To reliably form and maintain structures with specific functions, many multicellular systems evolved to leverage the interplay between biochemical signaling, mechanics, and morphology.We review mechanochemical feedback loops in cases where cell–cell contact-based Notch signaling drives fate decisions, and the corresponding differentiation process leads to contact remodeling. We compare different mechanisms for initial symmetry breaking and subsequent pattern refinement, as well as discuss how patterning outcomes depend on the relationship between biochemical and mechanical timescales.We conclude with an overview of new approaches, including the study of synthetic circuits, and give an outlook on future experimental and theoretical developments toward dissecting and harnessing mechanochemical feedback.  •The mechanics and geometry of cell–cell contacts affect Notch signaling interactions.•Mechanochemical feedback loops arise, when Notch, in turn, regulates contact remodeling.•Contact-dependent mechanochemical feedback coordinates fate patterning in diverse contexts. Multicellular processes, including development, regeneration, and homeostasis, require the precise spatial and temporal organization of a combination of mechanical, biochemical, and genetic factors ,2,60. Moin vivo contexts [in vitro where cell–cell configurations can be controlled by micropatterned bowtie-shaped wells [Drosophila, for example, is thought to involve Notch interactions through extended basal protrusions [When signaling interactions are restricted to direct cell–cell interfaces, feedback may arise naturally between cellular mechanics, morphology, and signaling a. Signalcontexts , as welled wells . Reactioed wells , and simed wells . Contacted wells ,14•. Fored wells . Over thed wells . Changesed wells ,18. Bristrusions ,20. Moretrusions . Similartrusions ,23••, retrusions —can affetrusions ,26.Figurin vivo, ex vivo, and synthetic contexts. We focus on examples that involve mutually inhibitory signaling interactions mediated by the Notch pathway or related synthetic circuits that lead to divergent cell fates .FiIn some contexts, mechanical processes—such as buckling  or contrIn the developing zebrafish heart, for example, proliferation-induced crowding of cells creates tension heterogeneities in the epithelium of the early myocardium ,38. CellRather than being temporally separated processes, mechanical rearrangements and signaling interactions occur on the same timescale during cardiac trabeculation, and their interplay regulates the spatial distribution of cell types. In the next section, we discuss how interlinked signaling and contact remodeling permits the coordination of distinct differentiation events within the same organ.In contrast to their mammalian counterparts (Section 1), mechanosensory epithelia in other vertebrates remain proliferative after patterning and, in some cases, exhibit remarkable regenerative capacity ,40. In tin vivo, cells receive a range of molecular signals that influence each other and are affected by ongoing external processes. It is challenging, for example, to decouple feedback effects between contact-dependent signaling and contact remodeling from the cross-talk with other signaling pathways [Within a dynamic environment pathways . Engineepathways ,48.This approach can link contact-mediated signaling to downstream effectors of cellular mechanics directly, that is, independent of other molecular pathways. Synthetic Notch receptors permit the design of custom input and output domains that can be used to program contact-dependent transcription ,50. IndeIn multicellular systems where cells exchange signals through direct physical contacts, signaling is affected by cellular rearrangements due to proliferation, migration, or cell shape changes. When signaling directly influences cell–cell contact mechanics, feedback effects can arise, which are harnessed in diverse ways toward generating spatial structures or dynamical states during development, regeneration, and homeostasis , 25•.ex vivo and in vitro platforms, allow us to monitor and manipulate the spatial dynamics of cells and their signaling states in unprecedented ways [Gaining a formal and predictive understanding of mechanochemical systems is a core goal of theoretical biological physics. It motivates novel combinations of theoretical approaches from cellular biophysics, the collective dynamics of active matter, and the nonlinear dynamics of cell–cell communication ,23••,51.ted ways ,53,54. Ited ways , 58, 59,Nothing declared.


Min/+ mice based on the effectiveness of the medicine. Ultra-high performance liquid chromatography-quadrupole/orbitrap/linear ion trap mass spectrometry (UHPLC-Q/Orbitrap/LTQ MS) in the AcquireXTM intelligent data acquisition mode, combining the exact mass and structural information, was established for the profiling and identification of the metabolites of DMU-212 in vivo, and the possible metabolic pathways were subsequently proposed after the oral dose of 240mg/kg for 3 weeks in the colorectal adenoma (CRA) spontaneous model ApcMin/+ mice. A total of 63 metabolites of DMU-212 were tentatively identified, including 48, 48, 34 and 28 metabolites in the ApcMin/+ mice’s intestinal contents, liver, serum, and colorectal tissues, respectively. The metabolic pathways, including demethylation, oxidation, desaturation, methylation, acetylation, glucuronide and cysteine conjugation were involved in the metabolism. Additionally, further verification of the representative active metabolites was employed using molecular docking analysis. This study provides important information for the further investigation of the active constituents of DMU-212 and its action mechanisms for CRA prevention.3,4,5,4’-Trans-tetramethoxystilbene (Synonyms: DMU-212) is a resveratrol analogue with stronger antiproliferative activity and more bioavailability. However, the metabolite characterization of this component remains insufficient. An efficient strategy was proposed for the comprehensive in vivo metabolite profiling of DMU-212 after oral administration in Apc Colorectal cancer (CRC) is a malignant tumor of the gastrointestinal tract that is highly likely to metastasize and recur, and it is associated with an extremely high mortality rate . ColorecMin/+ mice; the inhibitory effect of DMU-212 on CRA growth may be related to the interference with prostaglandin E-2 generation in cells +, or removed the methoxy group at the 4-carbon position to produce ions at m/z 270 [M + H-OCH3]+. Fragment ions at m/z 254 [M + H-OCH3-CH4]+ are the fragment ions corresponding to the loss of methyl and methoxy results in ring-closing at the 4, 5-carbon position from the [M + H]+ ions of DMU-212. The product ion at m/z 241 [M + H-2OCH2]+ is formed by the loss of two OCH2 molecules at the 3, 5-carbon position from the precursor ion, which further releases a CH2 molecule at the 4’-carbon position to form ions at m/z 227 [M + H-2OCH2-CH2]+. The fragment ion at m/z 209 [M + H-2OCH3-OCH2]+ corresponds to the [M + H]+ quasi-molecular ion with the loss of one OCH2 molecule and two methoxy groups. The ion at m/z 159 [M + H-C7H10O3]+ is formed by the benzene ring breakage between C-1, 6 and C-2, 3 and the ion at m/z 121 is formed by the double bond breakage between C-7 and C-8 from the [M + H]+ quasi-molecular ion. Based on the above analysis, characteristic fragment ions including m/z 301, m/z 286, m/z 270, m/z 254, m/z 241, m/z 227, m/z 209, m/z 159 and m/z 121 were serving as characteristic product ions for the identification of the DMU-212 metabolites. The proposed fragmentation pathways of DMU-212 and the MS2 spectrum are shown in DMU-212 generated a quasi-molecular ion [M + H]Min/+ mice, DMU-212 (M0) was detected in the blood, liver, colorectal tissues and intestinal contents samples, according to the retention time (t R = 7.454 min), the accurate quasimolecular ion, as well as the characteristic product ions. DMU-212 was detected in all samples in vivo. A total of 63 DMU-212 metabolites were detected and characterized from the ApcMin/+ mice’s blood, liver, colorectal tissues and intestinal contents samples according to the suggested transformation pathways, molecular formula, and mass spectrometry fragment ions by means of the UHPLC-Q/Orbitrap/LTQ MS method coupled with the established strategy. All the information on the 63 metabolites of DMU-212 detected using UHPLC-Q/Orbitrap/LTQ MS are listed in After intragastric gavage (i.g.) administration to the ApcDemethylated and/or desaturated metabolites:2) less than that of DMU-212, which indicated the loss of the methyl chain moiety, and the characteristic ions were in accordance with the speculative structure. Metabolites M34–36 showed the [M + H]+ ions at around m/z 299 (C18H18O4). Their molecular weights were all 2 Da (2H) less than that of the DMU-212, so this metabolite showed the characteristic fragments with m/z 254, 241, 209 and 121, indicating that they are the desaturated metabolites of DMU-212. Metabolites M9 and M10 showed the [M + H]+ ion at m/z 285 (C17H16O4) with a mass shift of 16 Da, with just the loss of one CH2(14 Da) and one H2 (2 Da) group. In the MS2 spectra, the characteristic fragment ions showed the ion at m/z 270, 254, 241, 227, 209, 159 and 121. Similarly, the molecular weights of metabolites M25 and M26 were all 30 Da less than that of DMU-212, this mass difference corresponds to the loss of the 2H and 2CH2 groups. In addition, the difference of M32 in mass (300 Da