The effect of antihypertensive drugs, especially drugs modulating the renin-angiotensin-aldosterone-system (RAAS), on neurodegenerative diseases still needs to be investigated. This study aimed to compare the effects of three different antihypertensive drugs  on neuroprotection and acetylcholine (ACh) levels against lipopolysaccharide (LPS)-induced injury in a differentiated SH-SY5Y cell line. Cells were treated with retinoic acid for differentiation to a neuronal phenotype. LPS 20 (μg/mL) was applied to the cells for one hour. Then, the cells were treated with 1, 5, and 10 µg/mL concentrations of telmisartan, perindopril, and nebivolol separately for 24 hours, except for the control and LPS alone groups. Cell viability was evaluated using a 3--2,5-diphenyltetrazolium bromide (MTT) assay. ACh levels were analyzed using an enzyme immunosorbent assay in the culture medium. Tumor necrosis factor-alpha (TNF-α), interleukin 1 beta (IL-1β), and nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) expressions were evaluated using western blot analysis. Telmisartan demonstrated the highest cell viability against LPS-induced injury, whereas the protective effect of perindopril was moderate. Nebivolol showed no neuroprotective effect. The protective effect of 10-µg/mL telmisartan was superior to 10 µg/mL perindopril (p=0.006), 5 µg/mL perindopril (p=0.001), 1 µg/mL perindopril (p=0.001), and 1, 5, and 10 µg/mL nebivolol (p<0.001). Among all the study drugs, only telmisartan provided a statistically significant increase in ACh levels after LPS-induced injury. Additionally, the administration of telmisartan provided a concentration-dependent reduction in TNF-α, IL-1β, and NFκB expression against LPS-induced neuroinflammation. These findings suggest that telmisartan has a superior neuroprotective effect against LPS-induced injury in neuron-like cells compared with both perindopril and nebivolol. In addition to the association with increasing age, it has been shown that cardiovascular diseases and hypertension are important and independent risk factors for cognitive impairment and dementia ,2. The rAcetylcholine (ACh) has an essential role in the pathophysiology of dementia and Alzheimer’s disease (AD). The results of post-mortem studies have shown that lower levels of ACh are associated with AD . SeveralIn our study, we selected an ARB, an ACE inhibitor, and a beta-blocker agent that can cross the blood-brain barrier (BBB) to compare their neuroprotective effects. In order to provide useful information for future neuroscience research, we chose telmisartan, perindopril, and nebivolol because all these drugs have lipophilic properties and can cross the BBB. Additionally, all these drugs are very commonly used in daily clinical practice. Telmisartan is an angiotensin II AT1 receptor blocker, whereas perindopril is an ACE inhibitor that inhibits the conversion of angiotensin I to angiotensin II. The last drug in the present in vitro experiment was nebivolol, which is a beta-blocker that selectively blocks the β1 receptor in the sympathetic nervous system and inhibits renin release in the juxtaglomerular apparatus.In the present study, we aimed to compare the effects on ACh release and the neuroprotective effects (based on cell viability) of three different classes of antihypertensive drugs against lipopolysaccharide (LPS)-induced injury in a differentiated human SH-SY5Y cell line , which provided an appropriate model to evaluate the neuroprotective effects of different compounds ,10. AddiCell culture, differentiation, and treatment2 and 95% air in DMEM/F12 medium with L-glutamine  containing 10% fetal bovine serum , 1% penicillin-streptomycin , and 1% amphotericin B  at 37 °C. The medium was changed twice per week, and cells were split at about 80% confluence. Cells were seeded in 96-well plates (3 × 103 cells/per well), 24-well plates (3 × 105 cells/per well), and 6-well plates (6 × 105 cells/per well) for the experiments [The human neuroblastoma SH-SY5Y cell line was obtained from the American Type Culture Collection . Cells were cultured under humidified 5% COeriments . Cells tStudy groupsFor the primary aim, the study was divided into ten groups: the LPS alone group, three nebivolol treatment groups, three perindopril treatment groups, and three telmisartan treatment groups. In the treatment groups, in addition to LPS (20 µg/mL), nebivolol, perindopril, and telmisartan were administrated in concentrations of 1, 5, and 10 µg/mL separately. Additional experiments for cell viability were repeated in the absence of LPS stimulation and were grouped as a control, three nebivolol treatment groups , three perindopril treatment groups , and three telmisartan treatment groups .In addition to direct comparison with control and LPS groups separately for each study drug, the cell viability (%) results of the study drugs against LPS-induced injury were compared with each other using statistical methods for multiple comparisons described in the statistical analysis section.3--2,5-diphenyltetrazolium bromide (MTT) cell viability assayThe viability of cells in each group with or without administration of LPS and/or study drugs was tested using an MTT assay. Cells were incubated at 37 °C with 0.1 mg/mL MTT  for two hours. The supernatants were decanted without being dispensed to the cells. Then, 100 µL DMSO (Merck) was added to the cells, which were then kept in the dark. After 30 minutes, optical densities were measured using a microplate reader  at 570 nm . The celACh ELISA assay5 cells in each group were cultured using a differentiation medium (choline-free medium). The cells were treated with 20 µg/mL LPS (except the control group) for one hour, and study drugs  were added separately for 24 hours. The relative concentration of ACh in the culture medium was calculated using an ACh ELISA Kit  with a microplate reader  at 570 nm. Two parallel wells were repeated in each group.An ACh enzyme-linked immunosorbent assay (ELISA) was performed according to the manufacturer’s instructions to detect the ACh release in the culture medium. Briefly, 3 × 10SDS-PAGE and western blotting6/well) were differentiated into neurons with RA, they were treated with telmisartan, perindopril, and nebivolol for 24 hours. Total protein was isolated using the RIPA Lysis Buffer  and then protein concentrations were determined using a Bradford assay (Merck). Samples were subjected to western blot analysis as previously described [Inflammation-related markers were evaluated using Western blot analysis after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) . After Sescribed . Equal aStatistical analysisStatistical analyses were conducted using the IBM Statistical Package for the Social Sciences (SPSS) for Windows version 21.0 software. Multiple comparisons between the treatment groups were performed using a one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test. For correlation analysis, Pearson's correlation coefficient was used. P-values of <0.05 were considered significant. Data are presented as mean ± standard deviation (SD), and the results of Western blot analysis are given as mean ± standard error of the mean (SEM).The effect of telmisartan, perindopril, and nebivolol on cell viability in the differentiated SH-SY5Y cell lineIn the first step, the effects of study drugs on cell viability were evaluated in the differentiated SH-SY5Y cell line, without LPS stimulation. The percentages of cell viability for 1, 5, and 10 µg/mL nebivolol were 93.86±3.28%, 95.19±4.00%, and 74.99±3.19%, respectively. The percentages of cell viability for 1, 5, and 10-µg/mL perindopril were 100.36±2.53%, 105.73±1.71%, and 99.85±6.24%, respectively. The cell viability percentages for 1, 5, and 10 µg/mL telmisartan were 102.25 ± 11.52%, 108.49±2.58%, and 107.37±1.57%, respectively. Among all the study drugs, only 10 µg/mL of nebivolol caused a significant decrease in cell viability compared with the control (p<0.001) . The potential neuroprotective effect of telmisartan on LPS-induced cell death in the differentiated SH-SY5Y cell line was investigated. When compared with the administration of LPS alone, the addition of telmisartan in increasing concentrations  significantly reversed the decrease in cell viability caused by LPS . Additionally, telmisartan in 5 and 10-µg/mL concentrations provided cell viability values similar to the control. The percentages of cell viability for LPS alone and LPS plus 1, 5, and 10 µg/mL telmisartan were 32.24±5.18%, 70.10±1.32%, 86.01±8.22%, and 87.11±10.49%, respectively . The percentages of cell viability for LPS alone and LPS plus nebivolol 1, 5, and 10 µg/mL were 32.24±5.18%, 31.96±5.97%, and 19.34±2.57%, 15.91±3.55%, respectively  value of 10 µg/mL telmisartan was significantly higher than the values of 10 µg/mL perindopril (p=0.006), 5 µg/mL perindopril (p=0.001), 1 µg/mL perindopril (p=0.001), and 1 µg/mL, 5 µg/mL, and 10 µg/mL nebivolol (p<0.001). There was no statistically significant difference between the different concentrations of telmisartan; however, the cell viabilities with both telmisartan 5 and 10 µg/mL were slightly higher than with telmisartan 1 µg/mL.The effect of telmisartan, perindopril, and nebivolol on ACh levels after LPS-induced injury in the differentiated SH-SY5Y cell lineIn addition to cell viability, the effect of study drugs on ACh levels was evaluated under the condition of LPS-induced injury in the differentiated SH-SY5Y cell line. Administration of LPS alone considerably lowered the ACh levels (p=0.001). After LPS-induced injury, only the administration of telmisartan provided a statistically significant increase in ACh levels; the administration of perindopril or nebivolol in different concentrations showed no statistically significant changes  Figure .Decrease in the inflammation-related protein expressions with telmisartan after LPS-induced neuroinflammation in the differentiated SH-SY5Y cell LineInflammation-related protein expressions were evaluated using Western blot analysis after SDS-PAGE. The expressions of TNF-α, IL-1β, and NFκB were evaluated to understand the underlying mechanisms related to inflammatory processes. Cells were treated with LPS for one hour in the presence or absence of telmisartan. LPS stimulation significantly elevated the levels of these proteins Figure . TreatmeHypertension is an independent risk factor for cerebrovascular diseases and neurodegeneration ,17. Its The BBB is a critical structure that prevents several substances in the blood from entering the brain. Drugs that can cross the BBB can directly show their effects in the treatment of CNS diseases, including neurodegenerative diseases . Local RPrevious studies illustrated the potential therapeutic effects of ARBs, especially telmisartan, against neurotoxicity -23. In aPerindopril, an ACE inhibitor, is in a different class of RAAS inhibitors and crosses the BBB . In a raOne of the most remarkable results of the present study was the lack of a neuroprotective effect with nebivolol administration against LPS-induced injury. Moreover, 5 and 10 µg/mL concentrations of nebivolol decreased cell viability, even though it was statistically nonsignificant. Additionally, among all the study drugs, only nebivolol at the 10 µg/mL concentration caused a significant decrease in cell viability in the absence of LPS stimulation. This result can only be explained by the direct effects of nebivolol. The main mechanism of action of nebivolol and other beta-blockers is based on the inhibition of sympathetic activity via beta-receptor blockade; however, the potential cytotoxic effect of nebivolol should not be ignored and this potential effect requires further investigation, especially in long-term treatment.As a promising beta-blocker in the treatment of coronary artery disease, nebivolol was reported to inhibit the proliferation and moderately induce apoptosis in human coronary smooth muscle cells and endothelial cells . The finACh is an important neurotransmitter and reduced levels of ACh are associated with the development of dementia and cognitive impairment. In earlier preclinical studies, a link between the cholinergic system, RAAS, and neuroinflammation, and the protective effects of RAAS inhibitors against LPS-induced neuroinflammation through angiotensin II AT1 receptor blockade and ACE inhibition have been demonstrated . In the All the drugs in the present study have a different mechanism of action; therefore, the effects of ACE enzymes and angiotensin receptors should be evaluated to understand the role of those pathways in signaling mechanisms in future research. As a limitation of the present study, the effects of the study drugs on ACh levels in the absence of LPS stimulation were not evaluated. Therefore, future studies that test the direct effects of the study drugs on ACh levels may provide new findings. Additionally, future research focusing on the effects of increased concentrations of the study drugs and prolonged duration can be conducted. Lastly, the present study was an in vitro study; therefore, the results should be evidenced by further in vivo preclinical and clinical studies.The present study showed that telmisartan potently and directly ameliorated LPS-induced injury and neuroinflammation and provided maintenance of ACh levels. The neuroprotective effect of perindopril was inferior to that of telmisartan. The only drug that showed no neuroprotective effect was nebivolol. Therefore, the protective effects of telmisartan and other ARBs, as well as the potential long-term effects of nebivolol and other beta-blockers on neurodegenerative diseases including cognitive impairment, dementia, and AD, should be thoroughly investigated in clinical studies.


