ALT), which can also be evaluated without snorkel , has been proposed; however, these methods have never been compared. Thus, this study (i) analyzed the effect of maturation stage on ACFS during maximal 400 m swimming (Part I), and (ii) compared AOD with ACALT and ACFS, determined in a maximal 400 m effort (Part II). In Part I, 34 swimmers were divided into three groups, according to maturation stages , and subjected to a maximal 400 m free-swimming to determine ACFS. In Part II, six swimmers were subjected to one 400 m maximal effort, and four submaximal constant efforts. The AOD was determined by the difference between the estimated demand and accumulated oxygen during the entire effort. The ACALT and ACFS (for Part I as well) was assumed as the sum of lactic and alactic anaerobic contributions. ACFS was higher in pubertal (3.8 ± 1.1 L) than early (2.1 ± 0.9 L) and middle pubertal group (2.4 ± 1.1 L). No difference was observed among absolute AOD (3.2 ± 1.3 L), ACALT (3.2 ± 1.5 L), and ACFS (4.0 ± 0.9 L) . Relative AOD (51.8 ± 12.2 mL·kg−1), ACALT (50.5 ± 14.3 mL·kg−1), and ACFS (65.2 ± 8.8 mL·kg−1) presented main effect , without posthoc difference. The bias of AOD vs. ACALT was 0.04 L, and AOD vs. ACFS was −0.74 L. The limits of agreement between AOD and ACALT were +0.9 L and −0.8 L, and between AOD and ACFS were +0.7 L and −2.7 L. It can be concluded that ACFS determination is a feasible tool to determine anaerobic contribution in young swimmers, and it changes during maturation stages. Also, ACFS might be useful to measure anaerobic contribution in swimmers, especially because it allows greater speeds.Evaluation of anaerobic contribution is important under swimming settings (training and modification through ages), therefore, it is expected to change during maturation. The accumulated oxygen deficit (AOD) method can be used to determine the contribution of nonoxidative energy during swimming; however, it requires several days of evaluation. An alternative method to estimate anaerobic contribution evaluation (AC This method considers the sum of the fast component of excess oxygen consumption postexercise , without differences in relative AnaALA . Pubertal group presented greater absolute AnaLAC than the other groups , while no differences were found between early-pubertal and middle-pubertal.LAC than early-pubertal . ACFS were greater in pubertal group than the other groups , and no differences were found between early-pubertal and middle-pubertal ].Pubertal showed greater relative Anapubertal . No diff−1) and VO2Peak was 3.07 L·min−1. The VO2-speed relationship presented values of angular, linear, and determination coefficients of 4.00 ± 1.22 (L·min−1)·(m·s−1)−1, 1.82 ± 1.06 L·min−1, and 0.94 ± 0.02, respectively. ALT, and ACFS.Speed ranged between 64.42 ± 0.93 and 80.30 ± 6.85% of 400 m performance in submaximal efforts. The mean time for 400 m was 330.59 ± 13.20 s (mean speed = 1.20 ± 0.04 m·s2) and ACALT (3.2 ± 1.5 LO2), and ACFS (4.0 ± 0.9 LO2) determined in the 400 m maximal effort . The relative AOD (51.8 ± 12.2 mL · kg−1), ACALT (50.5 ± 14.3 mL · kg−1), and ACFS (65.2 ± 8.8 mL · kg−1) values presented main effect ; however, post-hoc analysis did not indicate any differences among values . AOD was very strongly correlated with ACALT , and strongly correlated with ACFS .The agreement analysis between methods are shown in FS changes within maturation stages, and (ii) to compare conventional AOD with an alternative method to estimate anaerobic contribution using a single effort with and without snorkel . The main findings were that ACFS modifies within maturation stages, and the preliminary validation study did not show differences among AOD, ACALT, and ACFS, and that they were strongly correlated ; however, agreement analysis between AOD and ACFS showed greater lower limits (−2.26 L).The aims of the present study were (i) to confirm whether ACFS was sensitive to maturation stages in swimmers, with the pubertal group presenting significantly higher absolute ACFS than middle-pubertal and early-pubertal groups. The pubertal and middle-pubertal groups presented greater muscle mass than early-pubertal; however, the difference between middle-pubertal and pubertal was of ≈7 kg on average, which can have practical influence on performance, besides the absence of statistical differences. Thus, expressing ACFS values relative to total body mass and muscle mass is extremely important when comparing the anaerobic indices of swimmers of different biological ages.In accordance with our hypothesis, ACLAC and AnaALA, the latter only tended to be greater (p = 0.07) in pubertal than in the other groups. Thus, for swimmers, AnaLAC is the main variable differing between maturation stages. Therefore, the difference in absolute ACFS may be related to AnaLAC since no differences were found in AnaALA between maturation stages. Furthermore, no differences were detected in relative ACFS between maturation stages, indicating a possible influence of muscle mass on ACFS.These results agree with the findings of Kaczor et al. , which hFS is practical because it enable swimmers to swim freely; however, it was important to compare it with currently used anaerobic contribution determination methods .Due to its importance in swimming context, a feasible tool to evaluate anaerobic contribution would be important, and ACFS would be an even more interesting and applicable tool to evaluate the anaerobic contribution of swimmers without interfering on technique and speed.The measurement of energy cost in swimming has received great attention on swimming, since it is important for performance . These results were confirmed in breaststroke for 200 and 100 m   affects the precision and validity of the AOD model . Thus, although linear regression is the major concern for AOD calculation, this method is still considered the most acceptable for anaerobic evaluation  and a mean error very close to zero, which agrees with the present findings. Therefore, the difficulties implemented by the need for submaximal exercises to estimate VO2-speed relationship are overcome in the alternative method. Finally, determination of ACALT allows the calculation of AnaLAT and AnaALA separately, enabling the investigation of different training models on these two metabolisms.This is the first study to compare conventional AOD with ACi et al.  comparedALT decreases the number of evaluations and allows the evaluation of AnaLAT and AnaALA, it was still calculated with swimmers using snorkel during swimming. Besides changes in mechanics during swimming, the apparatus reduces the speed of the swimmers (330.5 ± 13.2 s vs. 303.6 ± 10.8 s), which might limit anaerobic contribution. Another important limitation refers to the impossibility of swimmers performing the turns and the underwater dolphin kick, a technique that has been commonly observed in swimming events. The use of snorkel also limits the use of “filipina” during breaststroke swimming, in addition to being uncomfortable for swimmers, limiting its use in practical settings.Even though ACFS with ACALT and AOD; however, a tendency was detected in absolute values and an effect was found for relative anaerobic contribution . This might have occurred due to the reduced sample size. It is important to note that the limits of agreement between AOD and ACFS highlighted a lower limit of 2.26 L. Four out of six presented significantly greater ACFS than AOD (mean difference of 1.24 L). Thus, even though no statistical differences were observed, free swimming anaerobic contribution evaluation (ACFS) might be recommended because it allows the athletes to perform in greater intensity, which is especially important since swimmers did not reach exhaustion during swimming.We have shown no differences between ACPart I which might have influenced the comparison between maturation stages, and the small sample size in Part II. It would be desirable to confirm these results with a larger sample size. Finally, for the AnaALA determination, 5 min of recovery was used. Bertuzzi et al.  were evaluated in i et al.  have obsn et al.  and Ozyen et al.  equationFS is sensitive to maturation stages, and no differences were detected with AOD and ACALT. Therefore, ACFS might be useful to estimate anaerobic contribution in swimmers, facilitating its determination in practical settings, because swimmers are able to swim freely, which increases the speed of swimming.Collectively, it can be concluded that the ACThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Human Research Ethics Committee - UNESP - Rio Claro/SP; Ethics Committee Number: 1413/2013. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.EC, MS, TA, CK-F, and RG collected the data. EC, CK-F, FM-G, and MP wrote the manuscript and delineated the study. All authors contributed to the article and approved the submitted version.This study was supported by Grant 2013/15322-31, São Paulo Research Foundation (FAPESP).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


P = .287); overall response rates were 20% and 27%, respectively. Conversely, median overall survival (OS) was 141.9 weeks and 83.3 weeks . Overall, 61% and 55% of patients in the perifosine and placebo arms reported grade 3/4 adverse events, including thrombocytopenia (26% vs 14%), anemia (7% vs 8%), hyponatremia (6% vs 8%), and pneumonia (9% vs 3%). These findings demonstrate no PFS benefit from the addition of perifosine to bortezomib‐dexamethasone in this study of relapsed/refractory MM, but comparable safety and OS.Perifosine, an investigational, oral, synthetic alkylphospholipid, inhibits signal transduction pathways of relevance in multiple myeloma (MM) including PI3K/Akt. Perifosine demonstrated anti‐MM activity in preclinical studies and encouraging early‐phase clinical activity in combination with bortezomib. A randomized, double‐blind, placebo‐controlled phase 3 study was conducted to evaluate addition of perifosine to bortezomib‐dexamethasone in MM patients with one to four prior therapies who had relapsed following previous bortezomib‐based therapy. The primary endpoint was progression‐free survival (PFS). The study was discontinued at planned interim analysis, with 135 patients enrolled. Median PFS was 22.7 weeks  in the perifosine arm and 39.0 weeks (18.3–50.1) in the placebo arm (hazard ratio 1.269 [0.817–1.969];  TheConsequently, perifosine was investigated clinically in MM and demonstrated encouraging activity in patients with MM when combined with bortezomib and lenalidomide , 21. In 22.19/L, absolute neutrophil count ≥0.5 × 109/L, hemoglobin ≥8.0 g/dL), renal (creatinine ≤3.0 mg/dL), and hepatic  function. Patients were excluded if they had previously received treatment with perifosine or an experimental proteasome inhibitor.Between March 2010 and March 2013, relapsed and/or refractory patients aged ≥18 years with a confirmed diagnosis of MM, who had measurable disease (serum M‐protein > 0.5 g/dL and/or > 200 mg/24‐h urinary M‐protein excretion) and an Eastern Cooperative Oncology Group performance status of 0–2, were enrolled at 48 study sites in the United States, Israel, Spain, South Korea, Canada, Russia, the Czech Republic, France, Ireland, and Slovakia. Patients were required to have received 