Müggelsee is a polymictic eutrophic lake located in the eastern suburbs of Berlin, Germany (52°25′–52°27′N, 13°37′–13°41′E). The lake has a mean depth of 4.9 m (max. 8 m) and a surface area of 7.3 km2 (Kohler and Hoeg, 2000 ). Every week between January 2015 and December 2019 (biweekly in January–February 2015), plankton were sampled from five locations in Müggelsee using a 5 L Friedinger sampler (Hydro-Bios Apparatebau GmbH, Kiel, Germany) and then combined to capture potential spatial heterogeneity in plankton communities. From this combined sample, a subsample (50–500 mL depending on plankton density as estimated by eye) was filtered through a glass fibre filter (Whatman GF/F, 25 mm diameter, 0.7 μm pore size) using vacuum filtration at 200 mbar. All filters were freeze-dried (Alpha 1–4, Martin Christ Gefriertrocknungsanlagen GmbH, Osterode am Harz, Germany) for 8 h at −45°C and then stored at −20°C until DNA extraction.
Alpha 1 4
Manufactured by Martin Christ
Sourced in Germany
The Alpha 1-4 is a laboratory centrifuge designed for general-purpose applications. It offers a compact and robust construction with a capacity of up to 4 x 100 mL sample tubes. The centrifuge provides a maximum speed of 4,500 RPM and can achieve a maximum RCF of 2,900 x g. The Alpha 1-4 is a reliable and versatile instrument suitable for a wide range of laboratory procedures.
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Lab products found in correlation
Ultra turrax, by IKA Group (1 mentions)
Xl30 esem feg, by Philips (1 mentions)
Jem 2010, by JEOL (1 mentions)
Jsm 5600lv, by JEOL (1 mentions)
Amicon ultra 4, by Merck Group (1 mentions)
Laborota 4000, by Heidolph (1 mentions)
D8 advance, by Bruker (1 mentions)
Multi element standard solution, by Merck Group (1 mentions)
Model 7900, by Agilent Technologies (1 mentions)
21 protocols using alpha 1 4
1
Plankton Sampling in Müggelsee, Germany
2
Quantification of Target Molecules
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DoE experiments 6, 9, and 15 were repeated with selected strains (N5670c, N4994d, N6000b, N1001, N5589c, N5594d, N5658a, 5996, N2820a, 5999, N6006d, 5985) to quantify the target molecules on a CDW basis. They were performed similarly to the DoE but in multiple executions for a higher biomass yield. The biomass was harvested, measured, filled into micro-reaction tubes, and frozen at −80 °C before freeze-drying (Alpha 1–4, Christ, Osterode, Germany) for 24 h at 37 Pa. The weight of the biomass was recorded before and after drying, and CDW was calculated. Extraction and measurement of the target molecules were performed as described above.
3
Quantitative Determination of Bacterial IAA
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The cultures were grown in Okon’s malate medium (34 (link)) added with tryptophan (100 mg/ L) as the precursor of indole-3-acetic acid. The cultures were incubated in a shaker at 160 x g at 30±2 oC for a week. The production of indole-3-acetic acid by the bacterial isolates was qualitatively determined by using Fe-HClO4 and Fe-H2SO4 reagents. For quantitative estimation, bacterial cells were harvested. The supernatant obtained by centrifugation at 8000 rpm for 8 minutes at 10 oC. The supernatant was reduced in volume from 70 to 15 mL using freeze dryer (Martin Christ, Alpha 1–4, Germany). The pH of the sample was adjusted to 2.8. Indole acetic acid was extracted by Tien et al. (35 (link)) method. Equal volume of ethyl acetate was added to cell free liquid culture medium (supernatant) and mixed in a separating funnel. Ethyl acetate fraction was then evaporated to dryness at room temperature in fume hood. The residues of each fraction were dissolved in 1 mL of ethanol. The samples were analyzed on HPLC using Turbochom software (Perkin Elmer, USA). The elution was performed by using licosorb-C18 column for IAA. Ethanol: acetic acid: water (30:1:70) was used as mobile phase at the rate of 1 mL/ minute for 30 minutes. IAA absorbance was detected on a UV detector at 280 nm wavelength. The concentration of IAA was calculated on the basis of peak height and peak area.
4
Quantifying Bone Marrow Dry Weight
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For each control and experimental group, five OCAs were assessed for dry weight as an indicator of the weight of bone marrow elements after removal. The OCAs were collected and immediately frozen at −80°C for 4 hours, placed in a vacuum freeze drier (Alpha 1–4, Christ, Germany), and then processed according to standard drying procedures. After 24 h, the OCAs were measured using an electronic balance.
5
Phytochemical Fractionation and Extraction
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The milled inflorescences (1.9 kg) were defatted with n-hexane by dynamic maceration for three days, with subsequent drying of the inflorescences at room temperature. Afterwards, acetone was used as extraction solvent at a proportion of 4% (w/v) in an Ultra-Turrax® (UTC115KT, Ika Works) for 5 min and then subjected to maceration for 15 h. Next, turbo-extraction was performed for 20 min, with intervals of 5 min (t < 40°C). The extract was filtered, concentrated under reduced pressure, frozen, and lyophilized (Alpha 1–4, Christ®) to give the crude acetone extract (CAE, 5.86%). CAE was fractionated according to Filho and Yunes [30 (link)]. Briefly, 105 g CAE was resuspended in 1 L of methanol : water (2 : 8, v/v) and partitioned with different solvent volume ratios. The yields were n-hexane (HF) 19.27%, dichloromethane (DF) 10.17%, ethyl acetate (EAF) 13.38%, n-butanol (BF) 36.59%, and aqueous (AQF) 15.02%.