Resistance to regeneration of insulin-producing pancreatic β cells is a fundamental challenge for type 1 and type 2 diabetes. Recently, small molecule inhibitors of the kinase DYRK1A have proven effective in inducing adult human β cells to proliferate, but their detailed mechanism of action is incompletely understood. We interrogated our human insulinoma and β cell transcriptomic databases seeking to understand why β cells in insulinomas proliferate, while normal β cells do not. This search reveals the DREAM complex as a central regulator of quiescence in human β cells. The DREAM complex consists of a module of transcriptionally repressive proteins that assemble in response to DYRK1A kinase activity, thereby inducing and maintaining cellular quiescence. In the absence of DYRK1A, DREAM subunits reassemble into the pro-proliferative MMB complex. Here, we demonstrate that small molecule DYRK1A inhibitors induce human β cells to replicate by converting the repressive DREAM complex to its pro-proliferative MMB conformation.  Type 1 (T1D) and type 2 (T2D) diabetes together afflict 463 million people globally  are able to induce adult human β cells to proliferate, to increase in numbers, and to enhance their differentiation and function , CDKN1C (encoding p57KIP2), CDKN2A (encoding p16INK4), and CDKN2B (encoding p15INK4), with the effect of driving entry into the cell cycle and progression through S and G2M phases. In this scenario, DYRK1A serves as a nuclear kinase that rephosphorylates NFAT transcription factors, thereby expelling them from the nucleus, and terminating their mitogenic effects. Thus, in this paradigm, NFATs are the activators of β cell proliferation, and DYRK1A is the “brake” on human β cell proliferation.The current understanding of the DYRK1A inhibitor mechanism of action in pancreatic β cells is derived from earlier experiments in T lymphocytes as well as rodent and human β cells  7–28). –28. 7–28This NFAT-driving proliferation scenario is supported in β cells by reports from several laboratories showing that DYRK1A inhibitors induce rodent and human β cells to replicate, that NFATs translocate to the nucleus in response to intracellular calcium increments, and that they bind to promoters of cell cycle regulatory genes –17. On t1/S pathway . This cluster of proteins assembles on consensus DP1, E2F4, p130 binding sites (CDEs) of, and thereby repressing, some 1000 target genes involved in G2M entry, progression, and other cellular activities  referred to as the “MMB complex”  38–45).–45.38–45Here, we report that DREAM plays a central role in enforcing replicative quiescence in the adult human β cell. We also report that small molecule DYRK1A inhibitors convert DREAM from its repressive configuration to the proliferative MMB conformation. This comprehensive model for control of cell cycle in the human β cell alters a well-established paradigm in the field of diabetes. Moreover, it provides a mechanism of action to explain how DYRK1A inhibitors induce human β cells to replicate. Finally, to the best of our knowledge it is the first time the biology of DREAM has been comprehensively defined in any nonmalignant human cell type.NFATs exist in 4 classical forms, NFAT1, -2, -3, and -4,  . While ahttps://doi.org/10.1172/JCI157086DS1). Taken together, these findings make it clear that NFATs can be effectively overexpressed, and that the constitutively active versions effectively translocate to the nuclear compartment. Surprisingly, however, wild-type and CA-NFATs were unable to match the higher rates of proliferation observed in response to harmine or other DYRK1A inhibitors . This resulted in a “Bisque4 module membership” group of 253 genes , MMB-FOXM1 , and RB-E2F complexes , suggesting that these pathways may serve as the key gatekeepers for β cell quiescence and modulators of insulinoma proliferation  we previously reported on our human insulinoma cohort in which we had identified 6 modules out of 52 that were significantly enriched in genes upregulated in insulinomas compared with FACS-isolated β cells , 47. Of nd NUSAP , we surm53 genes  46). We. We2/M ganalysis . Among tanalysis . We founferation . CollectTo assess these possibilities, we explored 3 different human β cell transcriptome data sets for members of the DREAM complex within normal human β cells  46, 48,,48, 49. 2/M genes involved in mitotic spindle formation, centriole separation, and other G2M processes, exemplified by AURKA, AURKB, PLK1, CENPA, CENPF, FOXM1, BUB1, BIRC5, CDC25A, CDC25C, MELK, and late-G1/S genes such as A- and E-cyclins  and CDK1 .–45.2/M gand CDK1 . Thus, i drivers , which a drivers , also pr drivers –32. Thusanalysis .LINs, RBBP4, E2F4, p130, etc). In contrast, harmine treatment did lead to increases in expression of essentially all of the canonical DREAM targets, including MYBL2 , the maximally effective dose for inducing human β cell replication  8–11, 1, 111, 15ng MYBL2 . RemarkaKI group . Once agKI group .1/S cyclins and CDKs   . As withferation , but res cyclins . In contnd MYBL2 . Thus, t28 in LIN52, an adenovirus expressing a V5-tagged LIN52, in which Ala28 was substituted for Ser28, was cotransduced with the HA-tagged p130 adenovirus  of DREAM target genes –45. Thusd CDC25A , and tar β cells , p130 ph β cells , and thiReasoning that quiescence in other islet endocrine cells may reflect the presence of the DREAM complex, we also assessed the presence of DREAM members in α cells in normal intact human pancreas. As observed in Here we report 4 fundamental advances in our understanding of mammalian cell cycle control. First, we provide the first example to our knowledge of the contribution of the DREAM complex in maintaining quiescence in a normal mammalian cell type. Second, we demonstrate the presence and the central role of the repressive configuration of the DREAM complex in maintaining quiescence in the adult human β cell, a cell type notorious for its resistance to proliferation, as well as the normal human α cell. Third, we demonstrate that the repressive DREAM complex configuration can be converted to its pro-proliferative MMB counterpart by DYRK1A inhibition, thereby moving adult human β cells from quiescence to active replication. Fourth, we provide a revised model explaining how human β cell regenerative drugs — the DYRK1A inhibitors — are able to coax previously quiescent human β cells to reenter the cell cycle. These observations have important implications for diabetes therapy, and also for cancer and developmental biology.The mammalian DREAM complex has been studied principally in continuously growing cell lines derived from common cancers, exemplified by breast, lung, prostate, esophageal, and ovarian cancers and certain leukemias, in which the repressive form of DREAM has been disrupted, and/or in which misexpression of MYBL1 or MYBL2 results in them acting as oncogenes –45. As aINK4A encoded by CDKN2A and p57KIP2 encoded by CDKN1C , and other islet endocrine cells? These questions and others will provide ample opportunities for future studies.To conclude, these studies provide a current working model to clarify the mechanisms of action whereby DYRK1A inhibitors induce human β cell proliferation . They alhttps://iidp.coh.org), Prodo Laboratories, The Alberta Diabetes Institute, and the Transplant Surgery Department, University of Chicago. Details and demographics of the 53 donors and islet preparations are provided in Isolated deidentified human pancreatic islets from otherwise normal organ donors were provided by the NIH Integrated Islet Distribution Program (IIDP, Complete methods are provided in the t test as described in the figure legends. P values less than 0.05 were considered to be significant. Detailed bioinformatics statistics are provided in the Results section, The human insulinoma transcriptome predicts the DREAM complex as a central enforcer of human β cell quiescence.Statistical analyses were performed using 2-tailed Student’s paired Human islets were purchased from the islet isolation centers listed above and in PW, EK, CA, HW, MB, HGR, XL, OW, HL, and LC performed experiments. PW, EK, CA, HW, JAP, DKS, LL, and AFS analyzed data. RIB provided human pathology samples. PW, CA, EB, DH, JAD, and AFS conceived of the studies. PW, EK, JAD, and AFS wrote the manuscript.