versus 256 Da) corresponds to the loss of the 2H and 3CH2 groups, indicating that they were all the desaturated (loss of 2 H) and demethylated metabolites of DMU-212.The molecular weights of metabolites M1-4, M16-22, M29 and M30 were all multiples of 14 Da (CHOxidized/Oxidized and desaturated metabolites:+ ions at around m/z 317 (C18H20O5), which is 16 Da (O) more than that of DMU-212, and the characteristic ions were in accordance with the speculative structure. Metabolites M39-40 (m/z 313) were the oxidized products in the benzene ring, with the four H atoms leaving. Metabolites M38 showed the [M + H]+ ions at around m/z 331 (C18H18O6), which is 30 Da (O) more than that of DMU-212. The difference in mass corresponds to the binding of two oxygen atoms and the loss of two hydrogen atoms. In the MS 2 spectra, these metabolites had common fragments with the parent drug, indicating they are the oxidized and desaturated derivatives of DMU-212.Metabolites M41-45, the oxidation products in the benzene ring, gave the [M + H]Demethylated and dehydrated (and reduced/desaturated) metabolites:+ ions at around m/z 255 (C16H14O3), which is 56 Da (C2H6O) less than that of DMU-212. This mass difference corresponds to the loss of one water molecule and two CH2 groups. In addition, the fitted chemical formula of M31 was C15H12O3, a difference of C3H8O with DMU-212, which indicated the loss of one water molecule and three CH2 groups. Furthermore, metabolites M14-15 generated a protonated [M+ H]+ ion at m/z 257 with the predictive molecular formula of C16H16O3. DMU-212 yielded a difference of C4H8O with the m/z 257 by successively losing H2O and two CH2 (14Da). Metabolites M14-15 further lost four H atoms via a desaturation reaction to form metabolites M27 with m/z 253 (C16H12O3). The characteristic ions were in accordance with the above speculative structure, suggesting that these metabolites could be the demethylated and dehydrated (and reduced/desaturated) metabolites of DMU-212.Metabolites M23 and M24 were detected at the retention time of 6.331 and 6.144 min, respectively. They both showed the [M + H]Demethylated and oxidized/reduced metabolites:m/z 303(C17H18O5), 2 Da more than that of DMU-212 (-CH2+O), suggesting that it is possibly an oxidized and demethylated product of DMU-212. Metabolites M13 and M28, with [M + H]+ ions at around m/z 275 and 261, were eluted at 5.533 and 5.743 min, respectively. They were 26 Da or 40 Da less than DMU-212 with the formula C16H18O4 or C15H16O4, suggesting that it lost two or three methyl moieties and added two hydrogen atoms. All these metabolites had common fragments with the parent drug.Metabolites M5-8 were, respectively, eluted at 6.349, 4.781, 5.923, and 6.784 min and all exhibited a protonated molecule ion at Other Phase I metabolites:17 H16 O5, with [M + H]+ ions at around m/z 301, a difference of (-CH4+O) with DMU-212, which indicated the loss of the alkyl chain moiety, oxidation in the benzene ring and desaturation, and the characteristic ions were in accordance with the speculative structure. Metabolite M12, with [M + H]+ ions at around m/z 305, was eluted at 5.554 min. The metabolite was 4 Da more than DMU-212 with the formula C17H20O5, which was yielded via demethylation of the alkyl chain and hydration, and the characteristic fragment ions at the MS2 spectra, such as m/z 241, 121 were able to be detected. Metabolite M59 was found at 5.846 min with [M + H]+ ions at around m/z 319 , so this metabolite showed the characteristic fragments and could be a hydrated metabolite of DMU-212.The fitted chemical formula of M11 was CGlucuronide-conjugated and/or methylated metabolites:23H26O10 (m/z 463 [M + H]+). The molecular weight of M56 was 162 Da more than the parent drug, it was a glucuronide conjugate, obviously. The fitted molecular formula for metabolite M57 was C23H26O11 (m/z 479 [M + H]+), indicating that oxidation happened on the basis of metabolite M35. Metabolites M47 (t R = 5.257 min) and M48 (t R = 8.077 min) have the same molecular formula of C19H22O4, deduced from their [M + H]+ ions at m/z 315. In addition, compared with the parent drug, M47 and M48 may bind to a CH2 group to form methylated metabolites. Metabolite M51–53 were, respectively, detected at 7.460, 8.936 and 7.912 min with the mass charge ratio 313. Furthermore, the fitted molecular formula was C19H20O4, just a difference of 2H with the M47 and M48. Accordingly, they were tentatively identified as the loss of 2H and a methylated product of DMU-212. Metabolite M58 generated the [M + H]+ ion at m/z 477 with the molecular formula C24H28O10, and its retention time was 6.302 min, a difference of C6H8O6 with DMU-212, which indicated the conjugation of glucuronide (C5H6O6) and a methyl chain moiety (CH2). Further, the characteristic ions were in accordance with the secondary fragments of the above metabolite.Metabolite M56 was eluted at 6.112 min and exhibited a molecular formula of CAcetylated (and methylated) metabolites:m/z 329 (C19H20O5), 28 Da more than that of DMU-212 (+CO), suggesting that it was an acetylated product of DMU-212. Metabolite M50 was eluted at 6.599 min, which exhibited the molecular formula of C19H20O6 , indicating that oxidation happened on the basis of M49. Metabolite M37 was speculated to be C19H18O5 according to the spectral data. It was 26 Da (+CO − H2) higher than that of DMU-212 and was deduced as an acetylated and desaturated metabolite of DMU-212. The fitted molecular formula for metabolite M60 (m/z 345 [M + H]+) was C20H24O5, this suggests that acetylation and methylation may have occurred on the basis of the reduced DMU-212. Importantly, the fragment ions of the above metabolites are consistent with the characteristic fragments of DMU-212.Metabolite M49 was detected at 5.873 min. It generated the protonated molecule ion at around Cysteine-conjugated metabolites:21 H27 N O7 S with the experimental [M + H]+ ions at m/z 438. Comparing their molecular formula with that of M0, it is presumed that they might undergo oxidation and combine with cysteine. Metabolites M62 and M63 were eluted at 5.564 and 5.647 min, respectively. Two of them gave the same theoretical mass (m/z 436) and formula (C21H25NO7S). They were 2 Da (H2) more massive than M54–55, indicating that they might undergo oxidation and combine with cysteine on the basis of desaturated DMU-212. Metabolite M46 was eluted at 3.867 min with the [M + H]+ ion at m/z 392 (C19H21NO6S). As it was 91 Da more massive than DMU-212, and the difference in mass corresponds to the loss of two CH2 groups and four hydrogen atoms, and its combining with cysteine, it was speculated to be a demethylated, desaturated and cysteine-conjugated product of DMU-212. The fragment ions of the above metabolites match the characteristic fragments of DMU-212, which is an important part of metabolite characterization.M54 and M55 were eluted at 5.442 and 5.532 min, respectively. The two metabolites possessed the common molecular formula of CGlycine or glutamine-conjugated metabolites:+ ion at m/z 330 (C18H19NO5), which was 29 Da (-H+NO) larger than DMU-212; its fragments were consistent with those of DMU-212, suggesting that M33 was a glycine conjugate. Metabolites M61, with [M + H]+ ions at m/z 399 (C21H22N2O6), were eluted at 5.002 min. A mass increase of 98 D corresponds to the loss of two CH2 groups, one oxygen atom and four hydrogen atoms, and its combining with glutamine. The fragments ions of M61 also coincide with the characteristic fragments of DMU-212.M33 eluted at 6.325 min with a [M + H]Min/+ mice’s serum and all of them can be observed in the ApcMin/+ mice’s liver (48 metabolites) except for two unique metabolites. Another three unique metabolites were also identified in the ApcMin/+ mice’s colorectal tissues and the rest of the 25 metabolites in colorectal tissues can be found in the serum, liver or intestinal contents, of which 2 metabolites can only be detected in the intestinal contents (50 metabolites) and 1 metabolite can only be obtained in the livers of the ApcMin/+ mice.A total of 63 metabolites with different structures were observed and identified in the biosamples in vivo A. Among Min/+ mice administrated with DMU-212. A total of 43 Phase I metabolites of DMU-212 were identified, most of which were produced through demethylation, oxidation, desaturation, dehydration, reduction and hydration. A total of 20 Phase II metabolites were identified. Among them, most were methylation, acetylation, glucuronide, cysteine, glycine and glutamine conjugation. As shown in Min/+ mice’s intestinal contents, the demethylated, dehydrated and demethylated reaction in the liver, and the demethylated, oxidized and demethylated reaction in the serum and colorectal tissue, were the primary metabolic steps according to the relative percentage of metabolite type. Additionally, the prototype DMU-212 was found in all serum, liver, colorectal tissues and intestinal contents. The parent drug that was most abundant in the liver, colorectal tissues and intestinal contents was not also the most abundant in the serum, and demethylated metabolite M1 was detected as the most abundant metabolite found in the serum. Except for the parent drug, metabolites M1, M51 and M44 were more abundant in the intestinal contents of the ApcMin/+ mice, and metabolites M1, M24 and M19 were most abundant in the liver; metabolites M3, M54 and M26 were most abundant in the colorectal tissues. The data are presented in Summarizing the metabolic pathways  of