Introduction: Breast cancer is the most common cancer in women, with roughly 10–15% of new cases classified as triple-negative breast cancer (TNBC). Traditional chemotherapies are often toxic to normal cells. Therefore, it is important to discover new anticancer compounds that target TNBC while causing minimal damage to normal cells. Receptor tyrosine kinase-like Orphan Receptor 1 (ROR1) is an oncofetal protein overexpressed in numerous human malignancies, including TNBC. This study investigated potential small molecules targeting ROR1.Methodology: Using AutoDock Vina and Glide, we screened 70,000 chemicals for our investigation. We obtained 10 representative compounds via consensus voting, deleting structural alerts, and clustering. After manual assessment, compounds 2 and 4 were chosen for MD simulation and cell viability experiment. Compound 4 showed promising results in the viability assay, which led us to move further with the apoptosis assay and immunoblotting.Results: Compound 4 (CID1261330) had docking scores of −6.635 and −10.8. It fits into the pocket and shows interactions with GLU64, ASP174, and PHE93. Its RMSD fluctuates around 0.20 nm and forms two stable H-bonds indicating compound 4 stability. It inhibits cell proliferation in MDA-MB-231, HCC1937, and HCC1395 cell lines, with IC50 values of approximately 2 μM to 10 μM, respectively. Compound 4 did not kill non-malignant epithelial breast cells MCF-10A (IC50 > 27 μM). These results were confirmed by the significant number of apoptotic cells in MDA-MB-231 cells (47.6%) but not in MCF-10A cells (7.3%). Immunoblot analysis provided additional support in the same direction.Discussion: These findings collectively suggest that compound 4 has the potential to effectively eliminate TNBC cells while causing minimal harm to normal breast cells. The promising outcomes of this study lay the groundwork for further testing of compound 4 in other malignancies characterized by ROR1 upregulation, serving as a proof-of-concept for its broader applicability. Breast cancer stands as a prevalent form of cancer commonly diagnosed among women, roughly 10%–15% of new cases as triple-negative breast cancer (TNBC) . In the Among various proteins, Tumor-Associated Antigens (TAAs) are expressed differently in cancerous and non-cancerous cells. These include Receptor Tyrosine Kinases (RTKs), which have been identified as critical regulators of various cellular activities, including cell-to-cell communication, proliferation, motility, differentiation, and metabolism . They arpostpartum tissues , and small chemical inhibitors have been investigated for targeting ROR1. However, only cirmtuzumab, a monoclonal antibody, has been clinically tested . ARI-1 hThe pseudokinase/kinase domain of the ROR1 protein, ID 6TU9, was retrieved from the PDB database . The strAutoDock Vina uses a Linux environment for docking. The program employed a gradient optimization approach for docking with random seeds. Random seed provides slightly different results each time the process is run on the same inputs, which aids in consensus voting . ExhaustWe used a Python script to eliminate compounds with high chemical reactivity, molecular fragments, and those activated with high chemical reactivity by human enzymes. With it, PAINS (Pan-Assay Interference Compounds) are also eliminated, which are physiologically active compounds that appear to be prospective therapeutic candidates during high-volume screening but are not . The K-m−1 nm−1. The NVT and NPT assemblies were assembled for 10 ns at 300 K and 1 atm pressure, respectively. The system temperature was first brought to equilibrium using a constant Number of Particles, Volume, and Temperature (NVT) ensemble. The pressure was then stabilized using the Number of Particles, Pressure, and Temperature (NPT) ensemble, which closely matched the experimental conditions. Finally, the production MD simulation was run for 200 ns to create trajectories. These trajectories determined the complex’s binding energies, fluctuations, and hydrogen bonds.MD simulations can provide detailed information on protein-ligand interactions that cannot be obtained using methods such as docking . We perf2.The study used the TNBC cell line MDA-MB-231 (ATCC HTB 26) and the HCC1395  which was cultured in DMEM/F12K containing 5% horse serum, insulin (10 μg/mL), hydrocortisone (0.5 mg/mL), EGF (20 ng/mL), penicillin (100 U/mL), and streptomycin (0.1 mg/mL). HCC1395 (ATCC CRL-2324TM) and HCC 1937 (ATCC CRL-2336™), two other TNBC cell lines, were grown in RPMI with 10% FBS, penicillin (100 U/mL), and streptomycin (0.1 mg/mL). The cell cultures were maintained at 37°C and 5% CO50 values.An MTT assay  assessed breast cell proliferation and viability. Briefly, 10,000 cells were seeded per well in a 96-well plate and allowed to develop for 12–24 h. ROR1 inhibitors dissolved in DMSO  were serially diluted into the cells in each column, except for the last, which was saved for the DMSO vehicle control group used in all analyses. After 72 h, the treatment media was replaced with fresh untreated media. The MTT dye was applied and incubated at 37°C for 2 h. Formazan crystals were dissolved in DMSO. Finally, the absorbance was measured at 540 nm. Cell viability was evaluated as a percentage of untreated control cells. GraphPad Prism software (version 8.0) was used to calculate IC50 or DMSO vehicle control for 72 h. Next, the cells were trypsinized, resuspended in an assay binding buffer, and stained with Caspase 3/7 to be analyzed for apoptosis using a Muse Cell Analyzer.To assess apoptosis after treatment with ROR1 inhibitors, Caspase 3/7 staining was performed using the Muse Caspase 3/7 assay kit (Luminex MCH100108). The cells were seeded at a density of 250,000 cells/well in a 6-well plate. The cells were treated with either ROR1 inhibitors at the ICAfter ROR1 inhibitor treatment (10 µM) and vehicle (DMSO) control treatment, the cells were collected by scraping. The proteins were lysed using RIPA buffer  with 1 mM PMSF . Bradfort-test or nonlinear regression, with p-values less than 0.05 considered significant. All statistical analyses were performed using GraphPad Prism 7.0. .Unless otherwise specified, all data are presented as the mean ± standard error. Statistical significance between groups was determined using a two-tailed Student’s 6TU9). After docking and consensus voting, 10,000 compounds remained. These compounds were filtered using Python scripts to eliminate structural alerts and PAINS. After this process, 250 compounds were clustered, and the top 10 compounds with the lowest LogP were chosen  interacted with LYS47, but compound 10 (CID648475) did not . Both coin silico results indicated that compound 4 (CID1261330) is expected to be potent against malignancies expressing high levels of ROR1.The Root Mean Square Deviation (RMSD) value of compound 2 (CID135399549) fluctuates for the initial 15 ns to reach 0.35 nm and then gets a stable trajectory for 85 ns at roughly 0.25 nm. Further, the structure stabilizes for the remaining 100 ns around 0.30 nm . In cont50) of approximately between 2 μM and 10 μM, respectively. Non-malignant epithelial breast cells MCF-10A were markedly unsusceptible to compound 4 (IC50 > 27 μM) (Compound 4 (CID1261330) inhibited proliferation and induced apoptosis of TNBC cells. The potency of ROR1 inhibitors compounds 4 (CID 1261330) and compound 2 (CID135399549) toward TNBC cells  was assessed using the MTT cell viability assay. Compound 4 (CID1261330) significantly suppressed cell proliferation in the TNBC cell lines, MDA-MB-231 (with high ROR1 expression), HCC1937 and HCC1395, at a low half-maximal inhibitory concentration (IC> 27 μM) , right. > 27 μM) . This re> 27 μM) , left. TROR1 activity induces cancer cell survival and inhibits apoptosis . TherefoTo determine if compound 4 targets ROR1, MDA-MB-231 cells were treated with 10 μM of Compound 4 for 72 h, and immunoblots were performed in whole cell lysates. Current therapeutic agents for TNBC, such as anthracyclines, taxanes, or capecitabine, are cardio-, neuro-, and hepato-toxic to patients, causing a host of disastrous side effects and poor prognosis . TherefoSeveral studies have consistently demonstrated that ROR1 exhibits limited kinase activity, primarily due to specific changes in highly conserved amino acids within its kinase domain’s GXGXXG motif. These alterations result in an inaccessible ATP-binding domain and the inactivation of the activation loop. Nevertheless, through the use of a kinase-dead mutation (K506A) and an autophosphorylation site mutation (YXXXYY to FXXXFF) in ROR1, researchers have discovered that the kinase domain of ROR1 still possesses crucial signaling capabilities, even in the absence of ATP binding. Interestingly, this modified ROR1 lost its ability to drive the growth of BaF3 cells. Moreover, constitutive phosphorylation of ROR1 has been observed in chronic lymphocytic leukemia (CLL) cells, indicating that ROR1 possesses inherent kinase activity . Based oin silico approach and validate the compounds using in vitro experiments. The validation shows the utility of the in silico pipelines. There are examples like; Gefitinib for non-small-cell lung cancer (NSCLC)  ; Erlotin (NSCLC) ; Sorafen (NSCLC) ; and Alp (NSCLC)  which weproaches . Researcproaches .We focused more on the protein-ligand interaction than the docking energy. Docking energy needs to be used as the filtration criteria, not the selection criteria . Hydrogein silico approach helped us filter down the 70000 compound datasets to a few hits and further narrowed it down to Compound 4 (CID1261330) following MD simulations. In contrast, compound 2 (CID135399549) showed a better docking score but poor stability in the binding pocket during MD simulations. The apoptosis test results supported this prediction. The primary reason for this could be the weak interaction of compound 2 with the ROR1 intracellular kinase domain and the presence of off-targets. Upon searching PubChem, BioAssay AID1768 showed that compound 2 targets Multiple Endocrine Neoplasia 1 (MEN1) and Lysine Methyltransferase 2A (KMT2A). Off-target compounds may cause cell death in all cell lines, impair average cell growth, and have low potency, making the compound non-selective for ROR1.The This study demonstrated that compound 4 (CID1261330) appears effective in limiting TNBC cells, inhibiting cell viability, and inducing apoptosis in TNBC cells (MDA-MB-231 and HCC1395 cells) at a half-maximal inhibitory dose of approximately 2–10 µM. We performed the same experiments in the non-tumorigenic cell line MCF10A  and observed no significant cytotoxic effects. Further corroborating these data are immunoblots showing that ROR1 in TNBC cells is successfully knocked down by Compound 4 (CID1261330). This suggests that the inhibitory effect of Compound 4 is specific to TNBC cells because of the presence of ROR1 receptors. Hence, we propose that compound 4 (CID1261330) specifically targets TNBC cells with the least harm to normal breast cells. We believe compound 4 (CID1261330) has therapeutic potential for treating TNBC. Our data sets the stage to further validate compound 4 as a ROR1-inhibitor and an anti-tumor agent.