Bovine respiratory disease is the leading cause of morbidity and mortality in cattle and is estimated to result in USD 800 to USD 900 million in losses each year. Unfortunately, few improvements have been made over the past 30 years to reduce the negative effects associated with bovine respiratory disease. Many naturally occurring factors, such as breed and temperament, have previously been observed to influence immune responses to infection. However, there is limited information on the influence of sex on the immune response to bovine respiratory disease. Thus, a study was conducted where castrated male (steer) and intact female (heifer) calves were administered a dual viral–bacterial respiratory disease challenge. In response to the challenge, heifer and steer calves produced distinct innate immune responses where steers produced a stronger early acute phase response, while heifers appeared to have a delayed response to the challenge. This suggests heifers may be more resilient to the viral–bacterial challenge when compared with steers. Therefore, the sexually dimorphic immune response should be considered when monitoring cattle for symptoms of bovine respiratory disease8 PFU/nostril) and Mannheimia haemolytica  administered 72 h later. Body temperature was lesser in heifers than steers (p < 0.01). There was a sex × time interaction (p = 0.05) for white blood cells where heifers had reduced concentrations compared with steers at −72 and 0 h but greater concentrations from 36 to 60 h post-MH. Concentrations of neutrophils were lesser in heifers compared to steers from 0 to 4 h, and from 8 to 12 h (p = 0.03). Lymphocytes were greater in heifers compared to steers at 12 h and from 36 to 60 h post-MH (p < 0.01). The neutrophil–lymphocyte ratio was lesser in heifers compared to steers from 2 to 24 h and at 48 h post-MH (p < 0.01). Monocytes were greater in heifers compared to steers from 24 to 60 h post-MH (p < 0.01), while eosinophils were greater in heifers compared to steers at 48 and 60 h (p < 0.01). Serum IL-4 was lesser in heifers compared to steers at 0 h and from 2 to 72 h post-MH challenge (p = 0.02). Non-esterified fatty acid concentrations were lesser (p < 0.01) in heifers compared to steers from 2 to 4 h post-MH challenge. Urea nitrogen concentrations were greater (p < 0.01) in heifers than steers at 36 h post-MH challenge. Data from this study reveal distinct differences in the acute phase response following a respiratory disease challenge where steers produced an early response, while the response in heifers appeared to be delayed.The potential for sexually dimorphic innate immune responses to respiratory disease was evaluated, where eight steers and seven heifers (280 ± 4 kg) were subjected to a viral–bacterial respiratory disease challenge utilizing bovine herpesvirus-1 (BoHV-1; intranasal; 1 × 10 Salmonella Typhimurium has been observed in weaned pigs, but there was no effect of sex on tissue Salmonella counts following oral challenge [Sexually dimorphic biological responses are not a novel observation, as clear sexual dimorphic phenotypes exist in nature . This cahallenge .Mannheimia haemolytica (MH). This challenge model has been demonstrated to mimic the pathogenesis of a typical viral–bacterial BRD infection and allows for the study of factors that may influence how cattle respond to the disease [Bovine respiratory disease (BRD) is the leading cause of morbidity and mortality in cattle  and is e disease . Whereas disease  and mana disease  have bee disease . Thus, tGuide for the Care and Use of Agricultural Animals in Research and Teaching and approved by the Institutional Animal Care and Use Committee at the Livestock Issues Research Unit (Protocol # 1606F).All experimental procedures were in compliance with the Mannheimia haemolytica bacterin was administered. Calves were transported approximately 1300 km (12 h) and upon arrival were housed in covered outdoor pens until processed where they had access to hay, a pelleted standard ration and water.Crossbred calves approximately 10 months of age  and 8 castrated males ) that were selected based on body weight, phenotype, temperament, health status, and serum BoHV-1 titers measured 2 weeks prior to challenge were used for this study. All calves were vaccinated against respiratory pathogens at 21 d before and at weaning using a combination 5-way respiratory modified-live virus vaccine. No g for 20 min at 4 °C. Triplicate aliquots were stored at −80 °C until analyzed for cytokines (interleukin-4 (IL-4), IL-6, and interferon-γ (IFN-γ)), haptoglobin, glucose, BoHV-1 titers, non-esterified fatty acids (NEFA), and serum urea nitrogen (SUN). The second sample collected for hematology analysis was collected into a 4 mL vacutainer containing EDTA and was analyzed using the ProCyte Dx Hematology Analyzer  using bovine-specific analogs. Following placement of the temperature devices and collection of blood samples, a visual nasal lesion score was recorded (described below). Lastly, calves were dosed intra-nasally with 1 mL per nostril of 1 × 108 pfu of BoHV-1 using an atomizer device  attached to a 3 mL syringe. Following administration of BoHV-1, calves were returned to the outside pens for 72 h. During this time calves were visually appraised for health daily as described below. At −72 h (d −3), calves were worked through an indoor working facility and were weighed, fitted with indwelling rectal  or vagin7 cfu MH was administered in 100 mL of PBS intratracheally. Immediately following these procedures, calves were moved into individual bleeding stalls in an environmentally controlled barn. Serial blood samples for serum isolation were collected into 10 mL Sarstedt tubes containing no additive  via the indwelling cannulas at 0 (immediately prior to MH administration), 1, 2, 3, 4, 5, 6, 7, 8, 12, 24, 36, 48, 60, and 72 h. Additionally, serial blood samples for hematology were collected into 4 mL vacutainers containing EDTA at 0, 2, 4, 6, 8, 12, 24, 36, 48, 60, and 72 h. These samples were processed as described above. While in the stanchions, calves were fed twice daily to maintain ad libitum feeding and had ad libitum access to water provided through paddle-type individual waterers  that were connected to a custom-built water intake monitoring system that allowed for the measurement of individual daily water intake as well as water intake bouts.After 72 h (d 0), calves were processed again through the indoor working facility. Each calf was weighed, fitted with an indwelling jugular vein catheter for collection of serial blood samples, and two blood samples were collected from the cannula for isolation of serum and measurement of hematology variables as previously described. Additionally, a nasal lesion score was recorded. Next, 1.3 × 10Following collection of the 72 h sample, steers were worked through the working facility where they were weighed, and temperature devices and jugular cannulas were removed. Additionally, a nasal lesion score was recorded. Lastly, calves were treated with a florfenicol  according to label instructions. Following these procedures calves were returned to the outdoor pens. Prior to shipment of cattle to a collaborator, cattle were processed through the working facility on d 7 for collection of a blood sample for BoHV-1 titers and recording of nasal lesion score.www.vetmed.wisc.edu/dms/fapm/fapmtools/8calf/calf_respiratory_scoring_chart.pdf, accessed on 01 October 2017). Scoring was performed by 2 trained observers throughout the study. Nasal lesion scores were assigned to calves on days 0, 3, and 7 of the challenge based on a score of 0 to 4 relative to the amount of naris covered by lesions. Specifically, 0 = no visible lesions within the naris, 1 = presence of lesions on < 10% of naris, 2 = presence of lesions on 11 to 25% of naris, 3 = presence of lesions on 26 to 50 percent of naris, and 4 = presence of lesions on >50% of the visible mucosa in the naris.Health scores were assigned to each calf daily based on the adaptation of the system developed by the School of Veterinary Medicine, University of Wisconsin. Calf respiratory scoring chart  were measured by custom bovine 3-plex sandwich-based chemiluminescence ELISA kits . The minimum detectable concentrations were 2.6, 0.1, and 3.3 pg/mL for IL-4, IFN-γ, and IL-6, respectively. All intra-assay coefficients of variation were less than 6.2% and all inter-assay coefficients of variation were less than 11.8% for all assays.Glucose concentrations were determined by modification of the enzymatic Autokit Glucose  to fit a 96-well format as previously described . The intConcentrations of NEFA were determined by modification of the enzymatic HR Series NEFA-HR (2) assay (Wako Diagnostics) to fit a 96-well format as previously described . The intConcentrations of SUN were determined according to the manufacturer’s directions using a colorimetric assay . The intra- and inter-assay coefficients of variation were less than 2.1% and 6.9%, respectively.Serum haptoglobin concentrations were determined using a commercially available ELISA kit according to the manufacturer’s directions . Concentrations of haptoglobin were determined by comparing unknown samples to a standard curve of known haptoglobin concentrations. The intra- and inter-assay coefficients of variation were less than 4.2 and 3.1%, respectively.p ≤ 0.05 considered significant and trends toward significance were considered at 0.05 ≤ p ≤ 0.10. All data are presented as the LSM ± SEM.Health, serum BoHV-1 titers, nasal lesion scores, and water intake bouts were analyzed by PROC GLIMMIX with sex, day, and sex × day included as main effects, day included as a random effect, and pen as the subject. Temperature data were averaged into 1 h intervals prior to being analyzed. Temperature, water intake volume, hematology, and serum analyses were analyzed as repeated measure over time with the use of the MIXED procedure of SAS . Sex, time, and the sex × time interaction were included as fixed effects with calf within sex included as the experimental unit. Covariance structure used was based on having the lowest AICC fit statistic value. When main effects were significant, means were separated using the PDIFF option in SAS, with p = 0.02) for health scores. Specifically, heifers had greater average health scores on days −2, −1, and 1 compared to steers (data not shown). Nasal lesion scores were not influenced by sex  or a sex × time interaction (p = 0.39), but they changed over time . Specifically, scores increased from d 0 to day 3 (2.19 ± 0.19 vs. 3.13 ± 0.22), before decreasing on d 7 (1.93 ± 0.22). Pre-screen BoHV-1 shedding analysis from nasal swabs was negative for all calves, while all calves were positive for BoHV-1 shedding on d 0 and 3, with the exception of one negative heifer on d 0. Additionally, there was no effect of sex (p = 0.22) or a sex × time interaction (p = 0.44) for serum BoHV-1 titers, although values changed over time  on body temperature measurements, where heifers had reduced body temperature compared to steers . Specifically, temperature increased gradually following the BoHV-1 challenge, with a more robust increase within 6 to 12 h following the MH challenge. There was no sex × time interaction (p = 0.54) for body temperature.There was an effect of sex (p = 0.06) for a sex × time effect for mL of water consumed per hour (data not shown). Specifically, steers consumer greater amounts of water than heifers at 10, 21, 30, 54, and 56 h relative to MH challenge. Additionally, water intake per hour changed over time (p < 0.01), but there was no main effect of sex (p = 0.27). The number of water intake bouts was similarly affected by time (p < 0.01), but there was no effect of sex (p = 0.17) or a sex × time interaction (p = 0.65).There was a tendency (p ≥ 0.34) on total red blood cells, hemoglobin, hematocrit, or platelet concentrations; however, these variables changed over time  for total white blood cell concentrations, where heifers had reduced concentrations compared with steers at −72 and 0 h (immediately prior to administration of BoHV-1) yet had greater white blood cell concentrations than steers at 36, 48, and 60 h post-MH challenge (p = 0.03) for neutrophil concentrations, where heifers had reduced concentrations compared with steers at 0, 2, 4, 8, and 12 h post-MH challenge, and tended (p = 0.09) to have greater neutrophil concentrations at 48 h post-MH challenge (p < 0.01), with heifers having greater lymphocyte concentrations than steers at 12, 36, 48, and 60 h post-MH challenge, and heifers tended (p ≤ 0.06) to have reduced lymphocyte concentrations at −72 h post-MH challenge (p < 0.01) for the neutrophil–lymphocyte ratio (p < 0.01), where values were greater for heifers than steers from 24 to 60 h post-MH challenge (p < 0.01), with heifers having greater eosinophils at 48 and 60 h post-MH challenge  for serum concentrations of IL-4, where heifers maintained reduced concentrations of IL-4 concentrations at 0 h and from 2 to 72 h post-MH challenge, with a tendency at 1 h  for heifers to have reduced IL-6 concentrations compared to steers (p < 0.01). There was no effect of sex on concentrations of IFN-γ (p = 0.27); however, concentrations increased over time .There was a sex × time interaction (o steers b. Concenver time c. Specifp = 0.14). However, concentrations of glucose decreased over time (p < 0.01), with greater concentrations observed from −72 to approximately 4 h following the MH challenge before decreasing. A sex × time interaction (p < 0.01) was observed for serum NEFA concentrations, where heifers had reduced concentrations of NEFA compared to steers from 2 to 4 h post-MH challenge (p < 0.01) for SUN concentrations, where heifers had greater SUN concentrations at 36 h and tended (p = 0.08) to have greater SUN concentrations at 60 h post-MH challenge  compared to males ,25. AddiThe hematological response between heifers and steers was very interesting, with significant differences observed in the WBC differential counts. Neutrophil concentrations increased in steers following the challenge with BoHV-1, as indicated by the greater concentrations at 0 h. In contrast, neutrophil concentrations appeared to begin increasing later in the challenge period in heifers. Thus, there appeared to be a quicker neutrophil response by the steers, suggesting steers may have been more sensitive to the challenge compared to heifers, which displayed a later response. In future studies, it would be interesting to extend the sampling period to see if neutrophil concentrations continue to increase in heifers following the 72-h sample. An increase in neutrophils in response to BRD infection has been previously reported . AdditioThe responses observed for cytokines, while not all statistically significant, further support a greater inflammatory response by steers. As discussed above, greater TLR4 expression on immune cells isolated from male rodents may be a driving factor associated with the greater pro-inflammatory cytokine response . Indeed,Pasteurella haemolytica. While there was no effect of sex or a significant sex × time interaction for the haptoglobin response in the current study, it is interesting to note the marked numerical difference between heifers and steers at 72 h, where haptoglobin appears to remain elevated in heifers, yet concentrations in steers appear to be decreasing. This would further support the theory of a quicker response from the steers resulting in recovery, yet a later or delayed response in the heifers. Thus, this aspect of the study bears repeating to determine if a relationship exists between sex and haptoglobin concentrations in response to a BRD challenge, as haptoglobin has been reported as one variable that can be used as a BRD diagnostic tool [Acute phase proteins are important for combating both viral and bacterial infections. In particular, haptoglobin has a role in reducing iron availability to bacteria through binding and sequestering hemoglobin . The littic tool .Available energy sources are extremely important for immune activation, the acute phase response, and recovery from an infection. A study by Kvidera et al.  found thDistinct differences exist in the acute phase response of heifers and steers to a viral–bacterial respiratory disease challenge. Steers produced an early response to the challenge, as indicated by the greater neutrophil, IL-4, and NEFA response, while the response from the heifers appeared later or delayed based on lymphocyte, monocyte, eosinophil, and SUN values. This suggests that the heifers may have been more resilient than steers and required a lesser inflammatory response to the challenge. However, a strong immune challenge that overwhelms the innate immune response may require medical intervention in heifers similar to steers, and these data are not indicative of the response of calves to therapeutic treatment for BRD. Thus, the sexually dimorphic immune response to respiratory disease should be considered when monitoring calves for symptoms of BRD.