the 6Microbial bile salt hydrolases contribute to the pathogenesis of colon cancer through the bile acid signaling pathway . BSHs maTM, a novel data-dependent acquisition workflow, was used to analyze DMU-212 metabolic profiles in vivo based on the effectiveness of the medicine. Through the AcquireXTM data acquisition workflow, background subtraction and method updating were performed in real-time. Hence, only the MS2 of potential metabolites were triggered using the updated method without any user intervention. In total, 63 DMU-212 metabolites were identified after the oral dose of 240 mg/kg for 3 weeks in CRA spontaneous model ApcMin/+ mice, including 48 metabolites in the intestinal contents, 48 metabolites in the liver, 34 metabolites in the serum, and 28 metabolites in the colorectal tissues, centering on the further reaction of the prototype, demethylated DMU-212 and oxidized DMU-212.Identifying drug metabolites can be crucial to finding potential drug targets, developing safe drug treatments in clinics, and even rationally modifying drugs . On one Min/+ mice’s liver, which inferred that the liver possessed high metabolic activity for DMU-212. In the liver, CYP450 plays an important role in drug metabolism [Min/+ mice’s liver but was also involved in many other reactions including Phase I, such as desaturation and dehydration, and even Phase II reactions, such as acetylation, glucuronide and cysteine conjugation, which were responsible for the identification of abundant DMU-212 metabolites. In general, the drug is metabolized in the liver and then distributed into the blood. During previous research, DMU-212 was rapidly cleared from the blood within an hour after administration, and a small amount of the prototype was detected [Min/+ mice orally administered with DMU-212, the parent drug was also low, which indicated that DMU-212 was metabolized by intestinal bacteria or was absorbed into the blood and underwent hepatic first-pass elimination, followed by Phase I and II metabolism to be quickly transformed into the other metabolites, especially the demethylated, dehydrated and oxidized metabolites involved in Phase I, and the acetylated, glycine/glucuronide-bound metabolites involved in Phase II observed in this paper.As we know, before oral drugs can be used to enter the blood circulation in the body, they must first be digested in the gastrointestinal tract in order to be absorbed in a suitable form . As a retabolism . A previtabolism . In contdetected . In thisMin/+ mice’s colorectal tissue, demethylated, desaturated and oxidized metabolites were also found. In addition, the oxidized and cysteine-bound metabolite M54 was also a major Phase II metabolite and found at a relatively high level in colorectal tissue. Phase II reactions convert compounds to more water-soluble and often less active or toxic derivatives to increase excretion [Min/+ mice (our unpublished data). Microbial BSHs, which initiate bile acid metabolism, are highly related to CRC, these enzymes have been considered a promising target in the manipulation of gut microbiota to benefit human health [Min/+ mice’s colorectal tissue, which may affect bile acid metabolism to prevent CRA by acting on the BSHs’ target. These provided a hint that DMU-212 metabolic products may be the pharmacodynamic material basis of DMU-212 to prevent CRA. However, it is still necessary to conduct further experiments to confirm this speculation.Medicines enter the bloodstream and are delivered to the target tissue where they exert their therapeutic impact. Colorectal tissue is the target tissue in which DMU-212 might prevent malignancy or delay its onset. In our study, in addition to the most abundant parent drug detected in the Apcxcretion ; therefon health . Therefon health , severalThe resveratrol analogue DMU-212 was synthesized according to the reference method . Its purMin/+ mice  were obtained from GemPharmatech Co. Ltd.  and housed under a standard 12-h light-dark cycle at 25 ± 2 °C and 60 ± 5% humidity with free access to water and a high-fat diet . At 7 weeks of age, the ApcMin/+ mice were randomly assigned to the vehicle-treated ApcMin/+ (MOD) and DMU-212-treated ApcMin/+ groups (DMU) (10 mice per group). The MOD group was orally administrated 0.5 % sodium carboxymethyl cellulose (CMC-Na) per day. The DMU group was orally administrated 240 mg/kg of DMU-212 per day. Drug treatment was performed once daily for 28 successive days. The efficacy of DMU-212 prevention for CRA was evaluated using blood feces, colon length, spleen index, adenoma number, histopathology and inflammatory cytokines, and CRA-related protein expression in colonic tissues. The data is being published in detail in a different publication.All of the experiments were approved by the Animal Care Welfare Committee of the Heilongjiang University of Chinese Medicine. In total, 20 Apcg rpm, 4 °C, 10 min). The supernatants were dried in vacuo and resuspended in 200 μL of methanol and centrifuged at 12,000 rpm for 10 min. A total of 0.2 g of the mice’s liver tissue and colorectal tissues were weighed, respectively, and homogenized in 1 mL of methanol, and then centrifuged at 12,000 g for 10 min at 4 °C. The supernatant was concentrated in vacuo and redissolved in 200 µL of methanol, and centrifuged at 4 °C at 12,000× g rpm for 10 min. Finally, the supernatants collected from the four kinds of samples were passed through a 0.22 μm membrane filter and a 2 μL sample was injected into the UHPLC-Q/Orbitrap/LTQ MS system for analysis.Blood, liver, colorectal tissues and intestinal contents samples were collected at 1 h after the last administration. The serum was obtained through the centrifugation of blood in a refrigerated centrifuge at 3000 rpm (4 °C). A 300 μL volume of the serum sample was added to 900 µL of methanol and vortexed for 1.0 min to precipitate the protein and the sample was centrifuged at 12,000 rpm and 4 °C for 10 min. The supernatants were then concentrated using a speed vacuum concentrator and the residue was redissolved using 100 µL of methanol and centrifuged at 12,000 rpm for 10 min. In total, 0.1 g intestinal contents were collected and resuspended in 1 mL of methanol followed by centrifugation  system hyphenated with a Q/Orbitrap/LTQ mass spectrometer system (Thermo Fisher Scientific). As a stationary phase, a Waters Corporation ACQUITY UPLCTM BEH C18 column  was used. Mobile phase A was 0.1% formic acid in water and mobile phase B was 0.1% formic acid in acetonitrile. The gradient was established as follows: 0–2 min, 1–10% B; 2–4 min, 10–40% B; 4–5 min, 40–65% B; 5–12 min, 65–100% B; 12–13 min, 100% B; 13–14 min, 100–1% B; and 14–17 min, 1% B. The flow rate was 0.3 mL/min and the column temperature was set at 35 °C.2 based on the AcquireXTM intelligent data acquisition technology (Thermo Fisher Scientific), with an Orbitrap resolution of 120,000 and a mass range of m/z 50–1200.A heated electrospray ionization interface, working in positive mode, was used in the mass spectrometer. The parameters were the following: spray voltage of 3.5 kV; sheath gas of 40 arb; and auxiliary gas of 15 arb. The ion transfer tube and the vaporizer temperature were at 350 °C. The scanning mode was full MS/DD-MSThe Xcalibur 4.0 workstation software (Thermo Fisher Scientific) was used for raw data recording and processing. Using Compound Discoverer 3.3 (Thermo Fisher Scientific), post-processing of the data was performed to extract the metabolite-related datasets based on the structural correlation between the drug and its metabolites. A maximum tolerance of 5 ppm was set for the mass error. Using blank samples, the workflow subtracted the chemical backgrounds, aligned the retention times, and found the expected compounds and metabolites. Fragment ion search (FISH) scoring was used, and each annotation was then manually evaluated based on the HCD, DDA spectra, molecular formula, and FISH coverage.https://www.rcsb.org/, accessed on 18 April 2023). The PubChem database  was used to download the SDF format files of the 2D structure of DMU-212. The structures of another core active component were made with ChemDraw . Molecular docking with the main active components of the main active ingredients and BSHs was performed using PyMoL 2.3.0 and AutoDock Vina 18. The binding activity was assessed using binding energy.The 3D structure of bile salt hydrolases (BSHs) was downloaded from the RCSB PDB (Min/+ mice’s serum, liver, colorectal tissues and intestinal contents were systematically and comprehensively investigated based on the effectiveness of the medicine. A total of 63 DMU-212 metabolites were determined and summarized through quick, sensitive and accurate UHPLC-Q/Orbitrap/LTQ MS combined with the data processing software “Compound Discoverer 3.3” and the AcquireXTM data acquisition workflow. In addition, further verification of the representative active metabolites was employed using molecular docking analysis. The Phase I metabolites of DMU-212 were mainly produced via demethylation, oxidation, desaturation, dehydration, reduction and hydration, while the major Phase II metabolites were methylation, acetylation, glucuronide, cysteine, glycine and glutamine conjugation products. As a result of this study, a simple method for studying drugs’ metabolism in vivo is provided, as is scientific and reliable support for a complete understanding of DMU-212’s metabolism and transformation. In addition, as a reference for the further development of new drugs, this is also important for a deeper understanding of the active constituents of DMU-212 and its action mechanisms for CRA prevention.In this paper, the metabolic profiles of DMU-212 in Apc


