This study proposes a disaccharide with a stable structure derived from ᴅ-psicose. The compound with a spiro-tricyclic core was generated at 32% conversion via caramelization of ᴅ-psicose under acidic anhydrous conditions. The compound was identified by high-resolution mass spectrometry and multi-dimensional nuclear magnetic resonance (NMR). The molecular formula was established as C12H20O10 from the molecular weight of m/z 324.1055. Twelve signals were observed by the 13C NMR spectrum. This compound, denoted di-ᴅ-psicose anhydride (DPA), exhibited a lower water solubility (40 g/L) and higher thermal stability (peak temperature = 194.7 °C) than that of ᴅ-psicose (peak temperature = 126.5 °C). The quantitatively evaluated metal ion scavenging ability of DPA was the best in magnesium (average 98.6 ± 1.1%). This synthesis methodology can provide disaccharides with high stability-reducing heavy metals.Demand for healthy diets has led researchers to explore new saccharide as sucrose alternatives.  Sugars satisfy an innate human preference for sweet tastes ,2, and aᴅ-Psicose is the C-3 epimer of ᴅ-fructose, which is rarely found in nature + m/z 347.0952  (13C NMR spectrum (C 105.86 (C-2), 97.80 (C-2′)], six oxygenated methines , and four oxygenated-methylene . The chemical shifts of carbon signals at C-1 to C-6 and C-1′ to C-6′ are shown in the 13C NMR spectrum. In 1H NMR signals were observed, except for C-2 and C-2′, which do not allow C-H bonding. The isolated compound was identified using HRMS and NMR. The molecular formula was established as C0.6 ppm) D. The unspectrum A, supporspectrum , indicatH 3.75  of the cross peak with a C-5 signal was observed as an H-5 signal of α-ᴅ-psicofuranose (starting compound). The two-dimensional NMR spectra are shown in 1H NMR signals were unambiguously confirmed. In particular, the H-6′ signal of ketohexose showed a unique coupling patterns, such as doublets of doublets and broad doublets. Several cross-peaks in the 1H−1H correlation spectroscopy spectrum confirmed a key correlation among the proton signals. The heteronuclear multiple bond correlation (HMBC) spectrum showed 3J correlations between H-1/C-3, C-2′ and H-1′/C-3′, C-2, and showed 2J correlations between H-1/C-2 and H-1′/C-2′, respectively. The structure of the isolated compound based on the NMR spectra and HRMS spectrum is shown in In the heteronuclear single quantum coherence (HSQC) spectrum , δH 3.75DPA was obtained as a white odorless powder. Its water solubility was determined by HPLC at room temperature (25 °C). After centrifugation, the initial 3.55 mM DPA solution was quantified to be 2.23 mM in the supernatant, incubating approximately 40 g DPA dissolved in 1.0 L water. ᴅ-psicose at room temperature. The peak temperature (TP), onset temperature (TO), and endset temperature (TE) were determined. The TO of DPA was approximately 74 °C higher than that of ᴅ-psicose. Similarly, DPA had a higher TP at 68 °C. During melting, the ΔH of DPA was approximately 32% of the value of ᴅ-psicose.p < 0.01) was performed based on the concentration by classifying the metal ions. The magnesium ions exhibited the largest reduction (average reduction 98.6 ± 1.1%). The iron ions exhibited the largest reduction at the concentration of 5 mM (92.4 ± 0.3%); however, there was no concentration-dependence observed. In particular, iron ions at 20 mM (78.3 ± 0.5%) had larger reductions than that at 10 mM (75.2 ± 0.5%). Copper ions had the lowest reduction obtained at 20 mM (7.7 ± 0.5%). In summary, the metal ions significantly reduced (p < 0.001) in the order of Mg > Fe > Cu at 10 mM, which was similar to the molar ratio of incubated DPA.ᴅ-psicose units linked together with two anhydride bonds  to that obtained at 25 °C and 6 days. This indicates that increasing the temperature can reduce the reaction time. Therefore, the optimal reaction temperature was noted to be higher than 4 °C. Considering the temperature, reaction time, and dehydration rate, DPA can be obtained under the optimal reaction conditions of 10–20 °C and 48 h.For the DPA generation, temperature was noted to have a more pronounced effect than reaction time. In particular, the DPA obtained at 4 °C and one day had no significant difference  of the peak (9.95 min) as that of DFA (m/z 324 comprises two water (m/z 36) dehydrated from two ᴅ-psicoses (m/z 360), which corresponds well with the NMR spectra. The twelve signals observed in the 13C NMR spectrum  can exist in four formations, namely α-ᴅ-psicofuranose, β-ᴅ-psicofuranose, α-ᴅ-psicopyranose, and β-ᴅ-psicopyranose by mutarotation in solution (in a ratio of 38:13:25:24) [α-ᴅ-psicofuranose was observed at low magnetic field, approximately 85 ppm in 13C NMR, and there was no signal at 75 ppm. In the HSQC spectrum (α-ᴅ-psicofuranose because the δH 83.30 (C-5) signal showed a cross-peak with δH 3.75 , the commonly reported signal of H-5 from α-ᴅ-psicofuranose. In addition, the coupling pattern (dd or broad-d) of the H-6′ signal in the 1H NMR spectrum , the structure of the isolated compound (peak 9.95 min) was identified as a novel compound, denoted di-ᴅ-psicose anhydride (ᴅ-psicose were bound [3:25:24) . Among tspectrum , the firspectrum B indicatspectrum  confirmenhydride . As mentre bound .ᴅ-psicose (291 g/100 mL) [w/w), which is 90–95% of the water solubility of sugar [ᴅ-psicose, its polarity is expected to decrease during this process.The water solubility of DPA (about 4 g/100 mL) is lower than that of /100 mL) . Similar/100 mL) ,20. Thisof sugar ,21. As Dᴅ-psicose and DPA were measured through DSC for compatibility with other studies. ᴅ-psicose. According to the study on the heat capacity of rare sugars, physical stability increases when two ketohexoses are combined in an anhydride structure [ᴅ-psicose, even in its crystalline state.DSC analysis is intrinsically dynamic and it is impossible to sense equilibrium . In thistructure . In addiᴅ-psicose can stabilize the ring conformation of ketohexoses. ᴅ-Psicose has more intramolecular hydrogen bonds than crystalline fructose and tagatose [P at 194 °C) suggests its potential as a food additive. ᴅ-Psicose, a monomer of DPA, increases the moisture content and reduces the tendency to crystallize in gelatin-based soft candy. The high-water association of ᴅ-psicose decreases the glass transition temperature value owing to the plasticizer effect [The intramolecular hydrogen bonding in tagatose . The ther effect .The DPA structure has an additional six-membered ring between the two ketoses. The 1,4-dioxane in the center of this spiro-tricyclic psicodisaccharide has a boat conformation, which participates in the complex stabilization with a metal cation. Together with the two oxygen atoms of 1,4-dioxane, the oxygen atoms of the hydroxyl groups at the C-3 and C-3′ signals simultaneously promote the bonding of the metal cation ,24. ThisThe reduction rate of DPA for each metal ion can be related to the electrochemical properties of the metal ions . The staᴅ-psicose (≥95%), hydrochloric acid (37%), sodium hydroxide, DMSO, iron(II) chloride (≥99%), magnesium chloride (≥99%), copper(II) chloride (≥99%), sodium phosphate monobasic, and sodium phosphate dibasic were purchased from Sigma Aldrich . Industrial grade ᴅ-psicose (≥95%) was obtained from Hengshui Haoye Chemical . HPLC grade water, methanol, and acetonitrile were purchased from Thermo Fisher Scientific .Analytical grade ᴅ-psicose was inspired by previous studies [ᴅ-psicose was dissolved in 10 mL cold (4 °C) hydrochloric acid by ultra-sonication for 5 min. The reaction was stored at −20, 4, 25, and 40 °C for 1, 2, 4, 6, and 8 days, respectively. The reaction was stopped by adding equal volumes of DMSO, vortexed for 1 min, and sonicated for 5 min. For the HPLC analysis, the reactants were diluted five-fold with 1 N sodium hydroxide and five-fold with 50% (v/v) methanol in water  equipped with a refractive index detector  and a Shodex Asahipak NH2P-50 4E C18 column . The mobile phase was 75% (v/v) acetonitrile in water. The total isocratic elution run time was 15 min. The flow rate and injection volume were 0.6 mL/min and 20 μL, respectively. The column oven temperature was set to 35 °C. Before the analysis, the samples were filtered through a 0.2 μm polytetrafluoroethylene filter .The samples were analyzed using a reversed-phase HPLC system . The mobile phase was 78% (v/v) acetonitrile in water. The isocratic elution was carried out at 30 °C at a run time of 30 min. The flow rate and injection volume were 10 mL/min and 5 mL, respectively. The effluent was collected (4.0 mL each) according to the elution profile using a fraction collector . The collected solvent underwent vacuum evaporation  at 40 °C in a water bath.For the HRMS and NMR analysis, the sample was filtrated using Whatman no.4 filter paper  under vacuum conditions. The filtered solution was performed using a preparative HPLC system , equipped with a refractive index detector . Separation was achieved on a YMC-Pack Polyamine II Cm/z. The HRMS parameters were optimized as follows: capillary voltage of 2.5 kV, cone voltage of 40 V, source temperature of 120 °C, and desolvation gas flow rate of 500 L/h.The molecular weight of a single fraction was analyzed using a time-of-flight HRMS system  with an electrospray ionization source operated via MassLynx 4.1 software. The sample was directly infused into the mass spectrometer through the electrospray ionization source. The mass spectrum was acquired in the positive ion mode and recorded in the range of 50–1200 d6. NMR analysis was conducted using a Bruker 600 MHz . One-dimension NMR analysis was performed for 1H (600 MHz), 13C (150 MHz), and DEPT. Two-dimension NMR spectra were recorded for the HSQC, correlation spectroscopy, and HMBC. All chemical shifts were given in ppm. The structure was assigned using MestreNova 11.0 .The obtained single compound was dissolved in 100 μL DMSO-g and 4 °C for 10 min. The supernatant of the DPA solution was filtered through a 0.2 μm polytetrafluoroethylene filter (Nanosep®). The amount of DPA was analyzed using HPLC.The assessment of the water solubility of the DPA followed the method described in a previous study . First, ᴅ-psicose were weighed to 4.5 mg, respectively, in a hermetic aluminum pan. The aluminum pans were sealed and placed in a measuring cell. An empty aluminum pan was used as the reference. The endotherm scans of the samples were obtained at a rate of 10 °C/min from 25 to 300 °C. Dry nitrogen was used to purge the sample at 20 mL/min.The DSC experiments were carried out using a DSC 4000 , equipped with a cooling system. DPA and 2, MgCl2, and CuCl2 were dissolved in deionized water at concentrations of 5, 10, and 20 mM, respectively. DPA was prepared in deionized water and diluted with the same solvent at 10 mM. Subsequently, 100 μL of DPA and metal ions was mixed and vortexed for 1 min. The mixed reactants were incubated in a thermomixer (Thermo Fisher Scientific) for 24 h at 37 °C and 400 rpm. The reacted solutions were diluted with a 2% (v/v) aqueous nitric acid solution before the metal ion analysis. The metal ion analysis was performed using inductively coupled plasma with an optical emission spectroscopy system . The three metal ions were quantified using the standard curve of each standard.The metal ions FeCl® 15 statistical software . One-way and two-way analyses of variance followed by the Tukey’s honestly significant difference test (p < 0.05) were applied to determine the significance of the differences among the means.The experiments were conducted in triplicate. The data were expressed as the mean ± the standard deviation of the values. The graphs were generated and drawn using GraphPad Prism 8.4.3 for Windows . Statistical analysis was performed using JMPᴅ-Psicose-derived compound with a spiro-tricyclic-disaccharide structure was obtained via a caramelization reaction. The compound, with a molecular weight of 324.1055, was identified as DPA via NMR analysis. Temperature and reaction time were found to be important factors for DPA production. The most DPA was produced (conversion of 32%) at 10–20 °C and 48 h. DPA has a lower water solubility than ᴅ-psicose, but has higher thermal stability. The spiro-tricyclic structure can bind metal ions (magnesium > iron > copper) indicating the potential of DPA for hazardous substance removal or mineral transport. The current study still has a limitation in that an efficient DPA production and purification methodology was not achieved. Nevertheless, this synthesis methodology can provide disaccharides with high stability, reducing heavy metals.Korea Food Research Institute has filed a patent (10-2022-0018883) that describes the synthesis of DPA by inventors Y.S.J and M.Y.