Swertia and Gentiana. Extracts of these plants have a long history of use in Japan as bitter stomachics because of their strong bitterness. Because the AG content directly reflects the potential activity of the extract, the quality control of these plant extracts through the quantitative analysis of AG is important. In the present study, we aimed to develop a quantitative analysis of AG using a monoclonal antibody (MAb) against AG (MAb 1E9) in the plant family Gentianaceae. Surprisingly, production of MAb 1E9 was successfully achieved by immunizing AG–bovine serum albumin (BSA) conjugates into mice although the number of AG bound to BSA was only one. The characterization of MAb 1E9 revealed that it shows high specificity to AG, enabling the development of an icELISA for the specific determination of AG. In addition, the detectable concentration of AG in the developed icELISA ranged from 1.95 to 62.5 ng mL−1 with a limit of detection of 1.28 ng mL−1, which is approximately 30–625 times higher in sensitivity compared with the conventional HPLC method. Validation analysis revealed that the icELISA using MAb 1E9 is precise  and accurate (recovery rates of spiked samples were between 91.0% and 106.4%). This method can be used for the quality control of plant extracts using AG as an index.Amarogentin (AG) is a naturally occurring secoiridoid glycoside produced mainly in the plant genera  MAb 1E9 was generated from AG–BSA conjugates possessing one AG molecule per BSA for icELISA. Swertia and Gentiana, both belonging to the family Gentianaceae. The extracts of members of this family have long been used as bitter stomachics because they contain bitter secoiridoid glycosides, including AG, amaroswerin (AS), swertiamarin, sweroside, and gentiopicroside ,11 high-performance liquid chromatography (HPLC),11,12 ultra-performance LC-electrospray ionization/MS,13 and capillary electrophoresis.14 However, these methods require large amounts of eco-unfriendly organic solvents, costly instruments, and time-consuming sample pretreatment.Amarogentin AG;  is a naticroside , all of 15In our previous studies, we focused on the quantitative analysis of plant secondary metabolites and developed various immunoassays, including enzyme-linked immunosorbent assays (ELISAs), fluorescence-linked immunosorbent assays, and immunochromatographic strip assays. These immunoassays exhibited several advantages over traditional analytical methods, such as HPLC, in terms of simplicity, selectivity, and sensitivity because the principle is based on a specific antigen–antibody reaction.via N,N′-carbonyldiimidazole (CDI) and were administered to BALB/c mice to induce an immunoresponse. Hybridomas secreting MAb 1E9 were obtained via the polyethylene glycol (PEG) method, and the MAb 1E9 were purified using affinity column chromatography. It is note that MAb 1E9 was successfully produced using AG–BSA conjugates possessing only one AG molecule per BSA molecule. The systematic characterization of MAb 1E9 revealed that it possesses high specificity to AG and high sensitivity, with a limit of detection (LOD) of 1.28 ng mL−1. To the best of our knowledge, this is now the most sensitive analytical method for determining the AG content in plant samples. Various validation analyses, including intra- and inter-assay tests, and spiked recovery tests alongside correlation analysis using HPLC support the accuracy and precision of the developed icELISA for the quality control of Gentianaceae through the quantitative analysis of AG.In the present study, we produced a specific monoclonal antibody (MAb) against AG (MAb 1E9) to develop an indirect competitive ELISA (icELISA) for the determination of AG. AG–bovine serum albumin (BSA) conjugates were prepared This paper demonstrated the production of MAb 1E9 and its application to icELISA.Amarogentin  was obtained from EXTRASYNTHESE . Bovine serum albumin , human serum albumin , and ovalbumin  were purchased from Sigma-Aldrich . Freund's complete and incomplete adjuvants were obtained from Difco . Goat F(ab) Anti-Mouse IgG H&L (HRP) (ab6823) was used as a secondary antibody and was purchased from Abcam . All the other chemicals were standard commercial products of analytical to reagent grade.S. japonica . S. japonica (∼200 g) were soaked in methanol (500 mL), sonicated for 1 h, and filtered. This extraction step was repeated five times, and the filtrates (∼2.5 L) were then concentrated under vacuum to obtain ∼53.5 g of extracts. The extracts were dissolved in 50% (v/v) MeOH and partitioned with hexane. The aqueous layer was then partitioned with CHCl3, followed by ethyl acetate, to obtain an ethyl acetate fraction (∼4.3 g) containing AS. Reverse-phase chromatography using Cosmosil 75C18-OPN  with 50% (v/v) MeOH and silica gel 60  chromatography with a mixture of CHCl3 and MeOH (CHCl3/MeOH = 8/2) was performed to obtain AS (∼210 mg). NMR spectra were recorded on a Varian Unity 500 plus apparatus  operating at 500 MHz for 1H NMR and 125 MHz for 13C NMR in methanol-d4.To evaluate the specificity of MAb 1E9 to AG, structurally similar compounds were required. Because AS  was commad libitum. The animal experiment conducted in the present study was approved by the Committee on the Ethics of Animal Experiments, Kyushu University , and the experimental procedures were performed in accordance with the Guidelines for Animal Experiments of the Graduate School of Pharmaceutical Sciences, Kyushu University.BALB/c mice  were purchased from KBT Oriental Co., Ltd. . A standard diet  and water were provided S. japonica (powdered), S. japonica (whole plant), G. lutea (powdered), and G. scabra (powdered) were commercially obtained from Uchida Wakanyaku Co. Ltd., . S. japonica , G. scabra I (root), and G. scabra II  were obtained from voucher specimens deposited at the Herbarium of the Faculty of Pharmaceutical Sciences, Kyushu University. Powdered samples were measured without any treatment, whereas whole plants and roots were ground into a fine powder and sieved through a 300 μm mesh filter. Plant powder (50 mg) was measured and immersed in MeOH (1 mL). Extraction was performed by sonication for 20 min, and the supernatant, after centrifugation at 10 000 rpm for 10 min, was collected in small test tubes. This step was repeated three times. The combined MeOH extract (3 mL) was concentrated to dryness under flowing nitrogen. The residue was then dissolved in MeOH (1.0 mL) and used as a sample for both icELISA and HPLC.Seven kinds of Gentianaceae were analyzed using icELISA and HPLC. via CDI, which is a zero-length cross-linking reagent.16 For the AG–BSA conjugates used as immunogens, AG (3.1 mg) and CDI (3.0 mg) were dissolved in dimethylformamide (0.6 mL), which was stirred at 30 °C for 3 h. Subsequently, this solution was added dropwise to another solution, where BSA (4.2 mg) was dissolved in distilled water (2.7 mL). The resulting mixture was then stirred at 30 °C for 20 h. The reaction mixture was dialyzed five times against distilled water at 4 °C for 2 days and then lyophilized to obtain AG–BSA conjugates (3.9 mg). AG–HSA (3.8 mg) and AG–OVA (3.6 mg) conjugates were prepared in the same manner using HSA (3.9 mg) and OVA (4.1 mg), respectively. All conjugates were dissolved in 50 mM Tris–HCl (pH 8.0) containing 8 M urea at 20 mg mL−1 and stored at −20 °C until use.AG possesses a hydroxyl group; therefore, AG was conjugated to proteins  17 At the first and second immunizations, Freund's complete and incomplete adjuvants were used to enhance the immune response by forming an emulsion with the immunogen (AG–BSA conjugates). The resulting emulsions, corresponding to 50 μg of AG–BSA conjugates, were administered intraperitoneally. Booster shots were administered three times by immunization with AG–BSA conjugates (100 μg) without using adjuvants. On the fifth day after the final booster, the splenocytes were prepared and fused with SP2/0 mice myeloma cells using the PEG method. The fused cells were then cultured in a selection medium containing enriched RPMI 1640-Dulbecco's-Ham's F12  medium supplemented with hypoxanthine–aminopterin–thymidine (HAT), RD-1 additives , and 10% (v/v) fetal calf serum  at 37 °C in a humidified incubator and a 5% CO2 atmosphere. Hybridomas secreting anti-AG antibody were then cloned by the limited dilution method and selected by direct ELISA and icELISA. The selected hybridomas (1E9) were scaled up in the same buffer without HAT until the volume became 1 L. Finally, the medium was exchanged to a serum-free medium (without FCS) and further cultured for 1 week.Immunization of BALB/c mice  was performed with AG–BSA conjugates every 2 weeks, as described previously.Purification of MAb 1E9 was performed using affinity column chromatography. Briefly, the culture medium containing MAb 1E9 was centrifuged, and the pH of the supernatant was adjusted to pH 7.0 with 1 M Tris–HCl buffer (pH 9.0). The culture medium was then filtered, and the filtrates were subjected to chromatography on a Protein G Sepharose 4 Fast Flow  column equilibrated with 10 mM phosphate buffer (pH 7.0). The bound proteins were then washed with 10 mM phosphate buffer (pH 7.0) and eluted with 100 mM citrate buffer (pH 2.7). Ten test tube fractions containing MAb 1E9 were obtained. To avoid inactivation of the eluted MAb 1E9, the eluates (4.0 mL) were immediately neutralized with 1 M Tris–HCl buffer . Subsequently, all fractions were collected, concentrated, dialyzed against distilled water at 4 °C for 1 week, and lyophilized to yield 79.0 mg of MAb 1E9.−1) in 50 mM carbonate buffer  were immobilized to the surface of a 96-well immunoplate  by incubation at 37 °C for 1 h. After washing the plate, the wells were then treated with PBS containing 5% (w/v) skimmed milk (300 μL per well) at 37 °C for 1 h to block and reduce non-specific adsorption of other proteins. The following step, using MAb 1E9, was different between indirect ELISA and icELISA. For indirect ELISA, various concentrations of MAb 1E9 (100 μL per well) in PBS containing 0.05% (v/v) Tween 20 (PBS-T) were allowed to react with the coated AG–HSA conjugates. For icELISA, free AG molecules (50 μL per well) in 5% (v/v) MeOH were mixed with MAb 1E9  in PBS-T, leading to a competitive reaction between free AG molecules and AG of coated AG–HSA conjugates against MAb 1E9. After incubation at 37 °C for 1 h and a subsequent washing step, MAb 1E9 binding to the immobilized AG–HSA conjugates was reacted with Goat F(ab) Anti-Mouse IgG H&L (HRP) (100 μL per well), which was 5000 times diluted with PBS-T, at 37 °C for 1 h. Finally, the 0.3 mg mL−1 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)diammonium salt substrate solution in 100 mM citrate buffer (pH 4.0) supplemented with 0.003% (v/v) H2O2 (100 μL per well) was added and incubated at 37 °C for 15 min to develop color.The binding reactivity of MAb 1E9 was evaluated by ELISA using AG–HSA as the coated antigen. The reactivity against AG–HSA conjugates and free AG molecules was investigated by indirect ELISA and icELISA, respectively. In both ELISA tests, AG–HSA conjugates . The cross-reactivities (CRs) of MAb 1E9 were evaluated using the formula described by Weiler and Zenk as follows:S. japonica (powdered). In the unspiked samples, 95.86 μg of AG was found per 50 mg of dried sample by icELISA. After spiking with AG standards, the amounts of AG were determined using the developed icELISA and the recovery rate was calculated as follows:Various amounts of AG  were spiked into MeOH solution (1 mL) extracted from et al., with slight modification.12 An LC-10AD VP HPLC pump connected to a SPD-10A UV/VIS detector (254 nm) and C-R8A chromatopac, all of which were Shimadzu products , were used in the analysis. After the samples (20 μL) were subjected to a reversed-phase column , equilibrated with a mobile phase comprising 20% (v/v) acetonitrile with 0.5% (v/v) acetic acid solution, the eluates were analyzed for 20 min. AG was detected at the retention time of ∼17.0 min when the flow rate was 1.0 mL min−1. The calibration curve for AG was drawn in the concentration range of 0.049–12.5 μg mL−1 with a LOD of 0.23 μg mL−1. All sample analyses were performed in triplicate.HPLC analysis was performed according to the method by Amakura m/z 66 451 and m/z 67 224, respectively . In this analysis, the BSA and AG–BSA conjugates appeared at y Fig. S1. By taki−1) in the serum were investigated by indirect ELISA and icELISA, respectively (405 = 2.01) and inhibition rate against AG (73%) increased enough to be used in ELISA after the fifth immunization , and this revealed that the MAb 1E9 was an IgG1 that possessed a κ light chain.