Introduction: Our aim was to evaluate the clinical and electroencephalographic effects of brivaracetam (BRV) in patients with drug-resistant focal epilepsy. BRV is a new antiepileptic drug (AED) with a high affinity for vesicle protein 2A (SV2A) and recently approved as adjunctive therapy for focal onset seizures.Methods: In this observational study of six-month duration, BRV (50-200 mg) was administered to 76 patients with drug-resistant focal epilepsy, who were ≥16-year-old and who suffered from daily, weekly, monthly and yearly recurrent seizures. At baseline and after six months of follow-up, we performed a neurological visit, neuropsychological tests: Quality of life in epilepsy-31 (QOLIE31), Epworth Sleepiness Scale (ESS), Intrapersonal Emotional Quotient (IEQ) and an electroencephalogram . Twenty-four patients underwent an overnight switch from levetiracetam (LEV) to BRV.Results: Seizure frequency of the 54 patients remaining at six months was reduced >50% in 29.6% of cases (responders), <50% in 31.5% (non-responders 1), while it remained unchanged in 38.8% (non-responders 2). Twenty-nine percent of patients early discontinued BRV because of lack of efficacy or minor adverse effects (AEs) like irritability, asthenia or headache. Neuropsychological tests in 28 patients demonstrated a significant improvement in I-EPI scores (p=0.04). Comparable results have been found in the subgroup of patients who switched from LEV to BRV. The EEG quantitative analysis showed a significant reduction of alpha absolute power at six months (p=0.03). Theta band power resulted significantly superior in non-responders than in responders (p=0.03). Furthermore, the δ+θ/α+β index resulted more elevated in patients with AEs than in patients without.Conclusions: BRV showed discrete results in terms of efficacy, safety and tolerability, with a good behavioural profile. BRV reduces the power of the alpha band, in correlation with its sedative effects but not with its minor efficacy. Furthermore, the increase in theta band power can be considered as a predictor of scarce response to treatment, while an increase in the δ+θ/α+β index could be a possible predictor of AEs occurrence. Drug-resistant epilepsy is defined by the International League Against Epilepsy (ILAE) as the failure of adequate trials of two tolerated and appropriately chosen and used antiepileptic drugs (AEDs) schedules, whether as monotherapies or in combination, to achieve sustained seizure freedom . This coWe conducted an observational study enrolling 76 consecutive patients admitted from July 2018 to July 2019 to our Epilepsy Center who were 16-years-old or more and with a diagnosis of drug-resistant epilepsy with focal onset seizures. We recorded demographical and clinical features at baseline (t0) relating to sex, age, history of epilepsy (years); etiology of epilepsy (2017 ILAE classification system); seizure frequency ; seizures type; number and type of AEDs tested and currently in use. BRV was prescribed at an initial dosage of 25 mg BID, subsequently titrated on the basis of individual needs to a maximum dosage of 200 mg per day. For patients who reported the absence of efficacy or intolerable AEs while on LEV or BRV therapy were started with a conversion ratio of 15:1 as proposed by Klein et al. . We perfOf 76 patients enrolled in our study, 41 (54%) were females, and the mean age was 42 ± 15 years, with no difference between males (43.8 ± 9.7 years) and females (40.9 ± 17.2 years). Mean epilepsy duration was 23.15 ± 13.2 years. 46% of patients had an underlying structural cause , 51.4% had an unknown cause and 2.6% had a genetic etiology. Regarding seizures type, 46 patients (60.5%) presented with the focal onset and focal to bilateral tonic-clonic seizures, 10 patients (13.2%) with focal onset aware seizures and 20 patients (26.3%) with focal onset impaired awareness seizures. At baseline (t0), seizure frequency was: −26.3% (20 patients) daily/daily recurrent; −26.3% (20 patients) weekly/weekly recurrent; −32.9% (25 patients) monthly/monthly recurrent (less than 4/month); −14.5% (11 patients) annual/annual recurrent (< 1 per month). Thirty-three patients (43.4 %) had already been on therapy with three or more drugs and the remaining 43 patients (56.6%) had already tried at least two drugs at optimal doses without benefit. During the study period, together with BRV, 22 patients were taking one other AED, 28 patients two AEDs, 18 patients three AEDs and 7 patients were on polytherapy with four AEDs. Carbamazepine (CBZ) was the most used AED in association with BRV (36.8%), followed by valproic acid (VPA) (32.9%) and lamotrigine (LTG) (31.6%). 34% of patients were on therapy with other sodium channel blockers , while benzodiazepines (BDZ) and phenobarbital (PB) were used by 23.7% and 22.4% of patients, respectively. Other drugs sporadically used in our population were perampanel (PER), topiramate (TPM), gabapentin (GBP) and acetazolamide  discontinued the study drug before six months follow-up. Of the remaining 54 patients, 16 patients (29.7%) were classified at six months as responders , 17 patients (31.5%) as non-responders 1 (seizure decrease < 50%) and 21 patients (38.8%) as non-responders 2  decreased (35.1% vs 52.6%). When confronting responders and non-responders - 1/2, we found that patients with baseline daily recurrent seizures had a lower response to BRV therapy (p=0.025). Moreover, 33 patients (43.4%) reported at six months a subjective decrease of seizure intensity, defined as a reduction of seizure duration and gravity. Conversely, we did not find any statistically significant difference between responders and non-responders-1/2 regarding age (p=0.266), sex (p=0.175), epilepsy type (p=0.606), involved hemisphere (p=0.503), seizure type (p=0.505), epilepsy duration (p=0.955), number of previous and concomitant AEDs . Mean dosage (+SD) of BRV was 131 + 38.3 mg per day in responders and 126.3 + 42.9 mg per day in non-responders - 1/2 (p=0.68). Particularly, of 16 responders, 6 were taking 100 mg per day, 7 were taking 150 mg per day and 3 patients were taking 200 mg per day. Among 16 responders, AEDs that were most frequently taken along with BRV were LCM (40%), LTG (33.3%) and TPM (20%). Contingency analysis did not show significant results in terms of BRV response when this was administrated in association with other drugs. Twenty-two of 76 patients enrolled (29%) early discontinued BRV, because of lack of efficacy in 3 patients (13.6%), AEs in 9 (40.9%) or both in 10 (45.5%). Thirty-three out of 76 patients (43%) reported AEs, of which the most frequent was irritability (52%). Other common AEs were somnolence (30%), asthenia (27%), headache (15%) and vertigo . However, we did not find any association between drug early discontinuation and other considered variables like age p=0.431), sex (p=0.132), epilepsy cause (p=0.752), seizure type (p=0.299), involved hemisphere (p=0.254), number of concomitant drugs (p=0.098), epilepsy duration (p=0.344) and seizure frequency at baseline (p=0.098). Conversely, treatment with previous AEDs was significantly associated with early drug discontinuation, with a higher drop-out rate for those who were taking more than three drugs (p=0.019). Finally, the number of previous or concomitant AEDs was not associated with a higher degree of AEs development  and contingency analysis did not show any statistically significant difference between relevant AEs and BRV association with CBZ (p=0.611) or VPA (p=0.459). A total of 37 patients underwent baseline and six months follow-up neuropsychological testing. When confronting mean scores at t0 and t1, we found a significant variation in I-EPI relative scores, with a reduction after BRV treatment . On the other hand, we did not find a significant difference for other administered tests  presented an unchanged EEG recording, 7 patients (26.9%) a better EEG recording and 4 patients (15.4%) a worse EEG recording. When a positive or negative variation was observed, it exclusively regarded the quantity of epileptiform discharges but not their site or morphology. About background activity, it was slower in 16 patients (61.5%) at six months, however, remaining in the same frequency range observed at baseline. Furthermore, we found greater stability and a higher trend in the diffuse transmission of background activity. In the other 10 patients, background activity remained unchanged. Regarding quantitative analysis, we herewith report the more relevant modifications. When confronting variables at baseline and at six months follow-up, we found that α absolute power was significantly reduced from a mean value of 19.01 ± 11.37 μV2 at t0 to a mean value of 13.19 ± 7.98 μV2 at t1 (p=0.03). We also found a trend towards reduction for β1 power, that passed from a mean value of 8.42 ± 8.1 μV2 at t0 to a mean value of 5.97 ± 5.17 μV2 at t1 . However, at t1, this difference was not evident anymore, while θ power differed significantly, being inferior in responders than non-responders-1/2 . Twenty-three patients underwent an overnight switch from LEV to BRV. Statistical analysis did not evidence significant differences at baseline between switch and non-switch patients regarding mean age (p=0.81), sex (p=0.31), epilepsy duration (p=0.55), epilepsy cause (p=0.10), seizure type (p=0.10), seizure frequency at baseline (p=0.15) and number of concomitant AEDs (p=0.44). After six months, four patients (16.7%) early discontinued the drug. Of the remaining 19 patients, 4 (21.1%) presented a seizure decrease >50%, 8 (42%) presented a seizure decrease <50% and 7 (36.8%) did not show any variation. Statistical analysis did not show any difference in terms of treatment response for the switch and non-switch patients (p=0.27). However, we found a seizure decrease in 21.1% of switch patients, but this reduction did not appear to be statistically significant (p=0.29). Four out of 23 switch patients (17.3%) discontinued treatment with BRV, one patient, because of lack of efficacy, one because of AEs and two because of both previous reasons. No difference was found between the switch and non-switch patients regarding discontinuation rate (p=0.15) and its cause (p=0.67). Thirteen out of 23 patients (56.5%) reported AEs, of which the most common were irritability (5), vertigo (4), insomnia (3), asthenia (3), somnolence (1), headache (1), tremor (1), gastrointestinal discomfort (1). No difference was found between the switch and non-switch patients (p=0.51). Neuropsychological evaluation at t0 and t1 was performed in 12 switch patients. When confronting scores at t0 and t1, we observed a significant reduction of I-EPI score . We did not evidence any significant difference between switch and non-switch patients at t0 in terms of testing results, while at t1 we observed a trend towards significance in I-EPI score (t 1 p=0.07 vs t0 p=0.23). We selected seven EEG recordings of switch patients for qualitative and quantitative analysis. However, they did not show any significant results.The population of our study presents typical features of drug-resistant epilepsy  defines drug-resistant epilepsy as “failure of adequate trials of two tolerated, appropriately chosen and used AED schedules (whether as monotherapies or in combination) to achieve sustained seizure freedom) ,3. A strIn our study, we enrolled a population of epileptic patients who presented baseline clinical features suggestive of disease gravity, like a long disease duration, focal onset to bilateral tonic-clonic seizures with weekly and/or daily recurrent frequency and a scarce response to previous numerous pharmacological therapies. Although its efficacy was partially and negatively influenced by the gravity markers that we have just described, BRV showed a discrete efficacy, mostly among patients with a seizure frequency less pronounced. Furthermore, we observed a better efficacy profile when BRV was associated with LTG and TPM. Its tolerability profile was also good, as we did not report serious AEs during the six months of treatment, but only minor AEs with an early discontinuation rate that was not inferior to that of other newer AEDs. Considering our results, we can also conclude that BRV has an acceptable cognitive-behavioural profile; despite irritability has been one of the more frequently reported AEs, this has not been confirmed by neuropsychological tests that, on the contrary, showed better results in this context. Either inspective or quantitative EEG analyses showed a background activity slowing after BRV, which correlated well with the sedative effects frequently reported in our population. Furthermore, the quantitative analysis highlighted an increased theta band’s absolute power which, in our opinion, could be a predictive marker of lack of efficacy of BRV, while the increase of delta+theta/alfa+beta index could be considered as a predictor of neurotoxicity. The switch from LEV to BRV, whether for lack of efficacy or for AEs, appeared as a feasible strategy. Indeed, our results permit us to conclude that a previous exposition to LEV is not predictive of lack of efficacy or a higher risk of AEs when switching to BRV. Moreover, LEV-naïve patients and patients who switched from LEV did not differ regarding their response to BRV and their tolerability to the new treatment. We can reasonably conclude that when it is necessary to suspend LEV because of AEs which are mostly behavioural, it is advisable to switch to BRV because it reduces those complications.


