Protein three-dimensional structure dynamically changes in solution depending on the presence of ligands and interacting proteins. Methods for detecting these changes in protein conformation include ‘protein footprinting,’ using mass spectrometry. We describe herein a new technique, PLIMB (Plasma Induced Modification of Biomolecules), that generates µs bursts of hydroxyl radicals from water, to measure changes in protein structure via altered solvent accessibility of amino acid side chains. PLIMB was first benchmarked with model compounds, and then applied to a biological problem, i.e., ligand (EGF) induced changes in the conformation of the external (ecto) domain of Epidermal Growth Factor Receptor (EGFR). Regions in which oxidation decreased upon adding EGF fall along the dimerization interface, consistent with models derived from crystal structures. These results demonstrate that plasma-generated hydroxyl radicals from water can be used to map protein conformational changes, and provide a readily accessible means of studying protein structure in solution. After labeling, proteins are analyzed using tandem MS and linkages or adducts are quantitatively mapped to specific regions, revealing areas and degrees of intermolecular interactions or solvent accessibility. The emergence of the quickly expanding protein therapeutics industry2 has generated a need for easy, sensitive, and reproducible methods for detecting changes in protein conformation, as part of the rigorous quality control process required for clinical use and for quickly detecting the regions of proteins involved in their binding to ligands and/or protein-protein interactions.The three-dimensional structure of proteins can be studied in solution using various methods of covalent and noncovalent labeling in which the readout is accomplished by mass spectrometry (MS). These procedures, collectively known as protein ‘footprinting’, all involve measuring the solvent accessibility of either the peptide backbone (hydrogen deuterium exchange) or amino acid side chains 3. HDX is so widely used that there now exist automated systems to improve throughput and reproducibility However, HDX is not technically easy to perform: time-sensitive back-exchange between deuterium and naturally abundant hydrogen occurs, so the technique must be rapidly performed at very low temperatures. In addition, HDX relies upon acidic conditions for quenching the exchange reaction, necessitating the use of the non-specific protease, pepsin, for digestion. Finally, the solvent accessibility information obtained is tied to the peptide backbone, rather than to amino acid side chains and while important, may not provide a complete picture of the protein’s three-dimensional state.One of the more prevalent means of noncovalent protein footprinting is Hydrogen Deuterium Exchange (HDX), which utilizes deuterium exchanged with hydrogen atoms along the peptide backbone to measure solvent accessibility4, trypsin-based methods for detecting changes in accessibility to proteolytic enzymes5, highly reactive carbenes6, and hydroxyl radicals1. Hydroxyl radical footprinting (HRF) for protein structure analysis is mainly performed with two well-known methods of generating hydroxyl radicals: fast photochemical oxidation of proteins (FPOP)7 and synchrotron-based radiolysis of water to form hydroxyl radicals8. It should also be noted that electrical discharge has previously been used for radical generation, but it has been limited to electrospray devices and has not been widely adopted10.Protein footprinting performed with chemical reagents, while similar in principle to HDX, results in covalent modification of amino acid side chains. A number of reagents for this purpose are available, such as EDC/GEE for carboxyl labeling7. Though both techniques can be used for labeling experiments, each has drawbacks that limit widespread adoption. Synchrotron experiments generate hydroxyl radicals via radiolysis of water and thus do not involve added chemicals8, but they nonetheless require access to one of the handful of major synchrotron radiation source facilities and with the current technologies labeling occurs on ms timescale9. While FPOP can be employed on a benchtop scale, the technique requires hydrogen peroxide and exposure to a 248 nm laser, within the absorption range of aromatic residues. Additionally, there is evidence that an FPOP-induced oxidative environment may occur over a millisecond, rather than a microsecond timescale11, and this longer-term exposure could potentially perturb protein structure, particularly in proteins that are rapidly altering their structure in solution.Whereas synchrotron radiation generates radicals directly from water, FPOP uses a 248nm-excimer laser to generate radicals from hydrogen peroxide added to a sample prior to exposure12. There is thus great interest in obtaining a firm handle on its three-dimensional structures under different conditions in solution. While a crystal structure of full-length EGFR has yet to be solved, various domains, including the externally located (ecto) domain that binds EGF on the outside of the cell, have been crystallized and had structures solved at high resolution. Given the great interest in this protein as both a diagnostic and therapeutic tool, we have tested our method using plasma-generated hydroxyl radicals to perform footprinting with the ecto domain of EFGR before and after the ligand, EGF, is bound.In this report, we describe a technique termed PLIMB (Plasma-Induced Modification of Biomolecules), in which HRF is performed using a plasma to generate µs pulses of hydroxyl radicals, from water, in solution. After calibrating this technology with free methionine, we then studied its use with bovine serum albumin (BSA), to benchmark PLIMB with a structured model protein. For a new application with a protein of importance in basic biological research as well as in clinical settings, PLIMB was then used to analyze which amino acids are involved in the conformational changes that occur when epidermal growth factor (EGF) is added to epidermal growth factor receptor (EGFR). EGFR is particularly important as a therapeutic target for cancer treatments10. In this report, we describe a technique geared towards biological experiments in which hydroxyl radicals, generated from plasma on a µs timescale, were used to label proteins dissolved in lightly buffered aqueous solutions, for solvent accessibility measurements. To do this, an instrument was built that generates a plasma, which is directed through a liquid sample, using a dielectric-barrier discharge  in solution suggested that PLIMB could also label proteins in solution. This was tested using Bovine Serum Albumin (BSA), a well-studied ~66 kDa protein with a known tertiary structure. BSA was dissolved in phosphate buffered saline at a concentration of 50uM, and treated with PLIMB for up to 60 seconds. We observed dose-dependent modification in multiple discrete regions of the protein, and oxidation on multiple amino acids Fig. , while BAny method using covalent protein footprinting reagents has the potential to perturb the tertiary structure and thus, we tested whether or not PLIMB maintains protein structure throughout exposure by repeating the experiment, but using BSA that has been digested with Trypsin/Lys-C prior to analysis (predigestion). Protein loads, solution buffers, and exposure times were controlled between experiments. If PLIMB is not perturbing the protein’s structure, and the less accessible residues remain buried inside its structure, heavier oxidation should be observed when we denature BSA prior to exposure. Indeed, the data revealed that denaturation via predigestion causes more oxidation to occur on many amino acids in BSA with PLIMB treatment Fig. . Importain vivo EGFR functionality involves 2 molecules of EGF binding to 2 molecules of EGFR, inducing homodimerization and ultimately stimulating a signaling cascade with multiple downstream responses14. Using the extracellular (ecto) domain of purified human EGFR and EGF, we mapped structural changes induced by allowing the protein and ligand to interact in vitro. A five and ten second PLIMB treatment of µs-long pulses was used, and oxidation was mapped and quantified in amino acids throughout EGFR treated with and without added EGF. After trypsin digestion and MS/MS analysis, we observed that a discrete subset of tryptic peptides showed statistically significant decreases in the plasma-induced oxidation state upon EGF addition , a protein with implications in many cancers, before and after binding one of its native ligands, epidermal growth factor (EGF). The currently accepted model for ion Fig. , suggest15  and secondary (+32 and −32) product formation rates. Constructing a concentration curve using purified methionine sulfoxide revealed that, based on the appearance of methionine sulfoxide over time, PLIMB is generating hydroxyl radicals at a rate of roughly 350 nmol/sec via spaced µs discharge bursts. This is taken to be the minimum value for radical generation, as we cannot rule out unobservable product generation  and radical recombination as side reactions that at this time have not been measured.PLIMB, which is workable on a benchtop, applicable to a range of protein concentrations and sizes, and generates µs bursts of hydroxyl radicals without added chemicals or reagents Fig.  has beenPLIMB was next used to treat a readily available model protein, bovine serum albumin (BSA), in solution. Two experiments were done with BSA–PLIMB treatment up to 60 seconds with BSA in its native, folded state, and alternatively, with BSA denatured by predigestion with trypsin prior to PLIMB exposure. The results suggest that PLIMB does not perturb protein structure during these exposure times, for several reasons. First, there are regions that exhibit virtually no modification in the native BSA that, when digested, become susceptible to oxidation with PLIMB treatment, such as peptides containing residues 310–318 and 387–399 Fig. . Second,17  domain of EGFR has been solvedted Fig. , and pre66 Table . Thus, tOther regions and residues