Antioxidant activity, which was determined by DPPH, ABTS, CUPRAC, and FRAP methods, was ameliorated by systems; the greatest results were obtained for RA–HP-γ-CD s.e. The inhibition of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) was increased from 36.876% for AChE and 13.68% for BChE to a maximum inhibition of the enzyme (plateau), and enabled reaching IC50 values for both enzymes by all systems. CDs are efficient excipients for improving RA physicochemical and biological properties. HP-γ-CD was the greatest one with potential for future food or dietary supplement applications. Rosmarinic acid (RA) is a natural antioxidant with neuroprotective properties; however, its preventive and therapeutic use is limited due to its slight solubility and poor permeability. This study aimed to improve RA physicochemical properties by systems formation with cyclodextrins (CDs): hydroxypropyl-α-CD (HP-α-CD), HP-β-CD, and HP-γ-CD, which were prepared by the solvent evaporation (s.e.) method. The interactions between components were determined by X-ray powder diffraction (XRPD), differential scanning calorimetry (DSC) and Fourier Transform infrared spectroscopy (FTIR). The sites of interaction between RA and CDs were suggested as a result of in silico studies focused on assessing the interaction between molecules. The impact of amorphous systems formation on water solubility, dissolution rate, gastrointestinal (GIT) permeability, and biological activity was studied. RA solubility was increased from 5.869 mg/mL to 113.027 mg/mL, 179.840 mg/mL, and 194.354 mg/mL by systems formation with HP-α-CD, HP-β-CD, and HP-γ-CD, respectively. During apparent solubility studies, the systems provided an acceleration of RA dissolution. Poor RA GIT permeability at pH 4.5 and 5.8, determined by parallel artificial membrane permeability assay (PAMPA system), was increased; RA–HP-γ-CD s.e. indicated the greatest improvement (at pH 4.5 from  Rosmarinic acid (RA) is a natural phenolic acid in many plants, including herbs and spices such as rosemary, oregano, sage, thyme, and lemon balm. It is a significant antioxidant with anti-inflammatory, antidiabetic, neuroprotective, anticancer, antimicrobial, and antiviral properties ,2,3,4. IMelissa officinalis extract containing 100 mg of RA in almost all healthy volunteers was not determined  grade) was provided by Merck , and other chemical reagents—including hydrochloric acid, dimethyl sulfoxide, sodium chloride, and potassium dihydrogen phosphate—were obtained from Avantor Performance Materials . Formic acid 98–100% was purchased from POCH . Prisma HT, GIT lipid solution, and acceptor sink buffer were supplied by Pion Inc .v/v) ethanol and evaporated using Rotavapor  at the temperature of 40 °C. The resulting systems were grounded in a mortar and stored with physical mixtures of the binary systems in a desiccator until further tests were performed.RA systems with HP-α-CD, HP-β-CD, and HP-γ-CD in a mass ratio of 1:1 were prepared by the solvent evaporation method. RA and CDs HP-α-CD, HP-β-CD, and HP-γ-CD were dissolved in 70%  at a tube voltage of 45 kV and a tube current of 40 mA. The angular range was 3° to 50° with a step size of 0.017° and a counting rate of 15 s/step. The OriginPro 8 software was used to analyze the acquired data . T. T−7 ± 7−7 ± 3.309 × 10−8 cm/s, which means it is poorly soluble  in the DPPH study was 59.483 ± 0.041 µg/mL and for ABTS 102.578 ± 3.427 µg/mL  for RA in CUPRAC was 13.677 ± 0.993 µg/mL and for Trolox 56.564 ± 0.664 µg/mL, while in FRAP RA the IC0.5 was 10.558 ± 0.203 µg/mL and for Trolox 41.941 ± 0.014 µg/mL.In the study of antioxidant activity, the RA turned out to be a strong antioxidant whose IC27 µg/mL . The ref50 value in DPPH was lowered to 57.398 ± 0.762 µg/mL and in ABTS to 87.766 ± 1.802 µg/mL. The systems with HP-α-CD and HP-β-CD also decreased the IC50 value and, thus, increased the scavenging activity; however, the changes were not statistically significant. DPPH and ABTS assays determine the ability of the substance to scavenge radicals, which is related to hydrogen-donating ability [The scavenging potential of the RA in the DPPH and ABTS methods increased significantly for the system with HP-γ-CD. The IC ability .50 (the concentration that inhibits enzymes by 50%). RA inhibited AChE by 36.876 ± 1.812% and BChE by 13.68 ± 0.519%. The increase in RA solubility due to its connections with CDs to 113.027 mg/mL, 179.840 mg/mL, and 194.354 mg/mL using HP-α-CD, HP-β-CD, and HP-γ-CD, respectively, is significantly above the IC50, which is for AChE 8.983 ± 0.162 mg/mL and for BChE 14.865 ± 0.358 mg/mL, and enabled the achievement of maximum enzyme inhibition. The RA tyrosinase IC50 is 1.591 ± 0.077 mg/mL, thus, achieving 50% inhibition of the enzyme within RA’s water solubility.RA at concentration 5.869 ± 0.140 mg/mL, which is the value of its water solubility, inhibited AChE and BChE; however, this value is below the ICM. officinalis extract comprising 500 mg of RA was assessed to be harmless [Orthosiphon stamineus leaves with the addition of ethanol was performed, and the optimization of the process to correlate the solubility of RA in supercritical carbon dioxide [The RA shows a wide range of biological activities from antioxidant, anti-inflammatory, hepatoprotective, neuroprotective, and photoprotective to antidiabetic and antimicrobial properties ,53. The harmless . The lim dioxide . The gre dioxide . In a ph dioxide .RA is unstable in gastric acidic pH, which is present in the fasted stomach ; thus, iThunbergia laurifolia extract was determined as better than pure RA. Moreover, appP values were increased after deconjugation by β-glucuronidase/sulfatase enzymes [L. johnsonii strains addition resulted in the (> 90%) hydrolysis of RA. This study attempted to overcome poor RA GIT permeability, which was confirmed in the PAMPA study, with a permeability coefficient in pH 4.5—6.901 × 10−7 ± 7.619 × 10−8 cm/s. However, the permeability was significantly increased by systems formation with HP-γ-CD to 1.085 × 10−6 ± 6.605 × 10−8 cm/s reaching a good permeability level. In pH 5.8, permeability was also assessed as low—appP 5.019 × 10−7 ± 3.309 × 10−8, whilst after preparation of the systems with the same CD it was significantly enlarged to 9.680 × 10−7 ± 7.235 × 10−8 cm/s, almost reaching a good permeability value (1.0 × 10−6 cm/s).In the study on Caco-2 cell monolayers, transport determined that RA mostly permeates via paracellular diffusion, and the intestinal permeability was <1% of its intake volume . The int enzymes . Additio enzymes . RA was During the connections of RA with CDs, hydrogen bonds between these molecules are formed, weakening RA intramolecular hydrogen bonds and subsequently increasing the hydrogen donation by the hydroxyl groups of RA. The stronger the interaction between system components, the greater the hydrogen donation of RA, as the covalent bonds between hydrogen and oxygen in the hydroxyl groups are weaker.50 from 59.483 ± 0.041 µg/mL to 57.398 ± 0.762); ABTS (IC50 from 102.578 ± 3.427 to µg/mL 87.766 ± 1.802 µg/mL); and FRAP (IC0.5 from 10.558 ± 0.203 µg/mL to 10.126 ± 0.132 µg/mL) assays, and the results were reported as prepared concentrations. Similar trends were reported in other studies. In Veras et al.’s study, RA complexes with HP-β-CD and M-β-CD that were obtained by freeze-drying assessed greater antioxidant activity than pure RA in the DPPH assay [50 = 2.8–3.7 μg/mL) than pure RA (EC50 = 10 μg/mL) [RA might play a vital role in the prevention of neurodegenerative diseases, as it has neuroprotective and neurodegenerative properties and impacts neuroinflammation in in vitro and in vivo studies ,61,62,63PH assay . The abiPH assay ,66. In aPH assay . All sys0 μg/mL) .50 and IC0.5 values are well below the water solubility of pure RA; therefore, the effect on the antioxidant potential is due to the systems formation. Very high CD concentrations might decrease the antioxidant potential of antioxidants, as the inclusion-complexation and systems formation equilibria of antioxidants may predominate those of their oxidation [0.5 for CUPRAC 13.823 ± 0.027 µg/mL, and FRAP 10.126 ± 0.132 µg/mL, which is statistically significant.In most cases, the increase in solubility of the complexed substance for antioxidant activity is not without significance. In the case of RA, the ICxidation . Thus, ixidation . SubsequCDs also increase the antioxidant activity of other biologically active compounds. Daidzein–CD–metal organic framework solid dispersion complexes prepared by the solvent evaporation method increased daidzein DPPH scavenging activity ~1.3 times . In anotThe antioxidant activity of RA will contribute to the reduction in oxidative stress, protecting cells’ structure and function and preventing excessive chronic inflammation, thus, being a neuroprotectant. Neurodegeneration might also be prevented by inhibiting enzymes such as acetylcholinesterase, butyrylcholinesterase, and tyrosinase among patients with dementia .50 for AChE was 8.983 ± 0.162 mg/mL and for BChE 14.865 ± 0.358 mg/mL. RA tyrosinase IC50 was determined within RA water solubility.To the best of the authors’ knowledge, there are no studies on the influence of amorphous or crystal systems influencing RA’s activity on AChE, BChE, and tyrosinase activity. Therefore, this study examined how the increase in the RA solubility affects the activity against these enzymes, since CDs do not leave GIT and do not directly affect the enzymatic activity. From 36.876 ± 1.812% of inhibiting AChE and 13.68 ± 0.519% of inhibiting BChE at the concentration 5.869 ± 0.140 mg/mL (water RA solubility), it was possible to obtain maximum enzyme activity (plateau) for each RA–HP-α-CD s.e., RA–HP-β-CD s.e., and RA–HP-γ-CD s.e. system, where the ICRosmarinic acid is a phenolic acid with excellent pro-health potential, but it is limited by physicochemical properties, which this study aimed to overcome. Interactions between the RA with cyclodextrins , especially in the case of HP-γ-CD, resulted in an increase in solubility, permeability, antioxidant activity, and the inhibition of acetylcholinesterase and butyrylcholinesterase.