Despite the significant advancements in the field, the optimal macromolecular crowder still remains elusive. Herein, the physicochemical properties of different concentrations of different molecular weights hyaluronic acid (HA) and their influence on equine adipose-derived stem cell cultures were assessed. Within the different concentrations and molecular weight HAs, the 10 mg/mL 100 kDa and 500 kDa HAs exhibited the highest negative charge and hydrodynamic radius, and the 10 mg/mL 100 kDa HA exhibited the lowest polydispersity index and the highest % fraction volume occupancy. Although HA had the potential to act as a macromolecular crowding agent, it did not outperform carrageenan and Ficoll Cell-assembled tissue engineering has the potential to revolutionise reparative biomedicine, as it takes advantage of the cells’ natural intrinsic abilities to fabricate tissues with complexity, precision and efficiency that human-made devices have yet to reach. Despite the repeatedly demonstrated safety and efficacy in the clinical setting , sulpha® ) and pol® ). Althou® , its que® ,34,35 im® . Althoug® ,38 in a ® . Herein,Cell culture plasticware and chemicals/reagents were purchased from Sarstedt  and Sigma-Aldrich , respectively, unless otherwise stated . HAs of different molecular weight (10 (10 to 20 kDa), 60 (66 to 99 kDa), 100 (100 to 150 kDa), 500 (301 to 450 kDa) and 1000 kDa (750 to 1000 kDa)) were purchased from Lifecore™ Biomedical . Equine fat tissues, from the mane, were provided from the School of Veterinary Medicine, Aristotle University of Thessaloniki, Thessaloniki, Greece.Clostridium histolyticum (C9722). Collagenase digestion was performed for 4 h at 37 °C in standard cell culture media , 10% foetal bovine serum  and 1% P/S). Fat tissue stromal vascular fraction (SVF) was obtained by centrifugation of digested samples for 5 min at 700 g (4 times). SVF was resuspended in standard cell culture media and passed through a Corning® nylon cell strainer of 70 μm (CLS431751). Nucleated cells from the SVF were counted using a Neubauer chamber and seeded at a density of 2,000,000 cells/cm2 in tissue culture flasks at 37 °C in a humidified atmosphere of 5% CO2. Media were changed every 3–4 days  until the cells reached ~ 80% confluency. Cell detachment was carried out using trypsin-ethylenediaminetetraacetic acid (EDTA) solution (T3924) for 5 min at 37 °C in a humidified atmosphere of 5% CO2. Cell suspensions were centrifuged for 5 min at 700 g and then seeded at a density of 5000 cells/cm2 in standard cell culture media  and conditions . At passage 2–3, cell culture flasks were tested for mycoplasma [6 cells in 1 mL 90% FBS /10% dimethyl sulfoxide (DMSO). The cryovials were frozen at −80 °C in a Nalgene® Mr. Frosty freezing container (C1562) and transferred to the liquid nitrogen storage after 24 h. Prior to use, cryovials were thawed in a thermostatic bath at 37 °C and cells were seeded in pre-equilibrated tissue culture flasks in standard cell culture media and conditions at 5000 cells/cm2. After 24 h, the media were changed to ensure complete removal of DMSO. From then onwards, the media were changed every 3–4 days until reaching 80% confluency and passage 4. All experiments were conducted with cells at passage 5.Fat samples were washed with Hanks’ Balanced Salt Solution  containing 1% penicillin/streptomycin , minced with sterile dissection material and enzymatically digested with 0.1% collagenase from coplasma . MycoplaCell morphology was assessed via bright field microscopy using an inverted Olympus IX73 microscope .Multipotency was assessed with established osteogenic, adipogenic and chondrogenic protocols, using cells in DMEM as negative controls, and surface markers were assessed with fluorescence-activated cell sorting (FACS) analysis, validating the antibodies with immunocytochemistry.2 were seeded in 6-well plates. Standard cell culture media was substituted after 24 h by osteo-induction media consisting of 100 nM dexamethasone (D4902), 100 μM L-ascorbic acid 2-phosphate (A8960) and 10 mM β-glycerophosphate disodium salt hydrate (G9422) in 10% FBS and 1% P/S. Osteo-induction media were changed every 3 days up to day 14. At day 14, cell layers were treated with methanol at 4 °C for 20 min and calcium depots were detected by adding 400 μL of 40 mM alizarin red S stain (A5533), pH 4.1. Excess dye was removed with PBS washes.For osteogenesis, 5000 cells/cm2 were seeded in 6-well plates. After 3 days the media were changed to adipo-induction media consisting of 0.1 μM dexamethasone, 500 μM 3-isobutyl-1-methylxanthine , 200 μM indomethacin (I7378), 15% horse serum (H1270) and 1% P/S. The adipo-induction media was changed every 3 days up to day 18. At day 18, cell layers were treated with 10% formalin for 15 min at room temperature and lipid droplets were detected by adding 400 μL of 0.3% red oil O stain (O1391) in 60% isopropanol. Excess dye was removed with PBS washes.For adipogenesis, 2500 cells/cmFor chondrogenesis, pellet cultures were performed. Approximately 500,000 cells were pelleted into 15 mL conical polypropylene tubes with chondro-induction media consisting of serum-free DMEM High Glucose, 100 nM dexamethasone, 100× ITS + 1  liquid media supplement , 40 µg/mL L-proline (P5607), 25 µg/mL L-ascorbic acid 2-phosphate, 10 ng/mL TGF-β3  and 1% P/S. The chondro-induction media were changed every 3 days up to day 21. At day 21, pellets were cryoprotected with increasing gradient (15 and 30%) sucrose solutions. Cryoprotected pellets were embedded in optimal cutting temperature compound and frozen in liquid nitrogen. Sections 5 µm thick were obtained using a Leica CM1850  cryotome. Sections were stained with Alcian blue (B8438), washed with 1% acetic acid, stained with nuclear fast red aluminium solution 0.1% (100121), washed with double-distilled water, dehydrated in 100% ethanol and mounted using DPX mountant for histology (06522). Images of osteogenic and adipogenic induction were captured with an Olympus IX73 microscope  and images of chondrogenic induction were captured with an Olympus BX51 microscope .6 cells/mL density in 2 mM EDTA/PBS buffer. 50 μL aliquots were tested in flow cytometry tubes and incubated for 30 min at 4 °C with the following antibodies: AlexaFluor® 488 anti-human CD29  [A FACScalibur™  device was used for cell surface markers analysis. Briefly, at 80% confluency at passage 5, cells were trypsinised and suspended at 2 × 10 TS2/16) ,42, FITC TS2/16) ,44,45, F TS2/16) ,44,45, P TS2/16) ,42,46 anTo validate the antibodies used for FACS analysis, immunocytochemistry was also carried out when the cells reached 80% confluency. Secondary antibodies were not used, as the CD markers used for FACS analysis were directly conjugated to the aforementioned fluorochromes. The antibodies were incubated at 4 °C overnight. Details of the immunocytochemistry analysis are provided below.In order to identify working concentrations of the different molecular weight  HAs, different concentrations  of each HA molecular weight were dissolved in standard cell culture media at 37 °C. Optimal concentrations from this experiment were also screened with cells in standard cell culture media under standard cell culture conditions after 4 days in culture (see below).Zeta potential, polydispersity index and hydrodynamic radius were assessed using dynamic light scattering . The crowding solutions were prepared in PBS to mimic physiological conditions. Fractional volume occupancy was calculated using the obtained values of hydrodynamic radius for the different MMC agents, as has been described before .2 density in standard cell culture media supplemented with 100 μM L-ascorbic acid 2-phosphate to induce collagen synthesis. After 24 h, media were changed and replaced with media containing the different concentrations of the different HA molecular weights. Media without MMC and media with carrageenan  and Ficoll®  were used as controls. All crowders were sterilised under UV light for 15 min and solubilised in standard cell culture media supplemented with L-ascorbic acid 2-phosphate at 37 °C until no particles in suspension were observed. Media changes were performed every 2 days until reaching day 8. The workflow is schematically illustrated in Cells were seeded at a 15,000 cells/cm® viability kit  was used to assess cell viability, as per manufacturer’s protocol. Briefly, at each timepoint, cells were washed with HBSS and incubated with a solution containing 2 µM calcein-AM and 4 µM ethidium homodimer in HBSS for 30 min at 37 °C in a 5% CO2 humidified atmosphere. DMSO was added for positive dead cell controls. Excess dye was removed by washing with HBSS. Samples were visualised using an inverted IX73 Olympus microscope . At least 5 regions of interest (ROI) per image were selected. Alive and dead cells were counted with ImageJ software  and percentage cell viability was calculated as alive cell number against total cell number.A Live/DeadCell proliferation was assessed via nuclei counting. Briefly, cell cultures were washed with HBSS at each timepoint and fixed with a filtered (0.2 μm sterile syringe filter) solution of 