Viral cell‐free DNA (cfDNA) in plasma has been widely evaluated for detecting cancer and monitoring disease in virus‐associated tumors. We investigated whether the amount of cfDNA of human T‐cell leukemia virus type 1 (HTLV‐1) correlates with disease state in adult T‐cell leukemia‐lymphoma (ATL). HTLV‐1 cfDNA in aggressive ATL was significantly higher than that in indolent ATL and asymptomatic carriers. Notably, patients with lymphoma type represented higher HTLV‐1 cfDNA amount than chronic and smoldering subtypes, though they had no abnormal lymphocytes in the peripheral blood. HTLV‐1 cfDNA can be a universal biomarker that reflects the expansion of ATL clones. ATL is classified into four clinical subtypes: acute, lymphoma, chronic, and smoldering types, based on clinical characteristics and the natural history of the disease [+ and cell adhesion molecule 1 (CADM1)+ cells [Human T‐cell leukemia virus type 1 (HTLV‐1) is integrated into the human genomic DNA and causes persistent and usually asymptomatic infections. However, some individuals experience a lymphoid malignancy of infected CD4 disease . Patient disease . In cont disease , C‐react disease , 7, the )+ cells , 9, and )+ cells . Also, A)+ cells , 12. HigCell‐free viral DNA in plasma has been widely evaluated as a liquid biopsy for detecting cancer and monitoring disease in patients with virus‐associated tumors. For instance, human papillomavirus (HPV) cell‐free DNA (cfDNA) in plasma is detectable and a good biomarker for tumor burden and presence of metastases in patients with HPV‐associated oropharyngeal or cervical cancer , 14. EpsWe enrolled 67 previously untreated patients: 42 asymptomatic carriers, nine smoldering types, seven chronic types, five lymphoma types, and four acute types . All patp = 0.01; lymphoma vs. smoldering, p = 0.001; lymphoma vs. asymptomatic carriers, p < 0.0001). The cfDNA levels of lymphoma type also tended to be higher than that of acute type, though there was no significant difference . The HTLV‐1 cfDNA levels of acute type were significantly higher than those of smoldering type and asymptomatic carries , but were not significantly different from those of chronic type (p = 0.164). However, the two patients with chronic type having the highest HTLV‐1 cfDNA amount , and acute and smoldering types (p = 0.199). These findings show that HTLV‐1 cfDNA is a more promising biomarker to evaluate disease states in HTLV‐1‐infected individuals compared to HTLV‐1 PVL of PBMCs.We first evaluated the amount of HTLV‐1 cfDNA according to clinical subtypes Figure . The medr = 0.773, p < 0.0001, Figure r = 0.195, p = 0.166, Figure r = 0.13, p = 0.31, data not shown), and the populations of CADM1+ CD7– in CD4+ cells . In contrast, HTLV‐1 PVL of PBMCs was correlated with the populations of CADM1+ CD7– in CD4+ cells , but not with sIL‐2R  and LDH . HTLV‐1 cfDNA was highly correlated with sIL‐2R, which reflects the overall tumor burden in ATL patients. The soluble form of IL‐2R is generated by the proteolytic cleavage of membrane IL‐2Rα from activated T cells [Next, we assessed the correlation between HTLV‐1 cfDNA and other clinical factors. HTLV‐1 cfDNA was well correlated with sIL‐2R  [+CD7– cells expands in patients who progress from indolent to aggressive ATL [HTLV‐1 cfDNA is a potential biomarker in HTLV‐1‐infected individuals from asymptomatic carriers to aggressive ATL. We demonstrated that HTLV‐1 cfDNA levels in patients with aggressive ATL were significantly higher than those in indolent ATL and asymptomatic carriers, indicating that HTLV‐1 cfDNA reflects the overall tumor burden and disease progression in HTLV‐1 individuals. HTLV‐1 PVL has been reported to be a predictive marker of ATL development from asymptomatic carriers. However, the PVL values do not correspond well with the overall tumor burden and the disease states as shown in Figure ent ATL) , while tsive ATL . HoweverThe histological features of lymph nodes in ATL patients are usually characterized by diffuse proliferation of atypical medium‐sized to large pleomorphic cells . HoweverIn conclusion, quantification of HTLV‐1 cfDNA may serve as a promising biomarker for the identification of ATL development and its disease states in HTLV‐1‐infected individuals. We assessed the utility of HTLV‐1 cfDNA in a limited number of patients, especially in acute and lymphoma types, and have not yet evaluated an association between HTLV‐1 cfDNA amount and the prognoses of ATL patients. These early data warrant a prospective large‐cohort study to further validate the utility of quantification of HTLV‐1 cfDNA as a predictive factor for ATL development and minimal residual disease detection after treatment.Hiroo Katsuya conceptualized the study, collected and analyzed data, and prepared the manuscript. Hideaki Nakamura and Aya Maeda performed the experiment. Keitaro Ishii, Toshiaki Nagaie, Haruhiko Sano, Haruna Sano, Hidekazu Itamura, Sho Okamoto, Toshihiko Ando, Toshiki Watanabe, Kaoru Uchimaru, and Eisaburo Sueoka provided clinical samples and data. Yorifumi Satou and Shinya Kimura conceptualized the study, and reviewed the results and manuscript. All authors have approved the submitted final version of the manuscript.The authors declare they have no conflicts of interest.This study was approved by the existing institutional review board at Saga University.All patients gave written informed consent in accordance with the Declaration of Helsinki.We permit to reproduce material from other sources.Supporting InformationClick here for additional data file.


Remarkably, these four genomic regions harbor many genes such as WDR19, RPL9, LIAS, UBE2K, DPF3, 5S-rRNA, 7SK, U6, and WDR7 that are related to sperm biology and male fertility. Overall, our findings suggest that inbreeding and increased homozygosity have a negative impact on male fertility in Italian Brown Swiss cattle. The quantification of ROH can contribute to minimizing the inbreeding rate and avoid its negative effect on fitness-related traits, such as male fertility.Intensive selection for improved productivity has been accompanied by an increase in inbreeding rates and a reduction in genetic diversity. The increase in inbreeding tends to impact performance, especially fitness-related traits such as male fertility. Inbreeding can be monitored using runs of homozygosity (ROH), defined as contiguous lengths of homozygous genotypes observed in an individual’s chromosome. The goal of this study was to evaluate the presence of ROH in Italian Brown Swiss cattle and assess its association with bull fertility. First, we evaluated the association between ROH and male fertility using 1,102 Italian Brown Swiss bulls with sire conception rate records and 572 K SNPs spanning the entire genome. Second, we split the entire population into 100 high-fertility and 100 low-fertility bulls to investigate the potential enrichment of ROH segments in the low-fertility group. Finally, we mapped the significant ROH regions to the bovine genome to identify candidate genes associated with sperm biology and male fertility. Notably, there was a negative association between bull fertility and the amount of homozygosity. Four different ROH regions located in chromosomes 6, 10, 11, and 24 were significantly overrepresented in low-fertility bulls (Fisher’s exact test,  Dairy cattle breeding programs currently face the challenge of achieving rapid genetic progress while maintaining adequate genetic diversity and low inbreeding levels. The intense selection for high-producing dairy cows commonly results in the widespread use of related individuals as parents of the next generation. Such selection practice leads to a decrease in the effective population size, a reduction in genetic diversity, and an increase in inbreeding levels. The major concern is that high levels of inbreeding can cause a reduction in fitness and overall performance, a phenomenon commonly known as inbreeding depression . SpecifiThe adverse effects of inbreeding negatively impact many economically relevant traits in dairy cattle. Some examples are male and female fertility , survivaTraditionally, inbreeding depression is estimated as a regression of the phenotype of interest on pedigree-based inbreeding coefficients . The inbAnother approach, involving runs of homozygosity (ROH), has been proposed to accurately capture alleles that are IBD using genomic information. This method relies on the determination of autozygous segments based on runs of consecutive homozygous genotypes, assumed to evolve from a common ancestor . Based oThe impact of inbreeding and increased homozygosity on fitness traits, such as cow fertility, has been studied in dairy cows. However, the impact of inbreeding and increased homozygosity on dairy bull fertility has been largely overlooked. Interestingly, there is a substantial variation in the conception rate among Italian Brown Swiss bulls, with a more than 20% conception rate difference between high-fertility and low-fertility bulls . The maiPhenotypic data consisted of sire conception rate (SCR) records from a total of 1,102 Italian Brown Swiss bulls. The sire conception rate is a phenotypic evaluation calculated using cow field data . BrieflyThe genotypic data for 572,528 single nucleotide polymorphism (SNP) markers located in autosomal chromosomes were available