Suspended solids were completely retained by the hollow fiber membranes while bioactive compounds, including polyphenols, anthocyanins, and resveratrol were recovered in the permeate stream. The clarified stream was then fractionated by nanofiltration (NF). Three different commercial membranes, in flat-sheet configuration , were selected and tested for their productivity and selectivity towards sugars and bioactive compounds, including phenolic compounds, anthocyanins, and resveratrol. All selected membranes showed high retention towards anthocyanins (higher than 93%). Therefore, they were considered suitable to concentrate anthocyanins from clarified wine lees extracts at low temperature. On the other hand, NF permeate streams resulted enriched in phenolic compounds and resveratrol. Among the selected membranes, the MPF36 exhibited the lowest retention towards resveratrol (10%) and polyphenols (26.3%) and the best separation factor between these compounds and anthocyanins.Polyvinylidenefluoride (PVDF) hollow fiber membranes prepared in laboratory through the inversion phase method were characterized and used to clarify an aqueous extract from red wine lees. Steady-state permeate fluxes of 53 kg/m The winemaking industry generates different residues characterized by high contents of biodegradable compounds and suspended solids . In partWine lees are deposits of dead yeasts, particulates and other precipitates formed at the bottom of wine containers after fermentation, during storage or after wine filtration. They represent approx. 2–6% of the total volume of wine produced .Conventional treatments of winery wastes are increasingly expensive, requiring significant amounts of effort, resources, and energy for safe discharge into the environment. On the other hand, wine lees contain phenolic compounds including low molecular weight acids, tannins, flavonols, anthocyanins, and resveratrol, well recognized for their potential beneficial effects on human health due to their antioxidant, antimicrobial, antiviral and anti-inflammatory activity . TherefoConventionally, the recovery of phenolic compounds from agro-food wastes and plant materials is based on the use of maceration assisted by liquid solvents , thus reIn this context, the development of “green” extraction and separation procedures is of great importance into industrial processes to reduce or eliminate the use and generation of hazardous substances. Innovative green analytical metrics, such as analytical eco-scale  and greeThe use of water as cheap, non-hazardous polar extraction solvent has attracted much interest in recent years. It has been shown to efficiently extract a wide variety of antioxidant compounds from plant materials . SimilarGiacobbo et al.  investigKontogiannopoulos et al.  evaluateGalanakis et al.  evaluateSustainable extractive technologies followed by membrane separation methods have been recently investigated to obtain commercial extracts of interest in the functional food industry, pharmaceutical and cosmetic fields from olives and artichoke by-products ,20.In the light of the aforementioned investigations, a combination of aqueous extraction and membrane operations was investigated on laboratory scale in order to develop a sustainable and environment-friendly approach for the extraction and purification of bioactive compounds from wine lees, in agreement with the principles of green chemistry. Specifically, the crude extract obtained from wine lees through an aqueous extraction at 45 °C was clarified by using poly(vinylidenefluoride) (PVDF) hollow fibers (HFs), prepared according to the phase inversion process. The clarified extract was then processed with three different commercial membranes in flat-sheet configuration and different molecular weight cut-off (MWCO) in order to evaluate and compare their potential to obtain high added-value extracts of interest in the formulation of natural antioxidant-based products. The performance of selected membranes was evaluated in terms of productivity and selectivity towards total phenols, anthocyanins, sugars, resveratrol and total antioxidant activity.®6010 supplied by Solvay Specialty Polymers . It is a white powder with a density of 1.78 gr/m3 and a melting temperature point between 165 and 177 °C. PVDF was chosen because it is an inert material suitable for use in contact with food products. Dimethylformammide (DMF), supplied by Sigma Aldrich , was used as a solvent for the solution. Polyvinilpyrrolidone  was supplied by BASF . Glycerol was supplied by Carlo Erba Reagents .Polyvinylidenefluoride (PVDF) was Solef®6010 25 wt.% in DMF. PVP Luviskol K17 was used as pore-forming additive, in the concentration of 35 wt.%. It was desiccated at 50 °C under vacuum overnight before use. The detailed conditions of HF spinning are resumed in PVDF HF membranes were prepared by the dry/wet spinning technique described by Figoli et al. . The polThe produced HFs were washed in distilled water at 50 °C for at least 24 h in order to remove residual solvent and soluble additive (PVP). Then, they were soaked in a glycerol/water solution (40 wt.%) for other 24 h in order to prevent pore collapse and finally dried at room temperature for 48 h.PVDF HFs morphology was evaluated by scanning electron microscopy (SEM) . Cross-sections were freeze-fractured in liquid nitrogen.mε), tensile stress at break (mR) and elongation at break (%) were measured using a ZWICK/ROELL Z 2.5 test unit, as described elsewhere [The mechanical properties of the HFs , and polD is the PVDF density (1.78 g/cm3).The average void fraction was calculated according to the following equation:A capillary flow porometer  was used for determining the pore size of the HFs, as described elsewhere . Tests w2) with epoxy resin .Pure water permeability (PWP) was determined by using a bench plant (DSS Lab Unit M 10) supplied by Danish Separation System  equipped with a membrane module prepared by embedding three HF membranes inside a 20 cm long glass tube  and at the exit (Pout) of the membrane module, a pressure control valve and a multitube heat exchanger fed with tap water. The water permeability was calculated from the slope of the straight lines fitted in the plot reporting the relationship between transmembrane water flux (J) and transmembrane pressure (TMP).Water permeability set-up was composed of a feed tank, a cross-flow pump (ECO type GA4-KDT-TTU), two manometers located at the entrance (PJ (expressed in kg/m2h) was calculated by using the following equation:s is the active membrane surface (m2), t is the operating time (h).w/w) and thermostated in a water bath at 45 °C for 1h . The aqueous extract was filtered on nylon cloth, and the procedure was then repeated until to obtain a final volume of 6.85 L of aqueous extract. It was used as feed solution in the clarification step.Red wine lees (Nero d’Avola variety) samples were collected during the 2015–2016 season from the bottom of a stainless steel wine stabilization tank. The collected wine lees were immediately transferred to the laboratory and stored in a freezer (−17 °C). After defrosting, about 800 g of wine lees were diluted with distilled water  in order to evaluate the effect of TMP on the permeate flux.f) of 580 mL/min and a temperature of 24 ± 2 °C. TMP was investigated in the range 0.3–1.2 bar. The duration of each experiment was 30 min.MF experiments were performed at an axial feed flowrate (Qf of 580 mL/min and a temperature of 24 ± 2 °C up to a weight reduction factor (WRF) of 4.5.The aqueous extract was clarified according to a batch concentration configuration (collecting separately the permeate stream and recycling the retentate stream in the feed reservoir) at a TMP of 0.8 bar, a QThe permeate stream was used as a feed solution in the fractionation step.After the treatment of the aqueous extract, PVDF membranes were cleaned with distilled water (at 40 °C for 30 min) in order to remove the polarized layer on the membrane surface. Then the fibers were cleaned with a solution of sodium hypochlorite  followed by a cleaning step with an enzymatic solution .0 and PWP1 are the water permeabilities measured before and after the treatment of the aqueous extract.The fouling index (FI) of PVDF membranes, expressed as percentage drop in water permeability, was estimated by measuring the water permeability before and after the treatment of wine lees extract, according to the following equation:4 is the water permeability measured after the enzymatic cleaning.The cleaning efficiency (CE) was evaluated by using the water flux recovery method, according to the following equation:The clarified extract was fractionated by using three different flat-sheet NF membranes with a molecular weight cut-off in the range 400–1000 Da. Membrane codes and technical specifications are reported in 2 and a processing capacity of 300 mL. It is capable of operating up to a maximum pressure of 68.9 bar. The stirred cell assembly consists of three primary components: A cylindrical body with removable top and bottom, a Teflon-coated stir bar assembly to provide mixing and a porous stainless steel membrane support disk. A nitrogen cylinder, equipped with a two-stage pressure regulator, was connected to the top of the stirred cell to supply the desired pressure for filtration experiments. The prevailing transmembrane pressure (TMP) was monitored by a manometer connected at the cell inlet. Stirring inside the cell was accomplished by using a magnetic stirrer.Filtration experiments were performed in dead-end filtration mode by using an experimental set-up comprising a high-pressure stirred cell filtration  with an effective membrane area of 13.85 cmAll experiments were performed by using 200 mL of a clarified extract with constant fluid agitation (350 rpm), at an operating pressure of 20 bar and at a temperature of 25 ± 2 °C, until to reach a WRF