6
Scanning Electron Microscopy of Mineralized Fiber Scaffolds
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For scanning electron microscopy (SEM), the cBSA- and BSA-fibers colonized with MSCs were fixed with 2.5% glutaraldehyde for 10 min and dehydrated in an increasing ethanol series (50% (v/v), 60% (v/v), 70% (v/v), 80% (v/v), 90% (v/v), two times 100% (v/v)) for 10 min each. Afterwards, the samples were frozen at −80 °C in 100% (v/v) ethanol and freeze-dried for 24 h (Alpha1–4, Martin Christ Gefriertrocknungsanlagen, Osterode am Harz, Germany). Scanning electron micrographs were obtained, using a Philips XL 30 FEG ESEM operated at an acceleration voltage of 10 kV at high vacuum or 20 kV at a chamber pressure of 133 Pa (1 Torr). To improve image quality, the fibers with and without cells were sputtered with a thin conductive layer (5 nm Au/Pd 80/20) using a BAL-TEC MED020 coating system. SEM images were pseudo-colored by using PS Adobe Photoshop CS5 Extended Version 12 (Dublin, Ireland). For energy dispersive x-ray (EDX) elemental analysis of the mineralized fibers a liquid nitrogen cooled Sapphire Si(Li) detecting unit from EDAX (Mahwah, USA) was used. For spectra collection, the microscope was operated at 20 kV using a 100 µm aperture. The measurement time was 120 s.
7
Chelerythrine Effects on Bacterial Morphology
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Chelerythrine (1 × MIC) was added with bacteria at the concentration of 109 CFU/mL for a shaking Table (150 r/min) for 1.5 h and 6 h at 37 °C, respectively. Distilled water was used as control. The depositions were collected by centrifugation and fixed with 2.5% glutaraldehyde at 4 °C overnight, and washed three times with 0.1 M phosphate buffer solution (PBS, PH 7.2). The samples were dehydrated in a graded series of alcohols (30, 50, 70, 85, 90 and 100%). Tert-butanol was used to replace the ethanol twice before coating onto the metal foil. Eventually, the cells were dried by freeze-drying apparatus (ALPHA1–4, Christ, Germany), and visualized under a scanning electron microscopy (SEM, JSM-5600LV, JEOL, Japan) and a transmission electron microscopy (TEM, JEM-2010, JEOL, Japan), respectively.
8
Synthesis and Characterization of Gal-DHLA-AuNCs
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To synthesize the probes for the in vitro and in vivo tests, DHLA-AuNCs (8.6 × 10−3 μmol) and D-(+)-Galactosamine hydrochloride (0.95 μmol) were dissolved and mixed in ddH2O; then, N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) (1 μmol) was added, and the mixture above was left at 20 °C to continue the reaction for 1 min. Then, methoxypolyethylene glycol amine (0.05 μmol) was added and was allowed to continue to react for 2 h, and the products were noted as Gal-DHLA-AuNCs.
The free ingredients were removed using centrifugal filter devices (Amicon Ultra-4, Millipore, Darmstadt, Germany), and the probes were lyophilized and stored at 4 °C. Fourier transform infrared (FTIR) spectroscopy was conducted to confirm the conjugation.
The Fourier transform infrared (FTIR) spectra were acquired on a NEXUS-470 FTIR spectrometer (Nicolet, Wisconsin, WI, USA) using KBr pellets in the range from 4000 to 400 cm−1. Samples were lyophilized using a freeze-drier (Alpha 1-4, Christ, Osterode, Germany). Data were collected at a resolution of 8 cm−1 with 256 scans.
The free ingredients were removed using centrifugal filter devices (Amicon Ultra-4, Millipore, Darmstadt, Germany), and the probes were lyophilized and stored at 4 °C. Fourier transform infrared (FTIR) spectroscopy was conducted to confirm the conjugation.
The Fourier transform infrared (FTIR) spectra were acquired on a NEXUS-470 FTIR spectrometer (Nicolet, Wisconsin, WI, USA) using KBr pellets in the range from 4000 to 400 cm−1. Samples were lyophilized using a freeze-drier (Alpha 1-4, Christ, Osterode, Germany). Data were collected at a resolution of 8 cm−1 with 256 scans.
9
Fecal Composition Analysis Protocol
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The stools were rapidly frozen at −80°C and freeze-dried in a lyophilizer (Alpha 1–4; Martin Christ, Osterode, Germany) for 48 h. Fecal water content was calculated by the weight difference between fresh stools (weighed immediately after collection) and dried stools. The dried stools were then ground and used for the analysis of protein, fat, total dietary fiber and ash using the AOAC official methods, 955.04, 920.39C, 985.29, and 923.03, respectively [26] .
10
Extraction and Preparation of Alpinia nitida Leaf Extract
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Dried leaves of A. nitida, sourced from Pingle city (Guangxi Province, China), were ground using a pulverizer (LX-04, Xi'an Jinzhen Machinery Co., Ltd, China) to acquire 40-mesh sieve powder. About 30 g of dried A. nitida leaf was dispersed in 900 mL of 70% ethanol with magnetic stirring by a water bath pot (HCJ-4E, Lang Yue Instrument Manufacturing Co., Ltd, Changzhou, China) at 50 °C for 1 h and then centrifuged at 4500 rpm at room temperature for 10 min by a centrifuge (LD5-10, Beijing Jingli Centrifuge Co., Ltd, Beijing, China). The supernatant was concentrated under vacuum at 45 °C and freeze-dried (Alpha 1–4, Christ, Germany) to produce the A. nitida leaf extract, ANE.
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