In the last decade, Nerve Growth Factor (NGF)-based clinical approaches have lacked specific and efficient Tyrosine Kinase A (TrkA) agonists for brain delivery. Nowadays, the characterization of novel small peptidomimetic is taking centre stage in preclinical studies, in order to overcome the main size-related limitation in brain delivery of NGF holoprotein for Central Nervous System (CNS) pathologies. Here we investigated the NGF mimetic properties of the human NGF 1–14 sequence (hNGF1–14) and its derivatives, by resorting to primary cholinergic and dorsal root ganglia (DRG) neurons. Briefly, we observed that: 1) hNGF1–14 peptides engage the NGF pathway through TrkA phosphorylation at tyrosine 490 (Y490), and activation of ShcC/PI3K and Plc-γ/MAPK signalling, promoting AKT-dependent survival and CREB-driven neuronal activity, as seen by levels of the immediate early gene c-Fos, of the cholinergic marker Choline Acetyltransferase (ChAT), and of Brain Derived Neurotrophic Factor (BDNF); 2) their NGF mimetic activity is lost upon selective TrkA inhibition by means of GW441756; 3) hNGF1–14 peptides are able to sustain DRG survival and differentiation in absence of NGF. Furthermore, the acetylated derivative Ac-hNGF1–14 demonstrated an optimal NGF mimetic activity in both neuronal paradigms and an electrophysiological profile similar to NGF in cholinergic neurons. Cumulatively, the findings here reported pinpoint the hNGF1–14 peptide, and in particular its acetylated derivative, as novel, specific and low molecular weight TrkA specific agonists in both CNS and PNS primary neurons. Nerve Growth Factor (NGF) is the prototypic neurotrophin primarily described for its ability to sustain survival and induce differentiation of peripheral neurons . Over thNTR) receptor, to regulate the growth, development, survival and repair of the nervous system. TrkA engagement by its ligand induces receptor dimerization and autophosphorylation at the tyrosine residue 490 (Y490), driving the activation of early and downstream signalling molecules, such as 1) phospholipase C-γ (PLC-γ) pathways leading to intracellular calcium release, ion channels and transcription factors expression, 2) neuronal SH2 containing protein (ShcC)-driven phosphatidylinositol 3-kinase (PI3k) activation, which is critical to inhibit c-Jun N-terminal kinase (JNK) induced apoptosis by activating AKT-mediated survival, and 3) mitogen-activated protein kinase (MAPK)/Extracellular Signal-regulated Kinase (ERK) route. MAPK/ERK promote neuronal differentiation and survival [NGF homodimers bind to a multireceptor complex composed of two main classes of receptors, namely the tropomyosin receptor kinase A (TrkA) and the pan neurotrophin p75NTR recepNGF has been implicated in the therapy of a broad range of age-related, neuroinflammatory or genetic neuropathologies, like Alzheimer’s Disease, Parkinson’s Disease, autism, glaucoma, multiple sclerosis, Down’s syndrome, spinal cord injury, traumatic brain injury, and type 2 diabetes ,7,8,9,10Accordingly, several attempts have been made to recapitulate NGF properties by means of short NGF native peptides as well as by designing synthetic products ,12,13. P2+ and Zn2+ metals [The small N-terminal 1–14 sequence of the human NGF (hNGF1–14) has been previously shown to bind TrkA-D5 similarly to NGF through several non-covalent interactions ,36,37. R+ metals ,36.We found that hNGF1–14 retains biological activity of the NGF protein, activating the early and late signalling intermediates of the NGF-TrkA pathway, and finally results in CREB-driven gene transcription and Immediate Early Gene c-Fos nuclear elevation. Among downstream effects, hNGF1–14 sustains ChAT level, excitatory neurotransmitter release, and promotes mature BDNF expression in cholinergic cultures. As a confirmation of its NGF-like activity, hNGF1–14 is also able to promote survival and differentiation of primary dorsal root ganglia (DRG) neurons in the classical bioassay. Overall, present and previous data envisage the in vivo preclinical studies with hNGF1–14 peptides as potent and selective NGF mimetics, and TrkA agonists for CNS and PNS in age-related or inflammation-driven human neurodegenerative diseases.4PIFH8RGESFV-NH2 (monomeric hNGF1–14 peptide), the acetylated monomer Ac-SSS H4PIFH8RGESFV-NH2 (Ac-hNGF1–14 peptide), the (SSS H4PIFH8RGESFC-S)2 hNGF1–15 dimer, and the hNGF1–14 scramble peptide (GFRESPHVSISSH-NH2) with the amidated C-terminal were synthesized at the University of Catania, Italy. Of note, the hNGF1–14 peptides contain two histidine residues (His4 and His8) implicated in TrkA binding and NGF biological activity [The human NGF sequence SSSHactivity . These tTM . The amino acid residues were coupled each other and to the TGR resin , by using 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium tetra-fluoroborate (TBTU) , hydroxybenzotriazole (HOBT) , N,N-diisopropylethylamine (DIEA) . All the reagents were solubilised in N,N-dimethylformamide (DMF). Removal of fluorenylmethyloxycarbonyl protecting group (Fmoc) during synthesis was achieved by means of 20% piperidine solution in DMF.Peptides synthesis, purification, and spectroscopic characterization and docking simulation have been previously described ,38,39. Bv/v) and DIEA (5% v/v) in DMF. The peptides were cleaved off from their respective resins and simultaneously deprotected by treatment with a mixture of trifluoroacetic acid (TFA)/triisopropylsilane (TIS)/H2O (95/2.5/2.5% v/v) for 2 h at room temperature (RT). Solution containing the resin-released peptide was filtered off and concentrated in vacuum at 30 °C. The peptide was precipitated with cold freshly-distilled diethyl ether. The precipitate was then filtered, dried under vacuum, re-dissolved in water and lyophilised.A monomeric derivative was obtained by N-terminal acetylation to remove the positive charge and prevent N-terminal degradation. N-terminal acetylation was performed by treating the fully assembled and protected peptide resins, after removal of the N-terminal Fmoc group, with a solution containing acetic anhydride . NGF protein was purified from rodent submaxillary glands, as already reported ,39,40.Reagents: picrotoxin and strychnine were purchased from Sigma  and were dissolved in ethanol  and water, respectively. Tetrodotoxin  was dissolved in water.Y490 , anti-Trk , anti-pShcY317 , anti-ShcC , anti-pPLC-γ Y783 , anti PLC-γ , anti-pMAPKY202/Y204 , anti-MAPK , anti-pPI3KY458/Y199 , anti-pAKTS473 , anti-AKT (11E7) , anti-pCREBS133 , anti-BDNF , anti-c-Fos , anti-ChAT , and anti-NeuN , anti-MAP2 . Secondary antibodies: anti mouse-HRP and anti rabbit-HRP ; Donkey anti mouse-Alexa546, donkey anti rabbit-Alexa488 .Primary Antibodies: anti-pTrkA Wistar rats  ,42. All 2 (Invitrogen), 87.5 ng/mL 5-fluoro-20-deoxyuridine, 37.5 ng/mL uridine, 50 U/mL penicillin and 50 mg/mL streptomycin (Sigma-Aldrich) at 37 °C in 5% CO2. After 2 days in vitro, DRG cultures were stimulated with 10 μM scramble peptide, 10 μM hNGF1–14 peptide, 10 μM Ac-hNGF1–14 peptide, 10 μM hNGF1–15 dimer or NGF  for 5 days. Unstimulated DIV2 neurons maintained in basal medium conditions for 5 days were considered as control DRG neurons (CTR). After the treatments, neurons were quickly washed with PBS, PFA fixed  and kept in PBS at 4 °C for immunofluorescent staining.Briefly, DRG were collected, incubated for 1 h at 37 °C with 0.125% collagenase (Sigma-Aldrich), mechanically dissociated and plated onto coverslips or Petri dishes pretreated with 10 mg/mL poly-l-lysine (Sigma-Aldrich), at a density of 50,000 cells/well of a 24 well tissue culture plate. The DRG cultures were maintained in low-serum medium, consisting of Dulbecco’s modified Eagle’s medium (DMEM)/F12  supplemented with 0.05% N6 cells on poly-l-lysine (Sigma) coated 35 mm plates  for western blotting analyses and 5 × 104 cells on glass coverslips in 24-wells plates  for immunofluorescence analysis. Cells were grown in Neurobasal (NB) medium supplemented with 2% B27 (Invitrogen) in a 5% CO2 incubator at 37 °C  for 10 days and then used for treatments (DIV10). Cholinergic neurons were treated with murine NGF , 10 μM scramble peptide, 10 μM hNGF1–14 peptide, 10 μM Ac-hNGF1–14 peptide or 10 μM hNGF1–15 dimer in NB medium supplemented with 2% B27. DIV 10 untreated cholinergic neurons were the control neurons (CTR) in this study. After the treatments, neurons were quickly washed with PBS and kept at −20 °C until biochemical analysis or PFA fixed  and kept in PBS at 4 °C for immunofluorescent staining.Cholinergic neurons were harvested from the septal region of E17 Wistar rat embryos, as previously described ,40 and dCultured neurons were digested in a RIPA buffer with “complete protease and phosphatase inhibitory cocktail”  and centrifuged . The supernatants were collected, and the amount of total protein was determined by Quick Start Bradford Dye Reagent. Each sample (40 μg) was separated by SDS-PAGE in precast 4–12% Bis-Tris Plus gels , transferred to nitrocellulose membranes , and incubated for 1 h at RT with 10% non-fat dry milk in TBS-T . The overnight incubation with primary antibody (4 °C) was followed by incubation with the appropriate HRP-conjugated secondary antibody  and the ECL substrate . The films were digitalized using a professional scanner (HP4050) and quantified by gel densitometry using the ImageJ software . Measurements were standardized between the experimental groups using the same calibration system and a fixed threshold over the background. To measure the effect of NGF/peptides on the activation of the pathway, the phosphoprotein level was normalized over the total protein level, with a further normalization over β-actin to correct for sample loading bias . Data arPrimary cultures were fixed for 20′ in PBS containing 4% paraformaldehyde, permeabilised with PBS plus 0.3% Tween, and quenched by ammonium chloride . Aspecific staining by the secondary antibody was blocked by incubation with normal donkey serum . The overnight incubation (4 °C) with primary antibodies was followed by the appropriate fluorescent secondary antibodies . For pAKT/pCREB double immunofluorescence, a mixture of rabbit anti-pAKT (1:500) and mouse anti-pCREB (1:100) antibodies was followed by incubation with a mixture of donkey anti-rabbit Alexa488 and anti-mouse Alexa546 secondary antibodies. The single c-Fos and pCREB immunostainings of cholinergic neurons were performed with rabbit anti-c-Fos (1:500) or rabbit anti-pCREB (1:200) antibodies, followed by anti-rabbit AlexaFluor-488 antibody . MAP2 staining of DRG neurons was achieved by means of mouse anti-MAP2 antibody  followed by anti-mouse Alexa546 antibody . The double pCREB/MAP2 immunostaining of cholinergic neurons were performed by a mixture of rabbit anti-pCREB (1:100) and mouse anti-MAP2 antibodies , followed by incubation with donkey anti-rabbit Alexa488 and donkey anti-mouse Alexa546 antibodies.Nuclei were counterstained with 4′,6-diamidino-2-phenylindole . Then, coverslips were mounted on Superfrost glass slides using the Prolong Gold Antifade Mounting (Life Technologies) and kept at −20 °C before image analysis.Immunofluorescence images were acquired with an epifluorescent microscope  equipped with a CCD camera (Leica) or by confocal microscopy with the laser scanning confocal microscope TCS SP5 (Leica Microsystems) using a 40× (NA = 1.25), and a 63× (NA = 1.4) oil-immersion lens. A UV Diode laser operating at 405 nm, an argon laser at 488 nm, a HeNe laser at 543 nm were used as excitation sources. NeuN widefield mosaic images were acquired with an inverted microscope (DMI6000 Leica Microsystems) equipped with a sCMOS camera  and a white light lamp. Image acquisition was controlled with Leica Application Suite . Mosaics were generated by merging several individual frames, using an automated spatial overlap of 15% with LAS X software . 