLR4/MyD88/NF-κB signaling pathway was activated in the model group, similar to previous studies (As an upstream target of NF-κ B and an important component of the innate immune mechanism, TLRs also play a crucial role in OA. With the discovery of the anti-inflammatory effects of TLR10 , the rol studies . The pro studies , the inhin vitro experiments, the high expressions of genes and proteins of TLR2, TLR4, and MyD88 in the IL-1β group were consistent with the results of in vitro studies (in vitro study demonstrated that the aggrecan 32-mer fragment induces the TLR-2-dependent gene to activate NF-κB in mouse and human chondrocytes, accelerating cartilage destruction (From our  studies  that dem studies . Korotkytruction . AltogetIt is worth noting the limitations of this study. First, ZFTG is produced by a Chinese patent medicine that uses modern equipment. However, the complexity of Chinese medicinal components makes it difficult to explore their effective mechanisms. Second, this study focused on the anti-inflammatory effect of ZFTG on KOA without comprehensively exploring the efficacy mechanism. Finally, further in-depth research is warranted to clarify the efficacy of ZFTG in treating KOA.The modified Videman method provided the experimental model of OA for this study. ZFTG inhibits IL-1β-induced inflammatory injury in rabbit chondrocytes by repressing the TLR4/MyD88/NF-κB signaling pathway, secreting inflammatory factors, and promoting the survival of chondrocytes while reducing the apoptosis of chondrocytes. In summary, TCM is a potential reservoir for prevention and treatment of KOA. Our study not only provides an important reference for treatment of KOA, which reveals that ZFTG can be used as a new drug and its curative mechanism, but also clarifies the vital function of the TLR4/MyD88/NF-κB signaling pathway in KOA. Our study can provide the direction for modern Chinese medicine research, provide the basis for the clinical treatment of KOA, and lay the foundation for the TCM treatment of KOA.


Microbial colonization of animal intestine impacts host metabolism and immunity. The study was aimed to investigate the diversity of the intestinal microflora in specific pathogen free (SPF) and non-SPF Beagle dogs of different ages by direct sequencing analysis of the 16S rRNA gene. Stool samples were collected from four non-SPF and four SPF healthy Beagle dogs. From a total of 792 analyzed Operation taxonomic units, four predominant bacterial phyla were identified: Firmicutes (75.23%), Actinobacteria (10.98%), Bacteroidetes (9.33%), and Proteobacteria (4.13%). At the genus level, Streptococcus, Lactobacillus, and Bifidobacterium were dominated. Among which, Alloprevotella, Prevotella_9, and Faecalibacterium were presented exclusively in non-SPF beagles, with potentially anti-inflammatory capability, which could protect non-SPF beagles from complex microbial environment. The number and diversity of intestinal flora for non-SPF Beagle dogs were the highest at birth and gradually decreased with growth, whereas the results for the SPF beagle samples were the opposite, with the number and diversity of intestinal microbiota gradually increases as beagles grow. In a nutshell, the microbial complexity of the rearing environment can enrich the gut microbiota of beagles, many of which are anti-inflammatory microbiota with the potential to increase the adaptability of the animal to the environment. However, the gut microbiota of SPF beagles was more sensitive to environmental changes than that of non-SPF beagles. This study is of great significance for understanding the bionomics of intestinal microflora in non-SPF and SPF beagles, improving the experimental accuracy in scientific research. Experimental animals played a key role in scientific and medical research. With the development of modern life science, the quality requirements of experimental animals are increasing. Conventional experimental animals have complex microbial states, which cannot meet the requirements of scientific research and production. Therefore, SPF experimental animals have become more important research work. Beagle as a standard non-rodent experimental animal has been widely applied due to its submissive behavior, medium size, long life span, and consistent genetic delivery. Therefore, it is very necessary to understand the biological characteristics of SPF beagles in research work.Gut microbiota, which is closely associated with host nutrition, metabolism, and immunity, acts as a “second genome” for modulating the health phenotype of the superorganism host , 2. As aHigh-throughput sequencing plays an increasingly important role in biological sample for studying the population structure, microbiome diversity, and evolution of the bacterial flora of humans and animals , 10, 11.Four non-SPF and four SPF newborn Beagle dogs from Qingdao Bolong Experimental Animal Co., Ltd were enrolled into this study. Throughout the study, none of the dogs received drugs, such as gastrointestinal disease drugs, antibiotics, or diabetes drugs, that could affect their gut microbiota, nor did they receive additional nutritional supplements. SPF beagles are bred in pairs in sterilized isolators and fed artificially sterilized breast milk until they are 1 month old, then gradually weaned on a dry diet. After weaning, they were fed sterilized maintenance commercial extrusion (dry-type) diet with free access to sterilized water. The isolator maintains a positive pressure of 118–127 Pa, humidity of 40–70% and 30 air changes per hour. The temperature inside the isolator is 34–35 degrees for the first week, and then decreases by 2 degrees every week until it reaches 24–26 degrees. Non-SPF beagles were raised in pairs in spacious indoor enclosure, kept at the same temperature and humidity as the SPF beagles' isolation unit, breastfed naturally until 1 month of age, then gradually weaned, after which they were fed the same sterilized maintenance commercial extruded (dry-type) diet as SPF dogs and were given free water. All dogs gain behavioral enrichment through interaction with each other, playtime with their caregivers, and access to toys.Fresh stool samples from newborn, 1 and 3-month-old non-SPF Beagle dogs or 1 and 