Composition, microstructure, and electromagnetic properties of the composites were characterized and analyzed. Results from TEM and XRD show that crystalline ZnO particles adhere to the surface of amorphous carbon, and the ZnO content increases as a function of a dosage of ZnNO3·6H2O. The as-prepared SiC@C-ZnO hybrids exhibit effective electromagnetic absorption, which is related to a synergy effect of different dielectric loss processes. The minimum reflection loss reached −65.4 dB at 11 GHz at a sample thickness of 3.1 mm, while the effective absorption bandwidth (EAB) reached 7 GHz at a sample thickness of 2.56 mm. Furthermore, the EAB of the samples can also cover the whole X band and Ku band at small sample thicknesses (2.09–3.47 mm). The excellent properties of the materials suggest great prospect as electromagnetic absorbers.A novel strategy is provided to improve the absorption of SiC nanomaterials through surface carbonization of SiC nanowires and hydrolysis. SiC@C-ZnO composites were synthesized with different dosages of ZnNO A good strategy is to form hierarchical heterostructures, characterized by diverse components, abundant heterogeneous interfaces, multiple reflective paths, and enrichment of structural defects [With increasing functionality of electronic devices, the widening of the working frequency bands, and the diversification of working conditions, new electromagnetic (EM) absorbing materials are gradually designed and fabricated to obtain thinner, lighter, wider, and stronger materials than the traditional materials such as carbonyl iron and ferrite –4. SiC h defects –20. Neve defects –23.min (−42.11 dB) [In our previous work, SiC@C nanowires have been successfully obtained by surface carbonization of SiC nanowires . Carbon 2.11 dB) . Growing2.11 dB) ,31–32, i3·6H2O) dosage on composition, microstructure and electromagnetic properties of the SCZ samples is discussed in detail.Herein, we describe a new strategy for preparing ternary hybrids  by growing ZnO particles on carbon surfaces derived from SiC nanowires. The influence of ZnO precursor , 50 mL H2O, and 8 mL PEG were mixed together. After that, 40 mg SiC@C nanowires was added to the above solution, followed by magnetic stirring for 20 min. At 60 °C, NH3·H2O was added dropwise until pH 9–10. After 2 h of continual stirring, the products were obtained after filtering, washed with distilled water, and dried at 50 °C under vacuum. The final samples were obtained by calcination at 600 °C for 4 h in a tube furnace . We labeled the samples as SCZx, where x indicates x mmol dosage of ZnNO3·6H2O.Different amounts of ZnNO3·6H2O. Also, a small peak at 26.5°, which corresponds to the (002) reflection of amorphous carbon, can be found in the SCZ samples, especially when the dosage of ZnNO3·6H2O is low. A carbon phase on the SiC surface was obtained through the removal of Si atoms from SiCnw in the carbonization atmosphere containing a low concentration of chlorine gas [All SCZ samples exhibit several strong XRD reflections , corresponding to the (100), (002), (101), (102), (110), (103), (200), (112), (201), and (202) planes of ZnO , respectively . Besidesrine gas .3·6H2O will lead to an increase in the density of ZnO particles on the carbon structure  have been determined by XPS in all SCZ samples, as shown in  of Zn2+ . BesidesZ0 and Zin according to the following equation:The microwave absorption of the SCZ samples is simulated based on the transmission line theory . The refZ0 and Zin are the impedance of free space and the input impedance, respectively. Their relationship can be expresses as:where r is the complex permittivity, εr = ε′ − jε″, μr is the complex permeability, μr = μ′ − jμ″, ƒ is the frequency, d is the thickness of the material, and c is the speed of light.where ε3·6H2O used for growing the ZnO particles. For SCZ0.5, the best RLmin value of −54.6 dB and an effective absorption bandwidth (EAB) of 6.6 GHz (11.36–17.96 GHz) are obtained for 25 wt % filler load at a sample thickness of 2.35 mm. For SCZ2 and SCZ4, the best RLmin values are, respectively, −63.9 dB and −58.1 dB, while their EAB values are 6.48 GHz (11.48–17.96 GHz) and 7.04 GHz (10.96–18.00 GHz), respectively. However, the filler loads should be increased to higher levels of 35–50 wt %. In general, SCZ1 exhibits the best performance among the samples. Its highest values of RLmin and EAB reach −65.4 dB and 7 GHz (10.96–17.96 GHz), respectively, at a small sample thickness and 30 wt % filler load. Furthermore, the EAB of other SCZ samples can even cover the X and Ku bands at small sample thicknesses (3O4) and other reported materials (such as ZnO-decorated SiCnw or graphene/SiC) [min, broader EAB, and less filler load. This might enable a wide application as microwave absorbers as elaborated in this study.The microwave absorption of the SCZ samples strongly depends on the dosage of ZnNOene/SiC) ,24,34–353·6H2O does not lead to an increased dielectric tangent loss, suggesting that a moderate content of ZnO precursor is needed to synthesize SiC@C-ZnO with relatively good dielectric performance for microwave absorption. Although the SCZ0.5 sample has the highest value of dielectric tangent loss (3·6H2O at the same filler load (20–50 wt %). This may be because both the real and imaginary parts of the carbon permittivity are higher than those of ZnO. Comparing the dielectric constant of SCZ0.5 and SiC@C at the same filler load of 30 wt %, the ε′ and ε″ values of SCZ0.5 are lower than the dielectric constants of SiC@C . These findings suggest that SiC@C-ZnO hybrids with diverse microstructures may have a bright future as EM absorbers.Based on the above analysis, it is considered that multiple loss mechanisms may contribute to the improvement of EM absorption for the as-prepared SiC@C-ZnO hybrids . First,  effects . Liao et3·6H2O. The amorphous carbon shell has a significant effect on the nucleation of crystalline ZnO particles, possibly due to oxygen-containing functional groups (such as carboxyl and hydroxy groups) and defects on the SiC@C surface, which both provide locations for the deposition of Zn2+ by electrostatic interactions. SCZ1 exhibits the best EM absorption properties. Its values for RLmin and EAB reach −65.4 dB and 7 GHz (10.96–17.96 GHz), respectively, at a small sample thickness and 30 wt % filler load. The effective EM absorption is related to the synergy of dielectric losses (including conductive loss and polarization relaxations) and multiple reflection or scattering losses, enabling a promising EM absorbing nanomaterial.A new strategy for the controllable fabrication of SiC@C-ZnO hybrids via carbonization and hydrolysis reaction is described. Morphology and permittivity of the hybrids can be adjusted by changing the dosage of ZnNOFile 1Additional experimental data.


In this paper, a semi-discrete fractional derivative complex coupled dispersionless system is proposed. The properties of M-fractional derivative are utilized to convert discrete M-fractional derivative system to a classical discrete differential system. Then the invariant subspace method (ISM) is utilized to find dark, bright, kink and W-shaped soliton solutions for the proposed system. Coupled dispersionless (CD) system is firstly modeled by Konno and Oono7 and it was solved using the inverse scattering transform. After that, CD systems is developed to include generalized CD and three dimensions Euclidean space8. Several methods have been used to solve both of real CD equations12 and complex CD equations14. In Refs.16, real semi-discrete coupled dispersionless (SDCD) system is analyzed and soliton solutions are found.Dispersionless integrable equations have many valuable applications in physics and mathematics such as in quantum fields17, the bright soliton solution, breather solution and rogue wave solution of the semi-discrete complex coupled dispersionless integrable system (SDCCD) are obtained using Lax pairs and Darboux transformation method. Also, the modulational instability of SDCCD is studied in20.In Ref.19. So, in this paper, we introduce the semi-discrete M-fractional derivative complex coupled dispersionless (FDSDCCD) system20.The nonlinear phenomena have been accurately described by the M-fractional derivative. Such as in wave propagation, gravity wave propagation, optics, and in air pollutant dispersion21, exponential function method23, square operator method25, similarity transformation method27, and neural network methods28. One of the effective methods for getting exact solution of D 32.There are many methods proposed in the literature for obtaining exact solutions of differential-difference equations (D  as Eqs.  and 1b)\documentIn this paper, we use the ISM to obtain some novel exact soliton solutions of the system  and 1b)1b). The This paper is organized as follows: In section “1832. The M-fractional derivative has the same properties of classical derivatives in linearity, multiplication, and division of two functions but what distinguishes the M-fractional derivative are the following properties32:The M-fractional derivative of In this section, we give a brief description of the ISM and how we can use it to get exact solutions of Eqs.  and 1b)1b).Consider the following D Step 1: Assume that the solution of Eqs. , ,  i i10b) inUsing two-dimensional invariant subspace , , 13b),  and Eqs.ting Eq. , (13b), ting Eq.  and solvting Eq. , (13b), ting Eq. , we find of Eqs. , 11b), , 16\docu of Eqs.  take theSubstituting Eqs. , 19b), , 19b),  and Eq. Comparing the coefficient of n in Eq. , we findSubstituting Eq.  into Eqs in Eqs.  and (20cSubstituting Eqs.  and 22)22) into Substituting Eq.  into Eq.Substituting Eqs.  and 24)24) into To Solve Eq. , we distCase 1: The first solution of Eq.  is isCase 1Substituting Eqs.  and 31)31) into The arbitrary parameters in Eqs.  and 33)33) can bFigures mentclass2pt{minimsolution  is obtaimentclass2pt{minimCase 2: LetSubstituting Eq.  into Eq.Solving Eq. , we find of Eqs. , 9b) is is36\docSubstituting Eq.  and Eq. Figure In this paper, we used the properties of M-derivative to convert FDSDCCD system  and 1b t