1–4 prior anti‐myeloma therapies, including at least two 21‐day cycles of either single‐agent bortezomib or bortezomib in combination with other agents, and patients had to have relapsed following their prior bortezomib‐based therapy, with progression occurring >60 days after last dose . Patients could have relapsed following or have been refractory to other non‐bortezomib‐based therapies. Patients also required adequate hematological  and disease status after last prior therapy . Dose reductions and delays were permitted for the management of toxicity.This was a double‐blind, placebo‐controlled, randomized phase 3 study (NCT01002248). Patients were randomized in a 1:1 ratio to receive perifosine 50 mg orally, once‐daily, or matching placebo, plus intravenous or subcutaneous bortezomib 1.3 mg/mThe primary endpoint was progression‐free survival (PFS). Secondary endpoints included overall response rate, overall survival (OS), and safety. Tertiary endpoints included perifosine population pharmacokinetics and evaluation of treatment effects on electrocardiogram (ECG) parameters. Serum and urine protein electrophoresis were performed by a central laboratory at the start of each 21‐day treatment cycle to assess disease status until confirmed disease progression. Response or progression determined according to parameters other than serum and urine M‐protein or determined by local laboratory readings were adjudicated by an independent reviewer blinded to treatment arms. All responses were assessed using modified European Group for Blood and Marrow Transplantation criteria  and Inte2.3Enrolment of 450 patients was planned in order to provide 265 events of disease progression or death for the final statistical analysis of the primary endpoint, PFS. Two formal nonbinding interim analyses were planned by the independent Data and Safety Monitoring Board (DSMB) to assess efficacy at the time of approximately 30% and 60% of the total PFS events having occurred, respectively, that is when approximately 80 and 160 PFS events had been observed. The results of the first planned interim analysis are reported herein, which was performed when 80 PFS events had been observed across the two arms. Following the recommendation of the DSMB, the study was discontinued at this interim analysis  due to absence of benefit in the primary endpoint, limited study logistics, and enrolment challenges, which had resulted in very slow patient accrual and thereby limited the sample size.The primary analysis was performed using the intent‐to‐treat population and included only those progression events defined by a computer algorithm and, when required, an independent reviewer. Progression determined by the investigator alone was not considered a progression event. Hypothesis testing between the two treatment arms was performed using a log‐rank test with an overall two‐sided .05 level of significance via a model including treatment effects. For each treatment arm, the median duration of PFS and the proportion of patients alive and progression‐free at 6 and 12 months were estimated using the Kaplan–Meier method. For each estimate, a 95% confidence interval (CI) was reported. The hazard ratio (HR) and its 95% CI were also reported. The secondary endpoint of OS was analyzed similarly to PFS. Other data were summarized using descriptive statistics.33.1At the data cut‐off for the first interim analysis March 12, 2013), a total of 135 patients had been enrolled and randomized – 69 to the perifosine arm and 66 to the placebo arm . The Kaplan–Meier distributions for PFS are shown in Figure The primary endpoint of PFS was assessed in the intent‐to‐treat population, with the exception of one patient in the placebo arm. This patient was not evaluable for time‐to‐event outcomes; subsequent to randomization, the patient was found not to meet eligibility criteria based on M‐protein levels required by the protocol, and so they were excluded from the analyses of PFS and OS. At the time of data cutoff for this first planned interim analysis, a total of 42 patients in the perifosine arm and 38 patients in the placebo arm had progressed or died, with 27 patients in each arm remaining alive and progression‐free after a median follow‐up of approximately 28 and 31 weeks, respectively. The median PFS was 22.7 weeks (95% CI 16.0–45.4) in the perifosine arm and 39.0 weeks (18.3–50.1) in the placebo arm; the HR for PFS in the perifosine arm relative to the placebo arm was 1.269  and 27% (18 patients) in the perifosine and placebo arms, respectively (Table P = ·356). The Kaplan–Meier distributions for OS are shown in Figure At the time of data cut‐off, a total of 17 patients in the perifosine arm and 19 patients in the placebo arm had died, with 52 and 46 patients in the respective arms remaining alive after a median follow‐up of approximately 48 and 40 weeks, respectively. The median OS was 141.9 weeks (95% CI 72.6–not estimable) in the perifosine arm and 83.3 weeks (51.1–not estimable) in the placebo arm; the HR for OS in the perifosine arm relative to the placebo arm was 0.734  of patients in the perifosine arm and 50 (75.8%) of patients in the placebo arm reported at least one AE; the most common events are summarized in Table A total of four patients (6%) on the perifosine arm and three patients (5%) on the placebo arm died during the study reporting period due to reasons other than disease progression, including respiratory infection, suicide, sudden death (treatment‐emergent grade 5 AE), and myocardial infarction (each n = 1) on the perifosine arm, and respiratory failure/sepsis (treatment‐emergent grade 5 AE), congestive heart failure, and pneumococcal sepsis (treatment‐emergent grade 5 AE) (each n = 1) on the placebo arm.4The findings from the first planned interim analysis of this randomized, double‐blind, placebo‐controlled, phase 3 study demonstrated no benefit in terms of the primary endpoint, PFS, with the addition of perifosine to bortezomib‐dexamethasone in patients with RRMM who had previously relapsed following bortezomib‐based treatment. Based on these data and the recommendation of the DSMB, and associated with concerns regarding slow accrual resulting in a limited sample size and regarding resource constraints and study logistics, the study was discontinued following this first planned interim analysis. Thus, with limited follow‐up, further interpretation of these findings is restricted. In particular, OS data were immature at the time of this analysis, with only 25% and 29% of patients in the perifosine and placebo arms, respectively, having died. Median OS was numerically longer in the perifosine arm, but these data were based on the tail‐ends of the Kaplan–Meier distribution curves and must therefore be interpreted with caution.The response rates reported in the present study appeared low compared to data from the previous phase 1/2 study of perifosine plus bortezomib‐dexamethasone in RRMM ; in the It is of interest to assess the overall response and clinical benefit rates in both arms in the context of the high proportion of unevaluable patients and patients missing response data ; if these patients were to be omitted from the calculations of overall response and clinical benefit rates, the rates would be similar to those previously reported for perifosine plus bortezomib‐dexamethasone in bortezomib‐relapsed RRMM patients and bortezomib ± dexamethasone retreatment in patients with relapsed MM . Nevertheless, these findings suggest the acute antitumor activity of the triplet combination in bortezomib‐relapsed patients was primarily driven by bortezomib‐dexamethasone, a hypothesis supported by data from a previous multicenter phase 2 study of perifosine ± dexamethasone in RRMM patients after a median of 4 prior lines of therapy , in whicA limitation of the present study is that, due to the dates between which it was conducted, a number of now‐commonly collected prognostic parameters were not recorded, including cytogenetics; thus, it is not possible to determine whether the patients in the study presented with an elevated rate of poor prognostic features, such as high‐risk cytogenetic abnormalities, which could potentially explain the poor response rates and outcomes seen in both arms. Further, no correlative studies related to the PI3K/Akt signaling cascade were prospectively planned; such analyses may have helped identify a patient population who did benefit from the combination .Reflecting the previous phase 1/2 study of perifosine plus bortezomib‐dexamethasone  the tripSubsequent to the discontinuation of this study, perifosine is no longer being investigated as a potential novel targeted agent for patients with RRMM. Reflecting these findings, a similar lack of OS benefit was seen with the addition of perifosine to capecitabine in patients with metastatic colorectal cancer in the phase 3 X‐PECT study . NeverthIn conclusion, perifosine at the dose tested did not improve response rates or outcomes in combination with bortezomib‐dexamethasone in patients with RRMM who have relapsed following previous bortezomib‐based treatment. Although the rational combination of Akt pathway inhibition with perifosine and proteasome inhibition with bortezomib demonstrated synergistic anti‐MM activity in preclinical investigation , this waP.G.R. was the principal investigator and takes primary responsibility for the paper; P.G.R., A.N., D.B.‐Y., A.B., P.N.H., R.H., I.S., H.K., R.L.B., S.‐S.Y., K.K., J.M.‐L., M.M., O.S., K.C., J.P.L., I.M.G., M.L., M.E.G., and K.C.A. collected data and/or recruited patients; P.G.R., A.N., P.B., T.H., P.S., and K.C.A. designed the study. P.S. and M.C. participated in the statistical analysis; P.G.R., P.S., and M.C. co‐ordinated preparation and analysis of the dataset; P.G.R. and P.S. interpreted the data and prepared the manuscript. All authors reviewed and approved the manuscript.P.G.R. received research support from Oncopeptides, Celgene, Takeda, and Bristol‐Myers Squibb and served as advisory committee member for Karyopharm, Oncopeptides, Celgene, Takeda, Amgen, Janssen, and Sanofi. P.N.H. received research funding from Takeda and Celgene, and served as a consultant for Takeda, Celgene, Bristol‐Myers Squibb, Janssen, Spectrum, Pharmacyclics, and Kite. R.H. received consulting fees, honoraria, and research funding from Amgen, Takeda, Celgene, Janssen, Abbvie, Novartis, PharmaMar, and BMS. H.K. served as a consultant and also served on a Speakers Bureau for Takeda. J.M.‐L. received research funding from BMS, Janssen, Novartis, and Celgene, and served as a consultant for Janssen, Novartis, Celgene, and Roche. P.B. was an employee of Aeterna Zentaris. K.C. served as a consultant for Celgene. I.M.G. served as a consultant for GSK, Sanofi, Janssen, Takeda, Celgene, Karyopharm, AbbVie, GNS, Cellectar, Medscape, Genentech, Adaptive, BMS, Aptitude, Curio Science, and Oncopeptides, and served as an advisor for GSK, AbbVie, and BMS. P.S. was an employee and had equity interest in Keryx Biopharmaceuticals. M.C. served as a consultant for Keryx Biopharmaceuticals. K.C.A. served as a consultant for Celgene, Millennium Pharmaceuticals, Bristol Myers Squibb, Janssen, Gilead, and Sanofi, and was a scientific founder of Oncopep and C4 Therapeutics.