2% paraformaldehyde (PFA) in PBS. Excess fixation solution was removed by PBS washes. Samples were then incubated for 5 min in a 4′,6-diamidino-2-phenylindole  in methanol at 0.5 μg/mL concentration solution. Excess DAPI solution was removed with PBS washes. Samples were visualised using an inverted IX73 Olympus microscope . Cell nuclei were counted with ImageJ software ; the watershed plugin was used to separate overlapped nuclei. At least 5 ROI per image were selected. Nuclei were counted to obtain cell number per area at the different timepoints.2 humidified atmosphere. Absorbance at 550 nm excitation and 595 nm emission was then measured using a plate reader . Cell metabolic activity was expressed as the% reduction in the alamarBlue® dye and normalised to the control group without MMC.Cell metabolic activity was assessed using the alamarBlue™  assay, as per manufacturer’s protocol. Briefly, at the end of each culture timepoint, cells were washed with HBSS and 10% alamarBlue™ solution in HBSS was added. Samples were incubated for 3 h at 37 °C in a 5% COAt each timepoint collagen extraction from the cell layers and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed . Briefly® 488 (A-32731) and goat anti-mouse conjugated AlexaFluor® 555 (A-32727); both ThermoFisher Scientific . As negative controls to subtract the background, the samples were incubated in PBS without primary antibodies and incubated with the corresponding secondary antibodies. Samples were washed 3 times with PBS and incubated for 5 min with DAPI at 0.5 μg/mL concentration in methanol. The samples were washed 3 times with PBS and mounted with 10 μL of VECTASHIELD® HardSet™ Antifade Mounting Medium . Glass coverslips with a diameter of 8 mm were placed on top of the samples. Images were taken with an Olympus IX73 inverted microscope . The Q-Capture Pro 7 software  was used for image acquisition. At least 5 ROI per image were selected. Fluorescence intensity was calculated using the ImageJ software , subtracting from every ROI the average fluorescence intensity value of the negative control. The values were normalised to the corresponding cell number of each ROI.At each timepoint, media were removed, samples were washed with HBSS and fixed for 15 min with filtered 2% PFA at 4 °C. Excess PFA was removed with PBS washes. Blocking of non-specific bindings was performed with a 3% BSA (A7638) solution at room temperature for 30 min. The samples were then incubated for 90 min at room temperature with the following primary antibodies: mouse monoclonal anti-collagen type I , rabbit polyclonal anti-collagen type III  and rabbit polyclonal anti-collagen type IV ; all Abcam, Ireland. The samples were washed 3 times with PBS and then incubated for 30 min with the following secondary antibodies at 1:500 dilution: goat anti-rabbit conjugated to AlexaFluor® Excel and statistical analysis was performed using SigmaPlot® 12.0 software . One way analysis of variance (ANOVA) followed by pairwise multiple comparison (Holm-Sidak’s method) was used when the distributions of the populations were normal  and the variances of populations were equal (Levene’s test). Non-parametric analysis was performed using Kruskal-Wallis followed by Dunn’s multiple comparison post-hoc analysis for pairwise multiple comparisons, when the assumptions of parametric analysis were violated. Statistical significance was accepted at p < 0.05.Numerical data are expressed as mean ± standard deviation (SD). Data were processed in MicrosoftThe extracted cells were mesenchymal in origin as trilineage analysis  revealedSolubility assessment in cell-free standard cell culture media at 37 °C  revealedp < 0.05) lower negative charge than carrageenan; and that carrageenan and all HA 60 kDa, HA 100 kDa (but the 0.5 mg/mL), HA 500 kDa (but the 0.5 mg/mL) and HA 1000 kDa concentrations exhibited a significantly (p < 0.05) higher negative charge than Ficoll®. Within the different HA molecular weights, for HA 60 kDa and HA 1000 kDa, the charge was not affected (p > 0.05) as a function of increased concentration, whilst for HA 100 kDa and HA 500 kDa, the charge was significantly (p < 0.05) decreased as a function of increased concentration  higher hydrodynamic radius than carrageenan; that the 0.5 mg/mL concentration of HA 100 kDa, the 0.5 and 1 mg/mL concentrations of HA 500 kDa and the 0.1 mg/mL concentration of HA 1000 kDa exhibited a significantly (p < 0.05) lower hydrodynamic radius than carrageenan; and that carrageenan and all concentrations of HA 60 kDa, HA 100 kDa, HA 500 kDa and HA 1000 kDa exhibited a significantly (p < 0.05) higher hydrodynamic radius than Ficoll®. Within the different HA molecular weights, for HA 60 kDa and HA 1000 kDa, the hydrodynamic radius was not affected (p > 0.05) as a function of increased concentration, whilst for HA 100 kDa and HA 500 kDa, the hydrodynamic radius was not significantly (p > 0.05) affected between 0.5 and 1 mg/mL concentration and was significantly (p < 0.05) increased as a function of increased (from 1 to 5 mg/mL and from 5 to 10 mg/mL) concentration  of all), HA 500 kDa and HA 1000 kDa.Polydispersity analysis  revealedp < 0.05) higher % fractional volume occupancy than Ficoll® and all HA 60 kDa concentrations, the 1, 5 and 10 mg/mL HA 100 kDa concentrations, the 5 and 10 mg/mL HA 500 kDa concentrations and the 5 mg/mL HA 1000 kDa concentration exhibited a significantly (p < 0.05) higher % fractional volume occupancy than carrageenan and Ficoll®. Within the different HA molecular weights, the highest concentration of HA 60 kDa (10 mg/mL), HA 100 kDa (10 mg/mL), HA 500 kDa (10 mg/mL) and HA 1000 kDa (5 mg/mL) exhibited the highest (p < 0.05) % fractional volume occupancy (p < 0.05) and the lowest (p < 0.05) % fractional volume occupancy, respectively  analysis  revealedccupancy . Among aectively .Bright field microscopy analysis  revealedQualitative  and quan® at all timepoints and the carrageenan, the 10 mg/mL HA 100 kDa, the 5 and 10 mg/mL HA 500 kDa and the 5 mg/mL HA 1000 kDa at day 8 significantly (p < 0.05) decreased cell number and increased cell metabolic activity.Quantitative cell proliferation  and metap < 0.05) collagen type I deposition at all timepoints and Ficoll® induced significantly (p < 0.05) higher collagen type I deposition than the non-MMC group only at day 4 and in some instances, at day 8, induced the lowest (p < 0.05) collagen type I deposition. The high concentrations (5 and 10 mg/mL) of HA 60 kDa (p < 0.05) higher collagen type I deposition than the non-MMC group only at day 4. The 5 mg/mL concentration of HA 500 kDa (p < 0.05) higher collagen type I deposition than the non-MMC group only at day 4. None of the HA 1000 kDa (p > 0.05) higher collagen type I deposition than the non-MMC group at any timepoint.SDS-PAGE and complementary densitometry analyses  revealedA 60 kDa  and HA 1A 60 kDa  induced  500 kDa  induced 1000 kDa  concentrp < 0.001) collagen type I deposition at all timepoints, Ficoll® induced higher (p < 0.05) collagen type I deposition than the non-MMC group only at days 4 and 6 and HA had no effect (p > 0.05) in collagen type I deposition at any timepoint, independently of the concentration and molecular weight.Immunocytochemistry  and comp® induced the highest (p < 0.001) collagen type III deposition  different to the Ficoll® group) and the carrageenan, the 10 mg/mL HA 60 kDa, the 5 mg/mL HA 100 kDa (only at day 6), the 10 mg/mL HA 100 kDa, the 5 and 10 mg/mL HA 500 kDa and the 5 mg/mL HA 1000 kDa groups induced higher (p < 0.05) collagen type III deposition than the non-MMC group at all timepoints.Immunocytochemistry  and comp® induced the highest (p < 0.001) collagen type IV deposition and the carrageenan, the 10 mg/mL HA 60 kDa, the 10 mg/mL HA 100 kDa, the 10 mg/mL HA 500 kDa (only at days 6 and 8) and the 5 mg/mL HA 1000 kDa (only at days 6 and 8) groups induced higher (p < 0.05) collagen type IV deposition than the non-MMC group at all timepoints.Immunocytochemistry  and compIn recent years, the use of MMC has been advocated for, for the accelerated development of tissue moduli for regenerative medicine  and drugStarting with solubility assessment, we found a molecular weight-dependent solubility, with the 1000 kDa molecular weight HA able to remain soluble up to 5 mg/mL concentration, whilst the 10 kDa molecular weight HA was found to be soluble up to 200 mg/mL concentration. In accordance with our work, similar HA concentrations/molecular weights have been used in the literature as media supplements , HA 500 kDa and HA 1000 kDa exhibited a polydispersity index of > 0.7, which indicates a high degree of heterogenicity, due to broad size  distribution or agglomeration or aggregation of the particles of a population [® 70 kDa and Ficoll® 400 kDa [® 70 kDa and Ficoll® 400 kDa cocktail (subject to the concentration of each molecule in the solution) and polyvinylpyrrolidone 40 kDa and polyvinylpyrrolidone 360 kDa (subject to the concentration of each molecule in the respective solution) [® is considered as a deformable sphere, as opposed to a compact sphere [With respect to biophysical analysis, all molecules assessed had a negative charge; the high negative charge of carrageenan can be attributed to its sulphate content , and themponents , and themponents ,69,70. Fmponents ,72,73. Cpulation . We attrpulation   ; and >10olution) ). Althout sphere ).