Mitochondria are involved in many vital functions in living cells, including the synthesis of ATP by oxidative phosphorylation (OXPHOS) and regulation of nuclear gene expression through retrograde signaling. Leigh syndrome is a heterogeneous neurological disorder resulting from an isolated complex I deficiency that causes damage to mitochondrial energy production. The pathogenic mitochondrial DNA (mtDNA) variant m.13513G>A has been associated with Leigh syndrome. The present study investigated the effects of this mtDNA variant on the OXPHOS system and cell retrograde signaling. Transmitochondrial cytoplasmic hybrid (cybrid) cell lines harboring 50% and 70% of the m.13513G>A variant were generated and tested along with wild-type (WT) cells. The functionality of the OXPHOS system was evaluated by spectrophotometric assessment of enzyme activity and high-resolution respirometry. Nuclear gene expression was investigated by RNA sequencing and droplet digital PCR. Increasing levels of heteroplasmy were associated with reduced OXPHOS system complex I, IV, and I + III activities, and high-resolution respirometry also showed a complex I defect. Profound changes in transcription levels of nuclear genes were observed in the cell lines harboring the pathogenic mtDNA variant, indicating the physiological processes associated with defective mitochondria. Mitochondria are dynamic cell structures that possess their own intrinsic complement of circular DNA (mtDNA), ribosomes, and proteins . MitochoMutations in human mtDNA have been associated with various multisystem disorders known as mitochondrial diseases, with the severity of these disorders depending on the mutant mtDNA load in cells . FurtherMT-TL1 m.3243A>G variant, showing that quantitative changes in mtDNA mutant levels can qualitatively regulate nuclear gene expression [Transmitochondrial cytoplasmic hybrid systems (cybrids) are created by fusing mitochondria-enriched platelets with mitochondria-depleted cells ,25. Thespression .MT-ND5 gene at 60% heteroplasmy level [Isolated complex I deficiency represents the most common mitochondrial respiratory chain defect, involved in mitochondrial disorders, and it has been associated with heterogeneous manifestations, including phenotypic overlaps of mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS), Leigh syndrome, and Leber’s hereditary optic neuropathy (LHON) . A previmy level . The preA patient with mitochondrial disease and healthy volunteers were recruited for this study. The study protocols were approved by the Central Committee of Medical Ethics of Latvia , which covers all consent- and data-handling-related issues for genetic research on human subjects. All participants and/or their legal guardians provided written informed consent prior to enrollment. The study conformed to the guidelines of the Declaration of Helsinki.2. Human 143B ρ0 cells were generated by long-term culture of 143B cells with 50 ng/mL ethidium bromide , 1 mM pyruvate (Gibco), and 50 μg/mL uridine  [Human 143B osteosarcoma cells were cultured in Dulbecco’s modified Eagle’s medium  supplemented with 10% heat-inactivated fetal bovine serum  and antibiotic–antimycotic solution (Gibco) at 37 °C in an atmosphere containing 5% COMO, USA) . The celMO, USA) .w/v; Sigma). The mixture of fused cells was allowed to recover for 1 week in ρ0 medium and cultured in pyruvate/uridine-free DMEM, a medium, in which even cybrids with very low Cox-II expression had been shown to grow. On days 14–30 after fusion, successful cybrid clones growing in this medium were isolated. The presence of exogenous mtDNA or specific mtDNA haplogroups in each cybrid was confirmed by Sanger sequencing of isolated DNA [Transmitochondrial cybrids were generated by fusing 143B ρ0 cells with human platelets in the absence of pyruvate and uridine, as previously reported  and modiated DNA .g for 10 min at 4 °C. The postnuclear supernatant was decanted and again centrifuged at 10,000× g for 10 min at 4 °C. Mitochondria-enriched pellets were washed with a buffer containing 250 mM sucrose, 2 mM EGTA, 40 mM KCl, and 20 mM Tris (pH 7.4); suspended in the same buffer; and stored at −70 °C.Mitochondria were isolated from cytoplasmic hybrid cells as previously described , with moEnzymatic activities of citrate synthase (CS), OXPHOS complexes I–IV, and complexes I + III and II + III in isolated mitochondria were measured spectrophotometrically  at 37 °C, as previously described ,33,34, wTo measure complex I activity, 40 µL of isolated mitochondria were first disrupted by resuspension in water and incubated for 3 min at 37 °C. Then, a reaction mixture containing 50 mM Tris (pH 8.1), 0.1% BSA, 0.3 mM KCN, and 50 µM decylubiquinone was added, and the reaction was initiated by adding 0.2 mM NADH. The NADH oxidation reaction was allowed to proceed for 10 min, and complex I activity was measured at 340 nm, after which 3 µM rotenone, a complex I inhibitor, was added, and nonspecific NADH oxidation was recorded.To measure complex II activity, 10 µL of isolated mitochondria was added to a reaction mixture containing 50 mM potassium phosphate buffer (pH 7.8), 2 mM ethylenediaminetetraacetic acid (EDTA), 0.1% BSA, 3 µM rotenone, 80 µM 2,6-dichlorophenolindophenol, 50 µM decylubiquinone, 1 µM antimycin A, 0.2 mM ATP, and 0.3 mM KCN. After preincubation for 10 min, the reaction was started by adding 10 mM succinate and allowed to proceed for 5 min, during which complex II activity was measured at 600 nm. Subsequently, 10 mM malonate was added to stop the reaction and determine nonspecific activity.Complex III activity was measured by adding 5 µL of isolated mitochondria to a reaction mixture containing 50 mM potassium phosphate buffer (pH 7.8), 0.08% Tween-20, 2 mM EDTA, 0.3 mM KCN, 100 µM cytochrome c, and 200 µM reduced decylubiquinol. Cytochrome c reduction was measured at 550 nm for 4 min, after which 1 µM antimycin A was added to inhibit the reaction and determine the nonspecific activity.The activity of complexes I + III was measured by incubating 10 µL of isolated mitochondria in water for 3 min at 37 °C, followed by addition of a reaction mixture containing 50 mM Tris (pH 8.1), 0.1% BSA, 0.3 mM KCN, and 50 µM cytochrome c. The reaction was started by adding 0.2 mM NADH and allowed to proceed for 3–4 min, with activity measured at 550 nm. The reaction was stopped by adding 3 µM rotenone, and nonspecific activity was determined.The activity of complexes II + III was determined by incubating 10 µL of isolated mitochondria with a reaction mixture containing 50 mM potassium phosphate buffer (pH 7.8), 2 mM EDTA, 0.1% BSA, 3 µM rotenone, 0.2 mM ATP, and 0.3 mM KCN at 37 °C. The reactions were started by adding 50 µM cytochrome c and 10 mM succinate. Activity was measured for 3 min, and 10 mM malonate was added to stop the reaction and determine nonspecific activity.Complex IV activity was measured as described , with mo2, 20 mM taurine, 10 mM KH2PO4, 20 mM HEPES, and 0.1% BSA essentially free of fatty acids) at 37 °C. Mitochondrial respiration was initially measured in intact cells (ROUTINE state), followed by the addition of 5 µg/mL digitonin to permeabilize the cells. Complex I-linked oxygen consumption was measured using 5 mM pyruvate plus 2 mM malate (LEAK state), followed by subsequent additions of 5 mM ADP (OXPHOS state) and 10 mM glutamate. Complex II-linked mitochondrial respiration was evaluated by adding 10 mM succinate and 0.5 µM rotenone. Residual oxygen consumption (ROX) was determined by adding 2.5 µM antimycin A, and complex IV-linked oxygen consumption was measured by adding 0.5 mM N,N,N′,N′-tetramethyl-p-phenylenediamine dihydrochloride (TMPD) and 2 mM ascorbate. Complex IV activity was inhibited, and chemical background determined by the addition of 100 mM sodium azide. The contribution of each substrate to the respiration rate was determined by calculating the flux control factor using the formula: Flux control factor = 1 − (respiratory rate before the addition of substrate)/(respiratory rate after the addition of substrate).Mitochondrial functionality was determined by high-resolution respirometry using an OxygraphO2k , according to the manufacturer’s instructions. All measurements were performed in Mir05 media , according to the manufacturer’s instructions. The integrity of the extracted RNA was evaluated by determining the RNA integrity number (RIN) using the Agilent 2100 Bioanalyzer system . Ribosomal RNA was removed from 500 ng aliquots of total RNA from each sample using the Low Input RiboMinus Eukaryote System v2 . cDNA libraries were prepared using the Ion Total RNA-Seq Kits v2  and sequenced using the Ion Proton System and Ion PI Chip , according to the manufacturer’s instructions. All experiments were performed on two biological replicates.The adapters were trimmed from sequencing reads with Cutadapt . For incg for 1 min, and the supernatants were used directly. Strand-specific absolute levels of gene transcription were analyzed using Selfie-digital PCR, as described previously [Cell culture media were aspirated from the culture flasks, and each cell layer was lysed in 100ST DNA/RNA/Protein Solubilization Reagent  at a concentration of 250,000 cells/mL, to preserve the nucleic acids in the samples. The lysates were incubated at 90 °C for 3 min with shaking and centrifuged at 5000× eviously . The revMT-ND5 gene on the OXPHOS system and nuclear gene expression in cells were investigated by constructing cybrid cell lines. Platelets from a patient harboring 60% of mutant mtDNA in blood were fused with mtDNA-free ρ0 cells. Heteroplasmic cybrid cell lines containing 50% and 70% of mutant mtDNA were generated, along with a cybrid cell line harboring only wild-type (WT) mtDNA.The effects of the genetic variant m.13513G>A in the p < 0.05).To determine whether different heteroplasmy levels of the m.13513G>A variant impaired the performance of the OXPHOS system and mitochondrial respiration, enzyme activities were measured, and high-resolution respirometry was performed. All mitochondrial OXPHOS complex activities were expressed relative to CS activity, as CS is a marker of mitochondrial content , as a rap < 0.05)  . Assessm < 0.05) . Moreove < 0.05) , indicat < 0.05) . In cont < 0.05) .The effects of the pathogenic mtDNA variants on differential gene expression were assessed by performing transcriptome analyses of cybrid cells containing 50% and 70% of the m.13513G>A variant and cells containing WT mtDNA. Principal component analysis revealed that most variability is explained by the different mutational loads (50% versus 70%) within the heteroplasmic cells A. Furthep < 0.05) by the m.13513G>A variant (ArrayExpress accession No. E-MTAB-11035). The corresponding volcano plot is shown in RNA sequencing analysis of both heteroplasmic cell lines versus WT cells showed that the levels of transcription of 84 nuclear genes were significantly affected , particularly calcium release activity (adj. p = 0.093). The HDKC1 gene was also highlighted in the GO enrichment analysis (adj. p = 0.093); it encodes kinase, which is involved in carbohydrate  metabolism, and is downregulated according to RNAseq results. Cluster analysis of the molecular functions of DEGs, based on GO terms, showed a cluster of genes related to calcium transport/release (p = 0.430).To determine the affected molecular pathways and biological functions, the DEGs were subjected to Gene Ontology (GO) enrichment analysis . Cells h/release . These f/release , althougp < 0.05; The RNAseq analysis of cells harboring 50% versus 70% of the m.13513G>A