137 L

From January 2011 to October 2014, 120 patients with colorectal adenocarcinoma (according to the 7^th edition of the American Joint Committee on Cancer Staging) staged I-III were collected at Hainan Hospital of PLA General Hospital. Patients with the following criteria were excluded: (1) age < 18 years old; (2) a history of previous T2DM or elevated FBG beyond the upper limit (ref: 3.4-6.1 mmol/L); (3) multiple or recurrent malignancies or in situ lesions; (4) a history of previous neoadjuvant therapy; (5) complications such as infection, obstruction, and bleeding after surgery; (6) unsuccessful oral feeding within 14 days after the operation; and (7) no record of postoperative FBG or a follow-up date. Clinicopathological parameters included age (<60 or ≥60 years old), sex, BMI, CEA (ref: 0-5 μg/L), and CA19-9 (ref: 0.1-37 μg/mL) values, and the results of routine blood tests including hemoglobin (HGB; males: 137-179 g/L and females: 116-155 g/L), absolute white blood cell (WBC) counts (ref: 3.5-10⁹/L), monocyte (MON) counts (ref: 0.10-0.8⁹/L), platelets (PLT; 100-300⁹/L), and serum albumin (ALB; ref: 35-50 g/L) were recorded before the operation. In addition, pathological results regarding tumor location, histological grade, invasive depth, maximum tumor diameter, etc., were recorded. Postoperative adjuvant therapies were recorded. The study was supervised by the ethics committee of Hainan Hospital of PLA General Hospital (approved ID: 301HLFYLL15), and written informed consent was not needed since this was a retrospective study.

Commercial human umbilical vein endothelial cells (HUVEC) were bought from the European Collection of Cell Cultures (ECACC, Porton Down, Salisbury, UK) and multiplied in RPMI medium, supplemented with 10% fetal calf serum (FCS), gentamicin 50 μg/ml, and amphotericin 100 μg/ml (Biochrom AG, Berlin, Germany) in a humidified CO₂ incubator at 37°C. Cell cultures in the 23rd to 26th passages were used. The analysis of surface markers was performed by flow cytometry (BD FACS Canto II flow cytometer, Becton Dickinson & Company, Franklin Lakes, NJ, USA) and used different monoclonal antibodies (ICAM-1, CD29, CD34, CD73, CD90, and CD105).
Cells seeded at a density of 10⁴/cm² in cell culture Petri dishes (TPP) were settled for 24 h in medium then exposed for 24 h to either Equisetum arvense L. extract (25 or 2.5 μg/ml), caffeic acid (10 or 1 μg/ml), and cathechin (10 or 1 μg/ml), as positive controls; afterwards, the cells were washed 3 times with PBS and incubated for 24 h either in normotonic (137 mmol/l) or hypertonic conditions (200 mmol/l). Cells were collected by scraping and treated with a lysis buffer containing IGEPAL-Nonidet 1% (Sigma) and 1% protease inhibitor complex (Sigma) in PBS for 1 h, on ice. The protein content was determined by the Bradford method (Bio-Rad, USA). All the experiments were performed in triplicate.