for all the 1,102 Italian Brown Swiss bulls with SCR records. This set of SNP markers is a subset of the markers available in the BovineHD Genotyping BeadChip . The SNP information, which included the chromosome and position, was based on the bovine reference genome ARS-UCD-1.2.The software PLINK (version 1.9) was used to detect segments of consecutive homozygous SNPs called runs of homozygosity (ROH) . BrieflyThe total number of ROH segments, the average and maximum length in kilobases, and the total number of SNPs per ROH were calculated for all the animals for the autosomal chromosomes. For each chromosome, the proportion of homozygous regions was calculated as the total ROH length  divided by the chromosome length to explore differences in homozygosity between the chromosomes. The Pearson’s correlation coefficient between ROH and pedigree inbreeding was also calculated. The pedigree inbreeding was estimated using a pedigree file with more than five generations . The potTo investigate the differences in homozygosity between low-fertility and high-fertility bulls, the entire Italian Brown Swiss bull population was split into two subsets with extreme phenotypes: the bottom 100 bulls (low-fertility bulls) and the top 100 bulls (high-fertility bulls) based on the SCR distribution. The average SCR (standard deviation) for the low-fertility and high-fertility groups was −11.6% (3.7) and 5.9% (1.5), respectively. The amount of homozygosity, calculated as the total ROH length, was compared between fertility groups.p-value ≤0.001) were mapped to the bovine reference genome ARS-UCD-1.2 to identify candidate genes associated with male fertility. The Bioconductor biomaRt R package was used to retrieve the list of genes within each genomic region of interest  using the following linear mixed model:programs .A total of 87,488 runs of homozygosity (ROH) were identified in the Italian Brown Swiss bull population . The mean ROH length was 4,672 kilobases, equivalent to 1,038 consecutive homozygous SNPs. The ROH length ranged from 1,000 to 88,219 kilobases, and the number of SNPs ranged from 100 to 18,277. The number of ROH segments per bull ranged from 47 to 108, with an average of 79. The percentage of homozygous regions for each chromosome is shown in β = −0.008, t-value = −4.3, p-value ≤0.01, β = −0.092, t-value = −6.5, p-value ≤0.01, We evaluated the relationship between homozygosity and bull fertility in two ways. First, we evaluated the total homozygous regions per animal  versus the sire conception rate. The linear regression of the sire conception rate on ROH revealed a negative association between bull fertility and the amount of homozygosity  were found between the two fertility groups. Indeed, the total ROH length average was equal to 387 Mb for the low-fertility bulls and 353 Mb for the high-fertility bulls  and t-values  are listed in For the validation process, we only considered the four most significant ROH segments identified in the enrichment analysis. The significant association between these four ROH segments and male fertility was validated using the entire Italian Brown Swiss bull population. Notably, all four overlapping ROH regions were significantly associated with sire conception rate records in the entire population . The position of the ROH segments along with n ≈ 79, was lower than what was found by n ≈ 99 and n ≈ 94, respectively, possibly because we used a minimum threshold of 100 SNPs rather than the 15 SNPs. Interestingly, Brown Swiss cattle consistently have the largest average ROH length when compared to other dairy breeds (The average length of the ROH (4.6 Mb) was similar to what was previously reported in other studies using Brown Swiss animals . The avey breeds . This maThe distribution of total homozygosity among chromosomes was relatively even but higher in chromosomes 5 and 6, which was as shown by The pedigree-based inbreeding coefficients were moderately correlated with the ROH values (0.65). They were consistently lower than the ROH estimates. We evaluated the impact of homozygosity on service sire fertility by assessing the association between ROH and SCR values. Our findings indicate that bulls with longer homozygous regions and/or a greater number of homozygous segments in their genome exhibit lower male fertility. Interestingly, We also investigated the differences in homozygosity between low- and high-fertility animals, the bottom 100 and top 100 Brown Swiss bulls. Notably, there was a significant difference in the levels of total homozygosity between the two fertility groups, measured as the sum of ROH. Moreover, 22 overlapping ROH segments in four different chromosomes  were significantly enriched in low-fertility bulls. Statistical validation of the four most significant ROH segments per chromosome was conducted on the entire population, indicating that these findings are not false-positives resulting from the population structure. Remarkably, the region identified in BTA6 had been previously reported as a region associated with bull fertility in Brown Swiss cattle . These rWDR19, RPL9, LIAS, and UBE2K. The gene WDR19 is a strong candidate for service sire fertility in Brown Swiss cattle. The gene WDR19 is a constituent of the intraflagellar transport complex essential for the physiological functioning of motile cilia and flagella, such as in sperm motility (WDR19 that is significantly associated with various semen traits, such as sperm motility and sperm abnormalities, insemination success, and SCR, in Brown Swiss cattle (RPL9 encodes a ribosomal protein, which belongs to the L6P family of ribosomal proteins. The gene RPL9 has been identified as differentially expressed in idiopathic male infertility patients, suggesting that it may play a vital role in male fertility (LIAS is a lipoic acid synthase gene and predominantly expressed in the testes. Recent research suggests that LIAS may play a critical role in spermatogenesis, as it was shown to be downregulated in cases of spermatogenic dysfunction in humans (UBE2K gene encodes a protein that is a member of the ubiquitin-conjugating enzyme family and is crucial for the ubiquitination pathways, which are essential to the later stages of spermatid development during spermiogenesis (The significant ROH segments in BTA6 contain several candidate genes, such as motility . Interess cattle . The canertility . The genn humans . The UBEogenesis . Malfuncogenesis . It is nogenesis .DPF3, a potential candidate gene related to bull fertility. This gene encodes a member of the D4 protein family of zinc finger proteins involved in histone modification. Studies have suggested that DPF3 may play a role in spermatogenesis through chromatin remodeling, as epigenetic factors such as histone modifications can potentially affect spermatogenesis and embryogenesis. Notably, variants within DPF3 have been strongly associated with male infertility and sperm morphology in diverse ethnic groups from the US and China (The region in BTA10 harbors nd China .WDR7, which is significantly associated with semen production traits in Holstein bulls (The region in BTA11 contains three non-coding RNAs: 5S-rRNA, 7SK, and U6. The 5S-rRNA, a ribosomal RNA, is part of a sperm-specific DNA organization that may be involved in specific patterns of DNA replication and transcription of the paternal genome in the embryo . Borowskin bulls .The assessment of ROH is a powerful tool for understanding population history, assessing genomic inbreeding, and identifying genes linked with critical economic traits. It can help minimize inbreeding rates and prevent the loss of genetic diversity in dairy cattle. The quantification of ROH and identification of homozygous regions might contribute to the design of mating programs aimed at minimizing inbreeding and avoiding the production of homozygous offspring, which may carry deleterious alleles for male fertility. Moreover, the association between ROH and dairy bull fertility helps us understand the impact of intense artificial selection on the trait and prevent even more inbreeding and the loss of genetic diversity in the dairy cattle population. In this study, we found a higher prevalence of ROH in low-fertility Italian Brown Swiss bulls than in high-fertility bulls, indicating that inbreeding and increased homozygosity negatively affect dairy bull fertility. Conducting genome-wide mapping of ROH enables the identification of potential genes affecting bull fertility, leading to a deeper understanding of the molecular mechanisms underlying male fertility in cattle. It is important to acknowledge that our bull fertility data set is based on cow field records and is subject to pre-selection, as only bulls meeting specific sperm quantity and quality criteria are included.In this study, we evaluated the presence of ROH in Italian Brown Swiss dairy cattle and assessed its association with bull fertility. Remarkably, there was a negative association between bull fertility and the amount of homozygosity. We observed a significant difference in the levels of total homozygosity between the low- and high-fertility groups. Interestingly, the ROH segments enriched in low-fertility bulls harbor genes that are directly involved in sperm biology and male fertility. Our findings suggest that inbreeding and increased homozygosity harm male fertility in Brown Swiss cattle. The quantification of ROH can be used to minimize inbreeding and avoid its negative effect on fitness-related traits, such as bull fertility.