that have not been implicated in dimerization were also revealed to have their oxidation states decreased when EGFR dimerizes. These include localized areas containing residues W140/R141, and W453/K454. We hypothesize that these residues may experience less solvent accessibility due to force exerted through the molecule upon dimerization, leading to a slightly more closed conformation in these areas. For example, W140/R141 lie in proximity to the region containing I190/C191, Q193-C195, and D206/C207, all of which have less oxidation upon dimerization, suggesting there may be a tightening of this region in solution when dimerized. Altogether, the PLIMB derived EGFR data suggest minor differences in solvent accessibility when compared to the structure of EGFR solved by crystallographic techniques, though areas revealed to undergo large conformational changes, and to be important for both EGF binding and activation upon EGF-induced dimerization, are similarly identified using both techniques.In summary, the techniques currently available for hydroxyl radical protein footprinting  are powerful, but they have yet to be implemented to a widespread degree. Here, we present PLIMB, a technique that generates µs bursts of hydroxyl radicals from water, for protein footprinting and structural feature determination in solution using an instrument that involves relatively low cost electronic components and is easy to operate. PLIMB was used to map the dimerization of EGF-induced EGFR dimerization, yielding results consistent with published crystal structures and demonstrating its promising utility for quick mapping of protein-protein interactions for applications in the clinical protein therapeutics field, as well as for basic research on the three-dimensional structure of proteins in solution under a wide number of conditions.Plasma treatments were performed in 0.25 mL PCR tubes, and sample volumes were 100 µL. Samples were kept on ice prior to and following plasma treatment, unless otherwise specified, and for treatment were transferred to an in-lab-built Peltier cooling block set to cool to between 2° and 4 °C. All treatment times are listed in methods below.L-methionine (Sigma M9265) was dissolved into 50 mM ammonium acetate  at a concentration of 50 µM and treated with a dose curve of plasma from 0 to 60 seconds, with three replicates per timepoint shown. The same stock of ammonium acetate, lacking methionine, was treated in the same fashion for background subtraction during processing. 2.5 µL per sample was directly injected into an ESI-TOF mass spectrometer (Agilent 6210 G1969A) at a flow rate of 40uL/min and a flow buffer of 50:50 acetonitrile:water. Data was collected over the sample injection peak and the corresponding treated background/blank described above. Instrument parameters were as follows: for ESI, voltage was set to 3.5 kV, gas temp was set to 325 °C, drying gas was set to flow at 9.0 l/min, nebulizer was set to 30 psig. For TOF, fragmentor was set to 130 V and skimmer was set to 60 V. For TOF data acquisition in positive mode, scan range was 50–3200 m/z, in profile mode, with 10,000 transients/scan and 0.89 cycles/second. Data was analyzed with Analyst QS (Sciex). Scans were averaged over the injection peak and subsequent treated blank injection per sample, and the treated blank injection peak was used for background subtraction to result in the signal intensity for the peak at m/z 166 as shown. L-methionine sulfoxide (Sigma M1126) was used to build a concentration curve analyzed using the same parameters as listed above. Concentrations and signal intensities are shown in Fig X.BSA (Sigma A7906) was dissolved into PBS  at a concentration of 50 µM. Samples were treated for 0, 15, 30, or 60 seconds, with three replicates per sample. Following exposure, protein was precipitated with ~10% TCA for 30 minutes on ice and pelleted by centrifugation at 4 °C and 16Kxg for 10 minutes. Supernatant was removed and 100 µL ice cold acetone was added and the solution was vortexed. The sample was pelleted by centrifugation at 4 °C and 16Kxg for 10 minutes. The supernatant was then removed, and the pellet was resolubilized into 8 M Urea in 50 mM ammonium bicarbonate. The sample was diluted to 4 M urea with 50 mM ammonium bicarbonate, reduced with 5 mM DTT for 45 minutes at 50 °C, alkylated with 15 mM IAA for 45 minutes in the dark at room temperature, and then diluted to 1 M urea with 50 mM ammonium bicarbonate. The samples were digested overnight for 16 hours at 37 °C with 1ug of Trypsin (Promega) and 1ug Lys-C (Wako) concurrently.Following digestion, samples were desalted and concentrated using Omix tips (Agilent) and the following protocol: tips were equilibrated with 100 µL of buffer B , then buffer A (0.1% formic acid in water). The sample was slowly run through the tip five times, then the tip was washed three times with buffer. The sample was then eluted with 100uL buffer B into a fresh tube, and dried down via speed vac. Dried down peptides were resuspended into 80 µL 0.1% formic acid in water, and 1 µL of peptides was injected for spectral analysis (details below).For the BSA digest experiment, conditions and processing were identical to that above, but an equivalent amount of BSA was digested into peptides, desalted as described, resuspended into PBS, plasma treated, then desalted a second time for spectral analysis.For SDS-PAGE analysis, shown in Fig. E. coli and tested for activity  was solubilized in PBS for a stock concentration of 32 µM. A mix of EGFR and EGF was made with a molar ratio of ~3:1 EGF:EGFR (~1.2 µM:430 nM)and reacted for 15 minutes at room temperature with gentle vortexing to mix every few minutes after which samples were aliquoted for treatment.The ecto domain of human epidermal growth factor receptor (residues Met1-Gly645), expressed in human cells , was solubilized in PBS for a stock concentration of 2.16 µM. This was diluted to ~500 nM for treatment. For reaction with EGF, human EGF expressed in In triplicate, EGFR alone or EGFR + EGF were treated with plasma for 0, 5 or 10 seconds. Samples were processed identically as described for BSA experiments, except protein was digested with 300ng each of Trypsin and Lys-C, resolubilized into 20 µL of 0.1% formic acid in water, and 2 µL was injected for spectral analysis.The amount of peptides that were injected listed per experiment described above. For the LC (Agilent 1100 series G2226A nanopump) method, buffer A was 0.1% formic acid in water , and buffer B was 0.1% formic acid in acetonitrile . For an overall 80-minute method, peptides were injected onto the column at a flow rate of 500nL/min from min 0–29 in 0% B. The gradient was then ramped to 95% B from min 53–58, then switched to 0% B from min 58–62, and flowed at 0% B at 500nL/min to min 65.https://chorusproject.org).With the above gradient conditions, peptides were injected onto a PepMap RSLC C18, 3 µm, 100 Å, 75µmx15 cm reversed phased analytical column (Thermo Scientific) set to 60 °C and ionized at 1.85 kV via electrospray ionization fed into an Orbitrap Elite mass spectrometer (Thermo Scientific) for analysis. Data-dependent acquisition settings were as follows. From the MS1 spectra acquired at a resolving power of 120 K, from the m/z 350–1800 in profile mode, the top 20 ions were selected for MS/MS analysis, rejecting + 1 charge states. Ions for MS/MS analysis were isolated with a 1.0 m/z isolation width, and fragmented with CID with a normalized collision energy of 35.0, an activation Q of 0.25, and activation time of 10ms. Dynamic exclusion was used, with a repeat count of 2, a repeat duration of 12 s, and an exclusive duration of 12 s. Raw data was uploaded to and made publicly available via the Chorus project (Raw data was searched using Proteome Discoverer 2.1 (Thermo Scientific) and the Mascot search algorithm (Matrix Sciences). A database consisting of common contaminants with the proteins of interest was used for searching. Trypsin was specified as the protease, with 2 missed cleavages allowed. Ten ppm tolerance was used for the precursor mass tolerance, and 0.6 Da was used for the fragment mass tolerance. All modifications were set to variable. These included deamidation (NQ), carbamidomethylation (C), and oxidation (FHIKLMPRTVWYACDENQS). Data was automatically filtered by PD to a 1.0% strict max FDR, and area under precursor ion chromatograms is calculated by PD.Data was output to Excel (Microsoft) for further manual processing. The output was filtered with the following conditions: protein of interest, confidence = high, rank and search engine rank = 1, and ion score ≥20. The oxidation events per PSM were then counted in a separate column, and the data was filtered for redundancy based on the data in total and modification count, rather than individual modification events. Note that this removes extracted ion chromatograms (XICs), which result from multiple PSMs under the same peak and prevents redundant summation while allowing multiple charge states to be considered in the summation. Next, areas under the XICs were summed per peptide for unmodified variants and modified variants, and percent modification was calculated based on this. These numbers make the basis for average and standard-deviation calculations shown in the tables and figures, as well as for the statistical tests performed. All statistical tests were two-tailed and unpaired with equal variance specified. To examine residues of modification, manual examination of the dataset was performed. Graphs and figures were made using Excel and Illustrator (Adobe), and crystal structures were obtained from the PDB archive. Crystal structure images were created using Pymol (Schrodinger).Supplementary Dataset 1Supplementary Dataset 2Supplementary Dataset 3