Furthermore, CD pathology can be mediated by Mycobacterium avium subsp. paratuberculosis (MAP) infection. However, the direct effect of folate (B9) and cobalamin (B12) deficiency during MAP infection remains uncharacterized. This study investigates how folate and B12 deficiency impedes macrophage apoptosis and exacerbates the inflammation in macrophages infected with MAP isolated from CD patients. Accordingly, we measured folate and B12 in ex vivo plasma samples collected from CD patients with or without MAP infection (N = 35 per group). We also measured the expression of the pro-inflammatory cytokines IL-1β and TNF-α, cellular apoptosis and viability markers, and bacterial viability in MAP-infected macrophages cultured in folate and B12 deficient media. We determined that MAP-positive CD patients have significantly lower plasma folate and B12 in comparison to MAP-negative CD patients . We further show that pro-inflammatory cytokines IL-1β and TNF-α are significantly upregulated during folate and vitamin B12 deprivation following MAP infection by several folds, while supplementation significantly reduces their expression by several folds. Additionally, depletion of folate, B12, and folate/B12 following MAP infection, led to decreased macrophage apoptosis from 1.83 ± 0.40-fold to 1.04 ± 0.08, 0.64 ± 0.12, and 0.45 ± 0.07 in folate-low, B12-low, and folate/B12-low cells, respectively. By contrast, folate and folate/B12 supplementation resulted in 3.38 ± 0.70 and 2.58 ± 0.14-fold increases in infected macrophages. Interestingly, changes in overall macrophage viability were only observed in folate-high, folate/B12-high, and folate/B12-low media, with 0.80 ± 0.05, 0.82 ± 0.02, and 0.91 ± 0.04-fold changes, respectively. Incubation of Caco-2 intestinal epithelial monolayers with supernatant from infected macrophages revealed that folate/B12 deficiency led to increased LDH release independent of oxidative stress. Overall, our results indicate that folate and B12 are key vitamins affecting cell survival and inflammation during MAP infection. Folate and vitamin B During cofactor . The metcofactor ,17.12 deficiencies are associated with exacerbated inflammation and damage in the brain, vasculature, immune system, liver, and gastrointestinal tract [12 may link hypovitaminosis and chronic inflammatory disease, particularly when the disease progression impedes vitamin uptake [Several studies have shown that folate and Bal tract ,25,26,27al tract ,29. Howeal tract . These eal tract . Therefon uptake ,32.E. coli infection [12 supplementation on pathogens associated with CD needs further investigation.Crohn’s disease (CD) is a chronic inflammatory bowel disease characterized by a pattern of relapse and remission and asymmetrical, segmental, and transmural inflammation . CD patinfection . HoweverMycobacterium avium subspecies paratuberculosis (MAP) is an obligate intracellular pathogen that is known as a causative agent in Johne’s disease (JD), which is a chronic inflammatory disorder affecting the intestines of ruminants [12 supplementation or deprivation on MAP infection and the resultant inflammatory response.uminants . In geneuminants ,39,40. Suminants ,42. In t2-EDTA tube) were collected from 100 CD patients (CDAI ≥ 220 and ≤450). The presence of MAP was subsequently evaluated via IS900 PCR as described earlier [12 (VB12) ELISA Kit  to determine levels of folate and vitamin B12, respectively. This study was approved by the University of Central Florida Institutional Review Board # STUDY00003468. All samples were de-identified before handling.Plasma samples from peripheral blood  with 10% fetal bovine serum . Pure folic acid and 1 mg/mL vitamin B12 solution in methanol  were then added to the medium to generate media with differential B-vitamin status , followed by 48 h of incubation at 37 °C. In MAP-positive treatment groups, monocyte-derived macrophages were infected with clinical MAP UCF4 (1 × 107 CFU/mL), followed by 24 h of incubation under the same conditions.THP-1 immortalized monocyte-like cells (ATCC TIB-202) were cultured in custom formulated folate and Bn status . The cel® Mini Kit  according to manufacturer protocols. RNA concentrations were measured using NanoDrop (OD at 260 nm). RNA was then reverse-transcribed to cDNA cDNA was synthesized from 1000 ng of each RNA sample using 5.8 µL master mix made from the High-Capacity cDNA Reverse Transcription Kit  and then topped up to a total volume of 20 µL with RNase-free water, according to manufacturer protocols. A thermal cycler  was used to perform the reactions for 5 min at 25 °C, 20 min at 46 °C, and 1 min at 95 °C. The cDNA samples were stored at −20 °C or used immediately for RT-qPCR analysis. Gene expression was measured using specific primers for GAPDH, TNF-α, and IL1β, obtained from Bio-rad , followed by quantitative reverse transcription PCR (RT-qPCR) analysis. For each sample, 5 µL of cDNA was mixed with 10 µL of PowerUp SYBR Green Master Mix , 1 µL primer mix, and 4 µL of DEPC-treated water. Samples were added in triplicate to a 96-well microamp RT-PCR reaction plate, and the experiment was run using the 7500 Fast Real-Time PCR System . GAPDH was the control used to obtain baseline CT readings. Relative mRNA expression levels were calculated using the equation (2−∆∆CT).RNA was isolated from each 2.0 mL sample of monocyte-derived macrophages following 24 h of infection with clinical MAP strain (UCF4). RNA was extracted using the RNeasy2 for 48 h. Macrophages were infected following this incubation with clinical MAP at 1 × 107 CFU/mL and maintained for 24 h. We then conducted one of two assays on the plated cells. First, we used the RealTime-Glo™ MT Cell Viability Assay , an ATP-based luminescence assay according to manufacturer protocols. Briefly, we mixed MT Cell Viability Substrate and NanoLuc® Enzyme to 2X concentrations in folate and B12-free RPMI 1640 media and added 100 µL to each well. Cells were incubated with the reagent for 1 h, and then luminescence was measured using the GloMax Navigator system GM-2000  Next, we used the RealTime-Glo™ Apoptosis Assay  an annexin-V-based luminescence assay. Briefly, we combined 2X concentrations of Annexin NanoBiT® Substrate, CaCl2, Annexin V-SmBiT®, and Annexin V-LgBiT in prewarmed folate and B12-free RPMI 1640. 100 µL of the assay mixture were administered to each well and incubated for 20 min, and then the same instrument was used to measure luminescence. Both luminescence readings were analyzed to determine cell viability and apoptosis.THP-1 macrophages were cultured in 100 µL media on a 96 well opaque-sided plate. All cultured cells used the specialty RPMI 1640 formulations described previously. Macrophages were administered 50 ng/mL PMA and kept in a humidified incubator at 37 °C and 5% CO2 incubator. 3 × 105 cells were seeded in the base of clear-bottomed, opaque-sided 96-well plates with 200 μL media. The cells were allowed to differentiate for 14 days before the media was changed to the RPMI 1640 media with variable levels of folic acid and vitamin B12  Cells were cultured in ATCC-formulated Eagle’s Minimum Essential Medium (EMEM) supplemented with 20% FBS  and maintained at 37 °C in a humidified 5% COamin B12 . On day +, respectively, according to manufacturer protocols. The heated lysate was then cooled to room temperature and administered the assay components. After a 30 min incubation, luminescence was again quantified with the GloMax™ Navigator. Cells were not reused between assays. All experimental groups were plated and assayed in triplicate.After preparation of the Caco-2 monolayers and administration of the infected THP-1 macrophage supernatant, we used the LDH-Glo™ Cytotoxicity Assay and the NADP/NADPH-Glo™ Assay  to quantify plasma membrane damage and oxidative stress, respectively. Briefly, for the former assay, all components were combined in warm media and administered to intact, adherent cells inside their respective wells. The cells were incubated for 30 min, and luminescence was measured with the GloMax Navigator™  For the latter assay, cells were lysed with 0.2 N NaOH solution with 1% DTAB, and the lysate was separated to be subjected to heat under acidic or basic conditions to decompose NADPH or NADPt-test was to assess significance between two groups of values. One-way ANOVA was used to assess significance in studies with multiple experimental groups, followed by Sidak’s multiple comparisons test. All data are expressed as average ± SD of the mean, and the difference between treated samples vs. controls was considered statistically significant at p-value < 0.05 and 95% confidence interval. All experiments save ELISAs of plasma samples were performed in triplicates unless noted otherwise.GraphPad Prism V.9.4.0  was used for statistical analysis. First, the Kolmogorov–Smirnov normality test was used to assess normal distribution for all values. Following this analysis, we used Student’s 12 deficiency, there is a lack of data on how MAP-positive and MAP-negative CD patient subsets compare. As such, we took plasma samples from patients previously confirmed to be MAP-negative or MAP-positive via IS900 PCR and compared their folate and B12 levels via ELISA. We found that average MAP-positive plasma folate levels were significantly reduced to 14.48 ± 13.88 ng/mL from MAP-positive 24.15 ± 25.74 ng/mL [12 to 414.48 ± 94.60 pg/mL from 512.86 ± 129.12 pg/mL in MAP-positive versus MAP-negative patients [12 deficiency may be correlated particularly with MAP infection in CD patients.While previous studies have established that CD patients are at an elevated risk of folate and B74 ng/mL A In addipatients B. Since 12-free RPMI 1640 and manually supplemented it with folate and B12 to yield folate-low media, folate-high media, B12-low media, B12-high media, folate/B12-low media, folate/B12-high media, and ordinary RPMI 1640. After 48 h of PMA stimulation and differentiation in the depleted media, we infected them with clinical MAP for 24 h and examined relative cytokine mRNA. folate, B12, and folate/B12 deficiency increased TNF-α expression in uninfected macrophages by 1.63 ± 0.08-fold, 1.91 ± 0.17-fold, and 1.79 ± 0.19-fold, respectively; however, these effects were not statistically significant [12-deficiency increased TNF-α expression from 20.37 ± 0.54-fold to 25.52 ± 1.17-fold [12 deficiency all had inhibitory effects on IL-1β expression in uninfected  and infected macrophages  [Next, we considered how differing vitamin levels in cell culture media alter macrophage cytokine expression. To examine this phenomenon, we took folate and Bnificant . In infe.17-fold . However ± 0.38) .12 in culture medium for infected macrophages reduced TNF-α expression from 12.49 ± 0.33 to 10.66 ± 0.39-fold, while folate had no significant effect (13.14 ± 0.50) [12 supplementation alone had no significant effect on TNF-α expression in uninfected macrophages  [12 supplementation increased TNF-α expression by 1.34 ± 0.04-fold in uninfected macrophages but reduced it from 12.49 ± 0.33 to 11.31 ± 0.10 in infected macrophages [12 reduced IL-1β expression from 1.00 ± 0.04 to 0.80 ± 0.01-fold in uninfected macrophages and from 10.75 ± 0.25 to 9.39 ± 0.09-fold in infected macrophages [12 supplementation’s effect is more uniform in IL-1β than