A low Neutrophil Lymphocyte Ratio (NLR) has been shown to be associated with good prognosis in Bell’s Palsy (BP). However, the effect of chronic diseases that may affect the NLR, including Diabetes Mellitus (DM), has not been clarified in this context. This study aimed to evaluate the relationship between NLR and Mean Platelet Volume (MPV) in BP according to whether it is accompanied by DM, and their relationship with prognosis.A prospective observational study was conducted from May 2014 to May 2020 in a tertiary referral center, of all 79 consecutive participants diagnosed with BP in department of otolaryngology and 110 consecutive healthy participants admitted to the check-up unit. Patients diagnosed with BP were divided into two groups according to whether they were diagnosed with DM: diabetic BP patients  and non-diabetic BP patients without any chronic disease . Neutrophil (NEUT) and Lymphocyte (LYM) counts, and Mean Platelet Volume (MPV) were assessed from peripheral blood samples, and the NLR was calculated. Prognosis was evaluated using the House–Brackmann Score (HBS) six months after diagnosis.p < 0.001). The mean NLR was 2.58 ± 1.83 in the nonDM-BP group, 3.23 ± 1.83 in the DM-BP group, and 1.69 ± 0.65 in the control group. The NLR was significantly higher in the nonDM-BP and DM-BP groups than in the control group (p < 0.05). The recovery was 90% according to the HBS. The optimal cut-off value was 2.41 (p = 0.5).The mean NLR was 2.85 ± 1.85 in BP patients and 1.69 ± 0.65 in the control group. The mean NLR was significantly higher in BP patients than healthy controls (The NLR was increased in both diabetic and non-diabetic BP and had similar prognostic value in predicting the HBS before treatment in diabetic and non-diabetic patients with BP. MPV wasn’t significantly different in diabetic and non-diabetic BP patients compared with the normal population. Bell’s Palsy (BP) is an idiopathic peripheral Facial Paralysis (FP) characterized by acute unilateral weakness of the facial muscles. BP is the most common cause of FP. The annual incidence is between 15 and 30 per 100,000 people.The House–Brackmann Score (HBS) is used to evaluate paralysis and is associated with prognosis.A high Neutrophil-Lymphocyte Ratio (NLR) is considered a reliable etiological indicator of disease severity in inflammatory disorders.DM is one of the diseases that have been implicated in the etiology and prognosis of BP. NLR has also been shown to be increased in patients with diabetes. Likewise, high Mean Platelet Volume (MPV) values demonstrate increased platelet activity in patients with diabetes and are associated with insulin resistance. Based on these findings, this study aimed to evaluate the NLR and MPV in BP according to whether it is accompanied by DM, and their relationship with prognosis.This study was approved by the Başkent University Institutional Review Board and Ethics Committee (Project nº KA 15/245) and was conducted in accordance with the Declaration of Helsinki and its later amendments. Written informed consent was obtained from all participants. The study prospectively included 79 consecutive participants diagnosed with BP between May 2014 and May 2020 and 110 consecutive healthy participants admitted to the check-up unit. Patients diagnosed with BP were divided into two groups depending on the concomitant presence of DM: diabetic BP patients (DM-BP) and non-diabetic BP patients (nonDM-BP). All patients with FP were evaluated by an otorhinolaryngologist. FP grade was determined using the HBS by the same two researchers. All patients were evaluated by MRI using internal acoustic canal gadolinium contrast. Neutrophil (NEUT) and Lymphocyte (LYM) counts, and Mean Platelet Volume (MPV) were assessed using an Abbott CELL-DYN Ruby System  from peripheral blood samples obtained from patients with FP developed within 3 days or less. The NLR, a marker of subclinical inflammation, was calculated by dividing the number of neutrophils by the number of lymphocytes. The percentage of glycated Hemoglobin (HbA1c) of patients in the DM-BP group was measured. All diabetic patients required subcutaneous insulin injections during the treatment for blood glucose control. Routine prednisolone treatment was administered to all patients . The prognosis was evaluated using the HBS six months after diagnosis. According to the final evaluation of facial functions, H-B grades I and II were considered as a recovery while HB grade over II considered as non-recovery.The exclusion criteria were as follows: polyneuropathies such as central induced paralysis, cerebellopontine corner tumor, herpes zoster oticus, otitis media, Guillain–Barre syndrome, recurrent BP, bilateral FP, traumatic FP, iatrogenic FP; more than 3 days from the onset of FP, follow-up period of less than 6 months, chronic renal failure, chronic liver failure, previous transplant, chronic arterial disease, inflammatory bowel disease, vasculitis, malignancies, asthma patients, pregnant women; botulinum toxin administration, and use of steroids, antihistamines, chemotherapy, antibiotics, or anti-inflammatory drugs; and grade II HBS at the time of admission.t-test was used for the parameters with normal distribution, Mann Whitney U test was used for the parameters not showing normal distribution, and Kruskall Wallis test was used for more than two variables. The relationship between quantitative variables was examined with Pearson and Spearmean correlation analyses. Analysis of variance (ANOVA) was used to check if the means of two or more groups are significantly different from each other. The optimal cut-off value was determined using Receiver Operating Characteristic (ROC) curve. The level of statistical significance was accepted as 0.05 in all tests.SPSS 23.0 package program  was used for statistical analysis of the data. Categorical measurements were presented as number and percentage while continuous measurements were expressed as mean and standard deviation . Chi-Square test and Fischer’s precision test were used to compare categorical variables. Shapiro–Wilk test was used to determine whether the parameters in the study showed normal distribution. In comparing the continuous measurements between the groups, the distributions were checked, and independent Student p < 0.001), (p = 0.454). The mean HbA1c in the DM-BP group was 6.90 ± 0.48% (range 5.78–8.10).A total of 189 patients, including 46 (26 men) in the nonDM-BP group; 33 (15 men) in the DM-BP group; and 110 (63 men) in the healthy control group participated in the study. There were no differences among the groups in terms of sex distribution. The mean age was 41 ± 15 years in the non-DM-BP group, 58 ± 15 years in the DM-BP group, and 43 ± 12 years in the control group. The mean age was significantly higher in the DM-BP and nonDM-BP groups than in the control group ( 0.001), . There wp < 0.001). The mean NLR was 2.58±1.83 in the nonDM-BP group, 3.23 ± 1.83 in the DM-BP group, and 1.69 ± 0.65 in the control group. The NLR was significantly higher in the nonDM-BP and DM-BP groups than in the control group (p < 0.05). The mean NLR was not significantly different between the DM-BP and nonDM-BP groups (p = 0.073), (p < 0.001). MPV and NEUT and LYM counts were not different among the groups (p > 0.05). NLR and HbA1c were positively correlated in the DM-BP group .The mean NLR was 2.85 ± 1.85 in BP patients and 1.69 ± 0.65 in the control group. The mean NLR was significantly higher in BP patients than healthy controls ( 0.073), . The leup > 0.05), (p = 1). The mean NLR was 2.73 ± 1.54 in BP patients with recovery and 3.91±3.58 in BP patients with non-recovery. The mean NLR was significantly higher in BP with non-recovery group than BP with recovery (p = 0.006). ROC curve analysis of NLR to predict recovery in BP showed the optimal cut-off as 2.41 (AUC 0.573 [0.331–0.815]), sensitivity 50% and specificity 54.9%), (p = 0.5). The NLR increased as the HBS increased before treatment in BP patients , (p = 0.089).The HB grade was classified as grade III in 28 patients, grade IV in 15 patients, and grade V in 3 patients in the DM-BP group. Five patients were classified as grade III, 11 patients as grade IV, 6 patients as grade V, and 1 patient as grade VI in the DM-BP group. There was no difference in either pre-treatment or post-treatment HB grades between the nonDM-BP and DM-BP groups (> 0.05), . The rec 0.054), . The proÖzler et al. reported that the NLR was higher in HB grade V patients before treatment compared with patients with HB grade III, IV, and VI.The variability of inclusion and exclusion criteria in studies investigating the role of NLR in BP affects the reliability of the results.Various factors affecting BP prognosis have been suggested, including age, DM, hypertension, and hypercholesterolemia. Takemoto et al. reported a relationship between poor recovery and DM.The NLR has been reported to be higher in patients with type 2 DM than in those without diabetes, and this increase has been reported to be associated with high insulin resistance.The MPV parameter, indicating platelet volume, can also be associated with microvascular thrombotic disease. The correlation between MPV and stroke has been demonstrated in the literature.NLR has a similar prognostic significance for diabetic and non-diabetic patients with BP, and a high NLR is associated with higher stage HBS before treatment. MPV was not significantly different in diabetic and non-diabetic BP patients compared with the normal population.The authors declare no conflicts of interest.


BP was obtained within a day of 24-h personal exposure measurements at baseline, when participants were using biomass for cooking. We used generalized additive models to characterize the shape of the association between BP and HAP, accounting for the interaction of personal exposures and age and adjusting for a priori identified confounders. A total of 418 women (mean age 52.2 ± 7.9 years) were included in this analysis. The interquartile range of exposures to PM2.5 was 42.9–139.5 μg/m3, BC was 6.4–16.1 μg/m3, and CO was 0.5–2.9 ppm. Both SBP and PP were positively associated with PM2.5 exposure in older aged women, achieving statistical significance around 60 years of age. The exact threshold varied by BP measure and PM2.5 exposures being compared. For example, SBP of women aged 65 years was on average 10.8 mm Hg (95% CI 1.0–20.6) higher at 232 μg/m3 of PM2.5 exposure (90th percentile) when compared to that of women of the same age with personal exposures of 10 μg/m3. PP in women aged 65 years was higher for exposures ≥90 μg/m3, with mean differences of 6.1 mm Hg (95% CI 1.8–10.5) and 9.2 mm Hg (95% CI 3.3–15.1) at 139 (75th percentile) and 232 μg/m3 (90th percentile) respectively, when compared to that of women of the same age with PM2.5 exposures of 10 μg/m3. Our findings suggest that reducing HAP exposures may help to reduce BP, particularly among older women.Elevated blood pressure (BP) is a leading risk factor for the global burden of disease. Household air pollution (HAP), resulting from the burning of biomass fuels, may be an important cause of elevated BP in resource-poor communities. We examined the exposure-response relationship of personal exposures to HAP —fine particulate matter (PM •Household air pollution (HAP) may be a risk factor for elevated blood pressure (BP).•We examined the link between HAP and BP in women across 4 resource-poor settings.