2; 20× objective) was measured. The analysis was performed after calibrating for particle pixels size (50-400 pixels) and applying a fixed threshold over the background. Nuclei were counted both by manual and automated counting methods (ImageJ) with comparable results. Data are expressed as percentage to control group (CTR) and presented as mean ± SEM.Immunofluorescent images were collected for direct comparison among experimental groups using fixed settings. The number of positive cells over the total number of DAPI stained nuclei per field  for 5′, and immunolabelled for MAP2, as described above. Neurons with neurites connected to the soma, longer than the mean neuronal soma diameter, typically around 25 μM, and with an identifiable axonal arborisation were quantified for a number of parameters. In detail, the number of neurons, total number of neurites, total neurite length , maximum neurite length (length of longest neurite), and total number of branch points were measured and compared by automated cell count by means of ImageJ.A threshold over the background was fixed by applying a contrast-based mask, followed by the calibration for particle pixels size (50–400 pixels) to exclude aspecific pins. Number of cell bodies was automatically counted by Image J, while neurites were drawn and quantified by means of the NeuronJ plugin (ImageJ). d-glucose (pH 7.4 with CsOH). The standard extracellular solution consisted of 130 mM NaCl, 3 mM KCl, 2 mM MgCl2, 1.5 mM CaCl2, 10 mM HEPES, 6 mM d-glucose, and 10 mM tetraethylammonium (TEA) Cl (pH 7.4 with NaOH). Whole cell patch-clamp recordings were performed from primary neurons in cholinergic cultures  to study the excitatory neurotransmission in different culture conditions. The recording pipettes were pulled from borosilicate glass with an outer diameter of 1.2 mm and had open tip resistances of 3–5 MΩ. The internal solution for filling pipettes consisted of 140 mM CsCl, 1 mM EGTA, 10 mM HEPES and 6 mM To isolate the miniature Excitatory Post Synaptic Currents (mEPSCs), 0.5 μM tetrodotoxin (TTX), 5 μM strychnine, 100 μM picrotoxin were added in the bath solution, in order to block voltage-dependent Na+ currents, glycine and GABAA receptors, respectively. Recordings were carried out for 5′ from each neuron and the last 2′ of each recording were analysed.In order to evaluate the effect of Ac-hNGF1–14 peptide on mEPSCs, Ac-hNGF1–14 peptide and NGF were added to the culture medium for 30′ and maintained at 37 °C. Specifically, recordings were obtained from neurons in control condition (Control), following treatment with the Ac-hNGF1–14 peptide  and following treatment with NGF . As negative control respectively for TrkA requirement and sequence specificity, neurons were incubated the TrkA specific inhibitor GW441756  followed by NGF treatment , or with the scrambled hNGF1–14 . The Ac-hNGF1–14 peptide, the scrambled hNGF1–14 and NGF were also added to the extracellular solution, according to the experimental condition.Experiments were performed at 22–24 °C, RT. Recordings were made using a MultiClamp 700B amplifier . pCLAMP 9.2 software was utilized for the data acquisition system . After the formation of a high-resistance seal (>1 GΩ), the capacitance and the resistance of the electrodes were compensated electronically. The whole cell capacitance was assessed online using the Membrane test function of pClamp9.2. Current signals were sampled at 100 kHz and filtered at 3 kHz. Neuronal viability was assessed by the 3--2,5-diphenyltetrazolium bromide (MTT) assay, according to manufacturer’s instructions , and with Trypan blue exclusion assay, as previously described , with coThe MTT assay is based on reduction of MTT to an insoluble formazan product by viable and metabolically active cells. DIV10 neurons, previously seeded on 96-wells microplates, were treated for 1 h with hNGF1–14 peptides at different concentrations , or untreated (CTR) DIV10 neurons were incubated with MTT  in Hank’s balanced salt solution (Life Technologies). The formazan crystals were dissolved in dimethyl sulphoxide (DMSO), and the absorbance at 570 nm wavelength was measured using a Bio-Rad microplate reader . The amount of MTT conversion was evaluated as a percentage of the absorbance measured in treated cells relative to the absorbance of control cells.Alternatively, the Trypan Blue test allows the count of living cells able of cytoplasmic dye exclusion. Briefly, DIV10 neuron seeded on glass coverslips in 24-wells plates and treated for 1 h with hNGF1–14 peptides at different concentrations , or untreated (CTR) DIV10 neurons were incubated with Trypan Blue , and then cleared, and mounted on glass slides. The number of Trypan blue positive dying cells was counted, converted into percentage of surviving cells and reported in the graphs .p value <0.05 was considered statistically significant. The graphs were generated using PRISM , and the data reported as the mean ± standard error of the mean (SEM). One-way ANOVA followed by Student’s t-test or Tukey-Kramer post-hoc was used to analyze the data, depending on the number of variables and groups . A p < 0.05.To analyse miniature Excitatory Post Synaptic Currents (mEPSCs), the 6.0.7 version of Mini Analysis Program  was used. mEPSCs were manually detected using a 8 pA threshold crossing algorithm. Frequency, event amplitude, kinetic characteristics (rise and decay time), and event area were compared in the different experimental conditions. Data were expressed as mean ± SEM. Fitting and statistical analysis were performed using SPSS 17.0.0 for Windows  and Origin 7.0 . Statistical tests were performed by using One-Way ANOVA followed by Bonferroni post-hoc correction. Statistical significance was taken as In order to address hNGF1–14 peptides as NGF signalling agonists, we resorted to a well-established and characterized in vitro method of cholinergic neurons culture ,42,44. WIn detail, cholinergic neurons were incubated with 10 μM scrambled hNGF1–14, 10 μM hNGF1–14, 10 μM Ac-hNGF1–14, and 10 μM hNGF1–15 dimer either for 7′ to study activation of the NGF specific neurotrophic receptor TrkA A,C and ep < 0.01), as well as hNGF1–14 , Ac-hNGF1–14 , and hNGF1–15 dimer . No statistically significant difference was found between the control (87.67 ± 9.50%) and the scrambled hNGF1–14  neurons.The analysis of TrkA activation A,C showep < 0.001), as well as with hNGF1–14 , Ac-hNGF1–14 , and hNGF1–15 dimer . No statistically significant difference was found between control (110.33 ± 6.06%) and scrambled hNGF1–14  neurons.The level of PLC-γ A,D was sp < 0.0001), as well as with hNGF1–14 , Ac-hNGF1–14 , and hNGF1–15 dimer . No statistically significant difference was found between control (CTR: 98.00 ± 1.16) and scrambled hNGF1–14  treated neurons.Also, the activation of pShc A,E was up < 0.01), as expected. Interestingly, also hNGF1–14 , Ac-hNGF1–14 , and hNGF1–15 dimer  were able to stimulate MAPK, as compared to control (CTR: 109.67 ± 6.12%).As for downstream signalling molecules, MAPK phosphorylation B,F was ap < 0.05), as well as with hNGF1–14 , Ac-hNGF1–14 , and hNGF1–15 dimer . No statistically significant difference was found between control (CTR: 100 ± 0%) and scrambled hNGF1–14  neurons.Also pPI3K level B,G increp < 0.01), as expected, as well as by the hNGF1–14 , and Ac-hNGF1–14 . No statistically significant difference was seen upon treatment with the hNGF1–15 dimer . The scrambled hNGF1–14 didn’t exert any effect on pAKT level , by comparison with control neurons (84.64 ± 9.29%).The survival factor AKT B,H was sp = 0.606, 5 μM: p = 0.427, 10 μM: p = 0.934, 50 μM: p = 0.597, 100 μM: p = 0.869), hNGF1–14 , Ac-hNGF1–14  and hNGF1–15 dimer  peptides. At 10 μM concentration the peptides fully exploit a NGF-like action  NGF, or 10 μM hNGF1–14, or 10 μM Ac-hNGF1–14, or 10 μM hNGF1–15 dimer, and number of pCREB A,B and pp < 0.05) and pAKT  positive cholinergic neurons (p < 0.01) and hNGF1–15 dimer , but not hNGF1–14  and scrambled hNGF1–14  augmented the number of pCREB positive neurons. Further, we observed that cholinergic neurons expressing pAKT were significantly increased also upon hNGF1–14 , Ac-hNGF1–14 , and hNGF1–15 dimer  incubation (p = 0.811) was not able to affect the number of pAKT positive neurons, as compared to control (100.00 ± 3.59%). To ensure that increase in activated neurons is a measure of NGF or peptide stimulations, and not the result of increased number of neurons, total NeuN positive neuronal nuclei were counted and compared (p = 0.922), hNGF1–14 , Ac-hNGF1–14 , hNGF1–15 dimer , and scrambled NGF1–14 , as compared to control conditions (99.26 ± 4.51%).We observed that NGF augmented the percentage of pCREB  also express MAP2 (red colour), pinpointing that CREB activation upon NGF or peptides incubation mainly occurs in neurons.Since cholinergic cultures are not pure neuronal cultures pCREB and MAP2 double immunofluorescent labellings were performed, in order to exclude any glial contribution to the neurotrophic effect. As shown in To evaluate the ability of hNGF1–14 peptides to elicit neuronal activity of cholinergic neurons, we incubated cholinergic neurons for 60′ with 10 μM scrambled hNGF1–14, or 100 ng/mL (3.84 nM) NGF, or 10 μM hNGF1–14, or 10 μM Ac-hNGF1–14, or 10 μM hNGF1–15 dimer. The number of nuclei positive to the immediate early gene (IEG) c-Fos, an established marker of cellular activation upon an acute stimulus was