The aim of this study is to identify the risk factors of nonobese nonalcoholic fatty liver disease (NAFLD) individuals under different insulin resistance status.  This cross-sectional study was conducted at the Medical Center of Beijing Chaoyang Hospital affiliated with Capital Medical University. NAFLD was diagnosed based upon ultrasonographic findings consistent with fatty liver disease. 2 were enrolled in the study. And all patients were divided into homeostasis model assessment of insulin resistance (HOMA − IR) > 1 group and HOMA − IR ≤ 1 group. When all the variables were adjusted in both the HOMA − IR > 1 group and HOMA − IR ≤ 1 group, older age (>50 years), higher BMI (23.0-24.9 kg/m2), higher AST (>18 U/L), higher TG (>0.9 mmol/L), higher GLU (>5.25 mmol/L), and higher HbA1C (>5.5%) were associated with higher risks of nonobese NAFLD. In patients with HOMA − IR > 1, lower homeostatic model assessment of β-cell function (HOMA-β) (<47.1%)  was associated with higher risks of nonobese NAFLD.  A total of 1257 nonobese adults (625 non-NAFLD and 632 nonobese NAFLD) with body mass index (BMI) 18.5-24.9 kg/mβ-cells may be the risk factor of nonobese NAFLD with insulin resistance, who should pay attention to further development of pancreatic β-cell dysfunction.s. Metabolic profiles  are risk factors of nonobese NAFLD, regardless of insulin resistance status. Decreased function of pancreatic  Insuline” NAFLD . However2 [2; (3) systolic blood pressure ≥ 140 mmHg; (4) diastolic blood pressure ≥ 90 mmHg; (5) significant alcohol intake ; (6) liver disease associated with other conditions including viral hepatitis, autoimmune hepatitis, hemochromatosis, or Wilson's disease; and (7) fatty liver associated with other conditions such as drug use or hereditary disease. Participants provided written informed consent, and the Ethics Committee of the Beijing Chaoyang Hospital, Capital Medical University, approved the present study.This was a cross-sectional study conducted from November 2018 to October 2019 at the Medical Center in Beijing Chaoyang Hospital affiliated with Capital Medical University. We consecutively recruited 1257 nonobese subjects. Inclusion criteria were as follows: (1) age ≥ 18 years, (2) BMI 18.5-24.9 kg/m2 . Patientweight (kg)/height (m2). The homeostasis model assessment of insulin resistance (HOMA-IR) index was calculated according to the formula HOMA‐IR = (FINS(uIU/mL) × FBG(mmol/L))/22.5. Homeostatic model assessment of β-cell function (HOMA-β) index was calculated according to the formula HOMA‐β = (20 × FINS(uIU/mL))/(FBG(mmol/L)‐3.5) [The general health checkup included a medical history, physical examination, lifestyle questionnaire, biochemical measurements, and abdominal ultrasonography. Blood specimens were sampled from the antecubital vein after more than 12 h of fasting. Colorimetric assays were used to measure total cholesterol (TC), low-density lipoprotein cholesterol (LDL-c), high-density lipoprotein cholesterol (HDL-c), and triglyceride (TG) via the use of an autoanalyzer Hitachi 7170). Fasting blood glucose (FBG), fasting insulin (FINS), and HbA1c levels were analyzed at the Central Chemistry Laboratory of Beijing Chaoyang Hospital affiliated with Capital Medical University. BMI was calculated as 170. FastAbdominal ultrasounds were performed by three experienced radiologists who were unaware of the purpose of the study and blinded to laboratory values. NAFLD was diagnosed as per the criteria of the Chinese Liver Disease Association . Fatty lt-test or the Wilcoxon signed-rank test for continuous variables and chi-square test for categorical variables. Collinearity diagnostic was conducted prior to further assessment. A stepwise multivariable logistic regression was performed  to identify risk factors linked with nonobese NAFLD. P values of <0.05 were considered statistically significant.SPSS 22.0  was employed for statistical analyses. The differences of covariates between the NAFLD status were compared using P < 0.001), higher BMI (P < 0.001), higher ALT (P < 0.001), higher AST (P < 0.001), higher TG (P < 0.001), higher LDL-c (P = 0.031), higher fasting glucose (P < 0.001), higher insulin (P < 0.001), and higher HbA1c (P < 0.001) values relative to patients without NAFLD. However, subjects with NAFLD were more likely to have lower HDL-c (P < 0.001) and lower HOMA-β (P < 0.001)  were men . Subject< 0.001) .2, P < 0.001) (P = 0.001) (P = 0.042) (P < 0.001) (P = 0.029)  compared to those without NAFLD  , higher = 0.001) , higher = 0.042) , higher < 0.001) , and hig= 0.029)  values rut NAFLD .2, P < 0.001) (P = 0.021) (P = 0.002) (P < 0.001) (P < 0.001)  compared to those without NAFLD  , higher = 0.021) , higher = 0.002) , higher < 0.001) , and hig< 0.001)  values rut NAFLD .2), higher ALT (>17 U/L), higher AST (>18 U/L), lower HDL-c (<1.36 mmol/L), higher TG (>0.9 mmol/L), higher LDL-c (>2.65 mmol/L), higher GLU (>5.25 mmol/L), higher HbA1C (>5.5%), higher FINS (>3.89 uIU/ml), and lower HOMA-β (<47.1%) were independent factors. When all the variables were adjusted, the results showed that older age (>50years) , higher BMI (23.0-24.9 kg/m2) , higher AST (>18 U/L) , higher TG (>0.9 mmol/L) , higher GLU (>5.25 mmol/L) , higher HbA1C (>5.5%) , higher FINS (>3.89 uIU/ml) , and lower HOMA-β (<47.1%)  were associated with higher risks of nonobese NAFLD , higher BMI (23.0-24.9 kg/mse NAFLD .2), higher ALT (>17 U/L), higher AST (>18 U/L), lower HDL-c (<1.36 mmol/L), higher TG (>0.9 mmol/L), higher GLU (>5.25 mmol/L), higher HbA1C (>5.5%), and lower HOMA-β (<47.1%) were independent factors. When all the variables were adjusted, the results showed that older age (41-50years) , age > 50 years , higher BMI (23.0-24.9 kg/m2) , higher AST (>18 U/L) , higher TG (>0.9 mmol/L) , higher GLU (>5.25 mmol/L), and higher HbA1C (>5.5%)  were associated with higher risks of nonobese NAFLD , higher BMI (23.0-24.9 kg/mse NAFLD .2), higher AST (>18 U/L), higher TG (>0.9 mmol/L), higher GLU (>5.25 mmol/L), and higher HbA1C (>5.5%) were associated with higher risks of nonobese NAFLD. In patients with HOMA > 1, lower HOMA-β (<47.1%) was associated with higher risks of nonobese NAFLD.We conducted this study to identify risk factors of nonobese NAFLD individuals under different insulin resistance status in the Chinese population. Our results revealed that when all the variables were adjusted in both the HOMA − IR > 1 group and HOMA − IR ≤ 1 group, older age (>50years), higher BMI  in the HOMA − IR > 1 group and lower HOMA-β  in the HOMA − IR ≤ 1 group. We found that lower HOMA-β (<47.1%) had significant higher risk for nonobese NAFLD  in the HOMA − IR > 1 group. Furthermore, Musso et al. reported that microsomal triglyceride transfer protein- (MTP-) 493G/T polymorphism is associated with pancreatic β-cell dysfunction in NASH [β of the lean group controls (BMI< 25 kg/m2) was nearly 10% [HOMA − β ≤ 40.12% [HOMA − β < 47.1% of our study, the participants all had normal glucose. It can be explained by the review of Gastaldelli, who revealed that even in the case of a 50% reduction, i.e., after experimental partial pancreatectomy, normoglycemia can be maintained by relatively small pancreas remnants. The system to compensate for changes a reduction in insulin sensitivity by increasing insulin secretion. T2DM develops when the beta-cell secretory capacity is not sufficient to overcome the insulin resistance of the tissues [HOMA-is grade . Musso econtrols . Meanwhi in NASH . Siddiquarly 10% . In addi≤ 40.12% . In spit tissues .HOMA − IR > 1 and HOMA − IR ≤ 1 groups. This is consistent with the prior findings from Wei, who determined that high BMI, and high HbA1c, and insulin resistance were independently associated with NAFLD in nonobese subject [It is known that NAFLD is highly associated with the metabolic syndrome . In the  subject . Chen et subject . Meanwhi subject .2) and middle-aged relative to patients without NAFLD. Xu et al. previously found that BMI was a primary risk factor linked to NAFLD incidence in nonobese subjects. Even slight increases in BMI within the normal range can elevate the risk of NAFLD development [2) individuals with and without NAFLD was 27.98 ± 2.84 and 26.69 ± 2.27 kg/m2, respectively [2 were at a higher risk of nonobese NAFLD  in the HOMA − IR > 1 and  in the HOMA − IR ≤ 1 groups. A study conducted in Japan also found that the prevalence of NAFLD increased gradually with age, with a peak prevalence of 23.3% in the 60-to 69-year age group, which was 3.4 times as high as that in the 30- to 39-year age group [age > 50years had higher risk for nonobese NAFLD in theHOMA − IR > 1 and in theHOMA − IR ≤ 1 groups. Moreover, Wei et al. showed that the prevalence of NAFLD among nonobese subjects increases with age [We found that nonobese NAFLD patients were more likely to be higher BMI 23.0-24.9 kg/m.9 kg/m2 ge group . We founwith age . In lineβ-cell dysfunction.These studies including ours consistently emphasize that nonobese NAFLD is closely associated with metabolic profiles. And people with insulin resistance and nonobese NAFLD should pay attention to further development of pancreatic Moreover, lean NAFLD has been associated with PNPLA3 polymorphisms. In particular, the substitution of methionine with isoleucine at the residual 148 would limit the access of the substrate to the catalytic serine in position 47, thus triggering an altered hydrolysis of hepatic triglycerides and a consequent increase in the content of cellular triglycerides. All these changes seem to be not associated with insulin resistance –29.β-cell over time. Another limitation of the study is that NAFLD was detected via ultrasound rather than via liver biopsy, although this approach is the most convenient and common means of diagnosing this condition. The ultrasound method is not quantified. Thirdly, lean NAFLD has been associated with PNPLA3 polymorphisms. The clinical utility of the polymorphisms involved in NAFLD is in part limited by low diffusion of genotyping methods in the routine clinical diagnostics and high cost [There are multiple limitations to the present study. For one, potential limitations of this study are cross-sectional studies where we are unable to determine the longitudinal relationship between nonobese NAFLD, its histological parameters, and pancreatic igh cost . Finallyigh cost . NAFLD iigh cost . Furtherβ-cells may be the risk factor of nonobese NAFLD with insulin resistance, which should be paid attention to further development of pancreatic β-cell dysfunction.In conclusion, we found that metabolic profiles  are risk factors of nonobese NAFLD, regardless of insulin resistance status. Decreased function of pancreatic