of 2.0.A schematic representation of the procedure used in this study is illustrated in Feed (F), permeate (P) and retentate (R) samples coming from filtration experiments were collected and analyzed in terms of suspended solids, total soluble solids, total polyphenols, total carbohydrates, total anthocyanins, and resveratrol.Suspended solids were determined by centrifuging 10 mL of a pre-weight sample at 2000 rpm for 20 min. The weight of settled solids was determined after removing the supernatant.Total soluble solids (TSS) measurements were carried out using a hand refractometer  with a scale range of 0–32 °Brix.The total polyphenols content was estimated by using the Folin–Ciocalteau method . In the Total carbohydrates were measured by using the phenol-sulfuric acid method . A sampl2S2O8) before the addition of the antioxidant [The total antioxidant activity (TAA) was determined by an improved version of the 2,2-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) assay  in whichioxidant . The decThe determination of total anthocyanins was carried out by spectrophotometric measurements ,30. Specv/v/v) [Resveratrol was assessed by high-performance liquid chromatography (HPLC) using a D-7000 HPLC system manager (Hitachi) equipped with a vacuum degasser, a quaternary pump, an autosampler, and a UV-Vis diode array detector. Chromatographic separation was performed by using a Luna C18(2) column  according to the following conditions: V = 1 mL/min, T = 25 °C, pressure = 90 bar, λ = 320 nm. Samples were eluted in isocratic mode by using a mixture of water/acetonitrile/acetic acid (70/29.9/0.1 v/v/v) .The peak areas in the chromatograms were plotted against calibration curves obtained from resveratrol standard solutions  in a concentration range of 50–300 ppm.p and cf are the permeate and feed solute concentrations, respectively.The rejection of selected membranes towards specific compounds was calculated according to the following equations, respectively:The cross-section of HF membranes showed a typical symmetric structure, mainly made up of parallel finger-like macrovoids a,b. ThisThe high percentage of PVP in the dope (35 wt.%) strongly increases its instability promoting faster coagulation and enhancing macrovoids growth (thermodynamic effect).In addition, the high percentage of both PVP and PVDF in the dope solution leads to very high viscosity at room temperature, which should delay the demixing process as a consequence of the kinetic hindrance. However, the HFs were spun at the temperature of 120 °C where the dope solution appeared visually fluid. Therefore, kinetic hindrance of macrovoids formation was not observed.As shown in The main properties of the prepared PVDF HFs are resumed in The produced fibers showed an average pore diameter of 0.13 μm which is typical of MF membranes. As reported in the literature, larger pore size can be obtained for fibers spun using water/solvent mixture as an inner coagulant and with slow solvent/non-solvent exchange .2hbar, in agreement with typical values of MF membranes.The measured water permeability of prepared membranes was of about 396 kg/mf) of 580 mL/min and a temperature of 24 ± 2 °C. The effect of TMP on the permeate flux was evaluated in experiments performed according to the total recycle configuration at an axial feed flowrate , of about 396 kg/m2hbar, was reduced to 174 kg/m2hbar after the treatment of the aqueous extract. The fouling index calculated according to Equation (3) was of 56.1%. The first cleaning with distilled water allowed to recover about 53% of the initial water permeability (213 kg/m2hbar), due to the removal of the reversible polarized layer. The chemical cleaning improved the water permeability up to 252 kg/m2hbar. The initial water permeability was totally recovered through the enzymatic cleaning , according to its lower MWCO. The MPF36 membrane showed a much higher productivity when compared with the NP010 membrane despite their similar MWCO. This behavior can be attributed to the higher hydrophilicity of the MPF36 membrane (contact angle 54°) in comparison to that of the NP010 one (contact angle 72°).The physicochemical composition of permeate and retentate streams obtained in the clarification of the aqueous extract is reported in Most of the bioactive compounds, including phenolics, anhocyanins and resveratrol were recovered in the clarified extract. Indeed, despite the high fouling index of the MF membrane (56.1%), the retention of phenolic compounds, anthocyanins and resveratrol were of 15.3%, 11.2%, and 14.2%, respectively. This can be attributed to the large pore diameter of the MF membrane allowing the diffusion of compounds with low molecular weights . Accordi2 and CF2 conferring a hydrophobic nature to the material. Therefore, they are less prone to form hydrogen bonds and Van der Waals interactions with the hydroxyl groups exhibited by polyphenols and, therefore, more resistant to fouling and highly permeable to phenolic compounds when compared with polysulphone membranes.Low retentions towards phenolic compounds (3.1%) were also measured in the clarification of pomegranate juice by using PVDF HFs with a pore size of 0.13 μm . PVDF meThe physico-chemical characterization of permeate samples produced in the treatment of the clarified extract with NF membranes is reported in The rejection coefficients measured towards different compounds for the investigated membranes are reported in As a general trend, the rejection towards all detected compounds increased according to the following order:Anthocyanins retentions were significantly higher than 93% for all selected membranes with the NP030 exhibiting the highest value (99%): Therefore, these membranes appear to be suitable for concentrating the clarified extract and producing an added value concentrate of anthocyanins. Similar results were found by Cissé et al.  in the cOn the other hand, the retention of selected membranes towards phenolic compounds and resveratrol resulted lower than 34% and 30%, respectively. Accordingly, most part of phenolic compounds and resveratrol can be recovered in the permeate fraction. In particular, the MPF36 membrane exhibited the lowest retention towards resveratrol (10%) and polyphenols (26.3%) when compared with the other two membranes and the best separation factor between these compounds and anthocyanins.Retention values for TAA were in the range of 29–35%, in agreement with those observed for polyphenols: These results corroborated the strict correlation between the phenolic content and the antioxidant activity measured in white and red wines .The rejection of phenolic compounds can be explained not only on the basis of the MWCO of the selected membranes. Indeed, these compounds generally include aromatic (benzene) ring structures that have aliphatic carbon groups which, if undissociated in pH conditions lower than their pKa values, can be readily adsorbed on the membrane surface . The incAccording to rejection data, the investigated membranes did not show a preferential rejection of phenolic compounds over sugars. Similar results were found by Diaz-Reinoso et al.  in the uThe whole results indicate that the combination of MF and NF membranes is an attractive alternative process for producing, at low temperatures, concentrated extracts of anthocyanins from wine lees without thermal damage before final concentration  or spray drying. They can be considered a new source of anthocyanins for various applications in the food industry, as a natural colorant in alternative to synthetic ones, as nutraceuticals or in the pharmaceutical industry for new functional formulations.Permeate streams containing resveratrol, and phenolic compounds appear also of interest for the formulation of natural products containing antioxidant compounds.2h were measured in the in optimized conditions of transmembrane pressure when the extract was processed according to a batch concentration configuration. Suspended solids were completely removed from the extract, producing a clarified solution enriched in sugars and bioactive compounds including phenolic compounds, anthocyanins, and resveratrol.A green integrated approach applying sustainable extraction of red wine lees and membrane-based clarification and fractionation of aqueous extracts was proposed. PVDF membranes in hollow fiber configuration prepared according to the phase inversion method were characterized and used to clarify the aqueous extract. The prepared membranes showed an average pore diameter of 0.13 μm and a high porosity (81.0 ± 4.5 %) in agreement with their finger-like morphology. Steady-state permeate fluxes of about 53 kg/mThe clarified solution was then fractionated by using commercial NF membranes in flat-sheet configuration. All selected membranes resulted suitable for concentrating anthocyanins from the clarified extract (retention higher than 93%) with the NP030 exhibiting the highest retention (99%). Therefore, these membranes appeared suitable to concentrate anthocyanins from clarified wine lees extracts at low temperature before final concentration by vacuum evaporation or spray drying. Final products could be of interest for the use as natural colorants and/or for nutraceutical applications. Among the selected membranes, the MPF36 showed the lowest retention towards phenolic compounds and resveratrol  with a production of a permeate fraction of interest for the formulation of natural antioxidant-based products.The innovative integrated approach allows to obtain individual fractions enriched in bioactive compounds of interest for specific applications in pharmaceutical, cosmetic, food, and functional food industries. Overall, it turns up as a clean and a low-cost method to valorize red wine lees through the recovery of natural ingredients within the logic of both process intensification and zero discharge strategies.


