Sulfurifustis and Thiobacillus), which are more abundant at greater depths, whereas the tailings of site A had a higher proportion of potential As-cycling and -resistant genera . A multi-variate statistical analysis showed that (1) distinct site-specific groupings occur for the Co # vs. Fe #, Co# vs. S#’s and for the microbial community structure and (2) microbial communities are statistically highly correlated to depth, S#, Fe#, pH and the average valence of As. The variation in As valence correlated well with the abundance of N, S, Fe, and methane-cycling bacteria. The results of this study provide insights into the complex interplay between minerals containing the critical element cobalt, arsenic, and microbial community structure in the Cobalt Mining Camp tailings.Mine tailings host dynamic biogeochemical processes that can mobilize a range of elements from the host material and release them into the environment through acidic, neutral, or alkaline mine drainage. Here we use a combination of mineralogical, geochemical, and microbiological techniques that provide a better understanding of biogeochemical processes within the surficial layers of neutral cobalt and arsenic-rich tailings material at Cobalt, ON, Canada. Tailings material within 30-cm depth profiles from three tailings sites  were characterized for their mineralogical, chemical and microbial community compositions. The tailings material at all sites contains (sulf)arsenides , and arsenates (erythrite and annabergite). Site A contained a higher and lower amount of (sulf)arsenides and arsenates than site B, respectively. Contrary to site A and B, site C depicted a distinct zoning with (sulf)arsenides found in the deeper reduced zone, and arsenates occurring in the shallow oxidized zone. Variations in the abundance of Co+As+Sb+Zn (Co#), Fe (Fe#), total S (S#), and average valence of As indicated differences in the mineralogical composition of the tailings material. For example, material with a high Co#, lo Fe# and high average valence of As commonly have a higher proportion of secondary arsenate to primary (sulf)arsenide minerals. Microbial community profiling indicated that the Cobalt tailings are primarily composed of Actinobacteria and Proteobacteria, and known N, S, Fe, methane, and possible As-cycling bacteria. The tailings from sites B and C had a larger abundance of Fe and S-cycling bacteria (e.g.,  Understanding the interactions between minerals and microorganisms is essential in the interdisciplinary and growing field of biogeochemistry, an integration of geology, chemistry and biology . StudyinAn understanding of the biogeochemical cycling of both elements in the mine tailings at the CMC is therefore critical in terms of the environmental impact of the current mine tailings, the addition of mine waste during future mining operations or their potential bioleaching from the existing mine waste.This study applied for the first-time a multi-variate statistical analyses to gain a critical understanding on the relationships between the abundance of Co-bearing (sulf)arsenides and arsenates, geochemical parameters such as pH and Eh, geomorphological parameters such as depth, and microbial community compositions in the near-neutral surficial tailings material at the CMC.3+-bearing minerals ; and (2) characterized the role of both biotic and abiotic processes involved in sulfide-mineral weathering.Numerous studies have characterized the relationship between microbial community structure and the variation of geochemical and mineralogical parameters in tailings material of an acidic pH e.g., . StudiesSphingomonas, Caulobacter, and Janthinobacterium genera), (b) siderite (FeCO3) formation was the result of Fe-reducing bacteria (IRB) such as Geobacter and Rhodoferax spp., and that (c) acidophilic microorganisms  were able to thrive in predominantly neutral-pH tailings environments, specifically within microenvironments around altered and/or decomposing sulfide grains.The characterization of microbial communities and their relation to geochemical and mineralogical factors in As-rich mine tailings of near neutral-pH are less commonly studied even though As-cycling is known to play an important role on the bacterial community structure and composition . For exaClostridium was the dominant bacterial genus within high As samples), with total organic carbon (TOC) and the concentrations of Fe and Mn having a less pronounced effect. The authors also showed that the bacterial communities developed an As-tolerance through phylum-level horizontal gene transfer, mainly within the Proteobacteria phylum.Additionally, Achnanthidium minutissimum and diatom populations that correlated to