Neisseria gonorrhoeae are responsible for over half of the symptomatic urethritis cases in U.S. men. Recently, clinics in Indianapolis, Columbus, Atlanta, and other U.S. cities began to note increasing numbers of men presenting with urethritis and Gram-negative intracellular diplococci in their urethral smears who test negative for N. gonorrhoeae. Many of these discordant cases, which have periodically reached highs of more than 25% of presumed gonococcal cases in some sexually transmitted infection clinics in the U.S. Midwest, are infected with strains in a novel urethrotropic clade of Neisseria meningitidis ST-11 (US_NmUC). However, no cultivation-independent tests are available for the US_NmUC strains, and prior studies relied on microbial culture and genome sequencing to identify them. Here, we describe a PCR test that can identify the US_NmUC strains and distinguish them from commensal and invasive N. meningitidis strains as well as N. gonorrhoeae. Our SimpleProbe®-based real-time PCR assay targets a conserved nucleotide substitution in a horizontally acquired region of US_NmUC strain genomes. We applied the assay to 241 urine specimens whose microbial compositions had previously been determined by deep shotgun metagenomic sequencing. The assay detected the single US_NmUC positive case in this cohort, with no false positives. Overall, our simple and readily adaptable assay could facilitate investigation of the pathogenesis and epidemiology of the US_NmUC clade.Urethritis, or inflammation of the urethra, is one of the most common reasons men seek clinical care. Sexually transmitted pathogens including  Urethritis is defined by the presence of polymorphonuclear cells in male urethral smear or urine specimens, and is often, but not always, associated with urethral and urinary symptoms –4. MilliNeisseria gonorrhoeae (NG). Men with urethritis whose Gram stain smears contain Gram-negative intracellular diplococci are diagnosed with GU, whereas men whose smears who do not are diagnosed with NGU. When a point-of-care test is unavailable, a negative nucleic acid amplification test for NG can indicate a diagnosis of NGU. Chlamydia trachomatis, Mycoplasma genitalium, and Trichomonas vaginalis are common causes of NGU [Many specialized sexually transmitted infection (STI) clinics use point-of-care urethral Gram stain smears to categorize urethritis into gonococcal urethritis (GU) and nongonococcal urethritis (NGU) cases because the optimal empiric antibiotic regimens for GU and NGU differ , 8. Press of NGU –12. SensNeisseria meningitidis (NM) is best known for its ability to invade and cause meningitis and septicemia, although these invasive infections are rare and asymptomatic nasopharyngeal NM colonization is more common [e common , 14. NM e common –22, but norB -aniA  locus from NG [Multiple STI clinics in the U.S. Midwest began to note an increased frequency of Gram-negative intracellular diplococci-positive, NG nucleic acid amplification test-negative, male urethritis cases starting in 2015 –25. Uret from NG . The eme from NG . US_NmUC from NG , 28, 29.C. trachomatis, T. vaginalis, and M. genitalium because culture of these microorganisms is costly, requires controlled transport conditions, and FDA-approved tests that offer increased sensitivity compared to culture of these organisms are now widely available. NM is not routinely cultured from urogenital sites, and since the sensitivity of NM culture from routine oropharyngeal specimens using optimized transport conditions can be as low as 72% [Nucleic acid amplification tests have mostly supplanted cultivation for diagnosis of NG, w as 72% , the sennorB that is present in US_NmUC clade strains, but absent in invasive NM strains, NG, and other commensal Neisseria species that colonize the urogenital tract. Assay sensitivity and specificity were evaluated using urine and urogenital swab matrices spiked with urogenital pathogens, representative US_NmUC clade isolates, and other pathogenic and commensal urogenital microorganisms. The assay also identified the only US_NmUC positive specimen in a collection of 241 male urine specimens whose microbiomes had been characterized by deep shotgun metagenomic sequencing. The US_NmUC Simpleprobe® assay could be easily adapted for other types of clinical specimens, is compatible with platforms used in contemporary diagnostic laboratories, and can be rapidly implemented for US_NmUC surveillance.Here, we describe a rapid real-time test for the US_NmUC clade strains using a SimpleProbe® assay . The assNeisseria perflava (ATCC-14799); Neisseria subflava (ATCC 49275); C. trachomatis [M. genitalium [T. vaginalis [Ureaplasma urealyticum, . HSV 1 and 2 control DNA was purchased from Acrometrix  and the HPV 16 plasmid was a gift from the Indiana University Infectious Disease laboratory.Unpublished NG and NM clinical isolates from Indianapolis area clinics were used to represent local strains; other strains were obtained from American Type Culture Collection (ATCC) and other sources. NM FAM18 ATCC 700532D-5), NM1 and NM2 ; NM3 andD-5, NM1 norB-aniA locus. The Indiana University/Purdue University-Indianapolis Institutional Review Board and the Marion County Public Department of Health approved this study.Males ≥18 years of age who presented to the Marion County Public Department of Health, Bellflower STI Clinic (BFC) in Indianapolis, Indiana with acute urethral symptoms or as healthy controls without symptoms, and provided written consent to participate, were enrolled in the idiopathic urethritis men’s project (IUMP) , 33. A GnorB gene that contains the target SNP in position 431 in the US_NmUC reference isolate NM1 [Fluorescein-SPC-CGTCATCAGCGATACGCG-Phosphate) was synthesized by FLUORESENTRIC . The PCR reaction mixtures contained LightCycler 480 Genotyping Master Mix (2x) , 0.5 μm of the forward primer F-NM1 (5’-GCTTGGCCGATGAATACC-3’), 1.5 μM of the reverse primer R-NM1 (5’-CGTAAACGCCGTGATAGT-3’), 0.2 μM probe (P-NM1), 5 or 2 μl DNA template, and 3 mM MgCl2 in a total reaction volume of 20 μl. Amplifications were initially performed on a Roche LightCycler 2.0 instrument and were subsequently scaled-up on a Roche z480 (z480) real-time instrument . Amplification conditions for the LightCycler 2.0 were: 95°C for 10 min, 45 cycles at 95°C for 10 sec (ramp rate of 20°C/s), 55°C for 20 sec in single acquisition mode (ramp rate of 10°C/s), 72°C for 20 sec (ramp rate of 20°C/s). Melting curve analysis commenced by raising the temperature to 95°C for 0 sec (ramp rate of 20°C/s), followed by annealing at 40°C for 2 min (ramp rate of 1.5°C/s). The temperature was increased stepwise to 95°C with continuous signal acquisition (ramp rate of 0.1°C/s). Cool down was performed at 40°C for 30 sec (ramp rate 20°C/s). Amplification conditions on the z480 were: 95°C for 10 min (ramp rate 4.4°C/s), 45 cycles at 95°C for 10 sec (ramp rate of 4.4°C/s), 55°C for 20 sec in single acquisition mode (ramp rate 2.2°C/s), 72°C for 20 sec (ramp rate 4.4°C/s). After amplification, melting curve analysis commenced by raising the temperature to 95°C for 1 min (ramp rate 4.4°C/s), followed by annealing at 40°C for 2 min (ramp rate 2.2°C/s). In the final annealing step, the temperature was raised to 90°C (ramp rate 0.06°C/s) with continuous signal acquisition. Cool down was performed at 40°C for 30 sec (ramp rate 2.2°C/s).A primer set was designed with Roche Applied Science Light Cycler Probe design software 2.0 to amplify a 117 bp segment of the 2,256 bp norB encompassing the region containing the target SNP were PCR amplified from NM1 or NG genomic DNA. The amplicons were cloned into the pCR2.1-TOPO vector  to create pNG-norB and pNM1-norB. The sequences of the norB inserts in the final plasmids were confirmed by sequencing. The limit of detection (LOD) of the SimpleProbe® US_NmUC real-time assay was determined using spiked mock urine and vaginal swab matrices that tested negative for NG, C. trachomatis, T. vaginalis, M. genitalium, and U. urealyticum. Serial dilutions of plasmids pNG-norB and pNM1-norB were added directly to mock urine and vaginal swab solutions, with the final plasmid concentrations ranging from 25 to 1000 copies/ml in the spiked urine matrices, and 250–4000 copies/ml in the spiked vaginal swab matrices. The contrived samples were processed, and DNA was extracted on the Roche Cobas 4800 automated extraction platform.To construct the control plasmids, 400 bp amplicons of metA gene was adapted from a prior study [A TaqMan assay for the NM or study . The assnorB-aniA locus via a horizontal gene transfer event with NG [norB in all 204 of the US_NmUC clade strains for which complete genomes were available (26). The norB sequences were identical in 194 of these 204 isolates. Alignment of the norB-ani locus of NM1, NG, NM FAM18, and other commensal Neisseria spp. revealed two nonsynonymous SNPs in norB, G431A and C1782T, that were unique to NM1, a representative of the US_NmUC clade strains  element and acquisition of a region of the  with NG , 26, 28. strains . Both SNted SNPs . We idennorB alleles  was matrix and platform dependent. The lower LOD on the Light Cycler 2.0 platform was 50 copies/ml with urine and 500 copies/ml with vaginal swabs. On the z480 platform, the lower LOD was 100 copies/ml with urine and 1000 copies/ml with vaginal swabs.We assessed the limit of detection (LOD) of our assay by spiking mock urine and vaginal swab matrices with serial dilutions of plasmids pNG-C. trachomatis, T. vaginalis, M. genitalium, N. meningitidis, N. cinerea, N. lactamica, N. perflava, N. subflava; genomic DNA from U. urealyticum, HSV 1, HSV2, HPV 16; or the control plasmids pNM1-norB and pNG-norB was tested in the Simpleprobe® assay. Contrived specimens spiked with the two US_NmUC isolates (NM1 and NM2), and the uNM plasmid control plasmid yielded a melting peak around 57°C, whereas none of the contrived specimens spiked with other Neisseria species or other urogenital microorganisms yielded a melting peak near this temperature  strains , one urethrotropic NM isolate (NM1), one Nm urethral isolate, and one N. lactamica (Nl) strain are included in the alignment. Sequences unique exclusively to the urethrotropic NM isolate are marked in bold red. The target SNP for our assay is boxed.Two (DOCX)Click here for additional data file.