variant identified 1247 nuclear genes whose transcription levels were significantly affected  of the thirty-one most pronounced DEGs were selected for further evaluation. These genes were chosen because of their putative capacity to be involved in cellular physiological and pathological processes. Genes involved in cancer physiology, however, were excluded, as changes in their expression might be associated with the cancerous nature of ρ0 cells used for the production of cybrid cells. The results of RNA expression analysis of the selected genes were validated using Selfie-digital PCR in freshly propagated cybrid cells , obtained a set of DEGs and identified NDUFA4L2 gene as a target for further functional studies [Little is known about changes in gene expression levels, associated with Leigh syndrome, and in particular with the m.13513G>A variant in the he brain . A previ studies .The RNA sequencing analysis of cells harboring 50% versus 70% of the m.13513G>A variant identified 1247 nuclear genes whose transcription levels were significantly affected, with 437 of them having expression levels altered more than four-fold.The patient, who participated in our research as a donor of altered mtDNA, which was used for creation of cybrid cell lines, had a heteroplasmy level of 60% in the blood and exhibited features of Leigh syndrome. Confirming this, our studies on OXPHOS system functionality revealed that cells with both 50% and 70% heteroplasmy levels showed reduced complex I and III activities as well as complex I related defect in oxygen consumption. Additionally, it was previously described that, cells harboring 20–30% of the m.3243A>G variant were found to have mutually similar transcriptional profiles . FurtherTCEA3, SEL1L3, ABCG1, FABP3, and GRM2) showed a trend in expression consistent with the results of transcriptome analysis. The expression of the TCEA3 gene, which encodes transcription elongation factor, A3 which releases RNA polymerase II from transcriptional arrest [TCEA3 is expressed at low levels, however, upon differentiation, TCEA3 is upregulated [TCEA3 expression in cells harboring the variant may serve as a compensatory mechanism for defects in muscle functions. The SEL1L3 gene, which encodes protein sel-1 homolog 3, suppressor of lin-12-like protein 3, is induced in response to mitochondrial damage [SEL1L3 was identified as one of ten common mitochondrial defect (CMD) genes, and was shown to contribute to CMD-mediated regulatory mechanism, resulting in mitochondrial defect and subsequent retrograde signals for transcriptional reprogramming during hepatocellular carcinoma progression [ABCG1 gene, which encodes ATP binding cassette subfamily G member 1, plays a prominent role in apolipoprotein mediated cholesterol transport and suppresses β-amyloid (Aβ) accumulation. Dysregulated Abcg1 expression is associated with intracellular cholesterol accumulation in the brains of the APP/PS1 mouse model [ABCG1 gene in cells harboring the m.13513G>A variant could be considered a compensatory mechanism, possibly unspecific, for Aβ induced early mitochondria-associated ER membrane dysregulation. The FABP3 gene, which encodes fatty acid binding protein 3, is critical for the uptake of alpha-synuclein and its aggregation into intracellular filamentous-shaped inclusion bodies in a mouse cell model, as well as being involved in MPP+-induced neuronal retraction, mitochondrial activity, and reactive oxygen species (ROS) formation [Fabp3 knockout mice showed decreased alpha-synuclein oligomerization and neuronal degeneration of tyrosine hydroxylase positive neurons in vivo. Published data indicate that FABP3 is critical for preventing synucleinopathies; therefore, its downregulation in cells harboring the m.13513G>A variant could lead to impaired alpha-synuclein uptake and formation of aggregates [GRM2 gene, which encodes glutamate metabotropic receptor 2, plays an important role in normal brain function, and is a potential target for the development of drugs for neurological and psychiatric diseases [GRM2 is negatively regulated by miR-342-p, one of miRNA expressed in the epileptic hippocampus. Upon pharmacological induction of tonic–clonic seizures in rats, the downregulation of miR-342-p and upregulation of its target gene GRM2 were observed [GMR2 in epileptogenesis. Seizures are one of the clinical manifestations associated with the m.13513G>A variant. Therefore, we propose that upregulation of GRM2 may play a role in their development.Selfie-digital PCR analysis of five genes (l arrest , is tissl arrest . In prolegulated . Becausel damage . SEL1L3 gression . The ABCse model . The uprormation . Fabp3 kgregates . This indiseases . In a reobserved . These rMT-ND5 gene showed defects in the OXPHOS system, which had a more pronounced effect in cells bearing 70% than 50% heteroplasmy. These findings are in agreement with results showing that higher levels of heteroplasmy cause more severe symptoms. The pathogenic mtDNA variant had pronounced effects on nuclear gene expression in cybrid cell lines, with DEGs in cells harboring the m.13513G>A mtDNA variant generally reflecting the physiological processes associated with damaged mitochondria.In summary, mutant cell lines harboring the m.13513G>A variant of the


Dear Editor,Measurable residual disease (MRD) is an important post-treatment biomarker that predicts progression-free survival (PFS) following fixed-duration treatment in chronic lymphocytic leukemia (CLL) , 2. MRD In this study, we used the IGHV leader-based next-generation sequencing (NGS) assay to longitudinally measure MRD in samples from the HOVON-139/GIVE trial , 6. The Of the patients treated in the HOVON-139/GIVE trial, 60/67 had ≥1 sample available for MRD measurement (Supplementary Table −5). MRD dynamics for the total cohort are depicted in Fig. −5) in 7/36 (19.5%) patients in induction cycle 1 (C1), 40/43 patients (93.0%) at EOiT, 45/53 (84.9%) patients at R+6 and 39/53 (73.6%) at R+12. MRD levels were comparable between the randomization arms and are depicted in Supplementary Fig. 2A,MRD was measured in 185 samples, representing four time points: during the fourth week of venetoclax ramp-up in induction cycle 1, at the end of the twelfth cycle of induction treatment (EOiT), and 6 and 12 months post-randomization (R+6 and R+12)  down to MRD < 10−5 in 7/36 (19%) patients. In patients with MRD measurements available at both C1 and EOiT (n = 25), high levels of MRD (>10−2) at C1 were associated with failure of reaching MRD < 10−5 at EOiT  , loss of MRD < 10−5 at R+12 exclusively occurred in patients with MRD ≥ 10−4 at C1  . Most importantly, no patient with MRD < 10−4 at C1 experienced disease progression during follow-up, resulting in superior PFS at 40 months, compared to patients with MRD ≥ 10−4 at C1   Fig. . In addi0%) Fig. , suggest48) Fig. . Reachin10 fold change in disease burden between baseline and C1 (ΔMRD). Mean ΔMRD was 5.0 log10 , ranging from 2.6 to 7.3. Patients reaching MRD < 10−5 at EOiT had higher ΔMRD, compared to patients that failed to reach MRD < 10−5 . The optimal ΔMRD cut-off to predict reaching MRD < 10−5 at EOiT was estimated at 4.0 log10 (AUC 0.93). ΔMRD was not associated with pre-treatment biomarkers of high-risk disease . Following obinutuzumab cessation at cycle 6 and treatment with 6 cycles of venetoclax monotherapy, mean IGHV diversity increased incrementally to 1.38 (95%CI 0.94–1.83) at EOiT, 1.84 (95%CI 1.31–2.37) at R+6 and 4.04 (95%CI 2.74–5.34) at R+12   Fig. .Fig. 2IG−5) in 93% of assessed patients, with 85 and 74% of patients retaining uMRD (<10−5) at 6 and 12 months after the end of induction therapy. Reaching low levels of MRD (<10−4) in the fourth week of induction cycle 1, having received 11 weeks of treatment, was associated with a 100% PFS rate in this cohort. Conversely, high MRD (>10−2) at C1 and a slow rate of early MRD eradication were associated with failure to achieve uMRD (<10−5) at the end of induction treatment.In this study, we characterized the MRD dynamics and IGHV repertoire reconstitution of treatment-naive CLL patients treated with venetoclax and obinutuzumab in the HOVON-139/GIVE trial. Induction treatment induced deep and durable levels of MRD, resulting in uMRD (<10−4 in this cohort), their response is shallower, culminating in the earlier loss of uMRD and a shorter time to progression. This notion is in line with the recent results of the UK TAP Clarity trial, where, in the relapsed and refractory setting, achieving a 100-fold or greater reduction of disease burden after 2 months of treatment with venetoclax and ibrutinib was associated with sustained low MRD and clinical response after a follow-up of 3 years [−5 after 1 year of treatment cessation [Very early MRD measurements are a proxy for the rate of disease eradication. Our data indicate that a rapid reduction in disease burden can be used as a biomarker which, irrespective of established genomic features, signifies therapy-sensitive disease, resulting in deep MRD levels at the end of treatment and a long time to progression. Conversely, slow disease eradication is a prognostically poor sign. Whereas these patients still reach deep levels of MRD  and a slow rate of MRD eradication, measured after 11 weeks of treatment, predicted failure to reach uMRD (<10−5) at the end of induction treatment. Conversely, patients with low levels of early MRD (<10−4) had a low probability of uMRD loss and disease progression. In addition, venetoclax consolidation treatment impaired polyclonal IGHV repertoire reconstitution after induction treatment with venetoclax and obinutuzumab, potentially associated with a higher chance of adverse infectious events.In conclusion, in treatment-naïve CLL patients treated with venetoclax and obinutuzumab, high levels of early MRD (>10For the methods, please refer to the Supplementary DataSupplementary Figure 1Supplementary Figure 2Supplementary Figure 3Supplementary Figure 4