3-month-old SPF Beagle dogs were collected immediately after spontaneous defecation, immediately frozen at −80 °C without any additives or pretreatment, and sent to Shanghai Majorbio Bio-pharm Technology Co., Ltd. for the 16S gene library construction, quantification, and sequencing. Twelve stool samples for non-SPF Beagle dogs were divided into three groups, named F1, F3 and F5, which were collected from newborn, 1 and 3-month-old non-SPF Beagle dogs, respectively. Eight stool samples for SFP Beagle dogs were divided into two groups, named F2 and F4, which were collected from 1 and 3-month-old SPF Beagle dogs, respectively.t-test or analysis of variance (ANOVA) on at least three independent replicates. P values of < 0.05 were considered statistically significant for each test .After sequencing, paired-end reads were assembled using FLASH software (v1.2.11) based on overlap. MOTHUR (v 1.30.2) software is used for quality control and filtering of assembled sequences. Uparse (v 7.0.1090) was used for OTU cluster analysis, and Usearch (v 7.0) was used for taxonomic analysis. Taxonomy based on the 16S rRNA gene sequence was assessed using the Ribosomal Database Project (RDP) classifier (v 2.11) against the Silva database at a confidence level of 0.7. Microbial diversity in the individual stool samples was estimated using rarefaction analysis. Alpha diversity and beta diversity indexes were calculated using MOTHUR. PICRUSt (v 1.1.0) was used to predict the KEGG and COG functions of 16S sequences. The alpha diversity for each sample was measured by OTUs using Sobs, Shannon, Simpson, ACE, Chao, and Coverage indexes. Statistical analyses were performed using Student's n = 12) and SPF (n = 8) Beagle dogs were assessed using high-throughput sequencing. After removing low-quality sequences and non-target regions, the number of cleaned sequences were 914,442 reads, approximately 45,722 reads per sample. Among them, 547,960 filtered sequences were obtained from the non-SPF Beagle dogs  and 366,482 filtered sequences were from SPF Beagle dogs .Stool samples of non-SPF . All the stool samples showed 792 OTUs, with an average of 423 bp high-quality sequences . For the non-SPF Beagle dogs, 726 OTUs, with an average of 422 bp , and for the SPF Beagle dogs, 347 OTUs, with an average of 426 bp  were acquired by sequencing.P = 0.007) between F1 (newborn) and F3 (1-month-old) groups; but no significant difference (P = 0.103) between F3 (1-month-old) and F5 (3-month-old) groups. For the SPF Beagle dogs, there was a significant difference in the number of OTUs (P = 0.003) between F2 (1-month-old) and F4 (3-month-old) groups.The number of OTUs for the non-SPF and SPF Beagle dogs fit a normal distribution. For the non-SPF Beagle dogs, there was a significant difference in the number of OTUs , as the number of sequences increased, the curve tended to be flat, indicating that the sequencing of each sample was sufficient to reflect the species diversity in the sample.These results indicated that the number and diversity of intestinal flora for non-SPF Beagle dogs were the highest at birth and gradually decreased with growth. In SPF beagles, in contrast, the number and diversity of intestinal microbiota gradually increases as beagles grow.Firmicutes (75.23%) was the highest, followed by Actinobacteria (10.98%), Bacteroidetes (9.33%), and Proteobacteria (4.13%).The results of microbiota analysis at the phylum level for the five groups of stool samples demonstrated that of the 4 phyla identified, the content of Firmicutes was the dominant taxa in all the five groups, the percentage of Firmicutes was different. In non-SPF beagles, with the dog's growth, the proportion of Firmicutes in the intestinal flora gradually increased, to 92.27% at the age of 3 months. At the same time, the proportion of Actinobacteria, Bacteroidetes, and Proteobacteria decreased gradually. In SPF dogs, the proportion of Firmicutes and Proteobacteria declined slightly with age, while the proportion of Actinobacteria and Bacteroidetes increased. Different from the previous reported that Fusobacteria ranks the third in the intestinal microbiota of healthy beagles , whereas the microbiota in the stool samples from SPF beagles is composed of 12 main genera . On the PICRUSt software was used to compare the species composition information obtained from 16S sequencing data, and to predict the composition of functional genes in the samples, to analyze the functional differences between different samples. KEGG pathway analysis predicted many basic metabolic function genes, among which the abundant genes were Metabolic pathways, Biosynthesis of secondary metabolites, Microbial metabolism in diverse environments, Biosynthesis of amino acids, etc. In general, the functional distribution of intestinal microbiota in non-SPF and SPF beagles was similar . For nonLactobacillus, Blautia and Prevotella in the non-SPF Beagle intestinal microflora (Lactobacillus was involved in carbohydrate and protein metabolism (Blautia was a major producer of short-chain fatty acids, especially acetic acid (Prevotella was involved in de novo synthesis of amino acids and the synthesis of several B vitamins (Bifidobacterium, Bacteroides, unclassified_o_Lactobacillales, Faecalibaculum and Pepto streptococcus with higher abundance in the intestinal microbiota of SPF Beagle dogs (Bifidobacterium and Bacteroides are known to have bile salt hydrolase enzyme-coding genes and are also participated in carbohydrate metabolism, Bacteroides, Lactobacillus, and Streptococcus were identified microbial species for their role in proteolysis or amino acid production (Bacteroidetes was the major group involved in the synthesis of vitamin B and conjugated linoleic acid, and Faecalibaculum was a major producer of SCFA (By comparing the functional distribution of intestinal microbiota of 1-month old non-SPF and SPF Beagle dogs, we found that the abundance of pathways related to Microbial metabolism in diverse environments and Biosynthesis of amino acids was higher in the non-SPF group than in the SPF group. This may be related to the high levels of croflora . It has tabolism , 14. Blatic acid . Prevotevitamins , 17. At gle dogs . Bifidoboduction , 14, 16. of SCFA . The abuIn this