Psychobiotics are a group of probiotics that affect the central nervous system related functions and behaviors mediated by the gut-brain-axis via immune, humoral, neural, and metabolic pathways to improve not only the gastrointestinal function but also the antidepressant and anxiolytic capacity.To assess the efficacy the combination of selective serotonin reuptake inhibitors antidepressants (SSRI) and probiotic supplement containing Lactobacillus Plantarum CECT7485 and Lactobacillus Brevis CECT7480 (PLANTARUM) in patients with mild-to-moderate depression.9 CFU once per day. Thirty patients received SSRI antidepressants only without probiotics intake. The severity of depressive symptoms was assessed using Hamilton Depressive Rating Scale (HDRS) and Patient Health Questionnaire (PHQ-9).Sixty patients with mild-to-moderate depression  were included in an 8-week open label study. Thirty participants received either SSRI antidepressants with PLANTARUM at a dose of 1.0 × 10After 8 weeks intervention, a clinically significant reduction of HDRS total score  was detected in patients with mild-to-moderate depression who received SSRI antidepressants and PLANTARUM , compared with participants who didn’t receive probiotics . A significant reduction of PHQ-9 total score  was identified in patients with mild-to-moderate depression who received SSRI antidepressants and PLANTARUM . However, the participants received SSRI antidepressant only didn’t meet a clinically significant reduction depressive symptoms  by PHQ-9 scale.The combination use of SSRI antidepressants and probiotic supplement PLANTARUM significantly reduced the depressive symptoms.None Declared


Short root anomaly (SRA), along with caries, periodontitis, and trauma, can cause tooth loss, affecting the physical and mental health of patients. Dental implants have become widely utilized for tooth restoration; however, they exhibit certain limitations compared to natural tooth roots. Tissue engineering-mediated root regeneration offers a strategy to sustain a tooth with a physiologically more natural function by regenerating the bioengineered tooth root (bio-root) based on the bionic principle. While the process of tooth root development has been reported in previous studies, the specific molecular mechanisms remain unclear. The Bone Morphogenetic Proteins (BMPs) family is an essential factor regulating cellular activities and is involved in almost all tissue development. Recent studies have focused on exploring the mechanism of BMP signaling in tooth root development by using transgenic animal models and developing better tissue engineering strategies for bio-root regeneration. This article reviews the unique roles of BMP signaling in tooth root development and regeneration. A tooth is composed of an enamel-covered crown and a cementum-covered root. The tooth root plays a critical role in transmitting occlusal forces through the periodontal ligaments (PDLs) to the jaw bones. While the mechanisms of tooth crown development have been extensively studied, those governing tooth root development are distinct . AdditioTooth root development involves complex process of epithelial-mesenchymal interactions, with various signaling pathways, such as Transforming Growth Factor β (TGFβ) and WNTs, being implicated in this process . Bone MoThe Bone Morphogenetic Proteins (BMPs) belong to the Transforming Growth Factor β (TGFβ) family of extracellular signaling proteins. The term “BMP class” has been used to refer to all named BMPs and Growth and Differentiation Factors (GDFs) in certain context . AdditioUp to now, researchers have identified at least 20 BMP members that are structurally and functionally related across various species. Based on similarities in the amino acid sequence and receptor specificity, BMP ligands in most mammals can be classified into five different subcategories: BMP-2/4, BMP-5/6/7/8, BMP-9/10, BMP-12/13/14 , and BMP-15 . BMPs caThe receptors involved in the BMP signaling pathway can be divided into two types, type I and typ e II Gip. Type I The canonical BMP signaling pathway involves the participation of type I and type II receptors and SMAD complexes . BMP ligIn addition to SMAD-mediated signaling activation, BMPs can also signal through a non-canonical pathway, which includes the activation of type I and type II receptors. This pathway operates independently of the SMAD proteins and instead relies on components of the MAPK and PI3K-AKT pathways . PreviouThere are three categories of BMP signaling inhibitors. (1) Extracellular antagonists, including noggin, chordin, follistatin, Tsg, and members of the Dan family  . (2) IntSimilar to tooth development, tooth root development is a complex process involving epithelial-mesenchymal interactions . After cMoreover, HERS functions as a signaling center by producing growth factors, such as Sonic Hedgehog (SHH), that contribute to maintaining the function of epithelial cells and inducing the differentiation of apical papilla. Multiple outcomes for HERS have been proposed, including undergoing apoptosis, transforming into the epithelial rests of Malassez (ERM), and undergoing epithelial-mesenchymal transformation to differentiate into cementoblasts .Although the roles of epithelial-mesenchymal interactions during root development are not fully understood, multiple signaling molecules, such as TGF-β/BMP, WNTs, SHH and fibroblast growth factors (FGFs), have been implicated in this process BMP signaling is involved in different stages of tooth root development. In this context, we will delve into the root development of the first mandibular molar in mice as an illustrative example. By postnatal day (PN) 3, crown development is completed, marking the onset of tooth root development. Subsequently, the location of active BMP/SMAD signaling gradually shifts from the crown to the root.Between PN3 and PN5, concurrent with tooth root development, stem cells crucial for tooth root development progressively emerge, accompanies by evident BMP/SMAD signaling within the dental pulp. Progressing to PN6-PN8, the HERS gradually forms and extends towards the root, simultaneously leading to the formation of the DAC. The distribution of BMP/SMAD signaling gradually extends from the coronal to the apical region, forming associations with blood vessels within the dental pulp and the DAC zone.During the PN9-PN14 phase, there is swift elongation of the tooth root alongside rapid mineralization of tooth root dentin. At this stage, robust BMP/SMAD signaling is notably detected within the root pulp and the apical region. As PN15-PN18 approaches, the rate of tooth root elongation slows down, indicating near-completion of root length development, even though the apical foramen remains wide. During this peiod, the activity of BMP signaling gradually weakens, but intensified in the apical region and the root pulp.Progressing to PN19-PN21, the molar gradually erupts into the oral cavity, achieving occlusion. Simultaneously, the narrow apical foramen gradually forms. Notably, BMP/SMAD signaling is only activated along blood vessels in the root pulp . For a cAdditionally, studies have reported that targeted downregulation of BMP signaling in the epithelium using keratin 14-Noggin transgenic mice leads to reduced sizes of the first and second maxillary molars, altered crown patterns, and failed formation of multiple roots .Collevetively, BMP signaling plays important roles throughout tooth root development .Bmp2 is detected obviously in odontoblasts, dental pulp cells, and osteoblasts, and weakly in ameloblasts, as demonstrated by in situ hybridization   . These fBmp6 expression is observed from postnatal days 1–11 in rats, as demonstrated by RT-qPCR. Moreover, BMP-6 promotes alveolar bone formation at the crypt’s base and contributes to tooth eruption  results show that ng roots . In situ in HERS . These rFor other BMP ligands, i.e., BMP-5/8/10/15, the expressions and roles in tooth root development have not been reported so far.Although various BMP ligands are involved in tooth root formation, the mechanisms of BMPs regulating tooth root development are not fully elucidated.In situ hybridization and Northern Blot results show that Acvr1 is expressed in dental pulp cells in adult rat, bovine, and human  rescues the ectopic cementogenesis caused by Bmpr1a depletion. This finding underscores the antagonistic interaction between BMP and Wnt signaling in epithelial cells, which governs the transition from crown to root formation at the onset of tooth root development  along with its associated periodontal tissues, providing a biological alternative to conventional dental implants.To simulate the natural morphology of a tooth root, researchers have developed a treated dentin matrix (TDM), which serves as a natural 3D graft material derived from dentin matrix . It is rIn an intriguing study, Jin and colleagues transplanted rat TDM together with rat dental follicle stem cells (DFSCs) into the alveolar fossa microenvironment, resulting in the formation of a tooth root . SubsequAlthough autologous TDM is limited in origin compared with xenogenic TDM, it demonstrates better capabilities in promoting dentin regeneration and odontoblast differentiation. In contrast, xenogenic TDM poses challenges due to transplant rejection . OverallTDM can be manufactured in various shapes, including sheets, slices, root-shaped forms, barrier membranes, and press-fitted configurations, tailored to the specific defect shape . NotablyTo our knowledge, no standard protocol for demineralization has been established for tooth root regeneration . TherefoStem cells play a crucial role in tissue engineering, particularly in achieving the regeneration of an ideal root . CurrentDFSCs are a type of dental mesenchymal stem cells that can be isolated from extracted third molars with minimal damage to subjects and without ethical controversy. Compared to DPSCs and PDLSCs, DFSCs demonstrate a superior capacity for proliferation and osteogenic-related differentiation . While tSimilar to DFSCs, DPSCs are also multipotential stem cells and can serve as seed cells for bio-root regeneration. In order to achieve a more comprehensive bio-root regeneration encompassing both the periodontium and pulp-dentin complex, a sandwich structure consisting of hDPSC/TDM/Matrigel is developed and transplanted