The genes gpd1 and gpp2 were co-expressed in K. pneumoniae with the dCas9 gene integrated in genome, and this recombinant strain produced 2 g/L glycerol in the shake flask. To minimize the glucose consumption by competing pathways including the EMP pathway, glycerol oxidation pathway, and by-products pathways, we developed an CRISPRi system in aforementioned recombinant K. pneumoniae strain to inhibit the expression of the glyceraldehyde-3-phosphate dehydrogenase gene (gapA) and 2,3-butanediol production gene (budA), resulting in a bi-functional strain harboring both glucose-to-glycerol pathway and CRISPRi system. Reverse transcription and quantitative PCR (RT-qPCR) results showed that this engineered CRISPRi system transcriptionally inhibited gapA and budA by 82% and 24%, respectively. In shake flask cultivation, this bi-functional strain produced 2.8 g/L glycerol using glucose as the carbon source, which was 46.6% increase compared to the strain without the engineered CRISPRi system. Moreover, this bi-functional strain produced 0.78 g/L 3-HP using glucose as the sole carbon source. In fed-batch cultivation, this bi-functional strain produced 1.77 g/L 3-HP. This study provides insights for co-utilization of distinct carbon sources.The recent decline of the international biodiesel industry has led to decreased production and therefore increased the price of glycerol, which is a major by-product of biodiesel but a substrate for production of 3-hydroxypropionic acid (3-HP), that is, glycerol as a feedstock has no advantage over glucose in price. Hence, we engineered glucose to the glycerol pathway and improved 3-HP production by CRISPR interference (CRISPRi). To begin with, we cloned the genes encoding glycerol 3-phosphate dehydrogenase ( Escherichia coli  is chemically active . As suchhia coli , S. cererevisiae , Corynebutamicum , Lactoba reuteri , and K. eumoniae . In aforeumoniae , glucoseeumoniae , and aceeumoniae . The glyeumoniae . The CoAeumoniae . Fortunaglycerol . To prodrevisiae . In addis regard , as it cs regard , its priS. cerevisiae, glucose is catalyzed into glycerol by GPD1 and GPP2  and gpp2 (GenBank accession No. DQ332890.1) were used to construct the shortest pathway for conversion of glucose to 3-HP. In addition, the CRISPRi system was developed to regulate the pathways affecting 3-HP production. To boost glucose conversion to glycerol, we engineered a CRISPRi system targeting glyceraldehyde-3-phosphate dehydrogenase gene (gapA) and α-acetolactate decarboxylase gene (budA). Reverse transcription and quantitative PCR (RT-qPCR) was performed to examine the expression of gpd1, gpp2, gapA, and budA. Shake flask cultivation of the recombinant strains were to determine the production of glycerol, 3-HP, acetic acid, lactic acid, 1,3-propanediol , and 2,3-butanediol . Fed-batch cultivation of the strain was to examine the production of 3-HP.Given aforementioned information, we conjecture that engineering a glucose-to-glycerol pathway in abolites . In doinK. pneumoniae strain with dCas9 integrated in genome (KP-dCas9) and plasmid ptac-15A was preserved in laboratory  medium containing the following components per liter: NaCl 10 g, tryptone 10 g, yeast extract 5 g, and chloramphenicol 34 mg. In CRISPRi and fermentation experiments, K. pneumoniae strain was grown in fermentation medium containing the following components per liter: glucose, 20 g; (NH4)2SO4, 4 g; K2HPO4·3H2O, 3.4 g; KH2PO4, 1.3 g; MgSO4·7H2O, 0.5 g; yeast extract, 3 g; CaCO3, 0.1 g; 1.25 ml of trace element solution; and chloramphenicol 34 mg. The trace element solution contained the following components per liter: CuCl2·2H2O, 1.88 g; FeSO4, 32 g; CoCl2·6H2O, 1.88 g; ZnCl2·6H2O, 2.72 g; Na2MoO4, 0.02 g; MnCl2·4H2O, 0.68 g; H3BO3, 0.24 g; and 40 ml concentrated HCl. Restriction enzymes, Taq DNA polymerase, and T4 DNA ligase were purchased from New England Biolabs . Primer synthesis and DNA sequencing were performed by Biomed Co., Ltd. RT-qPCR was completed by RuiBiotech Co., Ltd.The recombinant boratory . The plvboratory . E. coliglycerol . E. coligpd1 (GenBank accession No. Z74071.1) and gpp2 (GenBank accession No. DQ332890.1) genes were cloned by PCR from S. cerevisiae genome. Next, the gpd1 gene was cloned into ptac-15A at Noc I/Nde I sites, leading to a vector designated ptac-G1; the gpp2 gene was cloned into ptac-15A at Nde I/Nhe I sites, leading to a vector designated ptac-G2. Ligating gpd1 with the tac promoter and the RBS sequence from ptac-G1 to vector ptac-G2 at Not I/Xho I sites led to a vector designated ptac-G12 . Briefly, two complementary single-stranded target sequences were chemically synthesized and annealed to form a 23 bp double-stranded DNA owning cohesive ends matching the BspQ I-digested vector  with SYBR Green addition. The RT-qPCR data were analyzed using the ΔΔCT method with 16S rRNA as an internal control. All experiments were performed in triplicate.To examine the transcription levels of gpd1 and gpp2. Dehydrated tetracycline (aTc) at a final concentration of 2 μM was added to induce sgRNA expression. Fermentation broth was sampled every 3 h to examine cell growth, glucose consumption, and metabolites formation.In shake flask cultivation, strains KP-dCas9, KP-15A, KP-GS, KP-GG1, KP-GB2, and KP-GGB were pre-cultured in 4 ml LB medium overnight. Next, these strains were independently grown in 250-ml shake flasks each containing 100 ml 3-HP–producing medium and 8.5 mg chloramphenicol at 37°C and 150 rpm. After 3 h cultivation, IPTG at a final concentration of 0.5 mM was added to induce the expression of In fed-batch cultivation, the recombinant strain was grown in a 5 L bioreactor  containing 3 L fermentation medium to produce 3-HP. The strain KP-GB2 was pre-cultured in 100 ml of LB medium overnight at 37°C and subsequently inoculated into a bioreactor, containing antibiotics and IPTG. Air was supplied at 1.5 vvm, and agitation speed was set at 400 rpm. The temperature was set at 37°C, and the pH was maintained at 7.0 by adding 5 M NaOH. A total of 700 ml of 500 g/L glucose solution was added at a rate of 4.5 g/h to maintain cell growth after the glucose in medium was exhausted. The samples were taken out every 6 h to examine cell growth, glucose consumption, and metabolites formation.2O added in a cuvette. To measure metabolites, fermentation broth was centrifuged at 12,000 rpm for 10 min to remove bacteria. Major metabolites including 3-HP, lactic acid, and acetic acid in supernatant were analyzed by using the high-performance liquid chromatography (HPLC) system  equipped with a C18 column and an SPD-20A UV detector at 210 nm. The column was maintained at 25°C. The mobile phase was 0.05% phosphoric acid at a flow rate of 0.8 ml/min. 1,3-PDO and 2,3-BDO were analyzed by GC (Persee). Analytically pure 1,3-PDO and 2,3-BDO were used as standard for quantification. Residual glucose concentration was measured by an SBA-40E biosensor analyzer . A new immobilized enzyme membrane was installed on the electrode, 500 ml standard buffer was prepared with ddH2O, and the three-in-one standard solution of SBA was prepared . Glucose concentration in the supernatant was diluted to less than 1 g/L. After 25 μL standard solution was injected into the sampling port stabilization instrument with a microinjector, the glucose content in the sample was measured successively, and each sample was measured three times. Glycerol concentration was measured by the glycerin test kit (APPLYGEN Beijing). All samples were filtered through a 0.22 μm membrane filter.Cell concentrations were measured by using microplate reader at 600 nm with 200 μL fermentation broth and 1,800 μL ddHgpd1 and gpp2 were cloned from S. cerevisiae and independently subcloned into vector ptac-15A, resulting in vectors ptac-G1 and ptac-G2, respectively , while the strain KP-GB2 presented the lowest titer (1.185 ± 0.045 g/L). In addition, the strain KP-GB2 produced the highest 3-HP (0.734 ± 0.032 g/L). Interestingly, no significant differences were observed in the production of acetic acid, lactic acid, and 1,3-PDO in all strains.To maximize 3-HP and minimize 2,3-BDO, the glycerol synthesis genes gpd1 and gpp2 was able to synthesize glycerol from glucose and the strain KP-GB2 showed the highest 3-HP level in the shake flask, the strain was then cultivated in a 5 L bioreactor. Results showed that the strain did not consume glycerol until 12 h fermentation, at this time the glucose was exhausted. When glucose was supplemented, the glycerol level was further improved, indicating that the strain did not consume glycerol until glucose was exhausted. In addition, only a small amount of 2,3-BDO was produced in the initial stage of fermentation. However, in the middle and late stages of fermentation, 2,3-BDO production was increased presumably due to the rising pH value of medium and the failure of aTc induction in an alkaline environment, which hampered the inhibition of the CRISPRi system on by-product pathways. Due to the engineered glycerol synthesis pathway, the strain KP-GB2 produced 1.77 ± 0.04 g/L 3-HP in 72 h, with productivity of 0.025 g/L/h. This low productivity may be ascribed to the engineered CRISPRi system and genes gpd1 and gpp2, which imposed a heavy burden on cell growth. Indeed, the maximum OD600 was only 12.135 ± 0.005.Now that the strain co-expressing K. pneumoniae, and the resulting strain was subjected to CRISPRi-dependent gene regulation . As for the low 3-HP production, it was ascribed to the presence of glucose. In particular, the Crabtree effect hindered glycerol utilization. Future studies may focus on how to attenuate the Crabtree effect. To address this issue, an orthogonal expression system could be engineered to express glycerol utilization genes and minimize the Crabtree effect so that glycerol can be efficiently converted to 3-HP while cell growth is not compromised. Clearly, profound understanding of the dha regulon and Crabtree effect is a prerequisite for achieving this goal.In this study, the glycerol synthesis pathway was engineered in gulation . By co-eimproved . In addiThe Crabtree effect and Pasteur effect  are two gapA, an enzyme catalyzes 3-glyceraldehyde triphosphate into 1,3-diphosphoglyceric acid. CRISPRi was also used to inhibit budA, an enzyme required for 2,3-BDO production in K. pneumoniae (budA and gapA. In shake flask cultivation, the CRISPRi strain demonstrated slight enhancement in the glycerol level but significant decrease in 2,3-BDO production. However, the inhibitory effect on budA was significantly reduced when two groups of sgRNAs were linked in tandem. This might be ascribed to the low expression of dCas9, which was insufficient for binding two target genes simultaneously. Plasmid-based overexpression of dCas9 and multi-copy integration of dCas9 into genome may be feasible solutions. Overall, this study demonstrated the feasibility of glucose-to-glycerol as well as CRISPRi-dependent gene regulation in K. pneumoniae. We anticipate that better understanding of the metabolic networks is crucial for high-level production of 3-HP, 1,3-PDO, 2,3-BDO, and beyond.CRISPRi is usually not lethal to host cell, as it does not digest DNA. In contrast, gene knockout tools such as CRISPR editing, RecA homologous recombination, and Red homologous recombination are in most cases lethal to host cells. In present study, CRISPRi was developed to inhibit eumoniae . RT-qPCR


