Robot-assisted (RA) technique has been increasingly applied in clinical practice, providing promising outcomes of inserting accuracy and cranial facet joint protection. However, studies comparing this novel method with other assisted methods are rare, and the controversy of the superiority between the insertion techniques remains. Thus, we compare the rates and risk factors of intrapedicular accuracy and cranial facet joint violation (FJV) of RA, fluoroscopy-guided percutaneous (FP), and freehand (FH) techniques in the treatment of thoracolumbar fractures.A total of 74 patients with thoracolumbar fractures requiring pedicle screw instruments were retrospectively included and divided into RA, FP, and FH groups from June 2016 to May 2020. The primary outcomes were the intrapedicular accuracy and cranial FJV. The factors that affected the intrapedicular accuracy and cranial FJV were assessed using multivariate analyses.The optimal intrapedicular accuracy of pedicle screw placement (Grade A) in the RA, FP, and FH groups was 94.3%, 78.2%, and 88.7%, respectively. This finding indicates no significant differences of RA over FH technique (P = 0.062) and FP technique (P = 0.025), but significantly higher accuracies of RA over FP (P < 0.001). In addition, the rates of proximal FJV in RA, FP, and FH groups were 13.9%, 30.8%, and 22.7%, respectively. RA had a significantly greater proportion of intact facet joints than the FP (P = 0.002). However, FP and FH (P = 0.157), as well as RA and FH (P = 0.035) showed significantly similar outcomes with respect to the proximal FJV. The logistic regression analysis showed that FP technique (OR = 3.056) was independently associated with insertion accuracy. Meanwhile, the age (OR = 0.974), pedicle angle (OR = 0.921), moderate facet joint osteoarthritis (OR = 5.584), and severe facet joint osteoarthritis (OR = 11.956) were independently associated with cranial FJV.RA technique showed a higher rate of intrapedicular accuracy and a lower rate of cranial FJV than FP technique, and similar outcomes to FH technique in terms of intrapedicular accuracy and cranial FJV. RA technique might be a safe method for pedicle screw placement in thoracolumbar surgery.3 Thoracolumbar fractures account for most of the spine fractures, which can cause spinal instability and kyphosis, and often require surgical treatment. Pedicle screw reduction and internal fixation are the commonly used techniques. Since the launch of screw placement with freehand (FH) technique, numerous insertion techniques, such as fluoroscopy-guided percutaneous (FP), computer-assisted guidance, and robotic-assisted (RA) approaches have been introduced to achieve better surgical outcomes with increased inserting accuracy, reduced intraoperative blood loss, smaller incision, shorter surgical time, and better pain relief , 2. HoweRecently, a novel insertion technique of RA method has been increasingly applied in clinical practice; it provides promising outcomes of inserting accuracy, ranging from 93.2 to 98.2% . Many stBesides, many studies have found that the RA techniques resulted in lower cranial FJV rates (0–2.84%) than the FH methods (12.76–34.1%) , 18, 19.The thoracolumbar segments are in the transitional area of the facet joint anatomy, where the surfaces of facet joint change from the coronal to the sagittal planes. Thus, the thoracolumbar fractures might contribute to the dislocations in part of the facet joints and the shifts of normal anatomical landmarks for accurate placement of pedicle screw. Thus, the intrapedicular accuracy is reduced, and the rate of cranial FJV is increased. However, most literature regarding thoracolumbar fractures focused on the clinical efficiencies in different insertion techniques, but studies about the radiographic outcomes were rare –22. MeanThe retrospective study was approved by the hospital institutional review board. A total of 74 patients diagnosed of T11-L2 fractures with screw placement from T10 to L4 levels were included in the comparative study treated with robot-assisted (30 patients), fluoroscopy-guided percutaneous (14 patients), and freehand (30 patients) insertions during the period of June 2016 to May 2020. The diagnosis of T11 fracture accounted for 6.8%, and the T12, L1, and L2 fractures accounted for 27.0%, 45.9%, and 20.3%, respectively.Patients that satisfy the following criteria were eligible: (1) patients suffering from T11-L2 fractures in the treatment of pedicle screw insertion with or without interbody fusion; (2) patients receiving postoperative 3D CT examination. The exclusion criteria were as follows: (1) prior surgery at the instrumental levels; (2) pathological fracture caused by tuberculosis or tumor; (3) incomplete data in the review case.In the RA group, the TINAVI robot workstation was connected to the C-arm scanner. After scanning, the 3D image was transmitted to the TINAVI robot workstation for automated registration. According to the planned path, the final position of the robot arm was adjusted. Then, the working sleeve was inserted along the robot arm guide to touch the bony part of the vertebral body, and the guide wire was inserted with the electric drill along the direction of the working sleeve. The rod was placed after the insertions of the pedicle screw along the guide wire  Fig. 1)1).Fig. 1In the FP group, under the frontal and lateral fluoroscopy with C-arm X-ray, the puncture needle was inserted through bilateral pedicle . The core of the puncture needle was removed after confirming that the position was satisfactory with fluoroscopies. Then, the guide wire was inserted, and the outer sheath of the puncture needle was withdrawn . The metIn the FH group, the paraspinal muscles were striped from the midline approach to both sides, and then the lateral edge of the articular process was exposed. The transverse process, facet joint, and isthmus were used as the landmarks for entry points in the pedicle screw placement. Then, the appropriately selected screws were inserted and secured with rods , 25.The primary outcomes were intrapedicular accuracy and facet joint violation, which were assessed in accordance with the Gertzbein and Robbins scale  Fig. 2)2) and thThe secondary parameters were statistics of cases with the screw deviations of superior, inferior, medial, or lateral direction in the pedicle malposition, statistics on the screw parts of shaft, head, or rod causing cranial FJV, and complications including intraoperative revision due to screw malposition, postoperative revision due to screw malposition, wound infections, operation time, and estimated blood loss.The following variables were also collected for the analysis of possible risk factors associated with intrapedicular accuracy and cranial FJV: age, gender, body mass index (BMI), pedicle screw placement technologies, pedicle angle, facet joint osteoarthritis, and distance from skin to insertion point. The facet joint osteoarthritis was evaluated with Pathria classification . The radt-test was utilized to compare between two groups when the two sets of data were in accordance with Gaussian distribution. Otherwise, the Wilcoxon test was performed for analysis. The categorical variables were presented as the absolute (no.) and relative (%) frequencies, and the chi-squared test was used to compare two groups. Significance level was set a = 0.05. Moreover, continuous and categorical variables were analyzed with ANOVA and chi-squared tests, respectively, to compare the three groups, whereas the significance level was adjusted as 0.0167 with Bonfferoni correction in chi-squared test for the comparison of three groups. The factors that possibly affected the intrapedicular accuracy and cranial FJV were also assessed with multivariate logistic regression analysis.All statistical analyses were conducted using SPSS 23.0 software . The continuous variables were expressed as the mean and standard deviation. The 2. The baseline parameters showed no significant distances among the three groups in terms of the mean age, gender ratio, average BMI, and preoperative diagnosis (P > 0.05)  was 27/47, and the average BMI was 22.88 ± 3.78 kg/mThe optimal intrapedicular accuracy of pedicle screw placement (Grade A) in the RA, FP, and FH groups was 94.3%, 78.2%, and 88.7%, respectively. This finding indicates no significant differences of RA over FH technique (P = 0.062) and FP technique (P = 0.025), but significantly higher accuracies of RA over FP (P < 0.001). In addition, the clinically acceptable accuracy of pedicle screw placement (Grade A + B) was 97.5%, 93.6%, and 97.0%, indicating no significant differences among the three techniques  , FP (27.60 ± 7.91 min/screw) and FH (28.86 ± 8.10 min/screw) techniques showed no significant differences in average time per screw (P > 0.05). Besides, FH (59.94 ± 26.49 ml/screw) resulted in significantly more blood loss per screw than RA (11.75 ± 2.20 ml/screw) and FP (11.61 ± 2.41 ml/screw), but no significant difference was observed in RA and FP techniques (P > 0.05).The logistic regression analyses showed that FP technique  was independently associated with intrapedicular accuracy. Moreover, the age , pedicle angle , moderate facet joint osteoarthritis , severe facet joint osteoarthritis  were independently associated with cranial FJV (Table Pedicle screw placement has been widely used in spine surgery. It has gradually developed from free hand method in open surgery to percutaneous insertion in minimally invasive methods with different auxiliary equipment applied during the procedures of screw placement. However, in any case, the safety and accuracy of pedicle screw placement are essential. Thus, different instrumental techniques have been applied to increase intrapedicular accuracy and reduce cranial facet violation . RecentlFor intrapedicular accuracy, Han et al.  and FengCranial FJV has been regarded as a crucial risk factor of ASD , 6, 8. MThe outcomes might be attributed to the following factors. The first factor is the guidance equipment, the selection of which can directly affect the clarity of the anatomical landmarks during the operation. This factor influences the rates of insertion accuracy and violation of facet joint. Different from the FP method mainly relying on the 2D images, the RA system can automatically identify the entry point and preplan insertion trajectory in accordance with the 3D images, which show anatomical structures clearly . Thus, tThe characteristics of the adverse events among the three techniques were reported. In the FH and FP groups, most deviations of screws were medial, thereby resulting in severe complications, such as spinal cord and nerve root injuries. However, in the RA group, most deviations were lateral, thereby reducing the possibility of severe consequences and revealing a safer choice for screw implantation. In addition, more than 90% cranial FJVs were caused by the screw head in three groups. This finding reminded the surgeons to insert screws in less depth on the premise to ensure spinal stability for facet joint protection. Moreover, for the complications, no patients underwent postoperative revision caused by screw malposition, and 3.3% and 7.1% of patients received intraoperative revision caused by screw malposition in the FH and FP groups. These results were higher than those of the RA group. Furthermore, no patients suffered from wound infections after surgery in three groups.The factors that potentially affected the intrapedicular accuracy and cranial FJV need further investigation. Kim et al.  retrospeThe following limitations should be interpreted in this retrospective study. First, the clinical outcomes among the three techniques were not reported in the current study, and the relationship between the different grades of malposition and clinical outcomes need further investigation for the future work. Second, the clinical results were not discussed in this study, and the radiographic outcomes were obtained in short term after surgery without long-term follow-up. However, we assumed that the short-term results concerning the safety and accuracy of pedicle screw placement were crucial issues, thereby providing reference and reflection for surgeons. Third, the number of patients and screws included in the study is limited and the prospective trials with large sample size and high quality are needed in the future.RA technique showed a higher rate of intrapedicular accuracy and a lower rate of cranial FJV than FP technique, and similar outcomes to FH technique in terms of intrapedicular accuracy and cranial FJV. In addition, the FP technique was a risk factor for the intrapedicular accuracy. The RA technique might be a safe method for pedicle screw placement in spinal surgery.