subcutaneously in nude mice for a duration of 3 months . Due to SHED is another important category of DMSCs, exhibiting a notably higher proliferative potential compared to other types. In a series of experiments, researchers separately transplanted SHED along with TDM into the subcutaneous tissue and jawbone of nude mice and rats to detect the regenerative capacity of SHED in bio-root . FollowiCompared with DMSCs, adipose-derived stromal/stem cells (ASCs) have a wide range of sources and have been used in tissue engineering for many years. Recent study reports that regenerated dentin-like, pulp-like, and periodontal fiber-like tissues are found after transplanting ASCs with TDM into nude mice for 8 weeks . This fiStem cells play a crucial role in maintaining tissue homeostasis and facilitating tissue repair. The mobilization and homing of these cells provide an alternative approach for tissue regeneration. It is reported that successful root regeneration, encompassing PDL like tissues and cementum, has been achieved using autologous PRF and allogeneic TDM. This success is attributed to the facilitation of homing and differentiation of both PDLSCs and Bone marrow stem cells (BMSCs). Nonetheless, the prospect of tooth root regeneration through endogenous mobilization, homing, and differentiation of stem cells remains a promising approach that needs to be further investigated.Biomolecules constitute a crucial component of tissue engineering-mediated root regeneration. Although research on utilizing BMPs to regenerate bio-roots is still relatively uncommon, these proteins hold significant potential as bioactive factors for tooth root regeneration. This potential is based on the role of BMP signaling in various stages of tooth root development and its ability to promote the differentiation of DMSCs into odontoblasts, cementoblasts, PDL cells, and osteocytes, contributing to the formation of dentin and periodontal tissues.It has been reported that a biomimetic approach involving the incorporation of two crucial odontogenic growth factors (TGF-β1 and BMP-4) into the aforementioned TDM, which embodies a spatial interface gradient, facilitates the formation of a functional enthesis to promote bio-root regeneration . In a raBiomaterial scaffold-based bioengineered tooth roots have shown promise as a prospective approach for treating tooth loss. However, despite efforts to replicate the mechanical integrity of a native tooth root, the bio-root still faces limitations, including the challenge in achieving long-term functional stability due to the lack of an effective cervical seal. Targeting the BMP signaling pathway could offer potential solutions for enhancing tooth root regeneration therapy.It is evident that BMP signaling regulates the tooth root development by promoting the formation of HERS and the differentiation of odontoblasts and cementoblasts. Both ectoderm-derived and mesenchyme-derived BMP signaling are required for odontoblast differentiation and root formation. However, the mechanisms by which BMPs exert their functions are still largely unknown. For example, the reason behind the formation of massive osteodentin at the apical region of the root pulp when blocking BMP signaling in mice is still unclear. Additionally, the receptors that mediate the signaling pathway in the regulation of cell fate autonomous alterations of dental mesenchyme remain unknown. Furthermore, while the crucial roles of BMP signaling in periodontal tissues can be leveraged for the regeneration of bio-roots to treat tooth loss, research on utilizing BMPs to regenerate bio-roots is still relatively uncommon. While the task of complete tooth regeneration remains challenging, stimulation of third dentition by activating BMP signaling, as akin to anti-USAG-1 therapy, holds promise as a viable strategy in the future. Overall, targeting the BMP signaling pathway could offer potential solutions for enhancing tooth root regeneration therapy.


While thrombosis is the leading cause of morbidity and mortality in the United States, an understanding of its triggers, progression, and response to anticoagulant therapy is lacking. Intravital fluorescence microscopy has advanced the study of thrombus formation by providing targeted, multi-color contrast. However, photodegradation of fluorophores limits the application in longitudinal studies . Fluorescent nanodiamond (FND) is a fluorophore which utilizes intrinsic fluorescence of chromogenic centers within and protected by the diamond crystalline lattice. Recent developments in diamond processing have allowed for the controlled production of nanodiamonds emitting in green or red. Here, the use of FND to label blood clots and/or clot lysis is demonstrated and compared to commonly used organic fluorophores. Model ex vivo clots were formed with incorporated labeled fibrinogen to allow imaging. FND was shown to match the morphology of organic fluorophore labels absent of photobleaching over time. The addition of tissue plasminogen activator (tPa) allowed visualization of the clot lysis stage, which is vital to studies of both DVT and pulmonary embolism resolution. Thrombosis is the leading cause of morbidity and mortality in the United States  ,2. AlthoIn the care of patients with thrombotic conditions, understanding time-dependent changes in clot progression and/or resolution is critical. Residual thrombosis confers heightened morbidity and mortality in both deep vein thrombosis (DVT) and pulmonary embolism (PE). DVT patients with residual thrombosis have increased risk of recurrent DVT and PE and severe post-thrombotic syndrome ,4,5,6. ROver the past several decades, investigators have increasingly relied on intravital microscopy by optical fluorescence to study mechanisms of thrombus formation . FluoresFluorescent nanodiamonds (FNDs) are intrinsically biocompatible fluorescent labels where fluorescence arises from color centers encapsulated within the diamond lattice, providing truly infinite photostability. Different combinations of nitrogen (N) and lattice vacancies (V) are the basis for color centers in diamonds . Specifi18 e cm−2 to generate vacancies, and a subsequent two annealing regimes were used to generate specific color centers in the particles. Red FNDs were prepared by annealing at 850 °C for 2 h under vacuum to form NV centers. Green fluorescent particles were annealed at 1800 °C for 2 min using our recently reported [Synthetic type Ib HPHT synthetic nanodiamond particles  with average size 60 nm and 120 nm and initial substitutional nitrogen content of ~100 ppm were used for the production of red and green fluorescent colors in this study. Particles were irradiated with high-energy electrons (3 MeV) to fluences of 5 × 10reported . Using sreported . ParticlN-hydroxysuccinimide  and N-Ethyl-N′-(3-dimethylaminopropyl)carbodiimide hydrochloride  were added to particles at 1 mg/mL with sonication and were activated for 15 min. Particles were washed with DMF, then suspended in sterile deionized water with sonication. To the solution, 200 µg streptavidin (New England Biolabs N7021S) was then added. The solution was allowed to react overnight at 4 °C and then was washed three times with water. Finally, the solution was suspended in standard phosphate-buffered saline with 0.1% bovine serum albumin as a stabilizer at a diamond concentration of 10 mg/mL (1% wt).Avidin particles (either Avidin-FND-(Green) or Avidin-FND-(Red)) were produced by carbodiimide activation and reaction with streptavidin . BrieflyWhole blood (2 mL) was collected from unmedicated healthy donors into a serum separator tube  containing either 100 µL fibrinogen FITC alone  or avidin-biotin mixtures including 100 µL biotin-labeled fibrinogen  with 100 µL of FND-labeled avidin (avidin-FND Red or Green) or 100 µL biotin-labeled fibrinogen with 100 µL phycoerythrin-labeled streptavidin (avidin-PE) . Tissue plasminogen activator  was added to some PBS containing wells after clot formation as described below. Controls included avidin-FND alone without addition of labeled fibrinogen as well as clot alone.Serum separator tubes containing whole blood with and without labeled fibrinogen were inverted five times to allow for mixing and placed in a 37 °C dark chamber for 2 h to facilitate clot formation. To remove unincorporated fibrinogen or fluorescent labels, the clot was washed three times in 30 mL of phosphate-buffered saline  and resuspended in PBS in a tissue culture plate . For clot lysis experiments, 100 µL of tPA (1 mg/mL) was added to wells containing PBS. Clots either in PBS alone or tPA-containing medium were then removed after 1 h of treatment for subsequent epifluorescence microscopy. Clots were gently extracted from the wells and placed on clear slides for imaging.Widefield imaging was performed on an Olympus IX-71 inverted epifluorescence microscope using a mercury lamp excitation under 10×, 40×, and 100× objectives with the following filter sets (Semrock): Green emitting fluorophores (FITC and NVN containing FND-(green)) were excited by blue excitation (FF01-470/28) with green longpass emission (BLP01-488R). Red emitting fluorophores (PE and NV containing FND-(red)) were excited by green excitation (FF01-517/20) with red longpass emission (BLP02-561R). Images were taken with a CCD color camera fitted to the microscope  and processed in FIJI .Scanning electron microscopy (SEM) imaging was performed on the ex vivo clot using standard methods . BrieflyClotting occurs when fibrinogen is activated by thrombin to form fibrin polymers that enmesh erythrocytes and platelets . In thisTo determine the adequacy of FND to visualize clots, images of the clots needed to be benchmarked to existing commercially available fluorophores and compared to the possible autofluorescence of the blood itself. Epifluorescence microscopy on the clot with no added fluorophore A with thFor structure labeling, smaller-sized particles could have less steric hindrance in a developing clot and could increase image resolution. However, FND brightness is proportional to particle size, thus smaller particles will be dimmer. To assess these varying effects of smaller FND on clot imaging, two samples of avidin-FND (red) were used for labeling, varying only by size (60 nm or 120 nm) to determine if there was any resolution or fluorescence advantage of one of the