Camelus bactrianus) and horses, produced by Mongolian nomads for self-consumption. Peptides from skimmed fermented milks were separated by ultrafiltration and reverse-phase high-performance liquid chromatography. Then, their amino acid sequences were determined by matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry. Consequently, eleven peptides were identified in the fermented camel’s milk including four from β-casein (β-CN), three from αs1-CN, and two from both κ-CN and lactophorin. On the other hand, twenty-four peptides were identified in the fermented mare’s milk including nineteen from β-CN, three from αs1-CN, and one from both κ-CN and αs2-CN. According to previous reports on the bioactivities of milk-derived peptides, antibacterial and antihypertensive activities were promising in both the fermented camel’s milk and mare’s milk. In addition, potential antioxidant activity was conjectured in the fermented camel’s milk. Further investigations are currently needed to clarify the potential role of immunomodulatory peptides in the two fermented milks.To date, bioactive proteins and peptides from minor livestock milks and their fermented products have been scarcely reported. In Mongolia, nomads are commonly rearing five livestock animal species  for milking and other purposes. In this study, we analyzed the peptide composition in fermented milks of Bactrian camels ( Camelus genus includes two domesticated species of camels: Dromedary camel  and Bactrian camel . According to statistics from the Food and Agriculture Organization (FAO), in 2018, the total population of Dromedary and Bactrian camels and available worldwide camel milk production yield were 35.8 million heads and 3.15 million tons (http://www.fao.org/faostat/en/#home). More than 75% of camel milk is assumed to be consumed by nomads as raw milk and processed dairy products. In 2018, camel milk production in Mongolia was 114,830 tons, accounting for 3.6% of the available camel milk production in the world. For the nomads living in the Gobi region of Mongolia, camels are multipurpose livestock animals, e.g., they supply milk, meat, wool, and hide as well as transportation. Because nomads consume greater quantities of Bactrian camel milk than milk from other domestic animals, they have established processing methods specialized for Bactrian camel’s milk that were inherited across generations. They believe that Bactrian camel’s milk has an anti-swelling and therapeutic effect on intestinal and kidney diseases [The It was reported that the general constituents of a Bactrian camel’s colostrum obtained at 2 h postpartum were 14.23% protein, 4.44% lactose, 0.27% fat, and 0.77% ash , immunomEquus caballus) occurred approximately 5000 years ago, and it was assumed that its purpose was mostly for transport, which had a significant contribution to the development of rural societies [Domestication of horses . Amino acid sequences of the partially purified peptides were determined by matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI TOF-MS/MS), and two peptides were further identified by Edman degradation. The potential bioactivities of the peptides isolated from fermented Bactrian camel’s milk and mare’s milk collected in Mongolia are discussed according to their similarity to previously identified milk-derived bioactive peptides .A Centriprep YM-3 filter device was purchased from Millipore . Acetonitrile (ACN), trifluoroacetic acid (TFA), and protease inhibitor cocktail for mammals were purchased from Sigma–Aldrich . Buffer solutions for the RP-HPLC were filtered through a 0.2 μm membrane filter from Millipore prior to use. Peptide calibration standard II (1–3 kDa) used for calibration of the MALDI TOF-MS, MALDI target plate, and α-cyano-4-hydroxycinnamic acid (α-CHCA) were purchased from Bruker Daltonik GmbH . The HPLC system was a JASCO  and equipped with a PU-2089 pump, UV 2075 detector, MX 2080-32 dynamic mixer, and a CO-8020 column oven. The TSK gel ODS-80Ts (0.46 × 25 cm) column was from Tosoh Co. . All other chemicals were of analytical grade.Fermented Camel’s milk was produced from September 2015 to April 2016, and fermented mare’s milk from August to October 2016. Approximately 500 mL each of Bactrian camel’s fermented milks were collected from 3 different herders: two herders near Hamriin hiid, Ulaanbadrakh soum, Dornogovi aimag and one herder in Dalanzadgad soum, Umnugovi aimag in Mongolia on 16 October 2016. Approximately 500 mL each of the mare’s fermented milk were also collected from 3 herders located in a narrow region, 3–5 km apart from each other, in Adaatsag soum, Dundgovi aimag, Mongolia, on 2 October 2016. The numbers of milked animals in the herds ranged from 10–20 and 15–24 heads for camels and horses, respectively, at the time of sampling. It was confirmed that both the fermented camel’s milk and mare’s milk were free from contamination of milks derived from other livestock animals via investigation with the nomads who made them. All samples were collected and stored independently in sterile containers and kept at 4 °C in cooling boxes during transport (3 h at maximum) from the farms to the State Central Veterinary Laboratory in Ulaanbaatar and kept at −30 °C until frozen transport to Japan.g for 10 min at 20 °C. The fat and cells were removed and then a supernatant as a whey was centrifuged again at the same condition to remove residual cream. Residual caseins and insoluble compounds were removed by centrifugation at 26,900× g for 60 min at 4 °C. The supernatant, namely, acid whey, was subjected to ultrafiltration at 4 °C using the Centriprep YM-3  3000). Membrane permeate was lyophilized and dissolved in 0.1% of TFA at 80 mg/mL concentration. A 100 μL aliquot of membrane permeate was injected into the ODS-80Ts column (0.46 × 25 cm), pre-equilibrated with 0.1% TFA, and connected to the HPLC system. After 5 min of static flow with 100% solvent A (0.1% TFA in water), elution was performed by a linear gradient from 0% to 50% solvent B (0.1% TFA in ACN) for 90 min at a flow rate of 1.0 mL/min and at 30 °C. The eluent was monitored by an ultraviolet detector at the wavelength of 214 nm. Eluted peptide fractions were collected manually, concentrated by rotary and vacuum evaporation, and lyophilized. The lyophilized peptides were dissolved in 50 µL of 0.1% TFA and stored at −20 °C until used.Each of the fermented camel’s milk and mare’s milk samples (100 mL) was mixed with 1 mL of the protease inhibitor cocktail for mammals and defatted by centrifugation at 500× http://www.matrixscience.com/index.html). None of the fixed modifications were selected, but varied settings of the enzyme, variable modifications, peptide tolerance (±50–250 ppm), and MS/MS tolerance (±0.5–1.0 Da) were tested. Other settings were used as default. Peptides were identified only when their probability based Mowse scores showed statistical significance. In addition, amino acid sequence similarity was analyzed using protein BLAST interface at National Center for Biotechnology Information (NCBI) (https://blast.ncbi.nlm.nih.gov/).The peptide solution was desalted using ZipTip C18 pipette tips  according to the manufacturer’s instructions. A 1.0 μL aliquot of the desalted peptide solution and an equal volume of 10 mg/mL of α-CHCA saturated in 0.1% TFA/ACN  were mixed, and 1.0 μL of the mixture was loaded on a target plate . After the solvent dried, the target plate was mounted in an AutoflexII TOF/TOF mass spectrometer . Mass spectra were obtained using the pre-installed method, RP_1-3kDa (a reflector positive ion mode optimized to the mass range of 1–3 kDa). Peptide calibration standard II was used as an external mass calibrant. The acquired spectra were statistically analyzed using Flexanalysis 2.0 software . Amino acid sequences of the peptides were determined by fragmentation analysis of MALDI-generated ions using a technique of LIFT-TOF/TOF MS (MS/MS). The mass list of fragment ions was searched using the BioTools 3.0 interface  connected to the Mascot search engine . In brieN-terminal amino acid sequences of the peptides were determined by the Edman degradation method  and follLow molecular weight fractions (< kDa) prepared from fermented camel’s milk and mare’s milk using the Centriprep YM-3 were further separated with RP-HPLC. Fermented camel’s milk and mare’s milk showed distinct chromatograms with the RP-HPLC as shown in 76HQNQNPK83 and R75RHQNQNPK83, were identified in peaks C3–3 and C3–4, respectively  of the three individual fermented camel’s milks  showed a lower number of major peaks compared to those of the fermented mare’s milks  . Among C110PRPRPS116, could be aligned using protein BLAST with a C-terminal region of bovine para-κ-CN HPHPHLS, which is known to be produced by chymosin treatment during cheese manufacturing [75RHQNQNPK83 and R76HQNQNPK83, found in this study were embedded in the middle part of mature lactophorin. Ibrahim et al. [113NTMRETMDFLKSLF127 and A79TTLEGKLVEL89, in pepsin-digested camel milk whey hydrolysates that confer tolerance against H2O2-induced oxidative stress to yeast cells. The biological significance of the lactophorin-derived peptides found in the fermented camel’s milk in this study remains to be elucidated. One α2-CN-derived peptide, K16HNMEHR22, was characteristically found in fermented mare’s milk H1, but its bioactivity also remains unclear.In this study, 11 and 24 peptides were identified in the fermented camel’s milk C3 and the fermented mare’s milk H1, respectively. A high number of unidentified peptides could arise from presence of unknown post-translational modifications in their mother proteins. Peptides originated from caseins were dominant in both C3 and H1. Especially, a variety of β-CN-derived peptides accounted for the majority of the identified peptides due to the high abundance of β-CN  in camel or mare milks ,33. The acturing . This paacturing . To dateacturing  and GlyCacturing , respectm et al.  recently221PVPQP226, V194PYPQR199, and Q210EPVPDPVR218—which are similar to known ACE-inhibitory peptides PVPQP from human β-CN [196DTPVQAF203 and N145LRLPV150—which were similar to the reported ACE-inhibitory peptides RDMPIQAF from bovine β-CN [To date, a number of α- and β-CN-derived peptides are known to exhibit ACE inhibitory activity , antibacterial, antioxidant, immunomodulatory, mineral binding, and opioid agonists  activities ,40,41. Sman β-CN , AVPYPQRman β-CN , and YQEman β-CN —were fouine β-CN  and NLHLine β-CN —were fous1-CN R16PKYP20, β-CN Q192MVPYPQR199, β-CN V206LPFQEPVPDPVRG219, and β-CN L221HPVPQP226, isolated from fermented camel’s milk, showed antibacterial activity against Escherichia coli, Staphylococcus aureus, Staphylococcus faecalis, and Staphylococcus dysenteria [s1-CN R16PKYPLR22 and β-CN V194PYPQR199, Q210EPVPDPVR218, H221PVPQP226, were found in this study in the fermented camel’s milk C3; hence, it is highly possible that these peptides could show antibacterial activity. In addition, V194PYPQR199, found in fermented camel’s milk, is very similar to bovine β-CN-derived AVPYPQR, which has been reported as an antimicrobial peptide [Shigella sonnei and Salmonella Typhymurium, but the antibacterial substances are still to be identified (unpublished data). The peptide M157HQVPQ162, found in mare’s fermented milk H1, was similar to MHQPPQPL, which has been identified in goat β-CN as a peptide showing antimicrobial [It has been reported that a mixture of peptides, including αsenteria . Similar peptide . In facticrobial  and dipeicrobial .194PYPQR199 found in the fermented camel’s milk C3, owing to the high sequence similarity to AVPYPQR, which has been reported as an antioxidant peptide derived from bovine β-CN [Antioxidant activity could be expected to come from a β-CN-derived peptide Vine β-CN . Similarine β-CN ,43. Receine β-CN ; however210EPVPDPVR, was found in fermented camel’s milk fraction C3–11, as being similar (77.8% amino acid sequence identity) to a bovine β-CN derived peptide, Y208QEPVLGPVR217, which showed stimulatory activity against human peripheral blood lymphocytes [It is evidenced that casein-derived peptides, produced by the action of digestive enzymes, such as trypsin, pepsin, and chymosin, can exhibit immunomodulatory activities . In thisphocytes . TherefoVariations in peptides (<3000 Da) in Bactrian fermented camel’s milk and mare’s milk made by Mongolian nomads were partially illustrated in this study. It was confirmed that such traditional fermented milks were certainly attractive sources of bioactive peptides. Our results suggest that the presence of antihypertensive and antipathogenic peptides should be promising in Mongolian fermented camel’s milk and mare’s milk. In addition, the presence of antioxidant and antidiabetic peptides are likely in the fermented camel’s milk and mare’s milk, respectively. However, further investigation is needed to confirm the presence of immunomodulative peptides in the fermented milks. Moreover, further studies should be required for demonstrating the biological activities of the identified peptides in this study.