Introduction: The occurrence of fatty liver disease, resulting from the accumulation of excessive fat within the liver, has been showing a significant and rapid increase. This study aimed to evaluate the therapeutic effects of Cheong-sang-bang-pung-san extract (CB) on fatty liver disease, and to elucidate the underlying mechanisms.Methods: We used a high-fat diet (HFD)-fed fatty liver mice and free fatty acid (FFA) induced HepG2 cell lipid accumulation model. The levels of serum, hepatic, and intracellular lipid content were assessed. Histopathological staining was used to evaluate the extent of hepatic lipid accumulation. Real-time polymerase chain reaction and Western blotting were conducted to examine the expression of factors associated with lipid metabolism.Results: We demonstrated that treatment with CB dramatically reduced body weight, liver weight, and fat mass, and improved the serum and hepatic lipid profiles in HFD-induced fatty liver mice. Additionally, CB alleviated lipid accumulation in HFD-fed mice by controlling lipid metabolism, including fatty acid uptake, triglyceride and cholesterol synthesis, and fatty acid oxidation, at the mRNA as well as protein levels. In free fatty acid-treated HepG2 cells, CB significantly reduced intracellular lipid accumulation by regulating lipid metabolism via the activation of AMP-activated protein kinase.Conclusion: These findings provide insights into the mechanisms underlying CB’s effects on liver steatosis and position of CB as a potential therapeutic candidate for managing lipid metabolic disorders. The excess fatty acid cannot undergo oxidative metabolism; instead, it is directed toward triglyceride (TG) synthesis, causing hepatic TG storage to increase and very-low-density lipoprotein to be overproduced  in animal and cellular models of hepatic steatosis. We found that CB prevented high-fat diet (HFD)-induced fatty liver disease by regulating lipid metabolism. This study is meaningful in that it proposed the alternative therapeutic applications for dyslipidemia and expanded new indication of botanical medicine that is generally considered to be safe and effective agents.The botanical medicine materials were purchased from Omniherb Co. LTD. . Assurance of quality control for all the materials was validated according to the Korean Botanical Pharmacopoeia . CB-containing botanical medicines and the ratios were listed in Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and penicillin streptomycin were purchased from HyClone . Primary antibodies against sterol regulatory-element binding protein (SREBP)-1, α-tubulin, and β-actin antibodies were purchased from Santa Cruz Biotechnology, Inc . Primary antibodies against acetyl-CoA carboxylase (ACC), p-ACC, fatty acid synthase (FAS), carnitine palmitoyltransferase (CPT)-1α, AMP-activated protein kinase (AMPK) and p-AMPK were purchased from Cell Signaling Technology, Inc . SREBP2, proprotein convertase subtilisin/kexin type 9 (PCSK9), and LDL receptor (LDLR) antibodies were purchased from Invitrogen . Horseradish peroxidase-conjugated secondary antibodies were obtained from Jackson Immuno Research Laboratories, Inc. . THUNDERBIRD SYBR qPCR Mix was purchased from TOYOBO . Oligonucleotide primers for ACC 1/2, liver X receptor (LXR) α, SREBP 1/2, FAS, hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCoR), CPT-1α, LDLR, cluster of differentiation 36 (CD36), fatty-acid-binding protein (FABP), scavenger receptor class B type 1 (SR-B1) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were purchased from Cosmogenetech . To simultaneous analyzed CB, we obtained several metabolites according references that reported bioactivity and marker metabolites of CB. The reference standard compounds for simultaneous analysis, Baicalin and Baicalein, were purchased from sigma-aldrich . Wogonoside was obtained from Shanghai Sunny Biotech , Geniposide was provided by Wako  and Hesperidin was purchased from Biopurify . All solvents for analysis, tertiary distilled water, methanol, acetonitrile, were HPLC grade obtained Merck . Formic acid was purchased sigma-aldrich .Male fifty C57BL/6J mice  were purchased from DooYeol Biothech . Mice were acclimatized under constant conditions . After 1 week of adaptation to the feeding conditions, mice were randomly separated into 5 groups of 10 each: (1) normal diet (ND) group, (2) HFD  group, (3) HFD treated with atorvastatin (10 mg/kg) group, (4) HFD treated with CB (100 mg/kg) group, and (5) HFD treated with CB (200 mg/kg) group. With the exception of the ND group, all mice were fed an HFD. CB was dissolved in PBS and orally administered on a daily basis for the last 9 weeks along with HFD in the 100 or 200 mg/kg CB groups. Body weights were measured once weekly. After 14 weeks of experiment, the animals were fasted overnight and sacrificed. Blood samples were collected from mice via cardiac puncture under 2%–2.5% isoflurane anesthesia. After cervical dislocation, the liver tissues, and gastrocnemius muscle were excised, rinsed with PBS, weighed and directly stored at −80°C until analysis. All part of animal experiments were conducted in accordance with the Care and Use of Laboratory Animals of the National Institutes of Health of Korea and were approved by the Institutional Animal Care and Use Committee of Korea Institute of Oriental Medicine (KIOM) .g for 15 min at room temperature to obtain serum samples. The samples were stored at −70°C until use. Serum or liver homogenate levels of TG, total cholesterol (TC), low density lipoprotein cholesterol (LDL-c), high density lipoprotein cholesterol (HDL-c), alanine aminotransferase (ALT), and aspartate transaminase (AST) were determined by enzymatic methods using commercial kits.Blood samples were collected from each mouse and centrifuged at 1700 Collected liver tissues were fixed with 10% formalin. Paraffin-embedded tissue was cut into 3 μm sections and stained with hematoxylin and eosin (H&E). The stained sections were observed under a Leica DM IL LED microscope .To isolate total RNA from the liver tissues or cells, TRIzol Reagent  was used according to the manufacturer’s protocol. Total RNA was quantified using an NanoDrop spectrophotometers . Complementary DNA was synthesized from isolated total RNA (2 μg), oligo d(T)16 primer, and Avian Myeloblastosis Virus reverse transcriptase (AMV RT) with genomic DNA remover. Relative gene expressions of target gene versus reference gene were quantified using RT-qPCR analysis  with SYBR Premix. For calculate changes in gene expression as a relative fold difference, the comparative quantification cycle method was used. The Cq (Target gene–reference gene) values of target genes ACC1/2, LXRα, SREBP1/2, HMGCoR, CPT-1α, LDLR, CD36, and FABP were normalized to that of GAPDH.Total protein was extracted from liver tissues or cells using PRO-PREP™ protein extraction kit . The protein concentration was measured using the protein assay reagent . Proteins were separated by 10%–12% SDS-PAGE, and transferred to polyvinylidene difluoride membrane. The membrane was blocked for 1 h with 5% BSA followed by incubation with primary antibodies at 4°C overnight. After washing three times with Tween 20/Tris-buffered saline (T/TBS), the membranes were incubated with a horseradish peroxidase-conjugated secondary antibody  for 2 h at room temperature. The blots were again washed three times with T/TBS, and then bands were detected via enhanced chemiluminescence reagent (Bio-Rad).2. The cells were seeded at a density of 2× 105 cells per well into 6-well plate and then treated with 0.5 mM oleic acid  and 0.25 mM palmitic acid  dissolved in culture medium containing 10% BSA  with or without different concentrations of CB  for 24 h. Control cells were treated with 10% BSA only.Human hepatic cell line HepG2 was obtained from the Korean Cell Line Bank . HepG2 cells were grown in DMEM supplemented 10% FBS and 1% penicillin/streptomycin at 37°C under a humidified atmosphere of 5% CO5 cells per well. After incubation for 24 h, the cells were treated with different concentrations of CB  for 24 h. Afterwards, the cells were added with MTT working solution (5 mg/mL) and incubated for another 4 h. The supernatant from the plates was removed, and the purple formazan product was dissolved in DMSO. The absorbance was measured on an Epoch microplate spectrometer  at 540 nm.The cell viability was assessed using MTT assy. In brief, HepG2 cells were plated into a 96-well plate with concentration of 1×10After stimulation with OA + PA mixture, the cells were washed with PBS and fixed with 10% formalin in PBS for 1 h, followed by staining with Oil Red O working solution (3 mg/mL in 60% isopropanol) for 2 h. The cells were rinsed with distilled water and observed under a microscope. Next, the Oil Red O dye was extracted with isopropanol to determine the intracellular lipid content and was assessed using an Epoch microplate spectrometer at 520 nm.The CB was dissolved in water at 10 mg/mL concentration using ultrasonicator (JAC Ultrasonic JAC-3010) for 30 min. After dissolved, CB solution was filtered through a 0.2 μm PVDF membrane and then 10 μL of filtrate was injected to HPLC analysis. Stock solution of single compounds  were prepared at 1000 ppm with HPLC grade methanol and serially diluted at standard curve concentration of each. Standard stock solution was used for quantitative analysis after 0.2 μm PETE membrane filtration. Simultaneous determination of five metabolites  were conducted using using Dionex Ultimate 3000 system formed with column oven, an auto sampler, a binary pump and diode assay UV/VIS detector . HPLC condition were follows; column was Waters X bridge  connected to a C18 guard cartridge (4.0 mm × 3.0 mm), column temperature and auto sampler were kept 40°C and 20°C respectively, eluted condition was flow rate 1.0 mL/min with gradient of 0.1% formic acid (solvent A) and 0.1% formic acid acetonitrile (solvent B), solvent gradient method was applied; 0–40 min, 10–60%B; 40–50 min, 60–95%B; 50–55 min. 95%B, chromatogram detected under 280 nm and 245 nm. Calibration curves, assessed by standard solution and the limits of detection (LOD) and quantification (LOQ) under the chromatographic conditions, were determined by injecting a series of standard solutions. Each sample was three injected under same condition and data was processed using Chromeleon 7 software post hoc test, and p-values of less than 0.05 were considered statistically significant.Statistical analysis was performed using GraphPad Prism 5 software . Data are presented as the mean ± standard deviation (SD) of triplicate experiments. Statistical significance was determined using ANOVA and Dunnett’s in vivo experimental scheme is described in The Lipid metabolism and liver function are closely related. Therefore, we investigated whether CB treatment could inhibit hepatic steatosis and improve hepatic function. Liver weight significantly increased in the HFD group, but this increase was markedly blunted in the CB-treated groups . FurtherCD36 and FABP4, the lipogenesis markers ACC1, SREBP1, FAS, and LXRα, and the cholesterol synthesis markers SREBP2, ACC2, and HMGCoR  from Scutellariae radix , genipos Rhizoma , osthol  Rhizoma , and ber Rhizoma . As expe Rhizoma . FurtherIn conclusion, we demonstrated that CB effectively alleviated lipid accumulation in fatty liver disease. We uncovered a novel lipid-lowering mechanism wherein CB activated AMPK, thereby regulating the expression of transcription factors, its target enzymes, and β-oxidation-related genes. Based on the results of this study, CB could be a useful traditional medicine, and an effective candidate for preventing and/or treating fatty liver disease and associated metabolic diseases.