TNF-α, which shows a reduction from 1.00 ± 0.04 to 0.69 ± 0.02-fold in uninfected macrophages and from 10.75 ± 0.25 to 7.00 ± 0.10-fold in infected macrophages [12 supplementation have an anti-inflammatory effect on macrophage cytokine expression during MAP infection, with results varying by specific cytokine. By contrast, folate and B12 deprivation enhance the inflammatory response in identically treated cells.We followed these experiments with another analysis to examine supplementation with folate and B12. Tenfold elevation of B ± 0.50) . Folate ctively) . Interesrophages . Elevaterophages  Supplemerophages . Concurrrophages . These r12 would increase apoptosis to control the infection. We used two methods to assess the apoptosis of MAP-infected macrophages. First, we directly quantified apoptosis using annexin V luminescence. MAP infection increased apoptosis by 1.83 ± 0.40 fold compared to uninfected controls, as anticipated. Furthermore, folate-enriched media increased macrophage apoptosis during infection by 3.38 ± 0.07-fold, a significant increase compared to MAP infection in control media [12 supplemented media [12 alone. Depletion of folate, B12, and both folate and B12 had the inverse effect: annexin V luminescence decreased to 1.04 ± 0.08, 0.64 ± 0.12, and 0.45 ± 0.07-fold, respectively [Apoptosis of infected cells is a critical immune mechanism for countering intracellular pathogens, and MAP consequently uses a suite of anti-apoptotic proteins to impede it . Accordiol media A. An inced media A. We didectively A. 12 supplemented media, corresponding with the observed increases in annexin V luminescence [12, and folate/B12 deficient media did not correspond with a change in overall viability in these groups [12-low group, from 1.00 ± 0.05-fold to 0.91 ± 0.04-fold.We followed this experiment with an ATP-based viability assay. General macrophage viability after MAP infection declined to 0.80 ± 0.05 and 0.82 ± 0.02-fold in folate and concurrent folate/Bnescence B. Interee groups B. Howeve+/Total NADP in the monolayers.After assessing the effects of folate and vitamin deficiency on macrophage inflammation, we considered the impact of altered macrophage inflammation on enterocytes. We hypothesized that changes in macrophage survival and pro-inflammatory cytokine expression would have a detrimental impact on co-cultured Caco-2 cells. Accordingly, we differentiated Caco-2 cells into monolayers and maintained them in culture for 7 days in RPMI 1640 with altered vitamin levels. administered supernatant collected from macrophages cultured and infected in media with comparably altered vitamin levels. 24 h post-treatment, we examined LDH release and NADP12-low conditions increased LDH luminescence 7.73 ± 1.01-fold, which was significant compared to both infected and uninfected cells in control media [12 deprivation did not significantly alter LDH luminescence in culture. Treating Caco-2 cells with supernatant from infected macrophages did not significantly alter the percentage of NADPH compared with the control in any group, suggesting that oxidative stress did not mediate the damage observed in the LDH assay [Treating Caco-2 cells with supernatant from infected macrophages increased LDH luminescence 3.16 ± 0.95-fold compared with uninfected controls, though this effect did not reach statistical significance . By contol media . Similarol media . InteresDH assay .12 deficiency [12 supplementation on CD symptoms and inflammation. Furthermore, the state of the literature is murky on the effects of folate and B12 supplementation on common CD pathogens like MAP. Physicians with CD patients are left without guidance about the risks of prolonged folate and B12 deficiency in this patient subset and a potential tool to mitigate inflammation.CD patients are at an elevated risk of malabsorptive folate and vitamin Bficiency ,32. Furtficiency . Accordificiency . However12 intake. Rat models of methyl donor deficiency indicate that methyl donor-low diets impede bowel development and barrier function while aggravating induced colitis [E. coli in a mouse model of CD [Gastrointestinal health and resilience against infection have been correlated with folate and vitamin B colitis ,46. By c colitis . Additioel of CD . These fel of CD . In humael of CD ,50. Furtel of CD .12 alter inflammation during infection with a common CD pathogen. MAP-positive CD patients have significantly lower plasma folate and B12 than MAP-negative CD patients. We further show that pro-inflammatory cytokines IL-1β and TNF-α are significantly upregulated during folate and vitamin B12 deprivation in after MAP infection, while supplementation significantly reduces their expression. Macrophages in folate and B12-deficient media are less likely to undergo apoptosis during MAP infection, suggesting this key mechanism to counteract intracellular pathogens is inhibited during vitamin deficiency. Folate supplementation, by contrast, increased apoptosis of infected macrophages. Finally, the treatment of enterocytes with folate-low or B12-low supernatant from infected macrophages leads to increased LDH release without significant alterations in oxidative stress.The results of our investigation add to these findings by showing that folate and B12 in CD patient plasma cannot at this time be compared with normal clinical serum ranges for these vitamins, as the ELISAs used to quantify folate and vitamin B12 availability are not approved for clinical practice. However, these results indicate that even within a patient group at risk for folate and B12 deficiency, distinct subsets of folate and B12 availability can be characterized based on MAP infection. Furthermore, the data on cytokine availability indicate that folate and B12 levels affect macrophage-mediated inflammation after only 48 of h prophylactic exposure. Since monocyte-derived macrophages constitutively infiltrate the gut, these results suggest that clinical trials of folate and B12 supplementation may gradually impact inflammation patterns as older, vitamin-restricted cells are replaced [12 can be observed during an ongoing bacterial infection, not just after stimulation with a TLR agonist like LPS [12 supplements may experience reduced inflammation upon the initial macrophage encounter with MAP. Altered inflammation may be followed by apoptosis of the pro-inflammatory, MAP-infected cells, resulting in the removal of both pathogen and inflammatory mediator.Our findings on folate and Breplaced . Importalike LPS . Since t12 alter MAP infection in cell culture, but in animal and clinical studies. Our study centered on monocyte derived macrophages because prior studies have highlighted macrophages as a crucial innate immune mediator for both MAP destruction and proliferation [12’s anti-inflammatory effects. Furthermore, macrophages frequently interact with components of the adaptive immune system, thereby alter adaptive immunity. We do not explore how folate and B12 might affect this function to contribute to long-term inflammatory CD patterns. Accordingly, further investigation is warranted.Future studies are required to explore not merely how folate and Bferation . However


Long-term artificial selection for growth rate and lean meat rate has eventually led to meat quality deterioration. Muscle fiber type is a key factor that markedly affects meat quality. circRNAs have been reported to participate in diverse biological activities, including myofiber growth and development; thus, we herein compared porcine circRNA transcriptome between oxidative and glycolytic muscle tissues.2C12 cells but promote their differentiation.Longissimus thoracis muscle tissues were obtained from Lantang and Landrace pigs at birth  and 90 postnatal days . Hematoxylin and eosin staining and quantitative real-time PCR revealed that all structural traits of the muscle showed large variations between different breeds and growth stages. In total, 329 known miRNAs and 42,081 transcript candidates were identified; 6,962 differentially expressed transcripts were found to play a key role in myogenesis by gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses. In addition, 3,352 circRNAs were identified using five predicting algorithms, and 104 circRNA candidates were differentially expressed. Integrated analysis of differentially expressed miRNAs, mRNAs, and circRNAs led to the identification of 777, 855, and 22 convincing ceRNA interactions in LT1D vs. LT90D, LW1D vs. LW90D, and LT90D vs. LW90D, respectively. Finally, we identified a circRNA candidate circKANSL1L, which showed high homology between mice and pigs, and it was found to inhibit the proliferation of CWe identified genome-wide circRNAs in 0- and 90-day-old Lantang and Landrace pigs by RNA-seq and found that circRNAs were abundant, differentially expressed, and associated with myogenesis. Our results should serve as a reference for future studies on pork quality.The online version contains supplementary material available at 10.1186/s12864-022-08515-7. Over the past few decades, modern pig breeding programs have primarily focused on the genetic improvement of economically important traits . ConsequThe growth and development of myofibers involves ontogenesis during distinct embryonic stages, as well as hypertrophy and conversion in postnatal stage . Such biP < 0.01). To explain, the number of myofibers significantly decreased between birth and 90 postnatal days (P < 0.01), and the average cross-sectional area of myofibers showed an obvious increase during postnatal development (P < 0.01). Further, Lantang pigs showed higher number of myofibers than Landrace pigs at birth (P < 0.01), but there were no significant differences in terms of the cross-sectional area of myofibers. In comparison with Landrace pigs, Lantang pigs showed lower number and cross-sectional area of myofibers at 90 postnatal days (P < 0.01). We then calculated the proportion of different muscle fiber types based on the expression of myosin heavy chain isoforms , while the proportion of MyHC IIb myofibers was higher at 90 postnatal days (P < 0.01). At birth, the expression of MyHC I and IIa in Lantang pigs was significantly higher than that in Landrace pigs (P < 0.01), while the expression of MyHC IIb was higher in Landrace pigs (P > 0.05). Besides, at 90 postnatal days, higher amount of MyHC I was distributed in Lantang pigs (P < 0.01), and MyHC IIb showed the opposite trend between Lantang and Landrace pigs (P < 0.01).Hematoxylin and eosin staining was performed to characterize the structural traits of longissimus thoracis between Lantang and Landrace pigs at birth  and 90 postnatal days . The number of fibers per unit area and average cross-sectional area of myofibers were determined . We found that the structural traits showed large variations between different growth stages . The clean sequences were then annotated and assigned to 470.78 ± 86.52 thousand unique tags in each library by alignments to Rfam and porcine-specific sequences within miRBase, Repeat and Reference mRNA databases (Table S1B). We observed that only 3.16% ± 0.41%  unique reads belonged to known porcine miRNAs, and these unique reads represented 15.27% ± 0.69%  of total clean sequences. Using the miRDeep2 algorithm, 321, 311, 315, and 316 known miRNAs were identified in