Berberine hydrochloride (BRB) is an isoquinoline alkaloid with promising anticancer efficacies. However, application of BRB had been hampered by its poor aqueous solubility, low gastrointestinal absorption, and rapid metabolism. The present study takes advantage of small extracellular vesicles (sEVs) to increase both stability and efficacy of BRB. sEVs from immature dendritic cells were produced and loaded with BRB. Proliferation, migration and Matrigel assay were performed, cycle arrest and nitric oxide (NO) production were evaluated in human breast cancer cell line (MDA-MB-231) and human umbilical vein endothelial cells (HUVECs). sEVs loaded with BRB formed a stable and homogenous population with a drug entrapment efficiency near to 42%. BRB loaded into sEVs was more potent than free BRB for MDA-MB-231 and endothelial proliferation, migration, and capillary-like formation in HUVECs. The mechanisms involved a blockade of cell cycle in G0/G1 phase, increased S phase and decreased of G2/M in MDA-MB-231 and HUVECs, and inhibition of NO production in HUVECs. Altogether, sEV-loaded BRB displayed higher effects than free BRB on different steps leading to its antitumor activity and anti-angiogenic properties in vitro. Thus, sEV formulation may be considered as an innovative approach and promising delivery of BRB to prevent tumorigenesis and angiogenesis. Breast cancer is the most common type of cancer among women worldwide . Despite2.The murine dendritic cell line, JAWS II, was obtained from the American Type Culture Collection . JAWS II was cultured in the presence of alpha-minimum essential media  complemented with 20% fetal bovine serum , 1% penicillin-streptomycin , 4 mM L-glutamine (Lonza), 1 mM sodium pyruvate (Lonza), and 5 ng/mL of Granulocyte-Macrophage Colony Stimulating Factor . The cells were maintained in culture in a humified incubator at 37 °C and 5% CO2 at 37 °C in Dulbecco’s modified Eagle media (Lonza), supplemented with 10% FBS, 2 mM glutamine, 10% penicillin-streptomycin.The MDA-MB-231 human breast cancer cells were purchased from ATCC and cultured at 5% CO2 in endothelial basal cell growth medium, supplemented with 2% FBS, 1% streptomycin-penicillin, 0.1% ascorbic acid, 0.1% human epidermal growth factor, 0.1% heparin, 0.1% VEGF, 0.1% gentamicin-amphotericin B, 0.04% hydrocortisone, 0.4% human bFGF, and 0.1% R3-IGF-1 .Human umbilical vein endothelial cells (HUVECs) were purchased from Lonza and grown at 37 °C and 5% CO6 cells/175 cm2 in complete growth medium and they were deprived in FBS before sEV isolation. Supernatants were subjected to serial centrifugations . Then, the supernatant was centrifuged at 20,000× g for 30 min to remove large EVs, and the resultant supernatant was recentrifuged at 200,000× g  for 2 h to pellet sEVs. Finally, the pellet was washed with PBS recentrifuged at 200,000× g for 2 h and resuspended in 1 mL of PBS. sEVs were stored at −80 °C until further analysis. The amount of proteins associated with sEVs was quantified by Lowry method, using bovine serum albumin as reference.JAWS II cells were seeded at a density of 5 × 10g at 37 °C. sEVs loaded with BRB can be distinguished as a yellowish band in the pellet. BRB-loaded sEVs were collected, washed and resuspended in PBS and stored at −80 °C until use. The amount of BRB loaded and its entrapment efficiency (EE) within sEVs were quantified using a spectrophotometer . BRB absorbance was measured at 346 nm and standard curve with different concentrations (10–100 µg/mL) of free BRB was performed. The percentages of BRB EE and loading were calculated according to the following Equations:BRB  was loaded into sEVs with a simple impregnation method by suspending BRB powder (2 mg/mL) with sEV samples (250 µg/mL) in 1 mL absolute ethanol for 2 h. The mixture was vortexed, sonicated for 5 min and incubated at 37 °C for 2 h under magnetic stirring. Then, it was subjected to ultracentrifugation for 2 h at 200,000× Native sEVs and sEVs loaded with BRB were suspended in 0.9% NaCl and filtered (0.2 µm); then loaded into the sample chamber of NanoSight NS 300  to determine size distribution as previously described . Briefly® Nanoseries DTS 1060 . Formulations were diluted in 0.9% NaCl and filtered (0.2 µm). Results were analyzed as a number weighted distribution curve for the evaluation of the size of sEVs.Size distribution, polydispersity index (PDI), and zeta potential of sEVs and sEV-loaded BRB samples were quantified through dynamic light scattering on a ZetasizerNative sEVs and sEVs loaded with BRB were separated on 4–12% polyacrylamide gel electrophoresis. Then, proteins were transferred to nitrocellulose membranes and incubated with the following antibodies: Alix, TSG101, CD63 , and ß-Actin (Sigma-Aldrich). Each membrane was then incubated with the corresponding secondary antibody: anti-mouse or anti-rabbit (Santa Cruz), and visualized with the Chemi-Smart, 5000 Imaging System using Chemi-capt software .3 MDA-MB-231 and 104 HUVECs were seeded in a 96-well plate and allowed to attach for 24 h. Cells were treated with increasing concentrations (1.25 to 100 µg/mL) of BRB and sEV-loaded BRB for three different times . Concentration of BRB within sEVs was calculated taking in consideration the %EE of BRB. Then, the medium was removed and the dye-binding solution was added. Cells were incubated at 37 °C for 30 min. The fluorescence levels were read on a fluorescent microplate reader  with 485 nm excitation and 530 nm emissions filters. For the subsequent experiments, BRB and sEV-loaded BRB were used at the respective IC50 obtained for each cell type, being 20 µg/mL for free BRB and 5 µg/mL for sEV-loaded BRB for MDA-MB-231, and 40 µg/mL for BRB and 25 µg/mL for sEV-loaded BRB for HUVECs.Cell numbers were evaluated with the Proliferation Assay kit CyQUANT . Briefly, 5 × 105 (MDA-MB-231) and 3 × 105 (HUVECs) cells/well in 6-well plates, respectively, and allowed to attach for 24 h. MDA-MB-231 were treated with 10 µg/mL of native sEVs, 20 µg/mL of BRB, or 5 µg/mL of sEV-loaded BRB, whereas HUVECs were treated with 10 µg/mL of native sEVs, 40 µg/mL of BRB, or 25 µg/mL of sEV-loaded BRB. After 24 h, cells were washed in PBS, before adding 0.2 mL of nuclear isolation medium 0.6% NP40, 50 µg/mL propidium iodide, 100 µg/mL RNase in PBS . Cells were then incubated in the dark for 60 min and analyzed with FC 500 MPL flow cytometer (Beckman Coulter).Cells were seeded at a density of 2 × 106 cells/well in 12-well plates. When the cells reached about 90% of confluence, they were scratched with a sterile pipette tip, and the floating cells and debris were removed by washing with complete medium. MDA-MB-231 were then treated with 10 µg/mL of native sEVs, 20 µg/mL of BRB, and 5 µg/mL of sEV-loaded BRB, while HUVECs were treated with 10 µg/mL of native sEVs, 40 µg/mL of BRB, and 25 µg/mL of sEV-loaded BRB. Images of the plates were taken at T = 0 , at T = 24 h, and at T = 48 h. The area of the scratch was quantified using Image J software. The cell migration was quantified as percentage of the surface recovery.Cells were seeded at 2 × 103 cells/well in 15 µ-slide angiogenesis plate  precoated with Matrigel® (Sigma-Aldrich). Briefly, 10 µL of Matrigel® was added into a 15-well plate and allowed to solidify for 45 min at 37 °C. Then, cells were incubated with medium containing 10% FBS and allowed to adhere for 45 min, after which different treatments (phorbol myristate acetate as a positive control (20 ng/mL), native sEVs (10 µg/mL), BRB (40 µg/mL), and sEV-loaded BRB (25 µg/mL) were added for 24 h. Tube formation was observed with an optical microscope . Quantification was performed by measuring the number of capillary-like structures using Image J software 1.46r .HUVECs were seeded at 25 × 104 cells/well in 8 µ-slide plates (Ibidi). At 70–80% of confluence, cells were stimulated for 24 h and 48 h with 10 µg/mL of native sEVs, 40 µg/mL of BRB, and 25 µg/mL of sEV-loaded BRB. After 24 h or 48 h, cells were washed with PBS and incubated with a fluorescent NO indicator 4,5-diaminofuorescein diacetate  at 37 °C for 30 min. Then, cells were washed twice with cold PBS and fixed in paraformaldehyde at 4% for 20 min at room temperature in the dark. Cells were then washed with PBS and analyzed by confocal microscopy. From each well, a minimum of 4 randomly selected images were captured with a LSM 700 confocal microscope .HUVECs were seeded at 3 × 10p Values < 0.05 were considered significant.Data are expressed as mean values ± SEM. The significance of the differences between groups was determined by ANOVA, followed by Tukey’s or Sidak’s multiple comparisons test. To assess the feasibility of using JAWS II-derived sEVs as drug vehicles, the entrapment efficiency (EE%) of BRB within sEVs was calculated by determining the amount of remaining free BRB. BRB was indeed loaded into sEVs, as shown by the yellow colored of the loaded sEVs with BRB A. Table 50 of sEV-loaded BRB was 4 times lower than the one obtained with free BRB at any time studied . At higher concentrations, sEVs induced a slight reduction of cell proliferation. In MDA-MB-231, free BRB inhibited cell proliferation in a concentration- and time-dependent manner with a maximal effect reached at 40 µg/mL after 24 h, 48 h, and 72 h of treatment A. In the studied .50 of sEV-loaded BRB was significantly lower than free BRB (about 2 times) at 24 h and 48 h. These results suggest that when loaded into sEVs, BRB is more potent than free BRB in decreasing cell proliferation.In HUVECs, free BRB inhibited cell proliferation in a concentration and time-dependent manner with a maximal effect reached at 80 µg/mL after 24 h, and at 60 µg/mL after 48 h and 72 h of treatment B. The ef50 for each cell type.According to the previous results, for the following experiments, free BRB and sEV-loaded BRB were used at their respective IC0/G1 phase associated with an increased in S phase in both cell types. This effect was associated with a decrease in G2/M phase in the two cell types although it was not significant in MDA-MB-231  and sEV-loaded BRB  induced a diminution of GA-MB-231 . We concAs shown on In the absence of treatment, HUVECs restructured and formed capillary-like structures A. PhorboFinally, we examined endogenous NO production. As shown on 1 phase and cell migration in the two studied cell types. Interestingly, sEV-loaded BRB reduce the formation of capillary-like structures following the inhibition of endothelial NO release. These results show that sEVs may be considered as promising vehicles of BRB and as an innovative technology to target cancer and diseases characterized by an excess of angiogenesis.The present study shows that sEVs are potential stable BRB carriers with homogeneous particle size and steady zeta potential. More importantly, when loaded into sEVs, BRB is obviously more potent (up to 4-fold) than free BRB, regarding its ability to exert anti-tumor and antiangiogenic properties. These effects are associated with the increased inhibition of two major steps of tumorigenesis and angiogenesis, cell proliferation and migration. sEV-loaded BRB display anti-proliferative property accompanied by cell cycle arrest in Gkip1, an important regulator of cell migration, and cyclin A expression [One of the main technological challenges for the clinical employment of anticancer drugs is to establish a specific targeting of the bioactive molecules to their final cellular destination, to advance pharmacokinetics and long overdue of these drugs. Regarding BRB delivery, three types of delivery systems, i.e., solid lipid nanoparticles, nanoemulsions, and liposomes, have been described. Although the ratio of entrapment efficiency fluctuated between 78.5% to 97.6% ,15 depenpression . Such meIn this study, free BRB and sEV-loaded BRB decreased capillary-like formation in an experimental model of angiogenesis essential for blood vessel formation in tumors. sEV-loaded BRB was twofold more potent than free BRB. A previous study described that the antiangiogenic activity of BRB is mainly mediated through the inhibition of various pro-inflammatory and pro-angiogenic factors including HIF, VEGF, COX-2, NO, and TNF-α . We founIn summary, sEVs are potential carriers for BRB since they increased the efficacy of free BRB in inhibiting cell proliferation, cell migration, capillary-like formation, and NO release. Thus, sEVs represent a valuable delivery system with a good stability for BRB to treat cancer-related diseases by acting on both anti-tumoral and anti-angiogenic processes.