the abundance of As, during a paleolimnological and microbial study on several lakes within the CMC. The authors concluded that lake depth and pH were the significant drivers in community compositional changes and that the concentrations of As in the studied organisms had only minor effects on the compositions of the microbial communities.No prior microbiological studies have been completed on the tailings material in the Cobalt Mining Camp. However, 5+ was the dominant As valence in the lakes and streams that were affected by the As-contaminated tailings, and that pH, temperature, light and the presence of nitrate and chloride strongly affected the stability of As3+ in these waters. ; (2) As5+-species were reduced to more soluble As3+-species in the absence of soluble C, resulting in the competitive binding of arsenate and phosphate groups to adsorption site on Al-(hydr)oxides; and (3) that prolonged flooding periods, as well as the addition of sufficient soluble C, could stimulate the microbial reduction of As, favoring the stabilization of arsenide-bearing minerals.The speciation of As in natural waters and sediments around the CMC has also been studied. Thus, this study continued our efforts to elaborate the metal-mobilizing microbial communities naturally present in the CMC tailings materials. In a previous study, we identified important mineralogical-geochemical relationships within two tailings profiles. We could specifically show that tailings material became depleted in Fe and enriched in Co and As with increasing degree of alteration of the original tailings material (enriched in (sulf-)arsenides). The trend was visualized in plots with the Co-As-Sb-Zn number (Co#) vs. the Fe number (Fe#), and were defined as:andThe authors proposed that abiotic processes such as mineral replacement reactions and the complexation of Fe by carbonate species could have resulted in the mobilization and thus depletion in Fe with increasing degree of alteration. As the role of microbes during this process were not addressed by For this purpose, we focused this study on characterizing the mineral-microbial relationships in the CMC tailings, and determined whether variations in the geochemistry, mineralogy and average valence of As affected the composition of the microbial communities. These objectives were accomplished using geochemical, mineralogical and molecular biology tools to better correlate difference in microbial community composition within the natural gradient of conditions found within the CMC tailings.The Cobalt Mining Camp (CMC) in Northeastern Ontario contains a total of 16 distinct tailings sites . Three tThree tailings profiles were sampled in September 2019 (sites A and B) and October 2019 (site C) . The proDuring sampling, contact pH and ORP measurements were taken using a handheld field instrument, YSI Quatro . A 4-point pH standardization was completed before sampling using standardized solutions of pH 2, 4, 7, and 10. ORP standardization was done using Zobell’s ORP solution (228 mV). Both pH and ORP were completed with a 1:2 tailings to deionized (DI) water ratio. The ORP mV value was then converted to an Eh value simply by adding 200 mV to the ORP voltage .2(T)) and total sulfur (S(T)) at the Geoscience Laboratories, Sudbury, Ontario. The samples were analyzed with a LECO CS844 where approximately 0.2 g of soil was combusted in a stream of purified O2 gas and passed over a heated catalyst, oxidizing total S and C to SO2 and CO2, respectively. The latter two gasses were then detected by two non-dispersive infrared cells  of all 18 samples was carried out at the Perdue Central Analytical Facility, Sudbury, Ontario following the Environmental Protection Agency (EPA) method 3052 . A singl–1. All minerals identified both previous to this study and within this study, as well as their chemical formula, are listed in Three samples from each depth profile were prepared for X-ray powder diffraction. These samples were chosen based on their Co, As, and Fe concentrations or their average valence of As. Nine X-ray diffraction patterns were recorded with either a Philips 159 PW 1729 or a Bruker D 5000 X-ray diffractometer using Cu-Kα radiation (λ = 1.5418 Å) at 40 kV and 30 mA. Diffraction patterns were collected over a range of 5–75° and 5–65° 2θ with a step size of 0.02° and a counting time of 2 s stepA total of 7 samples were prepared for Scanning Electron Microscopy (SEM), two from sites A and B, and three from site C. The samples were embedded into epoxy pucks and polished using MicroPolish Alumina powder on 8-inch Nylon PSA Buehler discs. Six of the carbon coated epoxy pucks were analyzed using a JEOL 6400 SEM operating at 20 