LT1D, LT90D, LW1D, and LW90D libraries, respectively, and 302 miRNAs were common across all samples. The 10 most highly expressed miRNAs in each library accounted for 71.26% ± 1.15% of the total count of all identified miRNAs, and six miRNAs  were found across all libraries. Of them, ssc-miR-206 showed the highest expression level in LT1D and LW1D libraries, as well as ssc-miR-1 in LT90D and LW90D libraries. We further investigated differentially expressed miRNAs between different breeds and growth stages (Table S1C). In Lantang pigs, 89 miRNAs were differentially expressed between LT1D and LT90D libraries; 34 miRNAs were upregulated and 55 were downregulated in LT90D libraries. In Landrace pigs, 100 differentially expressed miRNAs were identified; 19 miRNAs were upregulated and 81 were downregulated in LW90D libraries. At birth, only seven miRNAs were differentially expressed between LT1D and LW1D libraries, including six up- and one downregulated miRNAs in LW1D libraries, and at 90 postnatal days, 11 miRNAs were differentially expressed between LT90D and LW90D libraries, including four up- and seven downregulated miRNAs in LW90D libraries.Sscrofa11.1 reference genome, including 78.88% ± 1.26% mapped reads with proper pair alignment (Table S2A). Transcript assemblies with StringTie revealed 138,278 isoforms across the 12 libraries, including approximately 24.98% identified candidates that completely matched Ensembl transcript regions (Table S2B). A comparison of known Ensembl transcripts revealed that 39,734, 39,909, 40,445, and 38,429 known transcripts were expressed in LT1D, LT90D, LW1D, and LW90D libraries, respectively; 42,081 transcripts existed in all libraries (Table S3A). Principal component analysis of globally expressed transcripts with fragments per kilobase of transcript per million mapped reads (FPKM) levels was performed, which showed that the differences between groups caused by breed or age were much greater than those between experimental individuals  in several biological processes associated with myogenesis, including skeletal muscle cell differentiation, muscle cell cellular homeostasis, positive regulation of smooth muscle cell proliferation, smooth muscle tissue development, muscle contraction, regulation of skeletal muscle satellite cell proliferation, and response to muscle stretch (Table S4A–D), and 25 myogenesis-related transcripts were identified between LT1D and LT90D samples  were subjected to Illumina sequencing after rRNA depletion, which led to the generation of approximately 1.49 billion reads (average of 124.57 ± 0.29 million reads per sample). After quality control/trimming, 122.86 ± 0.21 million valid reads were obtained, accounting for 98.63% ± 0.19% of raw reads in each library. On alignment of all valid reads, we found that over 84.45% ± 1.46% clean reads could be successfully mapped to the porcine We characterized circRNA landscape and expression by performing deep RNA-seq experiments using the 12 aforementioned muscle tissue samples. In total, 52,133 circRNA candidates were identified using five different predicting algorithms . On the contrary, 14 down–up–down regulation patterns were identified on comparing LW1D and LW90D: circKANSL1L_4 was downregulated in LW90D and could sponge miR-130a, miR-19a, miR-19b, miR-299, miR-376a-3p, miR-487b, and miR-493-5p to significantly downregulate ACTN2, COPS2, FBXO40, FOXN2, MYBPC1, SCN7A, TPM3, and TPM4 expression (Table S7I).The expression of circRNAs potentially plays a key role in physiological and pathological conditions by regulating endogenous RNA targets . We ther targets . This prTo verify the circular structure of circRNAs, differentially and highly expressed circRNA candidates that were correlated with myogenes were selected for further analyses. cDNA was amplified using a pair of divergent primers, which led to the identification of nine circRNAs: circPFKFB1, circKANSL1L-3, circLIMCH1, circKANSL1L, circ4082, circKANSL1L-2, circMYBPC2, circMYBPC2-2, and circNR1H3 . Sanger sequencing further verified their head–tail junction structure .2C12 cells using divergent and convergent primers . To further verify their structure, we analyzed them in Cers Fig. . Convergers Fig. A. On RNa2C12 cells, but the expression of the host gene KANSL1L was unaffected  and Cyclin D1 (P < 0.05) significantly decreased in C2C12 cells and that of Cyclin E showed the same trend (P > 0.05). After circKANSL1L knockdown, the expression level of PCNA (P < 0.01) and Cyclin D1 (P < 0.05) significantly increased in C2C12 cells and that of Cyclin E also increased, but the change was not significant , Cyclin D1 (P < 0.01), and Cyclin E (P < 0.01) significantly decreased, and when circKANSL1L expression was downregulated, the protein expression levels of PCNA (P < 0.05) and Cyclin D1 (P < 0.05) significantly increased and that of Cyclin E also increased, but the change was not significant  assay at 0, 12, 24, and 36 h. In comparison with the empty vector group, after circKANSL1L overexpression, absorbance (450 nm) significantly decreased at 24 h (P < 0.01); however, after circKANSL1L knockdown, absorbance (450 nm) significantly increased at 36 h . circKANSL1L knockdown promoted the progression of C2C12 cells to the S and G2 phases , MYOD1 (P < 0.05), MYOG (P < 0.05), and MyHC (P < 0.05), while circKANSL1L knockdown significantly decreased their expression levels . Western blotting was performed to detect MYOD1, MYOG and MyHC protein expression levels . We also assessed RNA expression levels of MyHC I and MyHC IIb. CircKANSL1L overexpression promoted MyHC I and MyHC IIb expression, while circKANSL1L knockdown inhibited MyHC I and MyHC IIb expression, but the results were insignificant  and MyHC IIb (P < 0.05) significantly increased, and when circKANSL1L expression was downregulated, the protein expression levels of MyHC I (P < 0.05) significantly decreased and MyHC IIb decreased but no significantly (P > 0.05), indicating that circKANSL1L has a regulatory effect on muscle fiber type differentiation; further studies are nevertheless warranted.RT-qPCR was performed to assess the relative expression levels of MYF5, MYOD1, Myogenin (MYOG), and MyHC Fig. A. circKA. (1997) reported that a decrease in the proportion of glycolytic fiber is beneficial to meat tenderness, color, and water-holding capacity  and induced to differentiate in a differentiation medium  when they reached 90% confluence. The cells were cultured in a humidified incubator at 37 °C and 5% CO2.The mouse myoblast cell line C2C12 cells using TRIzol, and cDNA was synthesized from RNA using the PrimeScript™ RT Reagent Kit with gDNA Eraser (Takara), according to manufacturer instructions. Genomic DNA was extracted from C2C12 cells using a kit . To verify the circular structure of circRNAs, we designed a pair of convergent and divergent primers and verified their head-to-tail splicing using PCR and Sanger sequencing (Sangon). The primer sequences used in the experiment are listed in Table S8. Moreover, 2 µg total RNA from C2C12 cells was incubated with 3 U/µL ribonuclease R (RNase R) at 37 °C for 10 min; total RNA without RNase R  was also incubated under the same conditions. Gene expression levels were determined using the 2−△△CT method. GAPDH served as the reference gene.Total RNA was isolated from the muscle tissue samples and C2C12 cDNA and subsequently cloned into pCD2.1-ciR  using the KpnI and BamHI (Takara) restriction sites (OE-circKANSL1L). The empty vector was used as the negative control (OE-NC). siRNAs targeting circKANSL1L junction sites (si-circKANSL1L) and negative control (si-NC) were designed and synthesized by GenePharma Co., Ltd. .To synthesize the full-length linear sequence of circKANSL1L, a primer was designed using Primer 5.0. This sequence was amplified using C2C12 cells were transfected with OE-circKANSL1L, OE-NC, si-circKANSL1L, and si-NC using Lipofectamine 2000 , according to manufacturer instructions, when they reached approximately 60% confluence. si-RNA transfection mix (20 pmol si-circKANSL1L or si-NC + 50 µL serum-free DMEM) or plasmid DNA transfection mix (1 µg OE-circKANSL1L or 1 µg OE-NC plasmid DNA + 50 µL serum-free DMEM) was prepared for each well, incubated at room temperature for 20 min, and subsequently diluted with transfection medium (1 μL lip 2000 + 50 μL serum-free DMEM). This mix was then added to each well, and the medium was replaced to GM after 6 h. The cells were harvested for protein and RNA analyses after 48 h to study cell proliferation. Further, the medium was switched to DM after 48 h, and the cells were collected for protein and RNA analyses at 96 h to study cell differentiation.C4 cells were seeded in 96-well plates. After they adhered to the wall, they were transfected with OE-circKANSL1L, OE-NC, si-circKANSL1L, or si-NC. Six hours after transfection was recorded as 0 h. CCK-8 was added at 0, 12, 24, and 36 h, followed by incubation for 1 h. Absorbance was then measured at 450 nm using a microplate reader .CCK-8  assay was used to evaluate cell proliferation. Approximately 102C12 cells were transfected with OE-circKANSL1L, OE-NC, si-circKANSL1L, and si-NC. After 48 h, the cells were collected, fixed with 75% ethanol, and stored overnight at − 20 °C. They were then resuspended in 500 μL PI/RNase staining buffer solution  and incubated at 37 °C for 30 min. A BD Accuri C6 flow cytometer and FACSDiVa software (BD Biosciences) were used to perform flow cytometric analysis.CThe cells were lysed using RIPA lysis buffer  to obtain proteins, which were then separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transferred to a 0.45-mm polyvinylidene fluoride membrane , and sealed with 5% skim milk for 2 h at room temperature. The cells transfected for 48 h were incubated overnight at 4 °C with the following primary antibodies: PCNA, Cyclin D1, Cyclin E, and Tubulin . Further, the cells transfected and differentiated for 96 h were incubated overnight with following primary antibodies: MYOG, MyHC, Tubulin (ZenBio), MyHC I, MyHC IIb, and GAPDH . After washing with Tris-buffered saline with Tween 20, the secondary antibody goat anti-rabbit IgG-HRP or goat anti-mouse IgG-HRP  was added, followed by incubation at room temperature for 1 h. Finally, enhanced chemiluminescence luminous fluid (Solarbio Life Sciences) was used for band visualization.t-test, and multiple comparative analysis was performed with one-way ANOVA. SPSS 20.0  was used for statistical analyses. P < 0.05 and P < 0.01 indicated different and statistically different, respectively.The comparative analysis of two groups was performed using unpaired independent Additional file 1. 12864_2022_8515_MOESM1_ESM.pdf.Additional file 2. 12864_2022_8515_MOESM2_ESM.pdf.Additional file 3. 12864_2022_8515_MOESM3_ESM.pdf.Additional file 4. 12864_2022_8515_MOESM4_ESM.pdf.Additional file 5. 12864_2022_8515_MOESM5_ESM.pdf.Additional file 6. 12864_2022_8515_MOESM6_ESM.xlsx.Additional file 7. 12864_2022_8515_MOESM7_ESM.xlsx.Additional file 8. 12864_2022_8515_MOESM8_ESM.xlsx.Additional file 9. 12864_2022_8515_MOESM9_ESM.xlsx.Additional file 10. 12864_2022_8515_MOESM10_ESM.xlsx.Additional file 11. 12864_2022_8515_MOESM11_ESM.xlsx.Additional file 12. 12864_2022_8515_MOESM12_ESM.xlsx.Additional file 13. 12864_2022_8515_MOESM13_ESM.xlsx.