Oryza sativa) germination was investigated to improve beverage qualities. Germinating brown rice has been shown to increase health-promoting compounds. Utilizing green processing, wholesome constituents, including bran, vitamins, minerals, oils, fiber and proteins should should convey forward into germinated brown rice beverages. Rapid visco-analyzer (RVA) data and trends established that brown rice, preheated brown rice and germinated brown rice had higher pasting temperatures than white rice. As pasting temperature in similar samples may be related to gelatinization, RVA helped guide the free-flowing processing protocol using temperatures slightly above those previously reported for Rondo gelatinization. Particle size analysis and viscometric evaluations indicate that the developed sprouted brown rice beverage is on track to have properties close to commercial samples, even though the sprouted brown rice beverage developed has no additives, fortifications, added oils or salts. Phenolics and γ-aminobutyric acid increased slightly in germinated brown rice, however, increases were not maintained throughout most stages of processing. Significantly lower inorganic arsenic levels (113 ng/g) were found in germinated (sprouted) brown rice, compared to Rondo white and brown rice, which is far below the USA threshold level of 200 ng/g.Plant-based beverage consumption is increasing markedly. Value-added dehulled rice ( Sprouted whole grain products have increased markedly in the food and beverage marketplace and re-invigorated health trends . Rice anWhen whole grain rice is eaten, bound and/or undigested phytochemicals are freed by digestive enzymes and micro-flora in the colon to release phenolic compounds that provide health-protective effects in sito and in other locations after transport and absorption ,16. GABAPrunus dulcis) and soy (Glycine max) milks command 70.1% and 10.6% of USA market shares, respectively [In recent years, the number of commercially available rice beverages has dropped substantially compared to 5–7 years ago, as alternate plant sources, and “sprouted” seed beverages diversified. However, only rice and soy offer agronomic crop acreages and yields , and redectively .Several methods and patents exist for making rice beverages and syrup from several starting materials, as outlined previously . HoweverConsumers are shifting toward clean product labels, with concerns about saturated fat levels, antibiotic and hormone residuals in dairy products, as well as environmental and animal husbandry issues . In respRough and milled Rondo rice were freight shipped to the Southern Regional Research Center (SRRC) and stored at 5 °C until use. Rice growing conditions, harvesting, shipping and moisture analysis evaluations were outlined previously . At the Methods used to germinate and optimize a free-flowing processing system for softening, milling, gelatinization and initial enzyme treatments were reported previously , and areAn RVA , using the 7.10 RVA Rice Method, according to the AACCI Method 61-02.01  was used3 and 5 mM KI) before measuring the absorbance at 600 nm on a UV-vis spectrophotometer. Approximate changes in starch concentration and percent soluble/insoluble starch were determined against a corn (Zea maize) starch standard curve. Analyses were completed in triplicate, per replicate.The USDA Starch Research method, which measures total, soluble, and insoluble starch concentrations in a variety of sugar products and by-products, was used to approximate the starch composition in the rice formulas upon processing . SamplesA Partica LA-950  laser scattering particle size distribution was used to assess differences between rice flours, WR, BRR and GBR processed samples. This equipment has a dynamic particle size range from 0.01–3000 µm and is capable of measuring 30 nanometers to 3 mm simultaneously in the same native liquid sample. The three aforementioned CRF were assessed for comparisons. Four commercial rice beverages (CRB) were also assessed. Ingredient labels indicated no added sugars however, the nutrition labels indicated from <1 to 10 g sugars, and an analyses indicated soluble solids ranged from 6.0–10.8° Brix . The anoThe viscosity of rice beverages processed using Rondo WR, BRR and GBR, along with the aforementioned commercial rice beverages, were determined using a Brookfield viscometer . Samples were maintained at 25 °C via a recirculating cooling system, and measured using the SC4-18 probe inside an SC4-13RPY sample chamber with 7 mL at 100 rpm. Commercial samples were run at 200 rpm as the torque fell below threshold (10% torque) and, therefore, output centipoise results were converted using Brookfield values for spring tension, spindle and experimental data to calculate centipoise using the equation: (100/RPM) × TK × SMC × Torque, where TK = viscometer torque constant, SMC is the spindle multiplier constant, and Torque and RPM are output data per run.2CO3 was added to the solution. They were incubated for 30 min at 40 °C in hot water bath, then, read at 760 nm . For GABA, a 10 mL solution was prepared with 89 mM Tris/Na2SO4 buffer, using 8.9 mL of buffer, 100 µL of 1M DTT, 10.66 mg of 1.4 mM NADP+, 1 mL of 2 mM α-ketoglutarate and 5.6 mg of GABase at 30 °C [2O (deionized), vortexed, allowed to settle, and the supernatant was added to designated blanks, samples and 96-well UV plate cells. Serial dilutions were run with 0–10 mM GABA of stock and water; a 10 mM stock solution (20.6 mg) and 10–20 mL deionized water. Blanks used 100 µL of GABase solution, and 90 µL GABase solution plus 10 µL was used for each sample/standard in each well. Plates were incubated for 1 h at 30 °C. The conversion of NADP+ to NADPH was measured as absorbance (340 nm) on a microplate reader . GABA concentrations in samples were calculated from calibration curves using the standard solutions. GABase was a mixture of γ-aminobutyrate glutamate amino transferase (EC 2.6.1.19) and succinic semialdehyde dehydrogenase (EC 1.2.1.16) from Pseudomonas fluoresces, purchased from Sigma . Microtiter 96-well UV plates were obtained from Corning .Rondo BRR and GBR were freeze dried into powders and WR was milled using a TekMar A-10 Analytical Mill  into rice powder and frozen (−20 °C) at the SRRC until testing. The Folin-Ciocalteu method was used to determine the phenolic content in the samples . Standarat 30 °C . Three e3 solution and heated at 95 °C for 90 min, diluted, centrifuged, filtered and analyzed by HPLC-ICP-MS. Arsenic species were separated using an isocratic anion-exchange with an arsenic-specific detector monitoring (m/z 75), per [Arsenic was measured based on the AOAC Method 2013.06, Arsenic, Cadmium, Mercury, and Lead in Foods ,40, and 75), per .® 13 PRO for Windows  and their distributions were verified by the software so that data points falling outside a normal distribution could be removed. Briefly, the data distribution histograms were viewed. Under the “Percent” option, “Normal Quantile Plot”  was selected with the “Continuous Fit” designated to “Normal”. The “Fitted Normal” was subjected to a “Goodness of Fit” utilizing the Shapiro-Wilk W hypothesis test to identify data outside the normal distribution, which were manually removed. Thereafter, data were submitted to ANOVA in JMP® 13 PRO for Windows. When statistically significant differences were found, means were compared against the control by the Dunnett’s test at p < 0.05. In the cases in which a control was not available, treatment differences were evaluated by Tukey-Kramer HSD (Honestly Significant Difference) test at p < 0.05.Data were analyzed in JMPn = 160), which corresponded with a high germination rate of 96.7 ± 0.8% (n = 600), and a balance between catabolic loss of macronutrients versus other endogenous physiological changes that might be beneficial, processing-wise and/or health-wise [Physicochemical changes resulting from sprouting and creating a brown rice beverage using green technologies and a free-flowing processing are detailed herein. Sprouting was terminated when coleoptile length averaged 2.24 ± 0.83 mm  flour preparation, which would be much lower or exceedingly low in commercially defatted white flour. Subsequently, the lower viscosities reported in BRR samples, compared to WR, in combination with the higher pasting temperatures, indicate that chemical and physical interactions between protein, oil and fiber changed the dynamics of these, and pre-heated BRR’s. The significantly highest pasting temperatures observed (93.8–94.7 °C) occurred in the pre-heated BRR samples (BRR had greater crude protein (7.41 ± 0.25%) and fat (3.59 ± 0.50%) compared to Rondo WR with 6.60 ± 0.04% protein and 1.19 ± 0.06% fat . RVA pro samples . DiffereIn BRR that was not germinated and subjected to the developed green processing protocol, the PWM samples contained 60.0 ± 2.3 g/100 mL total starch, of which, 97.8 ± 1.0% and 2.2 ± 1.0% were insoluble and soluble starch, respectively. After thermal softening, total starch was somewhat reduced, yet approached the initial level found in BRR (74.3 ± 1.1 g/100g), according to proximate analysis . After gGBR samples that were gelatinized, enzyme treated and passed a 140-mesh sieve (PNZ) generally had significantly larger particle size parameters than the post-wet milled (PWM) crude beverages that passed 30-mesh sieve prior to gelatinization . Not incIn general, the two native white rice flours had larger particle sizes than in-house WR samples. Yet, a fully gelatinized (cooked) white flour (Remy R500P) had the highest particle sizes compared against all other tabled means . The twoViscosity data indicate that both WR and BRR PWM samples were significantly more viscous than GBR PWM samples . MeanwhiIn general, aside from the viscosity measurement of the GBR CRBs, three of the four commercial rice beverages analyzed had very similar particle size parameters . This isHowever, CRB#2 had significantly higher viscometric measures compared to the other three commercial samples . AccordiDue to apparent interaction effects observed e.g., , rice tyGABA increased in all experiments when BRR was germinated into GBR, whereas phenolics increased in three of four experiments . For exaThe literature indicates clearly that health-beneficial compounds like GABA are enhanced in rice by various germination regimes ,57,58,59w/w) inorganic arsenic for polished (white) rice, and the European Union and USA FDA have proposed stricter limits, where infant rice food levels will be 100 ng/g [The Food and Agriculture Organization of the United Nations and Codex Alimentarius Commission of the World Health Organization have set a limit of 200 ng/g , soften, wet mill, gelatinize then saccharify brown rice into beverages was developed. The “green”sprouted brown rice beverage developed has no additives, fortifications, or added oils or salts. The method has very low inputs, is rather simple in terms of required equipment, and is applicable for both germinated brown and colored rice varieties. Based on rheological data, observations and preliminary evidence, protein and oil remain in the germinated brown rice beverages, and appear to be held in an unstable emulsion. Protein and oil characteristics in these beverages will be reported elsewhere, assessing levels retained in the developed beverages and the solubility properties. Although phenolic data were occasionally inconclusive, the GABA was found to increase slightly through germination, and significantly increase after saccharification of all in-house beverages. Whether or not health-beneficial compounds can be maintained without significant concentration loss in final beverages remains to be determined. Future analysis of other compounds besides GABA and phenolics should also help answer this question. The GBR post-enzyme-treated beverage had viscosity and particle size characteristics that were similar to commercial rice beverages. Inorganic arsenic was also found to be significantly reduced through germination (0.113 mg/kg), and should not be of concern, even at reduced threshold levels, after dilution and final beverage production. The process will next be scaled-up in the pilot plant to facilitate commercial-like pasteurization.