−1) was analyzed by indirect ELISA. The absorbance against log[MAb 1E9] was plotted for the reactivity response curve. The concentration of MAb 1E9 positively correlated with the absorbance in a logarithmic manner [−1 of MAb 1E9 gives an absorbance of ∼1.0 in the indirect ELISA test, suggesting that 900 ng mL−1 is optimal for MAb 1E9 as a primary antibody in icELISA because the volume of primary antibody in icELISA (50 μL) should be half of that in an indirect ELISA (100 μL), as mentioned above.The binding reactivity of MAb 1E9 against AG–HSA conjugates  was then added and incubated to allow for competition between free AG/AG–HSA conjugates and MAb 1E9. The increased free AG led to a decrease in the amount of MAb 1E9 that could bind to AG–HSA conjugates, and vice versa; thus, the absorbance decreased as the MAb 1E9 concentration increased in a logarithmic manner. In this icELISA, the detectable concentration of AG was in the range of 1.95–62.5 ng mL−1 with a LOD of 1.28 ng mL−1 [−1) and ∼30–625 times more sensitive than previously reported HPLC systems  MeOH, and applied (50 μL per well) to the immunoplate coated with AG–HSA conjugates. MAb 1E9 diluted with PBS-T . The inter-assay precision was evaluated by observing the variations from different plates (n = 5) at six AG concentrations within the detectable range . The maximum coefficient of variation in the intra-assay test was 3.86% whereas that in the inter-assay test was 8.75%, indicating that the developed icELISA possesses high repeatability for the determination of AG  were spiked into MeOH extracts obtained from powdered S. japonica and the recovery rate was calculated using the measured amount of AG by icELISA and the spiked amount  was determined using the newly developed icELISA method and HPLC ; however, the high sensitivity of icELISA enabled AG detection. With respect to G. scabra, small amounts of AG were detected by icELISA but no AG was detected by HPLC. To date, there have been no reports regarding the presence of AG in G. scabra; however, G. scabra has been reported to contain much gentiopicroside and sweroside (1–3% wt/dry wt).24 Note that gentiopicroside and sweroside had slight CRs to MAb 1E9 (G. scabra may result in a false positive for the detection of AG by icELISA. However, the presence of AG in G. scabra needs to be investigated further.The AG content in members of the plant family Gentianaceae (and HPLC . The AG  MAb 1E9 . These s20–22 very low hapten numbers may be sufficient for producing highly specific antibodies although some study concluded between 8 and 25 hapten number were optimal.19,25 A sensitive icELISA using MAb 1E9 was developed with a LOD of 1.28 ng mL−1, enabling the determination of small amounts of AG in members of the plant family Gentianaceae without the need for complicated pretreatment, which is commonly required in other analytical methods. MAb 1E9, which is specific to AG, could be useful for the quality control of Gentianaceae through quantitative AG analysis using the newly developed icELISA.In the present study, MAb 1E9, which is highly specific to AG, was successfully prepared using AG–BSA conjugates despite only one AG being bound to BSA. Considering our recent reports about specific MAbs prepared using BSA conjugates containing only two target molecules,The authors declare no competing financial interest.RA-008-C8RA02922A-s001


The optimized formulation displayed the desired criterium of a thin emulsion system, possessing the physicochemical properties and the stability comparable to those of commercially available products used in cosmeceutical applications.Driven by the customers’ growing awareness of environmental issues, the production of topical formulations based on sustainable ingredients is receiving widespread attention from researchers and the industry. Although numerous sustainable ingredients  have been investigated, there is a lack of comparative studies between conventional ingredients and sustainable alternatives. In this study, olive oil (30 wt.%) and α-tocopherol (2.5 wt.%) containing oil-in-water (O/W) emulsions stabilized with the bacterial fucose-rich polysaccharide FucoPol were formulated envisaging their validation as cosmetic creams. After formula composition design by Response Surface Methodology (RSM), the optimized FucoPol-based emulsion was prepared with 1.5 wt.% FucoPol, 1.5 wt.% cetyl alcohol, and 3.0 wt.% glycerin. The resulting emulsions had an apparent viscosity of 8.72 Pa.s (measured at a shear rate 2.3 s The global market demand for products based on innovative ingredients and technologies has compelled the cosmetic industry to rapidly increase the research and development of natural, organic, and eco-friendly formulations ,2,3. OneEmulsions are extensively used in cosmetic products to stabilize active substances, bioavailability, and sensory properties . Being c6–5.8 × 106 Da) bacterial fucose-rich polysaccharide, with a fucose content of 32–36 mol% and nearly equimolar contents of glucose (28–34 mol%) and galactose (25–26 mol%). Its structure also comprises glucuronic acid (9–10 mol%), and acyl groups: acetate (3.5–6.8 wt.%), pyruvate (3.7–14 wt.%), and succinate (0.6–3.0 wt.%) [FucoPol is a high molecular weight (1.7 × 10.0 wt.%) . The devvate 3.7– wt.%, an.0 wt.%) ,18,19,20vate 3.7– wt.%, an.0 wt.%) , photopr.0 wt.%) , and ant.0 wt.%) .In this study, the main objective was the development of O/W emulsions using FucoPol as a substitute for synthetic emulsifying agents commonly used in cosmetic products. RSM was used to define the optimal concentration ranges for FucoPol, cetyl alcohol, and glycerol. FucoPol-based cosmetic formulations were prepared and characterized in terms of physical stability, rheological, and textural properties, and compared with cosmetic emulsion-based products available on the market.With the objective of defining the composition resulting in emulsions with high EI after 24 h (E24), concomitant with high apparent viscosity, different FucoPol, cetyl alcohol, and glycerin concentrations were tested. High E24 values (≥95%) were obtained in most runs, except in runs 7, 8, 9, and 11, which were devoid of the FucoPol, irrespective of the cetyl alcohol and glycerin content that varied from 0.0–1.5 wt.%, and 1.0–3.0 wt.%, respectively. Higher FucoPol concentrations also conferred higher apparent viscosity to the emulsions, regardless of cetyl alcohol and glycerin concentrations. The emulsions presented a yellowish-white color, olive odor, and creamy/smooth texture, showing physical stability (as shown by the centrifugation test) for apparent viscosity values ≥90 Pa.s. ƞ values. The coefficients of multiple determination (R2) values of E24 and ƞ were 0.974 and 0.995, respectively. For ƞ, the R2 was in reasonable agreement with the adjusted R2 (0.989) and the predicted R2 (0.967). The adjusted coefficient of determination indicated that 98.9% of the variability in the response could be explained by the model. The quadratic model was significant , being supported by an insignificant lack-of-fit (p = 0.778) toward the response (ƞ), meaning that the error predicted by the model was above the error of the replicas [R2, adjusted R2, and predicted R2 were 0.974, 0.941, and 0.718, correspondingly. The difference between the predicted R2 and the adjusted R2 was higher than 0.2, which may indicate a large block effect or a possible problem with the model and/or data.ANOVA was used to define the working ranges for each variable resulting in the highest E24 and replicas ,25,26. Tƞ values. Moreover, FucoPol at 1.5 wt.% led to emulsions with ƞ values above 206 Pa.s. Increasing the concentration of FucoPol resulted in more viscous emulsions, and more stability against coalescence, avoiding emulsions’ phase separation [ƞ and E24 values were defined as: 1.5 wt.% FucoPol, 1.5 wt.% cetyl alcohol and 3.0 wt.% glycerin.The RSM results  suggest paration ,27. Thisparation . Based oThe freshly prepared formulations a presentstratum corneum barrier function [As presented in function . Given tfunction ,30,31, tfunction . Interes® 305 and stearic acid under the same conditions. In addition to acting as an emulsifying agent, FucoPol appeared to have a pH-lowering effect. Consumers prefer O/W emulsions due to their sensorial properties  [The conductivity value, which is indicative of the number of free ions and water present in the system , is used-greasy) ,38,39 re-greasy) .The formulations’ physical stability was also assessed by measuring the droplet size during the storage period at room temperature (~20 °C). The distribution profile of oil droplets and their size influences the emulsion’s stability, with smaller droplet sizes and lower PI values <0.3) being responsible for higher stability  being re,40,41,42As shown in All formulations exhibited a similar shear thinning behavior to the torque response, as the viscosity gradually decreased under increasing shear rates . The visCosmetic preparation stability over storage time is related to its tendency to exhibit changes in particle migration . In factAs shown in η values  of the prepared emulsified formulations are summarized in η values , where fSpreadability is an important texture parameter that infers on the product’s contact with skin  and ease of removal from packaging, which may affect utilization compliance ,53. This® hand cream showed phase separation. As shown in ® Xémose (face cream) (6.68) but lower than the other tested commercial products, such as Shiseido® primer (8.17) and Sephora® hand cream (8.18). These values are higher than the optimal pH range (between 4.0 and 7.0) compatible with human skin. Nonetheless, the droplet sizes of Shiseido® primer (22.0 µm) and Sephora® hand cream (27.9 µm) are very similar to that of Formulation C (24.2 µm). In terms of rheological parameters, Formulation C and Uriage® Xémose presented higher apparent viscosity values, 23.7 Pa.s and 25.9 Pa.s, respectively, and showed a similar viscoelastic profile to Shiseido® primer. Uriage® Xémose and Formulation C displayed very similar textural parameters, which suggests that Formulation C has adequate sensory characteristics for a face cream. Other polysaccharides presented similar behavior, as demonstrated by Miastkowska et al. [−1) when compared to tested market preparations  but higher spreadability. On the other hand, Danila et al. [Formulation C (after 60 days of storage) was compared to several cosmetic products available in the market in terms of pH, conductivity, droplet size, physical stability (by centrifugation test), and rheological and textural parameters. In the centrifugation test to assess the physical stability, both Formulation C and Sephoraa et al. , that dea et al.  found tha