® at all timepoints, the carrageenan at day 8, the 10 mg/mL HA 100 kDa at day 8, the 10 mg/mL HA 500 kDa at day 6 and 8 and the 5 mg/mL HA 1000 kDa at day 8 induced reduced cell proliferation and increased metabolic activity in comparison to the non-MMC control group. Previous studies have shown Ficoll® and/or carrageenan to not affect human bone marrow stem cell morphology, viability and metabolic activity [® after 24 h [® has been shown to affect cell morphology, to significantly increase metabolic activity only at day 1 and to significantly decrease DNA at days 4, 7 and 11 [® has been shown to not affect the morphology, viability and metabolic activity of human corneal fibroblasts [Moving into basic cellular function analysis, none of the MMC agents assessed affected cell morphology and viability, whilst the Ficollactivity ,77 . It is roblasts  and rabbroblasts ; and 2 mroblasts ). Collec® 70 kDa, Ficoll® 400 kDa, Ficoll® 1000 kDa and cocktails thereof, dextran sulphate 10 kDa, dextran sulphate 100 kDa, dextran sulphate 500 kDa and cocktails thereof [® 70 kDa and Ficoll® 400 kDa cocktail [® 70 kDa and Ficoll® 400 kDa cocktail [®, the highest (over the non-MMC control) collagen type I deposition was only observed at day 4. Although one study has shown the Ficoll® cocktail to result in lower collagen deposition than the non-MMC group after 14 days in human neonatal fibroblast cultures [® cocktail to enhance collagen type I deposition in various cell populations . This scocktail ,14. Withcultures , most stroblasts , human croblasts , human lroblasts , human broblasts  and humaroblasts ), albeitroblasts ,14,49; aroblasts ,93. Thusroblasts , 50 mg/mroblasts , 100 mg/roblasts , 500 mg/roblasts , 500 mg/roblasts , 500 androblasts ) and disroblasts , and 1 aroblasts ) with prem cells , human cem cells ,81, humaem cells ,98 and hem cells ,99 cultuem cells , human tem cells  and humaem cells  culturesem cells , whilst em cells . It is aem cells . It appeem cells ,38), it em cells ,49,78, wem cells ,102. Bioem cells ,104.®, customarily used crowding molecules, induced the highest collagen I, collagen III and collagen IV deposition, unless a biological benefit for hyaluronic acid is identified, we recommend their use as macromolecular crowding molecules for enhanced and accelerated extracellular matrix deposition.In the quest for the optimal macromolecular crowder for enhanced and accelerated extracellular matrix deposition, herein we ventured to assess the potential of different concentrations and molecular weight hyaluronic acids. As carrageenan and Ficoll


Bacillus subtilis subsp. subtilis MD 32 was isolated from kimchi. The strain was sequenced using an Illumina MiSeq platform, and the genome size was 4,238,856 bp with a GC content of 43.41 mol%. The genome encoded 4,396 proteins, with 45 tRNAs, 6 rRNAs, and 5 noncoding RNAs. Bacillus subtilis subsp. subtilis MD 32 was isolated from kimchi. The strain was sequenced using an Illumina MiSeq platform, and the genome size was 4,238,856 bp with a GC content of 43.41 mol%. The genome encoded 4,396 proteins, with 45 tRNAs, 6 rRNAs, and 5 noncoding RNAs. Bacillus subtilis is a Gram-positive, rod-shaped bacterium that can form heat-resistant spores but is not considered a human pathogen. B. subtilis has received much attention in the food industry because it is considered safe, it is involved in various fermented foods, and it produces a bacteriocin  for 1 min. The mixed samples were diluted with Ringer’s solution (Merck) in a 10-fold series, and 100 μl of the dilutions was plated onto LB agar and then incubated for 18 h at 37°C under aerobic conditions. A randomly picked colony was purified by streaking three times onto LB agar plates with aerobic incubation overnight at 37°C. The selected strain was presumptively identified as B. subtilis by 16S rRNA gene sequencing. The total genomic DNA of the strain was extracted from 5 ml of fresh culture grown aerobically in LB broth overnight at 37°C, with shaking at 200 rpm, using the peqGOLD bacterial DNA kit  according to the manufacturer’s instructions. A genomic DNA library of strain MD 32 was prepared with a Nextera XT library preparation kit , and paired-end sequencing was performed on a MiSeq sequencer (Illumina) with 2 × 250 cycles. In total, 734,725 paired-end raw sequencing reads were trimmed using the Trimmomatic (v. 0.32) pipeline (de novo assembled with SPAdes (v. 3.11.2) with parameters k-mer 77, careful, and minimum contig length of 500 bp  (v. 4.13) with default parameters  and aadk] were present. A sbo-alb  operon involved in bacteriocin production could be identified, which contained the subtilosin A gene (sboA) and the subtilosin immunity gene   accession number SRR12774112.The draft genome sequence of


The maintenance of pluripotency in mouse embryonic stem cells (ESCs) is governed by the action of an interconnected network of transcription factors. Among them, only Oct4 and Sox2 have been shown to be strictly required for the self-renewal of ESCs and pluripotency, particularly in culture conditions in which differentiation cues are chemically inhibited. Here, we report that the conjunct activity of two orphan nuclear receptors, Esrrb and Nr5a2, parallels the importance of that of Oct4 and Sox2 in naïve mouse ESCs. By occupying a large common set of regulatory elements, these two factors control the binding of Oct4, Sox2 and Nanog to DNA. Consequently, in their absence the pluripotency network collapses and the transcriptome is substantially deregulated, leading to the differentiation of ESCs. Altogether, this work identifies orphan nuclear receptors, previously thought to be performing supportive functions, as a set of core regulators of naïve pluripotency. Summary: Esrrb and Nr5a2, two orphan nuclear receptors, are identified as essential regulators of pluripotency in mouse embryonic stem cells. The uncommitted identity of mouse embryonic stem cells (ESCs) is maintained by the activity of a gene regulatory network that strictly depends on the function of Oct4 (Pou5f1) and Sox2 . BindingAmong all auxiliary factors, Esrrb is prominent , becauseOne simple hypothesis to explain how in 2i/LIF ESCs can bypass the requirements for Esrrb is that other TFs may perform a compensatory role. Of note, Esrrb is part of a broad family of TFs, orphan nuclear receptors, that show high sequence and structural homology, and have overlapping developmental functions . Among tFig. S1), we observed that Esrrb presents a broad distribution of expression levels in ESCs . Importantly, both genes were undetectable in a fraction of the ESC population. Next, we derived additional reporter lines in which the coding sequence for GFP and mCherry have been knocked-in after the Nr5a2 and Esrrb open reading frames , and found that Esrrb and Nr5a2 proteins show broadly overlapping patterns of expression, with cells negative for one TF also low or negative for the second . In 2i/LIF culture conditions, spontaneous differentiation was suppressed and the expression of Esrrb and other auxiliary TFs reinforced, becoming homogeneous. In line with this, double reporters revealed how in chemically defined medium Esrrb and Nr5a2 protein levels are elevated and their downregulation impeded . These results suggest that Esrrb and Nr5a2 respond similarly to signalling cues in ESCs. As the downregulation of Esrrb marks the commitment of ESCs to differentiate, it is possible that Nr5a2 is also relevant in this context, and that the concomitant downregulation of both TFs plays a causal role in the extinction of pluripotency.Expression of auxiliary TFs, including Esrrb, is heterogeneous in ESCs cultured in serum and LIF (FCS/LIF), which induces a metastable state permissive for spontaneous differentiation . Notably in ESCs A, as pree second B,C. NoneTable S1), revealing an extensive binding overlap in both conditions . Nr5a2 bound fewer loci in mouse ESCs , possibly owing to its lower expression, particularly in FCS/LIF, and was almost invariably found in association with Esrrb , revealing a preference of Esrrb for T and, more pronouncedly, of Nr5a2 for C , and vice versa. Interestingly, whereas perfect matches to the Nr5a2 motif variant accumulated at regions of high Nr5a2, optimal Esrrb motifs were more broadly distributed, and mildly enriched at regions bound by both TFs .To get an indication of whether Esrrb and Nr5a2 play overlapping functions in supporting pluripotency, we established their binding profiles across the genome, in FCS/LIF or 2i/LIF . EKOiE NrKO cells could be derived without special complications, indicating that in this context Nr5a2 is not strictly required for self-renewal . Moreover, we observed that genes responding to the loss of one orphan receptor, although not passing statistical tests of significance in response to loss of the other, still display concordant expression changes . As genes activated and