Panax ginseng C.A. Meyer and investigated their biological effects on human skin cells. GrEV or GcEV treatments improved the replicative senescent or senescence-associated pigmented phenotypes of human dermal fibroblasts or ultraviolet B radiation-treated human melanocytes, respectively, by downregulating senescence-associated molecules and/or melanogenesis-related proteins. Based on comprehensive lipidomic analysis using liquid chromatography mass spectrometry, the lipidomic profile of GrEVs differed from that of the parental root extracts, showing significant increases in 70 of 188 identified lipid species and prominent increases in diacylglycerols, some phospholipids , and sphingomyelin, revealing their unique vesicular properties. Therefore, our results imply that GEVs represent a novel type of bioactive and sustainable nanomaterials that can be applied to human tissues for improving tissue conditions and targeted delivery of active constituents.Ginseng is a traditional herbal medicine in eastern Asian countries. Most active constituents in ginseng are prepared via fermentation or organic acid pretreatment. Extracellular vesicles (EVs) are released by most organisms from prokaryotes to eukaryotes and play central roles in intra- and inter-species communications. Plants produce EVs upon exposure to microbes; however, their direct functions and utility for human health are barely known, except for being proposed as delivery vehicles. In this study, we isolated EVs from ginseng roots (GrEVs) or the culture supernatants of ginseng cells (GcEVs) derived from  Extracellular vesicles (EVs) are lipid bilayer-enclosed spheres that are released by all living cells (ranging from prokaryotic to eukaryotic cells) into the extracellular environment, and this process is developmentally conserved from bacteria to humans and plants . EVs appArabidopsis leaves [Citrus limon L. [Citrus limon L.-derived EVs [In contrast to EVs derived from mammalian or bacterial cells  root has been used as a traditional herbal medicine for over 2000 years in east Asian countries due to its various beneficial effects on human health, e.g., immunomodulatory, heart protective, anti-cancer, and neuroprotective properties [The Korean ginseng  could be purified from the extracts of ginseng roots or the supernatants of cultured ginseng cells  using a standard differential ultracentrifugation method. The GEVs exhibited morphologies and sizes typical of EVs and showed anti-senescence and anti-pigmentation effects in human skin cells. Through lipidomic analysis, we found that some lipid species were detected only in GEVs. In particular, diacylglycerol and several phospholipid classes were significantly enriched in GEVs compared to their parent root extracts, suggesting that the GEVs possessed a unique vesicular characteristic and can potentially be used as alternative and sustainable bioactive substances and delivery vehicles.P. ginseng C A Meyer) that were grown in the open field and purchased through the farm, and washed multiple times with water. The ginseng roots were ground, and ginseng root extracts (GrEXs) were prepared using a Hurom slow juicer . GrEXs were sequentially centrifuged at 500× g for 10 min and at 3000× g for 20 min to remove cell debris, fibers, and large particles. The clear juice (34 mL/tube) was loaded on the top of a sucrose cushion with two layers (1 mL of 0.8 M and 0.5 mL of 2 M sucrose) and ultracentrifuged at 100,000× g for 2 h . Following ultracentrifugation, the GrEV fraction (located between both layers of the sucrose cushion) was collected and assessed in terms of its biological effects in human skin primary cells. To purify GcEVs, the culture supernatants of ginseng cells were collected at 2 weeks post-cultivation in bioreactors. The supernatants were sequentially centrifuged at 500× g for 10 min and at 3000× g for 20 min to remove cellular debris. The resulting supernatants were ultracentrifuged at 100,000× g for 3 h, and the pellets were dissolved in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)-buffered saline (HBS).To purify EVs from ginseng roots (GrEVs), fresh ginseng roots (about 500–750 g) were collected from 5–10 plants of 4 year old Korean ginseng (w/v) solution of iodixanol in water. Next, 2.5 mL of control solution  or GEVs in 50% iodixanol was overlaid with 2.5 mL each of 35%, 20%, and 0% iodixanol solutions, which were prepared by mixing the OptiPrep solution with a homogenization medium , and then centrifuged at 200,000× g at 4 °C for 2 h . One milliliter fractions were collected from the top of the gradient of the control tube, and the density of each fraction was determined using a standard curve based on absorbance values at 340 nm. The EV fraction with a density of approximately 1.08 (fraction 3) was collected from the sample tube using a 1 mL syringe. To remove the iodixanol, the fraction was diluted in 60 mL of HBS, ultracentrifuged at 150,000× g at 4 °C for 2 h, and resuspended in 200 μL of HBS. The total protein concentration was determined by performing a Bradford assay . Distribution, average diameter, and particle numbers of GrEVs, GcEVs, and density-purified GrEVs were measured by dynamic light scattering (DLS) using a Zetasizer Nano ZS instrument  or by tunable resistive-pulse sensing (TRPS) using a qNano Gold-equipped NP80 nanopore . The purified GEVs were stored at −80 °C until use.To perform cryo-electron microscopy (cryo-EM) analysis and validate the biological effects of GrEVs, GrEVs were further purified via density gradient ultracentrifugation using OptiPrep solution  according to the manufacturer’s protocol with specific modifications and following previously described methods . BrieflyCryo-EM images were obtained as previously described . Briefly2) using a Bio-Sun irradiation system  and then cultured for 4 days. Human dermal fibroblasts (HDFs) derived from neonatal foreskin (Lonza) were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (FBS) (Lonza). Human epidermal melanocytes (HEMs) derived from neonatal foreskin of moderately pigmented donors  were cultured in Medium 254 supplemented with human melanocyte growth supplement . All culture media contained 100 unit/mL of penicillin-streptomycin , and all cells were cultured at 37 °C in the presence of 5% CO2.Neonatal human epidermal keratinocytes  were cultured in KBM-Gold medium supplemented with the components of the KGM-Gold Bullet Kit (Lonza) for 4 or 12 days, during which time normal growth and spontaneous terminal differentiation occurred. HEKs were subjected to ultraviolet B (UVB) irradiation (30 mJ/cm2) using a Bio-Sun irradiation system (Vilber Lourmat) over a 24 h period and subsequently cultivated for 2 weeks in Medium 254 supplemented with HMGS. Non-irradiated control cells were cultured under the same conditions.Replicative senescence in HDFs was induced by transferring confluent cells to two new culture dishes, expanding them to confluence, and doubling the population, as previously reported . Cellula3 HEM cells were placed in each well of 12 well plates and treated with different concentrations of GrEVs. Cell viability was tested after 48 h, according to the manufacturer’s instructions.Cell proliferation was measured using the WST-1 cell proliferation reagent . Briefly, 7 × 105), either non-treated or treated with GrEVs or GcEVs, were collected in PBS, and proteins were extracted using the M-PER mammalian protein extraction reagent (Thermo Fisher Scientific). After centrifugation at 3000× g for 15 min, 50 μL β-Gal assay reagent was added to 50 μL supernatant. The reaction proceeded for 30 min at 37 °C, and absorbance was measured at 405 nm with a Synergy H2 microplate reader .The SA β-gal activities of senescent HDFs or HEMs were determined using a Mammalian β-Gal Assay Kit (Thermo Fisher Scientific), according to the manufacturer’s instructions. HDFs or HEMs (2 × 10® reagent (Thermo Fisher Scientific) and quantified using a NanoDrop One spectrophotometer (Thermo Fisher Scientific). RNA (100 ng) was reverse transcribed using a RevertAid RT Kit (Thermo Fisher Scientific), following the manufacturer’s instructions. qPCR was performed using a 7500 Fast Real-Time PCR system  and TaqMan™ Gene Expression Master Mix (Thermo Fisher Scientific), per the manufacturer’s instructions. Thermal cycling was performed as follows: 5 min at 95 °C followed by 40 cycles of 15 s at 95 °C and 60 s at 60 °C. Primers and hydrolysis probe sets (TaqMan Gene Expression Assays) for tumor protein p53 (TP53), p21Cip1 (CDKN1A), p16INK4a (CDKN2A), matrix metallopeptidase 1 (MMP1), interleukin 8 (IL8), keratin 10 (KRT10), loricrin (LOR), and filaggrin (FLG) were purchased from Thermo Fisher Scientific. Gene expression levels were determined using the 2−ΔΔCq method, and all data were normalized based on ribosomal protein L13a (RPL13A) expression. Three independent treatments using two independent senescent cell lines from one donor were performed in HDFs, and three independent treatments were performed in HEKs.Total RNA was extracted from cells using the TRIzolg for 10 min. Protein concentrations in culture supernatants were quantified using a BCA Protein Assay Kit . Pellets containing melanin were dissolved in 1 N NaOH and incubated for 30 min at 60 °C. Protein concentrations and melanin contents were determined by measuring the absorbance at 562 nm and 450 nm, respectively, using a Synergy H2 microplate reader (BioTek). The melanin contents were normalized to the protein concentrations.HEMs were lysed by sonication in radioimmunoprecipitation assay (RIPA) buffer (0.1 M Tris-HCl (pH 7.2) containing 1% Nonidet P-40, 0.01% sodium dodecyl sulfate, and a protease inhibitor cocktail; Roche Applied Science) and centrifuged at 15,000× 6) were lysed in RIPA buffer  containing protease and phosphatase inhibitor cocktail (Sigma-Aldrich Corp.) for 30 min at 4 °C, followed by centrifugation at 3000× g for 30 min. Protein concentrations were determined using a BCA Protein Assay kit (Thermo Fisher Scientific), and 25 µg of total protein was loaded onto a 5–20% gradient gel. Subsequently, immunological analyses were performed using antibodies against p53 , tyrosinase (TYR) , TYRP1, TYRP2, glyceraldehyde 3-phosphate dehydrogenase , Ras-related protein 27 (RAB27), and high-mobility group box 1 (HMGB1) . Densitometry analysis of each band intensity was performed using ImageJ software (https://imagej.nih.gov/ij/).Cells  of 5 year old Korean ginseng plants as described previously ,44,45 wiThe following lipid standards used in this study were purchased from