These findings imply that the discovered phytoconstituents are promising in the search for anti-Omicron BA.1 drugs and should be investigated in future in vitro and in vivo research.(1) Background: SARS-CoV-2 Omicron BA.1 is the most common variation found in most countries and is responsible for 99% of cases in the United States. To overcome this challenge, there is an urgent need to discover effective inhibitors to prevent the emerging BA.1 variant. Natural products, particularly flavonoids, have had widespread success in reducing COVID-19 prevalence. (2) Methods: In the ongoing study, fifteen compounds were annotated from Echium angustifolium and peach (Prunus persica), which were computationally analyzed using various in silico techniques. Molecular docking calculations were performed for the identified phytochemicals to investigate their efficacy. Molecular dynamics (MD) simulations over 200 ns followed by molecular mechanics Poisson–Boltzmann surface area calculations (MM/PBSA) were performed to estimate the binding energy. Bioactivity was also calculated for the best components in terms of drug likeness and drug score. (3) Results: The data obtained from the molecular docking study demonstrated that five compounds exhibited remarkable potency, with docking scores greater than −9.0 kcal/mol. Among them, compounds  The World Health Organization (WHO) was informed on 31 December 2019 of a cluster of cases of pneumonia of unknown etiology first detected in Wuhan city, Hubei province, China −. However, MS data of compounds; 13 and 15 indicated that these two compounds could be identified as phenolic acid spermidine derivatives. Compound 15 produced molecular ions at m/z 468.3 [M + H]+, 466.2 [M − H]− and fragments in positive ionization mode at 436.3 [M + H − 31]+, 292.2 [M + H − 176]+, and 147 [M + H − 321]+, assisted the occurrence of feruloyl and coumaroyl moieties bonded to spermidine moiety. By comparing our results with the published data [13 was identified as diferuloyl spermidine that gave [M + H]+ at m/z 498.3, [M – H]− at 496.2, and fragmentation patterns at m/z 463.2, 322.3 [M + H – 176]+, 177.1 [M + H – 321]+, and 353.3 [M – H – 143]−, consistent with the attachment of two feruloyl moieties with spermidine −β−galactopyranoside (kaempferol 3−O−β−isorhamninoside), compound (3), −O−α−rhamnopyranoside, compound (7) [α−rhamnopyranoside, compound (8) [α−arabinopyranosyl−5− O−α−rhamnopyranoside, compound (9) [(10) [α−galactopyranosyl 7−O−α−rhamnopyranoside, compound (11) [α−rhamnopyranosyl (1→6) (3”−acetyl)−β−galactopyranoside, compound (12) [α−arabinopyranosyl−7−O−α−rhamnopyranoside, compound (14) [Besides; Twelve active components; ound (2) , Kaempfeund (3), , kaempfeound (4) , kaempfeound (5) , kaempfeound (6) , kaempfeound (7) , kaempfeound (8) , Kaempfeound (9) , Kaempfe9) [(10) , kaempfeund (11) , kaempfeund (12) , and kaeund (14) .A total of fifteen plant substances were subjected to molecular docking calculations using AutoDock Vina to find out the best active Cmds. The names of the identified Cmds and their docking scores are listed in In In addition, In detail, Cmd-1  formed a carbon-hydrogen bond with SER494 (3.35 Å), and SER 496 (3.75 Å). Interaction of pi-pi stacked type with TYR501 (4.74 Å). Interaction of pi-alkyl with TYR501 (4.45 Å) and HIS505 (4.57 Å). Cmd-1 formed six conventional hydrogen bonds with ASP405 (2.28 Å), ARG408 (2.80 Å), TYR453 , ARG498 (3.31 Å), and TYR501 (3.10 Å). Cmd-2, with a docking score of −9.5 kcal/mol, exhibited a featured pi-cation interaction with LYS403 (4.85 Å). It also formed pi-pi stacked and pi-alkyl interactions with TYR501 (5.50 Å) and ARG493 (4.58 Å), respectively. It also formed a carbon-hydrogen bond with SER496 (3.71 Å), a pi-donor hydrogen bond with the same residue at 4.02 Å, and several conventional hydrogen bonds with the following residues: LYS403 (2.99 Å), ASP406 (2.02 Å), GLN409 (3.08 Å), TYR453 (3.06 Å), SER494 (3.22 Å). Cmd-3, with a docking score of −9.2 kcal/mol, interacted with TYR501 in two ways: pi-pi stacked (4.41 Å) and pi-pi T-shaped (4.84 Å). It also interacted with HIS505 in a pi-pi T-shape (4.21 Å). Pi-alkyl interaction was observed with three different amino acid residues, VAL417 (4.29 Å), TYR453 (4.83 Å), and LEU455 (5.10 Å). It formed three main conventional hydrogen bonds with the following residues: LYS403 (3.22 Å), GLN409 (2.83 Å), and VAL417 (3.27 Å). Cmd-4 and Cmd-5 obtained the same docking score . However, they interacted with different residues inside the binding pocket. As in Cmd-2, Cmd-4 also showed pi-cation interaction with LYS403, but at different distances. Cmd-4 recorded one pi-pi T-shaped interaction with TYR453 (5.62 Å) and one pi-donor hydrogen bond with the same residue at 3.98 Å. One Carbon-hydrogen bond was detected with ARG493 at 3.67 Å. Cmd-4 exhibited eight conventional hydrogen bonds with ASP406 (2.13 Å), ARG408 (2.92 Å), GLN409 (2.90 Å), TYR449 (2.45 Å), SER496 (3.01 Å), ARG498 (3.09 Å), and TYR501 . Regarding Cmd-5, TYR453 and SER496 were detected as the main residues of interaction as they formed three conventional hydrogen bonds and three carbon-hydrogen bonds, respectively. One pi-donor hydrogen bond was noticed with TYR501, one pi-pi stacked interaction with the same residue, and one pi-pi-T-shaped interaction with HIS505 was also detected.For comparison, the best-ranked five compounds were also docked to the identified RBD residues of the wild-type SARS-CoV-2. Details of interacting residues of wild-type RBD-ligand complexes were examined in order to investigate various interactions in docked complexes. With respect to Cmd-1, six conventional hydrogen bonds were formed with the following residues; ARG403 (2.30 Å), TYR453 (2.25 Å), GLN493 (2.84 Å), ASN501 , and TYR505 (2.53 Å). Cmd-1 also made a carbon-hydrogen bond with SER 494 (3.49 Å), as well as a pi-donor hydrogen bond with GLY496 (2.94 Å).Contrary to Cmd-1, two additional types of interactions can be noticed, Pi-Alkyl interaction with TYR449 (4.60 Å) and Pi-Pi T-shaped interaction with TYR505 (4.92 Å). It also formed four conventional hydrogen bonds: ARG403 (2.92 Å), GLN493 (2.35 Å), SER494 (2.39 Å), one carbon-hydrogen bond with SER494 (3.65 Å), and one pi-donor hydrogen bond with GLY496 (3.26 Å).As in Cmd-2 docked complex, Cmd-3 demonstrated similar interactions with the wild RBD.Cmd-4, the second-best docking score inhibitor, interacted with the wild RBD via only one form of interaction: conventional hydrogen bonding. hydrogen bonding residues are: ARG403 , GLU406 , TYR453 (2.11 Å), GLY496 (2.51 Å), GLN498 (2.38 Å), and ASN501 (2.94 Å). Cmd-5, the least wild RBD inhibitors, with a docking score ≥ −9.0 kcal/mol, exhibited two different types of unfavorable interactions, unfavorable Acceptor-Acceptor with GLU406 (2.91 Å), and unfavorable donner-donner GLN493 (2.60 Å). It formed a double conventional hydrogen bond with ARG403  and GLN498 , one single hydrogen bond with GLU406 (2.11 Å), GLN493 (2.29 Å), and GLY496 (2.69 Å). Only one weak pi-alkyl interaction was also observed with TYR449 at 5.11 A distance.Molecular dynamics (MD) simulations investigate the stability of receptor-inhibitor complexes, structural details, conformational elasticities and the reliability of receptor-inhibitor affinities. Accordingly, MD simulations over 200 ns followed by binding energy estimates for the top five components complexed with the Omicron BA. 1 were performed. The binding free energies were calculated using the MM/PBSA technique. The corresponding binding energies were estimated and shown in Moreover, computed binding energies over 100 ns for Cmd-3, Cmd-4, and Cmd-5 were slightly lowered by −3.7, −8.0, and −6.6 KJ/mol, respectively. Longer MD simulations of 150 ns and subsequently 200 ns were accomplished for those five potent Cmds in complex with Omicron BA.1 to obtain more reliable results, and the related binding affinities were calculated.With respect to data from 200 ns MD simulation, Cmd-1 and Cmd-2 obtained binding energies of over −45.0 kJ/mol, whereas Cmd-4 had the highest stability compared to others, with a binding energy value of over −65.0 KJ/mol.To verify the consistency and behavior of the discovered Cmds, their complexes with Omicron BA.1 were structurally and energetically analyzed over 200 ns MD simulations and compared to those of the apoprotein structure. The structural stability of the investigated complexes was assessed by measuring root-mean-square deviation (RMSD), root-mean-square fluctuation (RMSF), the radius of gyration (Rg), and solvent-accessible surface area (SASA).As previously mentioned, the primary goal of the MD simulations is to examine the positional and conformational changes of ligands when they bind to the binding pocket, which provides insight into the binding stability. In order to analyze the structural stability of the best five plant substances in a complex with Omicron BA.1, the root-mean-square deviation (RMSD) of the complete complex backbone atoms was evaluated for comparison purposes. RMSD values indicate the stability of the structures generated during the simulation trajectories ,63. RMSDLower values of RMSD demonstrate an insignificant difference between the starting and final structures over the MD course.Throughout the 200 ns MD simulations, the estimated RMSD values for Cmd-2 were found to be 2.81 Å with a difference of 0.51 Å from the apoprotein structure (2.30 Å). As well, Cmd-3, Cmd-4, and Cmd-5 obtained an average RMSD of 3.22, 