repressed by both factors were enriched in terms such as ‘response to LIF’ (FDR=2.27e-24) and in terms linked to differentiation , respectively, we conclude that Esrrb and Nr5a2 cooperate to support pluripotency in FCS/LIF.We have previously derived ESC lines in which endogenous Esrrb is knocked out and Esrrb expression is rescued by a doxycycline (Dox) inducible transgene (EKOiE), such that upon Dox withdrawal the cells differentiate in FCS/LIF . In thes-renewal . Indeed,-renewal A,B. In c-renewal . Notably-renewal A,B. This-renewal C. These Klf4, Tbx3, Tfcp2l1 and Sox2 – and in the mild upregulation of differentiation markers . Yet, expression of Oct4 was unaffected, and colonies of undifferentiated ESCs readily formed. Similarly, the loss of Nr5a2 had detrimental effects, reducing expression of pluripotency genes, the clonogenicity of ESCs and triggering spontaneous differentiation. Nonetheless, it remained tolerated overall, as previously reported . This drastic phenotype was not exclusively observed after clonal plating. In the absence of Esrrb and Nr5a2, ESCs cultures rapidly deteriorated during regular passaging: evident morphological changes preceded a reduction in cell numbers, appearing after day 2 . In this context, defects in proliferation manifested only once the expression of pluripotency markers had dropped. Therefore, such effects can likely be attributed to differentiation. Immunofluorescence 7 days after Dox withdrawal, also confirmed the near-complete loss of Oct4, Sox2, Nanog and Klf4 in EKOiE NrKO ESCs . These results reveal a phenotype that is not predictable from the study of single knockouts because it is masked by the functional redundancy between the two orphan receptors. Altogether, we identify Esrrb and Nr5a2 as two arms of a single regulatory unit that is essential for maintenance of pluripotency.The extensive binding overlap between Esrrb and Nr5a2, their additive effect on gene expression, and the severe phenotype of their deletion in FCS/LIF prompted us to explore the effect of the concomitant loss of function in 2i/LIF, where the invalidation of Esrrb is compatible with self-renewal. In agreement with previous reports , clonal reported . In strireported E,F. In aTable S4). In EKOiE cells grown in the presence of Dox, 77,840 regions were bound by either Esrrb or Nr5a2 . As expected, Esrrb binding was ablated 2 days after withdrawal of the drug , we observed that, in the absence of support by Esrrb, Nr5a2 occupancy levels correlate more directly to the presence of a strong cognate motif at target regions . Hence, even though Esrrb and Nr5a2 can bind DNA independently, Esrrb facilitates Nr5a2 occupancy at sites of low affinity, indicating cooperativity in binding.The profound consequence of the loss of Esrrb and Nr5a2 prompted us to investigate in more detail how these TF conjunctly control pluripotency TF binding, as represented by Oct4, Sox2 and Nanog, in ESCs cultured in 2i/LIF . Moreover, although Esrrb and Nr5a2 also bind regions not targeted by the ensemble of other TFs . Strikingly, we observed that, at these common regulatory nodes, the loss of Esrrb and Nr5a2 leads to a global reduction of TF occupancy . Although the reduced levels of Nanog and Sox2 protein 2 days after depletion may partially contribute to these effects, Oct4 remained robustly expressed . Moreover, the reduction of Oct4, Sox2 and Nanog binding was significantly stronger at Esrrb/Nr5a2-bound regions than at the ensemble of sites irrespective of Esrrb/Nr5a2 occupancy . Overall, 43%, 67% and 74% of co-bound regions displayed reduced Oct4, Sox2 and Nanog, respectively, after depletion of Esrrb and Nr5a2. These effects are substantially more severe than those observed after depletion of Nanog in FCS/LIF . We conclude that Esrrb and Nr5a2 are conjunctly required to foster binding of Oct4, Sox2 and Nanog across thousands of shared target regions.Next, we assessed the overlap with other pluripotency TFs, and found that more than half of the regions bound conjunctly by Oct4, Sox2 and Nanog were also bound by Esrrb or Nr5a2, in line with the notion that the pluripotency gene regulatory network is extensively interconnected , Oct4/Sox2 composite motifs were more degenerate and more markedly enriched in proximity of Esrrb/Nr5a2 (<100 bp), compared with independent regions (group 5) E,F. ThusFig. S5I). Second, we observed that acting at the very same positions on DNA, Esrrb and Nr5a2 affect occupancy by other pluripotency TFs in a greater-than-additive manner . These results provide evidence of a direct functional cooperativity, which is distinct from the broad synergy between unrelated TFs.Our findings show that Esrrb and Nr5a2 operate conjunctly at target regulatory elements. Yet the two orphan receptors – as observed for other pluripotency TFs  – might Grhl3, Zic2/3/5 and Fgf5, and ‘neural development’ (FDR=1.3e-7). At these sites, Esrrb and Nr5a2 opposed binding of Oct4 and Sox2, which are also known to induce ESC differentiation when overexpressed  tend to be located close to known pluripotency regulatory elements, such as the enhancers of Klf4, Nanog, Esrrb, Sox2 and Oct4. Globally, these regions were enriched in proximity of genes associated with pluripotency, such as ‘response to LIF signalling’ , ‘blastocyst formation’  or ‘stem cell population maintenance’ . In contrast, cluster 5 was associated with genes linked to early epiblast differentiation, such as xpressed . These rTable S2). Nonetheless, uniquely responsive genes could also be identified. In line with the pronounced effect on TF binding, loss of both Esrrb and Nr5a2 affected a higher number of genes than the individual depletion of either factor . Of note, these transcripts showed less pronounced but concordant changes after individual depletions, confirming that Esrrb and Nr5a2 largely act in concert to control gene expression  after depletion of Nr5a2  or Esrrb , and highlighted that the targets of the two factors are largely overlapping (pression A. We thepression C. We obsTable S3). Notably, when we compared the effect of the loss of Esrrb and Nr5a2 with the response to LIF stimulation in ESCs  and in energetic and metabolic processes  . In agreement with this, we observed highly concordant transcriptional changes after the depletion of Esrrb and Nr5a2, and during the early stages of ESC differentiation, both when occurring spontaneously in FCS/LIF . When indicated, cells were grown in 2i-containing medium : 0.5× DMEM/F12 , 0.5×Neurobasal , 0.5% N2 supplement 100× , 1% B27 supplement 50× , 10 µg/ml insulin , 2 mM L-glutamine , 0.05% BSA , 100 μM 2-mercaptoethanol , 10 ng/ml recombinant LIF .ESCs were cultured on 0.1% gelatine  in DMEM+GlutaMax-I , 10% FCS , 100 μM 2-mercaptoethanol , 1× MEM non-essential amino acids  and 10 ng×mlNr5a2 (Nr5a2-205), encoding the first portion of the DNA-binding domain, was disrupted in EKOiE ESCs  at 4°C, washed and coated for 2 h with 10 μg/ml laminin  in PBS. Fixation was performed for 10 min in 1% formaldehyde  at room temperature (RT). After washing the cells twice in PBS, they were permeabilised with 0.1% v/v Triton X-100 in PBS (PBSTx) supplemented with 3% donkey serum  for 30 min at RT. Primary antibodies (diluted in PBSTx with 3% donkey serum) were applied for 2 h at RT or overnight at 4°C in a volume of 1 ml per dish. After three washes in PBSTx, secondary antibodies (2 μg/ml in PBSTx with 3% donkey serum) were applied for 2h at RT. Cells were washed three times in PBSTx, nuclei counterstained with 4',6-diamidino-2-phenylindole , and imaged with an inverted Leica SP8 confocal microscope using a 40× oil immersion objective. Acquisition was performed using the LASX acquisition software suite. Primary antibodies used were: 0.3 μg/ml anti-Nanog rabbit polyclonal ; 0.4 μg/ml anti-Oct4 mouse monoclonal - clone C10 , for staining in combination with anti-Sox2; 1 µg/ml anti-Oct4 rabbit polyclonal , for staining in combination with anti-Flag; 1:500 anti-Sox2 rabbit polyclonal ; 1 µg/ml anti-Klf4 goat polyclonal ; 1 μg/ml anti-Flag mouse monoclonal . Secondary antibodies used were: Alexa Fluor 594 AffiniPure Donkey Anti-Rabbit IgG (H+L) ; Alexa Fluor 488 AffiniPure Donkey Anti-Mouse IgG (H+L) ; Alexa Fluor 647 AffiniPure Donkey Anti-Goat IgG (H+L) .2). Images on single focal planes were acquired with a 63× oil immersion objective on an inverted LSM800 confocal Zeiss microscope, using the ZEN Blue acquisition software suite.For live-cell imaging, Nr5a2-GFP+Esrrb-mCherry ESCs grown in FCS/LIF or 2i/LIF in Ibidi plates, as described above, were incubated with 500 nM Hoechst-33342 for 20 min before imaging. During imaging, the cells were kept at 37°C in a humidified atmosphere , and EKOiE Nr5a2 KO Repair ESCs (c4.2 and c16.1) were plated in single wells of 6-well plates coated overnight with 0.01% poly-L-ornithine  at 4°C, washed and coated for 2 h with 10 μg/ml laminin  in PBS. After culture in FCS/LIF or 2i/LIF media in the presence or absence of 1 μg/ml doxycycline  for 7 days, cells were fixed and stained using an alkaline phosphatase staining kit  according to the manufacturer's instructions. Representative images of alkaline