counted and compared among experimental groups A,B.p < 0.0001) versus control (109.44 ± 11.38%). A comparable effect was observed following treatment with hNGF1–14  and Ac-hNGF1–14 , and scrambled hNGF1–14 .NGF incubation exerted a stimulating effect on nuclear c-Fos level in cholinergic neurons , as well as by Ac-hNGF1–14 , and hNGF1–15 dimer . No statistically significant difference was found between the hNGF1–14 , or the scrambled hNGF1–14  and control neurons (119.56 ± 10.24%).We also investigated the amount of mature BDNF (~14 kDa) by western blotting following NGF and hNGF1–14 peptides C,D. We fp < 0.05), hNGF1–14 , Ac-hNGF1–14 , and hNGF1–15 dimer  but not scrambled hNGF1–14  significantly stimulate ChAT level , Ac-hNGF1–14 , hNGF1–15 dimer , and NGF  treated neurons pre-incubated with the TrkA inhibitor GW441756  and then stimulated with 10 μM hNGF1–14, 10 μM Ac-hNGF1–14, 10 μM hNGF1–15 dimer or 100 ng/mL (3.84 nM) NGF. Peptides or NGF stimulation incubation lasted for 30′ for subsequent anti-pCREB A, or forGW441756 A.p = 0.259), Ac-hNGF1–14 , hNGF1–15 dimer , and NGF  treated neurons after GW441756 pre-treatment was comparable to control untreated 111.21 ± 13.23%) neurons  phosphorylation upon cholinergic neurons incubation with GW441756  followed by stimulation with hNGF1–14 , Ac-hNGF1–14 , hNGF1–15 dimer , and NGF  as positive control. Preincubation with GW441756 inhibited TrkA activation by NGF, as well as by hNGF1–14 peptides .To verify the status of excitatory neurotransmission in cholinergic neurons exposed to Ac-hNGF1–14 peptide, we have studied the miniature excitatory post synaptic currents (mEPSCs) in DIV10 control exposed or not to Ac-hNGF1–14 peptide and NGF.p < 0.001) showed mEPSC frequency (p < 0.0001), and comparable to that measured in neurons exposed to NGF , suggesting a presynaptic neurotransmitter release facilitation induced by both NGF and hNGF1–14 peptide.requency B signifip = 1; GW441756 + NGF: 0.65 + 0.06 Hz; p = 1), amplitude , rise time , decay time , and area .As control for the peptide sequence specificity and for TrkA-dependent NGF effect, the electrophysiological activity of scrambled hNGF1–14  and of NGF  in presence of the TrkA inhibitor GW441756  were analysed in another set of experiments. Any statistical significance was found for mEPSC frequency , hNGF1–14 , Ac-hNGF1–-14 , hNGF1–15 dimer  were statistically higher than CTR (16.00 ± 1.58%). The scrambled peptide did not exert any effect .Number of surviving neurons B in NGF p < 0.0001), hNGF1–14 , Ac-hNGF1–14 , hNGF1–15 dimer , but not scrambled peptide , were able to promote neurite elongation, as compared to CTR (4.57 ± 0.5%). The maximum neurite length , as well as of hNGF1–14 , Ac-hNGF1–14 , hNGF1–15 dimer  and comparable to, as compared to control CTR . Scrambled peptide did not exert any neurotrophic effect . Also, the number of total branch points was evaluated (p < 0.0001), hNGF1–14 , Ac-hNGF1–14 , and hNGF1–15 dimer . Notably, the number of branch points in the control condition and upon scrambled peptide incubation was equal to zero. The scrambled hNGF1–14 did not exert any neurotrophic action, resulting in lack of neurite elongation and reduced survival, with a trend comparable to control.Assessment of total neurites per field C showed e length D and tote length E were alvaluated F, as a mThe aim of this study was to evaluate in vitro efficacy of hNGF1–14 and its derivatives as potential drugs for in vivo neuroprotection, of interest for novel therapeutic approaches in neurodegenerative pathologies. Given the limitation of immortalized cell lines studies, to test NGF mimetic activity of the hNGF1–14 sequence we resorted to two types of well-known NGF-target primary neurons, as cellular paradigm of choice.In this study, we investigated and compared the hNGF1–14, its monomeric derivative peptide Ac-hNGF1–14, and the hNGF1–15 dimer, using NGF as positive and a scrambled peptide as negative controls. Here, we report that the Ac-hNGF1–14, hNGF1–14 and, to some extent, the hNGF1–15 dimer trigger a TrkA-dependent NGF-like signalling cascade in cholinergic neurons  and showThe rationale for using the N terminal sequence of the human NGF is mainly based on the biological implication of the N terminal domain for NGF-TrkA binding and pathway activation ,52,53. ITo deeply assess their ability to stimulate NGF/TrkA signalling and NGF typical bioactivity, we resorted to two elective cellular models of NGF-target neurons of the central (CNS) and peripheral (PNS) nervous systems, the cholinergic and DRG primary neurons, respectively. The cultured cholinergic neurons do not need NGF to survive, thus being a good paradigm to test the short-term effect of an acute NGF stimulus, e.g., signalling pathway and neuronal activity. Opposite, since DRG neurons require NGF/TrkA continuous supply to survive and differentiate, they represent the model of choice for in vitro survival assays with putative NGF mimetics.Here we show that the monomeric (hNGF1–14) acetylated monomeric (Ac-hNGF1–14) and dimeric hNGF1–15 peptides present NGF typical signalling properties, being able to stimulate its specific receptor TrkA, its early adaptors ShcC and PLC-γ, as well as its downstream PI3K and MAPK signalling molecules, finally leading to activation of survival promoting AKT pathways . Taken tp < 0.01), PI3K (p < 0.01) and AKT (p < 0.01), as compared to the optimal NGF dose . Also, we demonstrated that hNGF1–14 peptides mimic NGF in engaging an increased number of neurons responding by activation of key signalling intermediates, like CREB and AKT . Our data are in line with previous works indicating that NGF specifically affects cholinergic neurons in culture [Albeit glial cells are a minor component of embryonic septal cultures ,  and low  culture . OverallAs a result of CREB activation, a crucial hub in neurotrophic responses , the hNGFurther, our findings are in line with the previously reported increase of BDNF mRNA level and BDNF release upon hNGF1–14 peptides incubation of PC12 cells ,36. Of n50 = 2 nM) with more than 100-fold selectivity over other kinases, thus allowing the highly specific targeting of the NGF/TrkA pathway. The lack of hNGF1–14 effect in presence of the inhibitor strongly suggests that an active TrkA receptor is required for its CREB/c-Fos related neurotrophic action in cultured cholinergic neurons.Interestingly enough, the peptidomimetics property of hNGF1–14 peptides was abolished by previous incubation with the TrkA specific inhibitor GW441756 . As compared to other general tyrosine kinase receptors inhibitors used so far, like K252a and synthetic derivatives , the GW441756 is a potent TrkA inhibitor  , by augmSince TrkA activation is known to occur by NGF dimers binding, dimeric or cyclised peptidomimetics have been generally preferred to facilitate TrkA conformational rearrangement and subsequent autophosphorylation ,72. NoneInterestingly enough, peptidic as well as non-peptidic monomeric compounds, like D3, have been shown to induce TrkA dimerization and subsequent DRG differentiation . The exaTaking all these findings into account, preferring primary neurons to test hNGF1–14 peptides allowed full disclosure of their NGF-mimetic potential. At variance with studies performed on PC12 cell lines ,36, micrOverall, the findings here reported establish hNGF1–14 sequence as a linear monomeric TrkA agonist with full NGF-like bioactivity on NGF-target neurons. Further assessment of their efficacy as neuroprotective drugs in animal models of human CNS neurodegenerative pathologies is underway.The drawbacks and limitations of previous clinical trials and NGF-based therapeutic approaches also in recent times, paved the way for the screening of novel, potent and selective NGF mimetics. Nowadays, the characterization of novel small peptidomimetic is taking centre stage in order to overcome poor oral NGF bioavailability, size-related limitations of NGF brain delivery, as well as pain related side-effects of peripheral NGF administration for spinal cord injury or diabetic neuroinflammation. The use of small peptidic molecules or non-peptidic compounds with NGF activity is instrumental to non-invasive brain treatment by means of nasal ,75 and o


Objectives were to evaluate effects of ouabain-induced inhibition of Na+, K+-ATPase in equine blastocysts. Sixteen mares were ultrasonographically monitored, given deslorelin acetate to induce ovulation, and inseminated. Embryos (D7 and D9) were harvested and Na+, K+-ATPase inhibited for 1 or 6 h by exposure to 10-6 M ouabain, either natural ouabain or conjugated to fluorescein (OuabainFL), during incubation at 37° C. Evaluations included morphometric characteristics (bright field microscopy) and viability (Hoescht 33342 + propidium iodide). Blastocysts incubated for 6 h in Holding medium + ouabain (n=3) had, on average, a 45.7% reduction in diameter, with adverse morphologic features and no re-expansion after subsequent incubation in Holding medium for 12 h. In subsequent studies, even a 1-h exposure to Ouabain or OuabainFL, caused similar reductions, namely 38.7 ± 6.7% (n=5) and 33.6 ± 3.3% (n=7) for D7 and D9 blastocysts, respectively. Ouabain binding was confirmed after OuabainFL exposition and all embryos (n=12) lost viability. We concluded that Na+, K+-ATPase inhibition with ouabain caused death of equine blastocysts and therefore was not a viable method of reducing blastocyst size prior to cryopreservation.Although equine blastocysts ≤ 300 µm in diameter can be successfully vitrified, larger equine blastocysts are not good candidates for cryopreservation. As Na Cryopreservation of equine embryos has been a challenge due to capsule development, mitotic activity and in particular, embryo size . VitrifiThe first foal born after vitrification was reported by +, K+-ATPase activity, promoting embryo cavitation and modifying ionic gradients on trophectoderm epithelium, interfering with formation, distribution, and permeability of tight junctions (TJ) between trophoblastic cells . Sixteen mares and one Margalarga Marchador stallion, located in Viçosa-AL, Brazil  were used. These horses were raised in semi-extensive conditions, with free access to good quality water and grass, plus supplementation with 4 kg concentrate/head/d. All horses were deemed breeding sound and there was no indication of any reproductive disorder. Ovarian follicular growth and uterine characteristics of mares were monitored every 2 d, with transrectal ultrasonography. Mares in estrus with follicles > 35 mm and uterine edema grade 3 0-5;  were giv8 motile sperm, using routine procedures  covered by sterile mineral