•2.5 in women aged >∼60 years.Higher BP was associated with higher exposures to PM•Reducing HAP exposures may help to reduce BP, particularly among older women. In 20152.5), carbon monoxide (CO), black carbon (BC), hydrocarbons, nitrogen oxides and sulfur dioxide. Exposure to HAP is associated with endothelial dysfunction, and higher platelet activation, factor VIII levels, oxidative stress and subclinical atherosclerosis (2.5 exposure is associated with elevated blood pressure and adverse CVD outcomes (2.5 exposure and elevated blood pressure remains mixed (2.5 exposures and risk factors for hypertension including age (The burning of solid fuels resulting in household air pollution (HAP) is one of the factors that has been associated with elevated blood pressure in LMICs . HAP is clerosis , all of outcomes . Howeverns mixed . Reasonsding age  and bodyding age . Indeed,ding age . Finallyding age , 2017 ha2.5, BC and CO in 418 non-pregnant, non-smoking women aged 40–79 years in four resource-poor settings in Guatemala, Peru, India and Rwanda, and identify effect modification by age and body mass index. We used generalized additive models to capture non-linear interactions between continuous variables on blood pressure.Using baseline data from the Household Air Pollution Intervention Network (HAPIN) trial, , we sought to evaluate the cross-sectional association between BP measures and personal exposures to PM22.12.5 concentrations were an order of magnitude lower outdoors than in the kitchen, therefore ambient pollution contributed minimally to participants’ personal exposures  stove, continuous fuel distribution and behavioral messaging among 3195 households in four resource-poor LMIC settings: Tamil Nadu, India (T-IND); Jalapa, Guatemala (J-GUA); Puno, Peru (PER); and Kayonza, Rwanda (K-RWA). These four settings were specifically chosen for their low ambient air pollution levels. On average, 24-h PMxposures .The HAPIN trial is described in detail elsewhere . BrieflyClinicalTrials.gov (Identifier NCT02944682).The study protocol was reviewed and approved by institutional review boards at Emory University , Johns Hopkins University (00007403), Sri Ramachandra Institute of Higher Education and Research (IEC-N1/16/JUL/54/49) and the Indian Council of Medical Research-Health Ministry Screening Committee (5/8/4–30/(Env)/Indo-US/2016-NCD-I), Universidad del Valle de Guatemala (146-08-2016) and Guatemalan Ministry of Health National Ethics Committee (11–2016), Asociación Benefica PRISMA (CE2981.17), the London School of Hygiene and Tropical Medicine  and the Rwandan National Ethics Committee (No.357/RNEC/2018), and Washington University in St. Louis (201611159). The parent trial is registered with 2.2Systolic (SBP) and diastolic blood pressure (DBP) were measured on the right arm in triplicate, with at least 2 min resting between repeat measurements, using an automatic monitor, Omron HEM-907XL . The mean of all three measurements was used as the final value in our analyses. Measurements were taken after confirming that the participant had not smoked, consumed alcohol or a caffeinated beverage, or cooked using biomass within 30 min of the measurement. Participants were asked to sit in a chair in a quiet room for 5 min with legs uncrossed, back supported by a chair, and arm supported by a table prior to commencing measurements. Pulse pressure (PP) was calculated as the difference between SBP and DBP, and mean arterial pressure (MAP) was computed as orize BP : normal 2.3We used a directed acyclic graph to identify confounders of the relationship between BP and HAP (2).We measured weight and height in duplicate using seca 876 electronic scales and seca 213 stadiometer platforms , respectively. If the weight or height measurements differed by more than 0.5 kg or 1 cm, respectively, we collected a third measurement and the two closest readings were averaged. We computed BMI as the ratio between weight and height-squared (kg/mWe determined dietary diversity using the Minimum Dietary Diversity for Women (MDD-W) indicator, which is based on ten food groups . Diet diWe determined level of physical activity using the Global Physical Activity Questionnaire (GPAQ), which collects information on both vigorous and moderate-intensity activity in three settings: at work, traveling to and from places, and in recreational activities, as well as sedentary behaviour . From thWe used a dimension reduction technique known as principal component analysis (PCA)  to const2.42.5, CO, and black carbon (BC) over a 24-h period at baseline shortly after enrollment. PM2.5 exposures were measured using the Enhanced Children's MicroPEM , a combined nephelometric and gravimetric PM2.5 sampler. The ECM collects PM2.5 on a 15-mm diameter filter using a 0.3 L/min pump. We used polytetrafluoroethylene filters with a 2-μm membrane  and calibrated the volumetric flowrate of the ECM pumps prior to every sample collection with a TSI 4100 flowmeter  or a Gilibrator-2 primary calibrator . Filters were pre- and post-weighed in temperature- and humidity-controlled rooms using 1-μg resolution microbalances  at the University of Georgia  and at the Sri Ramachandra Institute for Higher Education and Research (for India). We collected four field blanks for every 100 samples and applied median blank corrections by site. Duplicates were collected in a subset of samples to determine monitor performance . Calibration checks for the CO monitors were performed every one to three months using a chamber to test the devices with clean air and CO span gas between 40 and 80 ppm . Both th2.5 filters was estimated via its black-body optical properties using SootScan OT21 Transmissometers , either at the University of Georgia for samples collected in Guatemala, Peru, and Rwanda, or at the Sri Ramachandra Institute for Higher Education and Research for samples collected in India. The instrument measures light attenuation through the filter at the 880-nm wavelength, which is then converted into BC mass deposition using the BC attenuation cross-section from similar source types on similar Teflon filters  and BP measures  adjusted for a priori confounders and accounting for effect modifiers. We used a directed acyclic graph to identify causal and non-causal paths between HAP exposure and BP and identified site and SES as two potential confounders of the relationship between personal exposures to HAP and BP  with tensor-product smooth and cubic regression splines  to model2.5 and age. These plots are visually useful to determine whether there are main effects and an interaction present. For example, if there is a positive association between SBP and PM2.5 but not with age, the 2D contour plot would only show an increased color gradient from red to white in the vertical axis . If there is a positive association between SBP and age but not with PM2.5, then the 2D plot would show an increased color gradient from red to white in the horizontal axis . If there are independent associations between SBP and PM2.5 and between SBP and age, then the 2D contour plot would show increased color gradients from red to white in both the vertical and horizontal axes, represented by angled, linear stripes. The degree of the angle of the stripes will depend on the relative strength of the association of the two factors. An interaction is likely present when patterns other than those described above are visualized, as seen in In 2.5 at any given age, and between SBP and age at any given level of PM2.5 exposure. As an example, we're plotting SBP vs PM2.5 at three specific ages in 2.5 exposure in 2.5 and SBP differs with age, indicating an interaction between PM2.5 and age. Similarly, 2.5 exposure.The 3D smooth surface function allows us to determine the associations between SBP and PM2.5 exposures of either 21, 43, 139 or 232 μg/m3 and 10 μg/m3, and report the results at three exact ages . These concentrations of PM2.5 exposures correspond to the 10th, 25th, 75th and 90th percentiles of observed values, and 10 μg/m3 was chosen as the lowest interim target (IT-4) recommended by the World Health Organization (WHO) . For BC, we measured expected differences in BP between personal exposures of either 3 (10th percentile), 6 (25th percentile), 16 (75th percentile) or 23 μg/m3 (90th percentile) and 1 μg/m3 . For CO, we measured expected differences in BP between personal exposures of either 0.1 (10th percentile), 0.5 (25th percentile), 2.9 (75th percentile) or 5.8 ppm (90th percentile) and 0 ppm . We verified goodness-of-fit of adjusted models by comparing expected and observed BP trajectories with age (We used the estimated models to measure expected differences in BP between PMon (WHO) . For comTo examine whether BMI was an important effect modifier of the association between BP and HAP, we constructed an additional model with a three-way interaction between HAP exposure, age, and BMI on BP adjusted for site and SES index. As supplementary analysis, we also constructed generalized additive models with age as a categorical variable with three age groups: <50; 50–64; ≥65 years. Further details on the models are provided in the Online Supplement.We conducted statistical analyses in R version 4.0.4 (Lost Library Book). .33.1We summarized baseline characteristics of study participants stratified by study site in 2.5, n = 103 for BC, n = 46 for CO). A final sample of 357 (85%), 314 (75%), and 362 (87%) participants was included in the PM2.5, BC, and CO models, respectively.In our analyses, we excluded seven participants because of missing BP measurements  to 111 μg/m3 (J-GUA), exceeding the WHO interim air quality target IT-1 of 35 μg/m3. The percentage of women with personal exposures below IT-1 was 7.7%, 13.5%, 21.1% and 35.2% in J-GUA, K-RWA, T-IND and P-PER, respectively. On average, BC constituted 14.2 ± 8.9% (mean ± SD) of the PM2.5 exposure. Median 24-hr BC exposures were highest in J-GUA (12 μg/m3) and lowest in P-PER (9 μg/m3), as with PM2.5. Personal exposures to CO on the other hand were highest in P-PER, with median 24-hr CO exposures ranging between 0.7 ppm in T-IND and K-RWA and 2.3 ppm in P-PER. Coefficients of variation for PM2.5, BC and CO were 0.98, 0.94 and 1.44, respectively.We plotted boxplots and cumulative fraction curves of 24-h personal exposures to all three pollutants for all participants aged 40–79 years at baseline and stratified by site in 3.42.5 at 40, 50 and 65 years of age in 3 of PM2.5, respectively, when compared to that at 10 μg/m3. PP at 65 years old was significantly higher for PM2.5 exposures ≥90 μg/m3  mm Hg and an increase in PP of 0.43 (95% pointwise CI 0.09 to 0.77) mm Hg at 65 years of age.Mean differences in the four BP outcomes for differences in PM2.5, mean differences in BP varied with age. However, none of the mean differences were significant for BC or CO. The exposure-response curves for our four BP outcomes with BC showed only a weak association between BP and BC exposure at 65 years of age, and no association or weak negative association at ages 40 and 50 years  compared to those with a BMI of 22 kg/m2 (2.8 mm Hg). At 232 μg/m3, mean differences in SBP compared to that at 10 μg/m3 were 4.6 mm Hg, 5.2 mm Hg and 14.7 mm Hg at BMIs of 22, 25 and 30 kg/m2, respectively. For PP, mean differences at 139 μg/m3 and 232 μg/m3 compared to 10 μg/m3 were 2.2 mm Hg and 3.9 mm Hg higher, respectively, at a BMI of 30 kg/m2 when compared to 22 kg/m2. While mean differences in DBP and MAP with higher PM2.5 exposures were not significant, similar trends with BMI were also found for these two BP measures.We also plotted the mean differences in BP with differences in PM30 kg/m2 . The exp30 kg/m2 . The res42.5 exposure and BP measures (SBP and PP) that increased linearly and then tapered off in women aged 65 years, whereas there was no relationship between PM2.5 and BP measures at 40 or 50 years of age. BMI was also an effect modifier of the relationship between PM2.5 exposure and BP. Specifically, when adding BMI in a three-way interaction with PM2.5 and age, we found that mean differences in BP with increases in PM2.5 exposure at age 65 were greater in participants with a higher BMI.In this cross-sectional analysis using data from four resource-poor settings, we found that the association between BP and HAP exposure in adult women aged ≥40 years not only varied with age but also with BMI. There was a positive association between PM2.5 exposure among older women  and which have important implications with regards to cardiovascular morbidity and mortality. Indeed, PP, which is an indicator of arterial stiffness, has been shown to be a strong determinant of coronary heart disease, stroke, and cardiovascular events in both hypertensive patients and the general population (2.5 exposure with PP than with other BP measures, suggest that HAP exposure may increase cardiovascular disease risk via an increase in arterial stiffness. Findings from other air pollution studies support this conjecture (Our analysis has