Harvesting of microalgae is a cost-consuming step for biodiesel production. Cellulose has recently been studied as a biocompatible and inexpensive flocculant for harvesting microalgae via surface modifications such as cation-modifications. In this study, we demonstrated that cellulose nanofibrils (CNF) played a role as a microalgal flocculant via its network geometry without cation modification. Sulfur acid-treated tunicate CNF flocculated microalgae, but cellulose nanocrystals (CNC) did not. In addition, desulfurization did not significantly influence the flocculation efficiency of CNF. This mechanism is likely related to encapsulation of microalgae by nanofibrous structure formation, which is derived from nanofibrils entanglement and intra-hydrogen bonding. Moreover, flocculated microalgae were subject to mechanical stress resulting in changes in metabolism induced by calcium ion influx, leading to upregulated lipid synthesis. CNF do not require surface modifications such as cation modified CNC and flocculation is derived from network geometry related to nanocellulose size; accordingly, CNF is one of the least expensive cellulose-based flocculants ever identified. If this flocculant is applied to the biodiesel process, it could decrease the cost of harvest, which is one of the most expensive steps, while increasing lipid production. With the development of various technologies, people have recently become interested in nano-sized environments and nanomaterials, which have a number of potential applications, including electronics, optics and biology. In biology, nanomaterials exhibit different characteristics and remarkable features when compared with macromaterials, while interacting with cells and tissues. Microalgae offer high potential for diverse nutrients, cosmetics, pharmaceuticals, and exploitation of biodiesel productionInorganic compounds, especially metal derived nanoparticles, have been processed and applied to diverse fields, including microalgal harvesting; however, these materials have been found to have many limitations, especially with respect to biocompatibility to humans and the environmentIn this study, we investigated the size effects of cellulose nanomaterials on microalgae flocculation and lipid metabolism. Cellulose nanofibrils (CNF) flocculated microalgae, despite having an anion surface, and lipid synthesis was highly upregulated via a mechanotransduction-mediated mechanism. CNF induced flocculation via a mechanical interaction based on geometric properties such as its nanocellulose size and hydrogen bonding . Based oTo confirm whether the hydrolyzed tunicates were separated into CNC and CNF according to their size, TEM and ELS analyses were conducted. As shown in Flocculation was the typical interaction between CNF and microalgae. When poured into microalgae culture, CNF aggregated the microalgae, but CNC did not . The OptTo investigate whether the flocculation mechanism is dependent on ionic strength of culture media, microalgal flocculation with CNF was compared between TAP growth media and DW. The flocculation rate was faster in DW for all CNF concentrations, but no significant difference was observed in flocculation at the final time . To deteWhile investigating the structural effects of CNF on microalgae flocculation, we found that free-moving CNF fibrils in static solution flocculated microalgae better than the pre-entangled CNF network . Moreove−1), of which hydrogen bonding were indicated 3414 and 3340 cm−1 intramolecular, and 3281 cm−1 intermolecular bond. Both had no significant difference in intramolecular hydrogen bondings but CNF in DW displayed more distinct peak than in TAP, implying that CNFs in DW interconnected themselves via OH bonding. Evaluation of the effects of sulfur contents revealed that desulfated CNF displayed 3340 cm−1 and 3414 cm−1 peaks that were sharper than the non-modified one, implying that desulfated CNF undergoes stronger hydrogen bonding in that region. Finally, agitated CNF presented sharper peaks than static ones at 3340 cm−1 and 3414 cm−1 peaks. These findings indicate that agitation induced strong hydrogen bonding of intra-CNF bonds.Crystallinity levels were determined for CNF and CNC  using XRLipid droplets were examined on microalgae in CNF by nile red staining. Microalgae cultured on CNF showed increased lipid bodies relative to control , and thiTo identify why lipid synthesis was upregulated in CNF environment in a transcript level, mRNAs of ACCase, DGAT, LPAAT, which are associated with fatty acid synthesis, were quantified. In To evaluate the mechanotransduction of microalgae cultured on CNF, intracellular calcium contents were measured by Fura-2 staining. Microalgae in CNF have stronger fluorescent intensity than in suspension . ChloropMicroalgal harvesting has high costs that must be overcome before it can be effectively applied for biodiesel production. This study was conducted to develop an unmodified CNF to both lower harvesting cost and increase lipid production, utilizing the size effect of CNF on its geometric structure and microalgal mechanotransduction. Conventional sulfuric acid treatment is a cost-effective way to yield cellulous nanomaterials and thus adequate to obtain cellulose nanomaterials, even though the reaction could be affected by acid concentration, acid type, reaction time, and temperatureCNF unexpectedly flocculated microalgae, which, to the best of our knowledge, has not previously been reported . Anion CAnother possible mechanism is formation of a CNF network, followed by mechanical entrapment of microalgae. CNF was shown to be entangled by its flexible nanofibrils . Similar3−1 to 3414 cm−1 than static CNF. This phenomenon demonstrated that agitation induces a tight network among CNF. This characteristic is related to the compact and faster network formation due to mechanical stress on CNF solution1417Generation of the CNF nanofibrous network via mechanical entanglement and attractive forces was evaluated in terms of amorphous structure and hydrogen bonding. In view of mechanical entanglement, the longer nanofibrils, which have more flexible morphology, lead to mechanical entanglement among nanofibrils more easily according to the shear-lag theory1315The mechanically entrapped microalgae naturally modify their physiology, adapting themselves to the flocculated environment. Mechanical stress in microalgae is transmitted through the mechanosensitive ion channel of calcium ionIn addition, GC-MS data showed that hydrocarbon of microalgae was also converted to long carbon chain. This upregulation and conversion of lipids could be the result of compensation for the downregulation of cell growth and sizeBiocompatible anionic CNF was studied for microalgae harvesting and high lipid production. CNF was transformed to a nanofibrous network by attractive forces such as hydrogen bonding and mechanical entanglement, followed by microalgal entrapment. This study is the first to report that anionic CNF could be a flocculant. In addition, flocculated microalgae excited mechanotransduction relevant to calcium ion influx, which drove metabolic flux to up-regulate syntheses of lipids and hydrocarbons, a self-defense pathway, at the expense of cell growth. Therefore, CNF is a biocompatible and low-cost flocculant that makes it easier to harvest microalgae while increasing lipid and hydrocarbon contents. Accordingly, this nanomaterial has the potential to dramatically decrease the economic cost of the microalgal biodiesel process.The integuments of tunicates were separated and dried in an oven, which were then immersed in 5% potassium hydroxide  aqueous solution at 80 °C for 3 days. Next, aqueous solutions of 0.17% acetic acid  and 0.34% sodium hypochlorite  were applied to the integuments at 60 °C and the solutions were exchanged hourly until the tunicate’s pigments were removed, after which they were washed with distilled water. Samples were then filtered with a 1 mm size sieve, after which the powered integuments were treated with 60% (w/v) sulfuric acid aqueous solution  and stirred for 20 min at 60 °C. Following the reaction, the temperature was reduced to 0 °C to stop the hydrolysis reaction. Residual sulfuric acid was subsequently neutralized with distilled water until pH 6, after which the samples were sonicated for 30 min Crude hydrolyzed cellulose was filtered using 0.7 μm whatman microfiber filters  under vacuum to separate CNF and CNC. For desulfurizing the integuments, 2N NaOH solution was added and reacted for 6 hr at 60 °C and subsequently dialyzed for 7 days to remove NaOH and remaining metal ions.The morphology and size of CNC and CNF were observed by transmission electron microscopy . Briefly, CNC or CNF were collected from 1 ml suspensions, then dyed with 1% uranyl acetate for 5 min in a dark room. After sequentially washing 3 times with DW, removing bubbles on the top of the grid with a pipette, and drying the TEM grid using a vacuum, TEM images were taken. Image J software  was used to calculate the size and width of CNC or CNF on TEM images.Surface charge and particle size of CNC or CNF was measured using zeta potential and dynamic light scattering . The crystallinity index of samples was examined by x-ray diffraction (XRD)  at 40 kV and 30 mA tube current at 2θ angles from 10° to 90°. Sulfuric contents were measured by elemental analysis .