oil (200 µl), and kept for 1 h at 37 °C for equilibration. Thereafter, embryos were morphologically evaluated  using an+, K+-ATPase specific inhibitor , under the same initial incubation conditions. Thereafter, blastocysts were withdrawn from ouabain-treated medium and incubated for 12 h in Holding media, to assess embryo re-expansion. During this interval, blastocysts were photomicrographed within 1 h and subsequent morphological and morphometric evaluations performed.In total, 17 embryos were recovered, all classified as Grade I. Two blastocysts D7 were incubated only in Holding medium for 24 h at 37 °C to evaluate the influence of culture system on embryo quality. Two D7 and one D9 blastocysts, after initial incubation in Holding medium (1 h), were transferred and maintained for 6 h in Holding medium + Na+, K+-ATPase inhibition and assess embryos using fluorescent probes. Propidium iodide , Hoechst 33342 , fluorescent-conjugated ouabain  and ouabain (10-6 M) were used after dilution in Holding medium. Analyses were performed using epifluorescence microscopy (Feldmann Wild Leitz- 3500T FL), with excitation/emission filters of 533/617, 361/497, 503/512 for PI, Hoechst 33342 and Bobipy™ FL ouabain (OuabainFL), respectively.Based on results of the first experiment, it was decided to evaluate shorter intervals of NaSix D9 blastocysts and one D7 were incubated  in Holding + ouabain, followed by Hoescht and PI staining to evaluate membrane integrity of trophoblastic cells. Moreover, to confirm ouabain-embryo binding, four D7 and one D9 blastocysts were incubated  in Holding + OuabainFL + Hoechst, followed by IP staining. Before and after incubation, blastocysts were photomicrographed in bright field, to assess morphology and diameter.t-test, with data presented as means ± SEM. Results were considered significant when P<0.05.Results of the present study are predominantly presented using descriptive statistics. Average diameter reduction (%) of D7 and D9 blastocysts exposed to either ouabain or ouabainFL were compared after normality analysis (Shapiro-Wilk test), followed by Student’s -6 M) during 6 h reduced their diameter 35.9, 48.7 and 52.5% , with no subsequent re-expansion after up to 12 h in Holding medium  of blastocysts exposed to ouabain or ouabainFL were positive for PI staining after incubation , with further re-expansion in ouabain-free medium in a pilot study, although that was not observed in the present study.Exposure of 3 blastocysts to ouabain for 6 h, resulting in blastocoel collapse and no subsequent re-expansion, even after 12 h incubated in Holding medium (without ouabain), indicated that Na+, K+-ATPase inhibitor, reduced diameter of D7 and D9 equine blastocysts, presumably by interfering with fluid exchange through the trophectoderm, similar to a previous report with D-9 equine embryos  or β  subunits that comprise the protein  was expected to inhibit the α1 subunit, which comprises Na+, K+-ATPase isoform less sensitive to ouabain  and briefer exposure to ouabain (1 h), and embryonic stage (D7 or D9 blastocyst), there was a reduction on blastocyst diameter and adverse effects on morphology after Namembrane , reinfor+, K+-ATPase resulted in increased intracellular Na+ concentrations, inhibition of Na+, Ca2+ ionic channels and an elevation of intracellular Ca2+ and AMPc, compromising E-cadherin/catenin system, leading to TJ disruption and impairment of cell membrane polarization control  as well as induced trophectoderm membrane damage.Blastocysts expand due to continuous fluid movement through trophectoderm, although that does not happen until tight junctions (TJ) are fully developed and functional between trophectoderm cells, maintaining blastocoel fluid and epithelial integrity . In prev control . We infe+, K+-ATPase with ouabain caused embryonic death and therefore, this approach is not a viable option for chemical reduction of equine blastocyst size prior to vitrification. Future studies, at molecular levels, should be undertaken to evaluate effects of Na+, K+-ATPase inhibition on tight junction structure and function in equine embryos.Inhibition of Na


Pueraria lobata (Wild.) Ohwi. (P. lobata) flowers known as ‘Kudzu flower’ contain isoflavonoids and essential oil components. They have a wide range of biological and pharmacological activities, including protective effects against non-alcoholic fatty liver disease, hyperglycemia, and hypolipidemia, anti-mutagenic effects, and benefits for weight loss. However, the molecular mechanism of these effects remains unclear. Our study aimed to systematically examine the effects of flos puerariae crude extract (FPE) as an anti-diabetic agent using in vitro assays. The cytotoxicity of FPE was evaluated using MTS assay in L6 rat myocyte and 3T3-L1 murine fibroblast cell lines. PPARγ binding activity and adipogenesis were examined using dual-luciferase and differentiation assays, respectively. For investigating the anti-diabetic activity, glucose utilization, including GLUT4 protein expression, glucose uptake assay, and GLUT4 translocation using immunofluorescence microscopy were conducted in L6 cells. Furthermore, we assessed the antioxidant and anti-inflammatory activities of FPE. Our results demonstrated the ability to augment glucose uptake in L6 cells and enhance glucose utilization activity by increasing the expression of glucose transporter type 4 (GLUT4). In summary, our findings suggest that FPE may be a potential anti-diabetic substance for the treatment of diabetic patients and can prevent inflammatory or oxidation-related diseases. Diabetes mellitus is a metabolic condition with a rapidly increasing prevalence worldwide, currently affecting hundreds of millions of people. This multifactorial disease is characterized by hyperglycemia, insulin deficiency, insulin resistance, and/or abnormal insulin secretion. Diabetes can be classified into four categories: type 1 diabetes (T1DM), type 2 diabetes (T2DM), gestational mellitus (GDM), and other specific types of diabetes due to other causes. T2DM is characterized by impaired insulin-mediated glucose clearance in the skeletal muscle, but not always accompanied by dysregulation of hepatic glucose production by insulin. Insulin has dual roles in controlling postprandial glycemia response to a meal and activation of glucose transport into skeletal muscle and adipose tissue, both of which are impaired in T2DM .DPPH  and ABTS  were used for the radical scavenging assay as previously described . The abs50 value, the half of the concentration of the sample that can scavenge 50% of the free radicals, were calculated.The vitamin C Equivalent Antioxidant Capacity (VCEAC) in mg/g dry weight extract and ICp < 0.05, ** p < 0.01, and *** p < 0.001. For screening of PPARγ ligand activity, analysis was performed using Dunnett’s multiple comparison test.Data are expressed as mean ± SD or SEM from at least three independent experiments. Statistical analyses were performed using the one-way ANOVA test. All comparisons were made relative to untreated controls and significant differences have been indicated as * To identify plant extracts exhibiting PPARγ binding activity, the selected plants extracts were tested for their effects on the luciferase activity of a construct containing three copies of PPRE linked to luciferase (pPPRE3X/tk/Luc) A. HCT-11Firstly, we examined if FPE exhibits toxicity on the growth of 3T3-L1 preadipocytes. No cytotoxicity was observed upon maintaining 3T3-L1 cells in growth medium containing 100 μg/mL FPE A. Next, L6 cells were treated with FPE to determine its cytotoxicity on the cells. After treatment with 100 μg/mL FPE for up to three days, there was no significant difference with respect to cell growth, indicating that FPE is a safe extract for L6 cells A. The ef50 of FPE was 882.267 ± 52.726 µg/mL and the percentages of DPPH scavenging activity were 9.361 ± 2.000, 16.052 ± 3.389, 28.618 ± 6.374, 48.905 ± 8.638, and 57.363 ± 9.587 for 62.5, 125, 250, 500, and 1000 µg/mL FPE, respectively  is a key molecule that increases the inflammatory status of cells leading to pathophysiological conditions by increasing prostaglandin formation. It also leads to excessive formation of reactive oxygen species . PersistThere are various molecular targets for T2DM treatments such as the IRS1/PI3K/Akt pathway, AMPK pathway, and the GLP-1 pathway. Our study showed that FPE activates the PPARγ nuclear receptor as a ligand and exhibits anti-inflammatory and antioxidant activities upon treatment in cells. It also affects glucose metabolism by increasing GLUT4 expression B. Overal


Escherichia coli that yields a highly pure, active and homogeneous single isoenzyme. We successfully developed a multi-step inclusion body process giving a final yield of 960 mg active HRP/L culture medium with a purity of ≥99% determined by size-exclusion high-performance liquid chromatography (SEC-HPLC). The Reinheitszahl, as well as the activity with 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) and 3,3′,5,5′-tetramethylbenzidine (TMB) as reducing substrates, are comparable to commercially available plant HRP. Thus, our preparation of recombinant, unglycosylated HRP from E. coli is a viable alternative to the enzyme from plant and highly interesting for therapeutic applications.Horseradish peroxidase (HRP), an enzyme omnipresent in biotechnology, is still produced from hairy root cultures, although this procedure is time-consuming and only gives low yields. In addition, the plant-derived enzyme preparation consists of a variable mixture of isoenzymes with high batch-to-batch variation preventing its use in therapeutic applications. In this study, we present a novel and scalable recombinant HRP production process in  Armoracia rusticana), where it is glycosylated usually at eight of nine asparagine sites [Escherichia coli (rHRP) it is unglycosylated resulting in a size of only 34.5 kDa [A. rusticana has a large variety of isoforms, with 34 different entries in the UniProtKB database (April 2020) [E. coli and therefore well suited for medical applications, as foreign glycosylation patterns can cause immune reactions in humans [E. coli follows two major routes: the protein of interest can either be soluble or insoluble in the form of protein aggregates, also known as inclusion bodies (IBs). In the E. coli cytoplasm, HRP forms IBs because the absence of glycans on the protein surface results in enhanced hydrophobicity and the disulfide bridges cannot be formed. Both production strategies pose several advantages as well as disadvantages and are dependent on protein properties as well as cultivation parameters [Horseradish peroxidase (HRP) EC 1.11.1.7) has high industrial relevance entailing an immense pool of published data, ranging from refolding of denatured plant enzyme to recombinant production in different hosts .11.1.7 h,4,5,6,7.ne sites ,10. Due il 2020) ,13,14. Til 2020) ,18,19,20il 2020) ,22,23. Iil 2020) ,27,28,29il 2020) ,31 or tail 2020) ,33. Howerameters ,36. Whilrameters ,38. Howerameters . In generameters , where aAsad et al.  publisheE. coli IBs with upscaling potential to industrial dimensions. Moreover, we assume that this procedure can be transferred to other peroxidases of the plant peroxidase superfamily, as all enzymes of class II and III are monomers and contain a non-covalently bound heme, two calcium ions and four disulfide bridges. As shown in In this study we present a novel production process for HRP from E. coli inclusion bodies using a series of multivariate experiments and an integrated approach. In this study, we developed a novel production process for recombinant HRP from In order to identify the optimal redox system for solubilization and refolding, a DoE for DTT and GSSG concentration was performed (“DoE 1”). During solubilization all disulfide bridges should be reduced, granting the complete solubilization of the protein. In the subsequent refolding step, the correct disulfide bridges have to be formed, requiring an oxidizing environment. We hypothesized that these two unit operations are dependent on each other, as the DTT concentration had an influence on the refolding yield even if the solubilization yield stayed constant. In order to provide an integrated approach, the final refolding yield (determined as the volumetric activity) was used as a response for both DTT and GSSG concentration. For the used dilution of 1:40 solubilization mix in refolding buffer, this is equivalent to a GSSG:DTT ratio of 7:1 in the refolding mix. The interaction of DTT and GSSG is clearly visible, with increasing DTT requiring increasing GSSG concentrations. This shows the importance of optimizing the redox system in an integrated approach including solubilization and refolding and using the final refolding yield as response. Note that the absolute values of DTT and GSSG presented here might vary depending on the properties of the IBs, which are influenced by several factors, such as fermentation conditions  and unit operations performed before solubilization .Based on these results, a second DoE (“DoE 2”) was performed, using DTT, GSSG and protein concentration as factors, and volumetric activity (U/mL) and specific activity (U/mg) as responses. These responses were chosen since lower protein concentrations usually lead to higher refolding yields . HoweverAsad et al.  previousThe experiments in DoEs 1–3 were all performed in 2 mL reaction tubes, resulting in a maximum volumetric activity of 56 U/mL after refolding. In order to identify possible scale-up effects, these optimized conditions were scaled up to a refolding vessel with a volume of 1200 mL (“refolding vessel experiment 1”), resulting in a higher volumetric activity of 84 U/mL. Due to the tendency of protein aggregation during refolding, we assumed that favorable stirring conditions and surface to volume ratio led to this increase in activity for the upscaled process. Overall, the presented optimization approach resulted in a scalable and robust process with high refolding yields.The concentration and time of hemin addition is another important parameter during refolding. Hemin has to be supplied in order to obtain holo-HRP; however, it is not necessary for the correct formation of apo-HRP. Previous studies showed that higher refolding yields could be achieved if hemin was added after the refolding step ,42,44,63A DoE approach (“DoE 4”) was chosen for the small-scale experiments, using the concentration of hemin as well as the time of hemin addition as factors. The volumetric activity (U/mL) after refolding was used as the response . When heThe optimal conditions were transferred to a bench-scale refolding vessel with a refolding volume of 1200 mL (“refolding vessel experiment 2”). For this experiment, hemin was added after 20 h of refolding to a final concentration of 20 µM. Additionally, the refolding kinetics was monitored during this experiment. Samples were taken every 2 h to measure the activity at-line . HoweverBased on these experiments, we assumed that a linear hemin feed starting after complete apo-enzyme formation might further improve the refolding yield. The hypothesis was that hemin is prone to self-aggregation as well as aggregation with incorrectly folded protein, leading to a reduced amount of hemin available to form holo-HRP. Moreover, hemin aggregates could potentially impede further downstream applications, e.g., through irreversible binding to chromatography columns. A steady supply of small amounts on the other hand can immediately be used for holo-enzyme formation while aggregates are kept at a minimum. Therefore, in “refolding vessel experiment 3” a linear feed commencing 8 h after refolding starts with a total feeding time of 12 h was applied. Again, samples were taken every two hours .At the beginning of the hemin feed, the volumetric activity , whereasAll results presented above were done at pH 8.5, since we performed these experiments after optimizing the redox system, but before, we identified pH 10 as most beneficial during refolding . TherefoThe last unit operation investigated in this study was the capture step after refolding. Several different approaches, including IMAC, CEX and SEC, have been reported thus far ,41,42,444)2SO4 and NaCl for protein precipitation with the goal of reaching efficient impurity separation and high HRP recovery in the supernatant at the same time. Due to their position in the Hofmeister series, the concentration was varied between 0 M and 1.5 M for (NH4)2SO4 and 0 M and 4 M for NaCl, respectively. The recovery of the volumetric activity after protein precipitation was 96% for 1 M (NH4)2SO4 and 95% for 4 M NaCl. Specific activity increased 2.5-fold for (NH4)2SO4 and 4.5-fold for NaCl . When refolding was done at pH 10, the refolding mix was brought to pH 8.5 before 4 M NaCl were added for salt precipitation. After centrifugation, the HRP solution was captured on a HiTrap Butyl FF column and eluted at 75% buffer B .E. coli cultivation broth. The produced recombinant HRP had a very high purity (≥99%) and the specific catalytic activity with ABTS as substrate was around 1500 U/mg for both pH 8.5 and pH 10.E. coli inclusion bodies.Finally, the kinetic parameters of the commercially available plant HRP from Sigma-Aldrich  were compared to refolded HRP rHRP; . In factE. coli inclusion bodies are summarized in The final process steps to prepare recombinant HRP from GSSG was purchased from AppliChem . ABTS was purchased from AppliChem or Sigma-Aldrich . Plant HRP Type VI-A (Cat. No.: P6782), hemin  and TMB were purchased from Sigma-Aldrich. DTT and all other chemicals were purchased from Carl Roth .hrp gene coding for HRP variant C1A was codon-optimized for E. coli and obtained from GenScript USA Inc. . The plasmid pET21d+ was used for HRP IB production in the cytoplasm. A stop codon was introduced so that the protein was produced without any tags. HRP was produced in E. coli BL21(DE3) in a 10 L Biostat Cplus stainless steel bioreactor . The pre-culture was grown in 0.5 L DeLisa medium [sq) was 0.333 g/g/h, which was set to 0.25 g/g/h after induction with 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG). After an induction phase of 8 h, the biomass was harvested by centrifugation and stored at −20 °C until further processing. All data collection and control of the process was done using a process information management system . All IBs used in this study were produced in one fermentation run.The a medium  at 37 °CThe biomass was resuspended using an IKA T10 basic ULTRA-TURRAX  in 5 mL buffer A/g wet biomass ) and homogenized at >1200 bar, three passages, cooled, using a GEA Niro Soavi Panda PLUS . The homogenized suspension was centrifuged , the supernatant discarded and the cell debris resuspended in 10 mL buffer B/g wet cell debris  and centrifuged again . This washing step with buffer B was performed twice. Afterwards, the pellet was resuspended in water (5 mL water/g wet cell debris), the suspension aliquoted into pre-weighed 50 mL reaction tubes, centrifuged  and the pellets were stored at −20 °C.For solubilization, an aliquot of the washed and frozen IBs was thawed, the wet inclusion body (wIB) weight was determined and the pellet was resuspended in the appropriate solubilization buffer to reach a wIB concentration of 100 g/L . After resuspension, DTT was added to reach a final concentration in the solubilization mix of 1 mM–28.44 mM and the solubilization mix was incubated (room temperature (RT); 0.5 h; slight agitation), followed by centrifugation . The supernatant was immediately used for refolding, and the pellet was discarded.2; 7% v/v glycerol; varying GSSG concentrations; refolding buffer 2: 20 mM glycine; pH 10; 2 M urea; 2 mM CaCl2; 7% v/v glycerol; varying GSSG concentrations), followed by incubation at 4 °C; 48 h; with slight agitation. A 1 mM hemin stock solution was prepared in 100 mM potassium hydroxide (KOH).Small-scale experiments were performed using DoE approaches. Planning and analysis of DoEs were done using Umetrics MODDE 10 . All small-scale refolding experiments were performed using 2 mL reaction tubes. The solubilisate was diluted 1:40 in the appropriate refolding buffer  and the GSSG concentration in the refolding buffer  were varied . A CCF wFor the second DoE, the DTT concentration for solubilization , the GSSG concentration in the refolding buffer  and the protein concentration in the refolding mix were varied . A CCF wFor the third DoE, the DTT concentration for solubilization, the GSSG concentration in the refolding buffer and the pH of the solubilization and refolding buffer were varied . For pH For this DoE, time and concentration of hemin addition were varied between 0–24 h after refolding start and 6–80 µM hemin, respectively . SolubilFor bench-scale refolding an Infors Labfors 5  with a vessel volume of 3.6 L was used. All data collection and control of the process was done using Lucullus . Temperature was kept constant at 10 °C during refolding using a Lauda Alpha R8 thermostat  connected to the double jacket of the glass vessel. The temperature was monitored using a sensor connected to the Infors Labfors 5. In addition, pH, dO2 (dissolved oxygen) and redox potential were monitored. The redox potential was monitored using a Hamilton