Ginkgo biloba L. (GB) and Selaginella tamariscina (P. Beauv.) Spring (ST), which displayed very strong inhibitory effects toward CYP3A4. The inhibitory effects of these biflavones on clinical drugs that mainly undergo CYP3A4-dependent metabolism were evaluated. The IC50 of GK toward tamoxifen, gefitinib and ticagrelor were found to be of 0.478 ± 0.003, 0.869 ± 0.001, and 1.61 ± 0.039 μM, respectively. These results suggest the potential pharmacokinetic interactions between the identified biflavones and clinical drugs undergoing CYP3A4-mediated biotransformation. The obtained information is important for guiding the rational use of herbal medicine in combination with synthetic pharmaceuticals.CYP3A4-mediated Phase I biotransformation is the rate-limiting step of elimination for many commonly used clinically agents. The modulatory effects of herbal medicines on CYP3A4 activity are one of the risk factors affecting the safe use of drug and herbal medicine. In the present study, the inhibitory effects of nearly hundred kinds of herbal medicines against CYP3A4 were evaluated based on a visual high-throughput screening method. Furthermore, biflavone components including bilobetin , ginkgetin (GK), isoginkgetin (IGK), and amentoflavone (AMF) were identified as the main inhibitory components of  Cytochrome P450 (CYP) enzymes are a large class of heme-thiolate protein superfamily serving the key enzymes in the first-pass metabolism of many foreign substances in humans . Among mDrugs mainly undergoing CYP3A4 metabolism are more susceptible to adverse drug reactions caused by drug-drug interaction with co-administered pharmaceuticals, and the inhibitory effect of herbal medicines on CYP3A4 can be one of risk factors affecting the safe use of drug and herbal medicine combinations . The comN-ethyl-1,8-naphthalimide (NEN) is a CYP3A4-specific fluorescent probe developed by our group . HydroxyGinkgo biloba L. (GB) and Selaginella tamariscina (P. Beauv.) Spring (ST) display the strongest inhibitory effects toward CYP3A4, and four biflavones were identified as the main inhibitory components of these herbal medicines. Subsequently, the inhibitory effects of these biflavones on clinical drugs mainly undergoing CYP3A4-dependent metabolism were evaluated. Results of these studies suggest the potential interaction between these biflavones and clinical drugs.In the present study, we employed rapid the screening of the inhibitory action of herbal medicines against CYP3A4 based on a visual high-throughput screening method using NEN. Our results indicate that 2, glucose-6-phosphate dehydrogenase, and D-glucose-6-phosphate (G-6-P) were obtained from Sigma-Aldrich . The pooled human liver microsomes (HLM) were purchased from Bioreclamation IVT .The prepared slices of Chinese crude medicines were purchased from Beijing Tongren Tang Co., Ltd. . Ginkgetin, isoginkgetin, bilobetin, and amentoflavone were purchased from Herbest Biotechnology Co., Ltd. . N-ethyl-1,8-naphthalimide (NEN) was synthesized by our lab, and was fully characterized by NMR and MS techniques. Midazolam, NADPNaMedical materials were ground to coarse powder, and then immersed into 10 volumes of 95% ethanol. Subsequently, the powdered materials were extracted twice with ultrasound (600 W) for 30 min. The filtered extracts were enriched by a rotary vacuum evaporator. The extracts were dissolved in DMSO as the stock solution (20 mg/ml) for the subsequent.2 (4 mM), NEN (50 µM), extracts of herbal medicine (20 µg/ml), or components. The incubation duration and HLM protein concentrations were 30 min and 0.05 mg/ml, respectively. The reaction was initiated by the addition of NADP+ and terminated by the addition of 100 µL ice-cold acetonitrile (ACN). NEN 4-hydroxylation were utilized as the probe reaction for CYP3A4, and fluorescence image of 96-well microplates were measured by a fluorescent image analyzer  under the excitation wavelength of 488 nm (Em: 570 ± 15 nm). The formation of NEHN was evaluated by the quantitative analysis of fluorescence signal for individual wells of microplates using the equipment supporting software. The percentage residual activity was calculated from percentage relative value of NEHN formation rate.The primary incubation mixture contains HLM, potassium phosphate buffer , G-6-P (10 mM), G-6-P dehydrogenase (1 U/ml), MgClGinkgo biloba L. (GB) and Selaginella tamariscina (P. Beauv.) Spring (ST) were analyzed based on UPLC with a photodiode array detector.The chemical fingerprints of Fractions were collected with an Acquity UPLC system  equipped with a photodiode array detector (2998), an auto sampler, a quaternary gradient pump, and a column compartment. An AQCUITY UPLC HSS C18  analytical column was used and kept at 30°C. The mobile phase consisted of 0.3% trifluoroacetic acid in water (A) and Methanol (B). The following gradient conditions was used: 0.0–5.0 min, 85% A, 5.0–38.0 min, 85–20% A, 38.0–41.00 min, 20% A, 41.0–44.0 min, 20–85% A, 44.0–47.0 min, 85% A for analysis of GB extract; 0.0–3.0 min, 65% A, 3.0–10.0 min, 65%–10% A, 10.0–15.00 min, 10% A, 15.0–18.0 min, 10%–65% A, 18.0–20.0 min, 65% A for analysis of ST extract. The precision of the fingerprinting method was previously confirmed by comparison of the HPLC spectrum mainly considering the relative retention time and peak area. Then, the LC fractions of crude extract  were collected consecutively at 30 or 60 s intervals, where the flow rate was set at 0.3 ml/min.in vacuum, and redissolved in DMSO. The fractions were evaluated for their activity moderating effect for CYP3A4 based on the high-throughput screening method. Then, the target fractions underwent further preparation and purification by semi-preparative HPLC.All LC fractions were dried Ki) values were determined using the equations for competitive inhibition . The high-throughput screening assay was performed, and the inhibition curve was fitted with GraphPad Prism (Version 8.0). The inhibition type was confirmed based on the R square of the following four equations and the Dixon and Lineweaver–Burk plots. The inhibition constant , tamoxifen (20 μM), ticagrelor (30 μM), respectively . The ExiGinkgo biloba L.  and Selaginella tamariscina (P. Beauv.) Spring  exhibited ultra-strong inhibitory effects toward CYP3A4 resulting in virtual elimination of the fluorescence signal in the corresponding wells of the 96-wells microplate. Quantitative analysis of the inhibitory effects of herbal medicine towards CYP3A4 demonstrate that the residual activities of CYP3A4 after treatment with the extracts of GB and ST were 6.3 and 5.0%, respectively (Tribulus terrestris L. (D2), Areca catechu L. (B10), and Fissistigma glaucescens (Hance) Merr. (F3).The inhibitory effects of 93 herbal medicines toward CYP3A4 were preliminary evaluated using the high-throughput screening system constructed with CYP3A4 selective fluorescent probe NEN . Inhibitectively . The quaWe attempted to reveal the inhibitory components of GB and ST since they exhibited strong inhibitory effect towards CYP3A4. However, the complexity and structural diversity of the two herbal medicines are very high, especially GB. In the present study, a combination of chemical fingerprinting-guided LC fraction collection and screening of the inhibitory potential was used to identify the inhibitory components of herbal medicine. The LC fractions of GB and ST were collected consecutively at appropriate time intervals (30 or 60 s) based on the reliable LC method which was confirmed by reproducible relative retention time of the main components of herbal medicine (RSD < 3%). Subsequently, the inhibitory components of GB and ST toward CYP3A4 were successfully identified by bioassay guided LC fractions studies utilizing high-throughput visualization screening. For GB, three inhibitory components were picked out from a comparison of the UPLC-UV fingerprint with the profile of CYP3A4 inhibition . Then, tIC50 values of DGK, GK, IGK, and AMF against CYP3A4 were evaluated as 0.162 ± 0.022, 0.106 ± 0.004, 0.982 ± 0.006, and 0.186 ± 0.004 μM, respectively. These findings suggested that the four biflavones exhibited a significant inhibition activity towards CYP3A4 and further confirmed the above conclusion that DGK, GK, IGK, and AMF were the main inhibitory components of GB and ST, respectively.The concentration range of DGK, GK, IGK, and AMF was used to depict the inhibitory plot and characterize the inhibitory effects of four identified biflavones toward CYP3A4. As shown in Ki) values of DGK, GK, IGK, and AMF toward CYP3A4 were further determined. As shown in Ki value was determined to be of 0.070 ± 0.018 μM. Correspondingly, the inhibition profiles of GK, IGK, and AMF were all fitted to mixed reversible inhibition equation with the Ki values of 0.034 ± 0.007, 0.278 ± 0.087, and 0.101 ± 0.030 μM , Areca catechu L. (B10), Fissistigma glaucescens (Hance) Merr. (F3) and Isatis indigotica Fort. (F11) displayed a moderate inhibitory effect toward CYP3A4, while the inhibitory effect of the remaining herbal medicines was insignificant. It is noteworthy that GB and Isatis indigotica Fort. are the most frequently used herbal medicines in Asia. Patients with ischemic stroke and neurological disorders used GB as a remedy (Isatis indigotica Fort. is one of the few well-recognized traditional herbs with anti-inflammatory, antibacterial and antiviral effects (Tribulus terrestris L. is currently approved as a dietary supplement due to its pro-sexual and androgen enhancing effects (Isatis indigotica Fort., Tribulus terrestris L. Our results provide important information for guiding the rational combination of herbal medicines and synthetic pharmaceuticals.Among the screened herbal medicines, GB and ST showed strong inhibitory effects on CYP3A4. a remedy , and Isa effects . Additio effects . The aboPrevious research reports on interactions between GB and CYP indicated that flavonol aglycones such as kaempferol, quercetin, and apigenin were the major CYP3A4 inhibitory components in GB . The disIC50 of 3.50 ± 0.034, 1.61 ± 0.039, and 10.2 ± 0.382 μM, respectively. Notably, the GB extract is commonly used for treatment in patients exhibiting angina pectoris, myocardial infarction and ischemic stroke, and for improving cardiac function, myocardium microenvironment and neurological function (IC50 values for the effect of AMF on the oxidation of tamoxifen and gefitinib are equal to 2.72 ± 0.093 and 5.20 ± 0.527 μM, respectively. Our results confirmed a significant inhibitory effects of the diflavonoids on the clinical drugs under study, and also pointed out that more attention should be paid to the herbal-drug interactions caused by the CYP3A4-mediated metabolic interaction between clinical drugs with biflavonoids and the respective herbal medicines.Biflavonoids are a class of compounds with outstanding anti-inflammatory and antioxidant biological activities, and have anti-microbial, anti-tumor and neuroprotective effects . In a vafunction . Therefofunction . The pot