Adipose-derived mesenchymal stem cells (ADSCs) have become a common cell source for cell transplantation therapy. Clinical studies have used ADSCs to develop treatments for tissue fibrosis, such as liver cirrhosis and pulmonary fibroma. The need to examine and compare basic research data using clinical research data derived from mice and humans is expected to increase in the future. Here, to better characterize the cells, the protein components expressed by human ADSCs used for treatment, and mouse ADSCs used for research, were comprehensively analyzed by liquid chromatography with tandem mass spectrometry. We found that 92% (401 type proteins) of the proteins expressed by ADSCs in humans and mice were consistent. When classified by the protein functions in a gene ontology analysis, the items that differed by >5% between human and mouse ADSCs were “biological adhesion, locomotion” in biological processes, “plasma membrane” in cellular components, and “antioxidant activity, molecular transducer activity” in molecular functions. Most of the listed proteins were sensitive to cell isolation processes. These results show that the proteins expressed by human and murine ADSCs showed a high degree of correlation. Mesenchymal stem cells (MSCs)  are a soBasic research to elucidate the characteristics and functions of ADSCs in detail using experimental animals such as mice is extremely important for confirming the safety and efficacy of ADSC cell therapy for humans. However, it is often noted that the results of basic research using mice contradict the results of clinical studies targeting humans. Thus, experimental results from the comparison of the proteins expressed in mouse and human ADSCs would likely be useful as preliminary data when cell therapy using ADSCs is performed in humans. Gene Ontology (GO), developed at the GO Consortium, provides genomic sequencing and annotation tools related to the functions of genes or “GO terms”. GO describes these functions based on their activity, location, and connected processes. In the past 18 years, the GO subsets have been well established and have been widely used in various studies, including (but not limited to) studies involving the use of mice, yeast, nematodes, and meta-genomics. GO facilitates the development of a common vocabulary to describe biological concepts . Since tThree types of cell, tissue, and organ transplantation are performed: autotransplantation, where a part of a person is transplanted to another part of the same person; allotransplantation, where a part is transplanted from one individual to another individual of the same species; and xenotransplantation, where a part is transplanted from an individual of one species to an individual of another species. Among these types of transplantation, allotransplantation is associated with a risk of an immune rejection reaction due to a blood type mismatch of leukocytes called human leukocyte antigen (HLA). In order to solve this problem, the usefulness of therapeutic cells using cells with HLA homozygotes, which are less susceptible to immunological rejection, has been indicated. In xenotransplantation, in addition to differences in HLA, there is a risk of severe immuno-elimination as a result of differences between the species of cell surface markers. Many causes of immune rejection among heterologous cells are due to species differences of different cell surface markers. There are cases in which cell surface marker variations differ and the protein surface structure of cell surface markers differ according to species differences. As examples of the former issue, there are reports of many species differences in hADSCs (CD45−/CD31−/CD34+) ,11,12 anIn this work, we studied adipose-derived mesenchymal stem cells (ADSCs) using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and categorically compared the characteristics with the available GO data on ADSC cells.hADSCs were cultured to 80% confluence using Dulbecco’s modified Eagle medium (DMEM) medium containing 10% fetal bovine serum (FBS). We observed no abnormalities in cell size, shape, or culture state with a normal microscope  were unique to hADSCs , DPP4 (Dipeptidyl peptidase 4), ENG (Endoglin), ENO2, FAP ), LMO7, MME, PAPSS2, SCRN1, STAT1, TLN2, TPD52L2). A total of 401 proteins in mADSCs were also identified in hADSCs (both hADSCs and mADSCs). Of the 413 proteins identified from mADSCs, 12 (3%) were specific to mADSCs . Thus, the highly expressed proteins of hADSCs and mADSCs were 92% identical .http://www.geneontology.org/) is a database of functional information aimed at describing biological phenomena in standardized terms. In addition, it is designed to capture biological phenomena exhaustively regardless of the species, with GO terms largely represented by three subcategories: biological processes, cellular components, and molecular functions. The proteins uniquely expressed in hADSCs were classified by a GO analysis  of the proteins expressed by ADSCs in humans and mice were consistent. Recently, in a comprehensive protein expression analysis using the iTRAQ measurement method , in whicIn this study, mADSCs were isolated from adipose tissue by almost the same protocol that is used to isolate hADSCs for clinical use. Then, using a medium composed of the same ingredients, cells that were passaged three times were used as samples for a comprehensive protein expression analysis. However, because of the small amount of adipose tissue in mice, the number of cell divisions in both hADSCs and mADSCs, even when the number of passages is the same. Indeed, it is well known that the culturing and passaging of adipose progenitors will lead to homogenization . Recentlhttps://www.proteinatlas.org/ENSG00000085733-CTTN/tissue), it can be confirmed that in addition to being expressed in adipose tissue, CTTN mRNA and protein are expressed in various organs of the body.A notable finding of this study is that hADSCs expressed eight proteins  related to cell adhesion, which was more than mADSCs . According to previous studies, the function of TLN2 expressed in hADSCs is classified as “migration and angiogenesis promoting”. It was reported that the expression of TLN2 in hADSCs was increased by the administration of IL-2 . PreviouIn addition, hADSCs expressed six proteins  related to locomotion, which was more than mADSCs . mADSCs also expressed one protein related to the rhythmic process (Mybbp1a). hADSCs expressed nine proteins classified as cellular components in the plasma membrane , while mADSCs expressed four such proteins . These results show that 92% of the highly expressed proteins corresponded between hADSCs and mADSCs, and a GO analysis of the 8% of mismatched proteins primarily turned up cell surface proteins involved in cell adhesion. The cell membrane proteins and cell adhesion-related proteins might have been affected by the methods that were used to isolate and separate hADSCs and mADSCs. The hADSCs and mADSCs express macrophage migration inhibitory factor (MIF) protein . MIF is In this study, mADSCs were prepared in the same way as hADSCs are prepared for human clinical studies, and the two cell types were cultured under the same conditions. As a result, the patterns of proteins expressed by hADSCs and mADSCs were highly similar. This result therefore provides a rationale for the utilization of basic research data obtained using hADSC  before researchers and doctors work on human clinical research using hADSCs. Nevertheless, the data from the present study cannot be used to predict whether the protein components expressed in MSCs derived from mice or other tissues taken from human beings will be similar, and additional research is necessary.A recent paper reported that symptoms in a mouse model of acute liver failure model were improved using a secretory protein of hADSCs . In thisFetal bovine serum (FBS) was obtained from BioWest . Dulbecco’s modified Eagle medium (DMEM)  was obtained from Wako Pure Chemical Industries . hADSCs  were purchased from PromoCell . The plastic dishes were obtained from TPP .ad libitum access to water. All efforts were made to minimize the suffering of the animals.All experimental protocols were performed according to the guidelines for the care and use of laboratory animals set by the Research Laboratory Center, Faculty of Medicine, and the Institute of Animal Experiments, Faculty of Medicine, University of the Ryukyus . The experimental protocol was approved by the Committee on Animal Experiments of University of the Ryukyus (permit number: A2017101). C57BL/6 male mice  were housed in a controlled environment . Animals were fed standard rodent chew pellets with g for 5 min followed by the removal of the upper fibrous layer and supernatant. The cell pellet was suspended in fresh DMEM containing 10% FBS to inhibit collagenase activity, followed by filtration through a 100-μm cell strainer. Filtered cells were then incubated in a T25 flask using DMEM containing 10% FBS. All mouse studies were approved by the Institutional Animal Care and Use Committee of the University of the Ryukyus.Adipose tissue specimens (ATs) were collected from the inguinal fat pad of 8-week-old C57BL/6 mice. The method of isolating MSC-AT from adipose tissue was in accordance with the ADSCs product standard document (RMRC-A 01: 2015) of the Ryukyus Regenerative Medicine Research Center. In brief, ATs were stored in cold Hanks’ balanced salt solution (HBSS) after collection and washed three times using HBSS before starting the digestion procedure. The ATs were then minced using scissors and tweezers to enhance enzymatic digestion. Digestion was performed in 2 mg/mL collagenase type IV  and HBSS in a 50-mL centrifuge tube at 37 °C in a shaker  at 20 rpm with manual shaking at 10-min intervals. After completing digestion, centrifugation was performed at 800× 2). The medium was completely exchanged every two days. The passage of cells was performed every 3 to 4 days after reaching 80% confluence. After reaching 80–90% confluence, the cells were washed with Dulbecco’s phosphate-buffered saline , then the adhered cells were dissociated using 0.25% trypsin and 1 mM EDTA . Subculturing was carried out in a non-coated T25 flask using DMEM containing 10% FBS. We performed three protein analyses of mADSCs and used their representative data in this experiment. The mADSCs were cryopreserved