EasyFerm Plus ORP Arc 425 . The hemin feed was applied using a LAMBDA PRECIFLOW peristaltic liquid pump  in combination with a Sartorius Entris scale , enabling a PID-Feed forward control using Lucullus. Final refolding volumes for the vessel were kept constant at 1200 mL (30 mL solubilizate and a dilution of 1:40).For this experiment, the solubilization mix contained 7.11 mM DTT and the refolding buffer contained 1.27 mM GSSG. Solubilization buffer 2 (pH 10) and refolding buffer 2 (pH 10) were used. Hemin was added 20 h after refolding start to a final concentration of 20 µM and then incubated for another 5 h, resulting in a total refolding time of 25 h.For this experiment, the solubilization mix contained 7.11 mM DTT and the refolding buffer contained 1.27 mM GSSG. Solubilization buffer 1 (pH 8) and refolding buffer 1 (pH 8.5) were used. Hemin was added 20 h after refolding start to a final concentration of 20 µM. Samples were taken every 2 h, hemin was added to a final concentration of 20 µM , the samples were incubated , and then enzyme activity was measured.For this experiment, the solubilization mix contained 7.11 mM DTT and the refolding buffer contained 1.27 mM GSSG. Solubilization buffer 1 (pH 8) and refolding buffer 1 (pH 8.5) were used. A constant feed  was applied 8 h after refolding start until 20 h (12 h feed time). Samples were drawn every 2 h and activity was measured. After the start of the hemin feed, samples were measured both directly (with a low hemin concentration at the start of the hemin feed), and again after the addition of hemin to a final concentration of 20 µM hemin and incubation for 2 h.4)2SO4 and NaCl. Several concentrations of both salts were tested in 2 mL reaction tubes (data not shown). The results obtained for the small-scale experiments were then validated using bench-scale experiments with a volume of 150 mL. The salt was slowly added under continuous stirring within 10 min, the solution was then incubated while stirring for 20 min at RT and then centrifuged . The capture step was developed in five HIC experiments using different resins, pH values and elution profiles  was used. Three wavelengths  as well as the conductivity were monitored. Two different salts were tested for the preceding precipitation step, namely  and 49 mL load were applied. After the load, a washing step with 8 column volumes (CV) buffer A was performed. Thereafter, a step elution was performed with 25% buffer B , 75% buffer B (10 CV) and 100% (17 CV) buffer B. Volumetric enzyme activity (U/mL) and protein concentration were measured for all fractions. The purity of the active pool was determined using SEC-HPLC and the Reinheitszahl.The load was prepared by adding 267 g NaCl/L refolding mix. A 1 mL HiTrap Butyl FF column  was used with a flow rate of 75 cm−1·h−1 with buffer A  and 751 mL load were applied. After the load, a wash step with 20% buffer B (20 mM Bis-Tris pH 7/1.5 CV) was performed. Thereafter, a step elution was performed at 75 cm−1·h−1 with 75% buffer B (3 CV) and 100% (3 CV) buffer B. Volumetric enzyme activity (U/mL) and protein concentration were measured for all fractions. The purity of the active pool was determined using Rz.After refolding at pH 10, the pH was adjusted to 8.5 with 2 M HCl under stirring. Afterwards, 267 g NaCl/L refolding mix were added. A column packed with 80 mL Butyl Sepharose 4 Fast Flow  was used. The column was equilibrated at a flow rate of 90 cmv/v glycerol). 170 µL of ABTS solution (5 mM ABTS in 50 mM KH2PO4 pH 5) were mixed with 10 µL of diluted sample in the well, after which 20 µL of hydrogen peroxide  were added to start the reaction. Immediately afterwards, the change of absorption at 420 nm over 2 minutes was recorded (at 30 °C). The volumetric enzyme activity was calculated using the following Equation (1):Enzyme activity: HRP enzyme activity was measured with a Tecan Infinite M200 PRO  using flat-bottom polystyrene 96-well plates. Depending on the concentration of correctly folded HRP, samples were diluted 1:50–1:200 in dilution buffer A/min … change in absorption (∆Abs 420 nm/min)∆Dilution … dilution of the samplesampleV … volume of sample (µL)d … length of the beam path through the cuvette (d = 0.58 cm)ε … extinction coefficient (ε420 = 36 mM−1 cm−1 [M−1 cm−1 ).ε420 = 36 mM−1·cm−1 [ε652 = 39 mM−1·cm−1 was used [Determination of kinetic parameters for final HRP preparation: The procedure was performed as described in . Enzyme M−1·cm−1 . For thewas used .Protein concentration: Protein concentration was determined using the method according to Bradford . In totaRP-HPLC: The HRP concentration in the samples was measured with RP-HPLC using a Polyphenyl BioResolve-RP-mAb 2.7 μm 3.0 × 100 mm column . The method was run for 10 min with the following program: 25% line B for 0.5 min, 55% line B in a linear gradient for 8 min, 55% line B for 0.5 min and then 25% line B for 1 min (Line A:MilliQ water with 0.1% trifluoroacetic acid (TFA); line B: acetonitrile with 0.1% TFA) at a flow rate of 1.2 mL/min. The column was kept at a constant temperature of 75 °C and the wavelengths 214 nm, 280 nm and 404 nm were monitored.SEC-HPLC: Purity of HRP was measured using a SEC-HPLC with a BEH 200A SEC 1.7 µm 4.6 × 300 mm, 3.5 µm  column. The method was run at 0.3 mL/min using 100% line A  for 18 minutes. The column was kept at a constant temperature of 30 °C and the wavelengths 214 nm, 280 nm and 404 nm were monitored.Reinheitszahl (Rz): The Reinheitszahl was calculated as the ratio of absorbance at 404 nm to 280 nm and the absorbance measurement was performed using a Hitachi Double Beam Spectrophotometer U-2900 .


Lipids play essential roles in numerous cellular processes, including membrane remodeling, signal transduction, the modulation of hormone activity, and steroidogenesis. We chose steroidogenic MA-10 mouse tumor Leydig cells to investigate subcellular lipid localization during steroidogenesis. Electron microscopy showed that cAMP stimulation increased associations between the plasma membrane (PM) and the endoplasmic reticulum (ER) and between the ER and mitochondria. cAMP stimulation also increased the movement of cholesterol from the PM compared to untreated cells, which was partially inhibited when ATPase family AAA-domain containing protein 3 A (ATAD3A), which functions in ER and mitochondria interactions, was knocked down. Mitochondria, ER, cytoplasm, PM, PM-associated membranes (PAMs), and mitochondria-associated membranes (MAMs) were isolated from control and hormone-stimulated cells. Lipidomic analyses revealed that each isolated compartment had a unique lipid composition, and the induction of steroidogenesis caused the significant remodeling of its lipidome. cAMP-induced changes in lipid composition included an increase in phosphatidylserine and cardiolipin levels in PAM and PM compartments, respectively; an increase in phosphatidylinositol in the ER, mitochondria, and MAMs; and a reorganization of phosphatidic acid, cholesterol ester, ceramide, and phosphatidylethanolamine. Abundant lipids, such as phosphatidylcholine, were not affected by hormone treatment. Our data suggested that PM–ER–mitochondria tethering may be involved in lipid trafficking between organelles and indicated that hormone-induced acute steroid production involves extensive organelle remodeling. Eukaryotic cells are comprised of fundamental structures known as organelles, most of which are compartmentalized within distinct membrane structures. These membranes create biochemical microenvironments that are specific to each organelle, and a number of essential biological processes take place at, or in, organelle membranes. Numerous studies over the past few decades have attributed the biological processes specific to each organelle to the distinct composition of proteins within the organelle membrane. For example, the plasma membrane (PM) is rich in receptor proteins, enabling both signal transduction and nutrient uptake , whereasTraditionally, lipid analyses were performed by extracting lipids from whole cells or tissues, followed by the separation and measurement of the lipid classes and their subspecies. However, this method eliminates all information about these lipids’ subcellular location, making it impossible to determine how lipid assemblies are organized. Therefore, to fully understand the biochemical reactions and pathways occurring at the subcellular level, it is essential to determine lipid localization and identify lipid assemblies by combining subcellular fractionation with the advanced mass spectrometry analysis of lipid species. Here, we used this strategy to perform the subcellular lipidomic analysis of cells involved in steroidogenesis to illuminate the dynamic changes in lipid profiles occurring in multiple organelles during the trafficking of cholesterol to the mitochondria for steroid biosynthesis.Steroid hormones are essential for proper development, reproduction, and behavior, and their synthesis involves a well-regulated, multistep process that effectively meets physiological needs in a tissue-specific manner . MitochoDuring acute steroid biosynthesis, large quantities of cholesterol must be trafficked to the cholesterol-poor mitochondria. Though lipid droplets rich in cholesterol have been considered the classic source for mitochondrial steroid production, recent studies have focused on the endoplasmic reticulum (ER) as another possible cholesterol source . The ER Clostridium perfringens, which can bind cholesterol in membranes with a high affinity, showed an increased movement of cholesterol from the plasma membrane during hormonally stimulated steroidogenesis in mouse Leydig tumor (MA-10) cells . P. P27]. PPrevious studies in the adrenal cortex have shown a rapid activation of sphingolipid metabolism upon ACTH or cAMP stimulation . This meAs with all “omics” studies, our map of the dynamic lipidome during hormonal stimulation can only be used as an effective hypothesis building system. Critically, this initial analysis allows us to propose several testable hypotheses, establishing the framework for future studies that could provide a deeper understanding of the various aspects of lipid metabolism, membrane dynamics, and steroidogenic cellular physiology. Overall, the results from this study strongly suggest that the formation of MAM and PAM associations, and their essential roles in multiclass lipid trafficking between organelles, may be critical for maintaining cellular homeostasis during steroidogenesis. Therefore, this work represents a first step towards enhancing our understanding of the roles of membrane associations and dynamic subcellular