Cyanidium caldarium was investigated. The efficiency of 30 min spark discharges was compared with standard treatment protocols, such as three times repeated freeze-thaw cycles, sonication, and pulsed electric fields. Input energy for all physical methods were kept constant at 11,880 J to ensure comparability. The obtained extracts were evaluated by photometric and fluorescent spectroscopy. Highest extraction yields were achieved with sonication (53 mg/g dry weight (dw)) and disintegration by spark discharges (4 mg/g dw) while neither freeze-thawing nor pulsed electric field disintegration proved effective. The protein analysis via LC-MS of the former two extracts revealed a comparable composition of phycobiliproteins. Despite the lower total concentration of phycocyanin after application of spark discharges, the purity in the raw extract was higher in comparison to the extract attained by sonication.Phycocyanin is a blue colored pigment, synthesized by several species of cyanobacteria and red algae. Besides the application as a food-colorant, the pigmented protein is of high interest as a pharmaceutically and nutritionally valuable compound. Since cyanobacteria-derived phycocyanin is thermolabile, red algae that are adapted to high temperatures are an interesting source for phycocyanin extraction. Still, the extraction of high quality phycocyanin from red algae is challenging due to the strong and rigid cell wall. Since standard techniques show low yields, alternative methods are needed. Recently, spark discharges have been shown to gently disintegrate microalgae and thereby enable the efficient extraction of susceptible proteins. In this study, the applicability of spark discharges for phycocyanin extraction from the red alga  The bright blue colour of the pigment phycocyanin is of great interest as a natural colorant in food industry. Its good water solubility especially allows for the processing of extracted phycocyanin as a food colorant for ice cream, jelly or chewing gum . Since cArthrospira sp. are used as phycocyanin source due to their widespread availability and cost-efficient cultivation [Cyanidium caldarium to live in extreme habitats such as hot, acidic springs [Cyanidium caldarium produce these accessory pigments in order to broaden the absorption spectrum of light used for photosynthesis. These pigments are characterised as phycobiliproteins [Cyanidium caldarium possess a dense cell wall [Limnothrix sp., indicating a three-times repeated freezing at −70 °C achieving the highest yields of phycocyanin. Major disadvantages of this method are the long treatment time, the high energy demand, and the number of freeze-thaw cycles strongly depending on the microorganism strain [Most commonly, cyanobacteria such as tivation . The out springs  qualify  springs . Furtherproteins  that canproteins ,13. C-phproteins . The chrproteins . The fluproteins . The smaell wall . A succeell wall ,18,19. Sm strain ,21. Altem strain  and the m strain ,23,24. Tm strain . Generalm strain ,27,28. Tm strain .Spark discharge treatment of algae suspensions has been shown to be an effective, but gentle tool for the disintegration of microalgae and with regard to pigment and protein extraction ,31,32. IThe aim of this study was to determine the efficiency of spark discharges compared to standard treatment protocols, e.g., freeze-thaw cycles, pulsed electric fields, and sonication. The results were evaluated in respect of scaling-up potential. To keep the comparability of all applied techniques, the total energy input was kept constant, except for freeze-thaw cycles. To examine the extracted phycocyanin for protein modifications caused by the disintegration method, protein analysis via LC-MS was performed for selected extracts.Cyanidium caldarium was used for all experiments. The algae were maintained in a modified Galdieria-medium in 100 L scale at 34 °C under continuous light (100 µmol/m2s) and harvested 21 days after inoculation, exhibiting a dry biomass equivalent of approximately 4 g/L. After harvesting, the biomass was concentrated by centrifugation to a dry biomass equivalent of 100 g/kg. The pH of the suspension was adjusted with sulphuric acid to 2.1, and the conductivity was 2.3 mS/cm.Algae suspension of For each of the individual conditions, a volume of 75 mL algae suspension was subjected to disintegration. Experiments were performed in triplicate.Safaei et al., with some modifications. First, the algae suspension was lyophilised . Subsequently, the dry biomass was resuspended in distilled water to the original volume of 75 mL and freeze thaw cycles were repeated three times. Freezing was performed at −70 °C for 24 h, followed by thawing the suspension at 4 °C for another 24 h. Without accounting for losses in the cooling device, the estimated energy input for all three cycles was 111,900 J in total.Freeze-thaw cycles were performed in accordance to the procedure suggested by The algae suspension was exposed to a sonicator  in a 100 mL beaker. The sonotrode was placed in the centre of the algae suspension, approximately 2 cm above the beaker ground. The amplitude was set to maximum, i.e., 120 μm elongation at a frequency of 20 kHz, resulting in a provided sonication power of 114 W. The algae suspension was treated for 1 min 44 s, which equals an energy dissipation of 11,880 J in the sonicator. Due to strong heating of the suspension, the treatment was conducted in two different ways. The total energy input was either applied at once (continuous sonication) or sonication was paused after each 30 s until the temperature of the suspension was <20 °C (intermittent sonication).Two plasma-polished stainless steel electrodes with a diameter of 32 mm were placed in parallel with a distance of 4 mm into a 3.22 µL treatment chamber as described by Eing et al. and Goettel et al. ,35. The Spark discharges were ignited in the algal suspension between the tips of two tungsten rods with a diameter of 1 mm and a gap distance of 0.5 mm. As schematically visualised in g for 10 min at room temperature. The supernatant was transferred to a new falcon and centrifugation was repeated. The remaining supernatant was taken as the raw extract and used for photometric evaluation  of phycocyanin content and purity, as well as for protein precipitation for further characterization.After the disintegration, the suspension was centrifuged at 3000× PC) was calculated according to Equation (1) [Calculations were performed as described by Abalde et al. and Bennett and Bogorad et al. ,36. A voEP) was estimated by the ratio of absorption at 615 nm and the amount of protein, given by the absorption at 280 nm, according to Equation (2) [Purity (tion (2) :(2)EP=ODg, 10 min, room temperature), the supernatant was discarded and the pellet was dissolved in 1 mL water (LC-MS-grade). The desalination was performed with PD-10 desalting columns , following the manufacturer’s instructions. The purified extract was analysed by fluorescence and absorption spectroscopy, and the protein fingerprint of phycocyanin was studied with LC-MS/MS-based protein analyses.An amount of 13 mg ammonium sulphate was added to the raw extract and mixed vigorously to then precipitate phycocyanin over night at 4 °C. The precipitated raw extract was stored in ammonium sulphate solution at 4 °C until further use. After centrifugation . Excitation wavelength was set to 620 nm and the emission was detected in a range of 600 nm to 750 nm with an increment of 0.5 nm. Spectra were recorded in triplicates.Fluorescence spectra of 1 mL purified extract were obtained with a FluorologAbsorption spectra were obtained with a spectrophotometer . The absorption was recorded in a range of 250 nm to 700 nm. Spectra were recorded in triplicates.v/v acetic acid (A) and acetonitrile with 0.1 % v/v acetic acid (B), both LC-MS-grade .For sample preparation, 25 µL trypsin with a concentration of 20 µg/mL  were added to an aliquot of 100 µL of the purified extract. Digestion was performed for 20 h at 37 °C. Nanoflow liquid chromatography mass spectrometry (nanoLC-MS/MS) was performed with an UltiMate 3000 RSLCnano system  with an Acclaim Pepmap C18 column  and the corresponding precolumn  for desalting and cleaning of the sample. The injection volume for each sample was 1 µL, corresponding to 0.02 µg protein. The gradient for sample separation see  consistem/z 300 to 1650 , and with the fragmentation of the top 15 most abundant precursor ions . Precursor ions were fragmented by using a normalized higher-energy collisional dissociation (27.5 eV) and were detected at a mass resolution of 17,500 at m/z 200. The automatic gain control target for full MS was set to 6.37 × 106 ions and for MS/MS to 2e5 ions with a maximum ion injection time of 100 ms for both modes. A dynamic exclusion was set to 60 s and ions with a charge state one, or above seven were excluded. To identify the peptides and potential post-translational modification, the MS raw data were analysed with the software Proteome Discoverer 2.4 . The modifications in the amino acid chain of the peptides were scanned with the software Byonic Version 3.6.0 , as a plug-in tool for the Proteome Discoverer. Detected modifications were filtered with a Byonic score > 250 and Delta Mod score > 5.Mass spectrometry (MS) acquisition was carried out in a data dependent mode with a full scan range of ® database [Arthrospira platensis (A. s.), Cyanidium caldarium (C. c.), Galdieria sulphuraria (G. s.), Porphyridium purpureum (P. p.), Polysiphonia urceolata (P. u.), and Synechococcus sp. (S. sp.). Abundances are given as averages of duplicates obtained by MS-protein analysis.Protein data for the phycobiliproteins (Allophycocyanin (APC), C-phycocyanin (C-PC), R-phycocyanin (R-PC), and phycoerythrin (PE)) were taken from Uniprotdatabase . The proCyanidium caldarium with standard extraction methods and spark discharges was determined by absorption and fluorescence spectroscopy with respect to phycocyanin, allophycocyanin and phycoerythrin content released to the supernatant. For freeze-thaw cycles, pulsed electric fields (PEF), and sonication with fixed temperature no increase in phycocyanin yield was detected, compared to untreated controls. The application of spark discharges resulted in a moderate increase of phycobiliprotein content, while sonicated samples showed a steep increase, when the total energy was applied at once. Protein analysis revealed that, independent of the disintegration method, the composition of phycobiliproteins is divers, including C-phycocyanin, allophycocyanin, R-phycocyanin, and phycoerythrin. Based on the rate of modifications in the amino acid chain of the peptides, the spark discharge showed to be the gentlest procedure with least modifications. Images of the raw extracts from freeze-thawing, sonication and spark discharge treatment can be found in the Phycobiliprotein extractability of By increasing the volume (from 1.5 mL to 75 mL) of algae suspension conducted to freeze-thaw-cycles at once, the supernatant after centrifugation remained colourless although an order of magnitude more energy, 111,900 J was dissipated. The phycocyanin content estimated by photometric determination did not differ from untreated control (0.2 mg/g dw) and was calculated with 0.5 mg/g dw see . The evaThe disintegration by sonication was divided into two different treatment schemes. First, the total energy  of the sonication process was applied at once, leading to a rise in temperature of the algae suspension above 45 °C. In order to avoid possible damages caused by the high temperature, the sonication process was paused after each 30 s in a second treatment scheme, allowing the algae suspension to cool down intermittently to 20 °C. By this process, the maximum temperature did not exceed 27 °C, comparable to the temperatures obtained after disintegration by spark discharges.The supernatant after centrifugation was of a dark blue colour. The photometric determination of phycocyanin content indicated a concentration in the raw extract of 53 mg/g dw see . The maxLimiting the rise of temperature by pausing of sonication process led to a marginal release of phycocyanin (data not shown). The supernatant after centrifugation was of a dark green colour without visible traces of blue. Since the high amount of impurities inhibited the purification, no further evaluation of phycocyanin