The significant improvement in solar tracking error compensation capacity with a highly efficient end-side-pumping configuration is meaningful because it reduces the cost of high-precision trackers for solar laser applications.We report a significant numerical improvement in multi-rod laser efficiency, with an enhanced solar tracking error compensation capacity for a heliostat-parabolic system. The solar laser head was composed of a fused silica conical lens and a single conical pump cavity ensuring multiple passes through four 4.55 mm diameter, 15 mm length Nd:YAG rods. 0.76° tracking error width at 10% laser power loss, and total multimode laser power variation of 0.05% at ±0.1° solar tracking error and 0.30% at ±0.2° solar tracking error were numerically calculated, being 1.27, 74.80 and 21.63 times, respectively, more than the experimental record in solar tracking error compensation capacity attained with a dual-rod side-pumping horizontal prototype pumped by the same heliostat-parabolic system. Additionally, the end-side-pumping configuration of the four-rod solar laser-enabled 43.7 W total multimode solar laser power, leading to 24.7 W/m The direct conversion of natural sunlight into laser light may be considered as one of the most promising technologies in renewable energy research, providing a cost-effective production of coherent optical radiation that could lead to numerous environmental and economic benefits ,2. As a 2 solar laser collection efficiency and 2.2% solar-to-laser conversion efficiency were achieved by exciting a 4.0 mm diameter, 25 mm length Nd:YAG rod with a Fresnel lens with 0.64 m2 collection area ) ) . SpectraThe pump flux distributions along five transversal cross-sections of the four Nd:YAG rods are given in ® analysis was then imported into the LASCAD® software to optimize the solar laser output performance. The material data of 1.0 at.% Nd:YAG crystal with a stimulated emission cross-section of 2.8 × 10−19 cm2, a fluorescence lifetime of 230 µs and an absorption/scattering loss of 0.002 cm−1 were set in LASCAD®. The mean absorbed and intensity-weighted solar pump wavelength of 660 nm was included in the analysis [L represents the separation length between the output end face anti-reflection (AR 1064 nm) coatings of each Nd:YAG rod and the respective PR 1064 nm output mirror. It is a key parameter for the efficient extraction of laser power due to the power-dependent thermal lensing of the laser material.The absorbed power in cubic matrix structure from the ZEMAX® and LASCAD® numerical analysis software, respectively, to achieve the highest multimode laser power from each laser rod. For such, a plane-concave laser resonator with a relatively small L was adopted to ensure that the energy of higher-order modes was not wasted by diffraction losses. The multimode laser power from each one of the four rods was numerically studied as a function of the rod diameter (RD), as represented in RL), were optimized for each DR. The maximum multimode solar laser extraction of 12.8 W was calculated for each of the four Nd:YAG laser rods with RD = 3.0 mm and RL = 20.0 mm for an absorbed solar power of 28.8 W, as presented in 2 collection efficiency and 3.1% solar-to-laser power conversion efficiency.All the design parameters of the four-rod solar laser head  and laser resonator were optimized by ZEMAXRD. For comparison, a single-rod scheme was also numerically optimized  considering the same pumping conditions . The tracking error width at 10% laser power loss (TEW10%) was chosen to analyze the solar laser tracking error compensation capacity.The movement of the Earth affects the collected solar radiation on solar systems, and consequently the solar pumping of the laser medium. Solar tracking error shifts the focal spot from its optimal alignment, resulting in less solar laser output power. Solar concentration systems such as point-focusing concentrators need to employ some form of STS with sufficient accuracy to enable higher concentration ratios, radiation flux and thus conversion efficiency . Consequ10% of 0.36°. For the four-rod scheme, the maximum total multimode solar laser power of 51.2 W was calculated with RD = 3.0 mm and RL = 20.0 mm. For this case, around 0.44° TEW10% was numerically calculated. As expected, for RD = 2.5 mm, RL = 22.5 mm, both total multimode solar power and solar tracking error compensation capacity were lower. Large diameter laser rods significantly improved the tracking error compensation capacity. For RD = 4.5 mm and RL = 15.0 mm total multimode solar power of 44.0 W and around 0.76° TEW10% were numerically calculated. Despite the better tracking error compensation capacity calculated for RD = 5.0 mm, RL = 15.0 mm, the total multimode solar power was not only lower but also presented less stability with the maximum of 40.7 W found for a solar tracking error of ±0.2°. For an accurate analysis of the solar laser tracking error compensation capacity of the four-rod scheme, the total multimode laser power as a function of the tracking error was analyzed in more detail for the for RD = 4.50 mm, 4.55 mm and 4.60 mm using a more accurate tracking variation of 0.05°, as shown in p±0.1°) and ±0.2° solar tracking error (∆p±0.2°) were calculated as a measure of power stability.The highest multimode solar laser power of 54.8 W was obtained with the single-rod scheme; however, it showed the lowest solar tracking error compensation capacity, with a TEWp±0.1° = 0.05% was achieved by using four Nd: YAG rods with RD = 4.55 mm. By considering the tracking error range of ±0.20°, the total multimode laser power calculated with RD = 4.60 mm showed better stability (∆p±0.2° = 0.16%), however its variation at ±0.10° tracking error was higher (∆p±0.1° = 0.23%).The slightest power variation of only ∆RD = 3.00 mm, RL = 20 mm and from the four-rod with RD = 4.55 mm, RL = 15 mm, respectively, as a function of the altitude and azimuthal solar tracking errors, ranging from 0.0° to 0.5°.RD = 4.55 mm, RL = 15 mm , the single-rod scheme  numerica = 15 mm . The fouRD = 4.55 mm, RL = 15 mm). RD = 3.80 mm, RL = 15 mm) as a function of the altitude and azimuthal solar tracking errors, ranging from 0.0° to 0.5°.In order to further study the properties of multi-rod end-pumping configurations on solar tracking error compensation capacity, a five-rod scheme was optimized as in the four-rod case above-described  achieved a maximum total laser power of 44.4 W, leading to 25.1 W/m2 collection efficiency and 2.6% solar-to-laser conversion efficiency. Despite slightly outperforming the four-rod scheme (RD = 4.55 mm and RL = 15 mm) in multimode solar laser power, the solar tracking error compensation capacity was found reduced, around 0.66° TEW10%, 0.05% ∆p±0.1° and 1.06% ∆p±0.2° were calculated. Moreover, the addition of one extra Nd:YAG rod, and the respective mechanics and resonator mirror, increases the system complexity and cost. The five-rod scheme , and for the four-rod scheme with the rod’s dimensions that achieved better tracking error compensation capacity  and higher total multimode solar laser power . The results are summarized in The thermal performance of the laser material under solar pumping is of paramount importance in solar-pumped lasers. Therefore, it was also carried out a numerical analysis in order to study the thermal conditions of the Nd:YAG media. In LASCAD2, while by using a four rod-scheme the maximum stress intensity was significantly reduced to 35 N/mm2  and 34 N/mm2 . Note that for both schemes the maximum stress intensity was lower than the stress fracture limit of 200 N/mm2 for Nd:YAG medium [For an end-side pump scheme, the uneven light flux distribution along the crystal is a serious thermal issue, particularly at the upper-end side of the rod. Despite having the highest total multimode solar laser output power, the 5.5 mm diameter, 25 mm length rod of the single-rod scheme has a more severe stress intensity, reaching a maximum value of 116 N/mmG medium .2 collection efficiency and 3.1% solar-to-laser power conversion efficiency, by using four 3.00 mm diameter, 20 mm length Nd:YAG rods. In 2019, Almeida et al. proposed a four-rod scheme able to achieve 59.0 W total multimode solar laser power in the numerical analysis, leading to 19.2 W/m2 collection efficiency and 2.0% solar-to-laser conversion efficiency, which is significantly less efficient than the four-rod scheme here proposed [2, being also significantly less efficient than the four-rod scheme here proposed [2 effective collection area, obtaining 107 W multimode solar laser power in the numerical analysis. Still, a complex multi-mirror resonator system should be implemented in order to simultaneously extract seven solar laser beams from the single solar laser head [10%, ∆p±0.1°, ∆p±0.2°) when compared to the previous numerical analysis on the side-pumping dual-rod scheme [proposed . Moreoveproposed . In thisproposed ,35, beinproposed . The sevser head . The conser head . Furtherd scheme .10%, 0.05% ∆p±0.1° and 0.30% ∆p±0.2° were numerically obtained, being 1.27, 74.80 and 21.63 times, respectively, more than the previous experimental record in solar tracking error compensation capacity obtained by using the dual-rod side-pumping horizontal prototype with the same heliostat-parabolic system [2 collection efficiency and 2.6% solar-to-laser power conversion efficiency, being 1.75 and 1.44 times, respectively, more than those measured with the dual-rod side-pumping prototype [c system . Note thrototype . Collectrototype .It is worth noting that this four-rod solar laser concept can be applied in the pumping of other solar laser materials, such as Cr:Nd:YAG ceramic medium  and Ce:N® and LASCAD® numerical analysis. Substantial improvement in multimode solar laser performance was numerically achieved with this concept. A maximum total multimode solar laser power of 51.2 W was calculated from the four 3.00 mm diameter, 20 mm length rods, leading to 29.0 W/m2 collection efficiency and 3.1% solar-to-laser power conversion efficiency, being 1.58 and 1.41 times, respectively, more than the previous multi-rod experimental records in multimode regime with the NOVA heliostat-parabolic system [10%, 0.05% ∆p±0.1° and 0.30% ∆p±0.2° were numerically calculated, being 1.27, 74.80 and 21.63 times, respectively, more than the previous experimental record in solar tracking error compensation capacity obtained by using the dual-rod side-pumping horizontal prototype with the same heliostat-parabolic system [RD = 4.55 mm, RL = 15 mm) also enabled an increase of 1.75 and 1.44 times in collection and solar-to-laser power conversion efficiencies, respectively, in relation to that from the experimental the dual-rod side-pumping prototype [A four-rod single solar laser head concept was proposed using the full collection area of the NOVA heliostat-parabolic system to concentrate the solar radiation towards the laser head. The second-stage conical fused silica lens further concentrated and distributed the solar radiation onto the four Nd:YAG rods. By redistributing the pumping through the four rods, very effective rod cooling was achieved, avoiding the serious thermal lensing and thermal stress issues associated with classical large diameter single laser media. The single conical pump cavity also ensured higher absorption efficiency since solar pump rays not completely absorbed by one of the rods were further absorbed by the other rods, increasing the path length of the rays through the active media, and hence the amount of energy absorbed. Optimum optical pumping and lasing system design parameters were found through ZEMAXc system . Moreovec system . In addirototype .Simultaneous solar laser emissions from the four rods could provide new solutions for multi-beam solar-powered lasers applications. Four hollow-core photonic crystal fibres can be used to deliver four solar laser beams or may also be combined by the incoherent beam combining technique to achieve higher solar laser power . Further


Our calculations suggest a lower threshold of airway P0.1 measured by the Hamilton C6 on quasi-occlusion than that which has been previously reported.We aimed to identify the threshold for P0.1 in a breath-by-breath manner measured by the Hamilton C6 on quasi-occlusion for high respiratory drive and inspiratory effort. In this prospective observational study, we analyzed the relationships between airway P0.1 on quasi-occlusion and esophageal pressure . We also conducted a linear regression analysis and derived the threshold of airway P0.1 on quasi-occlusion for high respiratory drive and inspiratory effort. We found that airway P0.1 measured on quasi-occlusion had a strong positive correlation with esophageal P0.1 measured on quasi-occlusion and esophageal pressure swing, respectively. Additionally, the P0.1 threshold for high respiratory drive and inspiratory effort were calculated at approximately 1.0 cmH During invasive mechanical ventilation, high respiratory drive may be associated with strong inspiratory effort and negative pleural pressure (high transpulmonary pressure), which may cause patient self-inflicted lung injury (P-SILI) . AlthougP0.1 measures the airway pressure 100 ms after the initiation of an inspiratory effort during an airway occlusion and is frequently adopted as a surrogate index for respiratory drive. This measurement utilizes the coincidence of changes in intrathoracic and intra-airway pressure due to airway occlusion. P0.1 measured with airway occlusion correlates with inspiratory effort and respiratory drive , but becThis is a single-center prospective observational study conducted from March to September 2021. Eligible patients were as follows: (i) adults (aged ≥ 18), (ii) diagnosed with COVID-19 with a positive real-time polymerase chain reaction for SARS-CoV-2, (iii) receiving invasive mechanical ventilation with patient’s spontaneous breathing and (iv) underwent insertion of an esophageal balloon catheter. We measured airway P0.1 on quasi-occlusion (P0.1aw), esophageal P0.1 (P0.1es) and esophageal pressure swing (ΔPes). These measurements were repeated multiple times for each patient and performed via Hamilton C6 ventilators  and NutriVent multifunction naso-gastric catheters . Reliability of the esophageal pressure measurements was confirmed using the dynamic occlusion test. The acceptable range of the ∆Pes/∆Paw ratio during occlusion test was defined as 0.8–1.2 . For eacP-value of less than 0.05 was considered statistically significant. Then, we conducted a linear regression analysis and derived the threshold of P0.1aw for high respiratory drive and inspiratory effort based on this regression model. To account for clustering within patients, a generalized linear model was linked to the analysis . High respiratory drive was defined as two distinct thresholds: P0.1es 3.5 and 4.0 cmH2O [2O [We devised scatter plots between (i) P0.1aw and P0.1es, and (ii) P0.1aw and ∆Pes. We analyzed whether there was a linear relationship between the two variables. Repeated measures correlation was performed because multiple measurements were taken on individual patients . A P-val.0 cmH2O . High inmH2O [2O , 8. Addir = 0.73, P < 0.001). Regression analysis (GEE) revealed that P0.1aw was equivalent to 0.24-fold of P0.1es (P0.1aw = 0.24 P0.1es + 0.04). The P0.1aw thresholds for high respiratory drive were calculated as 0.88 and 1.00 cmH2O from the regression equations. These two values were found for the two different P0.1es thresholds selected to define high respiratory drive of P0.1es 3.5 and 4.0 cmH2O, respectively. Figure We included 15 patients and collected 172 measurements. The characteristics of the patients are shown in Table r = 0.73, P < 0.001). The P0.1aw thresholds for high inspiratory effort were calculated as 0.78 and 1.15 cmH2O using GEE (P0.1aw = 0.074 ∆Pes + 0.04). These two values were found for the two different ∆Pes thresholds selected to define high inspiratory effort of ∆Pes 10 and 15 cmH2O, respectively.The relationship between P0.1aw and ∆Pes is shown in Fig. 2O from both regression equations. Additionally, P0.1aw was equivalent to a quarter of P0.1es.In the present study, we found that breath-by-breath P0.1aw measured by the Hamilton C6 on quasi-occlusion significantly correlated with P0.1es and ∆Pes. The threshold value of P0.1aw for high respiratory drive was calculated at approximately 1.0 cmH2O [2O to be associated with high respiratory drive [A previous study reported the nominal range for P0.1aw measured with airway occlusion to be between 0.5 and 1.5 cmH2O . Anotherry drive . In contIt should be noted that clinical threshold of the P0.1 value for high respiratory drive and inspiratory effort described in the present study is not universally applicable to other ventilators. This is because we exclusively assessed P0.1 measurements using a single ventilator (Hamilton C6). The generalizability would be limited since each manufacturer utilizes its own proprietary software to estimate P0.1. For instance, Getinge group Servo I and U ventilators also provide P0.1 values without airway occlusions. Beloncle et al.  showed t2O or higher may suggest a high respiratory drive and inspiratory effort. P0.1aw may be equivalent to a quarter of P0.1es on Hamilton C6.In patients with COVID-19, the evaluation of respiratory drive and inspiratory effort are critical due to concerns of P-SILI. A P0.1aw value measured on quasi-occlusion of approximately 1.0 cmH