several strengths. First, the HAPIN trial was conducted across multiple countries, which helps to provide better generalizability of our findings. Second, we collected a comprehensive set of biological and sociodemographic factors which allowed us to adjust for confounding. Third, we adopted a comprehensive statistical model that captures a non-linear interaction between age and HAP exposure with a smooth surface. Previous exposure-response analyses have not considered two- or three-way interactions between PMrization . Lastly,pulation . Recent pulation . Our resnjecture .This analysis also has some potential shortcomings. First, we used personal exposures and BP measurements from a single visit rather than multiple visits. Since there can be significant day-to-day variability in HAP exposures, a single measurement may not be representative of a participant's average daily HAP exposures. In addition to day-to-day variability, BP can also vary throughout the day. Future studies may benefit from the use of ambulatory BP monitors that capture BP continuously over a 24-h period. Second, although we adjusted for SES index and site, there may be other unmeasured confounders. Our analysis was also limited to a population of women with low prevalence of self-reported chronic conditions. Therefore, our results cannot be generalized to less healthy populations with underlying conditions such as hypertension or diabetes, or to men. Women, however, tend to have the greatest exposures to HAP as the primary cooks in the household. Finally, while our sample size was larger compared to most previous HAP studies, it may not have been sufficient to detect small mean differences in BP for the range of observed HAP exposures.2.5 and either SBP or PP among women aged around 60 years of age and older, and strong effect modification by BMI. Our findings suggest that reducing HAP exposures may help reduce BP, particularly among older women and those with a higher BMI. Randomized control trials of HAP mitigation like HAPIN are needed to confirm these cross-sectional findings.In conclusion, we observed a positive exposure-response association between personal exposure to indoor PMLaura Nicolaou: Conceptualization, Methodology, Software, Investigation, Formal analysis, Data curation, Writing – original draft, Writing – review & editing, Visualization.; Lindsay Underhill: Project administration, Writing – review & editing.; Shakir Hossen: Data curation, Visualization.; Suzanne Simkovich: Project administration, Writing – review & editing.; Gurusamy Thangavel: Project administration, Resources, Writing – review & editing.; Ghislaine Rosa: Investigation, Project administration, Writing – review & editing.; John P. McCracken: Investigation, Project administration, Resources, Writing – review & editing.; Victor Davila-Roman: Writing – review & editing.; Lisa de las Fuentes: Writing – review & editing.; Ashlinn K. Quinn: Writing – review & editing.; Maggie Clark: Writing – review & editing.; Anaite Diaz: Investigation, Project administration, Resources, Writing – review & editing.; Ajay Pillarisetti: Investigation, Resources, Data curation, Writing – review & editing.; Kyle Steenland: Investigation, Writing – review & editing.; Lance A. Waller: Data curation, Writing – review & editing.; Shirin Jabbarzadeh: Data curation, Writing – review & editing.; Jennifer L. Peel: Investigation, Writing – review & editing, Supervision, Project administration, Funding acquisition.; William Checkley: Conceptualization, Methodology, Investigation, Writing – review & editing, Supervision, Project administration, Visualization, Funding acquisition.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Exposure to ultraviolet B (UVB) leads to the overproduction of reactive oxygen species (ROS), causing higher risks of skin disorders. Luteolin (Lut) is a naturally occurring antioxidant that can absorb a broad range of ultraviolet light, but its water solubility and skin permeability are limited and insufficient. The aim of the current study was to develop a Lut-loaded self-emulsifying phospholipid preconcentrate (LSEPP) for enhancing the solubility, permeability, and photoprotective activity of Lut. The designed formulations were firstly examined for their droplet size, zeta potential, dispersity, and in vitro corneum permeability after dispensing the preconcentrate to form an emulsion; the optimized formulation was further characterized for its emulsified morphology, compatibility with excipients, stability in the preconcentrate form, and photoprotective activity by the HaCaT cell model under the emulsified status. The optimized LSEPP formulation attained a smaller droplet size (140.6 ± 24.2 nm) with the addition of 1,8-cineole and increased the permeability of Lut by 7-fold. As evidenced in the cell model studies, the optimized LSEPP formulation can efficiently deliver Lut into HaCaT cells after emulsification and result in a 115% better cell viability as well as a 203% stronger ROS scavenging capability, compared with those of unformulated Lut after UVB irradiation. To sum up, we have successfully developed an LSEPP formulation, which is a safe and promising topical delivery system for enhancing the photoprotective effects of Lut. Specifically, UVA (320–400 nm) and UVB (290–320 nm) are the major types of UV that cause severe skin damage  were purchased from Tokyo Chemical Industry . Castor oil, jojoba oil, and shea butter were obtained from Magie Fairy . PHOSAL® 50 PG  was obtained from LIPOID® GmbH . Kolliphor® EL  was supplied by BASF  and Tween® 20  was from Sigma-Aldrich Co. . 1,8-cineole  and nerolidol  were purchased from Alfa Aesar . The MultiScreen® Permeability Filter Plate, 0.4 µm, non-sterile (The skin PAMPA kit) was from Merck . All solvents used in this study were of analytical grade.Luteolin  with a purity >98%, Tween® 50 PG) and emulsifiers  was assessed. An excess amount of Lut was added to oils, then vortexed for 5 min and sonicated for 20 min. The mixtures were shaken in a thermostat water bath at 37 ± 0.5 °C and 50 rpm for 48 h to reach equilibrium. After that, these mixtures were centrifuged twice at 20,000× g for 15 min to remove undissolved Lut completely [The solubility of Lut in oils . Before HPLC-PDA analysis, the diluted solution was vortexed for 5 min and centrifuged for 15 min at 13,600× g prior to HPLC-PDA analysis.In the aspect of surfactants, an excess amount of Lut was added to surfactants, followed by the same step as in the previous paragraph. Mixtures were centrifuged twice at 13,600× ® C18 column . The composition of the mobile phase was ACN: deionized water = 20:80 (v/v) and the flow rate was 1.0 mL/min. Samples of 20 μL would be injected and monitored at 343 nm. The formula of the calibration curve were y = 31959x − 2591.7 (R2 = 0.9999) and y = 41716x − 34150 (R2 = 0.9992), with a linear range from 0.25 to 5 µg/mL and from 5 to 50 μg/mL, respectively. The method accuracy and precision were within 10%, and the limit of quantification was 0.25 μg/mL.The HPLC-PDA analysis system consists of an LC-10AT pump SHIMADZU, a SIL-10AF autosampler, and an SPD-M10A detector . The column was a Kinetex® 50 PG, Kolliphor® EL, and Tween® 20 as the oil phase, emulsifier, and co-emulsifier, respectively. To optimize the ratio of the three components, various formulations were evaluated. The compositions of all formulations are listed in The LSEPPs was composed of PHOSALw/w) to give F7 and F8, respectively. The mixture was stirred for 15 min and sonicated for 15 min under ambient temperature to produce the final products.To further improve the permeation, two terpenoids  as enhancers were added to the optimized formulation. The design of F7 and F8 were based on the composition of F5. Therefore, F5 was blended with 1,8-cineole and nerolidol in a ratio of 95:5 , and zeta-potential of the formulations were evaluated with dynamic laser scattering . Each formulation was diluted 100 times with distilled water 10–15 min before the evaluation, and these characteristics were determined.Each LSEPPs was diluted 20-fold with a pH 6.4 phosphate buffer to simulate the skin environment, and the time taken for complete emulsification and the appearance were recorded. Based on the emulsification time and appearance, formulations were classified into five grades. Grade C exhibited a slow emulsification to form an opaque emulsion, and the properties implied the appropriate water resistance and better skin occlusion in topical delivery. As a result, Grade C is regarded as optimal for topical uses. The emulsification times for grade C and D were less and more than 2 min, respectively, and the appearance of grade C or D was milky white .n-hexane and 35 wt% . Next, 300 µL of 1 mg/mL emulsified LSEPPs, which was dispensed with receptor solution 10–15 min before the experiment, was added to the donor plate. Sampling points were 1, 2, 4, and 6 h. UV/Vis spectroscopy was adopted as the analytical method. A SpectraMax iD3  was utilized; the detected mode was the endpoint, and the wavelength was 343 nm. The samples in plates would be shaken in an orbital mode for 10 s before analysis. The formula of the calibration curves were y = 0.0331x + 0.00003 (R2 = 0.9994) and y = 0.0293x + 0.0579 (R2 = 0.9992) with linear ranges of 0.25–10 μg/mL and 10–100 μg/mL, respectively, and the accuracy was within 100 ± 10%. The limit of quantification was located at 0.25 μg/mL.A skin Parallel Artificial Membrane Permeability Assay (skin PAMPA) model was applied to evaluate the permeability in the stratum corneum, and the method was modified based on the manufacturer guide and a previous study . Brieflyw/v) phosphotungstic acid solution (PTA), and the excess PTA was removed with deionized water. The stained grid was dried overnight and subjected to JEM-1400 Transmission Electron Microscope  at 120 kV [TEM was applied to observe the morphology of the optimized formulation. The formulation was dispensed 10–15 min before the experiment with deionized water and dropped on a 300-mesh copper grid coated with carbon. Consequently, it was stained with 2% (t 120 kV .w/w) ratio, followed by grinding and compressing into a tablet. Excipients and F7 were analyzed by ATR, and air was used as the control group. The range of wavelength was evaluated at 600–4000 cm−1.To clarify the interaction between Lut and excipients, FTIR was applied to ensure the structure of Lut did not change, which may cause the loss of activity. The FTIR system was FT/IR-6800 . Lut and KBr were dried overnight and mixed in a 1:100 . The HaCaT cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) with 4.5 g/L glucose, 2 mM G-glutamine, 100 U/mL Penicillin, 100 µg/mL streptomycin, 0.25 µg/mL amphotericin B, and 10% fetal bovine serum . Cells were maintained at 37 °C in a humidified incubator with 5% CO® assays. The cells were seeded into 96-well plates at a density of 1 × 104 cells per well for 24 h when 70% confluence was achieved. The raw Lut was dispersed in DMEM, and LSEPPs and SEPPs were dispensed with DMEM 10–15 min before the experiment. All samples were added into 96-well plates; then, the cells were incubated for the indicated times. AlamarBlue® reagent was added into the culture medium 2 h earlier than the indicated time and incubated in a cell incubator. The fluorescence with an excitation wavelength of 560 nm and an emission of 590 nm was read by using a SpectraMax iD3.The cell viability was determined by AlamarBlueTM, Thermos Fisher Scientific, Waltham, MA, USA) was dissolved in ethanol to form 10 mg/mL stock solution. An 18 µL aliquot of DiI C18(3) was added into PHOSAL® 50 PG to label phospholipid, and the preparative procedures were the same as described in 4 density overnight at 37 °C with 5% CO2; then, the emulsified DiI-loaded SEPPs were pretreated for 3, 6, and 24 h. At the predetermined time, cells were washed with PBS and fixed with 4% (v/v) formaldehyde for 15 min. After fixation, cells were washed with PBS again, and cell nuclei were stained utilizing Hoechst 33,258 solution (10 µg/mL) for 30 min. Finally, the cells were washed with PBS and kept at 4 °C. The results were imaged by ImageXpress® Micro Widefield High Content Imaging System .To evaluate the cellular uptake of SEPPs, DiI-loaded SEPPs was prepared. The preparation method of DiI- loaded SEPPs was slightly modified from a previous study . 