kV, equipped with both backscattered (BSE) and secondary (SE) electron detectors and an Energy Dispersive X-ray Spectrometer (EDS). Additional SEM work was carried out on one sample from site C with a FEI Quanta 650 FEG field emission scanning electron microscope at the Manitoba Institute of Materials at the University of Manitoba.Genomic DNA from the total microbial community within each of the 18 samples were extracted from 0.25 g of tailings material using the Sox DNA Isolation Kit . A method blank extraction (DNA extraction column loaded with sterile water) was also completed to determine whether background contaminants were present. Manufacturer protocols were followed, with an additional 10-min heating step at 70°C, after the addition of Solution Sox 1, to enhance DNA yields. The extracted DNA was quantified using a Take3 spectrophotometry system on a Synergy HI microplate reader . DNA yield and quality was then tested by polymerase chain reactions (PCR) using Phire Green Hot Start Master Mix and 27F and 1492R primers (10 umol) in sterile Milli-Q water. The following PCR protocol was used: initial denaturation at 98°C for 30 s, followed by 30 cycles with denaturation at 98°C for 5 s, annealing at 55°C for 5 s, elongation at 72°C for 25 s, and a final elongation step at 72°C for 7 min. Verification of the PCR products was accomplished by gel electrophoresis on a 1% agarose gel. Samples that were not deemed to be of high enough yield or did not amplify, were re-extracted from frozen samples.1, to remove primers from the forward and reverse reads. Using the DADA2 pipeline (version 1.8), the trimmed reads were processed, using the default filtering and merging parameters, with the sample interference being conducted using the pseudo-pooling method . Library preparation was completed following MetagenomBio Inc. protocols  and the g method . To assig method . Post tag method . Contamig method , using b0 = 11919 eV) using the maximum first derivative of the XANES spectrum of a gold foil standard. Samples were mounted at a 45° angle to the X-ray beam and the detector was positioned at a 90° angle to the X-ray beam. Three different spots on each sample were selected and each spot was scanned three times. The scan parameters of the XANES spectra with respect to E0 were from 11667 to 12166 eV with a step-size of 10 eV, 0.3 eV or 0.1 k for the pre-, near-, or post-edge region (respectively) per step, with a dwell time of 2 to 2.5s per step. Arsenic standards for As5+, As3+, As1–, and As3– were a Na-arsenate (Na2AsO4⋅7H2O), arsenic trioxide, arsenopyrite and gallium arsenide, respectively, and were scanned with a 1s dwell time (X-ray absorption near-edge spectroscopy (XANES) spectra were characterized at the Very Sensitive Element and Structural Probe Employing Radiation from a Synchrotron (VESPERS) beamline at the Canadian Light Source, Saskatchewan, CA, in order to determine the average valence of As in the collected tailings material. XANES scans were collected on powdered samples in fluorescence mode using a Canberra 13-element Ge detector. The energy position was calibrated to the gold L3 edge  was implemented using the environmental fit function of the vegan package in R software (version 1.3.959). The relative abundance of microbial data (ASVs) was log transformed and subjected to the Bray-Curtis dissimilarity calculation, using the betapart package in R software. Each point of an NMDS plot represents the microbial community composition of a sample within a reduced 3D multidimensional space, with the distance between two points  representing the difference between the two samples microbial community composition. Hence, the closer two points are together, the more similar is there microbial community composition .R-value associated with the similarities between the tested groups  is a statistical test that was conducted using the anosim function in the vegan package in R software. The use of the ANOSIM here was to identify whether similarities (or differences) exist between the tailings sites. An ANOSIM provides an d groups . An R-van groups . The ANOThe geochemistry and mineralogy of the tailings material at sites A and B were very similar to those studied by D) = 25 cm at site B and at D = 11 cm at site C, and a thick dark-colouration at D = 25 to 30 at site C. The average contact pH and Eh values for the materials were as follows at site A [8.4 (±0.2) and 0.28 V (±0.01)], site B ), and site C [7.6 (±0.2) and 0.36 V (±8.87E–03)] ] . However(T)) whereas the material at site C had higher concentrations of S(T) ((T)) content in the tailings material differed significantly between the sites with the lowest and highest content at sites