at passage 2, then thawed again and used for the experiments. mADSCs were cultured in a non-coated T25 flask (TPP 90026), with the appropriate culture conditions maintained . In brief, ADSCs (1 × 105 cells) were mixed with 0.5 mL of Perfusion Solution . Each antibody (1/100 of the volume) was added to the cell mixture, which was then incubated on ice for 30 min. After washing the cells with Brilliant Stain Buffer , Fluorescence activated cell sorting (FACS) was performed. The following primary antibodies were used: Brilliant Violet 421TM Rat Anti-Mouse CD44 (BD Biosciences), Fluorescein Isothiocyanate (FITC) Rat Anti-Mouse CD90.2 (BD Biosciences), PerCP/Cy5.5 Anti-Mouse CD34 , and PE/Cy7 Rat Anti-Mouse CD45 (BD Biosciences). Isotype-identical antibodies were used as controls.Mouse ADSCs (passage 3) were used for a cell quality inspection and LC-MS/MS. The quality of the cells was assessed using fluorescein-coupled antibodies against CD90, CD34, CD44, and CD45 with a Novocyte® flow cytometer . In brief, ADSCs (1 × 105 cells) were mixed with 0.5 mL of Perfusion Solution . Each antibody (1/100 of the volume) was added to the cell mixture, which was then incubated on ice for 30 min. After washing the cells with Brilliant Stain Buffer (BD Biosciences), FACS was performed. The following primary antibodies were used: BV421 Mouse Anti-Human CD44, FITC anti-human CD90 (Thy1) Antibody, PerCP anti-human CD34 Antibody, PE/Cy7 anti-human CD45 Antibody . Isotype-identical antibodies were used as controls.We regularly produce clinical hADSCs at our university. However, when patient cells are used in a protein expression analysis, the patient’s age, gender, and disease background are expected to influence the cell state. Thus, in this study, we used quality assured hADSCs that are commercially available as research hADSCs. The hADSCs were cryopreserved at passage 2, then thawed again and used for experiments. Commercially available hADSCs were cultured using DMEM containing 10% FBS under appropriate culture conditions. The medium was completely exchanged every two days. The passaging of cells was performed every 3 to 4 days after reaching 80% confluence. After reaching 80–90% confluence, the cells were washed with Dulbecco’s phosphate-buffered saline , then the adhered cells were dissociated using 0.25% trypsin and 1 mM EDTA . Passage three hADSCs were used for a cell quality inspection and LC-MS/MS. The quality of the cells was assessed using fluorescein-coupled antibodies against CD90, CD34, CD44, and CD45 with a Novocyte4 cells/well. On days 3 and 5, the cells were counted using a Luna™ Automated Hemocytometer . The relative cell proliferation rate was calculated by converting the number of cells on the day of cell seeding to 100%.The cells were seeded onto 24-well plates at a density of 5.0 × 10TM Mesenchymal Stem Cell Adipogenic Differentiation Medium  and a Lipid Assay Kit  according to the manufacturer’s instructions. Osteogenic differentiation was performed using OriCell™ Mesenchymal Stem Cell Osteogenic Differentiation Medium  and a Calcified Nodule Staining Kit  according to the manufacturer’s instructions.Adipogenic differentiation was performed using OriCellImmunofluorescence staining was performed using a method introduced in our previous paper . The hAD6 cells each after culturing, and then 300 μL of EzRIPA Lysis solution was added to the cell pellet. A protein solution of 3105 μg/mL was obtained from the mADSCs. A protein solution (3800 μg/mL) was obtained from hADSCs, and 6.0 μg of protein was used for sample preparation. Finally, 0.4 μg of protein was used for nanoLC-MS/MS. Whole-cell protein extraction was performed using an EzRIPA Lysis kit in accordance with the manufacturer’s instructions . The comprehensive expression analysis of proteins using LC-MS/MS was performed according to a previously reported method [Both mADSCs and hADSCs were pelleted at 3 × 10d method ,31. In td method ,33,34,35Tandem mass spectrometry was performed using the Proteome Discoverer software program . Charge state deconvolution and deisotoping were not performed. All MS/MS samples were analyzed using the Mascot software program . Mascot was set up to search the SwissProt_2017_12 database  using a protein sample that had been degraded to a peptide sequence by digestive enzyme trypsin. Mascot was searched with a fragment ion mass tolerance of 0.60 Da and a parent ion tolerance of 5.0 PPM. Deamidation of asparagine and glutamine, oxidation of methionine, and carbamidomethylation of cysteine were specified as variable modifications in Mascot.The Scaffold software program  was used to validate the MS/MS-based peptide and protein identifications. Peptide identifications were accepted if the Scaffold Local FDR algorithm established that the probability of achieving an FDR of <1.0% was 95.0%. Protein identifications were accepted if the probability was >96.0% and they contained at least 1 identified peptide. Protein probabilities were assigned by the Protein Prophet algorithm. Proteins that contained similar peptides and which could not be differentiated based on an MS/MS analysis alone were grouped to satisfy the principles of parsimony. Proteins sharing significant peptide evidence were grouped into clusters.http://www.proteomesoftware.com/products/scaffold/) with imported data ) [The protein GO analysis was performed using the GO analysis function of the Scaffold 4 software program  (r 2016))  from theProteins expressed in hADSCs and mADSCs were subjected to a comprehensive protein expression analysis using LC-MS/MS. GO classification was also performed using proteins with an emPAI value of 10 (high-concentration proteins). As a result, 92% of the expressed proteins were found in both hADSCs and mADSCs. Furthermore, most of the 8% mismatched proteins identified by the GO analysis were related to cell adhesion on the cell surface. The above results show that the types and functions of the proteins that were highly expressed in hADSC and mADSC are similar.


Nigella sativa, this systematic review aims to provide comprehensive information on the effects of Nigella sativa on glucose and insulin profile status in humans. A computerized database search performed through Scopus and Medline via Ebscohost with the following set of keywords: Nigella Sativa OR black seed oil OR thymoquinone OR black cumin AND diabetes mellitus OR hyperglycemia OR blood glucose OR hemoglobin A1C had returned 875 relevant articles. A total of seven articles were retrieved for further assessment and underwent data extraction to be included in this review. Nigella sativa was shown to significantly improve laboratory parameters of hyperglycemia and diabetes control after treatment with a significant fall in fasting blood glucose, blood glucose level 2 h postprandial, glycated hemoglobin, and insulin resistance, and a rise in serum insulin. In conclusion, these findings suggested that Nigella sativa could be used as an adjuvant for oral antidiabetic drugs in diabetes control.Diabetes mellitus is one of the most prevalent metabolic disorders that affect people of all genders, ages, and races. Medicinal herbs have gained wide attention from researchers and have been considered to be a beneficial adjuvant agent to oral antidiabetic drugs because of their integrated effects. Concerning the various beneficial effects of  Diabetes mellitus (DM) is one of the most prevalent metabolic disorders that occur throughout the world, which affects people of all genders, ages, and races. Type-2 diabetes mellitus (T2DM) accounts for 90% of all DM cases as a result of the interaction between genetic predisposition and environmental factors . T2DM isNigella sativa (NS) has also been considered to be safer compared to oral antidiabetic drugs [Besides that, T2DM can also be associated with cardiovascular risk factors such as dyslipidemia, hypertension, and obesity. Measurements of fasting blood glucose (FBG), blood glucose level 2 h postprandial (2hPG), and glycated hemoglobin (HbA1c) have been used to assess glycemic control in patients with T2DM . Medicinic drugs . NS is aic drugs ,9. NS waic drugs . Screening for novel bioactive compounds from NS has gained researchers’ attention due to its therapeutic effects. The therapeutic effects of NS are mainly contributed to thymoquinone, which is one of the major bioactive compounds that was discovered to have a protective effect against diabetes . PreviouConcerning the various beneficial effects of NS, this systematic review was performed to provide comprehensive information on the effect of supplementation of NS in combination with oral antidiabetic drugs on glucose and insulin profile status in humans. Besides that, it may help to establish NS as a beneficial adjuvant to oral antidiabetic drugs for diabetes patients. Nigella sativa (NS) on Type-2 diabetes mellitus (T2DM). To conduct a comprehensive search of biomedical science journals, two databases, namely Scopus (published between 1823 and November 2019) and Medline via Ebscohost (published between 1865 and November 2019) were used. The search strategy involved a combination of the following two sets of keywords Nigella sativa OR black seed oil OR thymoquinone OR black cumin AND diabetes mellitus OR hyperglycemia OR blood glucose OR hemoglobin A1CA systematic review of the literature was conducted to identify relevant studies about the reported effect of The results were limited to original articles that were published in English and included abstracts. Review articles, news, case reports, and other original articles not associated with NS and T2DM were excluded from the review. For this review, only studies that reported the association of NS and T2DM were included.For this review, only studies that reported the direct effect of NS on T2DM was included. The inclusion criteria were as follows: (i) Clinical human studies including but not limited to observational study, randomized controlled trial, AND double-blind randomized controlled trial or single-blind, non-randomized controlled trial design, AND (ii) studies involving human adult subjects, AND (iii) studies examining the effects of a crude plant preparation and not its bioactive components, AND (iv) research that reported sufficient information on at least one T2DM blood parameter, such as fasting blood glucose, HbA1c or blood glucose level 2 h postprandial (2hPG), AND (v) studies that examined the antidiabetic effects of any form of NS .The following exclusions were also considered: (i) studies that investigated the impacts of an individual bioactive component of NS, OR (ii) studies on NS in combination with other herbs of ingredients as a mixture, OR (iii) studies on animals, OR (iv) studies on Type-1 diabetes mellitus (T1DM) or gestational diabetes (GDM).Papers were screened in three phases before they were included in the review. In the first phase, studies that did not match the inclusion criteria based solely on the title were excluded. In the second phase, abstracts of the remaining studies were screened, and studies that did not meet the inclusion criteria were excluded. In order to standardize the data collection, all data extraction was performed independently with the use of a data extraction form. The following data were recorded from the studies: Type of NS used, treatment groups, biochemical parameters measured, a summary of T2DM related outcome measures, and a conclusion of the study.Nigella sativa (NS) or its bioactive compound and were not related to T2DM. The search was limited to original articles written in the English, with an abstract available, and studies only in humans with T2DM and combined treatment of an oral antidiabetic drug with NS. From the remaining nine articles, one duplicate article and one systematic review article was removed before the full papers were retrieved for thorough reading. A total of seven articles were retrieved for further assessment and underwent data extraction to be included in this review. A flow chart of the selection process, including reasons for exclusion, is shown in The literature searches identified 875 potentially relevant articles. Of those, 866 articles were excluded because they were not associated with n = 3) [n = 2) [n = 1) [n = 1) [The summary of the characteristics of all studies is displayed in n = 3) ,16,17, c [n = 2) ,19, powd [n = 1) , and tea [n = 1) . DiffereIn this study, we reviewed the analysis of blood samples such as fasting blood glucose (FBG), blood glucose level 2 h postprandial (2hPG), glycated hemoglobin (HbA1c), as well as insulin level and insulin resistance.From the seven studies reviewed, six studies measured the changes of the fasting blood glucose (FBG) parameter. According to Ahmad et al. (2009), FBG levels significantly decreased after treatment with NS oil for 40 days . Bamosa Of the seven studies reviewed, three articles studied the blood glucose level 2 h postprandial (2hPG) parameter. Bamosa et al. (2010) reported a significant decrease in 2hPG after 4 and 8 weeks of treatment with 2 g/day NS . El-ShamOf the seven papers reviewed, six articles investigated HbA1c. Bamosa et al. 2010) reported a significant decrease in HbA1c levels following 12 weeks treatment with 2 g/day and 3 g/day of NS . El Sham10 reportβ-cell function after 12 weeks of treatment [Of the seven papers reviewed, four articles studied insulin level and insulin resistance. Ahmad et al. (2009) showed that the insulin levels significantly increased after treatment with NS oil for 40 d . NS at dreatment . Accordireatment . Meanwhireatment .Nigella sativa (NS) has a promising future in the prevention and treatment of diabetic diseases due to its capacity to treat multiple diseases, as well as being cost-efficient and having fewer side effects compared to synthetic medicines [Plants have long been used as medicinal herbs in Arabian countries, Asia, and Africa . To dateedicines . In this systematic review, we have assessed the effects of NS supplementation on fasting blood glucose (FBG), blood glucose level 2 h postprandial (2hPG), glycated hemoglobin (HbA1c), insulin level, and insulin resistance. NS was given as an adjuvant therapy in patients with Type-2 diabetes mellitus (T2DM) in addition to their oral antidiabetic drug. The literature search revealed six studies that showed treatment with different forms of NS caused marked reduction in the level of FBG and HbA1c compared to the control group ,18,19,21It has been reported that chronic elevation of blood glucose promotes oxidative stress through overproduction of reactive oxygen species (ROS). Excessive levels of ROS cause an increase in insulin resistance and β-cell dysfunction, thus contributing to the advancement of diabetic complications ,31,32. PMost of the studies reviewed demonstrated NS supplementation combined with oral antidiabetic drugs enhanced glycemic control manifested by improvement in pancreatic β-cell function and insulin resistance compared to the corresponding baseline. Based on findings by Bamosa et al. (2010), the insulin resistance was significantly reduced by 2 g daily supplementation of NS . The samHypoglycemic effects of NS have been attributed to its antioxidant properties . AntioxiNigella sativa such as oil, water extract, and powder produce similar hypoglycemic effects, in which these treatments resulted in significantly reduced HbA1c levels. This finding suggests that other constituents in Nigella sativa seed also possess the same beneficial properties as thymoquinone which is a major component of NS oil fraction [This systematic review also revealed different that forms of fraction .Some limitations are notable in this systematic review and must be considered. This current study included a small number of relevant studies. Besides that, all included studies utilized different preparations, doses, and duration of supplementation. Thus, our findings on the efficacies of supplementation of NS in combination with oral antidiabetic drugs should be interpreted with caution.All studies included in this review reported positive effect of NS on T2DM. NS was showed to improve laboratory parameters of hyperglycemia and diabetes control with a significant fall in fasting blood glucose, and a significant rise in serum insulin. These findings suggest that NS could be used as an adjuvant for oral antidiabetic drugs in diabetes control. However, further studies are needed since it is difficult to determine effective type and dosage of NS in diabetes management due to chemical compositions of different sources of NS, dosage, and duration of intervention.


The micronutrients vitamins B9 and B12 act as methyl donors in the one-carbon metabolism involved in transmethylation reactions which critically influence epigenetic mechanisms and gene expression. Both vitamins are essential for proper development, and their deficiency during pregnancy has been associated with a wide range of disorders, including persisting growth retardation. Energy homeostasis and feeding are centrally regulated by the hypothalamus which integrates peripheral signals and acts through several orexigenic and anorexigenic mediators. We studied this regulating system in a rat model of methyl donor deficiency during gestation and lactation. At weaning, a predominance of the anorexigenic pathway was observed in deficient pups, with increased plasma peptide YY and increased hypothalamic pro-opiomelanocortin (POMC) mRNA, in line with abnormal leptin, ghrelin, and insulin secretion and/or signaling during critical periods of fetal and/or postnatal development of the hypothalamus. These results suggest that early methyl donor deficiency can affect the development and function of energy balance circuits, resulting in growth and weight deficits. Maternal administration of folic acid (3 mg/kg/day) during the perinatal period tended to rectify peripheral metabolic signaling and central neuropeptide and receptor expression, leading to reduced growth retardation. The nutritional methyl donors folate vitamin B9) and vitamin B12 are cofactors in the one-carbon metabolism that plays a critical role in transmethylation reactions ,4,5,6. B and vitaGiven our previous observations and according to the concept stating that an adverse perinatal environment programs the development of several tissues, with long-term consequences on physiology and health ,23, we hIn addition, we investigated whether a perinatal supplementation with folic acid is able to reduce growth retardation in deprived rats and its influence on neuropeptide expression.Taken together, our data showed that in utero and early postnatal deficiency in folate and vitamin B12 was associated with growth retardation, in line with misexpression of hypothalamic neuropeptides and related receptors regulating energy homeostasis, suggesting impaired development of hypothalamic networks. Importantly, perinatal and postnatal supplementation with folic acid helped restore mRNA levels and could be an effective strategy to reduce deficiency-related defects.The profiles of expression changes in response to B9 deprivation reflect the specificities of mouse and rat cell lines .RT-qPCR experiments led to two striking observations: the expression patterns of neuropeptides and receptors differed between the two cell lines on the one hand, and on the other, they varied as a function of time in a given cell line. We observed that the rat cell line is a hypothalamic cell line secreting ghrelin and expressing receptors for NPY, insulin and leptin whereas the mouse cell line is an NPY/AgRP-secreting cell line. After 24 h of B9 deficiency, the expression of the AgRP gene was increased in the mouse cell line while the expression of NPY and of the various receptors was decreased. In the rat cell line, a 24 h B9 deficiency induced an overexpression of the genes encoding ghrelin and the different receptors. These results reflect the direct effect of B9 deficiency on hypothalamic neuropeptides and receptors’ gene expression and show that receptor expression varied depending on the neuropeptide secretion by the cells.As previously documented, nutritional methyl donor deficiency starting 1 month prior to mating affected female ability to conceive. Globally, 45.7% gave birth to pups (vs. 84% in controls). The number of live fetuses per litter was consistently reduced (6.7 vs. 11.2) . In addip < 0.01). In response to maternal deficiency during gestation and lactation, plasma levels of folate and vitamin B12 were dramatically reduced in the rat progeny at weaning . In parallel, homocysteinemia was significantly augmented  and anorexigenic pathways (POMC mRNA) appeared to be stimulated. However, immunohistochemical analyses of the arcuate nucleus revealed that NPY mRNA in excess was not