et al. . In geneOlea europaea (olive) fruit oil was purchased from a local market. Olive oil is an anti-aging ingredient indicated for dermatology applications due to its acidity, antioxidant activity, and soothing effect [g effect ,24, prevg effect . α-tocopg effect ,58. Olivg effect .Enterobacter A47 (DSM 23139) on glycerol-supplemented medium, as previously described [FucoPol was produced by the bioreactor  cultivation (10 L) of escribed , and extescribed . FucoPolOther ingredients that were selected for the emulsions’ formulation were cetyl alcohol, glycerin, triethanolamine (TEA), and methyl paraben. Cetyl alcohol is a long-chain alcohol  commonly® 305, acquired from Sigma-Aldrich  and from SEPPIC , respectively, were tested for comparison with FucoPol. Stearic acid is a solid saturated fatty acid [® 305 is a synthetic hydrophilic polymer that provides increased viscosity and stability in cosmetic formulations [The commercial emulsifiers stearic acid and Sepigeltty acid  commonlytty acid ,75 in cotty acid . Sepigelulations ,77.Response surface methodology (RSM)  was applY is the predicted response; ix are the independent variables (n = 3). The parameter β0 is the model constant; iβ are the linear coefficients; iiβ are the quadratic coefficients and ijβ are the cross-product coefficients [® software package from Stat-Ease Inc., Minneapolis, MN, USA). The validated model was plotted in a three-dimensional graph, generating a surface response that corresponds to the best emulsification index and apparent viscosity. Analysis of variance (ANOVA) was used to determine the regression coefficients of individual linear, quadratic, and interaction terms.The mathematical relationship between the independent variables can be approximated by the second-order polynomial model equation:ficients . A full ® ME . The mixtures were emulsified by manual agitation for 40 s, followed by vortex agitation for 10 s. The emulsification index  was determined by the following equation [e (mm) is the emulsion layer height and hT (mm) is the overall height of the mixture after emulsification.The emulsions were prepared by heating the oil phase (1.63 g) comprising olive oil (30 wt.%), cetyl alcohol (0.0–2.0 wt.%) and α-tocopherol (2.5 wt.%), and the aqueous phase (3.37 g) comprising FucoPol (0.0–2.0 wt.%), glycerin (1.0–3.0 wt.%), TEA (0.5 wt.%), and methyl paraben (0.02 wt.%) at 75 °C in a recirculated heated water bath Thermomixequation :(2)EI =h® 305 as emulsifier agents . Formulation A was prepared with FucoPol as the sole emulsifier, while formulation B additionally contained 1.5 wt.% cetyl alcohol as the co-emulsifier. Formulation C was similar to formulation B but 3.0 wt.% glycerin was added to it as an emollient (® 305 (1.5 wt.%).Six formulations were prepared according to mollient . Three o® ME . The emulsification was performed by slowly adding the oil phase to the aqueous phase and mixing with a shear rate of about 11,000 rpm , for 3 min, followed by manual continuous stirring until room temperature was attained [For preparing the emulsions, the oil phase 32.5 g) and the aqueous phase (67.5 g) were heated at 75 °C in a recirculated heated water bath Thermomix2.5 g andThe organoleptic  and macroscopic appearance of each formulation were visually analyzed. The EI was evaluated during the storage period  as described in w/w) [v/v) Nile Blue A  and observed in a Zeiss Imager D2 epifluorescence microscope , with a magnification of 100×, through ZEN lite software . The physical stability was evaluated by centrifuging 1 g of the sample, at 4800 rpm, for 30 min [The pH and conductivity were determined by dispersing the formulation sample in deionized water  ,79,80. Tw/w) , by placr 30 min .w/w) in a disposable cell with a scattering angle equal to 90°. Cumulants statistics data analysis was performed to determine the hydrodynamic size and polydispersity. Zeta Potential measurements were performed in a graphite electrode cell with a 173° scattering angle [Dynamic Light Scattering (DLS) was performed to determine the average particle size, the polydispersity index (PI), and the Zeta Potential, using a nanoPartica SZ-100V2 series  with a laser of 532 nm and controlling temperature with a Peltier system (25 °C). DLS measurements were performed by diluting the samples , equipped with a CP35-2 cone-plate sensor system  and a P-PTD 200/AIR Peltier plate to keep the measurement temperature constant at 25 °C. Dynamic viscosity measurements were performed at shear rates between 0.01 and 1000 sep tests .The firmness, consistency, cohesiveness, and adhesivity of the attained formulations were determined using a texture analyzer  equipped with a 10 N load cell . The sample was placed in a female conic holder and compressed at 11 mm of depth (which represented a sample deformation of around 70%); this procedure was done twice by a male conic probe at a speed of 2 mm/s .This study demonstrates FucoPol’s suitability for the development of emulsified formulations with good physical and chemical properties for their utilization as cosmetic creams. The fucose-rich biopolymer has shown to possess great potential to replace stearic acid as an emulsifier, resulting in emulsions with similar/better stability, viscosity, firmness, spreadability, and droplet size, which were also shown to be comparable to commercial creams. Although further tests must be done to fine-tune the formulations, the results obtained substantiate the relevance of FucoPol in the development of topical formulations.


Cucurbita pepo L. seeds, whose oil is commercialized for the treatment of genito-urinary tract pathologies, has been selected. Supercritical CO2 technology has been employed as a highly selective “green” methodology allowing the recovery of compounds without chemical degradation and limited operational costs. Free fatty acids have been collected in mild conditions while an enrichment in sterols has been selectively obtained from sc-CO2 extracts by appropriate modulation of process parameters , hence demonstrating the feasibility of the technique to target added-value compounds in a selective way. Obtained fatty acids were thus converted into the corresponding ethanol carboxamide derivatives by lipase-mediated biocatalyzed reactions, while the hydroxylated derivatives of unsaturated fatty acids were obtained by stereoselective hydration reaction under reductive conditions in the presence of a selected FADH2-dependent oleate hydratase.The valorization of biomass residuals constitutes a key aspect of circular economy and thus a major challenge for the scientific community. Among industrial wastes, plant residuals could represent an attractive source of bioactive compounds. In this context, a residue from the industrial extraction of  Plants represent an inestimable source of bioactive compounds for humans. From ancient times, humankind has always exploited the vegetal variability for a widespread number of applications spanning from their use as food, textile manufacture, and for the cure of several diseases, posing the basis of modern medicine ,2. Start2 extraction of Cucurbita pepo L. (Cp) seeds, whose oil is commercialized for the treatment of genito-urinary tract pathologies, has been selected for its possible exploitation as a novel low-cost source of bioactive agents. Cp residues have been provided by Indena S.p.A. , a leading company in the identification, development, and production of plant-derived active principles for the pharmaceutical and health food industries.In the present research, a residue from a selective industrial supercritical COSerenoa repens fruit extracts for the treatment of the same pathologies and to prevent prostatic cancer + and [M−105]+, which correspond to the loss of trimethylsilanol and the methyl group plus trimethylsilanol, respectively. The relative abundances of revealed species are reported in Spectral data of Δ7-spinasterol, Δ7,25-stigmastadienol, Δ7-avenasterol having the same molecular masses , the Cp_sc-COinolysis .v/v) solution of ethanol in cyclohexane in the presence of 2 g L−1 of enzyme at 55 °C for 1 h. After recovering the mixture of fatty acid ethyl esters by filtrating the resin-supported biocatalyst and concentrating in vacuo, the desired ethanol amides were obtained in quantitative yields and conversion by reacting the recovered materials (45 g L−1) with ethanolamine, used as solvent, again in the presence of 2 g L−1 Novozym® 435. GC-MS analysis confirmed the complete conversion of the starting fatty acid mixture into the corresponding ethanol amides within 72 h , an enzyme showing high activity as well as a broad substrate scope [−1) was incubated with OhyA2 (0.1 g L−1) in the presence of TiIII-citrate as reducing agent for 24 h at 35 °C, thus promoting the complete conversion of the two most abundant UFAs, i.e., oleic acid and linoleic acid  possess the potential to be converted into interesting derivatives in virtue of their reactive C-C double bond(s). UFAs can in fact be used to produce short-chain carboxyl acids of industrial or pharmaceutical interest ,56,57 ante scope . Similarte scope ,59,60, Oeic acid , into theic acid .Cucurbita pepo L. (Cp) exhausted seeds were generously donated by Indena S.p.A. Carbon dioxide (CO2) was purchased from Sapio s.r.l  with a purity of 99.999%. BSTFA-TMCS (99:1) was from MACHEREY-NAGEL. HPLC grade and analytical grade acetonitrile, water, formic acids, Amberlite IRA-400, and beta-sitosterol standard were purchased from Sigma-Aldrich Chemicals . n-Hexane was purchased from Carlo Erba.After selective extraction of commercial oils by means of supercritical fluids, n-hexane in a 250 mL round flask and extracted under mechanical stirring at room temperature. After 6 h, the hexane phase was removed and an equal volume of fresh solvent was added to the biomass to continue the extraction procedure under the same conditions. In order to remove mucilage and coarse materials, the suspension was centrifuged at room temperature by using a rotational speed of 6000 rpm for 10 min. The extract was concentrated by a rotary evaporator .Twenty grams of Cp biomass residual were added to 80 mL of 3 stainless steel extractor inserted in an oven, a constant pressure piston pump (SFT-Nex10 SCF Pump) with a Peltier cooler, and a collection vessel. Dry industrial residuals were ground into fine powder by means of a Pulverisette 11 blender  for 30 s at 4000 g (powder diameter: 0.5–1 mm). Twenty grams of residuals were loaded in a 100 cm3 stainless steel vessel for supercritical fluid extractions. Restrictor temperature was set at 75 °C for each condition. The CO2 was pressurized in the vessel at a pressure of 380 bar and a temperature of 50 °C (dCO2 = 913 kg/m3). Once the set pressure was reached, a static period was maintained for 15 min; then, the valves were opened to collect the sample for 30 min in dynamic conditions . Static and dynamic periods were chosen according to previous studies and preliminary experiments on the biomass. Identical extraction conditions were applied for 9 successive cycles.Supercritical fluid extractions were performed using a pilot unit SFT110XW System supplied by Supercritical Fluid Technologies, Inc. . It consisted of a 100 cm3, B: 45; 220 bar; 830 kg/m3, and C : 50; 380 bar; 913 kg/m3 were applied one by one, keeping the material in the same vessel, to achieve complete extraction of oil components from Cp residuals. Pressures and temperatures were chosen in accordance with the results obtained in the single-step SFE process, in which the best conditions in terms of extraction yield were exploited to ensure complete exhaustion of the biomasses. The SFE process was divided into three