Larodan Fine Chemicals AB:TG (11:1-11:1-11:1), diacylglycerol (DG) (8:0-8:0), monoacylglycerol (MG) (15:1), and cholesteryl ester (CE) (10:0). Additional lipid standards were purchased from Avanti Polar Lipids, Inc., including phophatidylcholine (PC) (10:0-10:0), phosphatidylethanolamine (PE) (10:0-10:0), phosphoserine (PS) (10:0-10:0), phosphatidylglycerol (10:0-10:0), phosphatidylinositol (8:0-8:0), phosphatidic acid (PA) (10:0-10:0), lysophosphatidylcholine (LPC) (13:0), lysophosphatidylethanolamine (LPE) (14:0), lysophosphoserine (LPS) (17:1), lysophosphatidylglycerol (14:0), lysophosphatidylinositol (13:0), lysophosphatidic acid (LPA) (14:0), sphingomyeline (SM) (d18:1-12:0), and ceramide (Cer) (d18:1-12:0). Each lipid standard was dissolved in methanol or chloroform (in the case of CE), stored at −20 °C, and diluted to 1 µg/mL for extraction.g, 20 min at 4 °C. The supernatants were collected, and protein concentration was determined using a BCA Protein Assay kit (Thermo Fisher Scientific). Samples set to contain the same amount of 30 μg protein in 50 μL of 0.01% SDS in PBS were added to 990 µL methanol/chloroform  with 10 μL of internal standard (IS) lipids . Each sample was vortexed three times (30 s each) and incubated for 10 min on ice. After centrifugation , 950 µL supernatant were transferred to a new tube. The pellet was resuspended in 750 µL chloroform/methanol/37% (1N) HCl  and incubated for 15 min at room temperature with vortexing for 30 s every 5 min. Next, 250 µL cold chloroform and 450 µL cold 0.1 M HCl were added to the sample followed by vigorous vortexing for 1 min and centrifugation . The bottom organic phase was collected and pooled with the transferred supernatant from the first extraction. Subsequently, the sample was divided into two equal aliquots and dried using a SpeedVac concentrator. One aliquot was then dissolved in 50 µL solvent A/solvent B  for neutral and positive lipid analyses, and the other aliquot was reconstituted in 50 µL methanol for trimethylsilyldiazomethane (TMSD) methylation and anionic lipid analyses. For TMSD methylation, a solution of TMSD (2 mol/L) in hexane was added to the cellular lipid extracts to obtain yellow colored solutions. After vortexing for 30 s, methylation was performed at 37 °C for 15 min. Glacial acetic acid was added to quench the methylation reactions, yielding colorless samples. The samples were then subjected to LC–MS analysis.A two-step extraction method was performed for high efficiency extraction of polar and non-polar lipids from GrEXs and GrEVs according to previously reported methods . First, v/v/v) with 20 mM ammonium formate and 0.1% (v/v) formic acid, and solvent B consisted of 2-propanol with 20 mM ammonium formate and 0.1% (v/v) formic acid. The gradient elution program was as follows: 0–5 min, B 5%; 5–15 min, B 5–30%; 15–22 min, B 30–90%; 22–25 min, B 90%; 25–26 min, 90–5%; and 26–30 min, B 5%. The flow rate was 250 μL/min, and the injection volume was 2 μL for each run. The total run time was 30 min. Lipids were analyzed using a QTRAP 5500 (AB Sciex) hybrid, triple quadrupole, linear ion trap MS instrument equipped with a Turbo V ion source, together with Analyst software, version 1.5.1. Ultra-high purity (UHP) nitrogen gas was used for the collisions. The following parameters were used with the operating source: 3500 V positive mode of capillary voltage, 3000 V negative mode of capillary voltage, a sheath gas flow rate of 11 L/min (UHP nitrogen) at 200 °C, drying gas flow rate of 15 L/min at 150 °C, and a nebulizer gas pressure of 25 psi. Optimized multiple reaction monitoring (MRM) conditions were used to analyze the various lipid species.Lipid analysis was performed using an Acquity ultra performance liquid chromatography (UPLC) system . A Hypersil GOLD column  was used for separation of lipids. The temperatures of the column oven and the sample tray were set to 40 °C and 4 °C, respectively. Solvent A consisted of an acetonitrile:methanol:water mixture  of precursor ions, the m/z of product ions, and the retention time, were exported using Qualitative Analysis software, version B.06.00 . Next, an in-house database constructed using Skyline software  was applied to determine the peak area of each assigned lipid, and all data were normalized by respective IS lipid. Statistical analyses such as PCA and hierarchical cluster analysis were performed using the MetaboAnalyst website. The volcano plot was generated by plotting statistical significance ) in the y-axis and magnitude of change between two groups ) in the x-axis using Excel program.LC–MS data were obtained and processed using Agilent Mass Hunter Workstation Data Acquisition software. The MRM data of target lipids, including the mass-to-charge ratios .The data were analyzed using the Student’s 3) were collected and used for morphological and lipidomic analyses, using cryo-electron microscopy (cryo-EM) and liquid chromatography–mass spectrometry (LC–MS), respectively. GrEVs revealed a closed spherical structure with diameter below 100 nm according to the cryo-EM and the bio-EM image analyses (Ginseng root-derived EVs (GrEVs) were isolated from the cold-pressed ginseng root extracts (GrEXs) using a sucrose cushion and subsequent density gradient ultracentrifugation a. The Granalyses b,c and sanalyses d. These KRT10) and terminal differentiation makers such as loricrin (LOR) and filaggrin (FLG) were not significantly changed by GrEV treatment under either normal or UV-irradiated conditions, although LOR and FLG expression seemed to be slightly upregulated by GrEV treatment  irradiation, and the expression levels of epidermal differentiation markers were determined. The expression levels of the early differentiation markers keratin 10 , p21Cip1 (CDKN1A), p16INK4a (CDKN2A), matrix metalloprotease 1 (MMP1), and interleukin 8 (IL-8) were also gradually downregulated with increasing concentrations of GrEVs , tyrosinase-related protein 2 (TRP2), and Ras-related protein 27 (RAB27). The expression levels of these proteins following UVB irradiation appeared to be decreased by GrEV treatment in a dose-dependent manner c. In conP. ginseng root explants and grown on Murashige and Skoog medium supplemented with 2,4-dichlorophenoxyacetic acid , sucrose, and a gelling agent [In general, ginseng roots are harvested when the plants reach 4 to 6 years of age, peeled or steamed, and then dried to prepare white ginseng or red ginseng, respectively. Considering the time-consuming and the labor-intensive processes involved in long term field cultivation of ginseng roots, culturing cells and tissues from ginseng roots using a bioreactor represents an attractive alternative for producing ginseng biomass and active ingredients with a uniform quality and yield ,52. We ing agent   ratios and matching retention times, when compared to internal standards representing a specific lipid family  based on their mass: charge  (p < 0.05) and three (4.1%) showing decrease  in GrEVs compared to GrEXs  a. Of theto GrEXs a. These to GrEXs b, and thThe largest number of differentially increased lipids in GrEVs was represented by PC (22) followed by PE (13), DG (11), LPC (11), LPE (6), SM (5), TG (1), and MG (1) . The difWe further examined compositional differences among the lipid classes in GrEVs and GrEXs by accessing the proportion of each lipid class with respect to the total lipids . In particular, the proportions of DG and phospholipids  were higher in GrEVs, whereas those of MG, LPA, LPS, Chol, and CE were lower . These fAssessing the mol% of each lipid class in the lipid category revealed that GrEVs contained similar ratios of MG (57.1%) and DG (42.8%), whereas GrEXs contained a relatively high ratio of MG (95.9%) , which suggests that the GEVs possessed unique characteristics.As key mediators of cell-to-cell communications, the characteristics of EVs derived from mammalian or bacterial cells have been verified in terms of their structures, vesicular components, and pathophysiological functions. However, those of plant-derived EVs, especially in terms of the biological effects on human cells, are barely known. Here, we report for the first time that EVs derived from Ginseng possesses multiple beneficial properties in terms of human health, and ginsenosides are the major constituents responsible for these activities ,53. HowePlant-derived EVs have been considered as attractive vectors for delivering siRNAs, microRNAs, proteins, and therapeutic drugs in view of targeting delivery, safety, and the cost of large scale production ,56. EV lIn this study, we focused on the beneficial effects of GEVs (GrEVs and GcEVs) on human skin cells by investigating their effects in replicative senescent fibroblasts or UVB-induced senescent melanocytes ,42. In aINK4A-positive senescent melanocytes in older subjects, and is associated with senescence phenotypes such as the increased cytokine levels [In this study, the vesicular constituents responsible for anti-senescence effects in human skin cells were not verified. However, we found that HMGB1 protein levels markedly increased after GEV treatment in melanocytes with UVB-induced senescence. HMGB1 is a redox-sensitive and multifunctional protein with both harmful and beneficial activities, which is functionally regulated in different ways depending on its location in the nucleus, the cytoplasm, or the extracellular milieu ,61. In te levels ,67. There levels ,69.Given that GEVs play immunomodulatory roles by inducing M1 macrophage polarization, which can be also mediated by HMGB1 ,70, it iBased on a comprehensive lipidomic analysis of GEVs and the parental extracts, DG, some phospholipids , and SM were enriched by over 20-, 8-, and 7-fold in GrEVs, respectively, compared to the corresponding levels in GrEXs b. In genIn this study, we verified the spontaneous release of EVs from ginseng cells in ginseng roots and cultured cells as well as their functional effects on the recovery from cellular senescence in human primary skin cells. Most active constituents in ginseng are extracted using organic solvents, and the remaining constituents (including active biomolecules such as proteins and lipids) and culture supernatants are discarded. Therefore, GEVs represent a potent alternative source of bioactive substances derived from ginseng for food products and topical applications. Based on our lipidomic analysis of GEVs, we suggest that DG is a potential GEV lipid biomarker that can mediate intercellular communication and directly evoke cellular responses in human cells via PKC signaling.