−1 range.Fourier transform infrared spectrometer  was used to investigate the interaction of specific inter and intra cellular hydrogen bonding. Aggregated, desulfated and different solvent (TAP media) of CNF solution(1 mg) was embedded into KBR pellet and dried water in a oven during 12 hr and detected with OPUS 6.0 software in 4000 ~ 400 cmChlamydomonas reinhardtii strain cc-124 was cultured in a shaking incubator (80 rpm) at 23 °C under a 12 hr light and 12 hr dark cycle. TAP media (Gibco) was used as a basal media. Cell mass was estimated based on the optical density at 680 nm using a spectrophotometer . To identify aggregation of microalgae with CNC or CNF, various volumes of CNC or CNF were then added to the C. reinhardtii culture  to adjust it to a final cell culture volume of 10 ml. Sedimentation level was measured at 680 nm using a spectrophotometer.Cell viability was determined based on the cell number and chlorophyll contents every 24 hr for 3 days. Various concentrations of CNC and CNF were added into the culturing microalgae, after which each sample was collected and amended with 2.5% glutaraldehyde  in PBS, separated by centrifugation to remove the TAP media and glutaraldehyde. Total cell mass was confirmed using a spectrophotometer at 680 nm. In addition, cell number was determined by direct cell counting using a hemocytometer , while cell size was measured using coulter counter . Cell mass of microalgae were measured using spectrophotometer . Briefly, cell pellets were treated with 100% methanol for 24 hr at 4 °C. Optical density was measured at 680 nm to measure cell mass contents.Nile red staining was applied to monitor intracellular lipid contents of microalgae. First, microalgae cultured with CNF were fixed with 2.5% glutaraldehyde in PBS. After discarding the sample’s media solution using a centrifuge , the cell pellet was resuspended with PBS, then mixed with Nile red dye  at a 1:3 volume ratio (dye:sample) and finally incubated for 10 min at 40 °C in the dark. Intracellular lipid images were obtained by confocal microscopy  with excitation 543 nm and emission 630 nm wavelengths. Quantified intracellular lipid contents were confirmed using a spectrofluorometer . The same Nile red stained sample was used to measure the quantity of lipid contents under the following conditions: excitation slit 10 nm, emission slit 10 nm, photomultiplier 400 V, excitation wavelength 480 nm, emission filter wavelength 570–590 nm). Hydrocarbon contents of microalgae was examined using 6890N GC system . Details to process of GC operation was indicated below. J&W GC column DB-5 ms , Inlet temp 280 °C, Injection volume 1 μl, Oven temperature 30 °C to 300 °C at 10 °C/min. Mass spectrometry was measured by Pegasus III .+/K+ buffer  and 3 μM Fura-2 dissolved in DMSO. The sample was then resuspended in a 6 well plate, incubated for 24 hr at 23 °C and collected. An image of the calcium influx of microalgae was taken by confocal microscopy at an excitation of 380 nm and emission of 510 nm.The intracellular calcium concentration was detected by Fura-2 staining (Sigma Aldrich). Microalgae samples cultured with CNF were centrifuged at 13,500 g for 1 min, after which the media was exchanged with NMG(−∆∆2γ)) and quantified using the Rotor-Gene Q Series Software . The 2(−∆∆2γ) value was normalized against the expression level of CBLP, a housekeeping gene.Microalgal mRNA was collected using Trizol reagent . The extraction was conducted according to the manufacturer’s guidelines. Total mRNA preservation was checked by agarose gel electrophoresis using safe shine green . After synthesizing cDNA using a QuantiTect Reverse Transcription Kit . Real Time qRT-PCR was conducted according to the manufacturer’s instructions. Briefly, the specific volume ratio of the cDNA tablet, 2× SYBR in a QuantiFast SYBR Green PCR kit and primer were mixed and diluted to 20 μl. Data describing specific cDNA were expressed in the form of delta-delta Ct  to represent difference.How to cite this article: Yu, S. I. et al. Nanocellulose size regulates microalgal flocculation and lipid metabolism. Sci. Rep.6, 35684; doi: 10.1038/srep35684 (2016).Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Pediatric posterior fossa tumor (PFT) survivors who have been treated with cranial radiation therapy often suffer from cognitive impairments that might relate to IQ decline. Radiotherapy (RT) distinctly affects brain regions involved in different cognitive functions. However, the relative contribution of regional irradiation to the different cognitive impairments still remains unclear. We investigated the relationships between the changes in different cognitive scores and radiation dose distribution in 30 children treated for a PFT. Our exploratory analysis was based on a principal component analysis (PCA) and an ordinary least square regression approach. The use of a PCA was an innovative way to cluster correlated irradiated regions due to similar radiation therapy protocols across patients. Our results suggest an association between working memory decline and a high dose  delivered to the orbitofrontal regions, whereas the decline of processing speed seemed more related to EUD in the temporal lobes and posterior fossa. To identify regional effects of RT on cognitive functions may help to propose a rehabilitation program adapted to the risk of cognitive impairment. The mosTask efficiency  and Organization. These subscores were measured as given by the Childhood Cancer Survivor Study Neurocognitive Questionnaire. While these studies did not identify a relationship between radiation dose of PF and changes in cognitive scores, such associations have been reported in children with ependymoma  or RT and chemotherapy (N = 10). The remaining patients were treated with chemotherapy alone and were used as controls. All patients with RT received a CSI and a boost in the PF and were treated with three-dimensional conformal radiation therapy (Table Inclusion criteria were 1) PFT patients treated at Gustave Roussy Cancer Campus between 2000 and 2014; (2) 17 years of age or under at diagnosis (3) multiple (>2) IQ assessments after treatment onset (4); for the PFT patients treated with radiation therapy, the computed tomography (CT) scan, T1-weighted MRI, and dosimetric maps had to be available. Thirty patients  matched these criteria. Information was gathered from medical files about the history of the illness  and the type of treatment . The underlying malignancy of the 30 patients studied was medulloblastoma, ependymoma, astrocytoma, and embryonal tumor in 25, 3, 1, and 1 patients, respectively. Twenty patients had a localized disease and 10 had a metastatic disease. Complete tumor resection was achieved in 20 PFT. Post-operative complications occurred in 10 patients. No patient relapsed between the two evaluations, but the patient with an astrocytoma whose relapse before the first evaluation, was treated with chemotherapy alone. The mean age at diagnosis was 4.62 years . The mean delay between treatment and the last assessment was 4.60 years . Pre-operative hydrocephalus was present in 19 patients (63%). Seventeen patients were treated with RT alone ( PFT patiN (ΔFSIQ) = 30, N (ΔPSI) = 23, N (ΔWMI) = 14]. Patients were evaluated at variable time points after treatment onset. Thus, the delay (T) between two neuropsychological assessments varied from one patient to the other  were collected and processed to create individual dose distribution maps into selected brain regions of interest (ROI) covering the whole brain.To study regional dose effects on changes in cognitive scores, 3D-T1 MRI, CT scan, and absorbed dose maps of patients treated with RT . In this clinical retrospective study, two types of T1-weighted images were collected: 3D T1-weighted sagittal slices  and 3D-T1 weighted axial slices .Computed tomography scans were acquired on a SIEMENS Sensation Open scanner located in Gustave Roussy RT department . Radiation dose maps (RD maps) were computed with the ISOgray™ Treatment Planning System . The Clarkson–Cunningham model was used for dose calculation. Dose maps resolution was 3 mm × 3 mm × 1 mm.We designed a five-step preprocessing pipeline to identify anatomical ROI on dose maps Figure .Step 1: We chose three MRI templates specific to ages 0–2, 2–5, and 5–9 years  and three corresponding anatomical atlases  from the Neurodevelopmental MRI DataBase , even at equal total doses, the biological effectiveness of these two types of irradiation will be different  depending on the number of equally spaced fractions per day; the dose per fraction (d) and the sensitivity of the tissue (α/β). D  and d varied across patients. Based on the current literature, α/β was fixed to 2 and T1/2 to 3 h EQD2Step 2: After calculating the biological dose map of each patient, for all subjects and ROIs we computed the EUD that accounts for heterogeneity of dose distribution, as follows , EUD was highly correlated across brain regions  in a least square regression  = −0.64, p = 0.52; ΔPSI vs. ΔWMI: t(35) = −0.81, p = 0.42; ΔFSIQ vs. ΔPSI: t(51) = −0.37, p = 0.70]  = 2.36; p = 0.11; RT alone: M = −10.5 (±9.11), chemotherapy alone: M = 1.57 (±11.28), RT and chemotherapy: M = −2.0 (±11.03)]; and ΔWMI , and ΔPSI .At time of first neuropsychological assessment, the mean estimated IQ over the whole population was 87.5 . A declining performance over time was observed in 67, 64, and 48% of the patients for ΔFSIQ, ΔWMI, and ΔPSI, respectively. The remaining patients showed either preserved or better performance over time. However, there were no statistically significant differences between cognitive scores  or procThis study confirms two cases for which there is a relationship between the radiation dose in particular brain areas and specific cognitive decline. The first case shows a correlation between orbitofrontal radiation and working memory decline, whereas the second case portrays a correlation between temporal lobe and PF radiation and slower processing speed. As this study is exploratory, it does aim to provide information regarding brain regions to avoid, but to describe relationships between radiation and cognitive function. The relationship between the cognitive profiles and the irradiation of these regions should be further confirmed in a prospective randomized with both, a bigger cohort and different radiation protocols.This retrospective study was approved by the Comite de Protection des Personnes CPP no. 14973 . All patient’s parents gave a written informed consent in accordance with the Declaration of Helsinki.All authors carried a substantial contribution to the article and approved the final version of the manuscript. None of them has any conflict of interest. Guarantor of integrity of entire study: EDS, MN, and LH-P. Study concepts and design or data collection or data analysis and interpretation: all authors. Data preprocessing: EDS, CR, AG, and CP. Statistical analysis: EDS and MP-G. Drafting the work or revising it critically for important intellectual content: all authors.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. The reviewer, JB, and the handling editor declared their shared affiliation, and the handling editor states that the process nevertheless met the standards of a fair and objective review.