Lactobacillus paracasei HY7015 (HY7015) and Lycopus lucidus Turcz. (LT) extract on hair regrowth and protection. In vitro experiments were conducted to assess the effects of HY7015 and/or LT extract on human follicle dermal papilla cells (HFDPC) of cytoprotective functions such as proliferations, antioxidants, anti-inflammatory, and growth factor expressions. In animal experiments, we investigated hair regrowth rate, hair follicle formation and secretion of growth factors in telogenic C57BL/6 mice. We confirmed the cytoprotective effects of HY7015 and LT through regulations of proliferation, SOD and IL-1β in HFDPC. In mouse experiments, oral administration of HY7015 and LT promoted hair regrowth as well as hair follicle maturation in the dermal skin of C57BL/6 mice, and upregulated VEGF and IGF-1 growth factor levels in mouse serum. In summary, our data demonstrate that ingestions of HY7015 and LT can promote hair regrowth by enhancing cytoprotective effects and expressions of growth factors.Hair loss is a disease that requires accurate diagnosis and type-specific medical treatment. Many hair loss treatments have some side effects, such as hormone-related effects, so there is a need for safe and effective hair loss treatment. In this study, we investigated the effects of  Hair plays an important role in creating a physical barrier against the external environment to protect the scalp . Hair loLactobacillus species can improve intestinal health and intestinal inflammation, and have various other functions, including as antioxidants, immunity enhancements and anti-obesity measures [Lactobacillus species also affect hair growth: Lactobacillus plantarum expresses vascular endothelial growth factor (VEGF) and keratinocyte growth factor (KGF), which contribute to hair growth in vitro and in vivo [Lactobacillus paracasei HY7015 (HY7015), which investigates in this study, promotes secretion of hair growth factors in hair follicle dermal papilla cells and telogenic C57BL/6 mice [Probiotics are viable bacteria that improve the intestinal microbial balance and have beneficial effects on the health of the host ,7. Lactomeasures ,9,10. In in vivo ,12,13. AL/6 mice .Lycopus lucidus Turcz. (LT) is an herb belonging to the family Lamiaceae that has been used for centuries as a traditional Oriental medicine  than untreated controls. Additionally, treatment of HY7015 (135.13 ± 0.29%) and LT  raised relative to untreated controls. Proliferation of HY7015+LT was mostly increased   A. Hence,2O2 only (38.98 ± 13.27%) and was recovered by HY7015+LT (78.28 ± 19.59%). There was a tendency to increase to 52.63 ± 7.71% with HY7015 treatment, but it was not significant. IL-1β protein levels were 21.19 ± 0.58, 54.96 ± 1.94, 52.57 ± 3.41, 70.21 ± 13.14, 50.55 ± 1.65, and 46.96 ± 2.94 pg/mL in control, H2O2 only, MYE, HY7015, LT, and HY7015+LT groups , LT  and HY7015+LT  groups. We also confirmed mRNA expression levels of VEGF by HFDPC after treatment for 24 h. The value for the control group was set as one, with expression in the other groups calculated relative to the control. Expression mRNA levels of VEGF were 2.74-fold, 1.07-fold, 1.20-fold, 3.25-fold in HFDPC treated with MYE, HY7015, LT, and HY7015+LT (p < 0.05).We investigated the effects of HY7015 and/or LT influenced VEGF protein secretions by HFDPC in culture supernatants after treatment for 24 h. VEGF protein levels were 77.38 ± 8.56, 142.36 ± 2.96, 253.00 ± 73.78, 193.37 ± 28.99, and 290.96 ± 29.32 pg/mL in Control, MYE, HY7015, LT, and HY7015+LT groups in HFDPC A. It sigY7015+LT B. There p < 0.001). As shown in p < 0.05).Next, we investigated whether treatment with HY7015 and LT for 24 h affected VEGF protein and mRNA expression of growth factors in NIH3T3 fibroblasts prior to animal experiments. VEGF protein levels were 105.06 ± 0.17, 118.61 ± 1.56, 100.04 ± 8.59, 106.89 ± 1.65, and 116.44 ± 0.96 pg/mL in Control, MYE, HY7015, LT, and HY7015+LT groups C. VEGF sWe used a telogenic mouse model to examine the effects of oral ingestion of HY7015 and LT extract on hair regrowth. In our experiment, we assessed whether oral treatment with HY7015 and LT could promote hair growth for 5 weeks A. To obsp < 0.001) was higher than that in the control group (9.48 ± 4.66%). Further, rates of hair regrowth were increased by 62.20 ± 5.98% (p < 0.001), 67.82 ± 7.76% (p < 0.001), and 74.98 ± 7.23% (p < 0.001) in the HY7015, LT, and HY7015+LT groups, respectively. The HY7015 and LT groups had less hair regrowth than the positive control (MYE) group, whereas levels of growth in the HY7015+LT group were similar to those in the MYE group.As shown in p < 0.001) and 75.25 ± 4.21% (p < 0.001) in the 4th and 5th weeks, respectively, relative to the control group. In the HY7015 group, growth rates were 35.38 ± 7.69% (p < 0.01) and 62.20 ± 5.98% (p < 0.001) in the 4th and 5th weeks, respectively. LT extract led to similar hair regrowth to that observed in the HY7015 group up to the 4th week of ingestion, with rates of 34.55 ± 11.03% (p < 0.05) and 67.82 ± 7.76% (p < 0.001) in the 4th and 5th weeks, respectively. Finally, hair regrowth in the HY7015+LT-treated group showed a similar trend to that in the MYE-treated group over the 5 weeks, with rates of 40.49 ± 10.80% (p < 0.01) and 74.98 ± 7.23% (p < 0.001) in the 4th and 5th weeks, respectively.In addition, we compared the hair regrowth in mice every week for 5 weeks between groups C. There 4 hair follicles/μm2, p < 0.001) was significantly higher than that in the control group (4.13 ± 2.45 × 104 hair follicles/μm2), as were those in the HY7015, LT, and HY7015+LT groups, at 160.57 ± 7.97 × 104 (p < 0.01), 158.71 ± 7.86 × 104 (p < 0.01), and 163.80 ± 19.27 × 104 (p < 0.001) hair follicles/μm2, respectively; these numbers were slightly lower than those in the positive control group (MYE) but indicate a similar follicle-generating effect.Hair regrowth occurs in association with increases in the number of hair follicles and dermal skin thickness. Therefore, by measuring numbers of hair follicles and dermis layer thickness, the effects on hair regrowth can be predicted . To analp < 0.001), 58.51 ± 18.46 (p < 0.001), 54.44 ± 15.81 (p < 0.001), and 63.72 ± 19.33 (p < 0.001) μm, respectively, compared with the untreated group (21.75 ± 7.96 μm). Skin thickness in the HY7015+LT group was comparable to that in the positive control (MYE) group.Measurement of dermis layer thickness was performed using ImageJ software. As shown in These results indicate that administration of HY7015 and LT extracts combined affects hair regrowth by upregulating the number of hair follicles and dermal layer thickness, similarly to the positive control.p < 0.001), HY7015 , LT , and HY7015+LT  were similar, and were significantly higher than those in the untreated group (117.24 ± 3.16 pg/mL). Further, serum levels of IGF-1 were significantly increased in the MYE, HY7015, LT, and HY7015+LT groups, at 533.13 ± 12.31 (p < 0.05), 560.80 ± 14.58 (p < 0.01), 558.67 ± 10.32 (p < 0.01), and 601.52 ± 17.2 (p < 0.001) ng/mL, respectively , 3.95-fold (p < 0.001), and 1.47-fold (p < 0.05) in HFDPC treated with MYE, 5 μg/mL and 50 μg/mL of RA (To confirm whether rosmarinic acid (RA) affected VEGF mRNA expressions in HFDPC, rosmarinic acid was treated for 24 h. mRNA levels of VEGF were 3.89-fold  of HY7015 and component(s) of LT influence cell proliferation, hair regrowth-related gene expression, and hair cycle progression.In total, 100 g of dried natural plant and aerial LT samples  was ground, mixed, and stirred for 6–8 h at 95–98 °C in 1 L distilled water. Extract was concentrated under reduced pressure using a rotary vacuum concentrator , and the concentrated extract was freeze-dried at –80 °C  and then powdered. LT extract was stored at –20 °C until use.Lactobacillus paracasei HY7015 was isolated from Makgeolli, a traditional Korean fermented liquor made from rice, and maintained as frozen stock in MRS broth  containing 20% (v/v) glycerol at –80 °C. HY7015 was incubated in MRS broth at 37 °C for 18 h and then harvested by centrifugation . Cell pellets were washed and resuspended in sterile phosphate-buffered saline (PBS) for in vitro assays. Cells were stored at –20 °C until use. For in vivo assay, fresh cultured HY7015 was prepared by freeze-drying and added to feed. Strains were identified through 16S rRNA sequencing  using universal rRNA gene primers (785F and 907R). 16S rRNA sequencing results were compared to the Genbank database via the Basic Local Alignment Search Tool (BLAST) of the National Center for Biotechnology Institute (NCBI). The sequenced strain was named L. paracasei HY7015 and deposited with KCTC  with accession number KCTC14004BP.Minoxyl-S  was purchased and used as positive control. The main component of Minoxyl-S is medicinal yeast extract (MYE), which supplies essential nutrients for hair growth.2 atmosphere. Cells were seeded into 12-well plates at 4 × 104 cells/well. Attached cells were washed with phosphate-buffered saline, treated by adding Dulbecco’s modified Eagle’s medium (DMEM) with DMSO only (Control) or medicinal yeast extract , HY7015 (1 × 106 CFU/mL), natural plant extracts (50 µg/mL), for 24 h.Human hair follicle dermal papilla cells (HFDPC) were purchased from Cell Applications . Cells were maintained in HFDPC medium and supplement kit (Cell Applications) in T-75 flasks at 37 °C in a humidified 5% CO2 atmosphere. Cryopreserved cells were cultured in DMEM with 10% BCS and 1% P/S in T-75 flasks for 24 h to reach 80% confluence. Cells were seeded into 24-well plates at 1 × 105 cells/well, maintained in DMEM containing 2% BCS and untreated (Control) or medicinal yeast extract , HY7015 (1 × 106 CFU/mL), natural plant extracts (50 μg/mL), for 24 h.Mouse embryonic NIH3T3 fibroblasts were purchased from Cell Applications . Cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplement with 10% heat-inactivated bovine calf serum (BCS) and 1% penicillin-streptomycin (P/S) at 37 °C in a humidified 5% CO6 colony-forming units (CFU)/mL), LT extract (50 µg/mL), a HY7015+LT  were treated for 24 h. After treatment, HFDPC proliferation were evaluated using the cell counting kit-8 (CCK-8) assay . CCK-8 solution (10 µL/well) was added to each well of the plate and incubated for 4 h, 37 °C in a CO2 incubator, and then absorbance was measured at 450 nm .HY7015  Quantikine ELISA kits  were used according to the manufacturer’s instructions. NIH3T3 cells treated with samples for 24 h, and then the supernatant was collected, and protein was measured.To determine superoxide dismutase 1 (SOD1), IL-1β, and VEGF contents in HFDPC, the ab119520 -Superoxide Dismutase 1 Human ELISA kit , Human IL-1β ELISA Set , and Quantikine ELISA human VEGF (DVE00) kits  were used according to the manufacturer’s instructions. For HVegf; Mm00437306_m1). Expression data were normalized using those of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase .HFDPC and NIH3T3 cells treated with HY7015, LT, HY7015+LT, MYE and untreated group (Control) for 24 h were washed twice in PBS, and then total RNA was extracted from washed cells using the easy-spin Total RNA Extraction Kit . Extracted total RNA samples were quantified using NanoDrop 2000  and stored at –20 °C until use for gene expression analysis. Total RNA  was reverse-transcribed into cDNA using an Omniscript Reverse Transcription Kit , with dNTP mix, 10× Buffer RT, Omniscript reverse transcriptase, and RNase free water, and then incubated at 37 °C for 1 h according to the manufacturer’s instructions. cDNA samples were amplified using the QuantStudio 6-Flex Real-time PCR System  and Gene Expression Master Mix (Applied Biosystems). Real-time PCR (RT-PCR) was conducted using mouse-specific TaqMan Gene Expression Assays as follows: vascular endothelial growth factor A  fed with normal control diet or supplemented as follows: 200 mg/kg MYE, HY7015 (1 × 108 CFU/day), 100 mg/kg LT extract (LT), and HY7015 (1 × 108 CFU/day) with 100 mg/kg LT extract (HY7015+LT). After 5 weeks, mice were sacrificed, and blood samples were extracted . Female C57BL/6 mice, aged six weeks, were purchased from KOATECH . All mice were bred at constant temperature (22 ± 1 °C) and humidity (55 ± 10%) with a 12 h light/12 h dark cycle. After 1 week of acclimatization, mice were divided into five groups (xtracted . Blood w4/µm2).Dorsal skin tissue samples from each mouse were fixed in 10% formalin solution, and then sectioned and stained with hematoxylin and eosin (H&E) on slides by T&P Bio . Dorsal skin thickness was measured under a Zeiss Axiovert 200M microscope  at 100× magnification. Images of dorsal skin tissue were analyzed using ImageJ software  to measure the number of hair follicles per unit area  and Mouse/Rat IGF-I/IGF-1 (MG100) Quantikine enzyme-linked immunosorbent assay (ELISA) kits , according to the manufacturer’s instructions.2O (7:3), followed by ultrasonication at room temperature for 30 min. After centrifugation, it was transferred to a 25 mL volumetric flask and made up to volume with 70% methanol. Then, it was filtered through 0.22 μm syringe filter and analyzed. Rosmarinic acid  was used as the standard. 330 nm was selected as the wavelength for UV detection. A CAPCELL PAK C18  column was used and the injection volume was 10 μL. Elution was performed at a flow rate of 1.0-mL/min at 25 °C. Two mobile phases A and B were used. Mobile phase A was 0.1% (v/v) formic acid in water and mobile phase B was acetonitrile. The gradient used was: 0–11 min, gradient from 25% to 95% B; 11–15 min, gradient from 95% to 25% B.The content of rosmarinic acid was measured by HPLC-DAD referring to the previous methods ,61. Apprt test. p < 0.05 was considered significant.All data are presented as the mean ± standard error (SE) of independent experiments and were analyzed using GraphPad Prism . Comparisons between groups were performed using the Student’s In conclusion, our data demonstrate that treatment of HY7015 and LT improves hair regrowth rate by upregulating hair follicle formation, and secretion of growth factors, such as VEGF and IGF-1, in animal experiments. These improvements were mediated in part by proliferation effect of HY7015 and LT. HY7015 and LT also showed cytoprotective effects by regulating SOD1 and IL-1β expressions in oxidative stress-induced HFDPC. These results suggest that this hair regrowth effect is related to the rosmarinic acid content in LT and synergistic effects of HY7015. However, our research focused on hair growth rather than detailed mechanisms. Therefore, further studies are needed to clarify the mechanisms.