Pseudopimelodus is a Neotropical genus of bumblebee catfish, composed of four valid species occurring in both trans- and cis-Andean rivers of South America. The orogeny of the Andes has led to diversification in the genus Pseudopimelodus in Colombia. This study analyzed partial sequences of mitochondrial cox1 and nuclear rag2 genes to test the hypothesis that the species, nominally recognized as P. schultzi and P. bufonius in Colombia, correspond to more than two different evolutionary lineages. Results indicate high levels of genetic divergence among individuals of nominal P. schultzi and P. bufonius, from trans- and cis-Andean basins in Colombia. In addition, five divergent lineages of Pseudopimelodus were confidently delimited by using a single-locus species-discovery approach and confirmed by species tree analyses. Additionally, molecular-clock dating showed that most diversification processes in Pseudopimelodus took place during the Miocene, when Andean tectonic evolution was occurring in northwestern South America. The present study provides, for the first time, phylogeographic insight into this Neotropical genus. Batrochoglanis (four species), Cephalosilurus (one species) Cruciglanis (one species), Microglanis (three species), Pseudopimelodus (two species), and Rhyacoglanis (one species)  to the Caribbean slope rivers , Magdalena-Cauca, Orinoco and Amazon river basins, whereas the endemic species Pseudopimelodus schultzi  was endemic to the Caribbean slope rivers and Magdalena-Cauca River basin  of this species of the Sinú River and the Magdalena-Cauca basin.Pseudopimelodidae is a Neotropical catfish family that includes seven genera and at least 50 valid species, with a distribution ranging from the Atrato River in Colombia to Río de la Plata in Argentina . Colombispecies) . The occer basin . Conversr basins , while PPseudopimelodus have been conducted, leaving a gap in our understanding of the possible effects of geomorphological processes on the diversification of this genus. This gap is important, since phylogeographic analyses may reveal cryptic and deeply divergent lineages that are not reflected in the current taxonomy (Pseudopimelodus: (1) the uplift of central cordillera during the Late Oligocene-Early Miocene, (2) the isolation of the Atrato-Pacific slope and the Magdalena-Cauca basin with the uplift of the Western Cordillera during the Late Oligocene-Middle Miocene  the Miocene , and (4) Miocene . Thus, wcox1; COI), which exhibits high rates of molecular evolution, to explore recent divergence events within Pseudopimelodus; we then used the nuclear recombination activating gene 2 (rag2) to explore more ancient phylogenetic relationships among samples of this genus from northwestern South America. Both genes are commonly used in evolutionary studies, due to their adequate variability and ability to reveal phylogenetic relationships and identify freshwater fish species, including members of the superfamily Pimelodoidea  and Amazon (Orteguaza River and Vaupés River) are separated by the Vaupes Arch, the major drainage that divide the Llanos region of eastern Colombia , following the manufacturer’s protocol. For phylogeographic analyses, we amplified a partial region of cox1 gene in all 257 specimens, using the universal primer cocktail VF2, FishF2, FishR2 , 0.75 µl dNTPs (10 mM), 0.75 µl of the primer cocktail (0.25 pmol/µl each), 0.15 µl of Platinum™ Taq DNA Polymerase (5 U/µl), and 20.6 µl of sterile nuclease-free water (Amresco). The thermal profile for cox1 consisted of an initial cycle at 95 °C for 3 min, 32 cycles of 94 °C for 3 min, 25 s at 60 °C, and 30 s at 72 °C, and the final extension was omitted. A similar thermal profile was used to amplify rag2, but with changes in annealing temperature (58 °C) and extension time (45 s). PCR products were confirmed by agarose gel electrophoresis and EZ vision staining and sequenced in both forward and reverse directions using an ABI sequencer 3730XL. Sequences were checked and edited using Geneious v10.0.9 (https://www.geneious.com) software, and multiple alignment to create consensus sequences was performed with the MAFTT v7.308 (https://www.ebi.ac.uk/Tools/st/emboss_transeq/).Genomic DNA was extracted using the PureLink, FishR2 , and FR1, FishR2 , as prevT v7.308 . Both geπ) diversity  and nucleotide  methods in R v3.4.163, by using the scripts developed by https://github.com/legalLab/publications. These methods were implemented to define a priori clusters of putative species (lineages) of Pseudopimelodus as trait values for StarBEAST2 v14  theAST2 v14 , and (3)AST2 v14 . For allAST2 v14  choosingAST2 v14 . We ran AST2 v14  was usedAll three analyses were based on point estimates from the maximum clade credibility tree generated using TreeAnnotator v1.8.4 and calculation of confidence intervals from the posterior sample of 1,000 trees. The R packages bGMYC v1.0.2 , splits cox1 and nuclear rag2 markers to implement a dated species tree considering the molecular substitution model of each gene using StarBEAST2 v14 , defined by 72 polymorphic sites, 52 of which were parsimoniously informative. Values of haplotype and nucleotide diversities for cox1 dataset were h = 0.761 ± 0.020 and π = 0.046 ± 0.001, respectively. Additionally, sequence analysis of rag2 in the trans-Andean Pseudopimelodus revealed 26 haplotypes of 77 sequences analyzed , defined by 22 polymorphic sites, 16 of which were parsimoniously informative. Values of haplotype and nucleotide diversities were Hd = 0.878 ± 0.024 and π = 0.010 ± 0.000. According to the Bayesian Information Criterion, the optimal model for cox1 and rag2 datasets were HKY + G4 and HKY respectively. Collection information, voucher numbers and GenBank accessions for all new sequences obtained in this study are provided in Sequence edition yielded a final alignment matrix of a 357 bp and 520 bp fragment for P. mangurus (EU179816) and P. charus (EU179815). Pseudopimelodus mangurus was only correctly delimited by GMYC, while P. charus was correctly delimited by locMin and GMYC . Notably, lineage 1 and lineage 5, represented by a higher number of samples, showed unique haplotypes related to a common ancestral haplotype distributed in different sampling sites  instead of the alternatives bGMYC (MLE = −4042.52818) or locMin (MLE = −4080.28218) models. Moreover, the five evolutionary lineages of x1 genes . This anx1 genes –3 showed8.037016 Pseudopimelodus during Middle Miocene (16.21 mya 95% HDP 11.91–20.79). Similarly, a Middle Miocene split was detected between lineage 2 and the clade comprising lineages 3, 4 and 5 (12.37 mya HPD 8.98–16.23), while a Late Miocene divergence was detected between lineage 3 and the clade formed by lineages 4 and 5 (9.93 mya HPD 6.54–13.20), and between lineages 4 and 5 (5.28 mya HPD 1.61–8.20).Divergence time estimates  indicateP. schultzi and P. bufonius in Colombia correspond to more than two different evolutionary lineages, the divergence times of which agree with Andean tectonic evolution in northwestern South America. Our expectation was that geological events induced vicariance in Pseudopimelodus from northwestern South America. Despite the limitations of using short fragments, the use of nuclear and mitochondrial molecular markers, with different mutational rates, allowed us to infer ancient, as well as more recent, genetic divergence in this group of bumblebee catfish. In this study, we did not cover the complete geographical range where nominal P. bufonius occurs in Colombia; instead, we analyzed only a small number of individuals from the cis-Andean portion. Thus, the detected haplotypes may not represent all genetic lineages present in this geologically complex region.This study tested the hypothesis that the species nominally recognized as cox1 and nuclear rag2 analyses supported our hypothesis of divergent lineages of Pseudopimelodus inhabiting trans and cis-Andean rivers in Colombia. Indeed, the molecular evidence gathered in this study supports the non-monophyly of P. bufonius and P. schultzi, based on the strong genetic divergence among specimens identified as P. schultzi representing populations from Magdalena, Cauca, Atrato, and Sinú rivers, and among nominal P. bufonius from the Amazon and Orinoco rivers. Specifically, trans-Andean P. schultzi includes three divergent lineages: (1) Magdalena River lower and middle sectors, Cauca River lower sector and San Jorge River, (2) Magdalena River upper sector, Cauca River upper and middle sectors, and (3) Sinú and Atrato rivers. Moreover, P. bufonius populations from the cis-Andean region of Colombia include two divergent lineages: (1) Orinoco River hydrographic zone and (2) Amazon River hydrographic zone.Mitochondrial P. schultzi and P. bufonius, occur in Colombia. This outcome suggests that a robust taxonomic revision of Pseudopimelodus, and especially P. bufonius, is needed; future studies should include samples from the type locality , previously reported by Furthermore, the divergence between lineage 2 and the clade containing lineages 3, 4 and 5 was estimated to be at 12.37 mya (HPD 8.98–16.23). This estimated date is consistent with the first development of the Amazon River during the late-middle Miocene, when the paleo-Amazon River was still partly connected to the paleo-Orinoco, through a fluvio-lacustrine system . Then, ttrans-Andean lineages 4 and 5. This fluvial corridor may correspond to the area of the Garzón Massif (between latitudes 1°–4°N), located in the southeastern flank of the Eastern Cordillera in the Colombian Andes (The Late Miocene divergence (9.93 mya HPD 6.54–13.20) observed between lineage 3 and the clade containing lineages 4 and 5, agrees with the uplift of Eastern Cordillera 10 mya;  that peran Andes , which han Andes . In factMoreover, the splitting of lineage 4 from lineage 5 occurred during the late Miocene (5.28 mya HPD 1.61–8.20), concordant with the uplift of the Western Cordillera that caused the isolation of the trans-Andean Atrato-Pacific slope from the Magdalena basin during Late Miocene to Pliocene . In addiPseudopimelodus is currently underestimated, which can affect the estimation of species richness, jeopardize the understanding of ecological patterns, and may be harmful to species at a high risk of extinction . Finally, and independently of taxonomic implications, genetic divergence of Pseudopimelodus lineages must be considered in current conservation and management policies of Colombian bumblebee catfish fisheries.Altogether, the results obtained in this study provide a better understanding of the phylogeographic processes that govern the current distribution and diversification of freshwater