We prospectively compared visual and refractive outcomes in patients with high myopia and myopic astigmatism after small-incision lenticule extraction (SMILE) and photorefractive keratetctomy (PRK) with mitomycin C. Sixty-six eyes of 33 patients  were included . Preoperatively, no significant difference was noted in manifest spherical equivalent (p = 0.326), manifest sphere (p = 0.277), and manifest cylinder (p = 0.625) between both groups. At 1 month, there were significant differences in logMAR uncorrected distance visual acuity, efficacy index and manifest refraction spherical equivalent between SMILE and PRK (p ≤ 0.029). At 6 months, the logMAR corrected distance visual acuity (p = 0.594), logMAR uncorrected visual acuity (p = 0.452), efficacy index (p = 0.215) and safety index was (p = 0.537) was comparable between SMILE and PRK. Significant differences were observed in postoperative manifest spherical equivalent (p = 0.044) and manifest cylinder (p = 0.014) between both groups. At the end of 6 months, 100% of the eyes in SMILE group and 69% of the eyes in PRK group were within ±0.50 D of the attempted cylindrical correction. The postoperative difference vector, magnitude of error and absolute angle of error were significantly smaller after SMILE compared to PRK (p ≤ 0.040) implying a trend towards overcorrection of cylindrical correction following PRK. The correction of high myopia is challenging owing to the associated risk of treatment regression and corneal ectasia. Although LASIK provides quick visual rehabilitation, PRK has been shown to offer early stabilization of cornea while allowing for correction of higher myopic correction compared to LASIK3567The traditional management options for correction of high myopia include laser Sixty-six eyes of 33 patients with a mean age of 29.7 ± 5.6 years were included . Preoperatively, there was no significant difference in manifest spherical equivalent (p = 0.326), manifest sphere (p = 0.277), and manifest cylinder (p = 0.625) between both groups . CorneasAt 1 month, there were significant differences in UDVA, efficacy index and manifest refraction spherical equivalent between SMILE and PRK (p ≤ 0.029) . The logAt 6 months, the logMAR CDVA was 0.021 ± 0.055 and 0.013 ± 0.049 following SMILE and PRK, respectively (p = 0.594). The corresponding logMAR UDVA was 0.044 ± 0.058 and 0.060 ± 0.093 (p = 0.452). The efficacy index was 0.92 ± 0.12 after SMILE and 0.86 ± 0.19 after PRK (p = 0.215). The corresponding safety index was 0.96 ± 0.10 and 0.94 ± 0.13 (p = 0.537) . The disThe vector analysis results using the 6-month refractive data are shown in At 6 months, the correction index (CI), defined as the ratio of SIA to TIA, was 1.02 (95% CI: 1.00 to 1.04) in SMILE and 1.34 (95% CI: 1.29 to 1.40) in PRK. et al. reported a CI of 1.25 for PRK in eyes with myopic astigmatismThe current study showed that SMILE was more effective in low to moderate astigmatic correction compared to PRK at 6 months. Although both procedures were found to be similar in spherical correction and safety, visual rehabilitation was quicker after SMILE compared to PRK. This could represent a differential healing response between both procedures1113We found that SMILE was more effective than PRK for correction of low (1.0 to 1.5 D) myopic astigmatism. Slight overcorrection was observed in eyes with low astigmatism, while slight undercorrection was observed in eyes with high astigmatism. Ivarsen and Hjortdal reported that undercorrection of astigmatism increased with attempted treatment following SMILE81718Overall, SMILE demonstrated better astigmatic correction compared to PRK. The residual spherical equivalent and astigmatism postoperatively were lower after SMILE. The observed difference in astigmatic correction between both procedures was likely influenced by the difference in absolute AE, which indicated alignment of treatment. High correlation between absolute AE and IS has been reported as well as demonstrated in the current study819It is possible that differential healing response between SMILE and PRK could be potential reasons for treatment misalignment. PRK utilizes ultraviolet light from excimer laser to reshape the corneal curvature through direct photoablation. SMILE uses femtosecond laser in near infrared spectrum to achieve the cutting effect within the cornea through photodisruption. The amount of energy delivered to the cornea was much lower with femtosecond laser compared to excimer laser2629et al. showed that although both SMILE and Laser-assisted subepithelial keratomileusis alter corneal biomechanical strength, the changes induced by SMILE are more predictable than those induced by LASEK in terms of per unit tissue removedOur study results did not include the impact of SMILE and PRK on corneal biomechanics. The SMILE procedure has potential benefit over flap-based surgeries in terms of biomechanical stability. Mathematical models have predicted that the tensile strength after SMILE is better compared to PRK and LASIKThe current study was limited by a small sample size and non-randomized design. We did not have access to keratometry, epithelial thickness maps and contrast sensitivity values. We also acknowledge the relatively short follow-up duration, which precludes analysis of the long-term stability of our results. However, significant group difference in astigmatism correction was detected between SMILE and PRK in vector analysis during the early postoperative period. In conclusion, both SMILE and PRK were observed to be safe for correction of low to moderate astigmatism in eyes with high myopia. Predictability of refraction was superior in SMILE, which showed a better treatment alignment of astigmatism than PRK in vector analysis. There was a trend towards overcorrection of cylindrical correction following surface ablation.This study was a prospective audit of consecutive patients who received SMILE and PRK with mitomycin C at the Hong Kong Laser Eye Center and Chinese University of Hong Kong Eye Centre respectively. Patients were offered either PRK or SMILE in the respective surgery centres. All surgeries were performed between June 2014 and May 2015. The Joint Chinese University of Hong Kong-New Territories East Cluster Clinical Research Ethics Committee approved the study protocol. An informed consent was obtained from all study participants. The study adhered to the tenets of the Declaration of Helsinki.Patients with no ocular abnormality except myopia and myopic astigmatism, a corrected distance visual acuity (CDVA) of 20/20 or better, stable refraction for more than 1 year, manifest myopic refraction spherical equivalent of ≥6.0 D and astigmatism between −0.25 D and −2.50 D were included. All eyes had emmetropia as the target refraction. Patients with corneal thickness of <500 μm, suspicion of keratoconus on corneal topography , ocular inflammation, infection were excluded.SMILE was performed using the 500-kHz VisuMax femtosecond laser  using a standard surgical technique (Sekundo BJO 2011). The following parameters were used: cap thickness, 120 μm; cap diameter, 7.5 mm; lenticule diameter, 6.5 mm with a transition zone of 0.1 mm; cut energy, 1.4 μJ; spot and tracking distance, 2.0–3.0 μm. The back of the intrastromal lenticule was created from periphery to center of cornea. The anterior lamellar cut was subsequently created from center to periphery of cornea, which extended towards the surface to create a 2–3 mm incision, from which the stromal lenticule was extracted. A thin blunt spatula was used to separate the lenticule, which was then grasped with a pair of forceps and removed. The corneal interface was flushed with balanced salt solution.Alcohol-assisted corneal epithelial removal was performed over a 9.0-mm optical zone centered over the pupil. Ethanol 20% in distilled water was dropped into a 9.0-mm well and kept in contact with the epithelium for 40 seconds and then absorbed with a dry cellulose sponge. The eye was then washed with Balanced Salt Solution. A blunt spatula was then used to remove the loosened epithelium. Stromal ablation was performed with Allegretto Wave & Eye-Q 400Hz laser  using a 6.5 mm optical zone. The system has an infrared high-speed camera operating to track the patient’s eye movements that either compensates for changes in eye position or interrupts the treatment if the eye moves outside a preset predetermined range. After the stromal ablation, a circular cellulose sponge soaked with mitomycin C 0.02% (0.2 mg/mL) was placed on the cornea for 60 seconds. The eye was then copiously irrigated with a balanced salt solution to remove the residual mitomycin C. A bandage contact lens was placed over the cornea at the end of the surgery.Patients were