In recent decades, the world has faced an emerging problem, which is assuming increasingly impressive dimensions: the appearance of New Psychoactive Substances, characterized by peculiar pharmacological and toxicological properties, and are exceptionally dangerous for consumers health. Among novel psychoactive substances, synthetic cathinones have recently emerged on the market and, based on their low cost and availability in smartshops and online, their use has been growing extremely, particularly among adolescents and young adults. However, the understanding of neurotoxic mechanisms induced by synthetic cathinones is still lacking, particularly on neural stem/progenitor cell cultures. In the current in vitro study, the effects of increasing αPHP concentrations, one of major MDPV derivatives, on cell viability/proliferation, morphology/ultrastructure, and cell death pathways, have been evaluated after exposure in murine neural stem/progenitor cells, using a battery of complementary techniques. We revealed several different alterations after αPHP treatment, indicating the potential harmful effects of this synthetic cathinone on young brains. Hence, the present investigation could pave the way for a broadened understanding of the synthetic cathinone toxicology needed to establish an appropriate treatment for novel psychoactive substances and the potential consequences for public health.Increasing reports of neurological and psychiatric outcomes due to psychostimulant synthetic cathinones (SCs) have recently raised public concern. However, the understanding of neurotoxic mechanisms is still lacking, particularly for the under-investigated αPHP, one of the major MDPV derivatives. In particular, its effects on neural stem/progenitor cell cultures (NSPCs) are still unexplored. Therefore, in the current in vitro study, the effects of increasing αPHP concentrations (25–2000 μM), on cell viability/proliferation, morphology/ultrastructure, genotoxicity and cell death pathways, have been evaluated after exposure in murine NSPCs, using a battery of complementary techniques, i.e., MTT and clonogenic assay, flow cytometry, immunocytochemistry, TEM, and patch clamp. We revealed that αPHP was able to induce a dose-dependent significant decrease of the viability, proliferation and clonal capability of the NSPCs, paralleled by the resting membrane potential depolarization and apoptotic/autophagic/necroptotic pathway activation. Moreover, ultrastructural alterations were clearly observed. Overall, our current findings demonstrate that αPHP, damaging NSPCs and the morpho-functional fundamental units of adult neurogenic niches may affect neurogenesis, possibly triggering long-lasting, irreversible CNS damage. The present investigation could pave the way for a broadened understanding of SCs toxicology, needed to establish an appropriate treatment for NPS and the potential consequences for public health. In recent years, the entire world has faced an emerging problem which is assuming increasingly impressive dimensions. The appearance of New Psychoactive Substances (NPS), characterized by peculiar pharmacological and toxicological properties, are exceptionally dangerous for consumers health, therefore representing a pressing matter of concern for National Regulatory Agencies and International Health Regulatory Bodies. In recent decades more than 600 NPS have been identified, highlighting the exponential growth in the “legal” and illegal market of new NPSs, with a consequent increase in reports of cases of toxicity and adverse effects ,2,3,4,5.Hence, although the currently available data are neither exhaustive nor conclusive, the toxicity of NPSs is extremely worrying, being the cause of immediate death and/or irreversible functional alterations both at the CNS level, as well as at the systemic level and in peripheral organs. The clinical relevance found in cases of acute NPS toxidromes ,6,7,8,9 Synthetic cathinones (SCs), β-keto-amphetamine analogues, are one of the most prevalent designer drugs that are widely offered by the global illicit market ,11,12. TSCs, commonly known as “legal highs” and “bath salts” including 3,4-methylenedioxypyrovalerone (MDPV), Mexedrone (3-methoxy-2-(methylamino)- 1-(4-methylphenyl)propan-1-one), αPVP , and αPHP -1-hexanone), induce psychostimulant effects similar to those triggered by cocaine, amphetamine, and MDMA ,17.αPHP, a less common analogue of αPVP, is a one of the pyrovalerone derivatives (-pyrrolidinophenones) known to be a SCs subgroup chemically characterized by a pyrrolidine ring and a long side chain linked to the -carbon ,19. The Furthermore, several in vivo studies have been conducted to identify the neurotoxicity mechanisms underlying the SCs-triggered adverse clinical manifestations. Typical hallmarks comprise inflammation, disruption of monoaminergic neurotransmitters, alterations in thermoregulation, oxidative stress, and cytotoxicity ,17,28,29The pharmacological activity of pyrovalerone derivatives has been also investigated, revealing that αPHP was able to trigger a selective and potent inhibition of dopamine and norepinephrine reuptake, showing instead negligible effects on serotonin transporters and also lacking the ability to increase neurotransmitter release ,25,30,312+ homeostasis disruption and mitochondrial dysfunction as fundamental events involved in SC-induced neurotoxicity [Recently, in vitro studies attempted to identify the neurotoxic potential of several SCs, identifying oxidative stress, disrupting neuronal Catoxicity ,5,32.Nonetheless, the understanding of neurotoxic mechanisms is still lacking, particularly for some SCs that have been relatively under-investigated, for example αPHP. Hence, the urgent need emerged for further in-depth studies to broaden knowledge of their pharmaco-toxicological profile and neurotoxic effects ,33.To fill this gap, we intended to investigate αPHP neurotoxic effects using a relevant in vitro model, i.e., neural stem/progenitor cell cultures (NSPCs) isolated from 8 weeks old C57BL/6 mice, characterized by peculiar staminal properties. In particular, NSPCs are self-renewing cells which can differentiate into multiple neural lineages, during embryo development, perinatal period and adult life, located in some restricted regions of adult mammalian brain, e.g., the subventricular zone (SVZ) and the hippocampus . These cIn the current in vitro study, the effects of increasing αPHP concentrations (25–2000 μM), one of major MDPV derivatives, on cell viability/proliferation, morphology/ultrastructure and cell death pathways, have been evaluated after 72 h-exposure in murine NSPCs, using a battery of complementary techniques, i.e., MTT and clonogenic assay , flow cytometry, immunocytochemistry, TEM and patch clamp. We revealed several different alterations after αPHP treatment, indicating the potential harmful effects of this SC on young brains.g, the supernatant was discarded, and the pellet was re-suspended in proliferation medium (PM) [2.The procedure was performed as previously described ,42,43. Bium (PM) . Under tThe synthetic cathinone αPHP, purchased from LGC Standards , was kindly provided by Prof. Matteo Marti . αPHP was dissolved in phosphate-buffered saline (PBS)  up to 40 mM stock solution and stored at −20 °C until use.(i) Phase-Contrast Microscopy: NSPCs Morphology CharacterizationTo narrow down the range to be employed in the following analyses, as the first experimental step, a range of αPHP concentrations, i.e., 25, 50, 100, 200, 500, 1000 and 2000 μM, chosen based on previous literature data ,48,49,50®-coated , at a density of 15,000–20,000 cells per coverslip, left to attach for 45 min, then fixed in 4% paraformaldehyde, washed in PBS and kept at 4 °C until the following analysis.After αPHP exposure, cells were counted in order to determine their proliferation capability by means of the dissociation of the neurospheres. Single cells were plated on 10 mm coverslip CultrexNSPCs were observed under inverted phase contrast microscopy equipped with a 20× objective (Olympus CKX41) after αPHP exposure (25–2000 μM) for 72 h to evaluate possible cell morphology changes/alteration. Digital micrographs were shot with a camera , stored on a PC and processed with the Olympus Cell F software (version n. 3.1).2.The analysis of cell density was performed using ImageJ software 1.54 . Specifically, four random images in each well (excluding parts near the edge of the well) were acquired at 32× magnification for each replicate at each concentration, and then converted to binary images and analyzed with the “particle analysis tool”. The cell density was calculated as reported below: number of cells/area in mm(ii) Proliferation assay2 in a 1 cm2 well (48 well plate) and maintained in medium added with selected αPHP concentrations (25–2000 µM) for 72 h ; then, the neurospheres were dissociated and the cells were counted. The cumulative total number of cells for each passage was calculated by multiplying the proliferation rate (viable cell harvest number/inoculum cell number) by the cumulative total number of cells of the previous passage [Growth curves were obtained from NSPC cultures between the 3rd passage (P3) and the 10th (P10). At each passage, cells were mechanically dissociated when the neurospheres reached a dimension of about 0.1 mm. Therefore, they were plated at a density of 10,000 cells/cm passage . As a go passage ,52.(iii) MTT Assay: cell metabolic activity study2), the medium was carefully removed avoiding aspirating the neurospheres , and subsequently, the formazan crystals, formed by mitochondrial dehydrogenases, were dissolved in DMSO (100 μL/well) and quantified by measuring absorbance at 550 nm using a microplate reader  [The MTT assay was employed to evaluate cell viability by mitochondrial metabolism measuring the reduction of 3--2,5-diphenyl-2H-tetrazolium (MTT) bromide by mitochondrial dehydrogenases. MTT assay was performed as previously described  with sli, Italy) . As a ba, Italy) ,52.(iv) Clonal cell survival assayCells were plated in a 96 well plate at a density of 150–180 cells/well in 100 µL PM containing different αPHP concentrations (25–2000 μM) and incubated for 8 days. Then, the neurospheres were counted. Specifically, only the neurospheres having a dimension bigger than 50 µm were considered, since these latter resulted from the proliferation of a single NSC, whereas smaller-sized neurospheres most likely derived from the proliferation of progenitor cells ,54. PlatAfter counting clones, plating efficiency (PE) and survival fraction (SF) were calculated using the following formulas:Based on the obtained cell morphology, viability and proliferation data, a narrowed range of αPHP concentrations, ranging from 25 to 200 µM, was chosen to be used in the following analyses.Control and treated NSPCs were plated and processed as previously reported for Phase Contrast microscopy evaluation, but after fixation with 4% paraformaldehyde for 10 min, cells were postfixed with 70% ethanol at −20 °C for at least 24 h and stored at −20 °C until staining. The samples were rehydrated for 15 min in PBS and then immunolabeled with selected monoclonal and polyclonal primary antibodies (summarized in An Olympus BX51 microscope equipped with a 100-W mercury lamp was used under the following conditions: 330–385 nm excitation filter (excf), 400 nm dichroic mirror (dm), and 420 nm barrier filter (bf) for Hoechst 33258; 450–480 nm excf, 500 nm dm, and 515 nm bf for the fluorescence of Alexa 488; 540 nm excf, 580 nm dm, and 620 nm bf for Alexa 594. Images were recorded with an Olympus MagniFire camera system  and processed with the Olympus Cell F software (version n. 3.1).For confocal laser scanning microscopy, Leica TCS-SP system mounted on a Leica DMIRBE-inverted microscope was used. For fluorescence excitation, an Ar/UV laser at 364 nm was used for Hoechst 33258, an Ar/Vis laser at 488 nm was used for FITC and a He/Ne laser at 543 nm was used for Alexa 594. Spaced (0.5 μm) optical sections were recorded using a 63× oil immersion objective. The “colocalization” analysis was done considering 30 cells for sample, and three points of colocalization in at least 15 cells. Images were collected in the 1024 × 1024-pixel format, stored on a magnetic mass memory and processed by LAS-X Leica Microsystems CMS GmbH software (version n. 5.1.0).After assessing the αPHP-induced effects as above reported, we focused on the dose of 100 µM αPHP, chosen to be used in the following additional evaluations, i.e., cytofluorimetric analysis, Patch Clamp experiments, and ultrastructural characterization by Transmission Electron Microscopy (TEM).After 72 h exposure to 100 µM αPHP, the NSPCs were detached by mild trypsinization  to obtain single-cell suspensions to be processed for flow cytometry with a Partec Cy-Flow Space system Sysmex-Partec , equipped with argon ion laser excitation at 488 nm (power 200 mW). Data were analyzed with the built-in software .A set of NSPC samples was washed in PBS, fixed in cold (−20 °C) 70% ethanol for at least 60 min and stored refrigerated (+4 °C) until all samples were recovered. Subsequently, samples were processed for cell cycle assessment by flow cytometry: fixed cells were washed in PBS to remove ethanol and re-suspended in PBS containing RNase A 100 U/mL, Nonidet P 40 0.1%  and propidium iodide (PI) 50 µg/mL . Cells were kept in this staining solution for at least 24 h before flow analysis.