FucoPol is an acylated polysaccharide with demonstrated valuable functional properties that include a shear thinning fluid behaviour, a film-forming capacity, and an emulsion forming and stabilizing capacity. In this study, the different conditions  for alkaline treatment were investigated to deacylate FucoPol. Complete deacetylation and desuccinylation was achieved with 0.02 M NaOH, at 60 °C for 15 min, with no significant impact on the biopolymer’s sugar composition, pyruvate content, and molecular mass distribution. FucoPol depyruvylation by acid hydrolysis was attempted, but it resulted in a very low polymer recovery. The effect of the ionic strength, pH, and temperature on the deacetylated/desuccinylated polysaccharide, d-FucoPol, was evaluated, as well as its emulsion and film-forming capacity. d-FucoPol aqueous solutions maintained the shear thinning behaviour characteristic of FucoPol, but the apparent viscosity decreased significantly. Moreover, contrary to FucoPol, whose solutions were not affected by the media’s ionic strength, the d-FucoPol solutions had a significantly higher apparent viscosity for a higher ionic strength. On the other hand, the d-FucoPol solutions were not affected by the pH in the range of 3.6–11.5, while FucoPol had a decreased viscosity for acidic pH values and for a pH above 10.5. Although d-FucoPol displayed an emulsification activity for olive oil similar to that of FucoPol (98 ± 0%) for an oil-to-water ratio of 2:3, the emulsions were less viscous. The d-FucoPol films were flexible, with a higher Young′s modulus (798 ± 152 MPa), a stress at the break (22.5 ± 2.5 MPa), and an elongation at the break (9.3 ± 0.7%) than FucoPol . Given these findings, d-FucoPol arises as a promising novel biopolymer, with distinctive properties that may render it useful for utilization as a suspending or emulsifier agent, and as a barrier in coatings and packaging films. Enterobacter A47 (DSM 23139) composed of fucose, galactose, glucose, and glucuronic acid (2.0:1.9:0.9:0.5 molar ratio) with a high molecular weight (1.7–5.8 × 106 Da) and an intrinsic viscosity value of 8.9 dL/g . I,6. I2,6]η], of d-FucoPol was 2.45 ± 0.33 dL/g. This value is lower than that previously reported for FucoPol (11.0 dL/g) [η]  . In addiL/g) [η] .ƞ0 values are associated with higher estimated τ values, representing higher relaxation times. The highest η0 value was observed for the 3.0 wt% d-FucoPol solution (29.5 ± 0.09 Pa.s)  , which w0η, which increased from 26.1 ± 0.27 Pa.s for the 0.1 M solution to 86.9 ± 0.80 Pa.s for the 3.0 M solution, concomitant with a higher τ (from 0.62 ± 0.02 to 1.01 ± 0.04 s) and m (from 0.72 ± 0.01 to 0.76 ± 0.01). These results show that the d-FucoPol solutions’ viscosity was highly impacted by the ionic strength. This behaviour may result from the polymer aggregation probably induced by the higher salinity of the solution [As shown in solution . This besolution .For all the tested ionic strength values, at low frequencies, the loss modulus (G″) was higher than the storage modulus (G′) A, denotiη0 (40.1–44.7 Pa.s), τ (0.71–0.80 s), and m (0.73–0.78) values  and low pH (pH ≤ 3) values , d-FucoP) values . PossiblFor all the pH values tested, at low frequencies, the loss modulus (G″) was higher than the storage modulus (G′) B, implyiη0 decreased from 79.1 ± 0.46 to 2.66 ± 0.04 Pa.s, as the temperature increased from 10 to 80 °C. This effect was previously described by several authors, showing that high temperatures do not promote interactions between polymer molecules, which lowered the reported viscosity values [η0 and τ decreased with the increasing temperatures. The relaxation time (τ) decreased from 1.26 ± 0.02 to 0.06 ± 0.01 as the temperature increased from 10 to 85 °C. This fact indicates that less time is needed to form new interactions between polymer molecules at higher temperatures. Consequently, the transition from the Newtonian plateau to the shear-thinning regime is less evident and moves to the higher shear rate values [Similarly to the FucoPol solutions , increasy values ,44,45. Ie values .As expected, the G″ > G′ at low frequencies, indicating a liquid-like behaviour C. Moreov24 = 98%) to FucoPol (E24 = 81–98%) [The ability of d-FucoPol to form and stabilize emulsions was assessed and compared with that of FucoPol, which has been reported as a good emulsifier for several oils ,2,4. The 81–98%)  under si 81–98%) ,48. Addi 81–98%) ,50,51.η0 value observed for the d-FucoPol emulsion (13.92 ± 2.36 Pa.s) was lower than that of the FucoPol emulsion (46.5 ± 5.3 Pa.s) (Millettia speciosa Champ [As shown in .3 Pa.s) , thus susa Champ ,54. In fsa Champ . This alsa Champ .Nevertheless, the d-FucoPol (1.0 wt%) stabilized emulsion still displayed a significantly higher viscosity (13.92 ± 2.36 Pa.s) than the 1.5 wt% polymer’s aqueous solution (1.61 ± 0.01 Pa.s) . This faInterestingly, the d-FucoPol stabilized emulsion presented the G′ higher than the G″ across the whole frequency range (0.01–10 Hz) b and didThe d-FucoPol formed slightly opaque flexible films with a brownish tone a and a tThe d-FucoPol films showed a higher mechanical resistance  comparedThe acetyl and succinyl substituent groups were removed from FucoPol’s molecule by an alkaline treatment under the mild conditions of the temperature and alkali concentration. Although the deacetylated/desuccinylated polysaccharide, d-FucoPol, maintained the same sugar composition and molecular mass distribution as the native FucoPol, there was a significant reduction in the d-FucoPol aqueous solutions’ apparent viscosity, which was also significantly affected by the ionic strength of the solution and the temperature. Nevertheless, the solutions’ viscosity was not impacted by the pH, contrarily to the native FucoPol solutions. d-FucoPol kept the ability to form and stabilize the emulsions, although the resulting emulsions had a lower apparent viscosity. Additionally, the film-forming capacity of the FucoPol was also kept, presenting a higher mechanical resistance than the native FucoPol films. Given these features, d-FucoPol presents itself as a promising FucoPol derivative with distinctive properties that render it valuable for several applications, including in packaging, cosmetics, and in other areas as a suspending, thickening, or emulsifying agent.