characteristics were examined.The application of pulsed electric fields with a total energy of 11,880 J did not foster the release of phycocyanin from algae cells. The phycocyanin concentration in the raw extract corresponded to values of untreated controls, as illustrated in After the exposure of the algae cells to spark discharges in a continuous flow-through system, the raw extract was of an intense blue colour. The photometric determination of phycocyanin concentration showed a distinct deviation from untreated control. Compared to the phycocyanin concentration in the raw extract after sonication, the concentration was 13-times lower (≙ 4 mg/g dw) after disintegration by spark discharges. Detailed analysis of absorbance and fluorescence spectra of the purified extract revealed an absorbance maximum at 619 nm and a fluorescence maximum at 650.5 nm see . In the For a more detailed study of the extracted proteins, a protein analysis via LC-MS was carried out for selected extracts. Both blue coloured extracts (spark discharge and sonication) were compared to the extract obtained by freeze-thaw cycles, which served as reference method. Independent of the disintegration method, different phycobiliproteins were found in all samples. Besides allophycocyanin (APC) and C-phycocyanin (C-PC) protein subunits alpha and beta, lower amounts of R-phycocyanin (R-PC) and phycoerythrin (PE) were identified. Interestingly, the amount of APC was lower compared to C-PC for all types of disintegration. Particularly, after disintegration by spark discharges, no APC alpha chain was found in the samples see .In a second approach, the proteins were examined with regard to non-enzymatic modifications in the amino acid chain of the peptides. The analysis revealed that the relative rate of modifications of target proteins is the highest for the extract, obtained by freeze-thaw cycles. The proteins extracted with the help of spark discharges underwent the least modifications see A. The moCyanidium caldarium as extremophile alga is an interesting alternative source for phycocyanin, also in comparison to other algae and cyanobacteria, e.g., Arthrospira platensis. Usually, phycocyanin is extracted by freeze-thaw cycles with various protocols. However, the method is associated with disadvantages, such as long treatment times, strong thermal effects on the extractives, and a high energy consumption. In this work, spark discharges were applied as an innovative tool and evaluated for their potential with respect to the extraction of phycocyanin from microalgae compared to freeze-thawing as reference and other physical methods (PEF and sonication). The total energy input for every physical method was kept the same as a basis for a process-relevant comparison. In addition, high biomass concentrations were used to mimic realistic industrial conditions. Where applicable, scaling recommendations are given.Limnothrix sp. In this study, the application of freeze-thaw cycles to the red algae Cyanidium caldarium achieved only values of 0.5 mg/g dw  after 360 min treatment time, when 15 to 50 pulses of 3 µs were applied to the suspension. However, it was found that an incubation time, subsequent to the end of the treatment, of at least 150 min was required to detect phycocyanin in the sample solution. For phycoerythrin extraction from Porphyridium cruentum, the authors found an incubation time of at least 6 h was necessary to detect noteworthy amounts of phycoerythrin and achieved the highest yields after 24 h of incubation time. A possible explanation could be that the phycobiliproteins do not only have to cross the cell membrane, but also dissociate from the thylakoid membrane. In this study, no phycocyanin was detected after 120 min incubation time, which is comparable to the results of Martinez et al. Additionally, Akaberi et al. [Pulsed electric fields were already successfully applied for the extraction of phycobiliproteins from cyanobacteria and red microalgae. Martinez et al. ,23 achiei et al.  found a Chlorella vulgaris are well extractable with this technique. Moreover, these often heat-sensitive compounds were not affected by the sparks [Nannochloropsis oculata. The work presented here shows that also phycocyanin is extractable with competitive yields of 4 mg/g dw. This is higher than for PEF with comparable conditions, especially energy input and freeze-thawing. However, in comparison to continuous sonication with accelerated temperature development, the phycocyanin yield is much lower. Even though the temperature seems to have conductive effects to the absolute yield of raw extract, it might be detrimental to the quality of the extract. The rate of detected peptide modifications was slightly lower after spark discharge disintegration, compared to continuous sonication, indicating a more compliant extraction method. Furthermore, the purification of the raw extract resulted in a 4-time reduction of phycocyanin for the sonication extract while the purified extract after spark discharge disintegration was only reduced 1.3-times, compared to raw extract. This makes spark discharges an interesting method for industrial scaling in the field of pigment extraction from microalgae, potentially reducing the time and cost intensive purification of the raw extract.Spark discharges have been found to be effective against stiff cell walls due to the development of strong shockwaves . Accordie sparks ,31. Alsoe sparks  found thProtein analysis was conducted to evaluate what kind of phycobiliprotein was extracted with the respective extraction method and to detect protein modifications that might be characteristic for a specific extraction method, e.g., due to temperature effects. The presence of various types of phycobiliproteins in red algae and cyanobacteria is well described in the literature ,45,46. IThe fingerprint of protein distribution of the associated proteins shows no distinct deviation between the three extraction methods . The absence of the APC alpha chain after spark discharge disintegration was remarkable while this protein was present in both other extracts, although in low concentration. The prevalence of C-PC in the spark discharges extracts determined by protein analysis, is in good agreement with the analysis of absorbance spectra. While the maximum absorbance of APC was found at 650 nm, the C-PC absorbance peak was described within the range of 550 nm to 620 nm ,31, no eCyanidium caldarium comprises phycobiliproteins extractable by sonication and spark discharges. The most prevalent protein is C-phycocyanin, albeit allophycocyanin, R-phycocyanin and phycoerythrin were found likewise in both extracts. Sonication showed to be the most effective extraction method with regard to absolute yield of phycocyanin . However, protein analysis revealed exposure to spark discharge disintegration improved extracted protein integrity. Thus, spark discharge disintegration seems to be promising for targeted extraction of relevant high-purity phycocyanin amounts.The red alga Cyanidium caldarium. Compared to other disintegration techniques (freeze-thaw cycles and PEF), continuous sonication in particular showed to be the most effective extraction method with regard to absolute yield of phycocyanin (53 mg/g dw), although the extraction yield was lower (4 mg/g dw) in comparison to sonication, a positive effect of spark discharge disintegration towards the integrity of extracted proteins was revealed by protein analysis. Here, only minor modifications were detected, while a similar effect to such an extent was not found for sonication. Further investigations may address whether the high yield of phycobiliproteins after continuous sonication is transferrable to larger scale experiments. Adaptations of the spark discharge settings may lead to an equivalent yield with focus on a high-purity phycocyanin, saving time and material for purification.Disintegration by spark discharges, as well as disintegration by sonication facilitated the extraction of phycocyanin from the red algae


Blomia tropicalis (Blo t) extract were performed in the presence or absence of four sugar-based galectin inhibitors  and one peptide-based inhibitor, G3-C12. Results showed that TD139, GB1107, and G3-C12 did not display a specific inhibitory effect on GOS-induced basophil activation as compared to control allergen. An inhibitory effect of lactose and TDG on GOS-induced basophil activation was observed and varied between subjects with up to 100% inhibition at low doses of GOS. The results of competitive ELISA suggest that the inhibitory effects of high dose lactose and TDG on the basophil activation is likely due to the cross-reactivity of GOS-specific IgE to lactose and TDG. Basophil activation is performed using purified basophils suggested that cell surface receptors on other blood cells were not required to induce basophil activation. In conclusion, our results suggest that GOS, a low molecular weight sugar, is able to cross-link IgE independently.The prebiotics, galacto-oligosaccharides (GOS), are small carbohydrate molecules with 1–7 galactose units linked to glucose and have been shown to trigger IgE-mediated anaphylaxis in some cases following ingestion. It is still an unresolved question of how GOS cross-links IgE on basophils. In this study, we examined whether human galectins, a class of lectins that bind specifically to β-galactoside carbohydrates, are involved in GOS-induced basophil activation. Basophil activation test to GOS and control allergen,  Bacillus circulans consists mainly of β-1,4-linked GOS. Other β-glycosidic linkages between the monomer units such as 1-6 Gal, 1-2 Glc, 1-3 Glc, 1-4 Glc, 1-6 Glc, 1-2 Gal, and 1-3 Gal also exist and can form linear and branched linkages  are prebiotics, which are used widely supplemented in commercial infant formula and beverages for their ability to selectively promote the growth of beneficial gut bacteria in the human intestine, such as bifidobacteria . GOS is Blomia tropicalis (Blo t)  .GOS is a unique allergen as it is a pure carbohydrate. The conventional allergen is a protein or glycoprotein that is capable to initiate T cell responses and bind allergen specific IgE. Even though a vast diversity of anti-glycan IgE is detectable in circulation, the binding of this IgE to cross-reactive carbohydrate determinants have been shown to be weak in affinity and, therefore, clinically irrelevant . This covia a carbohydrate recognition domain . TDG binds to galectin-1,−2,−3,−8,−9 with slightly higher affinity than lactose (Kd at micromolar range). Inhibitors TD139 and GB1107 have similar targets as TDG but have a higher affinity to galectin-3 (Kd at nanomolar range) selectively. G3-C12 binds to galectin-3 specifically at high affinity. Anti-human monoclonal antibodies used for BAT assay were purified α-IgE , fluorescein isothiocyanate (FITC) labeled α-CD63 , allophycocyanin (APC) labeled α-CD203c , phycoerythrin (PE) labeled α-IgE PE . Vivinal® GOS syrups were obtained from FrieslandCampina, The Netherlands. A fraction of GOS with enriched allergenic GOS DP4 structures and Blo t extract were prepared as previously described , thiodigalactoside (TDG) , TD-139 , GB1107 & G3-C12 . Their structures and binding affinities to galectins were shown in n = 5) or subjects who developed allergic symptoms upon GOS oral challenge (n = 4). The clinical features of these subjects were shown in A total of nine subjects allergic to GOS were recruited in this study. They are either patients who developed allergic symptoms after consuming GOS containing milk  cells .Basophil activation assay was performed using sodium heparinized blood from subjects who were allergic