examined preoperatively and 1, 3 and 6 months postoperatively. Severity of corneal haze after PRK was evaluated by slit-lamp biomicroscopy according to Hanna’s scaleRefractive astigmatism at the spectacle plane was converted to corneal plane using vertex distance of 12 mm. It was then analyzed using vector analysis, with consideration of change in the astigmatic axis, measuring 3 vectors and relationship among them (Alpins JCRS 2001). The target induced astigmatism vector (TIA) was defined as the astigmatic change which the surgery was intended to induce; the surgically induced astigmatism vector (SIA) was defined as the astigmatic change which the surgery actually induced; and the difference vector (DV) was defined as the induced astigmatic change that would enable the initial surgery to achieve its intended target or the postoperative astigmatism. Magnitude of error (ME) is the arithmetic difference between the SIA and TIA. Angle of error (AE) is the angle between the axis of the SIA and TIA.Statistical analysis was performed using R 2.15.3 . Visual and refractive outcomes, efficacy and safety index were evaluated at different time points. Linear mixed effect models were used to adjust for correlation between fellow eyes. Comparisons of mean values were performed using repeated measures analysis of variance (ANOVA) at each time point. A p-value < 0.05 was considered as statistically significant. Bonferroni adjustments were considered for multiple testing.How to cite this article: Chan, T. C. Y. et al. Early outcomes after small incision lenticule extraction and photorefractive keratectomy for correction of high myopia. Sci. Rep.6, 32820; doi: 10.1038/srep32820 (2016).


Physicochemical water parameters and biomass of plankton were measured in enclosures to evaluate the suitable stocking density of silver carp for relieving internal nutrients and constraining cyanobacteria growth in Lake Taihu. It is found that the 35 g m−3 silver carp group and 70 g m−3 silver carp group presented lower total phosphorus, lower chlorophyll-a, and higher water transparency. Increased nitrogen to phosphorus ratio, which indicated the result of algae decline in fish presence enclosures, was attributed to decline of phosphorus. Phosphorus decline also exerted limitation on reestablish of cyanobacteria bloom. Crustacean zooplankton biomass and Microcystis biomass decreased significantly in fish presence enclosures. Silver carp could be more effective to regulate algae bloom in enclosures with dense cyanobacteria. Therefore, nonclassic manipulation is supposed to be appropriate method to get rid of cyanobacteria blooms in Lake Taihu by stocking 35 to 70 g m−3 silver carp in application.In order to control cyanobacteria blooms with silver carp in Lake Taihu, an in situ experiment was carried out by stocking silver carp at a biomass of 35, 70, and 150 g m Daphnia in these lakes, crustacean communities are not able to control algal bloom by zooplankton-target manipulation: indeed grazing pressure by zooplankton is useless [In freshwater ecosystems, fish affect the structure and dynamics of pelagic plankton communities by trophic cascading effects , 2, whic useless .Introduction of filter-feeding planktivorous fish to hypertrophic shallow freshwater lakes successfully is another effective method to regulate the algal community which is called nonclassic biomanipulation –10. As f Daphnia, to small ones, such as Limnoithona sinensis during the last 60 years [Lake Taihu is the third largest shallow freshwater lake located at the lower reach of the Yangtze River. Due to accumulative nutrient-rich sewage and agricultural run-off inflow, it became eutrophic with heavy cyanobacteria blooms from late spring to autumn every year in last decades . In this60 years . Hence, Some previous studies , 10 inve−1 (max 0.2 mg L−1) and 2.3 mg L−1 (max 5.6 mg L−1) in the northern east of Meiliang Bay [Our experiment was located at Meiliang Bay  were fixed to a cage of steel pipes. Bottom margins of each enclosure with heavy stone cages were sank into the lake sediment as possible to avoid water exchange between inside and outside of the enclosure during the study period. Its cost is about 3000 YUAN  per enclosure including all the materials and labour costs.Planktivorous fish silver carp (average weight 137 g ± 3 g) were obtained from a local aquatic farm and then acclimated in a nearby pond until they were transferred into the enclosures. The whole experiment aimed to explore the proper density of silver carp to control algae bloom.−3. The experiment lasted from 30th May to 23rd June 2011 with a sampling interval of 3-4 days depending on the local weather. The average cyanobacteria biomass before the experiment in each enclosure was approximate about 5.8 g L−1 before the fish were introduced (confirmed with 2010 Microcystis biomass).In this experiment, 12 enclosures were chosen and randomly divided into four groups with triplications representing for control group (CG), low fish density group (LDG), medium fish density group (MDG), and high density group (HDG). CG, LDG, MDG, and HDG had fish biomass at 0, 35, 70, and 150 g mZSd). Turbidity was measured by a turbidimeter .Integrated water sample was collected by a 5 liter modified Patalas's bottle sampler. We determined water temperature, dissolved oxygen (DO), total dissolved solids (TDS), and pH using YSI Professional Plus  water quality monitor. Water transparency was determined by using a 20-cm in diameter Secchi's disk and represented as Secchi's depth (# (69 μm) plankton net and then fixed with 1 mL saturated formalin. All individuals were counted after precipitation for 1 day by using an Olympus compound microscope  at total 4 × 10 magnification in the samples to calculate density and biomass. Copepods and cladocerans were identified based on these papers [ Microcystis spp. quantitative measurement. Colonial Microcystis was broken up to individual cells by an ultrasonic wave cell knapper  so that single cells could be counted. Fixed samples (0.1 mL) were tested using Olympus compound microscope under magnification of 40 × 10. Wet weight of Microcystis was calculated based on the formula of this paper [50 mL samples of quantitative crustaceans were collected by filtering 10 L integrated water samples through 25e papers , 17, ande papers , 19. Int4+), nitrate nitrogen (NO3−), total nitrogen (TN), dissolved inorganic phosphorus, total dissolved nitrogen (TDN), total dissolved phosphorus (TDP), total phosphorus (TP), and chlorophyll-a were determined based on [ Microcystis was measured at a sampling interval between two sampling points, and water chemistry was tested on every sampling point.Water chemistry including ammonia nitrogen  using post hoc multiple by comparisons test (LSD) [x + 1) transformation. Differences were measured against control values and considered to be statistically significant at P < 0.05. Statistical analyses mentioned above were undertaken using SPSS  13.0 for Windows. Statistical figures were output by R [Data were normalized and variance was adjusted for homogeneity before analysis. Means of dissolved oxygen, pH, total dissolved solids, and transparency were compared between control group and each treatment during whole experiment using independent st (LSD)  and exprput by R  and OrigIn this experiment, water temperature varied from 22.1°C to 26.2°C . pH and total dissolved solids did not show significant different between control group and each treatment. MDG showed the lowest dissolved oxygen in the whole experiment. Higher fish density group presented lower dissolved oxygen. In the LDG, transparency was measured to be significantly higher than in the CG whereas the lowest occurred in the HDG and even lower than in the CG .P < 0.05, df7,3, other statistics were shown in Nitrate and ammonia were found to be significantly higher . Regression analyzes in fish groups between chlorophyll-a to TN and TP showed that TP was positively related to chlorophyll-a  while TN was less relative  to chlorophyll-a fluctuate  while TP was not related to TN : TP .During this experiment, TN : TP ratio values were lowest in CG . Signifiluctuate . RegressZSd) was negative related to Microcystis , TP , and chlorophyll-a . But TN was not observed related to all these parameters  inhibited cyanobacteria blooms more efficiently. These enclosures performed as refined water quality, lower nutrient, and cyanobacteria density; relative higher zooplankton biomass than higher fish group.In the present study, fish at the lower density  and nitrogen (average TN > 1 mg L−1) in the study area were enough for the cyanobacteria growth.In the present study, the increasing value of total nitrogen to total phosphorus ratio in fish presence enclosures indicated that cyanobacteria bloom was alleviated by fish, by referring cyanobacteria bloom explosion usually results in decline of TN : TP ratio . In the −3 to 70 g m−3 could be effective in controlling Microcystis blooms, promotion of fish production, and ameliorating the aquatic environment. Nonclassic biomanipulation is a proper means to reduce nutrients and phytoplankton under conditions of (1) eutrophic or hypereutrophic water, (2) lack of large sized zooplankton, and (3) dominance of filamentous or colonial algae.The present study provide evidences that, in enclosure conditions, fish at a density of 35 g m