Clopidogrel is widely used to prevent and treat cardiovascular atherosclerosis and thrombosis. However, disturbance in the expression and activity of liver cytochrome metabolic enzymes significantly changes clopidogrel efficacy. Therefore, the effect of chronic unpredictable mild stress (CUMS)-induced depression on the expression of liver cytochrome metabolic enzymes and clopidogrel pharmacokinetics in rats were explored.Nine different CUMSs were selected to establish a rat model of depression. Open field experiment and sucrose preference test were applied to explore the depressive behaviors. The concentration of serotonin in the cortex of depressed rats was determined using enzyme linked immunosorbent assay (ELISA). All rats were given 10 mg/kg clopidogrel orally after 12 weeks, and blood samples were collected at different time points. The clopidogrel concentration and CYP2C19/ CYP2C9 activity in rat liver microsomes were assayed by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). The rat liver drug enzymes expression was determined by Real-Time Quantitative Reverse Transcription PCR (RT-qPCR).∗∗p < 0.01). The area under the curve of clopidogrel pharmacokinetics decreased by 33.13% (∗p < 0.05) in the depression rats, while distribution volume and clearance increased significantly (∗∗p < 0.01). The half-time and distribution volume did not significantly differ. The CYP2C19 and CYP2C9 activity of liver microsomes in the CUMS-induced depression group were significantly higher than that in the control group (∗∗p < 0.01). CYP2C11 and CYP1A2 mRNA expression up-regulated approximately 1.3 - fold in the depressed rat livers compared with that in the control, whereas that of CYP2C13 was down-regulated by 27.43% (∗∗p < 0.01).  CYP3A1 and CYP2C12 expression were slightly up-regulated, and that of CES1 did not change.Open field experiment and sucrose preference test indicated the successful construction of the CUMS-induced depression model. The concentration of serotonin in the cortex of depressed rats decreased by 42.56% (CYP450 activity and expression in depressed rat livers might contribute to the disturbance of clopidogrel pharmacokinetics.These results indicated that CUMS-induced depression altered clopidogrel pharmacokinetics, and the change in  Clopidogrel is a P2Y12 antiplatelet inhibitor that is indispensable in treating and preventing atherosclerosis and thrombosis  and can Several clinical studies have shown that the rate of psychological stress response in cardiovascular disease patients is high during the lifetime, especially in female . Stress It is well known that clopidogrel is safe in healthy volunteers, but it can induce recurrence of gastric ulcers in high-risk patients . Clinicain vivo  was purchased from molnova Co., Ltd. , and ticlopidine  was purchased from Sigma Trading Co., Ltd. Clopidogrel hydrogen sulphate tablets were obtained from Sanofi Pharmaceuticals Co., Ltd . Substrates of S-mephenytoin, 4-hydroxymephenytoin, tolbutamide, and 4-hydroxytolbutamide substrates (≥95%) were purchased from Sinco Pharmachem . Diazepam  was purchased from the China Institute of Food and Drug (China). High-performance liquid chromatography (HPLC)-grade methanol and ethyl acetate were purchased from Merck . 5-Hydroxytryptamine (5-HT) ELISA Kit of rat was purchased from CUSABIO . TRIzol Reagent was purchased from Sigma-Aldrich Trading Co., Ltd. . The PrimeScript TM RT Master Mix (No:RR047A) and SYBR Premix Ex Taq™ (No: RR420A) were purchased from TaKaRa Bio Inc .Female SPF SD rats  were purchased from Hunan SLAC Laboratory Animal Co., Ltd. . The rats were kept at the Laboratory Animal Center, Hunan Provincial People’s Hospital, Changsha, China , in a well-ventilated and clean laboratory environment . All rats had free access to obtain food and water for 1 week. Then rats were randomly divided into two groups (each group comprising nine rats): control and depression groups.ad libitum in a cage alone for 12 weeks. The stressors included restraint , hot water swimming , cold water swimming , tail clipping , cage stilting , horizontal shaking (10 min), damp padding (24 h), noise interference (10 min), and day/night inversion (24 h). Each stressor was randomly used 8–9 times  for 10 min.Before and after the model establishment, the open-field behavior of each rat was analyzed using a SuperMaze Video Analysis System . The apparatus comprised an opaque box (100 cm × 100 cm × 50 cm). The open-field area was divided into 33 × 33 cmSD rats were euthanized by cervical dislocation after completing the pharmacokinetic experiment. Brain samples were collected from each rat and frozen in liquid nitrogen as described previously method . When 5-All rats were fasted overnight but with free access to water, then administered 10mg/kg of clopidogrel orally . Blood s+) mode, capillary at 5,500 V, an ion-spray gas temperature of 500 °C, a gas flow rate of 11 L/min, and nebulizer at 10 psi. The parameters of clopidogrel and Sit were as follows: fragmentary voltages of 70 V and 66 V, collision energy of 20 units and 23 units, respectively. The multiple-reaction monitoring mode was selected to quantify clopidogrel and Sit, for which the precursor-to-product ion transitions were 322.1 → 212.0 and 264.3 → 154.1, respectively. The MassHunter Workstation software  was used to collect and process data.The clopidogrel concentration in rats’ plasma were was determined using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method . A totalThe control and CUMS-induced depression rats were fasted for 12 h and killed by cervical dislocation before the liver was excised, rinsed with ice-cold normal saline , weighed to 1 g, and homogenized with 4.0 mL in a 0.05 M Tris-HCl buffer (pH 7.4). The homogenate was centrifuged at 10,000 × g at 4 °C for 20 min, and the supernatant was further centrifuged at 105,000 × g at 4 °C for 60 min. Afterward, the helvus sediment was reconstituted with 1.0 mL Tris-HCl buffer and stored at −80 °C for further analysis. The total protein concentration of liver microsomal enzymes was determined using a Bicinchoninic Acid Protein Assay Kit. In addition, the CYP450 activity of the liver microsomes was determined using a cocktail of probe drugs in which the activities of CYP enzymes were assessed by determining probe drug production. Tolbutamide (TOL) or mephenytoin (MPT) were metabolized to 4-OH-TOL or 4-OH-MPT by CYP2C9 or CYP2C19, respectively, and these products were determined by HPLC-MS .−1) were added, subjected to vortex oscillation for 2 min, and centrifuged at 3,500 × g at 4 °C for 10 min. Thereafter, the supernatant was sucked into a 1.5 mL brown centrifuge tube and placed in a vacuum drying oven  for evaporation. Next, the supernatant was redissolved with 200 µL methanol, vortex oscillated for 1 min, centrifuged at 14,000 × g for 2 min, and 5 µL of the supernatant were sucked into an injection bottle for analysis. The whole experiment was conducted in darkness.The liver microsome working solution was thawed at 4 °C, then absorbed 100 µL, probe substrate 100 µL, phosphate buffer 250 µL in 5 mL brown centrifuge tube, vortex oscillation for 1 min, incubation in 37 °C water bath for 5 min, adding NADPH 50 µL to start the reaction, and incubation in 37 °C water bath for 20 min. After the reaction, the incubation system was placed in an ice bath, then 1.4 mL of ethyl glacial acetate and 20 µL internal standard . The parameters of 4-OH-TOL and 4-OH-MPT included a fragmentary voltage of 100 V and collision energies of 24 and 23 units, respectively. The monitoring ions were set as m/z 287.3 →170.8 for 4-OH-TOL and 235.2 →150.2 for 4-OH-MPT.Chromatographic separation was achieved using an ACE Excel 5 SuperC18 column . The mobile phase contained 0.1% formic acid water (A) - methanol (B) with a flow rate of 0.3 mL/min. The gradient elution included 50% A (0–2.4 min), 5% A (2.4–3.2 min), and 50% A (3.2–4.5 min). The optimized MS conditions were as follows: ESIβ-actin was used as an external standard. The primer sequences used were shown in 2−ΔΔCt method . The RNA concentration was determined using a NanoDrop Spectrophotometer  at an absorbance of 260 nm. Total RNA was transcribed to cDNA using a PrimeScript RT reagent Kit with gDNA Eraser (Takara Bio Inc.), and RT-qPCR was performed using an ABI 7500 Real-time PCR system with SYBR Green Quantitative PCR Master Mix. To normalize the mRNA expression, the housekeeping gene t method .t-Test. Data are expressed as the mean ± standard deviation (SD). ∗p < 0.05 or < 0.01 was considered statistically significant.Statistical and pharmacokinetics parameter analyses were performed using SPSS 22.0  and DAS 3.0 software . Differences between the two groups were analyzed using an independent samples ∗∗p <0.01, ∗∗p <0.01) in the depression group, respectively. However, the freezing time increased from 271.39 ± 50.74 to 523.53 ± 31.50s in the depression group. There were no change for these in the control group (p > 0.05). After the model establishment, the cortex 5-HT levels of rats were also reduced significantly (4.52 ± 0.99 vs. 2.59 ± 0.31 ng/mL) compared with the two groups.The sucrose preference test, behavior scores of open-field test, and 5-HT levels were shown in 2>0.99) by weight of 1/x2 over the concentration range of 0.35–49.93 ng/mL. The lowest limit of quantitation (LLOQ) was 0.35 ng/mL, and the matrix effects were approximately 96%. Intra-day accuracy and precision were 99.79%-106.46% and 2.18%−6.34%, respectively; for inter-day, these were 97.46%-106.25% and 2.54%−8.10%, respectively (0−∞), peak time (Tmax), peak concentration (Cmax), and mean residence time (MRTt0−) in the depression group decreased by 33.13% (from 82.56 ± 22.22 to 55.21 ± 21.91), 10.52% (from 0.38 ± 0.24 to 0.34 ± 0.17), 17.71% (from 12.42 ± 7.17 to 10.22 ± 4.63) and 11.89% (from 11.27 ± 3.03 to 9.93 ± 2.09), respectively, but only the area under curve (AUC0−∞) had a notable significance (∗p < 0.05). The distribution volume  and clearance  increased significantly (∗p < 0.01). No statistically significant difference was observed in half-time (T1/2) between the two groups.Clopidogrel and IS retention times were 2.74 min and 1.21 min, respectively . No signectively . The conectively . The are∗∗∗p < 0.001). The CYP2C9 activity also varied significantly, increasing from 0.948 ± 0.112 to 1.528 ± 0.151 ng/(min mg protein) (∗∗p < 0.01).The retention times of 4-OH-TOL and 4-OH-MPT were 3.38 min and 1.81 min, respectively. The CYP2C19 and CYP2C9 activities of liver microsomes in the depression group were significantly increased than those in the control group . The actCYP450 in rat livers was analyzed using RT-qPCR. The relative expression of CYP2C11 mRNA increased by 22.60% in CUMS group, while that of CYP2C13 mRNA decreased by 27.43%. CYP1A2 increased by 34.39% (∗∗p = 0.01) in CUMS group compared with control group. CYP3A1 and CYP2C12 mRNA relative expression increased slightly between the CUMS group, but did not reach the significance. CES1 mRNA relative expression did not change in CUMS group.The expression of etc. Compared with previous studies, these stresses can make experimental animals unable to predict the occurrence of stress, but the more intense stress model does not meet the reason for people’s depression  and AUCt)(0− of clopidogrel decreased significantly (*p < 0.05), while Vz/F and CLz/F increased significantly (∗∗p < 0.01). Disturbance of these parameters indicated that CUMS induced depression in SD female rats changed the pharmacokinetics of clopidogrel in vivo. The plasma concentration of clopidogrel in the depression group was lower than that in the control group, indicating that clopidogrel metabolism in the depression group was faster than that in the control group. The plasma AUC of rats in the depression group decreased significantly, indicating that the absorption rate decreased. The average residence time in vivo also decreased significantly, and the clearance rate accelerated, indicating that the elimination rate of clopidogrel accelerated. In conclusion, the absorption of clopidogrel decreased, metabolism accelerated, distribution increased, and half-life did not change in the depression group. Previous studies found that CUMS-induced depressive SD rats could alter the pharmacokinetics of repaglinide and sitagliptin, although the specific drug metabolic parameters were different (Clopidogrel is an oral irreversible P2Y12 receptor antagonist which inhibits platelet aggregation and is widely used for patients with acute coronary syndromes or after percutaneous coronary intervention . Previouifferent .Clopidogrel is a prodrug, and needs to be converted into active metabolites through CYP450 enzymes in the liver. The main metabolic enzymes involved in the metabolism of clopidogrel in humans are CYP2C19, CYP2C9, CYP1A2 and CYP3A4 . CYP2C19CYP2C11 and CYP1A2 mRNA expression increased approximately 1.3-fold in the CUMS group, while that of CYP2C13 mRNA reduced by 27.43%. CYP3A1 and CYP2C12 mRNA relative expression increased slightly between two groups, and CES1 mRNA relative expression did not change in depressed rat livers. Previous studies found that the activity of CYP450 enzymes in the liver microsomes of depressed GK rats induced by CUMS changed significantly (In the present study, rat liver microsomes were collected and incubated with substrates. The results showed that the activities of CYP2C19 and CYP2C9 increased significantly in the CUMS group compared with those in the control group, especially that of CYP2C19. A recent study also showed that stress altered CYP2C19 activity . Since tficantly . In brieCYP2C12 in these rats. The perturbation of clopidogrel pharmacokinetic parameters in depressed rats was significantly influenced by CYP450 enzyme activity and expression. Therefore, the disturbance of CYP450 enzyme expression and activity in the liver of CUMS rats has a certain reference significance for applying clopidogrel in depressed female patients. However, the current study has some limitations. First, there were certain individual differences in rats in their response to chronic unpredictable stress. Second, administering the antiplatelet drug clopidogrel in rats resulted in death during blood collection.In clinic, female patients with ACS are prone to comorbid psychological disorders, such as depression . HoweverIn conclusion, to the best of our knowledge, the present study is the first time to confirm that CUMS-induced depression perturbed the liver CYP450 enzyme expression and activity, disturbing the pharmacokinetics of clopidogrel in female SD rats. This phenomenon can be extrapolated to depressed patients. Therefore, it is recommended to analyze the concentration of clopidogrel in female patients comorbid with depression to prevent the change of drug exposure that result in adverse bleeding events.10.7717/peerj.14111/supp-1Supplemental Information 1The raw data for sucrose preference, average moving speed, total moving distance, freezing time, times entering the central area, cortex 5-HT level, plasma clopidogrel level, pharmacokinetic parameters of clopidogrel in rats’ plasma , CYP450 activity and expression in rat livers  and 3Click here for additional data file.10.7717/peerj.14111/supp-2Supplemental Information 2Click here for additional data file.