to GOS. The galectin inhibitors, basophil stimulants  were prepared at four times concentrated solution  cells . Fifty m5 cells/ml supplemented with 4 ng/ml of IL-3 (Miltenyi Biotec). To perform the BAT assay, 100 μl of purified basophils aliquots were pre-incubated at 37°C for 10 min. Thereafter, 100 μl of vGOS, Blo t extract, PBS (negative control), or anti-IgE (positive control) were added into the tube. Samples were incubated at 37°C for 15 min, stained, and analyzed for the expression CD63+ using protocols as described in the previous section.Peripheral blood mononuclear cells (PBMCs) were isolated from heparinized blood by Ficol-Paque density gradient centrifugation. Basophils were then isolated from PBMCs using basophil isolation kit II and LS column according to manufacturer's instruction . The purity of basophils was more than >80% as analyzed by flow cytometry. Purified basophils were suspended in 1% BSA/Dulbecco's DPB (D-PBS) in a concentration of 1 × 10® GOS syrup or the enriched tetrasaccharide GOS fraction (DP4) at 1 or 10 μg/ml . The pre-absorbed sera were then tested for the level of GOS-specific IgE by ELISA developed in-house as previously described  to GOS and other two subjects showed almost negative BAT responses (<10%) in the assay using purified basophils. Similarly, there was 26–81% reduction in the percentage of CD63+ cells upon anti-IgE stimulation, with one subject having almost negative BAT responses (<10%). There was also a reduction (4–40%), albeit to a lesser degree, of CD63+ cells when cells were stimulated with a potent and multivalent allergen, Blo t. These results suggest that GOS may directly act on basophils and induce IgE cross-linking without the participation of other cell types.To test whether IgE cross-linking by GOS on basophils required other cell types to act as adjuvants, purified basophils were used in the BAT assay. Purified basophils were stimulated with GOS and two control stimulants (anti-IgE and Blo t). As compared to the BAT performed using whole blood, purified basophils were less sensitive to GOS stimulation and a higher concentration of GOS was required to induce basophil activation . When bamulation . Two subGOS is a mixture of oligosaccharides consisting of variable degrees of polymerization of galactose units (up to 7) linked to a glucose terminal . In our Our first approach was to consider the possible role of lectins that may participate in GOS-specific IgE cross-linking on basophils. Basophils are shown to have surface expression of some C-type lectins such as CLECSF14, DEC205, Dectin-1, Dectin-2, and mannose receptor C 2 (MRC2) and I-type lectins, immunoglobulin-like lectin (Siglec)-8 , 23. HowIn this study, we examined the involvement of galectins in GOS-mediated basophil activation in whole blood assay using five different galectin inhibitors. Our results, however, showed that three of the five high affinity galectin inhibitors TD139, GB1107, and G3-C12 are used, failed to inhibit GOS-triggered basophil activation , hence, per se.Contrasting results were obtained with the remaining two galectin inhibitors, TDG and lactose. Both showed inhibitory effects on GOS-mediated basophil activation at low concentrations of GOS, but this inhibition was diminished/lost at higher GOS concentrations . Since bIn view of the structural similarities between GOS, lactose, and TDG, we explored the possibility that these galectin inhibitors inhibit the binding of GOS to GOS-specific IgE. Indeed, the GOS IgE ELISA inhibition showed that both lactose and TDG inhibited the GOS IgE binding in a dose-dependent manner, with inhibition up to 2–3 log concentrations higher for lactose and TDG than self-inhibition with GOS and enriched GOS fraction (DP4) .+ cells (one subject negative) to control stimulant anti-IgE. We, therefore, speculate that the purification steps reduced the density of IgE molecules on basophils and may increase the threshold for basophil activation, hence, affecting the responses to anti-IgE and GOS stimulation. These effects were less marked with Blo t stimulation, and a plausible reason is that Blo t allergens are multivalent whereas anti-IgE is divalent. The number of IgE epitopes per GOS is still unknown but it is likely fewer than Blo t in view of its molecular weight. Furthermore, for BAT using whole blood samples, basophil activation was achieved with the concentrations of Blo t were 10–100 times less than GOS , followed by anti-IgE (1 sample negative), and Blo t showed the least reduction . The twothan GOS  indicatiAlthough the IgE binding epitopes of GOS allergy are still unresolved, our competitive ELISA assay indicates that disaccharides (lactose and TDG) can bind GOS-specific IgE. Of note, lactose and TDG are disaccharides composed of different sugar molecules suggesting that GOS-specific IgE is not monoclonal, with higher affinity for the structure with two galactose rings (TDG) than one galactose plus one glucose (lactose).in vitro  to epitopes in close proximity can be blocked by steric hindrance, especially for small molecules . In thisin vitro . BrancheIn conclusion, we have demonstrated that GOS-mediated IgE cross-linking does not require galectin as carrier. GOS may be able to cross-link IgE independently.The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by Institutional Review Board of the National Healthcare Group, Singapore. Written informed consent to participate in this study was provided by participants/participants' legal guardian.LL conducted experiments and performed data analysis. SL conducted experiments, performed data analysis, and drafted manuscript. HW conducted experiments. JS, WC, YZ, ET, and WL contributed to the sample collection, data interpretation, and revised the manuscript. DD provided the materials required for the study and contributed to the data interpretation and revised the manuscript. BL contributed to the study design, data interpretation, and revised the manuscript critically. C-HH conceived the study and study protocol and participated in manuscript writing. All authors contributed to the article and approved the submitted version.This work was supported by the National Medical Research Council, Singapore (NMRC/CIRG/1487/2018).DD is employed by FrieslandCampina, Amersfoort, The Netherlands. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Objective: Idiopathic generalized epilepsy (IGE) involves aberrant organization and functioning of large-scale brain networks. This study aims to investigate whether the resting-state EEG microstate analysis could provide novel insights into the abnormal temporal and spatial properties of intrinsic brain activities in patients with IGE.Methods: Three groups of participants were chosen for this study . EEG microstate analysis on the resting-state EEG datasets was conducted for all participants. The average duration (“Duration”), the average number of microstates per second (“Frequency”), as well as the percentage of total analysis time occupied in that state (“Coverage”) of the EEG microstate were compared among the three groups.Results: For microstate classes B and D, the differences in Duration, Frequency, and Coverage among the three groups were not statistically significant. Both Frequency and Coverage of microstate class A were statistically significantly larger in the IGE-Seizure group than in the other two groups. The Duration and Coverage of microstate class C were statistically significantly smaller in the IGE-Seizure group than those in the other two groups.Conclusions: The Microstate class A was regarded as a sensorimotor network and Microstate class C was mainly related to the salience network, this study indicated an altered sensorimotor and salience network in patients with IGE, especially in those who had experienced seizures in the past 2 years, while the visual and attention networks seemed to be intact.Significance: The temporal dynamics of resting-state networks were studied through EEG microstate analysis in patients with IGE, which is expected to generate indices that could be utilized in clinical researches of epilepsy. Idiopathic (or genetic) generalized epilepsy (IGE) is a well-recognized subgroup of generalized epilepsy, referring to four epilepsy syndromes: childhood absence epilepsy, juvenile absence epilepsy, juvenile myoclonic epilepsy, and generalized tonic–clonic seizures (GTCS) alone . GTCS isThe multichannel electroencephalogram (EEG) is a widely used technique with major clinical importance in the diagnosis of epilepsy. The postsynaptic inhibitory and excitatory potentials are the main sources of EEG signals. EEG is used to measure the synchronous electrical activity of spatially aligned neurons directly and dynamically in cortical networks. Therefore, EEG captures useful information on brain activity. Topographical map of electrical potentials characterizes the EEG signals by the spatial configuration of the electric fields at the scalp, which considers the information of all electrodes. Microstate analysis is an alternative, and increasingly applied, EEG-representation based on topographic analysis. During EEG, the global electrical brain activity on the scalp remains semi-stable in successive short time periods, defined as EEG microstates, indicating the “quasi-simultaneity of activity among the nodes of large-scale networks” . These tA resting-state functional MRI (rs-fMRI) study revealed that the brain can be intrinsically organized into large-scale functional networks in which the hemodynamic signature is stable for about 10 s . SpatialScalp EEG is widely used in patients with epilepsy, most under task-free conditions. In most cases, EEG is only visually inspected for clinical use, and were analyzed by characterizing the temporal waveform morphology , 19 and/In the present study, patients were chosen from the clinic for epilepsy of the First Affiliated Hospital of Anhui Medical University in 2019. Diagnostic and syndromic classification were based on their clinical history and video-EEG findings, in accordance with the diagnostic criteria of the International League Against Epilepsy . We inclIn total, 22 right-handed patients with IGE  met the above criteria and were selected for this study. After detailed clinical and neurological examinations, all patients underwent continuous video-EEG recording for 24 h, as well as 3.0-Tesla high-resolution magnetic resonance imaging (MRI). Two of the patients were newly diagnosed with IGE and had not been treated with anti-seizure medicines (ASMs), while the other 20 patients were treated with ASMs. Nine patients were treated with valproic acid only, nine with lamotrigine only, and the remaining three with combination therapy. Of the 22 patients, 12 had been seizure-free for at least the previous two years (IGE-Seizure Free); the group with the remaining 10 patients was called “IGE-Seizure.” An additional HC group consisted of 17 right-handed, healthy participants who were matched to the patient group in terms of age, sex, and education.All study procedures were approved by the Anhui Medical University Ethics Committee and conducted according to the Helsinki Declaration (1975 and subsequent revisions). Informed consent was obtained for all participants.All patients were asked to complete the MoCA, Beijing version. The Beck Depression Inventory  and Hamilton Anxiety Scale  were also administered to obtain measurements of depressive and anxiety symptoms for all the participants.Resting-state EEGs were recorded in a soundproof, light-controlled recording room, while participants were seated in a comfortable chair. All participants were instructed to keep their eyes closed and to relax during the whole recording