−1 and then stained at room temperature with propidium iodide (PI) 50 μg/mL  1 h before flow cytometric analysis. For both analyses, PI red fluorescence was detected with a 610 nm long-pass emission filter. At least 20,000 cells per sample were measured to obtain the distribution among the different phases of the cell cycle and to assess the percentage of dead cells normalized to control.Another set of NSPC samples were processed for the identification of dead cells. Briefly, the NSPCs were quickly washed in PBS, permeabilized in 70% ethanol for 10 min, treated with RNase A 100 U mL®-coated coverslip glass  in PM. Whole-cell patch clamp was executed by using Axopatch-200B  patch clamp amplifier at the output cut-off frequency of 5 kHz. Data were sampled with a Digidata-1440 interface. The extracellular solution contained NaCl 140 mM, KCl 5 mM, HEPES 10 mM, glucose 10 mM, CaCl2 3 mM, and MgSO4 1.2 mM, and the pH was adjusted to 7.4 with NaOH. Patch pipettes were pulled from borosilicate capillaries  and had a mean resistance of 7 MΩ. The intrapipette solution contained potassium gluconate 126 mM, NaCl 4 mM, glucose 15 mM, HEPES 5 mM, MgSO4 1 mM, BAPTA 0.1 mM, CaCl2 0.02 mM, ATP 3 mM, and GTP 0.1 mM, osmolarity 280 OSM, and pH was adjusted to 7.2 with KOH. TEA 20 mM was used in patch-clamp experiments. All reagents were purchased from Sigma-Aldrich .Control and 100 µM αPHP-treated NSPCs were plated in a 6-multiwell plate containing a 22 × 22 mm Cultrex4  for 90 min at RT and dehydrated by increasing concentration of alcohol/acetone. Finally, the pellets were washed with propylene oxide and embedded in epoxy resin . After resin polymerization, samples were cut at 0.5 μm thickness and stained with methylene blue and safranin to morphologically select the field of interest. Ultrathin sections were collected on a 300-mesh copper grid and stained with uranyl acetate and lead citrate. The plates, after being developed, were examined under a transmission electron microscope (Philips EM 208S), then computerized through an Epson Perfection 4990 photo scanner at a resolution of 800 dpi and then processed using the Epson Scan software (version n. 3.771).Control and 100 µM αPHP-treated cells were harvested by mild trypsinization (0.25% trypsin in PBS containing 0.05% EDTA) and centrifuged to obtain a visible pellet. This was immediately fixed in Karnovsky solution  for 90 min at RT and washed with PBS (pH 7.2). Then, the pellet was embedded in agar, post-fixed in 1% OsOp < 0.05 (*), p < 0.01 (**) and p < 0.001 (***). For multiple comparison, the statistically significant differences were considered as follows: * compared to ctrl; Φ compared to vehicle; Ψ compared to 25 µM αPHP; # compared to 50 µM αPHP, § compared to 100 µM αPHP, Ω compared to 200 µM αPHP, γ compared to 500 µM αPHP, Ʃ compared to 1000 µM αPHP, Δ compared to 2000 µM αPHP. All statistical analyses were performed by using GraphPad Prism 8.0 .Data are presented as the mean ± SEM. The Anderson-Darling, D’Agostino & Pearson, Shapiro–Wilk and Kolmogorov–Smirnov Tests were used to establish and confirm the normality of parameters. Then, data were analysed to verify statistically significant differences. For data passed the normality test, the analysis was conducted using one-way ANOVA followed by Bonferroni’s post hoc test for multiple comparisons. Diversely, for non-normally distributed results, the analysis was performed employing the Kruskal–Wallis test followed by Dunn’s test. The differences were considered statistically significant for In the current study, murine NPSCs collected from the subventricular zone (SVZ), one of the largest germinal regions in the adult mammalian brain, which extends along the lateral wall of the telencephalic ventricles, were chosen as the in vitro model with the aim of investigating whether αPHP affects their peculiar characteristics . Our final goal was to achieve a clear understanding of which mechanisms play a key role, also bearing in mind that our in vitro model, derived from 8-week-old C57BL/6 mice, could mimic the human exposure during puberty .A wide αPHP concentration range (25 to 2000 µM) to be tested on NSPCs was chosen based on previous in vitro findings on SCs ,48,49,50Based on the morphological evaluation by phase contrast microscopy, together with viability/proliferation data obtained by the MTT assay and clonogenic assay findings, the concentrations ranging from 25 to 200 µM αPHP were selected and employed for the following immunofluorescence evaluations. Then, the αPHP concentration of 100 µM, leading to a mild reduction  in the number of living/proliferation cells, was selected as the suitable sub-toxic dose to be employed for cytofluorimetric, electrophysiological and ultrastructural investigations.The effects of αPHP on cell proliferation were evaluated by measuring the cell growth after 72 h-exposure to selected concentrations. 2 round coverslip and examined. In detail, the phase-contrast microscopy evaluation revealed the absence of morphological changes after exposure to the lowest αPHP tested concentration, i.e., 25 µM, even though a significant decrease of cell density (about 24%) was measured. At the concentration of 50 µM αPHP, a slight further decrease in cell density (about 29%) was assessed, paralleled by an evident morphological alteration of cells, with a manifest enhancement of round-shaped cells (about 35%), often characterized by the presence of cytoplasmic blebs  in the number of living cells . A worseFor the clonal analysis, the limited number of plated cells (150 per well) was crucial to avoid the formation of chimera spheres (formed by the aggregation of two or more cells), allowing to seek the assessment of αPHP-induced modification in self-renewal cells capability. Based on the above reported data, a narrowed range of concentrations (25–200 μM) was tested with the aim at evaluating immunocytochemically the possible αPHP-induced cellular alterations. Specifically, the investigation focused on the apoptotic pathway and NSPCs cytoplasmic structures, i.e., cytoskeleton, mitochondria and lysosomes.Firstly, concerning the assessment of the potential activation of cell death pathway, a triple immunofluorescence staining  was perfConfocal microscopy showed mitochondria and β-tubulin labelling homogeneously distributed in the cytoplasm, with the latter predominantly localized in the peripheral region, close to the cell membrane . FurtherThe cytoskeleton reorganization was perceptible at 50 µM αPHP, when β-tubulin aggregates were clearly observed in the marginal zone of NSPCs .Moreover, homogeneous mitochondria agglomerates were clearly detectable in control, vehicle, 25µM and 50µM exposed cells. However, after 100µM- αPHP exposure mitochondrial network disaggregated, and notably, the overlapping of mitochondria and caspase3 immunofluorescence signals were detected at the highest αPHP dose  .The quantitative evaluation revealed that β-tubulin expression levels were similar comparing control, vehicle and 25 µM αPHP-treated cells. However, a significant increase was observed at 50 µM, notably followed by a slight decrease at the highest tested αPHP doses. Nonetheless, these latter β-tubulin expression values still remained slightly higher compared to those measured in the control, vehicle and 25 µM αPHP-treated NSPCs .Similarly to the above-reported β-tubulin expression trend, any significant difference was measured in mitochondria immunofluorescent signal comparing control, vehicle and 25 µM αPHP-treated cells, while a slight increase of immunopositive cell OD was determined at 50 µM αPHP. Interestingly, at the highest tested doses, namely 100 and 200 µM, a significant lessening of mitochondria immunopositive cell OD was determined .Parallelly, fluorescence microscopy revealed the presence of immunolabelled lysosomes (red fluorescence) regularly distributed in the cytoplasm . HoweverRegarding the quantitative evaluation of lysosomal alterations, any significant difference in immunopositive cell OD was determined by comparing control and vehicle NSPCs. However, a significant increase was detected when measuring immunopositive cell OD at 25 and 50 µM αPHP. Conversely, a slight reduction was highlighted at increasing doses, i.e., 100 and 200 µM. Nevertheless, these latter lysosomal immunopositive OD values still persisted slightly higher compared to those measured in Control and vehicle NSPCs .Concerning deathly pathways, the potential αPHP-induced alterations were further investigated by assessing specific markers, namely AIF, BAX, LC3B and p62.Apoptosis Inducing Factor (AIF) is a mitochondrion-localized flavoprotein with NADH oxidase activity, encoded by a nuclear gene, which is pivotally involved in apoptotic cell death ,63,64. TRegarding the quantitative evaluation of AIF immunofluorescent signal, any significant difference was assessed comparing control, vehicle, 25 µM- and 50 µM-treated cells. Interestingly, an extremely significant increase in immunopositive cell OD was measured in NSPCs exposed to 100 µM αPHP. Conversely, at the higher dose of 200 µM αPHP, a slight decrease was determined, even though this value still remained faintly higher compared to those measured in all other experimental conditions .BAX is a member of the Bcl-2 family, able to cross the mitochondrial outer membrane, and key regulator of the intrinsic pathway of apoptosis ,66,67. CAny significant difference was revealed between control and vehicle cells when measuring BAX immunopositive cell OD. Differently, a significant increase was determined in NSPCs exposed to 25 µM αPHP. This BAX immunopositive cell OD enhancement still remained extremely significant even at 50 µM αPHP. A slight decrease was instead highlighted at 100 µM αPHP, further exacerbated in NSPCs exposed to 200 µM compared to both 25 and 50 µM αPHP. Nonetheless, these BAX immunopositive cell OD levels, measured at the highest doses (100 and 200 µM αPHP) persisted faintly higher compared to those determined in control and vehicle cells .The autophagy protein microtubule-associated protein 1 light chain-3B (LC3B) was assessed based on its crucial regulatory role in the autophagic-apoptosis pathways ,69,70. TThe subsequent quantitative analyses revealed similar LC3B immunopositive cell OD values comparing control, vehicle cells and NSPCs exposed to 25 µM αPHP. Interestingly, after 50 µM, 100 µM and 200 µM αPHP-exposure, a significant increase of LC3B immunopositive cell OD was detected, compared to control, vehicle and 25 µM αPHP. However, a slight decrease of immunopositive cell OD was measured in NSPCs exposed to 100 µm αPHP compared to 50 µM αPHP. A further slight immunolabeling reduction was observed comparing 200 µm αPHP and 100 µM αPHP .Concerning p62, this versatile protein finely regulates the delicate balance between survival and cell death, being critically involved in several diseases, including CNS injury and neurodegenerative disorders ,72,73,74The double immunofluorescence reaction aimed at determining the expression/localization of p62 (green fluorescence) and Lysosomes (red fluorescence) revealed the lack of co-localization of the two markers  in control, vehicle cells and NSPCs treated with 25 µm αPHP . HoweverIn regard to the quantitative analyses, any difference in p62 immunopositive cell OD was measured comparing control, vehicle cells and NSPCs treated with 25 µM αPHP. Conversely, a very significant increase of p62 immunopositive cell OD was determined in NSPCs treated with 50 µM αPHP, becoming even more significant after exposure to 100 µM αPHP. Interestingly, an extremely significant p62 immunopositive cell OD reduction was revealed at the highest dose  compared to the lower concentration (100 µM αPHP). As a consequence, any significant difference subsisted between p62 OD values measured in NSPCs treated with 50 µM or 200 µM αPHP, although immunofluorescence persisted significantly higher compared to those determined in control, vehicle and 25 µM αPHP-treated cells .One of the first molecule involved in DNA damage response is Histone 2AX (H2AX), whose rapid phosphorylation/activation to γH2AX occurred after exposure to DNA-damaging agents to avoid genomic instability ,76. The The cytofluorimetric analysis, revealed that, similarly to control, the 100 µM αPHP exposed NPSCs was normally scattered among the different cell phases  showing a physiological DNA content