3.87, and 3.96 Å, respectively. In contrast, Cmd-1 showed higher values of RMSD (avg. = 5.53 Å), which reflects the occurrence of some positional or conformational changes. As shown in A root-mean-square fluctuation (RMSF) analysis was executed in order to study the flexibility of the Omicron BA.1 residues over the 200 ns MD simulations. This is a numerical measurement comparable to RMSD, except that instead of expressing positional changes across complete structures over time, RMSF calculates individual residue flexibility or how much a specific residue moves (fluctuates) throughout a simulation .RMSF per residue is often displayed vs. residue number and can indicate which amino acids in a protein contribute the most to molecular movement structurally. A high RMSF value indicates greater flexibility, whereas a low RMSF value indicates limited mobility. The RMSF graph for the five studied components and the apoprotein structure is illustrated in The averages values of RMSF for apoprotein structure, Cmd-1, Cmd-2, Cmd-3, Cmd-4, and Cmd-5 were 1.50, 1.86, 1.53, 1.54, 1.55, and 1.57 (Å), respectively.The radius of gyration (Rg) is a parameter that shows how the protein’s structural compactness changes during simulation . An Rg aDuring the 200 ns simulation time, all Cmds demonstrated appropriate Rg behavior, as shown in Compared to the apoprotein (Rg avg. = 18.25 Å), the Rg values for the five Cmds ranged from 18.35 to 18.88 (Å). Solvent-accessible surface area analysis (SASA) was computed to obtain a deeper insight into interactions between the complex and the solvent throughout the 200 ns MD simulation. 2), respectively.The average SASA values for Cmd-1, Cmd-2, Cmd-3, Cmd-4, and Cmd-5 were 10,317.33, 10,412.28, 10,669.97, 10,437.59 and 10,500.047  is a crucial element that influences drug transport from the site of administration into the bloodstream. It is also well recognized that low medication solubility can result in poor absorption. The logS value should be greater than −4.0. The second factor is the partition coefficient between n-octanol and water, cLogP, which is used to determine the molecule’s hydrophilicity. For the molecule to be well absorbed, values of cLogP should be in the range of −4.0 to 5.0. Numerical values of the drug-likeness parameter should be in the positive range. A positive value indicates that the molecule primarily comprises fragments that are commonly found in commercial medications. The drug score combines all computed values of drug-likeness, cLogP, logS, Mwt, and toxicity risks into a single useful value that can be used to assess the compound’s overall potential to qualify for drug approval. Both Cmd-1 and Cmd-3, which had negative drug-likeness values, had the lowest drug scores when compared to others. Strikingly, Cmd-2 obtained the highest values of drug-likeness and drug score when compared to other Cmds.Another parameter to consider is toxicity, which indicates whether the molecule has a low risk (G), medium risk (Y), or high risk (R). The toxicity prediction is executed based on a precomputed collection of structural fragments that cause toxicity alerts if detected in the studied structure. As shown in Echium angustifolium and peach were successfully identified and screened for possible inhibition of the SARS-CoV-2 Omicron BA.1 variant using various in silico methods. According to a molecular docking study, five phytochemicals showed promising inhibition of Omicron BA.1, with a docking score greater than −9.0 kcal/mol. The dynamic behavior of the five best docked protein–ligand complexes was studied on a time scale of 200 ns. This revealed different effects of the ligands on docking to Omicron BA.1, which were assessed by their respective RMSD, RMSF, Rg and SASA. Moreover, the analysis of molecular properties such as solubility (logS), hydrophilicity (cLogP), toxicity, drug-likeness, and drug score revealed that the studied components had a high chance of becoming drug-like compounds. The results of docking, dynamics and physicochemical property evaluation suggest that the annotated phytoconstituents from Echium angustifolium and peach are promising drug candidates. Further developments, especially in vitro and in vivo studies, are an important step to test these compounds as effective anti-Omicron BA.1 drugs.In this work, fifteen compounds from


Thrombogenicity remains a major issue in cardiovascular implants (CVIs). Complete surficial coverage of CVIs by a monolayer of endothelial cells (ECs) prior to implantation represents a promising strategy but is hampered by the overall logistical complexity and the high number of cells required. Consequently, extensive cell expansion is necessary, which may eventually lead to replicative senescence. Considering that micro-structured surfaces with anisotropic topography may promote endothelialization, we investigated the impact of gratings on the biomechanical properties and the replicative capacity of senescent ECs. After cultivation on gridded surfaces, the cells showed significant improvements in terms of adherens junction integrity, cell elongation, and orientation of the actin filaments, as well as enhanced yes-associated protein nuclear translocation and cell proliferation. Our data therefore suggest that micro-structured surfaces with anisotropic topographies may improve long-term endothelialization of CVIs. Despite technological advances, the use of cardiovascular implants (CVIs) such as left ventricular assist devices (LVADs) or heart valves is associated with adverse events, including thromboembolic complications that may lead to stroke or systemic embolism , 2. Throin vitro endothelialization and (ii) in vivo endothelialization. In vitro endothelialization requires the ex vivo seeding of ECs on CVI surfaces from either autologous vascular tissue  or allogeneic sources , whereas in vivo endothelialization involves EC migration from the intact endothelium or by adhesion, proliferation, and differentiation of circulating endothelial progenitors  genitors . Both apgenitors , 9. For genitors , 11.Overall, patients who could benefit from CVI endothelialization usually have multiple co-morbidities and are of old age. However, these factors are directly linked to premature EC inflammatory and replicative senescence (RS), a major challenge in the formation of intact EC monolayers when using autologous cells . In gene2. The final concentrations of supplements were 2% fetal bovine serum, 1 μg/ml hydrocortisone, 10 ng/ml human epidermal growth factor, 3 ng/ml basic fibroblast growth factor, and 10 μg/ml heparin. During in vitro expansion, cells were either seeded on plates at a density of 20,000 cells/cm2, coated with 1.5% gelatine solution  or on a surface with gratings (described below) at a density of 5 × 103 cells/cm2 coated with glutaraldehyde cross-linked gelatin to increase cell adhesion. When cells were grown on the gratings, the medium was supplemented with a higher serum concentration of 10% fetal bovine serum. For both conditions, the medium was changed every 2–3 days and the cells were passaged at 80–90% confluence.Human umbilical cord vein endothelial cells (HUVECs) and human umbilical cord mesenchymal stems cells (HUMSCs) were isolated from umbilical cords that were obtained from the Department of Obstetrics and Gynecology at the Charité-Universitätsmedizin Berlin. The collection and use of the samples was approved by the institutional ethics board under application number EA2/178/13. In total, three different healthy donors were included in this study. All cells were cultured in Medium 200  supplemented with low-serum growth supplement  at 37°C with 5% CO6 cells were suspended in 1 ml flow cytometry buffer containing phosphate-buffered saline (PBS) with 5 mM EDTA, 25 mM HEPES, 1% fetal bovine serum, and 1% penicillin/streptomycin at pH 7.6. After centrifugation at 300 × g for 5 min, the cell pellets were incubated with the following antibodies for 30 min at 4°C in the dark: anti-human CD31/PE-Cy7 , anti-human CD105/PE , or anti-human CD90/FITC , each at a concentration of 5 μg/ml in PBS containing 5% bovine serum albumin (BSA). The samples were washed twice with flow cytometry buffer, fixed with flow cytometry buffer supplemented with 0.5% paraformaldehyde (PFA) and stored at 4°C until measurement using a FACS Canto II Flow Cytometer . Analysis was performed using FlowJo version 10.6.1 .Cells were harvested after reaching 70–80% confluence and approximately 1 × 10Cell proliferation rate was determined using a cell proliferation kit  according to the manufacturer’s instructions. Images were taken with the Keyence BZ-X710 fluorescence microscope  and the number of 5-ethynyl-2’-deoxyuridine (EdU)-positive cells was analyzed using Imaris software .The population doubling time (PDT) was calculated using the following equation: PDT = t⋅log2/[log(Nf)-log(N0)], where t is the culture duration, Nf is the number of harvested cells and N0 is the number of seeded cells.5 HUVECs per well were seeded in a 48-well plate and cultured for 24 h. Cells were washed twice with PBS at pH 7.4 and fixed with the fixative solution for 15 min at room temperature. After two washes with PBS, cells were incubated at 37°C without CO2 supply in freshly prepared SA-β-gal staining solution. After 12 h, the reaction was stopped by removing the SA-β-gal staining solution and cells were washed twice with PBS. To ensure a representative count, each tissue culture dish was divided into four