period.EEG signals were continuously recorded using the Curry 7 data acquisition system , with 64 scalp electrodes (Ag-AgCl) mounted on an elastic cap, in accordance with the extended International 10/20 system , 23, as http://sccn.ucsd.edu/eeglab/) to preprocess the EEG data; it is an open-source toolbox implemented in MATLAB . The continuous EEG data were downsampled to 500 Hz and segmented into 2-s epochs. Abnormal amplitude (≥100 μV) was defined as artifactual epochs and removed. Independent component analysis (ICA) was also used to correct artifacts such as eye movement, heartbeats, and muscle artifacts . Data were visually inspected to identify any epochs containing obvious eye-blink artifacts missed during the independent component analysis, and all epochs with interictal epileptiform discharges were rejected. Each participant's EEG data contain more than 30 epochs.We used EEGLAB  was used for all statistical analyses. Non-parametric tests were performed to explore statistically significant differences in the background tests between the three groups, and sex differences between the three groups were assessed with chi-square tests.Post hoc ANOVA comparisons were considered. The threshold for significance was p < 0.05 for the MANOVA and the alpha value for Bonferroni procedures was 0.004 [0.05/ (3*4) = 0.004167].Multivariate analyses of covariance (MANOVA) were performed for the three microstate parameters. Each MANOVA contained one between-subject factor for 3 groups , one within-subject factor for 4 microstate classes. When the main effects or interaction were significant, Demographic data and the results of baseline tests of the three groups are summarized in The four microstate classes accounted for 72.86% (standard deviation [SD]: 5.39%) of the global explained variance in the IGE-Seizure group, 75.43% (SD: 3.09%) in the IGE-Seizure Free group, and 74.11% (SD: 4.02%) in the HC group. * 4 microstates , only microstate classes C showed significant difference between the three groups  = 9.753, p < 0.001); microstate classes A, B and D showed no significant difference  = 3.395, 3.494 and 2.266 respectively, p > 0.05). Further analysis found that the duration of microstate class C in IGE-Seizure group were significantly shorter than in HC group (p < 0.001), but there were no significantly difference between HC and IGE-Seizure Free group, and between the IGE-Seizure and IGE-Seizure Free group (p = 0.429 and 0.025 respectively).For Duration, MANOVA found that there was a significant interaction effect of 3 groups * 4 microstates were also found by MANOVA , only microstate classes A showed significant difference between the three groups  =8.385, p < 0.001); microstate classes B, C and D showed no significant difference  = 0.219, 3.925 and 0.975 respectively, p > 0.004). Further analysis found that the frequency of microstate class A in IGE-Seizure group were significantly higher than in IGE-Seizure Free group (p < 0.001), but there was no significant difference between HC and IGE-Seizure Free group (p = 0.806), and between the HC and IGE-Seizure group (p = 0.009).For Frequency, a significant interaction effect of 3 groups * 4 microstates ,microstate classes A and class C showed significant difference between the three groups  = 24.199, p < 0.001 and F  = 26.168, p < 0.001 respectively); The coverage of microstate class A in IGE-Seizure group were significantly higher than in HC group (p < 0.001) and in IGE-Seizure Free group (p < 0.001), but there were no significantly difference between HC and IGE-Seizure Free group; The coverage of microstate class C in IGE-Seizure group were significantly lower than in HC group (p < 0.001), and also the IGE-Seizure Free were significantly lower than HC group (p < 0.001), but there were no significantly difference between IGE-Seizure and IGE-Seizure Free group(p = 0.111); These details are provided in For Coverage, which means the product of Duration and Frequency, MANOVA also found that there was a significant interaction effect of 3 groups In the present study, we investigated the alterations of brain functional state dynamics in IGE patients and HCs using EEG microstate analysis to ascertain the temporal characteristics of sub-second brain activity. Four classes of microstates were identified in this study, according to those described in the existing literature. The main findings of this study can be summarized as follows. First, the mean Duration, Frequency, and Coverage of microstates B and D were similar between the three groups. Second, the Frequency and Coverage of microstate A were larger in patients with IGE with seizures in the previous 2 years than in those without such seizures and in HCs. Finally, the mean Duration and Coverage of microstate C were smaller in patients with IGE who had experienced seizures in the past 2 years than in those patients without seizures and in HCs. Those results may provide some insights into the aberrant intrinsic brain activities in IGE patients.IGE patients presented no structural abnormalities on a standard brain magnetic resonance image, but they showed significant differences in the structural and functional connectivity when compared with healthy controls . The gloMicrostate class A was related to negative BOLD activations of both superior and middle temporal parietal cortices, and regarded as the auditory or sensorimotor networks , 17. SenMicrostate class B was related to negative BOLD activation in the striate, extrastriate, and both occipital cortices, and regarded as the visual network , 17. StuMicrostate class C was mainly related to positive BOLD activation in both inferior frontal cortices, the dorsal anterior cingulate cortex, and the right insular area, and regarded as the salience network . The salMicrostate class D exhibited correlations with negative BOLD activation in the right lateral ventral and dorsal regions of the frontal and parietal cortices, and regarded as an attentional network . The attThe present study also has some limitations. The sample size is small and there may be some sampling bias. The influence of anti-epilepsy drugs was not considered. Also, we did not consider of the influence of focal seizure. In the future, more subjects will be included and various possible influencing factors will be fully considered.Using resting-state EEG microstate analysis, the present study indicated that the salience network and sensorimotor network were altered in patients with IGE (GTCS only), especially in those who had experienced seizures in the past 2 years.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.The studies involving human participants were reviewed and approved by Anhui Medical University Ethics Committee. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.YBJ and KW: designed experiments. MYZ and YH: carried out experiments. YBJ: analyzed experimental results, sequencing data, and wrote the manuscript. All authors contributed to the article and approved the submitted version.This study was supported by a grant from the University Natural Science Research Project of Anhui Province, China KJ2019A0289.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


However, this study took place during neap tide, and it is likely that peak bottom velocities during spring tides are high enough to periodically “clear” under-farm sediment of recent deposits.The benthic impact of aquaculture waste depends on the area and extent of waste accumulation on the sediment surface below and around the farm. In this study we investigated the effect of flow on biodeposit transport and initial deposition by calculating a rough aquaculture “footprint” around an oyster aquaculture farm in the Damariscotta River, ME. We also compared a site under the farm to a downstream “away” site calculated to be within the footprint of the farm. We found similar sediment biogeochemical fluxes, geochemical properties and macrofaunal communities at the site under the farm and the away site, as well as low organic enrichment at both sites, indicating that biodeposition in this environment likely does not have a major influence on the benthos. To predict accumulation of biodeposits, we measured sediment erodibility under a range of shear stresses and found slightly higher erosion rates at the farm than at the away site. A microalgal mat was observed at the sediment surface in many sediment cores. Partial failure of the microalgal mat was observed at high shear velocity, suggesting that the mat may fail and surface sediment erode at shear velocities comparable to or greater than those calculated from As demand increases to feed the world’s growing population, many are looking toward dramatically expanding marine aquaculture as an efficient and sustainable means of food production . Growth Past reports of the local benthic impact of aquaculture range from dramatic alteration of bottom characteristics to apparently few effects depending on the cultured organism, the method of culture and local environmental conditions . The impThe footprint of an aquaculture farm is determined by the intensity of lateral flow and settling rate of the particles. In areas with low flow, biodeposits would be expected to accumulate under the farm, increasing the organic content and driving high influxes of oxygen and effluxes of nutrients as organic matter is degraded . Higher a (chl-a), binding surface sediments and altering nutrient and oxygen fluxes . Much of that increase is attributable to expansion of aquaculture in the Damariscotta River, which alone constitutes approximately two-thirds of the total oyster production in Maine and increased in harvest yield from ∼1.3 million to ∼9.4 million oysters between 2005 and 2019. Despite the industry’s rapid growth, there is little existing research examining the impacts of oyster aquaculture on the local benthos. Our objective was to describe the footprint of an oyster farm in the Damariscotta River, ME, and assess our estimate of the footprint by comparing sediments at sites under the farm and downriver of the farm but within the same depth and flow regime.The Damariscotta River Estuary, Maine, is an ideal system for this study, as it experiences strong tidal currents . Mean flr higher . Aquacul2) lease  are grown at the farm in mesh bags held in rigid, rectangular floating OysterGro™ cages that are strung together in rows. The farm sits above a sloping shoal with a mean water depth ranging from ∼2–4 m. We assessed our estimate of the footprint by comparing a site directly underneath the oyster cages (“Farm” site) in the southern portion of the farm with one downstream, ∼90 m south of the edge of the farm lease area (“Away” site). The Away site location was constrained by the geomorphology of the river; because the river bends and narrows downstream , where a large oyster aquaculture operation is located on a 24-acre , flow velocity near the bottom was measured over a tidal cycle with an Acoustic Doppler Velocimeter (ADV) and combined with laboratory measurements of particle settling velocities. Here we define the footprint based on the settling velocity of the mean size of biodeposits and the maximum distance transported over the tidal cycle during neap tide. Because mean water column height varied from ∼2–4 m across the farm, calculations were done with the maximum and minimum depths to provide a range. Thus, biodeposit particles smaller than the mean would travel beyond our defined footprint under peak flow velocities, but the footprint would encompass most of the tidal range in deposition. To demonstrate the effect of particle size on the footprint, we also calculated transport distance for the 5th and 95th percentile of particle