translated, since the protein labelling was lower than control. This could be the result of specific microRNA regulation as documented in the literature ,40. ReceThe hypothalamus develops early during gestation and continues during the postnatal period . These d2. For homogeneity, the same lot of serum was used throughout all experiments. The cells grew to form a monolayer culture, attached to the culture plate and were split when they reached 70–90% confluence, with a plate ratio of 1:5. Because standard DMEM does not contain vitamin B12, methyl donor deficiency was induced by using a poor medium  lacking B9 (folic acid), with addition of 2 mM glutamine , 3.7% sodium bicarbonate , 0.35% glucose , 10% fetal bovine serum and 1% penicillin/streptomycin. Cells were kept in B9-free conditions for 24 or 48 h before subsequent analyses.mHypoE46 (mouse) et rHypoE11 (rat) neuronal cell lines purchased from American Type Culture Collection (ATCC) were conditionally immortalized by transfer of a temperature-sensitive simian virus 40 large tumor (SV40 T) antigen to primary hypothalamic neuronal cell cultures obtained from fetal mice on embryonic days E15, E17 and E18, and from E18 fetal rats . Cells wn = 5 per experimental group matched to one male for two females for mating) were fed either a standard diet  or a diet deficient in folate and vitamin B12 and lowered in choline . Methionine content (~0.4%) was similar in both diets. For maternal supplementation, folic acid  diluted in condensed milk was given per os. at the dose of 3 mg/kg per day in a final volume of 1 mL to dams from embryonic day 13 to postnatal day 21 (weaning). Matched control dams received the same volume of vehicle  over the same period, as previously described [n ≤ 40 issued from at least three different litters per experimental group) were euthanized between 8:00 and 10:00 by excess isoflurane at 21 days of age and blood was withdrawn for subsequent plasma measurements. Individuals were weighed and evaluated morphologically. The brains were rapidly collected, weighed, and the hypothalamus was carefully dissected before freezing in liquid nitrogen and storage at –80 °C. For immunochemistry, brains were fixed with 4%-paraformaldehyde 4% (24–48 h) at 4 °C, dehydrated and included in paraffin. Sagittal brain sections (12 µm) were generated at the level of hypothalamus by means of a microtome. They were then mounted onto glass slides and stored at ambient temperature.Animal experiments were conducted by using a validated rat model of early methyl donor deficiency, as previously described ,44. Theyescribed   as previously reported . ConcentPlasma levels of total ghrelin was measured in duplicate after appropriate dilution by specific radioimmunoassays using commercial kits , as previously described . Leptin,® RNA Plus, Macherey Nagel, Düren, Germany). In the case of rat hypothalamus tissues, RNA extraction was performed with TRIzol®  according to the manufacturer’s instructions. The concentration and purity of RNA were determined at 260/280 nm by using a nanodrop spectrophotometer Multiskan GO .RNA was purified from cell cultures by using a commercial kit  following the manufacturer’s instructions. Primers are detailed in Ct) was determined from each sample and real-time PCR amplification efficiencies were expressed by calculating the ratio of crossing points of amplification curves. The expression of genes of interest was normalized to those of GAPDH/RPS29 for the rat species and Pol2/RPS29 for the mouse species using the 2Ct−ΔΔ method.RNA (300 ng) was then subjected to a two-step RT-qPCR using the PrimeScript™ RT Master Mix and SYBR® software .Immunohistological analyses were performed on brain sections at the level of the arcuate nucleus of the hypothalamus according to the Paxinos and Watson rat brain atlas . Nonspecp-value < 0.05 was considered to indicate significance.Data were analyzed with StatView 5 software for Windows . They were compared by using one-way analysis of variance (ANOVA) with Fisher’s test. A The results of the present study suggest that early methyl donor deficiency can impair the hypothalamus development, with altered expression of neuropeptides and related receptors regulating eating behavior and energy homeostasis, leading to growth retardation. If maternal supplementation with folic acid during the perinatal period could not fully reverse the deleterious consequences of deficiency, it was shown to exert beneficial effects.


Here we argue that, in the anoxic Archean oceans, pyrite could form in the absence of ambient sulfate from organic sulfur contained within living cells. Sulfides could be produced through mineralization of reduced sulfur compounds or reduction of organic-sourced sulfite. Reactive transport modeling suggests that, for sulfate concentrations up to tens of micromolar, organic sulfur would have supported 20 to 100% of sedimentary pyrite precipitation and up to 75% of microbial sulfur reduction. The results offer an alternative explanation for the low range of δ Marine chemistry during the Early Earth (over 2.7 billion years ago) is commonly inferred to have been inorganically sulfate-reducing. Here, the authors argue that organic sulfur cycling may have played a previously unrecognized, yet important, role in the formation of ancient Archean marine sulfides. In modern oceans, however, the petagram inventory4 of dissolved organic sulfur and the estimated 0.2–0.4 Pg of particulate organic sulfur4 are dwarfed by the 109 Pg of inorganic sulfate, which at 28 mM is the second most abundant anion in seawater. At these concentrations, the geologically important cycling of sulfur through sulfate reduction, precipitation of pyrite, and reoxidation of sulfides is carried out overwhelmingly by inorganic sulfur. Abundant sulfate, however, was rare through most of the Earth’s history. Proterozoic oceans were likely characterized by sub-mM to low mM concentrations6, and low-sulfate conditions returned episodically throughout the Phanerozoic7. In the Archean, before the beginning of ocean oxygenation 2.7–2.5 billion years ago9, marine sulfate was scarce in coastal and surface pelagic ocean, at no more than tens of µM10, and likely absent in ferruginous deep waters. The cycling of sulfur under these conditions was very different from the one in the modern ocean, and freshwater systems, particularly stratified lakes, are commonly used as better analogs10. Recent work11 has demonstrated that in low-sulfate lakes (<100 µM), mineralization of organic sulfur (OS) supplies a significant portion of the substrates for microbial sulfate reduction12, and a significant fraction of sulfide is traceable to an organic source14. In the well-oxygenated sediments of oligotrophic Lake Superior11, for example, mineralization of the settled particulate organic sulfur causes accumulation of sulfate in the upper oxidized layer, often in excess of the water column concentrations, and supports over 80% of sulfate reduction in the deeper anoxic sediment. Paradoxically, the organic component has not been considered in reconstructions of the Early Earth sulfur cycling. The histories of atmospheric oxygen and oceanic sulfate are widely inferred from the records of sulfur isotopes preserved in pyrites15, but non-hydrothermal pyrite formation was considered only from seawater sulfate16 or elemental sulfur17.Since the beginning of Life, sulfur cycled between the geosphere and biosphere as an essential component of all living matter. Organic S-bearing molecules, such as amino acids cysteine and methionine, have been ubiquitous throughout the planet’s historyHere, we argue that organic sulfur must have been a significant component of the early biogeochemical cycling, and its mineralization provided at least two major pathways to pyrite, which could operate even in sulfate-free environments. The effects of these processes on the geochemical and isotopic compositions of sedimentary sulfides are consistent with available evidence and warrant a re-evaluation of the presently accepted interpretations of the geochemical and isotopic proxies.18. It occurs at lower oxidation states in proteins such as amino acids cysteine and methionine, in coenzymes , as iron-sulfur clusters in metalloproteins, and in bridging ligands 2. Higher oxidation state compounds, such as sulfonates R–SO3–H, sulfones R–SO2–R, and organo-sulfates, can be found in lipids  and are components of cell walls and photosynthetic membranes. Molar S:C ratios in modern plankton18 typically range between 0.003 and 0.01, with freshwater values19 being more varied than in marine environments because of a wider range of geochemical conditions. Archean S:C ratios likely spanned a similar range, or were even higher if sulfolipids could be used in place of phospholipids21 to alleviate P limitation22. The Archean organic sulfur pool was likely dominated by reduced compounds23, which are thermodynamically easier to assimilate under anoxic conditions24. Assimilation of sulfate requires energy even at the stage of cellular uptake by sulfate-binding proteins24, and sulfate (+6) is rare in prokaryotic cells24, whereas key molecules contain sulfites (+4) or sulfonates (+4)26. Reduced sulfur appears in evolutionary key molecules such as methionine, cysteine, cystine, coenzymes M and acetyl CoA, aromatic sulfur and disulfides, and in primitive metabolic processes such as S oxidation in anoxygenic phototrophs. Hydrothermally supplied hydrogen sulfide (+2) in the presence of CO2 could form thiols, critical coenzymes, CS2 and dimethyldisulfide28.Sulfur makes up about 1% of dry weight of aquatic organisms3–H) would generate sulfite (SO32−), which in Archean oceans would add to the pool generated by the dissolution of volcanic SO2  to form iron sulfides, bypassing the traditionally assumed pathway of sulfate reduction.Mineralization of the reduced organic S pool (R-SH) would provide an even more important input of inorganic sulfur. Such mineralization generates hydrogen sulfide, which under ferruginous Archean conditions would react with dissolved iron  and comparable to the estimated Archean pyrite burial flux41 (~0.1–1 Tmol S year−1). Through reduction of oxidized OS and direct release of hydrogen sulfide from reduced OS, followed by a nearly quantitative conversion to pyrite under ferruginous conditions, this re-mineralization could generate a