quarters and at least one image was obtained from each quarter. The percentage of SA-β-gal-positive cells was determined by counting all SA-β-gal-positive cells under bright field illumination. The total number of cells was determined by 4′, 6-diamidino-2-phenylindole (DAPI) counterstaining  and subsequent fluorescence microscopy . Data analysis was performed using ImageJ software   activity was measured using a histochemical senescence detection kit  according to the manufacturer’s instructions. Briefly, 2–3 × 10th, USA) .2, 0.2 mM of each deoxyribonucleotide triphosphate, 1 mM dithiothreitol, 1 M betaine , 0.5 × SYBR Green I , 0.1875 U Platinum Taq DNA polymerase , 0.0625 × Titanium Taq DNA polymerase , and 900 nM of each primer . Primer sequences are given in Cawthon (Relative telomere length was measured using a modified version of the monochrome multiplex quantitative polymerase chain reaction (PCR) protocol as previously described , 18. In  Cawthon . AmplifiCyclic olefin copolymer (COC)-coated surfaces were obtained from Ibidi GmbH  and cut into rectangular slides of 3 cm × 1.5 cm. The slides were cleaned with 70% ethanol, washed three times with PBS, and coated with glutaraldehyde cross-linked gelatin to increase cell adhesion . In deta2). While the channel dimension and medium property were fixed in our experimental setup , the flow rate was controlled using a peristaltic roller pump  to apply a WSS of 1.4 Pa to the endothelial monolayer. A compliance element was inserted between the roller pump and the flow chamber to dampen flow pulsation. The flow chamber was connected to the pump with Tygon tubing  certified for low cytotoxicity. The COC slides containing a cell monolayer with a density of 4–5 × 104 cells/cm2 was placed in the flow chamber, which was then filled with medium and connected to the hydraulic circuit. The flow bioreactor and the medium reservoir were allocated in a chamber in which the environment was maintained at 37°C and 5% CO2. The cell monolayer was then exposed to the flow generating the desired shear stress conditions for 16 h.All flow experiments were performed using a specifically designed parallel plate flow chamber to apply a constant shear stress to the monolayer of HUVECs . The sheTo produce surfaces with gratings, template wafers with gratings of 1 μm depth and height were first fabricated at the cleanroom facilities of the Binnig and Rohrer Nanotechnology Center by a standard nanoimprint lithography process as previously reported . At the At the end of each experiment, cells were fixed with 4% paraformaldehyde (PFA) for 20 min, washed with PBS, permeabilized with 0.5% Triton-X100 for 10 min and blocked with 5% BSA in PBS for 1 h. For the biomechanical characterization of senescent HUVECs under flow conditions, the samples were incubated with the following primary antibodies overnight at 4°C: rabbit anti-vascular endothelial cadherin (VEC) antibody  and rabbit anti-GOLM1 antibody . After washing three times with PBS, cells were incubated with Alexa Fluor 568-labeled donkey anti-rabbit antibody  for 1 h at room temperature protected from light. To visualize cytoskeleton, F-actin fibers were directly stained with Alexa Fluor 555-labeled phalloidin . For the biomechanical characterization of senescent HUVECs after cultivation on anisotropic topography with gratings, in addition to the staining for VEC protein and F-actin fibers, the samples were incubated overnight at 4°C with mouse anti-yes-associated protein (YAP) primary antibody , and after washing three times with PBS, the cells were incubated with Alexa Fluor 555-labeled donkey anti-mouse antibody . To visualize focal adhesions (FAs), the samples were stained with the following primary antibodies overnight at 4°C: mouse anti-paxillin antibody  and rabbit anti-p-paxillin Tyr118 antibody . After washing three times with PBS, the cells were incubated with the following secondary antibodies: Alexa Fluor 488-labeled donkey anti-mouse antibody  and Alexa Fluor 555-labeled donkey anti-rabbit antibody , respectively. After washing three times in PBS, samples were mounted with medium containing DAPI (0.1 μg/ml). Images were taken with the Keyence BZ-X710 fluorescence microscope  and analyzed using ImageJ (version 1.52p) and MATLAB  .To quantify the number of phosphorylated histone H2AX (γ-H2AX) foci per nucleus, images were analyzed and processed using the FoCo algorithm . A nucleL, and the end-to-end distance, D, were measured. The tortuosity was then computed as the ratio of L/D. Tortuosity index values close to 1 indicate a straight junction, while higher values indicate highly convoluted junction topology , and for each junction, the contour length of the junction, topology . A measuTo quantify the level of cytoplasmic-to-nuclear YAP translocation, the ratio between the cytoplasmic and nuclear YAP mean signal intensity was calculated using MATLAB  and ImageJ  .p-value of less than 0.05 was considered significant. All quantitative measurements reported are expressed as average values plus standard errors of the mean. Analysis was always based on three or more independent experiments, as specified in each figure legend. For box plots, the size depicts the measured standard error of the mean and the whiskers indicate 5% and 95% confidence intervals.GraphPad Prism 9.0  was used for performing data analysis and generating graphs. The comparison of population means was done using a two-way analysis of variance and a in vitro conditions on population doubling level (PDL) 3  activity, and significantly shortened relative telomere lengths  3 . RS was  lengths , three t lengths , 27. Fury others , 3  according to the distribution of VEC signal across the cell outer membrane, measured through the CI , andc.the orientation and elongation of ECs according to the actin filament reorganization ,p = 0.001) and from 1.0 ± 0.1 (3 PDs) to 0.5 ± 0.03 (17 PDs) under flow conditions (p = 0.0001), respectively (p = 0.0001), but was similar to HUVECs at high passage with values of 1.96 ± 0.1 under static conditions and 1.76 ± 0.1 under flow conditions (p = 0.01). At 17 PDs, there was no significant difference in directionality under both static and flow conditions (p = 0.004)  and high-passage HUVECs (17 PDs) achieved counterflow polarization . Under bectively . At 3 PDectively . The aspnditions . Similarnditions . Our dat= 0.004) . In concp = 0.04). The linear density of activated FAs was also increased significantly after 17 PDs, from 0.12 ± 0.01 to 0.17 ± 0.02 FA/μm (p = 0.006). A concomitant increase in the size of inactivated and activated FAs was not observed (p = 0.01 for both conditions). Similarly, the activated linear FA density increased from 0.12 ± 0.1 to 0.19 ± 0.02 FA/μm after 3 PDs and from 0.17 ± 0.02 to 0.3 ± 0.03 FA/μm after 17 PDs (p = 0.001 and p < 0.001 respectively) when cultured on a surface with gratings. Accordingly, the inactivated FA size increased from 2.51 ± 0.1 to 3.2 ± 0.2 μm2 after 3 PDs and from 2.6 ± 0.1 to 3.1 ± 0.2 μm2 after 17 PDs (p = 0.008 and p = 0.03 respectively). In summary, our data suggests that both senescence and cultivation on topographies lead to formation and maturation of FAs in HUVECs. In addition, HUVEC monolayers cultured on the micro-structured surface with gratings adopted elongated shape with no evidence of monolayer disruption and formed mainly linear AJs parallel to the grating axis with tortuosity index values close to 1. In contrast, HUVECs cultured on the flat surface mainly formed zipper-like AJs with no preferred orientation and resulted in an increase in tortuosity index values after 3 PDs  and after 17 PDs  (p < 0.001) at 3 PDs, as well as after 17 PDs from 1.9 ± 0.06 to 3.7 ± 0.14 (p < 0.001) (p ≤ 0.001). The average actin filament directionality of HUVECs after 3 PDs was 36 ± 11° on the flat surface and 3 ± 0.04° on the gratings (p = 0.002). A similar change from baseline was observed for HUVECs after 17 PDs; the average directionality of actin filaments was changed from 36 ± 3° on a flat surface to 7 ± 1° on a surface with gratings (p = 0.005) (p < 0.001). A similar change was observed for HUVECs after 17 PDs: from 47 ± 6° on a flat surface to 10 ± 0.6° on a surface with gratings (p < 0.001)  . To quan 0.0001) , analysi< 0.001) . Intrigu= 0.005) . This da< 0.001) . There w< 0.001) . Overallp < 0.001) (p < 0.001)  . Senesce< 0.001) . Cells w< 0.001)  revealed< 0.001) . Interes< 0.001) .p = 0.003). At 10 PDs, the number of EdU-positive cells decreased to 12 ± 1.2% on flat surfaces vs. 16 ± 0.6% EdU-positive cells on gratings (p = 0.02). Finally at 17 PDs, 6 ± 0.5% of cells were EdU-positive on flat surfaces vs. 11 ± 0.6% on gratings . The mean PDT was 48 ± 3 h on the flat surface and 43 ± 2 h on the surface with the gratings (p < 0.05). More interestingly, after 10 and 17 PDs, cells cultured on the flat surface as well as on the surface with gratings displayed the same level of SA-β-gal activity (p > 0.05). On both surfaces after 17 PDs, an up to 40% decrease of telomeres initial length was observed (p < 0.001) and from 1.1 ± 0.1 to 0.59 ± 0.03 (p < 0.001) on the topographies . The patients/participants provided their written informed consent to participate in this study.TN-S, CG, AF, NCh, and ME: scientific oversight. TN-S, VE, CG, and AF: experimental design. VE, NCe, TN-S, CG, HM, KK, HR, SM, FP, and FK: experimentation. VE, TN-S, and SN: manuscript writing. All authors: interpretation of data and critical revision.