sizes.http://maine.loboviz.com).The ADV was horizontally mounted to a metal tripod mooring deployed at the Farm site, measuring velocity (8 Hz) 37 cm above the bottom. An Onset HOBO dissolved oxygen (DO) logger was also mounted to the mooring (50 cm above the bottom) to measure near-bottom DO to determine whether low oxygen occurred that might affect biogeochemical fluxes or infauna. A cinder block mooring was deployed at the Away site. Temperature and salinity measurements were collected near-surface and at bottom at both sites to measure stratification. Water level at the farm was measured by the pressure sensor on the ADV. Moorings were deployed for ∼23 h on August 2–3, capturing nearly two tidal cycles during a neap tidal period. Because the experiment was conducted over a short time frame, hydrographic data for the entire summer season was obtained from a Land/Ocean Biogeochemical Observatory (LOBO) surface mooring ∼250 m southeast of the farm . Six traps were deployed during two 24-hr deployment periods separated by 5 days, during which the tidal range varied by ∼0.5 m. All traps were deployed and retrieved at high tide. At least one trap was lost from each location during each deployment. During the first period, traps were deployed and recovered from the Farm site (n = 4) and the cages (n = 5). During the second period, sediment traps were deployed and recovered from the Away site (n = 5) and the cages (n = 5). After collection, sediments were dried for 24 h at 60 °C and weighed.Sediment traps were attached to the bottom moorings at both sites to measure sedimentation rates. The traps were made of 5.08 cm inner diameter PVC cylinders, 15 cm in length, and capped on one end, following Vt is settling velocity, µ  is the viscosity of the water, ρp and ρw are the densities of the particle and water respectively, g is acceleration due to gravity, L is particle length, and D is particle diameter. Particle surface area and angle of descent were compared to settling velocity using a multiple regression. Density was not included in this model, as it was calculated from the settling velocity.To measure biodeposit settling velocities, biodeposits were collected from adult, harvest-size oysters held in a flow-through seawater system. Biodeposits were spread in a petri dish to prevent particle aggregation, maintaining the size and cylindrical shape of freshly released biodeposits observed in both the lab and field. Deposits were then pipetted into the middle of a 37.8 L aquarium of seawater, at least 12 cm away from any wall to prevent wall effects. Videos of their descent were recorded using a Nikon D5300 DSLR camera equipped with an AF-S Micro NIKKOR 105 macro lens. It was not possible to distinguish between feces and pseudofeces from the observations, and we did not describe the composition of the biodeposit particles. The descents of 100 individual particles were recorded. Each particle’s dimensions  were measured in ImageJ and the angle  and rate of descent tracked with the ImageJ MtrackJ plugin . SettlinLf) was calculated as: us) measured with the ADV and the mean particle settling velocity (ws) calculated from the lab observations. The height of the water column (h(t)), or vertical distance traveled, and bottom horizontal velocity (us(t)) changed depending on the time (t) in the tidal cycle. To provide an upper and lower bound on the potential biodeposit tidal transport, calculations used the deepest and shallowest mean water depths at the farm (2 and 4 m). It should be noted that this calculation uses velocity measured with the ADV which is assumed to be in the bottom boundary layer, so the transport distances should be viewed as conservative estimates.An estimate of biodeposit tidal transport (To determine whether settled biodeposits were likely to be resuspended (Q2), we calculated bottom shear velocity throughout the flood/ebb tidal cycle and conducted laboratory erosion experiments across a similar range of bottom shear velocities.via three indirect methods  as described by e.g., τb) from both of these methods is then linked to shear velocity as ρ is water density and u∗ is shear velocity. As seen in other studies, all three methods produced results with similar patterns, but peak magnitudes showed considerable differences . Becausedforms; ) and the−1, 0.95 cm s−1, 1.34 cm s−1, 2.01 cm s−1, 2.32 cm s−1). Each shear velocity level was maintained for 20 min before increasing disc rotation and flow rates. For each 20 min shear velocity level, water and eroded material were removed by a pump at the center of the disc at 10 cm above the sediment surface and filtered through 47 mm Whatman GF/F filters (1.5 µm pore size). Effluent was replaced with the same seawater supplied to the core holding tank, so suspended mass in the pre-erosion core overlying water and the replacement water was likely similar. The lowest shear velocity, 0.30 cm s−1, was used as a flushing step and was not filtered for suspended sediment analysis (no resuspension was observed at this low shear). Filters were then dried at 60 °C for 24 h and weighed to obtain eroded sediment mass for each shear velocity level. One Away core was not used in the analysis due to accidental introduction of a large nereid worm from an unrelated experiment in the holding tank. Suspended sediment concentration, Cs (kg m−3), for each site at each velocity level was calculated from the dry mass (kg) of filtered sediment divided by the volume (m−3) of water filtered. Cs was converted to eroded mass per area : Vc is chamber volume (7.9 ×10−4 m3), and Ac is sediment surface area within the core (7.9 × 10−3m2). Due to small and unequal sample sizes, statistical comparisons between sites and stress levels were not performed.We conducted laboratory erosion experiments using a custom-built Gust erosion chamber to generate near-uniform bed shear . Triplicu∗15 is shear velocity at 15 °C (cm s−1), n is rotations per minute, and Q is pumping rate (mL min−1). Shear velocity at 15 °C was converted to shear velocity at the water temperature measured during the erosion tests (22 °C) as: u∗22 is shear velocity at 22 °C (cm s−1)  generated by the Gust chamber were 0.009 Pa, 0.09 Pa, 0.18 Pa, 0.41 Pa, and 0.55 Pa. These were calculated from shear velocities  as: ρ of 1,021 kg m−3 was used based on an average temperature of 22 °C and salinity of 31 psu.To generate specific shear velocities, cap rotation and pumping rate were set using calibration equations from the University of Maryland Center of Environmental Science Gust Erosion Microcosm System (U-GEMS) Manual : (4)\docThis field sampling occurred during neap tide, and also coincided with the highest surface water temperatures of the year when thermal stratification likely peaked. To gain a broader sense of the potential for sediment resuspension during other times in the tidal cycle, near-surface turbidity and velocity data from the LOBO surface mooring east of the farm were analyzed for longer-term patterns. While turbidity is not a direct measure of sediment resuspension, water clarity is typically related to sediment concentrations. Potential relationships among precipitation, water level, current velocity and turbidity were investigated using both the instantaneous data and 40-hour low-pass filtered signals. Near-surface velocity data were also used to estimate bottom shear velocities in higher-flow times in the spring-neap cycle. Near-surface velocity was converted to a proxy for near-bottom velocity based on a scaling factor determined from the relationship between the LOBO buoy and the ADV-measured velocities during the overlap period in August. This near-bottom velocity proxy was used to estimate shear velocity using a quadratic relationship between the longitudinal velocity and direct calculations of the shear velocity (using the covariance method). This fitted relationship was extrapolated to predict shear velocity expected from the highest flow in the spring-neap tidal cycle.−1 sediment).Sediments at both sites were evaluated for grain size distribution, water content, percent organic content, and concentrations of C and N. One diver-collected 10-cm diam core from each site was vertically sectioned. Water content was calculated as the difference between wet and dry masses after sediment was dried at 60 °C for 24 h. Percent organic content was calculated as the difference between dry and ash masses after burning sediment at 500 °C for 4 h. Burned sediment was then soaked in a 1% sodium metaphosphate dispersant solution for 24 h to rehydrate and disaggregate sediment prior to grain size analysis. Grain size distribution was determined using a Malvern Mastersizer 3000 particle analyzer . Data were analyzed using Gradistat . Preparation of C and N samples included an acidification step to remove inorganic carbon. Concentrated HCl was added to dried sediment samples and heated at 80 °C until the fluid evaporated as described by i.e., were saturated, additional organic matter would be expected to have minimal effect on nutrient and oxygen fluxes. The usefulness of the Amended cores therefore depended on “background”  respiration rates being relatively low.To determine whether the “Away” site exhibited similar impacts of oyster biodeposits on biogeochemical fluxes as the site directly under the farm, sediment core incubations were performed in the dark to measure nutrient, DIC and oxygen fluxes and sediment surface samples were collected for chlorophyll a (chl-a) analysis. In an additional treatment, “Amended” cores, we added oyster feces and pseudofeces onto the top of two cores from the “Away” site. Because of the lack of available data for this area on typical respiration rates, the Amended treatment was included to determine whether Away site sediments were being enriched with biodeposits; if sediments from the Away site were receiving large influx of organic matter from the farm, 4+ and PO43−) and dissolved inorganic carbon (DIC) fluxes. Only dark incubations were performed so that maximum respiration rates could be measured without the influence of microalgal photosynthesis. Our measurements are therefore not representative of daytime in situ fluxes. A total of 12 15.2-cm diam sediment cores were taken by divers during two collections (2 and 7 August 2018). During each collection, two cores were taken from the Farm site and four were taken from the Away site. Care was taken during collection that each core had at least 10 cm of sediment depth and at least four cm of overlying water. The cores were brought back to the lab and kept in tanks with flowing water from the Damariscotta River until use in the incubations (<36 h post-collection). Immediately before the start of the incubations, eight mL wet volume of oyster feces and pseudofeces were pipetted onto the top of two cores collected from the Away site and designated the Amended cores. Between the two samplings, this resulted in a total of four replicates each of Farm, Away and Amended cores.Incubations of sediment cores were performed to measure sediment oxygen flux and nutrient  were multiplied by the height of overlying water to obtain flux rates (mmol m−2 d−1). We were unable to obtain oxygen flux values for the Amended cores because they lacked a consistent oxygen trend. Some cores produced several successive oxygen slopes within the same range of oxygen concentrations because low-oxygen water was replaced with high oxygen water during the sampling for nutrients. To calculate a single oxygen flux for each of these cores, time-weighted average slopes were calculated. Shapiro–Wilk and Kruskal–Wallis tests were performed on nutrient and oxygen flux data to determine if there were differences in fluxes between sites  and the remaining material preserved in 95% ethanol and rose Bengal stain. Macrofauna were picked out of each sample, and individuals identified to family level. Statistical comparison of abundances between the two sites was not performed due to small and uneven sample size. Data from replicate cores were analyzed for Shannon diversity (H’) and Pielou’s evenness (J’), averaged by site, then Student’s t-tests were performed on the diversity and evenness metrics to compare the two sites. An NMDS analysis was performed on square-root transformed data (to decrease weighting of highly abundant groups) to compare community structure between sites .