sequential steps. Once the set pressure was reached, a static period was maintained for 15 min; then, the valves were opened to collect the sample for 30 min in dynamic conditions (CO2 flow rate = 10 SCFH).The same procedure described in Neutral and acid component separation was achieved by means of ion-exchange chromatography. Briefly, Amberlite IRA-400 was activated before use with sodium hydroxide solution, then water was removed by flushing methanol. Consequently, 50 mg of oil collected from each extraction were dissolved in methanol and added to 0.5 g of resin and kept under gentle magnetic stirring for 12 h to achieve the complete acid component adsorption. The mixture was then separated from the resin, and the non-adsorbed species were recovered by washing the resin with methanol. Retained fatty acids were eluted by adding a solution of acetic acid in methanol (1:1). Neutral components obtained from Cp residuals were then saponified following the Method Cd 3c–91/Method NGD C33-1976 . Briefly−1 to 200; 5 min holding time; then 20 min−1 to 300; 20 min holding time; total run time: 38 min. Helium was used as a gas carrier (1 mL min−1) [Before analyses, samples were derivatized by silylation following a procedure reported by Priestap et al. with some modifications. Briefly, 5 mg of dry extract were treated with 0.2 mL of N,O-Bis(trimethylsilyl)trifluoroacetamide (BSTFA) + 1% trimethylchlorosilane (TMCS) at 60 °C for 1 h . TrimethL min−1) . Ionizat−1 to 300 °C then held for 1 min; inlet temperature: 300 °C; ion source temperature: 250 °C; MS transfer line temperature: 280 °C. Helium was used as a gas carrier (1 mL min−1). Ionization mode: electron impact: −70 eV. Acquisition mode: full scan. As described, the NIST 2014 database was used along with commercially available standards to identify the chemical structures of the eluates. Esterification and amidation reactions were analyzed without any further derivatization, while samples from biocatalyzed hydrations were analyzed after derivatization as methyl esters using a (trimethylsilyl)diazomethane solution in hexane: 30 μL of reactant of and 20 μL of MeOH were added to 100 mL of ethyl acetate extracts and briefly shaken at room temperature. Retention times (min): (a) ethyl esters: palmitic acid ethyl ester = 10.41, oleic/linoleic acid ethyl ester = 11.25, stearic acid ethyl ester = 11.35; (b) ethanol amides: palmitoyl ethanolamide = 12.82, oleoyl/linoleoyl ethanolamide = 13.60, stearoyl ethanolamide = 13.63; (c) methyl esters: capric acid methyl ester = 6.37, lauric acid methyl ester = 7.92, myristic acid = 9.15, palmitic acid = 10.25, oleic/linoleic methyl ester = 11.17, 10-hydroxy-cis-12-octadecenoic acid methyl ester = 11.86, 10-hydroxystearic acid methyl ester = 11.93.Enzymatic reactions were monitored using an Agilent HP-5MS column (30 m × 0.25 mm × 0.25 μm) on a Finnigan TRACE DSQ GC-MS instrument . Elution conditions: 100 °C, 3 min; 20 °C minStenotrophomonas maltophilia was synthetized and cloned in the pET28a vector in frame with a C-term His-tag sequence by Twist Bioscience , obtaining the plasmid pET28aSmOhyA2. E. coli BL21(DE3) was transformed with plasmid pET28aSmOhyA2 using standard techniques and stored at −80 °C as glycerol stock.The gene coding for oleate hydratase OhyA2 (GenBank WP_017356052.1) from E. coli BL21(DE3) carrying plasmid pET28aSmOhyA2 was inoculated in 100 mL of LBKAN30 and incubated for 24 h at 37 °C and 220 rpm. After that, the bacterial culture (20 mL) was inoculated and grown in 1 L of LBKAN30 culture medium at 37 °C and 220 rpm up to an OD600 of 0.6–0.8. Once reaching the desired OD600 value, the expression was induced by the addition of 0.2 mL of 0.5 M IPTG  and the bacterial culture was incubated for a further 24 h at 16 °C and 200 rpm. The cells were harvested by centrifugation for 30 min at 4 °C and 5 000 rpm and resuspended in wash buffer (20 mM potassium phosphate (PB) buffer, pH 8.0, 500 mM NaCl, 20 mM imidazole). Then, the lysis of the bacterial cells was performed by ultrasonication , followed by centrifugation for 30 min at 4 °C and 10 000 rpm. The protein purification was carried out by IMAC affinity chromatography, taking advantage of the His-tag at the protein C-terminal. As first step of purification, the cell lysate was collected and incubated on ice with Ni-NTA Sepharose 6 Fast Flow resin  for 90 min, then it was loaded onto a glass column (10 m × 110 m) previously equilibrated and washed with 15 mL of wash buffer. Protein elution was performed using a 3-step gradient . Subsequently, the protein fractions were collected and protein content was quantified using the Bradford method. Fractions containing the recombinant enzyme were dialyzed o/n at 4 °C against 50 mM citrate-PB buffer, pH 6.0, to remove residual salts and imidazole, and then stored at −80 °C.Enzyme expression was performed as follows. A single colony of a) Lipase-mediated synthesis of fatty acid ethanol carboxamides. Novozym® 435 (2 g L−1) and 5Å molecular sieves were added to a solution of Cp oil (45 g L−1) prepared in a mixture of EtOH and cyclohexane . The obtained mixture was incubated in vacuo at 55 °C and 160 rpm for 1 h. After assessing complete conversion by TLC 6MoO4 42 g, Ce(SO4)2 2 g, H2SO4 62 mL, H2O 1 L) and GC-MS, the enzyme and molecular sieves were removed by filtration affording the ethyl ester derivatives after in vacuo concentration. The ester intermediates were than dissolved in ethanolamine (45 g L−1) and incubated at 75 °C and 160 rpm in the presence of 2 g L−1 Novozym® 435 and 5Å molecular sieves for 72 h, monitoring their conversion via GC-MS. The target fatty acid ethanol carboxamides were recovered after filtration and in vacuo concentration.(b) Biocatalyzed hydration. First, 0.1 g L−1 of OhyA2 were added to a solution of Cp oil (20 g L−1) prepared in 50 mM citrate-phosphate buffer, pH 6.5, containing 2% glycerol and EtOH, 0.15 mM FADH2 and titaniumIII citrate (5 mM). After assessing complete conversion of oleic and linoleic acids by TLC  and GC-MS (derivatization as methyl esters), pH was changed to 3 and the reaction mixture was extracted with EtOAc, recovering the transformed oil after drying over Na2SO4 and in vacuo concentration., allowing the enrichment of target high-value classes of compounds. In particular, by treating under medium sc-CO2 conditions (in terms of pressure and temperature), the amount of extractables reached yields up to 8%, which is rewarding considering that the provided biomass was a “waste”.Fractions with potential industrial interest have been obtained by means of supercritical fluids, mainly composed of free fatty acids and ∆7-sterols. Results obtained through sequential sc-COThe selected enzymatic manipulations investigated in this work and aimed at the future valorization of the valuable fatty acids still present in the considered industrial wastes could be carried out by exploiting very simple and scalable procedures, without any need for specific purification of the starting material, thus further demonstrating the applicability of biocatalysis in a circular economy view.


Shigella as a typical foodborne pathogen has strong survivability in the environment or food, leading to infectious diseases, yet its rapid detection technology with high selectivity and sensitivity remains challenging. In this study, complementary strand modified upconversion nanoparticles (UCNPs) can offer stable yellow-green fluorescence at 500–700 nm excited by a 980 nm laser. Importantly, Shigella aptamer modified gold nanoparticles (GNPs) formed by “Au−S” bond act as a fluorescence resonance energy transfer (FRET) donor and recognition element that can bind specifically to Shigella and significantly quench the fluorescence of complementary strand modified UCNPs. As a result, the fluorescence of our developed nanoprobe increased linearly with the increase in Shigella in a wide range from 1.2 × 102 to 1.2 × 108 CFU/mL and the detection limit was as low as 30 CFU/mL. Moreover, the fabricated upconversion fluorescence nanoprobe can achieve Shigella detection in contaminated chicken without enrichment in 1 h. Shigella-contaminated food is also a cause of inflammatory diarrhea and dysentery disease. Shigella, as a Gram-negative bacterium, can survive at infectives in many types of foods such as meat products, fruits, vegetables, high salt/low pH food products, and packaged foods due to its acid and salt resistance properties [Shigella infection occur each year, including 1.1 million deaths. In addition, 69% of the deaths were in children under the age of five [Foodborne pathogens continue to be a serious public health problem throughout the world . Among toperties ,3,4. Acc of five . ConsequDirect foodborne pathogens detecting strategies without a time-consuming culture process are urgently desired . TraditiThe fluorescent probe as a promising and alternative bacterial detection method has received more and more attention due to its high sensitivity, high selectivity, and short response time ,12. TradEscherichia coli (E. coli) in water and tea powder samples with high sensitivity (LOD =17 CFU/mL) and high accuracy (96.5–101.6%) [Listeria monocytogenes (L. monocytogenes) in ham samples was reported with a low detection limit (2.8 × 102 CFU/mL) and high selectivity [Salmonella typhimurium (S. typhimurium) DNA in chicken samples with high sensitivity (11 CFU/mL) and high selectivity. The above upconversion fluorescent probes have shown excellent performance in the detection of pathogenic bacteria in food. However, upconversion fluorescent probes for Shigella detection are rarely reported. Thus, developing a fluorescence probe for Shigella detection based UCNPs is important. Recently, the fluorescent probes constructed from a series of functionalized upconversion fluorescent nanoparticles were reported to determine a variety of harmful substances  ; a sandwectivity ; a UCNPsShigella in chicken samples. Shigella detection. First, the UCNPs−GNPs fluorescence probe was proved to be successfully fabricated by a series of characterizations (transmission electron microscopy (TEM), X-ray diffraction (XRD) spectra, FT-IR spectra). Then, we acquired fluorescence lifetimes spectra, UV-Vis, and fluorescence spectra of UCNPs−GNPs to verify the FRET mechanism. Afterwards, the upconversion fluorescence spectra of different components were obtained to confirm the feasibility of the detection method. The spectra of the UCNPs−GNPs probe with the addition of a series of different concentrations of Shigella and other non-target bacteria were obtained to analyze the sensitivity and selectivity of the constructed probe. Finally, we used the constructed probe to analyze the accuracy of the method by using the method of spike recovery. Here, an upconversion fluorescence probe based on FRET between aptamer1-modification GNPs and complementary strand modified UCNPs has been designed to determinate 3·6H2O, ErCl3·6H2O, GdCl3·6H2O and YbCl3·6H2O) obtained from Sigma-Aldrich are 99% pure. Ammonium fluoride (NH4F), sodium hydroxide (NaOH), alendronic acid (ADA), 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), hydrogen tetrachloroaurate hydrate (HAuCl4·3H2O), N-Hydroxysuccinimide (NHS), succinic anhydride, and other reagents were purchased from Alfa Aesar . All aptamers were obtained from Sangon Biotechnology Ltd. . The sequence of Shigella aptamer is as follows: 5′-SH-CCG GAC TAG GGC TGG TTA GCT TCA ATA CTG CTG GGC GAG G-3′ (apt1) and 5′-NH2-GGC CTG ATC CCG ACC AAT CGA AGT TAT GAC GAC CCG CTC C-3′ (apt2). All foodborne pathogens used in the experiment were supplied by Nanjing Agricultural University.All rare earth chloride hexahydrates  images were