lipid organization in cholesterol trafficking and steroidogenesis. Mouse tumor Leydig cells (MA-10) were kindly provided by Dr. M. Ascoli, University of Iowa, Iowa City, IA, USA. These were cultured with Dulbecco’s modified Eagle medium (DMEM)/Ham’s F-12 Nutrient Mixture (F12)  with 1% penicillin and 1% streptomycin, and the culture was maintained at 37 °C. The MA-10 medium was supplemented with 5% fetal bovine serum and 2.5% heat-inactivated horse serum. For time-course experiments, cells were incubated with DMEM/F-12, without serum supplementation, in the presence of appropriate drug treatments.http://rsbweb.nih.gov/ij, accessed on 10 January 2021) [Cells were seeded onto 6-well plates at an initial concentration of 3 × 105 cells per well and immediately transfected using the TriFECTa Kit with DsiRNA duplex  and Lipofectamine RNAiMAX Transfection Reagent . The following small interfering RNA (siRNA) duplexes (150 nM) were used for the knockdown of Atad3 (NM_179203): duplex 1, 5′-CCAUCGCAACAAGAAAUACCAAGAA-3′; duplex 2, 5′-CCAGUUUGACUAUGGAAAGAAAUGC-3′; and duplex 3, 5′-AGGACAAAUGGAGCAACUUCGACCC-3′. Gene expression and target gene knockdown were evaluated by qRT-PCR , and quary 2021) . Statistry 2021) .g for 5 min, and the pellet was fixed with 2.5% glutaraldehyde  overnight at 4 °C. Fixed cells were washed three times with dH2O and then stained with OsO4 1% Osmium  for 2 h at 4 °C. Stained cells were then dehydrated by incubation in increasing acetone concentrations ranging from 30 to 100%. These were subjected to infiltration with acetone-epon ), the samples were embedded in pure epon , and polymerization was performed by incubation at 60 °C for 48 h. Blocks were cut into 100-nm slices, collected on 200 mesh copper grids , and post-stained with 4% uranyl acetate for 5 min and then with Reynold’s lead for 5 min. Cells on grids were observed with an FEI TECNAI 12 transmission electron microscope , operated at 120 KV. Images were collected on a charge-coupled device (CCD) Camera (AMT XR 80 C) at the McGill University electron microscopy facility.MA-10 cells cultured in 6-well plates were grown for 48 h and then treated with 1 mM dbcAMP for various lengths of time . Treated cells were washed once in a sodium cacodylate buffer (0.1 M and pH 7.4)  and gently scraped to harvest. These were then centrifuged at 1500× 9 cells from each group), were used as raw material for the multistep fractionation procedure. Cells were grown in 150 mm dishes to 80% confluence. These were then washed with phosphate-buffered saline (PBS) and incubated in serum-free media with or without 1 mM dbcAMP or 1 mM dbcAMP with 0.2 mM cycloheximide for 2 h. Cells were washed again with PBS, harvested using cell scrapers, and then centrifuged twice at 600× g for 5 min at 4 °C. Pellets were resuspended in 10 mL of ice-cold isolation buffer 1 , and the cells were homogenized using a Teflon pestle, attached to a motorized overhead stirrer, and run at 4500 rpm, with cell integrity checked every 25 strokes. Once 90% cell damage was observed, the homogenates were centrifuged twice at 600× g for 5 min at 4 °C. The supernatant was collected and centrifuged at 8000× g for 20 min at 4 °C. The resulting supernatant, containing the plasma membrane, PAM, microsomes, ER, and cytosolic proteins, was processed as described below.PAMs, MAMs, plasma membranes, and mitochondria were isolated from MA-10 cells following the protocol published by Suski et al., with some modifications outlined in g for 20 min at 4 °C centrifugation twice to eliminate excess contamination. The obtained pellet was resuspended in mitochondrial reaction buffer , and 0.5 mM EGTA) and layered onto a Percoll solution , 1 mM EGTA, and 30% Percoll (v/v)), followed by ultracentrifugation at 95,000× g for 30 min to yield crude MAM and mitochondria fractions. The samples were further purified by centrifugation at 6300× g for 10 min and further centrifuged at 95,000× g for 60 min, yielding pure MAM and mitochondrial fractions. The purity of the sample was verified using immunoblotting with antibodies to ACSL4 , COX IV , and VDAC .The pellet, which contained the crude mitochondrial fraction, was resuspended and further subjected to 8000× g for 20 min at 4 °C. The supernatant containing the ER and cytosol was centrifuged at 95,000× g for 180 min, resulting in a pellet containing the ER and cytoplasm. This pellet was resuspended and centrifuged again at 95,000× g for 60 min at 4 °C, which resulted in the isolation of the ER (pellet) and cytoplasmic fractions (supernatant). ER membranes were resuspended in isolation buffer 1, and the purity was verified by immunoblotting with calnexin antibody (Abacm). The pellet containing the plasma membrane and PAM fractions was subjected to ultracentrifugation at 25,000× g for 20 min at 4 °C to eliminate further ER and cytosol contamination. The thus obtained pellet was resuspended and layered onto a 4 mL 38%, 4 mL 43%, and 3 mL of 53% sucrose gradient before being centrifuged at 95,000× g for 180 min at 4 °C. This step ensured the separation of the plasma membrane and PAM fractions that formed two different layers in the sucrose gradient. The plasma membrane layer and PAM layer was separately collected, diluted in a 10 mL SB buffer and centrifuged at 10,000× g for 10 min at 4 °C. The collected supernatants were further centrifuged at 95,000× g for 1 h at 4°C to obtain pure PAM and plasma membrane pellets that were resuspended in 50-μL isolation buffer 2 . The enrichment of the samples was verified by immunoblotting using antibodies to TRPC3/6/7 (Santa Cruz Biotechnology) for the plasma membrane fraction and both ACSL4  and ORAI1  for the PAM fraction.To isolate ER membranes from supernatant containing the plasma membrane, PAM, microsomes, ER, and cytosolic proteins, the sample was centrifuged at 25,000× v/v). These were vortexed for 1 min and then kept at room temperature (RT) for 10 min. A 1:1 (v/v) mixture of chloroform–NaCl (154 mM) was then added to the samples, and they were vortexed and centrifuged at 1800 rpm for 5 min at RT. The bottom phase, containing the lipids, was extracted using a Pasteur pipette and dried by being subjected to nitrogen gas. These lipids were then resuspended in chloroform–methanol, 1:1 (v/v). Samples were loaded as a 0.8-cm band onto a chloroform–methanol, 1:1 (v/v), prewashed, and heat-activated (100 °C for 1 h) HPTLC plate . Lipid standards, 10 μg of cholesteryl ester 17:0 and 10 μg of cholesterol , were added in separate lanes. The plate was developed in a hexane:diethyl ether:acetic acid (70:30:1) mobile phase and then sprayed with primuline stain . Spots were visualized under UV light and quantified with a BAS 5000 image analyzer .Lipids from isolated membranes were extracted using the Bligh–Dyer method . BrieflyTo quantify cholesterol in subcellular organelles, fractions were first spiked with a d6-cholesterol internal standard. Samples were then extracted twice with 1 mL of methyl tert-butyl ether (MTBE). Aliquots were combined, dried in a Thermo Speed Vac™, re-dissolved in 500-µl aliquots of MTBE, vortexed, and dried again. Dried samples were re-suspended in 200-μL 50% (aq.) methanol, vortexed, and transferred into auto-sampler vials . The resolubilized samples were stored at –20 °C until ready for LC–MS analysis. The sample analysis was performed on an AB SCIEX  Triple-TOF 5600+ Mass Spectrometer coupled with a Shimadzu  Nexera XR UHPLC system. A binary mobile phase consisting of water with 0.1% formic acid (Mobile phase A) and acetonitrile with 0.1% formic acid (Mobile phase B) was utilized, and 2-µL aliquots of each sample were injected into the LC–MS. Analytes were chromatographically resolved by isocratic elution (90% (B) at 350 µL/min) on an Agilent Eclipse Plus C-8 analytical column (50 mm × 2.1 mm ID × 1.8 µm particle). The total run time was 10 min, and the mass spectrometer was operated in a positive HESI mode, with a vaporization temperature of 550 °C and a spray voltage of 5.5 kV. For quantification, a calibration curve was analyzed along with each batch of samples. After the acquisition, the data were analyzed using the MultiQuant™ software .v/v) and delivered to the source by isocratic flow at 7 μL/min of methanol:isopropanol, 3:1 (v/v), with 5 mM ammonium acetate, using a Shimadzu Prominence XR UFPLC autosampler and LC pump .Lipid extracts from subcellular fractions were analyzed by flow injection analysis using the MSMSall method . Approxi® TF 1.5.1 software, with MS/MSALL mode activated to perform a series of product ion scans. A high resolution TOF MS1 scan was run from m/z 200 to 1200, at an accumulation time of 300 ms. This was immediately followed by 1000 product ion scans for 1000 precursors evenly spaced from m/z 200.051 to 1200.051 (at unit mass resolution on the quadrupole) measuring across an MS/MS mass range of 100–1500, accumulated for 300 ms each, and collected in order from low to high m/z. The total time to perform one MS/MSALL acquisition in positive or negative mode was 5.48 min, with system flushing and equilibration for an additional 6 min. Solvent blanks (50 μL) containing the running buffer were run in between samples. All acquired TOF-MS and MS/MSALL data were processed with the LipidView™ 1.1 software (SCIEX) [The positive mode ion spray voltage was set at 5000 V, and the negative mode ion spray voltage was set to −4000 V, with a declustering potential at 40 V and an ESI source operating temperature of 150 °C. An atmospheric-pressure chemical ionization (APCI) probe and inlet were connected to a calibrant pump that delivered mass calibration solution. Sequentially, 50 μL of each sample were injected twice to complete both a positive mode and a negative mode based on the instrument manufacturer’s instructions. Positive and negative ion MS and sequential precursor ion fragmentation acquisitions were performed on a TripleTOF™ 5600+ System (AB SCIEX), controlled with Analyst (SCIEX) .t-test were used to calculate statistical significance—* p < 0.05; ** p < 0.01; *** p < 0.001.Data are shown as means ± SEM of at least three independent experiments performed in triplicate. GraphPad Prism  was used for graphic presentation. Two-way ANOVA, followed by Bonferroni’s post-hoc test (*) and the Student Taken together, these results revealed that hormone-induced acute steroid hormone production involves a dynamic reorganization of membrane lipids and extensive organelle remodeling in MA-10 Leydig cells. The data also suggested that plasma membrane–ER–mitochondria tethering (PAM interactions) may compose a route for cholesterol movement and supply during the early phases of steroidogenesis.