phosphatase-stained colonies formed by EKOiE, EKOiE Nr5a2 KO and EKOiE Nr5a2 KO repair ESCs were acquired using a Zeiss Discovery V8 Stereo microscope, and the ZEN blue software suite.6 EKOiE or EKOiE Nr5a2 KO ESCs were plated in single wells of 6-well plates and passaged every 2 days in the absence or presence of doxycycline, replating each time 0.75×106 cells. At each passage, cells were set aside for RNA extraction.0.75×102) in FCS/LIF medium and cultured for 3 days before analysis. After trypsinisation, cells were resuspended in FCS/LIF without Phenol Red, and analysed using a LSR II flow cytometer system or a Luminex Image Stream MK2 instrument with a 60× magnification objective. Data were analysed using the FlowJo software suite.E14Tg2a, Esrrb-T2a-GFP or Nr5a2-T2a-GFP ESCs were plated at low density  for 50 min at followed by 10 min in 1% formaldehyde PBS . Chromatin preparation, immunoprecipitations and library preparations are described in the supplementary Materials and Methods. Antibodies: anti-Esrrb mouse monoclonal , anti-Nanog rabbit polyclonal ; anti-Oct4 rabbit polyclonal ; anti-Sox2 rabbit polyclonal ; anti-Flag mouse monoclonal ; anti-HA mouse monoclonal .TF binding was assessed in E14Tg2a Flag-Nr5a2 ESCs cultured in FCS/LIF, and EKOiE, EKOiE NrKO or EKOiE HA-AID-Nr5a2 ESCs cultured in 2i/LIF. Approximately 105 EKOiE or EKOiE NrKO ESCs were cultured in wells of 6-well plates with/without 1 µg/ml doxycycline  for 2 days before RNA extraction with 500 μl TRIzol  according to the manufacturer's instructions. To eliminate any genomic DNA contamination, this was followed by an additional DNAse I treatment  for 20 min at 37°C followed by phenol:chloroform purification. RNAs were resuspended in Ultrapure DNAse/RNase Free Distilled Water . Stranded, poly-A selected RNA-seq libraries were prepared and sequenced (paired-end 150bp reads) by Novogene.2×10supplementary Materials and Methods.RNA was prepared with a Nucleospin RNA kit, performing DNAse I treatment . One microgram of RNA was used for cDNA preparation using a Transcriptor First Strand cDNA synthesis kit , using random hexamer priming. Real-time RT-PCR reactions were performed in triplicate in 384-well plates with a 480 LightCycler (Roche) using the LightCycler 480 SYBR Green I Master mix  and incubated for 2 h with 2500 U/ml benzonase  at 4°C. Samples were boiled in Laemmli Buffer , and 20 µl of each sample was loaded onto 10% Mini-PROTEAN TGX gels  run in glycine/SDS buffer and transferred onto a nitrocellulose membrane , which was blocked, and incubated overnight with primary antibodies in PBSTw (0.1% Tween-20 in PBS) with 5% BSA, washed and incubated with secondary antibodies. Membranes were then washed in PBSTw and, where relevant, incubated with PIERCE ECL2 Western Blotting Substrate  before detection. A list of primary and secondary antibodies used, and a detailed description of the method is available in the supplementary Materials and Methods.10supplementary Materials and Methods.Full details on the computational methods used in this study are available in the De novo motif discovery on Esrrb/Nr5a2-bound regions in FCS/L used the Regulatory Sequence Analysis Tools (RSAT) with standard parameters (rsat.sb-roscoff.fr) . Motif occurrences were identified and the frequency of T or C at the seventh base determined. To calculate enrichments of Oct/Sox motifs (MA0142.1) at a given distance from Esrrb/Nr5a2 motifs, we took all motifs with a score greater than the median and calculated the absolute distance between all pairs of Esrrb and Oct4/Sox2 motifs over the regions. We then compared the observations to a randomised background to derive a Poisson P-value for the observed number of pairs at each distance.ChIP-seq was performed in triplicate, except for duplicate Nr5a2 binding profiles in 2i/LIF. Paired-end reads were trimmed, aligned with Bowtie2  to the mcoff.fr) . To locacoff.fr) . AlternaP-values for the association between differentially expressed genes within x bp of a ChIP-seq peak belonging to a cluster, compared with a background of all genes within x bp of a cluster peak, for x in  bp, using the Julia package ProximityEnrichment.jl (https://github.com/owensnick/ProximityEnrichment.jl). Data visualisation was made in R using ggplot2 , and data plotted with the FlowJo software suite. Data from Oct4 ChIP-seq datasets from


Amphidinium carterae ACRN03, successfully cultured in an LED-illuminated pilot-scale (80 L) bubble column photobioreactor operated in fed-batch mode with a pulse feeding strategy. We report on the isolation of new structurally related AMs, amphidinol 24 , amphidinol 25  and amphidinol 26 , from a singular fraction resulting from the downstream processing. Their planar structures were elucidated by extensive NMR and HRMS analysis, whereas the relative configuration of the C-32→C-47 bis-tetrahydropyran core was confirmed to be antipodal in accord with the recently revised configuration of AM3. The hemolytic activities of the new metabolites and other related derivatives were evaluated, and structure–activity conclusions were established. Their isolation was based on a straightforward and high-performance bioprocess that could be suitable for the commercial development of AMs or other high-value compounds from shear sensitive dinoflagellates. The demand for valuable products from dinoflagellate biotechnology has increased remarkably in recent years due to their many prospective applications. However, there remain many challenges that need to be addressed in order to make dinoflagellate bioactives a commercial reality. In this article, we describe the technical feasibility of producing and recovering amphidinol analogues (AMs) excreted into a culture broth of  Dinoflagellates are a well-recognized source of bioactives exhibiting wide diverse functionality and distinctive chemical structures that show great potential for use in the fields of biology, biomedicine, pharmacology and toxicology . HoweverAmphidinium 25D +13 ; UV (MeOH) λmax 230 nm (ε 27123); IR νmax 3264, 2931, 2366, 2345, 2034, 2011, 1978, 1608 and 1020 cm−1; 1H and 13C NMR data (CD3OD) see m/z 1363.7606 [M + Na]+ . Amphidinol 25 (2): Yellow oil; [α]25D +1 ; IR νmax 3266, 2940, 2867, 2364, 2167, 2034, 1976, 1614 and 1022 cm−1; 1H and 13C NMR data (CD3OD) see m/z 1521.6678 [M − H]− .Amphidinol 26 (3): Colorless amorphous solid; [α]25D −30 ; IR νmax 3262, 2832, 2366, 2167, 2034, 1976, 1613 and 1022 cm−1; 1H and 13C NMR data (CD3OD) see m/z 1191.6482 [M + Na]+ .The known compounds luteophanol D and amphidinol 20B were identified by detailed analysis of the NMR and MS spectrometric data  and compSparus aurata) grown in a fish farm . Serial methanolic dilutions of AMs 24–26 (1–3), luteophanol D and AM20B were placed in a microwell and air dried. The concentrations of AMs in microwells ranged from 0 to 5.5 × 104 ng mL−1. A erythrocyte concentration of 45 × 106 cells per well was used. Negative controls consisted of erythrocytes incubated in Mediterranean seawater. Positive control, i.e., 100% hemolysis, was obtained using distilled water. The dose–response curves (percentage of hemolysis (PH) vs. log of number of A. carterae cells per well (x)) were interpreted in terms of the Hill Equation (1):PH is the percentage of hemolysis; x is the concentration of AM per well; maxPH represents the maximum percentage of hemolysis equal to 100%; EC50 is the concentration of AM per well giving 50% hemolysis and η is the Hill slope. Saponin , was used as a positive control obtaining an EC50 value of 10.7 × 106 ± 1.06 × 106 pg per well through Hill equation. An equivalent saponin potency (pg per AM pg) was calculated by dividing the EC50 for saponin by the EC50 for AM.Erythrocyte lysis assay was performed as described elsewhere . ErythroAmphidinium carterae, successfully cultured in a pilot-scale bubble column photobioreactor illuminated with multicolor light-emitting diodes (LEDs) operated in fed-batch mode with a pulse feeding strategy. The structures were established by extensive spectroscopic methods, while the relative configurations of the C16 common central core (C-32→C-47) were determined by comparison of the NMR data of AM24 with those of two synthetic intermediates of AM3 [Three new related amphidinol analogues, named amphidinol 24, amphidinol 25 and amphidinol 26, were obtained from the marine dinoflagellate s of AM3 . The resA structure–activity relationship study of the new metabolites and other related derivatives against hemolytic activity was carried out, and it was observed that they are dramatically affected by the hydrophobicity  of the polyene chain. The lack of activity of the molecules concerned can be correlated with their highly hydroxylated structures, additional presence of sulfated groups, or the shortening of the crucial amphipathic polyenic terminus. This work reveals, in terms of acclimation, growth rates, biomass productivity, downstream processing and excellent recovery yields of excreted AM analogs, a useful bioprocess strategy that may be adaptable to a suitable production of other super carbon chain compounds or biotoxins from marine dinoflagellates.