significant portion of the pyrite that was eventually preserved in Archean sediments. Like sulfate reduction, pyrite precipitation likely occurred primarily in organic rich coastal regions, including microbial mats42, where the contributions from organic sulfur could have been high. For a concentration of total inorganic sulfur in the ocean water on the order of 10 μM10, mineralization at the rate of 0.3–1 Tmol S year−1 implies that the entire oceanic pool of sulfur could cycle through the organic pool in under 10,000 years. In surface sediments , the abundance of organic sulfur  would compete with the low μM availability10 of inorganic S from the overlying water column, and could be the only source of reactive sulfur in deeper sediment.The geochemical fluxes and transformation rates sustained by the mineralization of organic sulfur must have been quantitatively significant. The inventory and cycling rates of organic matter are poorly constrained for the Archean, but assuming, as an order of magnitude estimate16 assumed that sulfate was transported from the surface ocean into the deep waters or sediments where it underwent microbial reduction to sulfide, which in the presence of ferrous iron precipitated to eventually form pyrite. In contrast, the oxidized inorganic sulfur compounds produced from the oxidized fraction of OS could support S reduction even when sulfate was absent from ambient water. Likewise, the hydrogen sulfide produced from the reduced OS could generate pyrite even in the absence of sulfate reduction.Generation of sulfite and dissolved sulfide from organic S within the sediments and water column radically changes the picture of the Archean sulfur cycling. Traditional view and previous numerical models11 and applied to Archean conditions9 (see Methods section). For the sake of concreteness, we performed simulations in sediments. While the transport mechanisms in water column may be more varied and three-dimensional, similar arguments should apply, at least qualitatively, to a water column where sulfate and particulate OS reach the anoxic ferruginous deep waters from the surface mixed layer10. For a more straightforward comparison with previous models that did not consider sulfite, and to extend the results to potentially oxygenated conditions of the Neoarchean, the model uses sulfate as the oxidized form of inorganic sulfur; this does not change the generality of the argument. Mineralization of reduced OS was assumed in the model to generate hydrogen sulfide  contribution of OS to sulfate reduction increases also with the sediment organic matter content, as organic matter supports OS mineralization rates, even though it also stimulates sulfate reduction and the drawdown of sulfate from overlying water  compared with those for sulfate (>30‰), they are consistent with observations of small ∆34S. The hydrogen sulfide produced from the more abundant reduced OS compounds would generate solid sulfides, bypassing microbial reduction. Rather than carrying an isotopic signature of redox processes, these sulfides could instead carry the isotopic signal of hydrolysis. The magnitudes of the fractionations during hydrolysis of organic sulfur are not well constrained, but thermodynamic considerations45 and laboratory investigations46 limit them to less than 17‰, consistent with small observed ∆34S. Small isotopic fractionations are similarly consistent with the evidence in modern sediments where care was taken to analyze the hydrolyzable fraction of organic sulfur48. In particular, depth variations in the isotopic composition of hydrolyzable organic sulfur pool seem to indicate a preferential loss of isotopically light organic sulfur during the early stages of diagenesis47. Incorporating these considerations into an isotopic model of sulfur diagenesis  only by a few permil  when the ∆33S composition of organic sulfur is similar to that of seawater sulfate.The contribution of organic component to pyrite formation profoundly changes the accepted interpretations of the Archean isotopic signals. Microbial sulfate reduction depletes the sulfide in 34SFeS records are equally consistent with sulfite reduction and formation of pyrite from reduced organic sulfur, neither of which requires sulfate, the isotopic evidence for an early (3.47 Ga) onset of sulfate reduction50, suggested based on ~10‰ isotopic fractionations51, may need to be re-evaluated. Sulfur isotopes are the only reliable tracer for the sulfate reduction metabolism, as preserved cellular structures are not readily identifiable for sulfate-reducers, while molecular fossils (biomarkers) do not seem to survive over geological times49. In a low-oxygen world where sulfate was produced in limited quantities52 by atmospheric photochemical reactions, and at somewhat greater but uncertain rates by disproportionation of reactive sulfur intermediates52, dissimilatory sulfate reduction could have become globally competitive for the first time when sulfate concentrations increased in the Neoarchean, following the initial marine oxygenation around 2.7 Ga53. The observed expansion in ∆34SFeS beginning around that time9 thus may reflect not only a more vigorous redox cycling of sulfur but also increased isotopic fractionations54 associated with the expanded range of redox states. Similarly, while anoxygenic phototrophs nearly universally can oxidize sulfide to elemental sulfur24, evidence for the evolution of groups capable of completing the oxidation to sulfate seems to appear first around 2.7 Ga55. As pyrite could be formed in non-hydrothermal settings from relatively abundant reduced organic sulfur . Diagenetic formation of elemental sulfur was considered through oxidation of sulfide by iron oxides, which is the pathway that is thermodynamically favorable at low sulfide concentrations9. Thiosulfate was not considered because at low sulfide availability aerobic oxidation of sulfide proceeds largely without intermediate redox compounds24.Mineralization of organic carbon and the distributions of iron within the sediment were modeled as described in ref. 3H groups) such as in sulfonates and esters was assumed to generate sulfite or sulfate. As the S:C ratios in the Archean ecosystem are not established, the S:C ratio was varied in the model within a range of literature values for modern marine ecosystems56. The S:C ratios may reflect, for example, factors such as organism physiology (specific sulfur requirements for biomass) and environmental conditions such as nutrient limitation. For example, in modern systems, P limitation in plankton is known to stimulate substitution of sulfolipids (and N-based lipids) for phospholipids21, strengthening the sulfur deposition into sediments.Mineralization of reduced OS was assumed in the model to generate hydrogen sulfide  of sediment S reduction supported by mineralization of organic sulfur, we used the ratio of the depth-integrated rates of sulfate generation and sulfate reduction, corrected for the in-sediment recycling of sulfur by the disproportionation of elemental sulfur and re-oxidation of hydrogen sulfide. To quantify the fraction (β) of pyrite precipitation supported by organic sulfur, we used the ratio of the depth-integrated rates of sulfide generation to iron sulfide formation, corrected for the production of sulfide through elemental sulfur disproportionation and reduction of the mineralized oxidized OS. Using R* as the notation for depth-integrated rates of the respective reactions , and elemental sulfur disproportionation rate constant. The parameters that affected the values of α and β most strongly included the sediment organic matter content, sulfur to carbon ratio (S/C), and the proportion of organic sulfur present in oxidized (fSO4) vs. reduced form (fH2S). The sensitivity of α and β to the S/C ratio and organic matter content, as well as the typical sediment geochemical profiles are illustrated in Supplementary Figs. 10, the sensitivity of the current model was further investigated by considering production of methane through methanogenesis and consumption of methane through aerobic and anaerobic oxidation (AOM)  during diagenesis, including during organic sulfur hydrolysis. Supplementary Fig. 9, the total rate of sulfate reduction obtained from the geochemical model was partitioned in terms of the individual isotope rates:The isotopic model tracked the transformation of sulfur isotopes  and 34SO4 (~4%), the individual isotope rates were calculated as:Approximating 32SO4] and [34SO4] are concentrations. The concentration ratio η = [34SO42−]: [32SO42−] is related to the standard δ34S notation as34S/32S-ratio of the standard troilite, an iron monosulfide from the Canyon Diablo Meteroite. The value of η at the beginning of iterations was calculated using the δ34SO42− of 12‰. For α, we conservatively imposed a fractionation factor of 30‰ (α = 1.030), typical for sulfate reducing bacteria58, and decreased it linearly to zero below 6 µM, which reflects sulfate limitation16, similarly to previous models10. Constant fractionation factors (α) were used for other reactions: organic sulfur hydrolysis (1.015), sulfide oxidation16 (1.005) and elemental sulfur disproportionation59 (0.988 for sulfate and 1.007 for sulfide).Here, the fractionation factor33SO42− was calculated as:α′ = 0.515α. The initial value of ηʹ =  [33SO42−]: [32SO42−] was calculated using the δ33SO42− of 2.5‰:The reduction rate for To considering isotopic fractionation during organic sulfur hydrolysis, the total rate of hydrolysis obtained from the geochemical model was partitioned in terms of the individual rates of oxidized and reduced organic sulfur:9.The rates and concentrations for the individual isotopes were calculated iteratively analogously to the process described above for sulfate reduction, using defined fractionation factors. Calculations for other reactions in Supplementary Table To calculate vertical concentration profiles, net rates were expressed as follows:2 was then found by integrating the FeS2 precipitation rates for individual isotopes. Based on the diagenetic equations with Di=0:The vertical gradients and concentrations for each isotope were then computed by integrating the diagenetic equations over depth with the rates given by Eqs. –19. The 9, isotopic fractionation during organic sulfur hydrolysis affects the isotopic composition of pyrite. Specifically, preferential mineralization of isotopically light organic sulfur results in an isotopically lighter pyrite. As a result, the isotopic composition of pyrite deviates more from that of the seawater sulfate, resulting in a greater ∆34SFeS2 (by up to 7‰) , characteristic of Archean pyrites. The atmospherically produced MIF-S signal is transmitted from elemental S to pyrite through diagenesis and is influenced by the isotopic composition of iron sulfide and porewater sulfideSupplementary Information