1,1′-Di2 with a UV peak at 302 nm. After exposure, the cells were incubated in a fresh medium without samples in the incubator for 24 h, and their viability was analyzed as ® Assays described. Quercetin and all-trans retinoic acid were chosen as positive control owing to their excellent antioxidant and anti-inflammatory effects [The procedures and conditions of cell culture, plating, and sample preparation are the same. The LSEPPs were pretreated for 6 h in a cell incubator. The cells were washed and incubated with PBS for UVB irradiation . The energy of UVB irradiation was 20 mJ/cm effects .4 density for 24 h. The cells were pretreated with samples for 6 h in a cell incubator and then the cell-permeable 2′,7′-dichlorodihydrofluorescein diacetate  was loaded and incubated for 30 min in a dark room. Consequently, the cells were washed with PBS and incubated for UVB irradiation (20 mJ/cm2). Immediately after irradiation, the fluorescence was recorded by SpectraMax iD3. Fluorescence with an excitation of 495 nm and an emission of 520 nm was utilized. The positive control was the same as HaCaT cells were plated in 96-well plates in a 1 × 10p < 0.05 was considered as a significant difference.The results were displayed as mean ± standard deviation. This data in this study were analyzed by one-way analysis of variance with a post hoc test of Tukey, and the analysis was conducted with SPSS v19 . ® 50 PG could solubilize Lut up to 15 mg/g, which was seven times higher than that of castor oil (approximately 2 mg/g), as shown in ® 50 PG was chosen as the oil phase.Castor oil, jojoba oil, and shea butter were commonly used oils in external preparations, and the solubility of Lut in castor oil, jojoba oil, and shea butter was 1.850 ± 0.016 mg/g, 0.090 ± 0.004 mg/g, and 0.061 ± 0.004 mg/g, respectively. PHOSAL® 20 (63.21 mg/g), Tween® 80 (52.33 mg/g), Kolliphor® EL (38.49 mg/g), and Span® 80 (9.76 mg/g). Taking safety into consideration, Kolliphor® EL was chosen as the surfactant and Tween® 20 as the co-surfactant.The rank from highest to lowest was Tween® 50 PG, possessed much smaller droplets and a narrower distribution. Notably, formulations containing terpenes (F7 and F8) had significantly smaller droplet sizes with a narrow PDI. In addition, formulations with more PHOSAL® 50 PG, F5–F8, needed longer emulsification time and presented a more turbid appearance after being emulsified by pH 6.4 phosphate buffer. In this section, LSEPPs was evaluated in the emulsified status. ® 50 PG could bring better permeability. To prove the role of phospholipids in permeability, the composition of the control group was the same as PHOSAL® 50 PG, but the phospholipid was replaced with deionized water followed by the dispersal of Lut to form a 1 mg/mL solution. The other control group was Lut (1 mg/mL) in 10% (v/v) EtOH to maintain the same concentration gradient. Obviously, the permeability of the control group was much lower than that of other formulations. The incorporation of the terpenoid as an enhancer contributed significantly to Lut permeation, and 1,8-cineole in F7 had better permeability improvement than nerolidol in F8. Therefore, F7 was chosen as the optimized formulation.The skin PAMPA model was utilized to evaluate the capability of corneum permeability. −1 (O-H vibration), 1665 cm−1 (C=C vibration), 1340 cm−1(Phenolic-OH bending vibration), 1159 cm−1 (C-O-C stretching), 1033 cm−1, and 1002 cm−1(C-OH vibration) [® 50 PG presented at 1736 cm−1 (P-O bond), 1454–1636 cm−1 (N-O bond), 1228 cm−1 (P=O bond), and 1102–1041 cm−1 (Amide bond conjugation) [−1 (O-H vibration), 1736 cm−1 (ester bond), and 1103 cm−1 (C-O bending) [® 20 were observed at 3477 cm−1 (O-H vibration), 2924–2857 cm−1 (C-H stretching) and 1733 cm−1 (O-H vibration) [−1 (CH3 deformation) and 983 cm−1  [® 50 PG had shifted slightly in the F7 group, showing that possible intermolecular forces may exist. According to previous studies, it is likely that hydrogen bonding between the phosphate group of phosphosphatidylcholine and the phenolic groups of Lut occurred [FTIR was employed to investigate the compatibility between Lut and excipients from the aspect of the characterization of functional groups, as shown in bration) ,31; the ugation) ,33,34; tbending) ,36; the bration) ; the dis2 plane) . −OH viboccurred . The chaTo mimic the realistic storage conditions of formulation, the stability of original LSEPPs was evaluated. As no water exists in the SEPPs, the excellent stability of SEPPs was expected. In this study, heating–cooling cycles, freeze–thaw cycles, and centrifugation tests were used to estimate the stability of F7. After three cycles and centrifugation, the content of Lut was measured by UV/vis spectroscopy. The amounts of Lut compared to the original were 99 ± 5%, 96 ± 4%, and 96 ± 2% in the heating–cooling cycles, freeze–thaw cycles, and centrifugation test, respectively. As expected, the loss of Lut was less than 10%, which suggested the great stability of F7.In this part, all groups were freshly dispensed to the indicated concentrations with DMEM–HG before the experiment. ® 50 PG, presented red fluorescence as shown in Hoechst-stained cell nuclei presented blue fluorescence, and DiI-stained phospholipid, a composition of PHOSAL2), and it is obvious that each group has significantly greater cell viability compared to the viability of the UVB irradiation group (UVB only group), which means that the energy of UVB indeed resulted in injury to HaCaT cells. Lut and excipients protect cells from UVB injury, and F7 was more capable of preventing cells from UVB injury (p < 0.05). Compared with positive control groups, Lut and excipients of 40 μM were comparable with quercetin (Que) of 20 μM, and F7 of 40 μM was comparable with all-trans retinoic acid (atRA) of 0.1 μM. The same pattern was also shown in the clearance of intracellular ROS, as In this section, all groups were dispensed to indicated concentrations with DMEM 10–15 min before the study. Phospholipids are well-established pharmaceutical excipients, which act as an emulsifier, solubilizer, and wetting agent. The structures of phospholipids vary their physicochemical properties, and this multifariousness widens the application on formulations in any administration routes . In topiEmulsified formulations with a high ratio of phospholipid (F5–F8) had smaller droplet sizes and ideal topical dispersity, which provides appropriate skin occlusion and water resistance in topical delivery . Notably2) and human epidermis (670.85 ± 189.05 μg/cm2) were close, and the lower standard deviation in skin PAMPA was noted. The skin PAMPA model is regarded as a golden tool for evaluation of permeability and has been applied in the formulation screening of a nanocomplex of low molecular-weight protamine [10H16) performed much better than sesquiterpenes (C15H24), and this theory was proven in this study again as the permeation effects of 1,8-cineole are better than that of nerolidol. Some previous studies supported our findings in this study. William et al. [For the in vitro permeability study, the skin PAMPA model was utilized as it is suitable to evaluate permeability among formulations in a short period of less than 24 h . PAMPA irotamine , ibuprofrotamine , and microtamine . Thereforotamine . Though rotamine , the hydrotamine , and it rotamine . Furtherm et al.  found thm et al.  also poim et al. .w/w) 1,8-cineole, and the concentration of 1,8-cineole was equivalent to 265 µM. Such a high concentration had been proven safe in this and a previous study [Lut is a flavonoid with low aqueous solubility, and it cannot permeate the stratum corneum easily, which limits its topical application. As SEPPs have been proven to be successfully accepted by HaCaT cells, the dramatically decreased cell viability in F7 of 80 μM at 48 h seemed reasonable, especially when raw Lut at 40 μM slightly reduced the cell viability. The composition of F7 contained 5%  presented desired physicochemical properties, including droplet size, distribution, dispersity grades, and permeability. The droplet was shaped in a self-assembled way after emulsification through the TEM image. In HaCaT cell models, F7 was delivered into HaCaT cells successfully and showed better photoprotective effects in the UVB-induced cell injury studies for which credit went to the synergistic effects of Lut and phospholipid.


Enterobacter phage myPSH1140 was characterized in silico, in vitro as well as in vivo using the purified protein. Gp105 contains a T4-type lysozyme-like domain (IPR001165) and belongs to Glycoside hydrolase family 24 (IPR002196). The putative endolysin indeed had strong antibacterial activity against Gram-negative pathogens, including E. cloacae, K. pneumoniae, P. aeruginosa, S. marcescens, Citrobacter sp., and A. baumannii. Also, an in vitro peptidoglycan hydrolysis assay showed strong activity against purified peptidoglycans. This study demonstrates the potential of Gp105 to be used as an antibacterial protein to combat Gram-negative pathogens.Bacteriophages and bacteriophage-derived peptidoglycan hydrolases (endolysins) present promising alternatives for the treatment of infections caused by multidrug resistant Gram-negative and Gram-positive pathogens. In this study, Gp105, a putative lysozyme murein hydrolase from  Enterobacteriaceae family include Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae, but also other nonfermenting GNB such as Pseudomonas aeruginosa and Acinetobacter baumannii. These bacterial pathogens have been declared a priority by the WHO, with the Disease Society of America creating the acronym “ESKAPE” pathogens that comprise Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter species. These bacteria are known to cause the most critical infections in humans and the development of treatment possibilities is urgent . In this study, a lysozyme murein hydrolase (Gp105) has been identified in the phage and was first characterized in silico, revealing the protein to contain a T4-type_lysozyme domain that belongs to the Glycosyl hydrolase family 24. This protein family is so far uncharacterized in bacteriophages infecting Enterobacter species. In order to evaluate the potential of the protein to be used as a therapeutic, the coding gene was cloned and overexpressed in E. coli. Subsequent tests using the purified protein, which could be obtained in high yield, showed that Gp105 is able to hydrolyze purified peptidoglycan in vitro and exhibits strong antibacterial properties against a wide range of pathogens, including E. cloacae, K. pneumoniae, P. aeruginosa, S. marcescens, Citrobacter sp., and A. baumannii.robacter . The genEnterobacter phage myPSH1140 .In Gram-negative bacteria, endolysins are released into the periplasm after their synthesis, making it necessary to cross the inner membrane. Often, specialized pore-forming proteins, including holins, facilitate this process. The outer membrane is a barrier to endolysins when added to cells from the outside, thus protecting the peptidoglycan layer from degradation. To assess if Gp105 also has antibacterial activity, we performed two tests with 12 Gram-negative bacteria belonging to eight genera, including Acinetobacter, Pseudomonas, and Serratia strains that were tested and to a slightly lesser extent with the Enterobacter, Klebsiella, and Citrobacter isolates. In contrast, we observed that neither the tested Salmonella nor the Escherichia strains showed inhibition zones in this semiquantitative well-diffusion assay, as was also observed in the case of the Gram-positive Enterococcus and Staphylococcus isolates.In the first assay, we assessed the growth of bacteria on solid media with a reservoir of the endolysin present, slowly diffusing into the agar . No inhiE. cloacae, K. pneumoniae, P. aeruginosa, S. marcescens, Citrobacter sp., and A. baumannii  for the Gp105 protein . No growaumannii . CorrelaE. cloacae at endolysin concentrations of 0.05, 0.25 and 0.5 mg/mL. When measuring the growth by determining the optical density at 600 nm, a significant reduction in bacterial growth was