The morphological defects and apoptotic cell deaths in the embryo and yolk sac placenta of the nicotine group were significantly increased. Exposure to nicotine resulted in reduced superoxide dismutase (SOD) activity and cytoplasmic SOD and cytoplasmic glutathione peroxidase mRNA levels, but increased lipid peroxidation level in embryos. Moreover, treatment with nicotine resulted in aggravated expressions of the mRNA or protein level of antiapoptotic , anti-inflammatory , and vasculogenic  factors in the embryo and yolk sac placenta. However, all the parameters were significantly improved by treatment with lycopene, as compared to the nicotine group. These findings indicate the potential of lycopene as a protective agent against embryonic anomalies and yolk sac vasculogenic and placenta-forming defects induced by nicotine through modulations of oxidative, apoptotic, vasculogenic, and inflammatory activities.Identification of a new agent from natural products for the protection of embryonic anomalies is potentially valuable. To investigate the protective effect exerted by lycopene against nicotine-induced malformations, mouse embryos in embryonic day 8.5 with yolk sac placentas were cocultured with 1 mM nicotine and/or lycopene (1 × 10 Maternal smoking during early pregnancy has undesirable effects on the fetal organs and tissue development . MoreoveThe yolk sac and placenta of rodents are structurally and functionally quite similar to those of humans –10. Befoβ-carotene and other carotenoids in removing singlet oxygen and ROS [Lycopene, an acyclic nonprovitamin A carotenoid, is plentiful in red fruits and vegetables, especially tomatoes, and is one of the most effective antioxidants among the dietary carotenoids because of its many conjugated double bonds –20. Some and ROS , 22. Lyc and ROS , 24. HowThe present study, therefore, examines the adverse effects of maternal smoking on the embryo during embryonic organogenesis and investigates the suitability of lycopene as a natural substance in preventing nicotine-induced embryonic malformation.Eight-week-old ICR mice (Koatech) were used for mating. In the evening (20:00 hrs), male and female mice were placed in a cage, and the vaginal plug in the vulva was checked the following morning (08:00 hrs); this day was considered as embryonic day (E) 0.5 during further embryo culture. All experiments were performed as per the guidelines of the animal study guide .μm filter before use.Ten-week-old male Sprague–Dawley rats  were used to prepare serum for culture medium . Briefly−6, 1 × 10−5 μM) were selected. Animals were randomly assigned to four experimental groups: control (Con), nicotine (N), nicotine and lycopene 1 × 10−6 μM (N+Ly10−6), and nicotine and lycopene 1 × 10−5 μM (N+Ly10−5). The culture bottles were gassed with a mixture of O2, CO2, and N2 .The embryo culture system used in this experiment  referred to a previously existing model . Brieflypost hoc test.Embryos were evaluated immediately after the 48 h culture period, by applying the embryonic total morphology assessment . The morμm thickness and subsequently stained with hematoxylin and eosin (H&E).After the relevant culture period, samples were separated into embryos and yolk sac placentas and fixed in 4% paraformaldehyde. Paraplast-embedded tissues were sectioned at 5 μg/ml) for 10 min. Preequilibrated slides were labeled with TdT reaction mixture for 60 min at 37°C. The slides were washed in 2x SSC and blocked with 0.3% hydrogen peroxide for 5 min. After washing in 0.05% PBS-T (Tween 20), the slides were incubated with streptavidin/horseradish peroxidase-conjugated antibody (1 : 5000 in PBS) for 30 min. Apoptotic cells were visualized using DAB, and the nucleus of the cell was counterstained with hematoxylin.Apoptotic cells were analyzed using the DeadEnd™ colorimetric TUNEL assay kit , according to our previous method . BrieflyTotal protein was isolated and assessed from cultured embryos. Briefly, embryos were homogenized and the resultant supernatant was separated and mixed with 8.1% SDS, 20% acetic acid, and 0.75% 2-TBA; the mixture was heated for 30 min at 95°C. After cooling, the mixed solution was centrifuged, and the absorbance was measured at 532 nm and calculated from the 1,1,3,3,-tetramethoxypropane standard curve. The embryo protein content was calculated from the bovine serum albumin standard curve .Superoxide dismutase (SOD) activity in cultured embryos was measured using a SOD assay kit , according to our previously described method . The resα), transforming growth factor-beta 1 (TGF-β1), hypoxia inducible factor-1 alpha (HIF-1α), insulin-like growth factor-1 (IGF-1), alpha smooth muscle actin (α-SMA), B-cell lymphoma-extralarge (Bcl-xL), tumor necrosis factor-alpha (TNF-α), SOD1, cytoplasmic glutathione peroxidase (GPx1), BCL2-associated X protein (Bax), caspase 3, and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). In addition, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primers were used as an internal standard (Treated embryos and placenta (including the yolk sac) were subjected to real-time PCR analysis, as per the protocol of our previous paper . Brieflystandard . Data weμg) were resolved on SDS-PAGE using 10% polyacrylamide gels and subsequently transferred to nitrocellulose membranes . Membranes were blocked for 1 h and immediately incubated with primary antibodies for VEGF-α (Abcam), TGF-β1 (Santa Cruz), and GAPDH (Santa Cruz). Probed membranes were subsequently incubated with the anti-rabbit secondary antibody . Reactions were visualized using a Western Lightning Chemiluminescence reagent . Immunoreactive bands were quantified and normalized using an ImageJ software .Western blotting analysis was performed according to a previous paper . BrieflyP < 0.05). However, morphological scores altered by nicotine treatment were significantly inhibited in embryos cotreated with lycopene (Tables P < 0.05).Exposure to nicotine induces significant decreases in embryonic growth  and deveEmbryos treated with nicotine showed growth retardation, pale color, immature organization, and abnormal yolk sac morphology with marked reduction in size, impaired vascular branching, and a ring of blood islands around the chorioallantoic border in the placentas , B. In cIn addition, the nicotine-exposed yolk sacs exhibited defects in histopathological as well as morphological evaluation. The nicotine group showed blood island, intracellular vacuoles, and abnormal blood vessel formation, such as absence of hematopoietic cells and disordered endodermal cells. In contrast, normal blood vessels filled with embryonic nucleated erythrocytes were observed in the lycopene cotreated groups. Likewise, nicotine-induced placenta showed deficiency of blood vessels where the allantois connects with the chorionic plate; moreover, the labyrinth, a layer filled with placental villi for the regulation of nutrient transport, was thin and poorly developed compared with control. In contrast, lycopene improved all defects of yolk sac placentas induced by nicotine treatment Figures , C and DTUNEL-positive cells in the yolk sac placenta treated with nicotine were fetal red blood cells and trophoblast giant cells , B and F−6 or 1 × 10−5 μM) revealed significantly increased values   , A.α mRNA levels were significantly increased to 1.24 in the nicotine group, which significantly decreased to 0.50 in response to cotreatment with lycopene  significantly recovered the values to 1.25 or 1.46, respectively (Hif-1ectively .−6 or 1 × 10−5 μM) significantly recovered the values to 0.85 or 0.89, respectively  was significantly decreased as compared to that of the control group. In contrast, treatment with lycopene  significantly decreases the α-SMA levels to 1.20 or 0.98, respectively  than in the control group; the mRNA levels significantly increased to 1.45 or 1.63 of levels obtained in the control group .VEGF- Figures  after trβ1 protein level was significantly increased in yolk sac placentas exposed to nicotine, but cotreatment with lycopene significantly decreased these levels. Also, the mRNA expressions in yolk sac placentas were increased in the nicotine group, but treatment with lycopene slightly recovered the damage caused by nicotine exposure  significantly reduced these levels to 11.1 ± 0.57 or 10.4 ± 0.81 nmol/mg, respectively (−6 or 1 × 10−5 μM) and nicotine exposure resulted in increased SOD activity (0.55 ± 0.01 or 0.66 ± 0.05 U/mg), SOD1 (0.96 or 0.98), and GPx1 mRNA (0.74 or 0.79) levels, as compared to the nicotine treatment group  when compared to the control group (12.1 ± 0.22 nmol/mg). However, treatment with lycopene  resulted in decreased mRNA levels of Bax  and caspase 3  , as well as significant increase in the TNF-α mRNA level (2.77) when compared to the control group. However, the addition of lycopene (1 × 10−6 or 1 × 10−5 μM) decreases the NF-κB  and TNF-α  mRNA levels  with nicotine significantly increased most of the growth and development scores. These results indicate that lycopene is effective in protecting against prenatal morphological defects due to nicotine exposure.The first trimester in pregnancy is considered an important period during development, since the embryo is sensitive to environmental influences and is active for organogenesis , 32. Theα and Bcl-xL. These findings indicate that lycopene improves the defects of yolk sac vascularization, formation of the labyrinth, and fetal hematopoiesis induced by nicotine exposure.Yolk sac placenta developments are important during organogenesis , 13. Yolα in the nicotine-exposed embryos results in abnormal organogenesis. Furthermore, the nicotine-induced abnormal Hif-1α transcription in yolk sac placenta subsequently upregulates TGF-β1 and downregulates VEGF-α and IGF-1. These abnormal gene expressions result in defects of vasculogenesis and formation of the labyrinth. However, cotreatment with lycopene significantly improved these defects.During early organogenesis, the uterine environment is relatively hypoxic . Hypoxiaα and NF-κB). Furthermore, since oxidative stress induces apoptosis and inflammation during early organogenesis caused by nicotine, it is important to protect the embryos from exposure to oxidative stress during early embryogenesis. In the current study, lycopene cotreatment with nicotine improved lipid peroxidation, SOD activity, and antioxidant enzyme  mRNA expressions. These findings indicate that lycopene reduces the nicotine-induced oxidative stress, but increases antioxidative activities in embryos.Some studies revealed that nicotine increases the ROS expression, causing oxidative damage and growth retardation in embryos . OxidatiIn conclusion, the findings from our study indicate that lycopene plays an antioxidative, vasculogenic, antiapoptotic, and anti-inflammatory roles in both embryos and yolk sac placentas damaged by nicotine exposure during early embryogenesis, which is an important stage of development. Hence, lycopene has the potential to be effectively used in the pharmaceutical market as a natural preventive agent that is efficacious in protecting embryos from smoking-induced risks during pregnancy.