−1 (−1 throughout the sampling period (data not plotted).The velocity data were strongly tidal during the deployment, with a clear semi-diurnal cycle. The water level had a 2.86 m range, with a mean water depth of 3.4 m at the away site and 3.5 m at the farm site. The ADV measured near-bottom velocities ranging ± 20 cm s−1 . While t−2 d−1, n = 4). The cage-area normalized rates of deposition calculated from the traps attached to the cages were  for the first deployment and  for the second. These rates were calculated by multiplying the deposition rate calculated from the beneath-cage traps by the percent area of the farm occupied by gear (40%), following the methods of −2 d−1, n = 5), where deployment and repositioning of the cinder block mooring may have resuspended sediments.The rate of deposition calculated from sediment traps attached to the bottom mooring beneath the farm was  cm s−1 increase in settling velocity  (p = 0.72). Using the modified Stokes equation, mean pellet density was calculated to be 1.42 ± 0.18 g cm−3with a range of 1.13 to 2.26 g cm−3.The biodeposits were cylindrical in shape, with an average length of 0.64 ± 0.14 mm and average diameter of 0.11 ± 0.02 mm. Biodeposit surface area ranged from 0.12 mm = 0.03) . The ang−1. At this velocity, a particle would take ∼5-10 min from release to deposition across the range of typical site depths (2–4 m). Oyster biodeposits had a mean tidal transport range of 72 m upstream at peak flood and 62 m downstream at peak ebb in the shallow (2m depth) section of the farm  was within this range, and the higher shear velocities tested extended above the maximum field observations, with values up to 2.32 cm s−1 (−2) was over twice as high as that away from the farm (3.22 ± 0.34 g m−2). Additionally, eroded mass appeared to increase more with increasing shear velocity at the Farm than at the Away site, but only at the highest shear velocity did error bars not overlap between the two sites . Using the scaling factor, bottom velocities during spring tides were estimated as ∼30 cm s−1 during ebb and ∼25 cm s−1 during flood, with the maximum at ∼40 cm s−1. Neap tide bottom velocities were ∼20 cm s−1 during both ebb and flood, corresponding well with the bottom velocities measured by the ADV. During the spring tides, the calculated shear velocity approached three cm s−1, higher than the highest shear velocities tested in the erosion tests or encountered during the experiment  was 11.2 at the Away site and 11.48 at the Farm site (−1 dry sediment and Away sediments were 7.3 and 22.5 µg chl-a g−1 dry sediment. Phaeopigment concentrations were lower than chl-a in both samples from both the Farm site (5.88 and 6.80 µg g−1 dry sediment) and Away site (11.05 and 8.74 µg g−1 dry sediment).Sediments at both sites were poorly sorted coarse silt to fine sand  Folk & . Organicarm site . Surfacen = 4 for each treatment, p = 0.69) (p = 0.66) but were highly variable, especially among the Away cores  (2 and DIC (p = 0.36), NH4+ (p = 0.44) or PO43− ( p = 0.51)  . DIC fluay cores . NH4+ anctively) , 9D. The = 0.51) , 9E, 9G.−2 d−1). A large nephtyid worm was found in an Away core that had the next highest oxygen flux measured (−60.6 mmol m−2 d−1). Removing these two cores from the sediment flux analyses did not result in significant differences among treatments  or significant relationships between nutrients and oxygen flux .A large nereid worm was found in a core taken from directly underneath the farm. This core also had the highest oxygen flux of all cores measured , surface deposit-feeding polychaetes  and the burrowing, carnivorous polychaete, Nephtyidae. Abundance and taxa richness were highly variable at both sites. Family richness did not appear to differ between the sites, with 3.25 ± 1.89 families (mean ± st. dev.) at the Farm site and 4.0 ± 2.0 families at the Away site. Abundance appeared to be higher at the Away site, with 3,411 ± 3,902 individuals m−2, compared to 459 ± 397 individuals m−2 at the Farm site, but variability was high with the abundance of most families having a greater standard deviation than the mean . Pielou’s evenness (J’) at the Farm site was 0.86 ±  0.18 and at the Away site was 0.54 ± 0.36 and was not significantly different between sites . There was no distinct partitioning in community structure between Farm and Away samples, although several of the Away site samples that shared relatively high abundance  of nephtyids were > 60% similar  illustrates that either the relationship between surface area and settling velocity is nonlinear near zero, or that other parameters such as biodeposit density vary in a non-random way. We hypothesize that the scatter of data and the related poor fit likely results from differences in density among biodeposits, specifically that smaller biodeposits settle relatively faster because they are more dense than larger biodeposits. Oysters separate labile organic matter from refractory material and nonorganic material like sand and excrete these materials in feces and psuedofeces, respectively (−3) are likely composed primarily of organic matter, while those at the upper end (2.26 g cm−3) likely consist primarily of nonorganic minerals. Presumably, the relative makeup of these deposits depends on the composition of the suspended material in the water column at the time of filtration, affecting the densities of both feces and pseudofeces. We were unable to differentiate between feces and pseudofeces in this study, and did not measure the density of individual pellets, preventing stronger correlations between biodeposit characteristics and settling velocity.The weak correlation between settling velocity and surface area  in additectively . Biodepo−1 and varied based on the size of the individual. These higher settling velocities would result in an area of impact smaller than that calculated here based on oyster biodeposits, which may lead to greater rates of accumulation, particularly if individual bivalves were larger and produced more biodeposits. We did not account for individual size in this study, which could have been a source of variability in both the mean settling velocity and mean deposition rate.The biodeposits of different species of bivalves also appear to have different settling velocities, and larger bivalves tend to produce more biodeposits than smaller ones . It is t−2 d−1  were higher than the highest measured by −1 dry sediment) in sediments from a deeper area of the Damariscotta. Chl-a concentrations were also higher than concentrations of phaeopigments (which indicate degraded chlorophyll often due to grazing), implying abundant living microphytobenthos . However, it is important to note that, because of the incubation equipment malfunction, the oxygen flux measurements and nutrient sample collections for the second set of cores did not occur simultaneously, so conclusions about the relationship between oxygen and nutrient fluxes should be drawn with caution.The “Amended” cores that were enriched with biodeposits prior to incubation showed no differences in nutrient fluxes from unenriched Away site cores, which suggests that these sediments were already saturated with organic matter. The amount of biodeposits added was comparable to the deposition at the farm site over a day  and suspension-feeding infaunal bivalves . Relative dominance of suspension feeders suggests that flow is slow enough to avoid sediment destabilization but fast enough to maintain sufficient suspended food particle flux e.g., . Additio−1) falls within the broad range of shear velocities at which biofilms fail. −1. −1 for clean sands to around 4 cm s−1 on sands with biofilms.The partial mat failure during the erosion experiments suggests the possibility that these sediments are at least periodically eroded, decreasing the impact of biodeposits on sediments under the farm. The high variability in erosion at the higher shear velocity levels  suggestsin situ bottom flow  surface velocity from the LOBO mooring and bottom velocity from the ADV for the duration of the experiment. (B) surface turbidity from the LOBO mooring.Click here for additional data file.10.7717/peerj.11862/supp-2Supplemental Information 2Blank cells represent no data. *very close to zero.Click here for additional data file.10.7717/peerj.11862/supp-3Supplemental Information 3−2 d−1. *no data.Due to equipment malfunction during the second incubation (2a), it was repeated (2b) to gather oxygen flux data. Positive values indicate flux out of the sediment and all units are in mmol mClick here for additional data file.10.7717/peerj.11862/supp-4Supplemental Information 4Numbers in parentheses indicate standard deviation. Letters beside family names indicate phylum/subphylum . Numbers are rounded to nearest whole number.Click here for additional data file.10.7717/peerj.11862/supp-5Supplemental Information 5Dimensions (measured length and width), angle of descent, calculated surface area and settling velocity of sample biodeposit particles.Click here for additional data file.10.7717/peerj.11862/supp-6Supplemental Information 6Raw data, calculations of fluxes, and correction for water volume for fluxes of DIC from incubations.Click here for additional data file.10.7717/peerj.11862/supp-7Supplemental Information 7Concentrations, flux calculations, and fluorometric measurements of ammonium from incubations.Click here for additional data file.10.7717/peerj.11862/supp-8Supplemental Information 8Raw data oxygen measurements for both incubations and calculations of dissolved oxygen fluxes.Click here for additional data file.10.7717/peerj.11862/supp-9Supplemental Information 9Concentrations, flux calculations, and spectrophotometeric absorbances for phosphate from incubations.Click here for additional data file.10.7717/peerj.11862/supp-10Supplemental Information 10n = 2 for each treatment).Calculations of Chl-a and phaeopigment concentrations for samples taken from the Farm, the Away, and the Amended cores used in biogeochemical incubations  and Away (n = 2) sites.Eroded mass at increasing shear stress for cores from the Farm (Click here for additional data file.10.7717/peerj.11862/supp-12Supplemental Information 12Counts of macrofaunal taxa, down to family where possible.Click here for additional data file.10.7717/peerj.11862/supp-13Supplemental Information 13Contains water content, C:N, and grain size fractions for sectioned cores from the Farm and Away site. Data pooled into surface and subsurface for figures.Click here for additional data file.10.7717/peerj.11862/supp-14Supplemental Information 14Dry mass of accumulated sediment in traps attached to cages, on bottom under the farm and at the Away site. Data was collected during 2 deployments.Click here for additional data file.10.7717/peerj.11862/supp-15Supplemental Information 15Contains files with velocity data from the ADV, salinity and oxygen data from a the onset hobo DO loggers, and data from the YSI instruments. A metadata file is included.Click here for additional data file.