Results revealed no changes in Flanker performance  or P3 (latency and mean amplitude) following either HIIE conditions compared to seated rest. Together, these data suggest inhibitory control and neuroelectric underpinnings are not affected by different modalities of HIIE at 30 min and 85 min post-exercise. Such findings reveal that engaging in short bouts of different HIIE modalities for overall health neither improves nor diminishes inhibitory control and brain function for an extended period throughout the day.Acute aerobic high-intensity interval exercise (HIIE) has demonstrated positive effects on inhibitory control and P3 event-related potential (ERP) in young adults. However, the evidence is not well established regarding the effects of different HIIE modalities that incorporate aerobic-resistance training on these cognitive and neurocognitive outcomes. The purpose of this investigation was to examine the transient effects of HIIE-aerobic and HIIE-aerobic/resistance on P3 and Flanker task performance. Participants ( Researchers have sought to establish a dose–response relation between parameters of acute exercise  and cognitive outcomes, with the intent of providing a universal ‘prescription’ to achieve optimal cognitive benefits. Indeed, early meta-analytical data suggest that intensity may be a significant moderator of cognitive benefits revealing an inverted-U relation, with the greatest benefits observed following moderate-intensity compared to both low-intensity and high-intensity bouts ,2,3. Howmax with intermittent 1 min bouts of walking); continuous aerobic running ; and seated rest, undertaken on three separate days and followed by completion of a Flanker task and P3 assessment. Results revealed improvements in performance for HIIE and continuous aerobic exercise compared to seated rest, with greater improvements observed specifically for HIIE. Additionally, a larger P3 amplitude was observed following continuous aerobic running compared to HIIE and rest. However, HIIE demonstrated a shorter P3 latency suggesting improved processing speed and stimulus classification. These data suggest that HIIE is comparable to traditional moderate-intensity exercise when it comes to cognitive benefits, and that modality may be a significant factor that impacts neuroelectrical and behavioral changes following acute exercise.Executive function is responsible for selection, scheduling and maintenance of goal-directed behavior ,18,19. ITo date, acute HIIE laboratory protocols have focused primarily on sustained aerobic intervals . A limitation of this style of research is the modality disconnect compared to typical HIIE protocols performed in real-world settings. That is, a majority of HIIE protocols developed by fitness professionals integrate forms of resistance exercise and calisthenics that are designed as a holistic high-intensity strength and conditioning program. Furthermore, recent research has sought to evaluate the feasibility of effective HIIE protocols that can be accomplished in workplace and university environments, revealing that a combination of aerobic and muscular fitness protocols is preferred and enjoyable for working employees  and univThe timing of cognitive assessments following HIIE may also serve as a significant moderator of cognitive changes. That is, research suggests that cognitive performance declines immediately after exercise but improves after a delay period . For exaThe first aim of the present study was to examine the effects of a single bout of HIIE-aerobic and HIIE-aerobic/resistance on inhibitory control performance and neuroelectrical measures of the P3 in young adults. Based on the findings outlined previously, it was predicted that HIIE-aerobic would improve behavioral performance compared to HIIE-aerobic/resistance and seated rest. Regarding the P3, it was predicted that measures would replicate Kao and colleagues , with den = 2) or second (n = 3) laboratory visit, poor performance on the Flanker task (<50% accuracy) and insufficient ERP trials in one or more conditions (n = 1).Young adults were recruited from the University of North Carolina at Greensboro. All participants provided written consent approved by the Institutional Review Board. Participants were sent the following electronic questionnaires via Qualtrics: a questionnaire concerning their health history and demographics; a Physical Activity Readiness Questionnaire (PAR-Q) ; and theA modified version of the Erikson Flanker task  was utilEEG activity was recorded from 64 Ag/AgCl sintered electrode sites organized in accordance with the international 10–10 system  using a Offline data were processed using MATLAB (R2017a) with EEGLAB  and ERPLStimulus-locked epochs were created at −200 ms to 1000 ms encompassing congruent and incongruent trial types. Epochs were baseline corrected using pre-stimulus intervals and low pass filtered at 30 Hz. Epochs were rejected if a moving window peak-to-peak amplitude exceeded 100 mV (100 ms window width and 50 ms window step); if the overall variance of the epoch exceeded ±3 SDs of the mean of local (by electrode site) and global  accepted epochs; and/or by visual inspection. ERP-averaged waveforms were created separately for trial type (congruent and incongruent), for each condition  and at each time point (35 min and 85 min). After artifact rejection, individual ERP waveforms for congruent and incongruent trials across each condition and time were created from an average of 125 epochs (95% CI: 118 to 132). P3 latency was quantified using peak latency, and P3 amplitude was quantified using mean amplitude within the post-stimulus latency window 300 ms to 600 ms.max) for the exercise conditions. Days 2, 3 and 4 were counterbalanced across sessions. At the beginning of each session participants were fitted with a heart rate (HR) monitor and completed practice trials for the Flanker task. They then undertook 9 min of the intervention, were fitted with an electroencephalography (EEG) cap, and completed the Flanker task at 30 and 85 min following each intervention condition, while recording continuous EEG measures. These measurement periods were determined based on prior research revealing a delayed cognitive benefit following HIIE [max (determined by cardiorespiratory fitness assessment) separated by 1 min walking, followed by a 1.5 min cool-down walk. Speed and incline were manipulated by the researcher to obtain 90% of HRmax. The purpose of selecting the timing and duration of each set was to replicate the methodology of prior research revealing changes in inhibitory control and P3 following 9 min of HIIE [max separated by 1 min walking, followed by a 1.5 min walking cool-down on the treadmill. The development of this HIIE protocol was based on prior research evaluating feasibility of real-world HIIE protocols designed for workplaces [National Geographic video . A research assistant measured HR during all three intervention conditions at time points that aligned with the transition periods from moderate intensity to high intensity , from high intensity to moderate intensity  and at the cessation of HIIE (9 min).Using a within-participants cross-over design, participants attended the lab on four separate days (with approximately one week between each testing session). Participants were instructed to avoid vigorous physical activity and to maintain typical daily behaviors  12 h prior to testing. During the first visit, participants reviewed the informed consent with the experimenter, completed practice trials for the cognitive task, and performed a cardiorespiratory fitness assessment on a motor-driven treadmill following a modified Balke Protocol  to detering HIIE  with therkplaces  and univrkplaces  and currp ≤ 0.05) were performed using Greenhouse–Geisser correction statistics with Bonferroni-corrected t-tests for post hoc comparisons. Reporting of main effects and interactions included partial η2 . Flanker RT and accuracy analyses were conducted utilizing a 3  × 2 (Time: 30 min and 85 min) × 2 (Type: Congruent and Incongruent) repeated measures ANOVA. Flanker interference scores for RT and accuracy were analyzed using a 3  × 2 (Time: 30 min and 85 min) model. P3 analyses for amplitude and latency were conducted using a 3  × 2 (Time: 30 min and 85 min) × 2 (Type: Congruent and Incongruent) × 5  repeated measures ANOVA.Statistical analyses were conducted using SPSS . With a sample size of 24 participants and a power of 0.88, the present study was sufficient to detect repeated measures effects computed using G * Power 3.1.9.6 . RepeateF’s  ≤ 1.79, p’s ≥ 0.15), across Flanker and P3 measures. Therefore, subsequent analyses were collapsed across session order. All mean (±SD) values for Flanker performance and P3 measures are reported in The preliminary analyses were performed on session order to determine if order effects influenced outcomes associated with the treatment. The results did not reveal any significant interactions with mode (t’s (23) ≥ 20.95, p’s ≤ 0.05), but no difference between HIIE conditions at each time point (t’s (23) ≤ 1.6, p’s ≥ 0.12)  see .F  = 174.65, p < 0.01, ηp2 = 0.88), which was superseded by a Time × Type interaction  = 6.07, p < 0.01, ηp2 = 0.21). Post hoc tests indicated shorter RT at 30 and 85 min for congruent trials compared to incongruent trials (t’s (23) ≥ 11.07, p’s ≤ 0.05). No significant difference was observed across time for congruent and incongruent trials (t’s (23) ≤ 1.34, p’s ≥ 0.19). Additionally, mean RT interference revealed a main effect of Time  = 6.07, p = 0.02, ηp2 = 0.21), revealing greater interference at 30 min compared to 85 min.The omnibus analysis for mean RT revealed a main effect of Type  = 9.20, p < 0.01, ηp2 = 0.29), indicating greater accuracy at 30 min compared to 85 min. Also, a main effect of Type  = 37.71, p < 0.01, ηp2 = 0.62) revealed greater accuracy for congruent trials compared to incongruent trials. Lastly, accuracy interference revealed no main effects or interactions  ≤ 1.26, p’s ≥ 0.25).The omnibus analysis for Flanker accuracy revealed a main effect of Time  = 68.62, p < 0.01, ηp2 = 0.75) and Site  = 25.82, p < 0.01, ηp2 = 0.53), which were superseded by a Type × Site interaction  = 6.83, p < 0.01, ηp2 = 0.23). The post hoc tests indicated shorter P3 latency for congruent compared to incongruent trials across all sites (t’s (23) ≥ 5.71, p’s ≤ 0.05). Lastly, the P3 latency revealed no main effects or interactions for Condition  ≤ 2.02, p’s ≥ 0.09, ηp2’s ≤ 0.08).The omnibus analysis for P3 latency indicated a main effect of Type  = 74.41, p < 0.01, ηp2 = 0.76) and Site  = 31.84, p < 0.01, ηp2 = 0.58), which were superseded by a Type × Site interaction  = 9.92, p < 0.01, ηp2 = 0.30). The post hoc tests indicated larger P3 mean amplitude for incongruent compared to congruent trials across all sites (t’s (23) ≥ 5.21, p’s ≤ 0.05). Additionally, a Condition × Type interaction was observed  = 