distribution. In detail, only a slight modification in the different cell cycle phases was gauged in αPHP-treated NPSCs, arguably ascribable to biological variability.Differently, concerning the cell death assessment, the biparametric cytograms evaluation revealed that αPHP was able to decrease the number of living cells (about 6%) already after exposure, with a further lessening after 15 min (about 9%) . Notablyn = 17) and treated (n = 15) cells displayed similar passive properties, evaluated as membrane capacitance  located in cell culture dishes (35 mm × 10 mm), were cultured in PM, added with Matrigel, with or without 100 µM αPHP, and were recorded after about 72 h. Control  and treated (n = 15) cells displayed both transient and persistent currents, without any statistical differences. In particular, Regarding active currents, the totality of the control  and medium-sized mitochondria, located in the perinuclear area .Differently, after 100 µM αPHP exposure various ultrastructural alterations were clearly detected. Specifically, NPSCs presented typical changed features related to the activation/progression of different types of cell death pathways. In detail, enlarged nuclei were observed, often characterized by chromatin condensation, absence of the nuclear envelope and partial cytosol degradation. At the cell periphery, the plasma membrane formed some blebs, enclosing cytoplasmic components as a consequence of cytoskeletal damage. Treated NPSCs also showed (i) an elevated number of vacuoles containing cell debris, which could represent distinctive elements of autophagy activation, (ii) a reduced density of mitochondria which appeared severely damaged, and (iii) degraded RER. In addition, several NPCSs exhibited a poorly visible cell membrane .Nowadays, the overflow NPS phenomenon is taking on public health relevance, alongside and often overlapping with that of traditional drugs. This trend is even more worrying considering the pervasive infiltration of offers on the internet-driven social networks, particularly frequented by adolescents and young adults. In this social setting, the use of SCs is linked to an intensive and repetitive administration pattern in which other drugs are concomitantly consumed, including alcohol . The cliA crucial link exists between neurogenesis and several psychiatric disorders, but the effects and mechanisms of action of psychoactive substances, including NPS, on neurogenesis still remain unknown or even controversial. Evidence accumulated in the last decade indicating that psychoactive substances negatively influence neurogenesis in the adult, in particular supporting the existence of a mutual relationship between postnatal neurogenesis and addictive behaviors ,79. BaseHence, in the current in vitro study, we investigated the unexplored effect of αPHP on proliferation, survival, cell mechanisms, electrophysiological features, and ultrastructure by using a murine cell-based model, namely NSPCs.In particular, NSPCs obtained from the SVZ of 8-week-old C57BL/6 mice, were employed to evaluate the toxic effects of αPHP by assessing cell viability/proliferation, clonal capability and selected cellular endpoints, i.e., cell morphology, mitochondrial function, and cell death pathways, also exploring membrane ion channels changes. We revealed that αPHP was able to induce a dose-dependent significant decrease of the viability, proliferation and clonal capability of the NSPCs, paralleled by the resting membrane potential depolarization and apoptotic/autophagic/necroptotic pathway activation. Moreover, ultrastructural alterations were clearly observed.Concerning the chosen model, it has to be mentioned that, compared to various available cell lines, e.g., SH-SY5Y , NT2, anFor clearness, it has to be underlined that mammals brain neurons arise from a small number of neural stem and progenitor cells (NSPCs). NSCs are able to generate all the cell types in the brain, whereas neural progenitors (NPs) have a more restricted potential ,83. As aA bulk of literature provides evidence on the neurotoxic mechanisms of SCs highlighting that these NPS can cross the brain-blood barrier, acting like psychostimulants in terms of their effects on neurotransmitter levels in the different brain regions. Intoxication cases and fatalities have been reported in humans, in addition to toxic effects revealed both in lab animals  as well as in in vitro models. The disclosed toxic effects are often dose- and time-dependent, with different injury extent depending on the diverse tested SC, thus revealing a close relationship between structure and activity .Several in vitro and in vivo studies have been undertaken regarding the toxic SCs outcomes, particularly related to mephedrone, methylone, MDPV and α-PVP exposure, while the effects of αPHP are still scarcely explored. Therefore, we paved the way to fill the gap in the knowledge of αPHP toxicity, by using NSPCs as a useful approach to test SCs.Accordingly, a recent study testing SCs, demonstrated a significant Mephedrone-induced decrease in the proliferation of NSPCs, cell cycle arrest, paralleled by the enhancement apoptotic and necrotic cells percentages; mephedrone 20–640 µM), also triggered a significant reduction in the number and diameter of neurosphere-forming cells was determined [ µM, alsoOur present findings demonstrate for the first time to our knowledge that αPHP induces toxicity in NSPCs of mouse origin, characterized by cell proliferation lessening and apoptosis/autophagy, morphological and ultrastructural alterations, and resting membrane potential depolarization. Notably, the main cytotoxic effects induced by 72 h-exposure to increasing αPHP doses (25–2000 μM) occurred in a concentration-dependent manner.In particular, the morphological evaluation by phase contrast microscopy, together with viability/proliferation data obtained by proliferation and MTT assays revealed that αPHP caused a dose-dependent cytotoxicity, showing a significant marked effect already at 100 µM, which became more pronounced at the higher doses, i.e., 200 and 500 µM, with the worst damages assessed at the highest concentration, i.e., 1000 and 2000. In particular, the αPHP concentration of 100 µM was able to induce a mild cell injury, causing a decrease of about 35% in the number of living proliferating cells. In addition, the clonogenic assay results revealed that long-lasting (8 days) exposure to increasing αPHP concentrations induced a dose-dependent NSCs colony formation inhibition, with a significant decrease already after exposure to the 100 µM dose and a further clonogenic reduction at 500 μM αPHP, with the highest tested doses, i.e., 1000 and 2000 μM αPHP, able to completely hinder the NSCs clonal capability.Furthermore, concerning the immunocytochemically evaluated markers, we demonstrated that αPHP was able to dose-dependently affect cell death pathways, altering the expression levels of different proteins crucially involved in these processes, i.e., caspase3, BAX, AIF, LC3B and p62, which showed different dose responses to NPS exposure.In particular, a significant increase in caspase3 expression levels was determined already at a dose of 100 µM αPHP. At the higher dose of 200 µM, a further enhancement was gauged, notably accompanied by the reduction of mitochondrial expression level , characterized by the overlapping of the two fluorescence signals, therefore suggesting the contribution of caspase3 in damaging mitochondrial function, through outer membrane permeabilization, and consequent increase in ROS species production, as typical events occurring during apoptosis . ConcernBased on these data, we could assume that the mitochondrial depolarization as the result of the rapid increase in active caspase3 levels and the significant cytoskeletal shrinkage represent manifest indicators of apoptotic phenomena occurrence. In support of this hypothesis are our current findings related to BAX and AIF, being key regulator of the apoptotic pathway. In detail, we presently demonstrated that αPHP boosted BAX expression levels already at the lowest tested doses, i.e., 25 and 50 µM, with a diminishing expression level trend at the higher concentrations. In a similar manner, AIF was significantly augmented by αPHP at the concentration of 100 µM, moving from the mitochondria to the cytoplasm, accompanied by tubulin cytoskeletal alterations, then showing a decreasing trend at the higher doses.Our data are in line with previous studies reporting that mitochondrial outer-membrane permeabilization, by pro-apoptotic Bcl-2 family members, plays a crucial role in apoptosis induction, causing the release of AIF, which seems to define a ‘caspase-dependent’ mitochondria-initiated apoptotic DNA degradation pathway. Further in the intricate balance existing among cell death pathways BAX-mediated mitochondrial release of AIF could be a critical player in early steps of another type of death mechanism, i.e., necroptosis , also shThen, it has to be mentioned that the mitochondrial integrity loss could lead to the formation of autophagic compartments and vacuoles. The formation of these structures is an indicator of autophagy, evidencing the initiation of a mitochondrial-mediated autophagy process . In lineOur findings are in accordance with several in vitro data, showing that SCs provoke cellular stress and elicit mitochondrial dysfunction, leading to apoptosis and cell deaths consistent with autophagy, and neurodegeneration ,44,45,91Therefore, in line with abundant literature, clearly indicating the existence of an intricate control of cell survival/death modulated by oxidative stress, apoptosis and autophagy in SCs-induced injury, our present data support the occurrence of a complex interplay among deathly mechanisms, i.e., apoptosis/autophagy/necroptosis, involved in αPHP-caused toxicity.As regards to the potential αPHP capability to alter DNA and cell cycle, our flow cytometry data indicated that 100 µM αPHP unaffected DNA content distribution in exposed NPSCs, displaying a physiological distribution among the different cell phases . About this data, it has nonetheless to be mentioned that, even though apoptosis can be clearly detected, flow cytometry technique cannot distinctly discriminate other form of cell death, i.e., necroptosis , whose oMoreover, in regard to the hypothesized NPS genotoxic activity, we interestingly revealed that αPHP increased γH2AX expression levels in NPSCs already at a dose of 50 µM, with a further increase at 100 µM. Since the detection of this phosphorylated histone variant has emerged as a highly specific and sensitive molecular marker for monitoring DNA double-strand damage, this finding suggested that αPHP is able to produce a significant genotoxic insult in NPSCs ,96. ThisOverall, the expression levels of all evaluated immunofluorescent markers tended to decrease at the αPHP highest doses, probably due to the massive αPHP-induced cell death/damage/degeneration, which in turn could trigger a complete alteration of physiological cell maintenance mechanisms and related molecules.+/K+-ATPase. Interestingly, apoptotic events [+/K+-ATPase [Lastly, the electrophysiology experiments confirmed the membrane passive and active biophysical properties  of untreated NSPCs already reported in the literature ,98,99. Ic events ,101, succ events , trigger+-ATPase ,103. It +/K+-ATPase-depolarization axis. On the other hand, Na+/K+-ATPase suppression by ouabain significantly enhanced mitochondrial toxin-induced cell apoptosis [Accordingly, the presence of broad-spectrum caspase inhibitor  or, morepoptosis . In agrepoptosis .Therefore, the resting membrane potential of exposed NSPCs could be due to a deep mitochondrial damage, as suggested by TEM ultrastructural analysis and by Caspase 3, BAX, and AIF immunocytochemistry data. We suggest that αPHP could be considered a mitochondrial toxin.Taken together, our current findings demonstrated for the first time to our knowledge that αPHP provokes toxicity in murine NSPCs, causing cell viability loss, apoptosis/autophagy/necroptosis activation, morphological and ultrastructural alterations, and resting membrane potential depolarization. These concentration-dependent cytotoxic effects were measured after 72 h-exposure to increasing αPHP doses (25–2000 μM). Our data demonstrated that αPHP, which are able to damage NPCSs, the key players in the neurogenic niche, may affect adult neurogenesis, possibly triggering long-lasting, irreversible CNS damages. As a needed continuation, our ongoing studies are still assessing whether and how this SC affects differentiated cells, exploring if different effects occurred on diverse cellular subtypes, e.g., neurons, oligodendrocytes, and astrocytes, with the final aim of contributing to broadening the knowledge of SCs toxicology, which is necessary to establish an appropriate treatment for NPS and the potential consequences for public health.