study, the fecal microbiota of non-SPF and SPF Beagle dogs, and different ages were characterized to understand the biological characteristics of intestinal microbiota in different rearing environment.The alpha diversity was compared by the Coverage index, Sobs index, Shannon index, Simpson index, ACE index, and Chao 1 index for the fecal microbiota of the non-SPF and SPF beagles . The resFirmicutes, Bacteroidete, Proteobacteria and Actinobacteria. Firmicutes were the main flora of the two groups of beagles, which was consistent with many studies on animal intestinal flora (Firmicutes increased gradually, and reached 92.27% by the age of 3 months. In SPF beagles, the proportion of Firmicutes decreased slightly with growth. This was contrary to the trend of fecal flora diversity (Firmicutes, Bacteroidetes, Fusobacteria, Proteobacteria and Actinobacteria in dog's gut (Fusobacteria was not the dominant phylum in the beagle's intestinal flora in present study, which may be caused by animal breeds, ages, diets, living environments or experimental methods. We believe that the most important reason may be the age of the beagle dogs. Most of the studies used beagle dogs as adults, but this study was conducted until the beagle dogs were 3 months old, focusing on the impact of living environment on the intestinal flora construction process of the puppies. The predominant phyla in healthy human intestinal flora are Firmicutes, Bacteroidetes, Actinobacteria and Proteobacteria, suggesting that the intestinal microflora of young dogs may be more similar to that of humans than that of adult dogs.There was no significant difference in the composition of fecal microbes between the two groups of beagles at the phylum levels, with predominantly al flora , but theiversity . Most ofog's gut , 14. In Streptococcus, Lactobacillus and Bifidobacterium were the main fecal flora of both non-SPF and SPF beagle dogs. The three dominant genera are bacteria genera that widely exist in the digestive tract of animals, some strains of Lactobacillus and Bifidobacterium can also act as probiotics to improve the distribution of intestinal flora and antagonize the colonization of harmful bacteria, so as to protect intestinal health (Based on the analysis at the genus level, l health , 21.Burkholderia-Caballeronia-Paraburkholderia, Alloprevotella, Faecalibacterium, Prevotella_9, [Ruminococcus]_gnavus_group and Blautia were present exclusively in non-SPF beagles. Alloprevotella, Prevotella_9 and Faecalibacterium can produce SCFAs (Blautia is a relevantly abundant taxonomic group present in the microbiome of mammalian gastrointestinal tracts, which plays certain roles in host metabolism, inflammation and biotransformation, and has potential probiotic properties (Burkholderia-Caballeronia-Paraburkholderia, Prevotella_9, Faecalibacterium and Blautia could be a consequence of complexity of microbial environment, protecting them from inflammations and non-infectious intestinal diseases.Moreover, ce SCFAs , 23, whoce SCFAs , 25. Blaoperties . We specVeillonella (Escherichia-Shigella (llonella  and EschShigella  peculiarBacteroidetes, also the SCFA producing bacteria (bacteria , 29, werLactobacillus, Blautia and Prevotella in intestinal microbiota. However, at 3 months of age, the abundance of many functional genes in SPF beagles was higher than that in non-SPF beagles, which may be due to the higher content of Bifidobacterium, Bacteroides, unclassified_o_Lactobacillales, Faecalibaculum and Pepto streptococcus in the intestinal flora of SPF Beagles. By participating in the metabolism of carbohydrates, proteins, SCFA, bile salts and vitamins in the gut, these bacteria help the host decompose food and obtain nutrients, improve the utilization of proteins, carbohydrates and vitamins.KEGG pathway analysis predicted many basic metabolic function genes of intestinal flora. The abundance of functional genes in intestinal flora of non-SPF Beagle dogs decreased with growth, while that of SPF beagle dogs increased with growth. At 1 month of age, the abundance of functional genes in intestinal microbiota of non-SPF Beagles was mostly higher than that of SPF beagles, which may be related to the high concentration of The experimental animals used in this study were newborn beagles in general environment and SPF environment. In order to obtain newborn puppies with similar genetic background of the same strain in the same breeding farm at the same time, only 4 animals were obtained in each group, 2 females and 2 males. The number of experimental animals is relatively small but representative. In addition, due to the limitation of experimental conditions, we failed to collect fecal samples from newborn SPF beagles, which is a pity of this study.This study laid the foundation for the study of SPF and non-SPF Beagle fecal microbiota. By comparing the composition and diversity of the fecal microbiota of non-SPF and SPF beagles, and different ages, we found that living and dietary exposure to the large variety environmental microbes could increase the potentially beneficial bacterial genomes, enriching the microbiome, enhancing the anti-inflammatory ability of intestinal flora. This may be the result of beagles that adapt to the complexity of microbial environment, indicating a role of microbiota in protecting beagles from pathogens. The gut microbes of SPF beagles were more sensitive to environmental changes than that of the non-SPF beagles, which may lead to a weaker environmental adaptability of SPF beagles. It is of great significance for understanding the bionomics of intestinal microflora in non-SPF and SPF beagles, improving the experimental accuracy in scientific research.The data presented in the study are deposited in the NCBI repository, SRP402573, accession number PRJNA890408.The animal study was reviewed and approved by Laboratory Animal Ethics Committee of Shandong Laboratory Animal Center.CY retrieved literatures, provided tables, figures, and wrote the manuscript. ZG retrieved literatures and provide methods and techniques. KW proposed the topic and provided outline. ZL revised the manuscript. XM and SC reviewed the final manuscript. All authors contributed to the article and approved the submitted version.