acquired on a Tecnai G2 F30. The zeta potentials of ADA−UCNPs, UCNPs−COOH, apt2−UCNPs, GNPs, and apt1−GNPs materials were measured in a neutral water solution with a Malvern Zeta sizer Nano . Siemens D5005 instrument was used to observe the crystal structure of UCNPs. Ultraviolet–Visible (UV–Vis) absorbance spectra were recorded on a UV-1800 . Fourier transform infrared (FTIR) spectra were collected by a Nicolet IR200 FTIR spectrometer . Upconversion fluorescence spectra were obtained by the assembled upconversion fluorescence detection system.3/HCl (volume ratio = 1:3) was used to wash all glassware used in the production of synthesis of the GNPs. HAuCl4 (16.98 mg) was mixed in a 250 mL flask containing 50 mL of deionized water. Then, the mixture was heated to 100 °C in a water bath under vigorous stirring. Subsequently, 5 mL of trisodium citrate solution (1%) were added to the above mixture. Then, the mixture was boiled in a water bath under vigorous stirring for 10 min. Finally, the yield GNPs were cooled down to room temperature and stored at 4 °C in the refrigerator. The apt1−GNPs were synthesized by a previously reported method with appropriate modifications [The GNPs were fabricated according to the previous literature with appropriate modifications . A mixedications . A total4: Yb, Er UCNPs was fabricated using a reported solvothermal method [3·6H2O  dissolved in 4 mL of methanol was added to a 250 mL three-necked round-bottom flask containing 14 mL of 1-octadecene and 6 mL of oleic acid. Next, the above mixture solution was heated to 160 °C under a nitrogen atmosphere and vigorous stirring for 30 min. After the reaction, the mixture solution was cooled down to 50 °C and added dropwise 20 mL of the mixed methanol solution (2.5 mmoL NaOH and 4 mmoL NH4F) under vigorous stirring, and then the mixture solution was transferred to a water bath at 50 °C and kept reacting for 40 min. Subsequently, the mixture was heated to 70 °C for 50 min to evaporate methanol. Finally, the mixture solution was heated to 300 °C in a nitrogen atmosphere and vigorously stirred for 60 min. After the reaction, the mixture solution was cooled down to room temperature. The yield OA−UCNPs were washed three times with cyclohexane-ethanol and dried in a vacuum oven at 55 °C for 8 h.Oleic acid (OA)-capped NaYFl method . RECl3·63 by sonication for 30 min. Next, the UCNPs solution was transferred to a 100 mL centrifuge tube containing 4 mL of ethanol and 6 mL of 8.3 mg/ mL ADA solution. Then, the mixture was adjusted to pH 2–3 using a 1M HCl solution and kept reacting for 1 h. Finally, the yield ADA−UCNPs were washed three times with deionized water–ethanol (1:1) and dried in the vacuum oven at 60 °C overnight. Alendronic acid-modified upconversion nanoparticles (ADA−UCNPs) were synthesized by a previously reported method with appropriate modifications and the detailed procedure was as follows : 200 mg Carboxyl-modified upconversion (UCNPs−COOH) was prepared according to the previous literature with appropriate modifications. Firstly, 200 mg ADA−UCNPs were dispersed in 15 mL methylbenzene by sonication for 30 min, then 5 mL of succinic anhydride (0.16 M) solution were mixed into the ADA−UCNPs solution. Then, the mixture was heated to 80 °C in a nitrogen atmosphere and vigorously stirred for 12 h. After the reaction, the mixture solution was cooled down to 35 °C and washed three times with deionized water. Finally, the yield UCNPs−COOH was dried in a vacuum oven at 60 °C overnight.The procedure of aptamer-functionalized UCNPs was adapted from a previously reported method with minor modification . FirstlyShigella (ATCC 12022), E. coli (ATCC 43889), S. typhimurium (ATCC 14028), L. monocytogenes (ATCC ATCC19111), Staphylococcus aureus , Pseudomonas aeruginosa , and Streptococcus thermophila . Shigella (ATCC 12022) as target bacteria was detected using the fabricated upconversion fluorescence probe. Meanwhile, S. typhimurium (ATCC 14028), L. monocytogenes (ATCC ATCC19111), S. aureus (ATCC 130), P. aeruginosa (ATCC 27853), and S. thermophila (ATCC 03872) as control groups were selected to confirm the specificity of an upconversion fluorescence probe. All bacteria used in this research work was cultured in a Luria–Bertani (LB) broth at 37 °C overnight. The bacterial cells were washed three times with deionized water under centrifugation (8000 rpm). The plate counting was used to calculate the concentration of the bacteria.All bacterial strains used in this experiment were Shigella (1.2 × 108 CFU/mL), and the experimental groups were prepared with the Shigella suspension concentration gradients of 1.2 × 101, 1.2 × 102,1.2 × 103, 1.2 × 104, 1.2 × 105, 1.2 × 106, 1.2 × 107 and 1.2 × 108 CFU/mL. The upconversion fluorescent probe was fabricated as follows: a total of 140 μL of a series of concentrations of Shigella were added to 140 μL of 100 nM apt1−GNPs and reacted for 25 min; then, 20 μL of 4 mg/mL apt2−UCNPs were added to the above mixture and continued reacting for 11 min. Finally, the mixture solution was detected using the assembled upconversion fluorescence detection system.Firstly, we used plate counts to calculate the cell concentration of 3–105 CFU/mL) of Shigella was spiked in chicken samples. The detailed procedure of Shigella detection in the chicken samples was as follows: Firstly, the macroaggregates in chicken sample solution were removed by standing for 30 min. Then, the 0.45 μm filtration membrane was used to filter the chicken samples’ supernatant. Finally, the filtrate solution was determined by the developed upconversion fluorescence probe and the traditional plate counting method, respectively.Chicken samples were purchased from a local supermarket in Nanjing, China. In order to eliminate the interference of target bacteria or other non-target bacteria in the chicken samples as much as possible, we used sterile saline to wash chicken samples (25 g) and exposed them to a 30 W UV lamp for 30 min. Then, the concentration range  [−1, 1572 cm−1, 2849cm−1, and 2934 cm−1 disappeared, and a new stretching vibration band at 1093 cm−1 appeared, which was associated with the NH2-stretching vibration, validating successful modification of ADA (curve b) [−1 disappeared, and two new stretching vibration bands at 1502 cm−1 and 1701cm−1 appeared. This is because of the successful graft of COOH groups onto NH2- groups (curve c) [0.57Yb0.39Er0.04) YF4 material.TEM characterizations of OA−UCNPs, ADA−UCNPs, UCNPs−COOH, and GNPs were displayed in curve a) . After scurve b) . After scurve c) . Hence, Shigella relies on a FRET process between apt2−UCNPs acting as a fluorescence donor and apt1−GNPs acting as a fluorescence acceptor and target bacterial recognition element. As demonstrated in Shigella aptamer modification GNPs were fabricated by the bond of “Au−S”. OA−UCNPs is sequentially modified by alendronate and carboxyl groups to generate UCNPs−COOH, and the complementary strand was conjugated on UCNPs−COOH. In the absence of Shigella, the UCNPs−GNPs complex formed by the complementary pairing of aptamer, causing the distance between GNPs and UCNPs to be less than 10 nm, led to the decreasing fluorescence. Upon addition of Shigella, Shigella was captured by apt1 and fell from the surface of GNPs. The bare gold nanoparticles cannot be adsorbed on the upconversion surface by base complementary pairing, leading to fluorescence recovering. To verify the FRET mechanism between apt2−UCNPs and apt1−GNPs, the upconversion fluorescence spectra and absorption spectra of the mixed UCNPs−GNPs were tested. As seen in 9/2 → 4F15/2, 4S3/2 → 4F15/2, and 4I11/2 → 4F15/2 transitions of Er3+, respectively [The detection of ectively . The GNPectively . In ordeShigella apt1 modification, the zeta potential of GNPs changes into −25.7 mV, revealing that the bare GNPs (−18.9 mV) or apt1−GNPs cannot be adsorbed on apt2−UCNPs (−13.2 mV) by electrostatic adsorption. In addition, the fluorescence spectra of apt1−UCNPs with the addition of GNPs, apt1−GNPs, and apt1−GNPs−Shigella were tested to confirm the feasibility of the upconversion fluorescence probe for detection of Shigella. As shown in Shigella occurred for recovery. Based on the above fluorescence changes, the developed upconversion fluorescence probe can be used to detect Shigella. The fluorescence spectra of OA−UCNPs, ADA−UCNPs, UCNPs−COOH, and apt1−UCNPs at the same concentration were determined to confirm the stability of upconversion fluorescence. As seen in Shigella. Firstly, the fluorescence intensity at 545 nm was significantly affected by the concentration of apt1 when the concentration of apt2−UCNPs was fixed. The upconversion intensity at 545 nm successively decreases as the concentration of apt1 increases, and the fluorescence intensity tended to be stable when the apt1 was 100 nM  increased, and the ΔFL remained stable after 4 mg/mL. Thus, 4 mg/mL was selected as the best concentration  the concentration of apt1, (B) the incubation time of apt1−GNPs, (C) the concentration of apt2−UCNPs, and (D) the incubation time between the nanoprobe and s 100 nM A. The res 100 nM B. The adntration C. The inion time D.Shigella. As seen in Shigella. This phenomenon was ascribed to the apt1 firmly adhered to the surface of bacteria, and the GNPs fell from the surface of apt−UCNPs. The higher the Shigella concentration, the more GNPs fell from the apt2−UNCPs surface; thus, a higher fluorescence signal can be acquired. A linear relationship was obtained between the upconversion intensity at 545 nm and the Shigella concentration. Density ranged from 1.2 × 102 to 1.2 × 108 CFU/mL . Furthermore, in comparison with the linear range, LOD and detection time of the developed upconversion fluorescence probe with other reported methods [Shigella detection with high sensitivity and meets the requirements for contaminated sample detection , E. coli (ATCC 43889), S. typhimurium (ATCC 14028), L. monocytogenes (ATCC 19111), S. aureus (ATCC 130), P. aeruginosa (ATCC 27853), and S. thermophila (ATCC 03872)) into the UCNPs−GNP probe. As shown in Shigella (ATCC 12022) induced a dramatic fluorescence recovering at 545 nm. In addition, we tested the fluorescence intensity of our developed probe with the addition of other non-target bacterial and found that E. coli (ATCC 43889), S. typhimurium (ATCC 14028), L. monocytogenes (ATCC 19111), S. aureus (ATCC 130), P. aeruginosa (ATCC 27853), and S. thermophila (ATCC 03872) in this upconversion fluorescence probe had no significant effect on fluorescence intensity at 545 nm, which verified that the upconversion fluorescence probe had excellent specificity.In order to evaluate the specificity of this developed upconversion fluorescence probe for The practicability of the proposed upconversion fluorescence probe in the analysis of chicken samples was further analyzed by spiked food samples, and the results are shown in Shigella based on apt1 modified GNPs (fluorescence acceptor) as reorganization elements and complementary strand modified UCNPs (fluorescence donor). The formation of apt1−Shigella complex resulted in the recovery of upconversion fluorescence intensity at 545 nm, and Shigella was detected based on the increase in upconversion fluorescence at 545 nm. A linear response was calculated in the range of 1.2 × 102 to 1.2 × 108 CFU/mL with an LOD of 30 CFU/mL. By the analysis of the interference of non-target bacteria with the fluorescence probe, it was found that the probe had good specificity. The study also provides one concept for design and synthesis of a UCNPs−GNPs fluorescence probe for Shigella detection in the food safety field.A UCNPs@GNPs fluorescence probe was developed for rapid sensing of