Chronic infection by hepatitis C virus causes impairment in neurocognitive function in up to 50% of patients which may not be detected by clinical tests.Early detection of neurocognitive impairment in chronic hepatitis C patients and investigating the cognitive function in HCV patient by p300 and clinical test.The study included 60 patients with chronic hepatitis C and 30 healthy controls. Participants were subjected to a biochemical, hematological assessment, mini-mental state examination, Montreal Cognitive Assessment, P300, polymerase chain reaction (PCR), and fibroscan made for hepatitis C patients.The digit span, attention, concentration, and memory were significantly lower in patients than controls. The delayed P300 peak latency and the reduction of its amplitude were significantly evident in patients with liver fibrosis than the controls and patients without fibrosis. These abnormalities were significantly higher with increasing the grade of fibrosis. All patients with cognitive impairment  had abnormal P300-evoked responses. P300 could detect neurocognitive impairment in some patients with normal neurocognitive functions by clinical test. P300 had sensitivity (100%), specificity (59.26), positive predictive value (75%), negative predictive value (100%), and accuracy (81.67) in the detection of neurocognitive impairment in HCV patient.Patients with chronic hepatitis C infection had significant impairment in their cognitive functions. This impairment increases with the increase in grade of hepatic fibrosis. P300 can detect minimal and subclinical impairment of cognitive function at early stages of chronic hepatitis with accuracy (81.67).PACTR201804003215168.PACTR on 19 march 2018 retrospectively. Identification number for the registry is  Impairment of cognitive functions increases with the increase in grade of hepatic fibrosis in HCV patients. P300 can detect minimal and subclinical impairment of cognitive function at early stages of chronic hepatitis.Hepatitis C virus (HCV) infection is a serious global health problem that affects 180 million people worldwide. Hepatitis C virus causes acute and chronic hepatitis which can eventually lead to permanent liver damage and hepatocellular carcinoma . HCV is This study aimed at early detection of neurocognitive impairment in chronic hepatitis C patients and investigating the cognitive function in HCV patient by p300 and clinical test.The study included 60 untreated chronic hepatitis C patients in addition to 30 healthy persons matched for age, sex, and educational level. The majority of participants achieved a high level of education (secondary school to university). All subjects did not give a history of cognitive dysfunction. We excluded patients with liver cirrhosis, other causes of chronic liver diseases rather than HCV, psychiatric disorders patients, patients with conditions that limit the fibroscan technique (such as ascites), obese patients with BMI ≥ 28, and patients with renal failure, diabetes, or history of drug abuse in the last 6 months. Fibroscan was done in HCV patients to detect the grade of liver fibrosis. The HCV group was subdivided into three groups according to the grade of liver fibrosis . A written informed consent was taken from patients and control before participation according to the guidelines of 1975 declaration of Helsinki. The patients were randomly selected from the outpatient clinic of the tropical medicine department and internal medicine department Minia University Hospital. The controls were randomly selected from outpatient clinic and relatives of the patients admitted. Both patients and control groups were subjected to the following clinical assessment and laboratory investigations, especially liver and kidney functions, blood sugar, HCV-AB (antibodies), HCV PCR, HIV (human immune deficiency virus), and abdominal ultrasound.Regarding FibroScan, we used M probe for measuring liver stiffness. The device generates a 50-Hz shear wave. The patients were in a supine position. Transducer probe was placed on the skin, between the rib bones at the level of the right lobe of the liver. The patient was holding breath and the operator pressed the probe button to take the measurements. Ten measurements were taken to detect liver stiffness which was expressed in kilopascals (kPa) . Cut-offMontreal cognitive assessment scores (MoCA) version A  was doneRegarding P300, oddball task auditory-evoked potentials (peak of p300 latency) using a Nihon Kohden Neuropack MEB-9204 was done. The electrophysiological activity was recorded from three different positions: Fz (forehead midline), Cz , and Pz . Impedances were maintained between 5 and 10 kΩ. Artifacts were automatically rejected at ± 50 μV. The stimuli were presented through headphones at 90 dispel in both ears. The subjects were seated in a comfortable chair, quiet and properly illuminated room, awake and exposed to a series of standard auditory stimuli and small amounts (15 or 20%) of deviant stimuli. Subjects were asked to detect or count the target stimuli (active task) reporting the total at the end of the session. The test was initiated only when the subject demonstrated a complete understanding of the task. The P300 component was defined as the largest positive-going peak occurring as a reaction for the deviant (rare) stimuli within a specific latency window (250–450 ms). Peak amplitude was measured relative to the prestimulus baseline (100 ms), and peak latency was measured from the time of stimulus onset .t test was used to compare data between two groups for normally distributed data, while ANOVA was used to compare data between the four groups. Qualitative data were analyzed by chi-square (χ2) test. Statistical significance was defined as p values less than 0.05.All analyses were performed with version 20 of Statistical Package of Social Science (SPSS). Qualitative data were expressed as proportions, while quantitative data were expressed as mean + standard deviation (SD). Unpaired Student’s We calculated sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy of p300 where sensitivity is the probability that a test result will be positive when the disease is present, specificity is the probability that a test result will be negative when the disease is not present, and positive predictive value is the probability that the disease is present when the test is positive. Negative predictive value will be negative when the disease is not present. Positive predictive value is the probability that the disease is not present when the test is negative. Accuracy is the overall probability that a patient will be correctly classified.P = 0.029). Meanwhile, the frequency of delay was increased with increasing the stage of fibrosis  showed delay in latency of P300 than control, but it was statistically not significant (p = 0.0919)  respectively while F0-F1 had a reduction in amplitude of P300 which was statistically not significant . P300 had sensitivity (100%), specificity (59.26), positive predictive value (75%), negative predictive value (100%), and accuracy (81.67) in the detection of neurocognitive impairment in HCV patient  and NNA-glutamate are raised in the basal ganglia and prefrontal cortex. These metabolites are raised as a compensatory mechanism to HCV inflammation. When these compensatory mechanisms fail, fatigue and possibly other signs of neurocognitive impairment take place [Cognitive dysfunction in patients with chronic hepatitis C virus (HCV) infection is a distinct form of minimal hepatic encephalopathy (MHE). In fact, the majority of HCV-positive patients, irrespective of the grading of liver fibrosis, display alterations of verbal learning, attention, executive function, and memory when they are evaluated by suitable neuropsychological tests. Similarities between the cognitive dysfunction of HCV patients and MHE of patients with different etiologies are unclear , 13, 14.ke place –20. NeurIn the present study, the F0-F1 group had no cognitive impairment (MMSE score 25–30). Fourteen patients had mild cognitive impairment , eight of them in the F2 group and six in the F3 group; 19 patients had moderate cognitive impairment , seven of them in the F2 group and 12 in the F3 group, and none had severe cognitive impairment. There was also a significant difference in impairment in digit span, attention, concentration, and memory between patients and control which increased with increasing the grade of fibrosis evidenced by MoCA and MMSE. In agreement with these results, Hamdy et al.  found thP = 0.029) and that the delay of P300 peak latency and reduction of P300 amplitude were significantly evident in F2 and F3 groups than control group while F0-F1 showed delay in latency of P300 and reduction in amplitude of P300 than control but they were statistically not significant. The prolonged latency and amplitude reduction of P300-evoked responses were significantly increased with increasing the stage of fibrosis. These results were agreed with that of Kramer [In our study, we also detected the cognitive function by p300 method. We found that the frequency of delayed P300 peak latency was increased in the three groups of chronic hepatitis than controls P = 0.09 and thaf Kramer  involvinf Kramer  suggestef Kramer  had alsoPatients with CHC infection has significant impairment of digit span, attention, concentration and memory and this cognitive impairment increases with severity of hepatic fibrosis. Patients without fibrosis show no cognitive impairment by psychometric tests while showing impairment by event-related potential. P300 can detect minimal and subclinical impairment of cognitive function at early stages of chronic hepatitis (F0) with a sensitivity 100% and accuracy 81.67% even with normal standard psychometric tests. A future study is needed with larger F0 chronic hepatitis patients to confirm this suggestion.