sizes over the other. In vivo, clots are broken down gradually through fibrinolysis, a process initiated by tissue plasminogen activator (tPA). To examine the utility in monitoring the structural changes of clots undergoing fibrinolysis, our ex vivo clots labeled with avidin-FND or FITC fibrinogen were subjected to tPA treatment. Photobleaching is a major limitation of current fluorophores used in imaging applications but does not occur with FND. A practical example is highlighted in To compare photobleaching effects, in a separate experiment we compared avidin-FND to the commonly used fluorophores FITC and phycoerythrin (PE), such that both the green and red diamonds could be compared. Imaging conditions were the same for the FND and organic fluorophore in their respective channels (green vs. red). Both PE A,B and FFND imaging of whole blood clots provides several potential advantages over conventional fluorophores used in biological imaging. As demonstrated in this series of experiments, FND can be conjugated to biological targets via the avidin–biotin bond. In this environment, their lack of photobleaching allows for temporal imaging and quantification within whole blood and clots. This stability extends to how the reagents are handled compared to other fluorescent materials: quantum dots, for example, are subject to photooxidation which influences surface functionality and emission properties . This chOrganic fluorophores still achieve better labeling resolution as compared to that of 100 nm particles. Two factors can contribute to this. First, due to their size, particles are sterically less able to label small analytes. Given that fibrils in the clot network are under 100 nm in size , 120 nm Despite these limitations of particle fluorophores, their utility for imaging to reveal all essential structural features of the clot was demonstrated. The small size and ability to easily conjugate FND to avidin allows for targeted binding of many antigens within whole blood and the clots that develop. FND is still small enough to allow for penetration and binding to the biomarkers as the clot forms. As FND sizes are ~3% the diameter of platelets and ~1.6% the diameter of erythrocytes, these structures are still readily labeled. Future studies can examine the use of avidin FND (NV) linked to a biotinylated antibody with other avidin FND (NVN) linked to another antibody. This will allow for simultaneous viewing of two separate antigens within a clot over time.A key advantage demonstrated in these experiments is that FNDs do not photobleach. Conventional fluorophores are limited in their ability to be examined serially within biological tissue by their rapid photobleaching. Both the FITC and PE fluorophores utilized in these experiments began to have photobleaching within seconds of confocal microscopic examination. Moreover, imaging artifacts were noticed during re-imaging in experiments on clot dissolution, when the dark “holes” caused by photobleaching looked similar in appearance to the web-like pattern present in the clot exposed to tPA . FND, onThrombosis and fibrinolysis are dynamic processes in a clot, which often are not routinely examined together in the in vivo setting. A potential application of FND for imaging thrombus formation and dissolution would be clot imaging studies in murine models of thrombosis. In particular, the inferior venous cava (IVC) stenosis model in mice lends itself to such studies, as clots can be monitored over time for several weeks . In thisThe use of tPA to accelerate fibrinolysis in our experiment demonstrates that FNDs are able to reflect the changes within the structure of the clot as breakdown occurs. We were able to visualize the clot lysis stage, which is vital to studies of both DVT and pulmonary embolism. Being able to quantify temporal changes with individual donor variability may lead to clinical interventions in the future. The ability to better prognosticate those affected by either DVT or PE who are more likely to develop chronic complications could allow for more aggressive management in the acute phase.Here, the use of FND for clot imaging was demonstrated for both traditional red (PE) and green (FITC) emission channels using, respectively, NV- and NVN-containing nanodiamonds. Photostability was assessed in comparison to these organic dyes, demonstrating reliable unchanging fluorescence over time. Such a capability is expected to translate to better clinical bedside patient care by allowing for longitudinal assessment of clot lysis in individuals.


Previously, we reported a reduction in Ki67-immunoreactive nuclei of human glioblastoma cells after GPR55 activation in general by THC and in particular by LPI. In the present study, we investigated intracellular mechanisms leading to an altered number of Ki67+ nuclei after stimulation of GPR55 by LPI and THC. Pharmacological analyses revealed a strongly involved PLC-IP3 signaling and cell-type-specific differences in Gα-, Gβγ-, RhoA-ROCK, and calcineurin signaling. Furthermore, immunochemical visualization of the calcineurin-dependent transcription factor NFAT revealed an unchanged subcellular localization after THC or LPI treatment. The data underline the cell-type-specific diversity of GPR55-associated signaling pathways in coupling to intracellular G proteins. Furthermore, this diversity might determine the outcome and the individual responsiveness of tumor cells to GPR55 stimulation by cannabin oids.GPR55 is involved in many physiological and pathological processes. In cancer, GPR55 has been described to show accelerating and decelerating effects in tumor progression resulting from distinct intracellular signaling pathways. GPR55 becomes activated by LPI and various plant-derived, endogenous, and synthetic cannabinoids. Cannabinoids such as THC exerted antitumor effects by inhibiting tumor cell proliferation or inducing apoptosis. Besides its effects through CB GPR55 is an orphan G-protein-coupled receptor (GPCR) and has been found within the central nervous system (CNS) in numerous brain regions, such as the hippocampus, striatum, cerebellum, hypothalamus, and cortex, as well as on glial cells and neurons of the dorsal root ganglia ,2,3,4,5.1) and CB2-dependent mechanisms have been reported for anti-tumor activities of THC on glioblastoma cells i) via the activation of PLC and formation of IP3 [2+]i was observed after stimulation with 5 µM THC and 3 µM LPI i, although CB1-dependent Ca2+ release via WIN 55,212-2 was detected i was found after GPR55 activation by LPI or AM251 i when GPR55 is coupled to RhoA-ROCK signaling. In murine fibroblast cell line NIH 3T3, a disrupted cytoskeleton altered the spatial interaction between PLC and IP3-sensitive receptors, thus affecting PLC-dependent Ca2+ signaling i was measured. Moreover, increased [Ca2+]i in the neurons of dorsal root ganglia after GPR55 activation was attributed to dual Gα12/13 and Gαq signaling  gamma-amino-butyric acid (GABA) by a Gβγ-dependent mechanism i by PLC and ROCK signaling in rat mesenteric arterial bed endothelial cells stimulated with LPI or AM251 I I ,30. To iAT [2+]I ,61. Calcomplexes . GBM #4 but supports partial calcineurin-independent effects observed in GBM #10. The pronounced background activity of CsA and FK506 did not enable a clear conclusion and was unlikely to be attributable to the inhibition of basally active NFAT. After CsA and FK506 treatment, NFAT2 and NFAT3 were constitutively present in the nucleus, suggesting calcineurin-independent mechanisms in these tumor cells. Notably, the effects of both are not limited to calcineurin but also regulate the transcriptional activity of NF-κB or proteasomal degradation i activates CREB-directed gene transcription via activation of CaMKIV and subsequently induces inactivation of CREB via calcineurin-mediated activation of protein phosphatase-1 [2+ signals can induce dephosphorylation of CRTCs by inhibition of salt-inducible kinases  or by the induction of calcineurin, respectively. Dephosphorylated CRTC translocates into the nucleus, binds to the bZIP domain of CREB, and operates as a co-activator [2+-dependent CREB activation is still not understood but might result from cell-type-specific differences and the amplitude, duration, and subcellular localization of Ca2+ signals [Similar cell-type-dependent effects were observed in cAMP-mediated signaling pathways induced by PTX and FSK, which give rise to speculation of additional signaling pathways capable of reducing the number of Ki67EB genes . Intereshatase-1 . Alternahatase-1 . Under bctivator . The res signals . These h+ nuclei of patient-derived glioblastoma cells were investigated. A strong involvement of the PLC-IP3 pathway was observed. Cell-type differences in Gβγ and RhoA-ROCK signaling were found, probably explained by differences in the coupling of GPR55 to intracellular G proteins. Additionally, GPR55-mediated effects required at least partial activation of calcineurin. Calcineurin-related activation of transcription factor NFAT was not evident after THC or LPI treatment, as its subcellular localization remained unchanged. However, the analysis of additional transcription factors, which are influenced by IP3-driven Ca2+ release and/or calcineurin, should be elucidated in future investigations to shed light on the precise mechanism of the GPR55-mediated reduction of Ki67+ nuclei in glioblastoma cells. As the present study showed similarities and differences in GPR55-associated signaling compared to the literature, we may anticipate a better understanding of the complex pathways by which THC and GPR55 affect tumor cell biology in the context of glioblastomas. The data underline the diversity of GPR55-associated signaling pathways that are distinct in various cell types and from other cannabinoid receptors, including CB1 and CB2.In the present work, signaling pathways related to GPR55 that might account for previously described THC-mediated reduction in the number of Ki67Furthermore, this diversity might account in part for the individual responsiveness of tumor cells to GPR55 stimuli by cannabinoids. Nevertheless, the identification of the precise signaling pathway might be useful for the purpose of finding prognostic markers and defining the conditions in which THC and GPR55 may be beneficial or unemployable as novel therapeutic options.