Isolation of populations of resident lung progenitor cells (LPCs) has been described by multiple protocols, and some have been successfully applied to healthy human lung tissue. We aimed at understanding how different cell culture conditions may affect, in vitro, the phenotype of LPCs to create an ideal niche‐like microenvironment. The influence of different substrates  and the impact of a three‐dimensional/two‐dimensional (3D/2D) culture switch on the biology of LPCs isolated as lung spheroids (LSs) from normal adult human lung biopsy specimens were investigated. We applied a spheroid culture system as the selective/inductive step for progenitor cell culture, as described in many biological systems. The data showed a niche‐like proepithelial microenvironment inside the LS, highly sensitive to the 3D culture system and significantly affecting the phenotype of adult LPCs more than culture substrate. LSs favor epithelial phenotypes and LPC maintenance and contain cells more responsive to specific commitment stimuli than 2D monolayer cultures, while secreting a distinctive set of paracrine factors. We have shown for the first time, to our knowledge, how culture as 3D LSs can affect LPC epithelial phenotype and produce strong paracrine signals with a distinctive secretomic profile compared with 2D monolayer conditions. These findings suggest novel approaches to maintain ex vivo LPCs for basic and translational studies. S A lung niche‐like microenvironment was created ex vivo inside the pneumosphere. Pneumospheres have a distinctive secretomic profile compared with two‐dimensional culture. Three‐dimensional culture switch significantly affects lung progenitor phenotype. Lung progenitors in pneumospheres were more committed toward epithelial lineages.Respiratory diseases are among the leading causes of morbidity and mortality worldwide, with lung infections, lung cancer, and chronic obstructive pulmonary disease together accounting for almost 10 million deaths worldwide during 2008 [The biology of airway stem cell compartments in the adult has become a rapidly advancing field. Multiple progenitor populations have been identified as either specialized or facultative, depending on the conditions analyzed, such as physiological turnover or response to injuries , 4. It iIsolation of different mesenchymal or epithelial populations of resident lung progenitor cells (LPCs) has been described by multiple protocols and criteria, with only few of them having been successfully applied to healthy human lung tissue , 13, 14,The biological complexity of the lung requires careful consideration of the conditions used to mimic ex vivo the ideal microenvironment for LPCs. Phenotype control of isolated cells represents a delicate task to manage in vitro, for both basic and translational research. Multiple stimuli play synergic roles on lung stem cell niches, modulating proliferation, commitment, epithelial‐to‐mesenchymal (EMT) or the opposite mesenchymal‐to‐epithelial transition, and crosstalk with stroma , 19, 20.Thus, it is mandatory to understand how different cell culture variables, such as substrate or a 3D system, may affect in vitro the phenotype of LPCs so an ideal microenvironment for their isolation can be created that could be readily adjusted for modulating their commitment and phenotype. In this study, we have investigated the influence of different substrates and a 3D/2D culture switch on the biology of LPCs isolated as spheroids from normal adult human lung biopsy specimens, whose translational relevance has been recently highlighted , characthttp://www.ethicon.com). Cell culture protocol was based on other spheroid culture systems [3 pieces, digested for 15 minutes in 0.05% trypsin‐EDTA, divided into equal amounts, and then plated as primary explants on Petri dishes coated with fibronectin , gelatin (GEL), or laminin  in complete explant medium (CEM). CEM medium is made of Iscove’s modified Dulbecco’s medium (IMDM) supplemented with 20% fetal bovine serum (FBS), 1% penicillin‐streptomycin, 1% l‐glutamine, and 0.1 mM 2‐mercaptoethanol. After 3–4 weeks, outgrowth explant‐derived cells (EDCs) were harvested by serial washes with PBS, EDTA, and mild trypsinization, and plated on poly‐d‐lysine‐coated multiwell plates at 6,000 cells per cm2 in pneumosphere growth medium (PGM). PGM medium is made of 35% IMDM plus 65% Dulbecco’s modified Eagle’s medium (DMEM)/F‐12 mix, supplemented with 3.5% serum, 1% penicillin‐streptomycin, 1% l‐glutamine, 0.1 mM 2‐mercaptoethanol, 1 unit/ml thrombin (Sigma‐Aldrich), 1:50 B‐27 , 80 ng/ml basic fibroblast growth factor, and 25 ng/ml epidermal growth factor . After 1 week, lung spheroids, here named pneumospheres (PSs), were collected by pipetting and centrifugation , and plated as PS‐derived cells (PDCs) on the corresponding coating in CEM media. Secondary PSs (IIPSs) were obtained again by replating PDCs on poly‐d‐lysine coating. For differentiation stimuli, PDCs and PSs were cultured for 1 week in bronchial/epithelial growth medium . The isolation procedure is summarized in The study was approved by the Ethics Committee of the “Sant'Andrea” Hospital, “La Sapienza” University (Protocol no. 5195/2013). After institutional review board approval and signed informed consent, lung tissue was obtained from 3 patients  who underwent lung surgery for benign disease at the Division of Thoracic Surgery, “Sant’Andrea” Hospital . Lung tissue biopsies were obtained during video‐assisted thoracoscopic surgery procedures using a mechanical stapler , Adipogenesis (1 × 104 cells/cm2) and Osteogenesis (5 × 103 cells per cm2) Differentiation kits . Cells were then fixed and stained with Alcian Blue, Oil Red O solution, or Alizarin Red (Sigma‐Aldrich), respectively, according to the manufacturer instructions, as previously described [Lung mesenchymal stem cells (L‐MSCs) were isolated from three lung biopsy specimens that were collected as reported above and as previously described , 31. Briescribed , 31.2 in 100‐mm Petri dishes) for 2 weeks, then fixed and Giemsa stained (Sigma‐Aldrich). Dishes were imaged and semiautomatic colony counting was performed with ImageJ software  for cloning efficiency calculation. Clusters of cells with a diameter >5 mm were considered as clones. PSs yield was calculated 1 week after seeding by semiautomatic particle count performed by ImageJ software on at least 8 random fields per condition. PSs dimension was calculated by ImageJ measurements of the average diameter on at least 20 random PSs per condition.For clonogenesis experiments, PDCs were cultivated at very low density (5 cells per cmhttp://www.abcam.com), and CD31 (Sigma‐Aldrich). Cells were then incubated for 15 minutes with anti‐rabbit or anti‐mouse Alexafluor‐488 (Thermo Fisher) secondary antibodies. Also, directly conjugated PE‐CD326/EpCam  and FITC‐CD90  antibodies were used, together with 7AAD labeling (BD Biosciences) to exclude dead cells. Appropriate isotype‐matched immunoglobulins were used as controls, and appropriate preliminary experiments were performed for compensation settings. A total of 10,000 events were recorded with a FACSAria II cytometer (BD Bioscience) using DiVa Software . Analysis was performed using Flowing software .Cells were collected by trypsinization and stained for 30 minutes with the following primary antibodies: CD105, CD44, CD45, CD117, CD133 . Reverse transcription was performed on a 500‐ng starting total of RNA (Thermo Fisher) in a 20‐µl reaction, and complementary DNA product was then subjected to real‐time polymerase chain reaction (PCR) with Sybr Green Mix in a 7900HT Fast Real‐Time system (Thermo Fisher). All primer sets were previously tested for optimal efficiency and all reactions were analyzed at the end by melting curves to confirm product specificity. Each reaction was performed in triplicate. The comparative cycle threshold method was used for relative quantification, using 18S as the housekeeping gene; reference conditions used for normalization are specified in each data set. Supplemental online Table 1 lists primer sequences.RNA was extracted with a