The differences in nutritional intake of total calories and carbohydrates were lower in the monozygotic twin group than in the dizygotic twin group . The differences in total body fat were lower in monozygotic twins than in dizygotic twins . Monozygotic twins had more similar dietary habits for total calories and carbohydrate intake. Other nutritional factors did not show differential similarity between monozygotic and dizygotic twins. Total body fat was more concordant in monozygotic twins.The present study aimed to investigate the coincidence of obesity and nutritional intake in monozygotic twins compared to dizygotic twins. The data from the Korean Genome and Epidemiology Study (KoGES) from 2005 through 2014 were analyzed. Participants ≥ 20 years old were enrolled. The 1006 monozygotic twins and 238 dizygotic twins were analyzed for differences in self-reported nutritional intake, total body fat, and body mass index (BMI) using a linear regression model. The estimated values (EV) with 95% confidence intervals (95% CI) of the difference in dietary intake, total body fat, and BMI score were calculated. The monozygotic twin group and the dizygotic twin group showed similar differences in nutritional intake, DEXA fat, and BMI (all  Obesity is a common disease whose prevalence is estimated to be approximately 36.9% in men and 38.0% in women in the worldwide adult population . In addiMultiple factors can contribute to the occurrence of obesity. Genetic predispositions to obesity have been suggested in twin studies . HoweverPrevious studies reported a number of genetic factors that result in obesity. In addition, because nutritional intake is a critical factor for obesity, it was questioned whether nutritional intake can be a contributor to obesity as an inherited trait. This study aimed to estimate the inherited portion of obesity compared to the shared environmental factors. To examine these current questions, twin cohorts were analyzed for differences in total body fat, body mass index (BMI), and nutritional intakes. This study is novel in analyzing nutritional intake in twin cohorts and comparing BMI and total body fat. The findings of the current study may enhance the knowledge on the inherited trait for the occurrence of obesity.The current research was permitted by the ethics committee of Hallym University (2021-03-004). The ethics committee exempted the written informed consent. The Korean Genome and Epidemiology Study (KoGES) from 2005 through 2014 was used ,10,11,12Among a population of 1300, the participants who did not completed the survey on nutritional intake, dual-energy X-ray absorptiometry (DEXA) exam, and sleep time were excluded. As a result, 1006 monozygotic and 238 dizygotic twin participants were enrolled . The dif2) was measured by the automated height-weighing machine in Frankfort Horizontal Plane. The income group was classified based on their household income. Education level, marriage status, and physical activity, walking time, and sitting time were surveyed. Smoking and the frequency of alcohol consumption were self-reported. Sleep time was surveyed with categorized questionnaire of ≤5 h/day, >5 and ≤7 h/day, >7 and ≤9 h/day, and >9 h/day.The self-reported surveys were conducted for the nutritional intake of total calories , protein (g), fat (g), carbohydrate (g), calcium (mg), phosphorus (mg), iron (mg), potassium (mg), vitamin A (mg), sodium (mg), vitamin B1 (mg), vitamin B2 (mg), nicotinic acid (mg), vitamin C (mg), zinc (ug), vitamin B6 (mg), folic acid (ug), retinol (ug), carotene (ug), ash (mg), fiber (g), vitamin E (mg), and cholesterol (mg) by trained interviewees using a validated questionnaire . Total bThe absolute difference in dietary intake including fat intake, total body fat, and BMI score between the matched twin participants were estimated.The categorical variables were compared using chi-square test. The continuous variables were compared using Wilcoxon rank-sum test.We calculated the estimated values (EV) (absolute difference between monozygotic twins—Absolute difference between dizygotic twins) with 95% CI of the absolute difference of dietary intake, total body fat, and BMI score using a linear regression model.p values < 0.05 were regarded as statistically significant. SPSS v. 24.0  was used.p > 0.05, p < 0.05). The levels of income and education, marital status, obesity, smoking status, alcohol consumption, and sleep time were not different between the two groups.The nutritional intake, DEXA fat, and BMI were not different between the monozygotic and dizygotic twin groups .The difference in dietary intakes of twin pairs was compared between the monozygotic and dizygotic twin groups . The difThe difference in total body fat was higher in dizygotic twins than in monozygotic twins (adjusted EV = 2427.86 g, 95% CI = 1777.19–3078.53 and adjusted EV = 1.90%, 95% CI = 1.33–2.46, The total body fat was more concordant within monozygotic twin pairs than in dizygotic twins. In addition, the concordances in the nutritional intakes for total calories and carbohydrate intakes were higher in monozygotic twins than in dizygotic twins in this study. The current results suggest the inherited portions for the development of obesity and nutritional intake. This study improved the evidence on the inherited contribution of obesity by analyzing monozygotic and dizygotic twins.The intake of total body fat was more concordant in monozygotic twins than in dizygotic twins in the current study. The metabolic pathways related to body fat storage and adipocyte lipolysis were reported to be related to shared genetic loci . MoreoveIn addition, nutritional intakes of total calories and carbohydrates were more similar between monozygotic twin pairs than between dizygotic twins in this study. Eating behavior is one of the main determining factors for weight gain . Thus, nHowever, there was no higher concordance of BMI in monozygotic twin pairs compared to dizygotic twins in the present study. In advance with genome-wide association studies, multiple predisposing genetic factors have been announced for obesity . In addiThis study evaluated the differences in nutritional intake, total body fat, and BMI in monozygotic and dizygotic cohort populations. Total body fat was measured using DEXA, and BMI was calculated based on the measured weight and height. In addition, socioeconomic factors of income level, education, and marital status were considered in the analyses. Furthermore, lifestyle factors of physical activity, smoking, alcohol consumption, and sleep duration were assessed to minimize potential confounding effects from these variables. The KoGES HTS data are generated and regularly monitored by statisticians in the Korean government. However, the cross-sectional study design limited the causality of obesity and nutritional intakes with monozygotic twins in this study. In addition, although validated questionnaires were used to examine nutritional intake , there cThe nutritional intakes of total calories and carbohydrates were more similar in monozygotic twin pairs than in dizygotic twins. Total body fat was more similar between monozygotic twins than between dizygotic twins.