observed within 5 min after the addition of 0.25 and 0.5 mg/mL of Gp105  peptidases , as demonstrated by our experiments in which we determined the number of viable cells . SimilarE. coli allowed the high-yield production of a functional protein with yields of approximately 1–2 mg of purified protein per mL of culture. The high yield expression is possibly due to the inactivity of the protein toward E. coli, as also shown in the results of the well-diffusion assay. The observed molecular weight of 21 kDa is similar to that of other endolysins previously reported which exhibit sizes between 16 and 25 kDa  were used for cloning and expression studies, respectively. The clinical strains of Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae, Acinetobacter baumannii, Salmonella Typhi, Pseudomonas aeruginosa, Serratia marcescens, Citrobacter sp., Enterococcus sp., and Staphylococcus aureus were used from the Antibiotic Resistance and Phage Therapy Laboratory, VIT, Vellore. T4 DNA ligase and restriction enzymes BamHI and XhoI were obtained from New England Biolabs (USA). Oligonucleotide primers were synthesized by Eurofins Scientific India Pvt. Ltd. (India). The plasmid- pET28a was purchased from Novagen (Germany). Ni-NTA agarose was purchased from G-Biosciences (USA), and plasmid isolation and gel extraction kits were obtained from TaKaRa Biotech (USA). All analytical grade chemicals and antibiotics were purchased from Himedia Chemicals (India). Standard recombinant DNA techniques were used as described elsewhere , and the mixture was incubated at 37°C for 24 h in a shaking incubator (150 rpm). The mixture was centrifuged at 6000 × g for 15 min and the supernatant was filtered through a 0.22-μm syringe filter. The filtrate was tested for phage activity using a spot test and double agar overlay method as previously described  was added, mixed well and incubated overnight at 50°C. An equal volume of phenol: chloroform: isoamyl alcohol (25:24:1) was added and centrifuged at 12,000 × g for 15 min (repeated twice). The aqueous phase was extracted into a new tube and an equal volume of 100% isopropanol was added to precipitate the DNA at −20°C for 6 h. The precipitated DNA was pelleted, washed with 100% ethanol and air-dried. The purified phage DNA was visualized on 0.8% agarose gels.Phage DNA was extracted from the enriched phage lysate using the phenol-chloroform (24:1) method. Briefly, the phage particles were treated with DNase (20 mg/mL) and RNase (0.5 mg/mL) for 1 h at 37°C to remove any host DNA and RNA. To the mixture, 20% PEG and 1.6 M NaCl were added and stored at 4°C for 1 h. The content was centrifuged at 12,000 × http://www.ncbi.nlm.nih.gov) for designing gene-specific primers. PCR was performed using the genomic DNA of Enterobacter phage myPSH1140 as a template. The gene was amplified using gene-specific primers listed in table S1, containing the BamHI (Forward primer) and XhoI (Reverse primer) restriction sites. The gene was amplified under the following conditions: 95°C for 5 min, 25 cycles , and a final extension of 72°C for 10 min. The PCR products were analyzed on a 1.2% agarose gel.The nucleotide sequence of the gp105 gene was retrieved from NCBI (E. coli DH5α cells and the colonies obtained were screened using T7 Promoter and T7 Terminator primers (table S1). The recombinant plasmid was isolated from the positive colonies using a plasmid miniprep kit and verified by DNA sequencing.The PCR products generated were gel purified, digested, and ligated into the pET28a vector. The ligation mixture was transformed into E. coli BL21(DE3) cells for expression studies. A single colony of E. coli BL21(DE3) containing pET28a-gp105 was inoculated in 5 mL of LB medium containing kanamycin (50 μg/mL) and grown at 37°C overnight. 1 mL of this culture was added to a 100 mL fresh LB medium containing kanamycin (50 μg/mL) and incubated at 37°C to reach the log phase. To the growing culture of E. coli BL21(DE3) containing pET28a-gp105, IPTG was added to a final concentration of 1 mM and incubated at 37°C for 3 h. After induction, cells were harvested by centrifugation at 8,000 × g for 10 min and the total protein was analyzed (both the control and induced cells) in 12% SDS-PAGE. The proteins were visualized by staining the gel with Coomassie blue (0.05%) for 30 min in a rocking shaker.The recombinant plasmid pET28a-gp105 was transformed into E. coli BL21(DE3) cells transformed with pET28a-Gp105 were grown at 37°C in LB broth containing kanamycin (50 μg/mL) until the log phase and the expression was induced by adding 1 mM IPTG for 3 h at 37°C. The cells from the 100 mL culture were resuspended in 5 mL of lysis buffer . After one freeze-thaw cycle, the cells were lysed by sonication (30s pulse × 30s pause). The soluble fraction was separated by centrifugation at 15,000 × g for 30 min at 4°C and loaded onto a prepacked 2 mL (bed volume) Ni–NTA agarose column. After washing the column with 30-bed volumes of Wash buffer A  and Wash buffer B  at a flow rate of 1.0 mL/min, the bound protein was eluted in Elution buffer . The purified proteins were quantified using the Bradford assay.Enterobacter cloacae and Staphylococcus aureus was obtained as described earlier by Santin et al. . The bacterial cells were broken by passing (3 to 5 times) through a syringe needle (0.4- by 20 mm needles) and the cell wall was collected by ultracentrifugation at 90,000 × g for 45 min at 4°C . To the pellet, 10 mL of 8% SDS was added and incubated at 96°C for 1 h. The peptidoglycan fraction was collected by ultracentrifugation at 90,000 × g for 45 min at 25°C and the obtained peptidoglycan was resuspended in 10 mL of 0.5 M NaCl and 10 mL of 8% SDS. The peptidoglycan was mixed thoroughly and incubated at 96°C for 30 min. The peptidoglycan was then pelleted by ultracentrifugation at 90,000 × g for 30 min at 25°C and resuspended (twice) in 10 mL of sterile distilled water. Then, the peptidoglycan was resuspended in 10 mL of buffer II  and incubated at 37°C overnight. After incubation, 10 mL of 8% SDS was added and incubate for an additional 1 h at 96°C. The peptidoglycan was pelleted by ultracentrifugation at 90,000 × g at 25°C for 30 min and the obtained peptidoglycan fraction was resuspended (twice) in 10 mL of water. The pellet obtained in the final step was resuspended in 1 mL of water. The peptidoglycan was stored at 4°C until use.The bacterial peptidoglycan (PG) from n et al. . BrieflyThe hydrolysis of peptidoglycan at different concentrations of endolysin Gp105 was determined using a peptidoglycan hydrolysis assay . Brieflyg at 25°C for 30 min and the absorbance (A595nm) was measured. The graph was plotted using the absorbance measured after 1 and 4 h when the RBB-labeled peptidoglycan was incubated with buffer and endolysin Gp105.The peptidoglycan was labeled with Remazol Brilliant Blue (RBB-PG) as described earlier by Uehara et al.  and the E. cloacae) was grown in LB broth at 37°C to reach log-phase culture. The bacterial cells were collected by centrifugation at 6000 × g for 10 min and washed once with sterile distilled water. For EDTA treatment, the bacterial pellet was resuspended in LB broth and EDTA was added to 2 mM, and incubated for 5 min at 37°C. After incubation, the cells were centrifuged at 6000 × g for 10 min and washed twice with sterile distilled water. Then, the bacterial pellet was resuspended in LB broth and used for further experiments, with controls that were not treated with EDTA but otherwise processed identically. Well-diffusion assay was performed on LB agar plates. Here, 200 μL of up to 0.8 mg/mL of endolysin was added into mechanically excised holes, “punched” wells (radius: 1 mm) and incubated at 37°C for 16 h. The inhibition zone, where no growth was observed, was recorded.The anti-bacterial assay was performed as described previously . This asE. cloacae treated with 2 mM EDTA) were incubated with 0.05, 0.25 and 5 mg/mL of endolysin Gp105 in LB media at 37°C for 24 h. The optical density was measured at 600 nm at every 5 min interval for 4 h. In parallel, a 100 μL aliquot of bacterial cells was removed at 0, 2, 4, 6, 8, 12, 16, and 24 h, followed by determining the number of cells (CFU) by plate counting. All the experiments were repeated three times for statistical significance.A time course analysis of antibacterial activity was performed to determine the reduction of bacterial cell numbers over time. Briefly, log-phase bacterial cells (E. coli, K. pneumoniae, E. cloacae, A. baumannii, S. typhi, P. aeruginosa, S. marcescens, and Citrobacter sp. The four Gram-positive bacteria include two Enterococcus sp. and S. aureus.To test the broad-spectrum antibacterial activity of purified Gp105 protein, a total of 12 Gram-negative bacteria belonging to eight genera were chosen. This includes The MIC of endolysin Gp105 was determined to analyze the activity. Accordingly, the microbroth dilution method  was foll


P < 0.05). Compared with the control, the mRNA expression of orexigenic factor genes including neuropeptide Y (npy) and agouti-related protein (agrp) were significantly increased in larvae fed the diet with 0.005% GL, while the mRNA expression of anorexigenic factor genes including thyrotropin-releasing hormone (trh), cocaine and amphetamine regulated transcript (cart), and leptin receptor (lepr) were significantly decreased in larvae fed the diet with 0.005% GL (P < 0.05). The trypsin activity in larvae fed the diet with 0.005% GL was significantly higher than the control (P < 0.05). The alkaline phosphatase (AKP) activity in larvae fed the diet with 0.01% GL was significantly higher than the control (P < 0.05). A clear increase of total glutathione (T-GSH) content, activities of superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) was observed in larvae fed the diet with 0.01% GL compared with the control (P < 0.05). Moreover, the mRNA expression of interleukin-1β (il-1β) and interleukin-6 (il-6) (proinflammatory genes) in larvae fed the diet with 0.02% GL were significantly lower than the control (P < 0.05). In conclusion, the supplementation of 0.005% -0.01% GL could enhance the expression of orexigenic factor genes, activities of digestive enzymes and antioxidant capacity, ultimately improving the survival, and growth performance of large yellow croaker larvae.A 30-day feeding trial was conducted to determine the effects of dietary glycyrrhizin (GL) on survival, growth performance, expression of feeding-related genes, activities of digestive enzymes, antioxidant capacity, and expression of inflammatory factors of large yellow croaker larvae with an initial weight of 3.78 ± 0.27 mg. Four 53.80% crude protein and 16.40% crude lipid diets were formulated with supplementation of 0%, 0.005%, 0.01%, and 0.02% GL, respectively. Results indicated that larvae fed diets with GL had higher survival rate and specific growth rate than the control ( Glycyrrhizin (GL), also known as glycyrrhizic acid (GA), is an important effective component of licorice, which is a traditional Chinese herbal medicine –3. GL isLitopenaeus vannamei), Asian seabass (Lateolabrax maculatus), and blunt snout bream  prove that GL could improve the anti-inflammatory capacity [Hypophthalmichthys molitrix) [Several studies indicated that many main components of Chinese herbal medicines have the advantages of natural, nontoxic, and pollution-free and could be added into animal diets to increase the survival and growth performance of cultured animals. –18. In rcapacity –23. In politrix) .Larimichthys crocea) is popular in China [Because of the high nutritional and economic value, large yellow croaker  and isolipidic (16.40% crude lipid) microdiets were formulated with the supplementing levels of 0%, 0.005%, 0.01%, and 0.02% of GL, respectively . Pure GLμm nylon mesh. Then, the mesh of all ingredients was thoroughly mixed with water and fish oil to produce stiff dough. Extrude and round the dough with axial single screw spherical extruder and spheronization to obtain the pellet. Then the pellet was dried at 60°C in a constant temperature for 24 hours. After drying, the microdiets were ground into 250 to 380 μm and 380 to 500 μm particle sizes. Subsequently, the microdiets were packed in transparent