6.32, p < 0.01, ηp2 = 0.22). The decomposition of this interaction revealed larger P3 mean amplitude for incongruent trials compared to congruent trials across all conditions (t’s (23) ≥ 5.70, p’s ≤ 0.05). Lastly, a Time × Type interaction  = 5.20, p < 0.01, ηp2 = 0.18) revealed a larger P3 amplitude for incongruent trials compared to congruent trials across both time periods (t’s (23) ≥ 6.47, p’s ≤ 0.05)  see .The present study evaluated inhibitory control and P3 following different HIIE conditions across an extended period. In contrast to our a priori hypotheses, these data suggest that neither HIIE-aerobic nor HIIE-aerobic/resistance enhance or inhibit behavioral and neural markers of cognitive functioning beyond 30 min compared to seated rest.The present behavioral results are inconsistent with recent meta-analytical findings ,31, whicAnother possible explanation for this lack of replication of previous behavioral results concerns individual differences in psychological factors related to mental context experienced prior to and during the exercise bout . HIIE exercise is a difficult routine to accomplish, especially for those unfamiliar or inexperienced, and this may increase heterogeneity in psychological preferences and attitudes toward the task . This asRegarding P3, the present findings revealed maintenance of P3 latency and amplitude regardless of the HIIE condition. These data are not in line with our hypotheses and are contrary to the findings of similar protocols revealing decreases in P3 amplitude after acute HIIE ,9. For iDespite the novelty of the present investigation, limitations are present. First, as addressed previously, the duration of the HIIE bouts may have contributed to the null findings. Future research should include protocols with varied timing to determine if duration moderates the longevity of cognitive benefits post-exercise. Secondly, cognitive and neuroelectrical measurements were not performed immediately after the HIIE and rest conditions. Our protocol may have elicited cognitive improvements that returned to baseline prior to the first assessment. Nevertheless, these data do not support a persistent timing effect for improved cognition and brain functioning associated with HIIE. Therefore, further research is needed to determine cognitive task timing effects following different exercise modalities. Next, the different HIIE modalities were primarily aerobic in nature with the HIIE-aerobic/resistance protocol incorporating arguably only two resistance activities . Given that the HR data revealed no difference in intensity, it is possible that both conditions constituted high-intensity aerobic exercise. Lastly, no pre-test assessment was conducted to control for day effects across each testing condition. However, participants in the present study were counterbalanced across sessions with the seated rest condition serving as the control. Regardless, future research should include a baseline measure to mitigate timing limitations, both for duration of the physical activity protocol and the timing of cognitive task performance.In conclusion, these data suggest that inhibitory control and neuroelectric underpinnings are not affected by different modalities of HIIE after 30 min. However, our findings do have useful implications for considering time-efficiency in relation to exercise, and for promoting short bouts of physical activity that do not interfere with cognitive functioning. The findings from this investigation expand the current understanding regarding acute effects of HIIE on cognition, which is important for future research addressing public health concerns from a psychological and physiological perspective in relation to HIIE. HIIE is a time-efficient physical activity modality that may be tailored to individual fitness levels, and which can improve cardiorespiratory fitness and reduce the risks of cardiovascular disease and Type II diabetes. Therefore, the current findings warrant additional investigation to further examine the impacts on cognition and other public health risks following acute bouts of HIIE to improve optimal health and well-being.


This dataset aims to evaluate the effect of different concentrations of amorphous silicon dioxide treatments on soilless-grown Kale . Data were obtained from fresh and dry samples. Total antioxidants capacity, total phenolic content, ascorbic acid, chlorophyll-a, chlorophyll-b, total carotenoids concentrations, total nitrogen, growth parameters and germination percentage varied in response to the concentrations of the used treatments (amorphous silicon dioxide compounds). Spectrophotometry, coulometric analyzer, capillary zone electrophoreses were the principal involved methods. Data of antioxidant capacity, total phenolic, and ascorbic acid contents can provide significant physiological health benefits as a functional superfood. Total carotenoids, Chlorophyll a and b concentrations, total nitrogen content, dry matter content, plants height, fresh weights, and the percentage of seeds germination contribute to the understanding of biometric and physiological plants growth parameters that indicates the effectiveness of the used treatments. This dataset compares the influence of soaking Kale seeds in different concentrations of amorphous silicon dioxide on the content and accumulation of phytonutrients including ascorbic acid, total nitrogen content, total antioxidant capacity, total phenolic content, pigments concentrations, dry weight, and germination percentages in Kale microgreens.22.1−1, 100 mg.L−1, 200 mg.L−1, 333 mg.L−1, respectively. Petri dishes were placed inside a dark closed constant climate cabinet  under a temperature of 22°C. Seeds were sown in growing substrates based on jute with dimensions of (17.5*47 cm) and placed in plastic pots and then placed in closed phytotron (ISR 0.1) conditions. The experiment was carried out on the 20th of August 2021 at Plekhanov Russian University of economics in the department of goods commodity and expertise of goods products, Moscow-Russian. Kale plants were drip-irrigated with mineral water Chernogolovka®  of the following contents in mg.L−1: HCO3− 122, Cl− 11, SO₄²- 36, Mg2+, K+ 28, Na+ 22. The pH of mineral water ranged between 6.7-6.9, The electrical Conductivity recorded 1.9 mS.cm−1 and total dissolved solids 261 mg.L−1. Upon the third day, germinated seeds were exposed to a photoperiod of 18 h and 23/16 °C (day/night) temperature inside the phytotron. Photosynthetic photon flux density (PPFD) of 50 µmol m−2 s−1with red: green: blue = 1:1:1 light-emitting diodes (LEDs). The experiment comprised of randomized complete block design (RCBD), replicated six times for each treatment. Kale seeds were grown for 14 days after germination. Upon harvesting, the cotyledon stems of Kale microgreens were cut using sterile scissors.One hundred Kale seeds were treated for 8 hours inside Petri dishes. In every petri dish Kale  seeds were soaked in 15 ml of gradual concentrations of amorphous silicon dioxide (SD) solutions, where: SD1, SD2, SD3, SD4 refers to 50 mg.L2.2Kale seeds were placed on dry Whatman filter papers in 28 cm diameter Petri dishes under 22°C temperature in a dark closed chamber. Exactly 100 seeds of Kale . Then 15 mL of each treatment solution was slowly poured inside the Petri dish. Petri dish covers were placed on the bottom parts and left for 3 days. Germination percentages were performed by calculating the number of germinated seeds. 6 replicates were used for each treatment.2.3Dry weight contents were measured by placing exactly 1.00 g of whole Kale microgreens in a moisture analyzer  until reaching constant weight under a constant temperature of 105 °C.2.43BO3 solution. Three drops of a mixed indicator containing methyl red and methylene blue were added to the distillate in a 50 mL Erlenmeyer flask and then titrated with 0.01 M HCl acid solution. The percent of total nitrogen (N) was calculated from the titer value and then was converted to grams of nitrogen per kilogram of dry weight (DW).Kale microgreens leaves were removed from their shoots, dried at a stable temperature of 70 °C for 72 h, and then macerated in a Wiley Mill before being used for the analysis. The total nitrogen content in the dried Kale microgreens samples was determined by the acid digestion method described by Bremner 2.5Galvanostatic coulometric analyzer  was used for total antioxidants capacity (AOC) in Kale microgreens samples using methods coulometric titration method with slight modifications 2.6Pigments concentrations in Kale microgreens leaves were determined spectrophotometrically using the methods and equations of Lichtenthaler and Wellburn ,2. Exact2.73 and thoroughly mixed for 10 min in the dark then it was filtered and placed in Eppendorf tubes and centrifuged twice under 12000 rpm for 10 min to ensure the absence of any impurities. The supernatant was replaced into the device for analysis under the conditions of high positive voltage polarity. The internal diameter of the silica capillary is 50 µm and has a total length of 50 cm. Sodium tetraborate 10 mM was used as a buffer pH 9.2. Samples injected applying a pressure of 450 mbar.s-1, Voltage: +20 kV. Ascorbic acid was detected at a wavelength of A254 nm, at 24°C.The content of ascorbic acid (Vitamin C) for the fresh samples was determined by capillary zone electrophoresis method ,7 using 2.82CO3 was poured into the mixture and incubated at 45°C for 20 min with intermittent agitation. After 90 min of incubation in the dark at ambient temperature, the absorbance was read at A765 nm using a spectrophotometer . Total phenolic content was expressed as mg gallic acid equivalents (GAE) per gram of dry mass of Kale microgreens samples using a gallic acid calibration curve with regression of (R2=0.978).The total phenolic contents for the dry Kale samples were analyzed using the modified Folin–Ciocalteu colorimetric method 2.9Raw Data analysis was subjected to one-way analysis of variance (ANOVA) using the IBM SPSS 25.0 version software . Means separation for all treatments were done by Duncan's test at 5% level of significance for separation of treatment means within each measured parameter There is no funding for the present effort. There is no conflict of interest. The data is available in public domain.Ali J. Othman: Conceptualization, Visualization, Methodology, Software, Formal analysis, Writing – original draft. Ludmila G. Eliseeva: Conceptualization, Visualization, Resources, Formal analysis, Writing – review & editing. Polina G. Molodkina: Methodology, Software, Formal analysis. Nazirya A. Ibragimova: Methodology, Software, Formal analysis. Fatima M. Duksi: Writing – original draft, Formal analysis, Writing – review & editing.The authors declare that they have no known competing financial interests or personal relationships which have, or could be perceived to have, influenced the work reported in this article.