ichthyofauna in northwestern South America. Moreover, as shown by SLSD and species tree results, the taxonomic diversity within tinction . In lighPseudopimelodus in Colombia comprises at least five different, clearly delimited, evolutionary lineages. The lineages exhibit phylogenetic histories that agree with the geological history of northwestern South America. We recommend that future genetic studies include more genetic markers and a greater number of samples, from additional localities, to examine more thoroughly and provide a deeper understanding of the phylogeographic patterns of this genus in South America. These results contribute to the phylogeographic knowledge of Pseudopimelodidae inhabiting cis and trans-Andean river systems in Colombia and provide an important benchmark for future phylogenetic studies in this diverse and widespread Neotropical catfish family.This study provides strong mtDNA evidence that the genus 10.7717/peerj.9028/supp-1Table S1Asterisks indicate samples used for phylogenetic analyses. Vouchers are indicated in parentheses. ANDES: Museo de Historia Natural - Universidad de los Andes, Bogotá; CIUA: Colección de Ictiología de la Universidad de Antioquia, Medellín, Antioquia; CP-UCO: Colección de peces de la Universidad Católica de Oriente, Rionegro, Antioquia; IMCN: Colecciones biológicas de referencia científica del Instituto para la Investigación y Preservación del Patrimonio Cultural y Natural del Valle del Cauca –Inciva, Cali, Valle del Cauca.Click here for additional data file.10.7717/peerj.9028/supp-2Table S2Asterisks indicate samples used for phylogenetic analyses. Vouchers are indicated in parentheses. CP-UCO: Colección de peces de la Universidad Católica de Oriente, Rionegro, Antioquia; IMCN: Colecciones biológicas de referencia científica del Instituto para la Investigación y Preservación del Patrimonio Cultural y Natural del Valle del Cauca –Inciva, Cali, Valle del Cauca.Click here for additional data file.10.7717/peerj.9028/supp-3Figure S1Pseudopimelodus. Haplotype is indicated in parenthesis.Colors denote different lineages of Click here for additional data file.10.7717/peerj.9028/supp-4Figure S2Pseudopimelodus. Haplotype is indicated in parenthesis.Colors denote different lineages of Click here for additional data file.10.7717/peerj.9028/supp-5Figure S3Pseudopimelodus.Colors denote different lineages of Click here for additional data file.10.7717/peerj.9028/supp-6Supplemental Information 6Click here for additional data file.10.7717/peerj.9028/supp-7Supplemental Information 7Click here for additional data file.10.7717/peerj.9028/supp-8Supplemental Information 8Click here for additional data file.10.7717/peerj.9028/supp-9Supplemental Information 9Click here for additional data file.


Sleep deprivation increases rates of forgetting in episodic memory. Yet, whether an extended lack of sleep alters the qualitative nature of forgetting is unknown. We compared forgetting of episodic memories across intervals of overnight sleep, daytime wakefulness, and overnight sleep deprivation. Item-level forgetting was amplified across daytime wakefulness and overnight sleep deprivation, as compared to sleep. Importantly, however, overnight sleep deprivation led to a further deficit in associative memory that was not observed after daytime wakefulness. These findings suggest that sleep deprivation induces fragmentation among item memories and their associations, altering the qualitative nature of episodic forgetting. Why are some memories remembered and others forgotten? Retroactive interference accounts of forgetting argue that learning and mental activity that occurs after encoding contributes to memory loss . ConsistGiven that forgetting is reduced by sleep, it is unsurprising that extended periods of sleep deprivation give rise to severe impairments in memory recall . In humaTo date, studies of sleep deprivation and memory have typically assessed forgetting for single items , sleep deprivation might open the door to fragmented forms of memory loss and, ultimately, alter the qualitative nature of forgetting.Across two experiments, we investigated the impacts of sleep deprivation, as compared to sleep and routine daytime wakefulness, on memory for items and their associations. In Experiment 1, 27 healthy adults  entered a within-subjects crossover design  followed identical procedures to Experiment 1, with the exception that retesting (T2) always took place in the morning following a night of sleep or total sleep deprivation. In both conditions, participants rose by 08:00 on the morning of the first session (∼12 h before encoding) and remained awake throughout the day (confirmed via wristwatch actigraphy). Resultantly, by T2 of the sleep deprivation condition, participants had been awake for ∼24 h. Across both experiments, we predicted that overnight sleep deprivation (vs. sleep and routine daytime wakefulness) would amplify adjective and category forgetting.Experiment 2 ; permitting investigation of potential relationships between sleep stages and forgetting. Electrodes for electroencephalography (EEG) were attached at eight standardized locations: F3, F4, C3, C4, P3, P4, O1, and O2, each referenced to the contralateral mastoid (A1 or A2). Electrooculography (EOG) and electromyography (EMG) electrodes were also attached. Sleep data were segmented into 30 sec epochs and scored as wake, N1, N2, N3, or REM sleep in accordance with standardized criteria .In both experiments, a follow-up test (T3) was administered 2 d after T2 (∼10:00) to assess item and associative memory loss following opportunities for recovery sleep. Participants completed the Stanford sleepiness scale  and a psn = 51, 4 male; mean ± SD age = 19.96 ± 5.29 yr). Emotional ratings  were significantly lower for negative images (mean ± SEM = 2.98 ± 0.09) than neutral images  = 26.00, P < 0.001, d = 3.64). All adjectives were emotionally neutral.All behavioral tasks were implemented on a PC with MATLAB 2017a and Psychtoolbox 3.0.13 . At encot(26) = 0.43, P = 0.67] or Experiment 2 . Each adjective-image pair was presented once, and participants were required to make each of their responses within 10 sec.Each encoding trial began with a 1.5 sec fixation period. A randomly selected adjective was then displayed above a randomly selected object or scene image for 5 sec. Participants were instructed to visualize the adjective and image interacting, and then to indicate via keyboard press whether the mental image they generated was realistic or bizarre  for item memory. Uncertainty data and analyses are available in Supplemental_Table_S3.docx and Supplemental_Analysis_S2.docx, respectively.A hierarchical approach was used at each test phase, permitting a distinction between item memory (adjectives) and associative memory (images associated with adjectives). T1 included 180 adjectives: 120 targets presented at encoding and 60 foils. Each trial began with a 1.5 sec fixation period, after which a randomly selected adjective was displayed for 3 sec. Participants were required to indicate whether the adjective was “old” (they recognized the adjective from encoding) or “new” (they did not recognize the adjective) within 10 sec. They were also able to indicate uncertainty by pressing “?”. This ensured that participants were reasonably confident in their “old”/“new” responses and discouraged guessing. Note that inclusion of the “uncertain” response at adjective recognition precluded calculation of the sensitivity index . For “new” or “uncertain” responses to adjectives, participants moved immediately to the next trial. The procedures for T2 and T3 were identical to those of T1, except that a new set of foil adjectives were used in each test.For each “old” response, participants indicated whether the image associated with that adjective at encoding was an object or scene, or pressed “?” if they were uncertain. After each “object” or “scene” response, participants provided a brief typed description of the image  = 2.44, P = 0.02, d = 0.47; Drawing on data from Experiment 1, we first investigated whether item memories were forgotten to a greater extent across a day of wakefulness relative to a night of sleep. To address this question, we isolated adjectives that were correctly recognized at the immediate test (T1) and then calculated the proportion of these adjectives that were forgotten (incorrect or “uncertain” responses) at the delayed test (T2). As expected, the resultant item loss proportion scores were higher after wakefulness than sleep .To assess whether overnight sleep deprivation was more conducive to item forgetting than routine daytime wakefulness, item loss proportion scores from both experiments were applied to a 2 (Delay: Sleep/Wake) × 2 (Experiment: One/Two) mixed ANOVA. A trend for the Delay*Experiment interaction suggested that the effects of wakefulness (vs. sleep) on item forgetting were amplified in Experiment 2 (overnight sleep deprivation) relative to Experiment 1  was correctly retrieved at T1. We then calculated the proportion of these adjectives for which the image category was forgotten (incorrect or “uncertain” responses) at T2. The resultant fragmentation scores for Experiments 1 and 2 were submitted to separate 2 (Delay: Sleep/Wake) × 2  repeated-measures ANOVAs.F = 0.15, P = 0.71; F = 0.03, P = 0.86; Emotion*Delay interaction: F = 0.74, P = 0.40].In Experiment 1, fragmentation scores were comparable after daytime wakefulness and overnight sleep .Strikingly, however, fragmentation scores in Experiment 2 were significantly higher after sleep deprivation than sleep . Sleep duration (mean ± SEM) was 430.20 ± 6.45 min in the sleep condition of Experiment 2. There were no significant correlations between item or associative forgetting and time (min) spent in any stage of sleep [all P > 0.05].Consistent with the view that memory fragmentation was more prevalent after sleep deprivation than daytime wakefulness, a 2 (Delay: Sleep/Wake) × 2 (Experiment: One/Two) mixed ANOVA (collapsed across image emotion) revealed a significant Delay*Experiment interaction . Note that the same effect was observed when comparing the wake and sleep conditions in Experiment 1 . To compute fragmentation scores between T1 and T3 in Experiment 2, we first isolated adjectives that were correctly recognized at T1 and T3, and for which the associated image category was correctly retrieved at T1. We then calculated the proportion of these adjectives for which the image category was forgotten at T3. Importantly, fragmentation scores were higher in the sleep deprivation (vs. sleep) condition . Taken together, our findings suggest that the memory deficits associated