Candida (NAC) species remarkably increase azole resistance in developing countries. We aimed to study candidemia trends and associated risk factors in oncology patients since they vary geographically, and rapid and appropriate treatment improves outcomes. Vitek 2 was used to identify the Candida species, and the E-test determined their susceptibility to azoles. Candida was the cause of 3.1% (n = 53/1701) of bloodstream infections (BSIs) during a 1-year study. Candida tropicalis was the most predominant species among the 30 candidemia episodes studied (36.7%), followed by C. albicans (33.3%). However, C. krusei, C. guilliermondii, C. pelliculosa, C. parapsilosis, C. famata, and C. inconspicua accounted for 30.0% of the isolates. An increased risk of NAC BSI was significantly associated with chemotherapy and leucopenia . However, the multivariable analysis revealed that leucopenia was the only independent risk factor (P = 0.048). Fluconazole and voriconazole resistance were 58.3% and 16.7%, with NAC species showing higher resistance rates than C. albicans. Both fluconazole and voriconazole minimum inhibitory concentration (MIC) median values were higher in NAC than in C. albicans, but only voriconazole was significantly higher . In conclusion, the increased prevalence of NAC BSIs and incredibly high fluconazole resistance rates in cancer patients emphasize the necessity of antifungal stewardship to preserve voriconazole effectiveness, continued surveillance of candidemia, and future studies into azole resistance molecular mechanisms.Candidemia is a life-threatening invasive fungal infection in immunocompromised patients. The widespread use of azoles and the shift toward non-albicans The online version contains supplementary material available at 10.1007/s00284-023-03468-w. Candida bloodstream infections (BSIs) have emerged as a global cause of invasive fungal infection in critically ill patients in healthcare settings. Death rates caused by this life-threatening infection range from 35 to 53% and have been linked to higher morbidity and hospital costs .In vitro antifungal susceptibility results and MIC values of ed Table . AccordiC. albicans, 6 C. tropicalis, and 1 C. parapsilosis, besides 2 C. krusei strains that have an inherent resistance to fluconazole. Despite a high median MIC for fluconazole against all Candida species of > 256 ranging from 0.190 to > 256 μg/ml, C. albicans had a lower resistance rate, median MIC value, and range than NAC species  versus > 256 (0.190–> 256) μg/ml with P = 0.981). Furthermore, the MIC50 and MIC90 of voriconazole and fluconazole in NAC species were higher than in C. albicans  .The overall resistance rate for fluconazole was high , including 5 Candida was the third-leading cause of BSIs in the United States and the seventh cause in Europe [,Candida species isolates represented 3.1% of all positive blood cultures. This isolation rate was parallel to the results of Lim et al., where 3.0% of all positive blood cultures were fungal infections, with the majority being Candida species (95%) [C. albicans as the pathogenic Candida species, a global rise of NAC species has been reported in recent decades. Studying local trends in Candida species distribution and antifungal susceptibility is essential since they differ considerably among countries or institutions and are influenced by patients' underlying conditions [Candidemia is one of the most common invasive fungal infections in immunocompromised cancer patients. n Europe , 16. In es (95%) . Despitenditions .Candida tropicalis was the most common species in our patients, followed by C. albicans (36.7 and 33.3%). But C. krusei, C. guilliermondii, C. pelliculosa, C. parapsilosis, C. famata, and C. inconspicua were less isolated. Candida species have shown variation in different geographical areas, with more reported C. albicans in some regions [C. tropicalis across Africa, Asia, and Latin America [Candida species distribution among patients having certain underlying disorders has been noticed. Similar to our results, a higher proportion of C. tropicalis and C. krusei was consistently recorded among candidemia patients with hematological malignancies [ regions , 18, 19, America –22. Howegnancies , 24.C. famata and C. pelliculosa [C. famata was also detected in children with cancer in a study by Vasileiou et al. [C. pelliculosa was documented primarily in neonates, it has also been reported at a lower frequency in other age groups, consistent with our findings [Some studies have implicated broad-spectrum antibiotics and immunosuppression as risk factors for candidemia caused by certain non-albicans species, such as liculosa , 26. Intu et al. . Althougfindings . The incCandida [P < 0.001) and cancer and transplantation settings (P < 0.001) and was ten times more prevalent in larger institutions (P < 0.001) [The worldwide rise in azole resistance has caused severe therapeutic challenges. This rise may be because of the widespread use of azoles, particularly in low-income countries, and their fungistatic mode of action against Candida , 5. A pr< 0.001) . Additio< 0.001) .Candida isolates with no established CBPs, fluconazole and voriconazole resistance accounted for 58.3% and 16.7% of total Candida BSIs, respectively. All species showed high resistance and MIC values to azoles, notably fluconazole, which showed higher resistance rates in NAC than in C. albicans (64.3% versus 50.0%). These results agreed with an Egyptian study that found high fluconazole resistance rates from NAC and C. albicans species causing BSIs in pediatric patients at Cairo University pediatric hospitals  [C. albicans, C. krusei, C. glabrata, C. tropicalis, and C. parapsilosis  in ICU patients at Ain Shams University Hospital [C. albicans species, respectively. It was conducted on pediatric candidemia patients at Egypt's Mansoura University Children's Hospital [Fluconazole and voriconazole were the most frequently utilized antifungals in our patients. Such azoles were used in 87.5% of prophylactic and 73.3% of antifungal treatments. After we excluded the six ctively) . AnotherHospital . A previHospital . TherefoC. tropicalis isolates were resistant to fluconazole and voriconazole, respectively [C. tropicalis (47.6%) as well as in uncommon Candida species (45.5%) [Several studies