The activity of ABC-transporters is generally modulated at gene transcriptional level: the presence of toxic compounds leads to higher transcription. In order to assess this topic, larvae of An. stephensi at the third instar were exposed to permethrin and the expression of ABC-transporter genes was monitored in the surviving larvae by quantitative RT-PCR twice after insecticide treatment: 24 h (e.g. the time at which the LD50 has been estimated) and 48 h following the study of Figueira-Mansur
[29 (link)] that found increased expression of ABC transporters in the mosquito Ae. aegypti. The larvae were treated with the LD50 (0.137 mg/l) of insecticide estimated by bioassays as described above and two pools, of ten larvae each, were collected after 24 and 48 h of insecticide-treatment. All pools of larvae were stored in RNAlater for molecular analysis and, controls (water + acetone) were collected following the same time frame.
RNA was extracted from each pool of larvae using the RNeasy Mini Kit (Qiagen, Hilden, Germany) including an on-column DNase I treatment (Qiagen, Hilden, Germany), according to the manufacturer’s instructions. Total RNA was eluted into nuclease-free water and the concentration of RNA was determined at 260 nm
[30 (link)] using a NanoDrop ND-1000 (Thermo Scientific, Delaware, USA). cDNAs were synthesized from 250 ng of total RNA using a QuantiTect Reverse Transcription Kit (Qiagen, Hilden, Germany) with random hexamers. The cDNA was used as template in RT-PCR reactions using the primers designed from the sequences of identified ABC genes (Table 
1). The amplification fragments, obtained using standard PCR conditions and the thermal profile indicated below, were sequenced in order to confirm the specificity of the amplification.
Quantitative RT-PCRs on the target ABCs were performed using a BioRad iQ5 Real-Time PCR Detection System (Bio-Rad, California, USA), under the following conditions: 50 ng cDNA; 300 nM of forward and reverse primers; 98°C for 30 sec, 40 cycles of 98°C for 15 sec, 59°C for 30 sec; fluorescence acquisition at the end of each cycle; melting curve analysis after the last cycle. The cycle threshold (Ct) values were determined for each gene, in order to calculate gene expression levels of target genes relative to rps7, the internal reference gene for An. stephensi[31 (link)]. The expression of the ABC transporters genes in the control group was considered as the basal level (equal to 1). The estimates of the expression level of each gene in the treated larvae are reported as the means ± standard deviation (SD) in Additional file
1: Table S1.

ACS-grade sodium nitrate (NaNO₃), dipotassium phosphate (K₂HPO₄), and ammonium chloride (NH₄Cl) were used and purchased from Sigma Aldrich, ON. The pH was corrected with 0.5 N nitric acid (HNO₃) and 0.5 N sodium hydroxide (NaOH) solutions purchased from Fisher Scientific. Sophorolipid (SL) biosurfactant was produced from Candida bombicola cultivated on a mixture of rapeseed oil and glucose [1 ,2 (link),3 (link),4 (link),5 (link),8 (link),47 (link)] and purchased from Belgium’s Ecover Company. It was composed of 30% acidic SL and 70% lactonic SL. Concentrations of 137.1 mg/L NaNO₃, 183.4 mg/L K₂HPO₄, and 296.5 mg/L NH₄Cl in double distilled water were prepared to obtain a stock solution of 100.0 mg/L NH₄⁺, PO₄³⁻, and NO₃⁻. The decrease in NH₄⁺, PO₄^3−, and NO₃⁻ by sophorolipid at various pHs and sophorolipid concentrations was studied in batch studies. To achieve equilibrium, the prepared samples were agitated at 60 rpm for 24 h, then centrifuged and analyzed.
In this work, we attempted to maximize the percentage of nutrient removal under suitable test conditions by maintaining appropriate permeate flux and maximizing the factors influencing permeate flux. For this, experiments were carried out at a flow rate of 200 mL/s by keeping the peristaltic pump at 70 rpm, the transmembrane pressure at 120 kPa with a molecular weight cut-off (MWCO) of 10,000, and an initial ion concentration of 100.0 mg/L at 22 °C, with a sophorolipid concentration of 0.30%.

All nutrients of the dietary treatment met or exceeded the NRC requirements [28 ]. The formula and chemical composition of the experimental diet are presented in Table 1 as follows: (1) a control diet with antibiotics as a growth promoter (AGP) (amoxicillin and colistin) at 300 mg/kg from the suckling phase to the grower phase and no supplement during the finisher phases; (2) a control diet without antibiotics as a growth promoter (NAGP); (3) a control diet with HK L-137 at 20 mg/kg from the suckling phase to the starter phase and no supplement from the grower to the finisher phases (HKL1); and (4) a control diet with HK L-137 at 20 mg/kg from the suckling phase to the weaner phase, at 4 mg/kg from the starter phase to the finisher 1 phase, and no supplement during the finisher 2 phase (HKL2).

Heat-killed Lactobacillus plantarum L-137 (HK L-137) was prepared based on the method previously described [21 (link)]. In this study, LP Pro^TM (House Wellness Foods Corp, Itami, Japan) that contained 10% of HK L-137 and 90% of whey protein, dextrin, and sunflower lecithin was used. It contained 1 × 10¹¹ cfu/g of Lactobacillus plantarum in the dry product stored at room temperature (information from product instruction).