and sealed plastic bags, respectively, and stored at -20°C until use. The particle size of the formulated diets ranged from 250 to 380 μm for fish from 15 to 25 days after hatch (DAH) and 380 to 500 μm for fish thereafter.Ingredients were ground into fine powder through 150 Brachionus plicatilis) (3-7 DAH), brine shrimp (Artemia nauplii) (5-10 DAH), copepods  (8-14 DAH), and microdiets from (12-14 DAH). During the experimental period of 15 to 45 DAH, larvae were completely fed with experimental diet, respectively. Larvae with the initial weight of 3.78 ± 0.27 mg and the initial body length of 5.98 ± 0.39 mm were randomly distributed into 12 breeding plastic tanks (water volume 220 L), and each tank had 2500 larvae. Each diet was randomly assigned to triplicate cages . During the experiment, larvae were fed seven times  daily. The water temperature was maintained between 23 to 25°C by heating rods and temperature controllers, the pH was maintained between 7.80 to 8.20 and the salinity was maintained between 23 to 27 g/L by renewed water in tanks. The concentration of dissolved oxygen in aquaculture waters should be kept at about 6 mg/L by the gas stones. The daily aquaculture water renewed was maintained between 100 to 200% in each tank.All larvae in this trial were purchased from Marine and Fishery Science and Technology Innovation Base, Zhejiang, China. Before the feeding trial, the larvae were acclimatized for 3 days by feeding live copepods and microdiets to adapt to the trial feed. Larvae were fed with rotifers , initial body weight (IBM) by vernier calipers, and microbalance. After the feeding trial, the survival rate (SR) was calculated by counting the surviving larvae in each tank. Meanwhile fifty larvae of 45 DAH were collected to measure the final body length (FBL) and final body weight (FBL) by vernier calipers and microbalance. The remaining larvae were sampled after 24 hours of fasting to the empty intestine. Thirty larvae from each tank were dissected on ice to separate the head segment (HS) and visceral mass (VM) containing a crude mixture of pancreas, liver, heart, spleen, and intestine. The HS and VM were put into 2.0 ml RNase-free Cryogenic Vials then immediately frozen in liquid nitrogen for gene expression assays. The pancreatic segments (PS) and intestine segment (IS) were separated maintained at 0°C under a dissecting microscope and placed on a glass plate for the assay of activities of digestive enzymes. The remaining larvae in each tank were immediately frozen for body composition analysis.The samples of diet and larvae were laid inside an oven at 105°C for about 24 hours and dried to constant weight for moisture calculation. The crude protein and lipid contents of diet samples and larvae samples were measured by the method of AOAC (2003) with automatic Kjeldahl apparatus  and Soxhlet extraction apparatus .α-amylase, trypsin, and lipase. The brush border membranes (BBM) of the intestine of larvae were extracted by the method of Crane et al. [α-amylase, trypsin, and LAP were detected by a colorimetry, while activities of lipase and AKP were detected by a microplate reader. The unit of α-amylase was (U/mg·prot), 1 U/mg · prot was defined as 10 mg starch hydrolyzed per mg protein at 37°C for 10 minutes. The unit of trypsin was (U/mg · prot), 1 U/mg · prot was defined as producing 1 μg of tyrosine at 37°C for 1 minute. The unit of lipase was (U/g · prot). 1 U/g·prot was defined as in this reaction system. 1 μmol substrate was consumed by reacting with the substrate for 1 minute. The unit of AKP was (K/g · prot). 1 K/g·prot was defined as 1 mg phenol produced per g protein at 37°C for 15 minutes. The unit of LAP was (U/L. 1 U/L) was defined as 1 nmol p-nitroaniline produced per L at 37°C for 1 minute. Activities of α-amylase, trypsin, lipase, and AKP were determined in strict accordance with the instructions of assay kit , and the activity of LAP was determined by the method of Maroux et al. [About 0.1 g PS or IS of larvae were weighed, separately homogenized in 1 ml PBS and centrifugated with a high-speed freezing centrifuge. The supernatant was prepared for the assay of e et al. . The BBMμmol H2O2 decomposed per mg protein per second. The unit of T-AOC was (mmol/g · prot), which was defined as total antioxidant per g protein. The unit of MDA was (nmol/mg · prot), which was defined as the content of MDA per mg protein. The unit of GSH-Px was (U/mg · prot). 1 U/mg · prot was defined as reducing 1 μmol/L GSH in the reaction system (deduct nonenzymatic reaction) per mg protein for 1 minute. The unit of T-GSH and GSH was (μmol/L). The unit of NO was (μmol/g · prot), which was defined as the content of NO per g protein. The unit of T-NOS and iNOS was (U/mg · prot). 1 U/mg · prot was defined as producing 1 nmol NO per mg protein for 1 minute. All of above enzymes were determined in strict accordance with the instructions of assay kit . Three replicates were made for each sample.About 0.1 g VM of larvae were weighed, separately homogenized in 1 ml PBS and centrifugated with a high-speed freezing centrifuge. The supernatant was prepared to detect activities of antioxidant and innate immune enzymes. Activities of superoxide dismutase (SOD), total antioxidant capacity (T-AOC), content of malondialdehyde (MDA), total glutathione (T-GSH), glutathione (GSH), and nitric oxide (NO) were detected by a microplate reader, activities of catalase (CAT), glutathione peroxidase (GSH-Px), total nitric oxide synthase (T-NOS), and inducible nitric oxide synthase (iNOS) was detected by a colorimetry. The unit of SOD was (U/mg · prot). The amount of enzyme corresponding to the sod inhibition rate of 50% in this reaction system was 1 activity unit. The unit of CAT was (mmol/g · prot). 1 mmol/g · prot was defined as the amount of 1 β-actin. All of the primer sequences in this study were synthesized or designed on the basis of the published sequences from Huang et al. [μl in total containing 10 μl SYBR® Premix Ex Taq™ II , 7 μl of RNase-free water, 2 μl of cDNA, 0.5 μl of upstream, and downstream primers. The program of qPCR was as follows: 95°C for 2 mins, followed by 39 cycles of 95°C for 10 s, 59°C for 10 s, and 72°C for 20 s. The mRNA expression of genes was calculated with the previous method (2ΔΔCT–) [About 0.03 g HS or VM of larvae were weighed and separately added into 1 ml Trizol reagent . Total RNA was extracted according to the manufacturer's protocol. The integrity of RNA was evaluated by electrophoresis. After that, the total concentration of extracted RNA was measured by Nano Drop® 2000 spectrophotometer . Then, the RNA was reversed to cDNA with Prime Script-RT reagent kit . Finally, the RT-qPCR was carried out by a quantitative thermal cycler . The reference gene in this experiment was g et al. , Wu et ag et al. , and Gen(2–ΔΔCT) . Three rP <0.05. All results were presented as means ± S.E.M (standard error of the mean).SPSS Statistics 19.0 was used to conduct all data in this study, and then one-way analysis of variance (ANOVA) and Tukey's test were used to analyze the data. The significance level was selected as P < 0.05). Compared with the control, the FBL, FBW, WGR, and SGR were significantly increased in larvae fed diets with GL (P < 0.05). Furthermore, the FBW and SGR of larvae fed the diet with 0.005% GL were significantly higher than larvae fed diets with 0.01% and 0.02% GL (P <0.05). There were no significant differences among dietary treatments in the body composition (P > 0.05)  .npy and agrp increased followed by decreased (npy and agrp were remarkedly increased in larvae fed the diet with 0.005% GL compared with the control (P < 0.05). For the anorexigenic factor genes, with the increase of GL from 0% to 0.02%, the mRNA expression of trh, lepr, and cck showed a decreasing trend. The mRNA expression of trh and lepr of larvae fed diets with GL was significantly lower than the control, while the mRNA expression of cck in larvae fed the diet with 0.02% GL was significantly lower than the control (P < 0.05). The mRNA expression of cart was significantly decreased in larvae fed diets with GL compared with the control (P < 0.05). No significant differences were found among dietary treatments in gene expression of ghrelin, pomc, and leptin (P > 0.05).For the orexigenic factor genes, with GL level in the micro-diet increasing from 0% to 0.02%, the mRNA expression of ecreased . The mRNα-amylase, and lipase in both PS and IS of larvae increased firstly and then decreased (P < 0.05). Meanwhile, larvae fed the diet with 0.005% GL had significantly higher activity of α-amylase and lipase in IS than the control (P < 0.05). However, no significant differences were found among dietary treatments in activities of α-amylase and lipase in larval PS (P > 0.05). For intestinal development index, larvae fed diets with GL had significantly higher activity of AKP in brush border membranes than the control (P <0.05). No significant differences were detected in the activity of LAP among dietary treatments (P >0.05).For digestive enzymes activities, with the increase of GL in diets, activities of trypsin, ecreased . Larvae P < 0.05). The activity of GSH-Px in larvae fed the diet with 0.01% GL was remarkedly increased compared with the control (P < 0.05). The content of T-GSH in larvae fed diets with 0.01% and 0.02% GL was significantly higher than the control (P < 0.05) (P >0.05)  . However Figures .P < 0.05) (P < 0.05). However, there were no significant differences among dietary treatments in iNOS (P > 0.05). For the proinflammatory cytokine genes, with increasing GL supplementation, the mRNA expression of thf-α, inf-γ, il-1β, and il-6 showed decreasing trends, while the mRNA expression of il-8 increased firstly and then decreased (il-1β and il-6 in larvae fed the diet with 0.02% GL were remarkedly decreased compared with the control (P < 0.05). However, the mRNA expression of tnf-a, ifn-γ, and il-8 showed no significant difference among different treatments (P >0.05). For the anti-inflammatory cytokine genes, larvae fed diets with GL had significantly higher mRNA expression of il-10 than the control (P <0.05).With the increase of GL from 0% to 0.02%, the NO content, T-NOS activity, and iNOS activity in larvae ascended firstly and then descended Figures . The con < 0.05) . Larvae ecreased . The mRNPelteobagrus fulvidraco) [The main components of many Chinese herbal medicines can be used as feed additives to improve the survival and growth performance of cultured animals , 10, 32.vidraco) . These rnpy, agrp, and ghrelin) and anorexigenic factor genes  [npy and agrp, while GL supplementation could decrease the mRNA expression of trh, cart, and lepr. Similarly, licorice extract supplementation had been confirmed to increase the growth performance in poultry by stimulating digestion and appetite [In the early stage of growth, appetite stimulation plays a vital role in increasing the survival and growth performance of larvae , 43. Theand cck) –46. The appetite . In geneα-amylase activity, and the lipase activity in intestinal segments. Similarly, silver carp  fed the diet with GL had higher digestive enzymes activities [Hypophthalmichthys molitrix) [Digestive enzymes activities are widely used as an index to study the nutritional status of fish larvae. –52. In ttivities . Especiaolitrix) . In summLarvae are in a period of rapid growth and are prone to oxidative stress, which will produce oxygen free radicals, eventually leading to body damage –56. The il-1β and il-6) and increase the mRNA expression of anti-inflammatory factor (il-10), which probably due to the higher proportion of GL supplementation could better reflect the anti-inflammatory capacity of GL. Similar results have been confirmed in studies of silver carp [The immune system of larvae was undeveloped, which lead to high mortality in the larval stage –60. In tver carp , Asian sver carp , blunt sver carp , and yelver carp . In summIn conclusion, results of the present study confirmed that the supplementation of 0.005% -0.01% GL could enhance the expression of orexigenic factor genes, activities of digestive enzymes, and antioxidant capacity, while the supplementation of 0.02% GL might alleviate inflammation.