column‐based kit , KDR . Slides were mounted in Mowiol medium (EMD Millipore). Fluorescence imaging was performed on a Leica DMI4000B automated inverted microscope , and confocal fluorescence imaging was performed on an Olympus IX83  equipped with FV10‐ASW 4.2 software. Incubation with secondary antibodies alone did not give any detectable background signal. Percentage of positive cells for ki67, Oct4, and TTF1was calculated by random field count of positive cells normalized to the number of total nuclei in the field.Cells were fixed for 10 minutes with 4% paraformaldehyde at 4°C, permeabilized with 0.1% Triton X‐100 (Sigma‐Aldrich) in PBS with 1% bovine serum albumin, then blocked in 10% goat serum, incubated overnight at 4°C in 1% goat serum with primary antibodies, and then incubated for 2 hours at room temperature with Alexa‐conjugated secondary antibodies (Thermo Fisher) and 4′,6‐diamidino‐2‐phenylindole nuclear dye (Sigma‐Aldrich). Primary antibodies were ki67, Oct4, TTF1, cytokeratin 5 (KRT5), prosurfactant‐C (pro‐SFTPC), aquaporin 5 , vimentin (VIM), cytokeratin 18 , according to the manufacturer’s instructions. Densitometric analysis was performed by ImageJ software, and data were presented either as optical density values or PS‐to‐PDC ratio.After 5 days of culture on poly‐t test, and a final value of p < .05 was considered significant.All results are presented as mean value ± SEM unless otherwise specified. Significance of difference between any two groups was determined by two‐sided Student d‐lysine formed PSs, consistently with a recognized stemness feature [We tested at first how different substrates—GEL, FN, and LAM—affected the efficiency of the protocol to isolate LPCs as spheroids, namely PSs and as PDCs. We also performed preliminary optimization of cell culture media: F12 and DMEM low‐glucose media could not successfully promote explant outgrowth (data not shown), whereas CEM on each coating yielded sufficient outgrowth EDCs after 2–3 weeks . A signi feature , whose yPSs were analyzed by immunofluorescence staining and confocal microscopy, and no differences were observed in protein abundance or distribution among PSs derived from different coatings (data not shown). Representative panels for PS staining are shown in TTF1, Oct4, SFTPA1, KRT18), gelatin coating was associated with slightly, albeit significantly, higher levels than fibronectin and laminin, with other genes  showing similar biological trends without reaching statistical significance. PDCs on fibronectin coating had significantly higher RNA levels of AQP5, with a clear trend toward downregulation for SFTPC, suggesting a preferential commitment toward an alveolar type 1 (AT1) phenotype.PDC monolayers were highly proliferating and undifferentiated, with comparable doubling times between 2 and 2.5 days on different coatings . ComparaPDCs contained a subpopulation with clonal growth capacity, yielding a comparable cloning efficiency of approximately 9% on all coatings . The PDCTTF‐1, CCSP, NGFR, and p63), pneumocytes , endothelium , and basal epithelium . This suggested a spontaneous 3D organization in a microtissue‐like structure, inducing epithelial committed phenotypes. The significant, albeit slight, upregulation of Snai1 and β‐catenin also suggested that partial EMT mechanisms are involved in PS formation, as already described for other spheroid culture systems [AQP1, KDR) were upregulated, although endothelial proteins were never detectable by immunofluorescence (data not shown) and cytofluorimetry  could be observed between 3D  and 2D (PDCs) culture by immunofluorescence staining, with the opposite switch for vimentin expression . To further characterize the phenotype, we performed a double‐labeling EpCam/CD90 for flow cytometry. Cells were mainly CD90+ at all stages . Results showed that a small EpCam+/CD90+ double‐positive population was present in a constant proportion among EDCs, PSs, PDCs, and secondary PSs stages , thus suggesting the stromal, albeit plastic, nature of the cell population overall.To fully elucidate the apparent mesenchymal traits acquired in 2D culture by PDCs, we also performed a mesodermal multilineage differentiation assay on EDCs and PDCs for the three mesenchymal lineages: osteogenic, chondrogenic, and adipogenic . EDCs displayed an incomplete mesenchymal potential, because osteogenic differentiation was not detectable. PDCs selected after the spheroid stage, however, contained a subpopulation with partial ability for multilineage mesodermal transdifferentiation. Nonetheless, such potential was dramatically lower than that of lung mesenchymal stem cells (L‐MSCs), independently isolated from lung parenchyma, shown as the positive control in supplemental online Figure 2. Thus, PDCs significantly differed from resident L‐MSCs, and the mesenchymal‐like traits acquired in 2D seemed consistent with a transient mesenchymal transition of progenitors induced when not in optimal 3D culture conditions.TTF‐1, CCSP, NGFR, Oct4, and p63), AT2 markers , and basal epithelial genes  were significantly upregulated compared with control media. AQP5, which was spontaneously upregulated in PSs, was downregulated after BEGM exposure; this was consistent with the induction of a preferential bronchiolar versus alveolar phenotype by this culture media. Endothelial markers were modulated, with a further increase in KDR levels but a decrease in AQP1, which was downregulated in a similar proportion in PDCs as well. PDC gene expression levels were mostly unaffected by specific BEGM media treatment except for a slight decrease in p63 expression and increase in β‐CAT expression. PSs cultured in BEGM were also analyzed by immunofluorescence and confocal microscopy  screening to define their specific secretomic profile compared with monolayer PDCs. PSs produce and release a wide panel of humoral factors, some of which are already described , includiThe lung is considered a very plastic organ, harboring multiple cell populations with stemness features, and able to significantly contribute to homeostasis as well as repair and regeneration after injuries , 4, 39. We aimed at studying the effects of different culture conditions on gene and protein expression in PSs and PDCs, mirroring the epithelial and mesenchymal plasticity of the organ in vivo , 45, 46.Results show that PSs and PDCs contain a stromal lung progenitor population with clonal growth capacities , togetheFGF9 is also produced at higher levels by PSs, in combination with TGFβ2. These factors play key roles in regulating lung development , and theUnder the conditions of our study, PSs and PDCs contained a majority of CD90+ cells , but we have shown that vimentin positivity marks a significant difference between 3D and 2D culture, suggesting a mesenchymal‐like potential for PDCs, which, nonetheless, do not display a standard lung mesenchymal stem cell phenotype , highlighting the relevant biological and functional differences between PS/PDC and lung MSCs. Thus, PDCs likely represent a single bipotent stromal progenitor population expressing minimal epithelial features but capable of significant epithelial commitment when cultured in optimal spheroid conditions, consistently with similar phenomena observed in vivo . In fact3D growth has been shown to affect differentiation of bronchospheres , and modOur data suggest a proepithelial niche‐like microenvironment inside the PS, highly influenced by the 3D culture system and able to modulate cell phenotype. This feature makes PSs more prone to LPC maintenance and more responsive to specific epithelial commitment stimuli than 2D monolayer cell cultures. Also, we have shown for the first time how PSs produce strong paracrine signals and have a distinctive secretomic profile significantly affected by the 3D culture switch. Thus the modulation of 3D growth alone could be a powerful tool to control stemness and epithelial commitment in in vitro models of lung progenitor cells.I.C.: conception and design, collection and/or assembly of data, data analysis and interpretation, manuscript writing, final approval of manuscript; F.P. and G.M.: collection and/or assembly of data, data analysis and interpretation; F.A., C.S., and V.P.: collection and/or assembly of data; E.D.F.: data analysis and interpretation, manuscript writing; M.P. and R.C.: data analysis and interpretation; M.I.: provision of study material; G.B.‐Z.: financial support, data analysis and interpretation; E.M.: conception and design, manuscript writing; G.F.: conception and design, financial support, provision of study material, manuscript writing, final approval of manuscript.The authors indicated no potential conflicts of interest.Supporting InformationClick here for additional data file.