Neisseria gonorrhoeae is seriously threatening the treatment and control of gonorrhea globally. Novel treatment options are essential, coupled with appropriate methods to pharmacodynamically examine the efficacy and resistance emergence of these novel drugs. Herein, we used our dynamic in vitro hollow fiber infection model (HFIM) to evaluate protein-unbound lefamulin, a semisynthetic pleuromutilin, against N. gonorrhoeae. Dose–range and dose–fractionation experiments with N. gonorrhoeae reference strains: WHO F , WHO X , and WHO V  reported), were performed to examine lefamulin gonococcal killing and resistance development during treatment. The dose–range experiments, simulating a single oral dose of lefamulin based on human plasma concentrations, indicated that ≥1.2 g, ≥2.8 g, and ≥9.6 g of lefamulin were required to eradicate WHO F, X, and V, respectively. Dose–fractionation experiments, based on human lefamulin plasma concentrations, showed that WHO X was eradicated with ≥2.8 g per day when administered as q12 h (1.4 g twice a day) and with ≥3.6 g per day when administered as q8 h (1.2 g thrice a day), both for 7 days. However, when simulating the treatment with 5–10 times higher concentrations of free lefamulin in relevant gonorrhea tissues , 600 mg every 12 h for 5 days  eradicated all strains, and no lefamulin resistance emerged in the successful treatment arms. In many arms failing single or multiple dose treatments for WHO X, lefamulin-resistant mutants (MIC = 2 mg/l), containing an A132V amino acid substitution in ribosomal protein L3, were selected. Nevertheless, these lefamulin-resistant mutants demonstrated an impaired biofitness. In conclusion, a clinical study is warranted to elucidate the clinical potential of lefamulin as a treatment option for uncomplicated gonorrhea .The emergence and spread of antimicrobial resistance in  Neisseria gonorrhoeae is the causative agent of the sexually transmitted infection (STI) gonorrhea, and 82 million new global gonorrhea cases were estimated to occur among persons aged 15–49 years in 2020 (N. gonorrhoeae as an urgent threat and a priority 2 (high) pathogen for which new antimicrobials are urgently needed  or orally (600 mg every 12 h for 5 days)  at the amino acid positions 145, 148, 149, 152, 155, 157, 158, and 159 and in L4 (rplD) at position 68 altered and sterically hindered the correct positioning of lefamulin within the A and P sites of the PTC  proteins, such as vga(A–E) and lsa(E), which rescue the translation apparatus from ribosome-targeted antimicrobials, has been described. Finally, resistance by the acquisition of cfr encoding the Cfr methyltransferase has been detected, originally identified not only in coagulase-negative staphylococci (CoNS) from animals and mostly in livestock-associated staphylococci but also in a limited number of cfr-positive MRSA from humans  in 2019 and subsequently in other territories, including the European Union, Canada, and Taiwan. Lefamulin underwent two successful phase 3 randomized controlled clinical trials, i.e., Lefamulin Evaluation Against Pneumonia 1 and 2 (LEAP-1 and LEAP-2) . Lefamul 5 days) . Safety  5 days) . Lefamul 5 days) . Due to  5 days) . Mechani the PTC . Furtherm humans .in vitro activity against N. gonorrhoeae, i.e., with modal MIC, MIC50, MIC90, and MIC range of 0.5 mg/l, 0.25 mg/l, 1 mg/l, and 0.004–2 mg/l, respectively  against C. trachomatis and MIC50 of 0.063 mg/l (range 0.002–0.063 mg/l) against M. genitalium  or correlation between in vitro susceptibility, pharmacodynamics (PD), and treatment outcome have been reported. Appropriate correlations between the in vitro susceptibility, PD, and clinical efficacy of lefamulin are imperative to guide the optimal dosing ideal for N. gonorrhoeae killing and suppression of resistance emergence. For the obligate human pathogen N. gonorrhoeae, for which no ideal animal model exists, a dynamic in vitro hollow fiber infection model (HFIM), which has been stated as imperative  lefamulin concentrations in human plasma and in urogenital tract tissues (main gonorrhea infection sites), which was estimated based on a rat model , WHO X , and WHO V [high-level (MIC>256 mg/l) resistant to azithromycin and with the highest lefamulin MIC (2 mg/l)] detected in a previous study (The us study  (Unemo eus study  were exawww.clsi.org), on GCVIT agar plates  supplemented with 1% IsoVitalex ] and microbroth dilution (in triplicates) in the HFIM medium, i.e., modified Fastidious Broth (mFB) was performed, as previously described .For determination of lefamulin MICs (mg/l), agar dilution, according to Clinical and Laboratory Standards Institute (CLSI) guidelines   of therapeutic antimicrobials against  States) .1/2) of lefamulin and free (protein-unbound fraction) lefamulin concentration–time profiles. Sampling for quantitative cultures [colony-forming units (CFUs)/ml] for total N. gonorrhoeae burden and possible lefamulin-resistant N. gonorrhoeae population and measurement of lefamulin concentrations were performed over 7 days. On the first day, 0.5 ml of N. gonorrhoeae cultures (18–24 h) from GCAGP agar plates  supplemented with 1% hemoglobin , 1% IsoVitalex , and 10% horse serum) was inoculated in 49.5 ml of mFB and incubated at 36°C in a humidified 5% CO2-enriched atmosphere to mid-log phase. Then, 10 ml (∼105–106 CFUs/ml) of the N. gonorrhoeae suspension was inoculated into each HFIM cartridge to mimic a clinically relevant N. gonorrhoeae load  . Furthermore, because the lefamulin concentration in the main STI tissues  in rats has been shown to be ≥ 5–10 times higher than the concentration in plasma  simulated, based on free lefamulin concentrations in human plasma, lefamulin single oral dose regimens of 0.4–1.6 g against WHO F, 0.4–3.6 g against WHO X, and 0.4–10 g against WHO V, and all experiments were followed in 7 days. Dose–fractionation experiments (n = 2) simulated, based on free lefamulin concentrations in human plasma, lefamulin oral daily dose of 1.2 g  and 1.5 g  against WHO F; 1.2–4.0 g against WHO X; and 1.2 and 1.5 g against WHO V; administered as one-half of the total dose given at 0 h and at 12 h (q12 h) repeatedly for 7 days, and 2.4–3.6 g against WHO X as one-third the total dose administered at 0, 8, and 16 h (q8 h) repeatedly for 7 days. Additionally, dose–fractionation experiments that simulated lefamulin oral dose regimens based on free lefamulin concentrations in urogenital tissues of rats were conducted, which have been shown to be ≥ 5–10 times higher than in plasma (n = 2) included lefamulin daily dose of 1.2 g against WHO F and WHO X and lefamulin of 1.2 and 1.5 g for WHO V, administered as one-half of the total dose given at 0 h and at 12 h (q12 h) repeatedly for 7 days.Dose–range experiments  was sampled from each HFIM cartridge at time points 2, 9, 24, 48, 72, 96, 120, 144, and 168 h for the dose–range experiments; at 2, 9, 12, 14, 24, 26, 33, 36, 38, 48, 72, 96, 120, 144, and 168 h for the q12 h dose–fractionation experiments; and at 2, 8, 10, 16, 18, 24, 26, 32, 34, 40, 42, 48, 72, 96, 120, 144, and 168 h for the q8 h dose–fractionation experiments. Samples were serially diluted in mFB and quantitatively plated on GCAGP agar plates and GCAGP agar plates containing 3×MIC of lefamulin, resulting in a detection limit of ≥100 CFUs per HFIM cartridge, as previously described .To determine the escribed . Colony To confirm that the predicted lefamulin PK time–concentration profiles were observed in the HFIM, broth samples (500 µl) were collected at time points 1, 2, 9, 19, 24, 48, 72, 96, 120, 144, and 168 h for the dose–range experiments; at 1, 2, 9, 12, 14, 21, 24, 26, 33, 36, 38, 45, 48, 50, 72, 74, 96, 98, 120, 122, 144, 146, and 168 h for the q12 h dose–fractionation experiments; and at 1, 2, 8, 10, 16, 18, 24, 26, 32, 34, 40, 42, 48, 50, 72, 74, 96, 98, 120, 122, 144, 146, and 168 h for the q8 h dose–fractionation experiments.All lefamulin concentrations were determined using liquid chromatography–tandem mass spectrometry (LC-MS/MS), with instrumentation and sample preparation as previously described , but theWithin-laboratory imprecision was estimated by analyzing five samples each on 5 days at three different concentrations. The coefficient of variation (CV) was calculated to 2.7% at 87 mg/L, 2.6% at 1.7 mg/L, and 4.6% at 0.035 mg/L. Linearity of response was confirmed over a range of 200–0.020 mg/L with 17 concentrations. Linear calibration was employed with weighting 1/x. Matrix effects were found to be slightly positive, on the order of 10% at 10 and 0.1 mg/L. The internal standard compensated for this effect with a slight suppression of 5% at a low level.5–106 CFUs/ml. Equal volumes (5 ml/strain) of the suspensions of each strain were inoculated into the same HFIM cartridge. Aliquots (1 ml) were sampled at 24, 48, 72, 96, 120, 144, and 168 h; serially diluted in mFB; and quantitatively plated on GCAGP agar plates and GCAGP agar plates containing 3×MIC of lefamulin, as previously described . The competitive index  compared to the lefamulin-susceptible WHO X parent strain, competition experiments using co-culture were performed in the HFIM. Briefly, bacteria were harvested from GCAGP agar plates and suspended in mFB to a quantity of ∼10escribed . Colony ndex CI;  was deteN. gonorrhoeae burden, and burden of N. gonorrhoeae with lower susceptibility/resistance to lefamulin (selected during treatment). Population modeling was performed employing the non-parametric adaptive grid (NPAG) program of We simultaneously modeled three system outputs for the analysis of the experimental data. The system outputs were as follows: concentration of lefamulin, total Whole-genome sequencing (WGS) was performed, as previously described , on seleN. gonorrhoeae reference strains WHO F , WHO X , and WHO V (high-level resistant to azithromycin and with the highest lefamulin MIC (2 mg/l) detected in a previous study  are sum11 CFUs/ml and WHO V around 109 CFUs/ml. WHO X subsequently maintained the growth around 1010–1011 CFUs/ml throughout all 7-day experiments. WHO F and WHO V both displayed a decrease in bacterial density at the 48-h time point to 109 and 107 CFUs/ml, respectively, and then stabilized at approximately 108–1010 CFUs/ml from the 72-h time point and onward  eradicated WHO F. No lefamulin-resistant subpopulations of WHO F growing on the 3×MIC lefamulin-containing agar plates were found  lefamulin in human plasma and with five and 10 times higher concentrations of free lefamulin, i.e., based on the measured ≥5–10 times higher concentrations in the urogenital tract tissues than in plasma in a rat model  that evolved during failing lefamulin treatments, both in the dose–range and dose–fractionation experiments, especially for WHO X. Similar mutations in rplC have also been shown to result in lefamulin resistance in other bacterial species (N. gonorrhoeae was earlier investigated, and significantly decreased MICs of lefamulin, i.e., 4- to 6-fold, were shown when the MtrC-MtrD-MtrE efflux pump was inactivated. On the contrary, inactivation of the MacAB efflux pump or NorM efflux pump did not impact the activity of lefamulin (Previously, no data regarding lefamulin resistance in any STI agents have been reported. In the present study, we report lefamulin-resistant mutants containing a C395T SNP in the  species . Fortunaefamulin .N. gonorrhoeae in our dynamic HFIM based on both the free lefamulin concentrations in human plasma and estimates of free lefamulin concentrations in urogenital tissues, i.e., based on PK data from a rat model (M. genitalium and/or C. trachomatis (In conclusion, nonclinical PK/PD studies and analysis like HFIM are exceedingly valuable to support optimal dose selection decisions for novel antimicrobials and can reduce the likelihood of drug development failures and, most importantly, guide the approved dosing regimens associated with optimized patient outcomes, including complete eradication of infectious bacteria and suppression of bacterial resistance. However, the absence of human PK data from the main infection sites for STIs, such as the anogenital tract and oropharynx, is a limitation, and more appropriate human PK data from these sites are imperative to generate for novel antimicrobials, such as lefamulin, and currently recommended therapeutic antimicrobials. In the absence of lefamulin PK data in STI tissues, we examined the PD of lefamulin in the treatment of at model , which pat model . Finallychomatis , lefamul