from other countries also found high MICs and resistance rates for azoles, mainly for fluconazole. However, these high values were detected only in NAC species and at lower rates. According to a study conducted in China, 50.0% and 56.5% of ectively . Another (45.5%) . Additio (45.5%) , Saudi Apicalis 4.6% as we (45.5%)  showed hCandida isolates with and without acquired resistance based on their phenotypes. An organism with a MIC greater than the ECV is assumed to have mutational resistance (non-WT). ECVs should not replace the published CBPs in clinical practice since they do not categorize isolates into sensitive and resistant (treatable and untreatable) isolates. However, it can guide physicians in making clinical decisions when CBPs are unavailable [Epidemiologic cutoff values can identify vailable . Furthervailable .Candida isolates showed increased rates of non-WT phenotypes for azoles, with MIC values higher than ECVs. Fluconazole and voriconazole non-WT phenotypes were identified in 88.5% and 48.0% of all isolated Candida species. Furthermore, we found severe azole cross-resistance among our isolates. All voriconazole-resistant Candida isolates were fluconazole-resistant (100%). In addition, all isolates that showed non-WT phenotypes for voriconazole were fluconazole non-WT phenotypes (100%). Most of our isolates showed elevated MIC values for azoles compared to published studies from other countries. Non-WT phenotypes of fluconazole and voriconazole were observed in 61.9% and 33.3% of C. tropicalis, 60.0% and 45.0% of C. glabrata, and 20.8% and 16.7% of C. albicans in Korea [C. guilliermondii, 16% and 7% of C. lusitaniae, and 8% and 17% of C. kefyr in the USA [C. tropicalis, and 5.6% and 5.6% of C. albicans in Algeria [Our in Korea ; 17% and the USA ; 36.9% a Algeria . Consist Algeria .Cancer patients are a unique population of patients subjected to multiple courses of antibiotics and antifungals whenever they manifest a persistent fever that does not respond to antibiotics. Thus, they are more at risk of developing resistance. Because fluconazole has been linked to high rates of resistance in many parts of the world, it should be used cautiously in high-risk patients who are unsure of their response. Furthermore, based on the high resistance rates of fluconazole shown in our study and other Egyptian studies, a stewardship program is needed to preserve voriconazole so as not to be subjected to increasing resistance with misuse.Candida to the species level. Therefore, conventional techniques are insufficient for Candida species identification, which causes invasive candidiasis. Broader spectrum techniques, such as matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF), more advanced ones, such as PCR-restriction fragment length polymorphism (PCR-RFLP), and sequencing or combined methods are recommended whenever applicable, particularly in patients at high risk for candidemia.In this study, although sn-PCR and CHROMagar results showed a significant agreement with those of Vitek 2, sn-PCR and CHROMagar could not identify 26.6% and 30.0% of Candida infections were all observed as risk factors in our patients . Several studies have also revealed these variables as significant factors in the development of candidemia [Candida BSIs in patients with hematologic and solid tumors equally. Surprisingly, in studies by Zheng et al. and Liu et al., solid cancers were more prevalent than hematological malignancies  [Candida infections is critical for optimal patient care. Candidemia caused by NAC species was significantly associated with chemotherapy and leucopenia. On the other hand, in adult patients, surgeries, prolonged hospitalization, and multifocal infections were predictive factors linked to candidemia caused by C. albicans. In multivariable analysis, leucopenia was the independent factor associated with an increased risk of NAC BSIs. Likewise, hematological malignancies, chemotherapy, leucopenia, and neutropenia were identified by other studies as increasing the risk of NAC, whereas surgeries, old age, and catheters increase the risk of C. albicans BSIs [Broad-spectrum antibiotics, prolonged hospital stays, ICU, chemotherapy, neutropenia, recent surgeries, coexisting bacteremia, and multifocal ndidemia , 38, 39.C. auris is rare in our country and the tested species account for more than 90% of cases, we focused on the prevalence of common Candida species rather than C. auris among our patients. A recent study in our country failed to detect C. auris in 414 candidiasis-causing isolates [The current study has some limitations. Although the NCI treats cancer patients from all over the country, our findings were limited to a single institute. Our study's sample size was relatively small, which may impact the risk factor analysis. Moreover, since C. tropicalis being the most prevalent species. NAC candidemia was significantly linked to chemotherapy and leucopenia. We believe that chemotherapy-induced leucopenia and neutropenia in cancer patients are strongly linked to NAC BSIs rather than an underlying tumor, such as hematological malignancies. Our findings demonstrated an increased reduction in fluconazole and voriconazole susceptibility, particularly in NAC species. The general critical condition of cancer patients and numerous risk factors, such as prolonged hospitalization, all contributed to these susceptibility trends. Furthermore, the extensive use of azoles at our institute enhanced the selection pressure for resistance. The high fluconazole resistance rates and the increasing prevalence of NAC suggest a potential decrease in therapeutic efficacy and point to the necessity of stewardship programs to preserve voriconazole efficacy. Moreover, this alarming rise in azole resistance rates and elevated MIC values highlights the importance of physician awareness, early detection, and continued future surveillance of candidemia, especially in this critically ill group of patients. Moreover, investigating the underlying molecular mechanisms of azole resistance is highly advised, as this may improve the understanding of this critical clinical situation.We revealed the predominance of NAC-causing candidemia in oncology patients, with Supplementary file1 (DOCX 369 KB)Below is the link to the electronic supplementary material.