with sleep deprivation were not simply due to excessive tiredness or stress at T2. It is nevertheless possible that high stress levels during consolidation contributed to a long-lasting fragmentation of memory.In Experiment 2, alertness levels at T2 were reduced in the sleep deprivation (vs. sleep) condition, as indicated by the Stanford sleepiness scale and psychomotor vigilance test . Item loss proportion scores calculated between T2 and T3 (the proportion of correctly recognized adjectives at T2 that were forgotten at T3) were comparable between the wake and sleep conditions in Experiment 1 . However, sleep deprivation (vs. sleep) led to a trend toward additional item forgetting in Experiment 2 . Fragmentation scores calculated between T2 and T3  were applied to a 2 (Delay: Sleep/Wake) × 2  repeated-measures ANOVA. However, no significant effects emerged in Experiment 1  or Experiment 2 .We next investigated whether postlearning wakefulness or sleep deprivation (vs. sleep) led to any t(26) = 0.79, P = 0.44] or Experiment 2 . Associative memory performance was calculated as the proportion of correctly identified “old” adjectives for which the associated image category was also correctly retrieved. A 2 (Delay: Sleep/Wake) × 2  repeated-measures ANOVA revealed no significant effects in Experiment 1  or Experiment 2 .Finally, we examined item and associative memory performance at T1 to ensure that the above effects were not driven by between-condition differences at baseline. Item memory performance was calculated as the proportion of “old” adjectives that were correctly identified as “old.” No differences were observed between the sleep and wake conditions in Experiment 1 [Taken together, our findings suggest that sleep deprivation prompts a qualitative change in the nature of episodic forgetting. In Experiment 1, a routine day of wakefulness increased item-level forgetting relative to a night of sleep, but had no impact on associative memory when the base items survived. In Experiment 2, by contrast, overnight sleep deprivation (vs. sleep) not only increased item-level forgetting, but also increased associative memory loss when the base items remained unscathed. Hence, sleep deprivation appears to induce fragmentation among episodic representations that are typically forgotten in an all-or-none manner .Proactive and retroactive interference are thought to contribute to forgetting . Hence, Wakeful experience is associated with a net increase in synaptic strength . A putatPrevious work has suggested that emotionally salient memories are more resistant to the effects of sleep deprivation than neutral memories . In the In conclusion, our findings suggest that sleep deprivation not only amplifies item-level forgetting, but induces fragmentation among item memories and their associations. Such fragmented memory loss might be due to a combination of proactive and retroactive interference, leading to severe and irregular impairments in episodic memory retrieval. More broadly, our findings offer novel insights into the cognitive impairments posed by insufficient sleep; an issue that is particularly pertinent when considering the global prevalence of chronic sleep deprivation , which ihttps://osf.io/s35f9/.Study data are freely available via the following link:


Posterior capsule opacification (PCO), a complication of extracapsular lens extraction surgery that causes visual impairment, is characterized by aberrant proliferation and epithelial-mesenchymal transition (EMT) of lens epithelial cells (LECs). Curcumin, exerting inhibitive effects on cell proliferation and EMT in cancer, serves as a possible antidote towards PCO.  Cellular proliferation of LECs after treatment of curcumin was measured with MTT assay and flow cytometry. The transcriptional and expressional levels of proteins related to proliferation and EMT of LECs were quantified by western blotting and real-time PCR. 2/M arrest via possible inhibition of cell cycle-related proteins including CDK1, cyclin B1, and CDC25C. It had also inactivated proliferation pathways involving ERK1/2 and Akt pathways in LECs. On the other hand, curcumin downregulated the EMT of LECs through blocking the TGF-β/Smad pathway and interfering Notch pathway which play important roles in PCO.  Curcumin was found to suppress the proliferation of LECs by inducing G This study shows that curcumin could suppress the proliferation and EMT in LECs, and it might be a potential therapeutic protection against visual loss induced by PCO. Cataract is a vision-impairing disease in which opacification and turbidness of the lens are formed. Various factors contribute to the pathological process of cataract, while the only solution is performing ophthalmic surgery extracting a muddy lens out of the capsule with or without the intraocular lens implantation. However, these surgeries are risky with complications. Posterior capsule opacification (PCO) is a common complication after cataract surgeries and causes setback of sight during postoperation period . PCO occα-smooth muscle actin (α-SMA), fibronectin, and N-cadherin, in addition to the upregulated expression of transcription factors including Snail, Slug, ZEB, and Twist [β) is the best known. The TGF-β family causes biological reactions by mediating Smad transcription factors and regulates gene's expression. It also activates non-Smad pathways that exert nontranscriptional roles including epithelial junction dissolution, cytoskeletal reorganization, motility, and translational control. There are three isoforms of TGF-β, and they are all identified in humans. Among them, TGF-β2 is the predominant isoform in ocular media and the most important factor contributing to transdifferentiation and pathologic fibrosis of LECs [Epithelial-mesenchymal transition EMT, a main characteristic process of PCO, includes the acquisition of mesenchymal cell-like morphology, diminished intercellular junctions, reduced expression of epithelial markers such as ZO-1 and E-cadherin, and accumulation of EMT markers including nd Twist . Among d of LECs , 6, 7.Curcuma longa), 6-heptadiene-3,5-dione,1,7-bis(4-hydroxy-3-methoxyphenyl)-1,-1 with the CAS number: 458-37-7, has been medically used in Asian for centuries [Curcumin as a component of turmeric  containing 10% fetal bovine serum . Cells were grown in a humidified atmosphere containing 5% CO3 cells/well and grown overnight. Then, cells were treated with different doses  of curcumin  for 24 h, 48 h, and 72 h, respectively. The following procedures were conducted as described previously [The proliferation of LECs under curcumin treatment was assessed with MTT assay. LECs were seeded into 96-well plates at a density of 4 × 10LECs treated with different concentrations of curcumin for 48 h were collected and washed with PBS; cell apoptosis and cell cycle were analyzed by flow cytometry  with the MultiCaspase Kit and the Cell Cycle Kit . All procedures were proceeded according to the manufacturer's protocols as we previously did , 22. Theβ2 or curcumin for 48 h was isolated with TRIzol reagent . cDNA synthesis and real-time RT-PCR were underwent as previously described [ΔΔCq− method with normalization to GAPDH.Total RNA of cells treated with TGF-escribed , 23. Datα-SMA , Notch1, Notch2, Smad2/3, p-Smad2/3 , p38, p-P38, Akt, p-Akt, ERK1/2, and p-ERK1/2 .Cell proteins were extracted with a lysis buffer solution containing phosphatase inhibitor from the LECs. The western blot procedures were referred to our previous work , 23. GAPP < 0.05 is regarded as statistically significant.Each experiment was performed in triplicate with same protocol. All data were expressed as mean ± S.E.M, and statistical analyses were processed with SPSS version 22.0 . One-way analysis of variance was used to compare differences among groups. μM had led to approximate OD values after the time point of 24 h, which suggested that the LEC cell growth arrest might be induced by 10+ μM of curcumin in vitro. On the other hand, curcumin had induced apoptosis of LECs. Flow cytometry showed that curcumin had raised cell apoptosis to (88.4 ± 6.09) % and (97.5 ± 0.90) % at the concentration of 20 μM and 40 μM after 48 h, compared with the control group (2.53 ± 0.32) % .To investigate whether curcumin can prevent the aberrant proliferation of LECs, we used gradient concentrations of curcumin on SRA01/04 for 72 h. The MTT assay revealed that the proliferation of LECs was suppressed by curcumin in a time-dose-dependent manner . Meanwhi Figures . HoweverμM had increased cell numbers in the G2/M phase (43.6 ± 5.75)%, while 5 μM curcumin did not affect the LEC cycle in the G2/M phase (28.9 ± 1.04)% (P > 0.05), compared with the control (29.7 ± 2.58)% (P < 0.05)  . The celficantly . But intβ2-induced EMT level in LECs at the concentration of 10 ng/ml for 48 h. Firstly, gene expression markers of EMT including fibronectin, collagen I, collagen IV, and N-cadherin, and transcription factor Snail were all elevated correspondingly  and TGF-β2 of 10 ng/ml, the transcriptional levels of fibronectin, collagen I, and collagen IV were decreased in a dose-dependent manner  inhibits proliferation of colorectal cancer cells by inducing senescence rather than apoptosis [β2 could activate the Akt pathway in human subconjunctival fibroblasts, and Akt along with the MAPK pathway contributed to TGF-β2-induced upregulation of Jagged-1 in retinal epithelial (RPE) cells [β treatment was related to ERK1/2 blockage and Akt inhibition but not p38  cells , 33. We  not p38 . As all β is an effective EMT inducer in both normal and pathological conditions [β was also demonstrated to induce EMT in lens, which increased the possibility of PCO onset [β2 successfully induced EMT in LECs by elevating protein markers including fibronectin, collagen I, and collagen IV. After treatment of curcumin, the RNA transcriptional levels of the markers fibronectin, collagen I, and collagen IV were downregulated  and long noncoding RNA (>200 nts), and they can both act as tumor suppressors or tumor oncogenes in tumorigenesis . Oncogen2/M cell cycle arrest and blocking ERK1/2 along with Akt pathway but also showed that the curcumin had inhibited TGF-β-induced EMT through suppressing the TGF-β/Smad pathway and interfered with the Notch pathway. Though there are still works to be done to further understand the effects and functional mechanisms of curcumin on cellular pathology, our work has illustrated its possibly curative effect in PCO.In conclusion, this study has revealed the potential value of curcumin in PCO treatment for the first time. It has not only provided evidence that curcumin could suppress LECs proliferation by G