We performed coarse-grained molecular dynamics simulations of DNA polymers pushed inside infinite open chiral and achiral channels. We investigated the behavior of the polymer metrics in terms of span, monomer distributions and changes of topological state of the polymer in the channels. We also compared the regime of pushing a polymer inside the infinite channel to the case of polymer compression in finite channels of knot factories investigated in earlier works. We observed that the compression in the open channels affects the polymer metrics to different extents in chiral and achiral channels. We also observed that the chiral channels give rise to the formation of equichiral knots with the same handedness as the handedness of the chiral channels. Polymers are long molecules consisting of many building units called monomers . The uniThe way the monomers are connected defines the polymer’s topology. A relatively new topology is represented by polymer knots . The knoIt is difficult to pinpoint when and how this scientific interest in knotted molecules began, whether it was sparked by imagination or, as is often the case, by observing nature. But now, it is clear that the topological state of molecules has strong biological and technological effects and implications. While in biology, the knots can be very harmful on a genome ,5,6,7,8 As pointed out above, polymers, especially with regard to their topology, represent a problem suitable to be studied using computers. While the progress in polymer synthesis enabled controlled preparations of knotted molecules of up to eight crossings , in compAs we also mentioned, the knots are formed naturally by biological processes ,14, but The next layer of complexity is added by investigating polymer knotting in confined spaces. The confinement state is the most typical state where polymers occur in nature ,20. ConfConsequently, the level of the complexity of the problem can be extended by adding external forces into consideration that induce compression of the confined polymer. The compression of polymer chains under confinement was studied by means of Monte Carlo (MC) ,24,25 anIt has been demonstrated that the theoretical and computational insights into polymer compression within nanochannels can be experimentally validated by confining DNA within nanofluidic channels and inducing compression through specific experimental setups ,42. MoreChirality is a prominent property of knots. As mentioned above, knots are formed by a substantially long polymer chain winding around itself. One of the parameters to characterize knots is the crossing number that quantifies how many times the polymer winds around itself. The direction of the polymer winding around itself defines the chirality of the knot. The crossing number is a combinatorial property, and the number of possible knot types that can be constructed with a given crossing number increases substantially. This is why it is feasible to create as many as 1.7 million prime knots with up to 16 crossings, out of which fewer than 2000 knots are achiral . The chiIn the context of knot formation, a recent computer simulation was devised to explore an intriguing scenario of whether and how knotting could be induced by simply pushing DNA inside nanochannels, without the need for the more complex lab-on-chip nanofluidic experiments . This scIn our current study, we are exploring the formation of knots in polymers as they are pushed into open, infinitely long nanochannels with varying sizes and geometries. To simulate chiral environments, we designed these channels with a helical geometry and induced different chirality by altering the winding direction of the helical loops within the nanochannels. We developed a novel computational approach to identify the chiral properties of the knots that form in the DNA strands as they are pushed inside these channels. Our method utilizes Knoto-ID , a topolσ corresponding to 2.5 nm  + 1/4 if |rij| < 21/6σ and Uex(rij) = 0 otherwise, where |rij| is a distance between a pair of beads position vectors ri and rj, where i ≠ j.The dsDNA is modeled as a discretized beaded chain consisting of N = 300 beads representing DNA portions with a width of 1 o 2.5 nm . The beamolecule . The nonRch and central axis described by the equation of a helix given as t is a periodic parameter in radial space and ω gives a subtended angle as t increases  = 2.5 nm and [τ] = 74 ns × (η/η0) ns [η0 is the viscosity of pure water, η0 = 1 cP, and η is the viscosity of the actual buffer used in the nanofluidic experiment. The buffers in nanofluidic applications often consist of a solution containing polymers, saccharose, agarose, etc., to increase the hydrodynamic drag of the media on the molecule [The span is shown as a function of velocity of the piston pushing the polymer along the infinite channels. The push by the piston is realized by applying an external force to the piston. Hence, the velocity of the piston was obtained from the simulated trajectories as a total distance traveled by the center of the piston from the beginning of the simulation to its final position at the end of the simulation over the simulation time η/η0) ns , where ηmolecule ,42, whilmolecule . The corWe would like to note that we opted for realizing the push intermediated by an applied external force instead of directly moving the piston by a constant distance at a time, as simulated in some existing works . This alD/P = 0.5. The difference seems to be higher than in the compression in channels with an impenetrable wall. The difference in polymer span also seems to disappear at weak confinement strengths in terms of D/P, especially if strong compressive forces are applied.The comparison of polymer metrics pushed in helical and cylindrical geometries on In γ. As the external force acting on the particle increases, the hydrodynamic drag force remains proportional to the velocity of the particle  [The comparison of the computed span shows, in general, smaller compaction of the polymer when compressed by pushing inside the open channels than in the case when the polymer is compressed against the impenetrable wall in the nanochannels. We understand the obtained results as follows. Given the form of the equations of motions in the Langevin dynamics, provided in the Methodology section , the hydhe span) , this meWe also showed in the previous work that the helical confinement in narrow channels acted to a certain extent like cylindrical channels with a smaller diameter, i.e., channels with higher confinement strength. The conformation of the DNA molecule is determined by a balance of several ongoing forces: the confinement force, elastic force, and hydrodynamic force intermediated by the pushing force . This isD/P and small compressive forces, the existence of a special regime was discovered, forming a shoulder on the dependencies of the span versus compressive forces [As investigated in the existing studies, the compaction of the polymer under external forces is relevant and related to conformational changes ,25,36, ae forces ,25, thatThe polymer metrics provide one-dimensional information on the polymer behavior and the effects of confinement and compressive force. Another convenient property that is directly accessible from computer molecular simulations to represent the situation of a dynamically moving polymer molecule is distributions of monomers. Here, we analyze the radial and axial distributions of monomers across the major axes of inertia of the confining channels. dH = 2πkσ, where k = D/2π in helical channels, and dH simply equals D. There are five heatmaps corresponding to five settings of the external force, from the bottom up following the increasing velocity of pushing, as shown in the inset of The heatmaps indicate expulsion of monomers into the lateral sides of the channel with an increasing velocity of pushing. This effect is numerically captured in the graphs showing radial distribution functions that are displayed adjacent to the heatmaps in each of the panels. The radial distributions show a similar shape to those already obtained for cylindrical ,68 and sx-axis also reflect the direction of push in the simulations, which went from right to left in Cartesian coordinates. The shape of the concentration profiles is very similar to what is observed experimentally in the dynamic nonequilibrium segmental concentration profile of a single nanochannel-confined DNA molecule [x-coordinate). This kind of projection was used in some existing works studying polymers in nanochannels by other authors [Fσ/ε0 = 0.1 and 5. Each panel also shows a representative snapshot from the simulation showing polymer in the channel with a particular geometry obtained at the end of the simulation for the setting of Fσ/ε0 = 1.To the right of the radial distributions on each panel, we also show the axial distribution of monomers along the main axis of inertia of the channels. These distributions show the number density or concentration of monomers from the position of the piston. In the axial distributions, the position of the piston is always at the origin. The values on the molecule ,43. In omolecule ,35,70, i authors ,35. The D/P = 0.5, 1, and 2. We can see that in the absolute numbers, a larger maximum on the axial distribution near the position of the piston surface is achieved in the case of cylindrical channels. Also, the observed flattening of the radial distributions is more extensive in the case of cylindrical channels. This indicates that when pushing the polymers inside open channels, the force is less effective in compressing the polymers, and the monomers do not fully explore the helical grooves of the helical channels. Consistently with the polymer metrics shown in In this section, we evaluate the topological state of DNA polymers under compression induced by pushing through open infinite channels. The knotting probability is evaluated as the frequency of finding knots in ten runs along the trajectories that contain 5000 structures each for topological analyses. The occurrence of knots was evaluated by Knoto-ID software  (v1.3.0)D/P ratio. D/P = 1. The analysis shows that, in general, the probability of knotting increases with the velocity of pushing. Also, it is shifted towards the occurrence of more complex knots with increasing velocity of pushing. This observation is consistent with previous investigations of knotting in DNA pushed through square channels  and alsoOn the other hand, the distinctive feature of the pushing inside the infinite channels seems to be apparent lower knotting probability in nanochannels with helical geometry as compared to the cylindrical channels. As discussed in k = D/2π, which determines the distance between helical loops or size of the helical turns, dH = 2πkσ. It is important to note that the current setting of the pitch was chosen based on our previous work where we investigated chiral effects in terms of mobility of localized knots with a given chirality. It is probable that for the current experimental setting of the polymers pushed inside the open channels, the pitch has to be fine-tuned, perhaps towards larger values above the deflection length λ = (D2/P)1/3 [dH > 2πkσ for k fixed to k = D/2π. The decreased knotting probability in helical channels can, however, be due to the specific geometric parameters of the helical channels determined by the pitch D2/P)1/3 , so thatdH = 2 P, and analyzed the knotting probability, as shown in D/P = 0.5) and very small compressive forces (Fσ/ε0 = 0.1). Since detailed refinement and thorough exploration of the parameter settings of the channels’ geometry are clearly beyond the scope and extent of a single work, we will readdress it in future works, providing more information on polymer behavior for various parameter settings of the helical channels, polymer chain length, and persistence lengths.In order to gain insight into this behavior, we simulated DNA polymer pushed into a helical channel with the size of helical loops well above persistence length, set to The midsection of D/P = 0.5 and D/P = 2. This might be related to the fact that for both of these settings, the knottedness is lower than in the case of D/P = 1, and the amounts of existing twist and torus knot types as a function of crossing number do not evolve equally; in other words, with increasing crossing number, there are more twist knots than torus knots. For the cases outside intermediate confinement, D/P = 0.5 and 2, we see an abundance of unknots. In the case of the strong confinement, D/P = 0.5, the unknots can be related to lower degrees of compaction and prevailing effects of confinement keeping the chain extended. In the case of weak confinement, D/P = 2, chain length effects or timescale and velocity effects might be taking place. The polymer at its given length is much more diluted; hence, on one hand, it leads to much more spooling, but also it might have not enough time to explore the geometrical spaces of the larger channels at the fixed rate of pushing. This may lead to the higher extent of writhing indicated in Note that there are only 20 amphichiral knots out of 801 knots that can be constructed from knotted lines up to 11 crossings . The pophttp://kymoknot.sissa.it/) [We further investigate whether the effect of the handedness of the helical channels on the knot chirality is preserved to some extent, and if channels with helical geometry and given handedness can be used to control handedness of the knots that are created during the pushing of the DNA through the channels. In the previous study , we probssa.it/) . In the current work, we directly use the information on the chirality of the knots provided by Knoto-ID software . Howeverℓ = 1 σ, there would be no need to construct a closure. Moreover, mathematically, knots occur only on closed curves, and the algorithms for finding knots often involve some kind of closure method that constructs a connection between the free ends of the linear polymer chain. In order to eliminate the possible bias coming from the closure method, we evaluated only the knots found in conformations with an arbitrarily chosen very short end-to-end distance. It is noteworthy that although we do not know how the end-to-end distance and the bias from the closure method are related quantitatively, one intuitively expects that the number of entanglements introduced by closing the arc grows with the distance spanned by the added closing segments . If we cσ based on the average variation of the end-to-end distance found in consecutive frames in simulated trajectories, and we consider it a ligation distance. After computing numbers of right-handed and left-handed knots, we evaluated their statistics, which are summarized in the last column of Furthermore, evaluating knots at short end-to-end distances can be of practical relevance, as the knots could be chemically embedded in the polymer by closing the polymer ring chemically. The distance was set to 10 In addition to the information on knotting statistics, computer simulations can also help understand the mechanisms by which knotting occurs ,75. In tAn earlier work investigated this by inspecting positions of emerging knots along the polymer chains , and fouThe occurrence of backfolding is evident from the evolution of the polymer metrics in terms of the chain span, investigated in Additionally, we show the evolution of the writhe throughout the simulations in By means of coarse-grained molecular dynamics simulations, we studied the behavior of polymers in terms of polymer metrics, monomer distributions and topology of the polymer chain pushed inside infinite nanochannels with both cylindrical and helical geometries, using a DNA biopolymer model system. The simulations showed that the polymer undergoes a compaction upon increasing pushing velocity that could be used for controlling knottedness. When compared to simulations of polymers compressed in finite channels, distinct features emerge. Primarily, the geometry of the channels exerts varying effects on the extent of polymer chain compaction. Consequently, when the polymer is pushed inside open channels, it forms fewer knots compared to when it is compacted by compression within finite channels against an impenetrable wall. The confined environment of the open channels limits the polymer’s ability to explore helical loops of the chiral nanochannels, but it still generates equichiral knots and equichiral writhe. The lower degree of knotting observed in helical loops during the pushing inside helical channels may seem inconsistent with Ralf Metzler’s argument regarding enhanced knotting resulting from irregularities in nanochannels . HoweverOur findings also prompted additional simulations involving variations in the pitch of the channels and the radius of the helix. Some of these simulations were included in the discussion of the results and provided as