A and C, respectively ((T) and C(T) varied, similar to Eh and contact pH, with depth. For example, material with the lowest contact pH at site B (D = 20 cm) also had the highest amount of S(T) and C(T) ((T) for all characterized tailings material occurred at D = 30 cm at site C with 0.56 wt% S(T)  . The totectively . The conand C(T) . The higwt% S(T) .D = 25 cm at site A, at D = 20 cm at site B, and D = 30 cm at site C)  s of interest such as Co, As, Fe and the average valence of As also varied with depth . A commo site C) . This de site C) . Tailing site C) . The mat site C) . A notab = 30 cm . The perm site C .Similar to the Co-Zn-Sb-As number (Co#) (Eq. 2) and Fe number (Fe#) (Eq. 3) , we defiD = 30 cm.Although, Co#, Fe#, and S# number vary with depth at all sites A, B, and C , the taiD = 15 to 25 cm at site A was chosen for SEM characterization as we were interested in mineralogical changes between material of high and low Fe# . At site B, arsenates were the dominant As phase over (sulf-)arsenides at both depths  (At site A (sulf-)arsenides were the dominant As-bearing phase at oatings) .D = 20 and 25 cm at site C, arsenates were also predominant over (sulf-)arsenides, but the ratio of arsenates to (sulf-)arsenides is smaller than at site B  (D = 30 cm of site C contained three distinct groups of: (1) sulf-arsenides; (2) arsenides; and (3) sulfides (At oatings) . Materiasulfides . The forsulfides  or textuAlthough the sampling in this study was at a lower resolution than the study by (1)Tailings material at site B had a higher Co#, lower concentrations of Fe and higher proportions of secondary arsenate phases in the form of mineral surface coatings than the material at site A , 6;(2)Tailings material at all sites had only minor amounts of Fe-(hydr)oxides;(3)At sites A, B and C, tailings material with higher Co#’s had lower amounts of Fe than those with lower Co#’s ; and(4)At sites B and C, material with a high S# generally coincide with a lower As valence  , 5.D = 30 cm) had lower average valences of As with +2.2 and +0.25, at sites B and C, respectively  content, Co#, S# and concentrations of As and Co, a decrease in the average As valence, and an increase in pH, Fe# and S(T), i.e., there was a gradual oxidation of (sulf-) arsenides, leaching of metal(loid)s and (slight) increasing acidity toward the surface.The tailings material at site C, which was not characterized by ectively . The lowectively . Hence, ectively . This zoIn order to better characterize trends in the microbial data, taxa with a relative abundance below 2% (or 0.5% for Species-level description) were grouped together into a single category, and only taxa with a relative abundance >2% are described here. Additional taxonomic abundance graphs  can be found in the D = 30 cm) in the material at site C relative to those at sites A and B.Within materials from all three tailings sites, the bulk microbial community composition consisted mainly of the Proteobacteria (14-75%) and Actinobacteria (5-48%) phyla, with lesser amounts of Chloroflexi (5-21%), and Acidobacteria (3-9%) . AdditioD = 0, 5, and 15 cm an abundance of Gaiellales (5-25%), Solirubrobacterales (7-13%), and Sphingomonadales (6-31%) occurred. At D = 20, 25, and 30 cm a greater abundance of Gemmatimonadales (6-14%), and Rhizobiales (18-40%) was present. The Bacillales, Betaproteobacteriales and Acidoferrobacterales were consistent throughout site A ranging from 3 to 12%. Corresponding genera that could be identified from the above Orders included: Sphingomonas present throughout the tailings profile at site A and up to 30% at D = 15cm, Gaiella (2-7%), Nocardiodes (20% at D = 20), Rubrobacter (10% at D = 0 cm), Sulfurifustis (3-7.5%), Methylocapsa (3-25%), and Methylovirgula (5-10%), with the latter three being more abundant in the lower half of the tailings profile (D = 20 to 30 cm)  .D = 20 cm), Rhizobiales (2–20%), and Micrococcales (6–12%) dominated. Corresponding genera that could be identified from the above Orders included: Gallionella (3–5% at D = 20–30 cm), GOUTA6, Gaiella (4%), Nitrospira (2–6%), Hyphomicrobium (2.5%), Methylobacter (2–16%), Methylocystis (4%) and Methylovirgula (5%), Sideroxydans (6%), Sphingomonas (3–6% in D = 0–15 cm), Sulfurifustis (5%), and Thiobacillus (9% at D = 25 and 30 cm)  .D = 0–15 cm, Acidoferrobacterales (3–6%), Betaproteobacteriales (3–10%), Gaiellales (7-12%), Micrococcales (2–7%), Rhizobiales (7–12%), and S085 (3–5%) dominated. At D = 20–30 cm, Acidoferrobacterales (3-25%), Betaproteobacteriales (13–18%) increased in abundance with similar amounts of Gaiellales (4–16%) and Rhizobiales (3–7%) as in the surficial layers. Notably, the class Thermodesulfovibrionia (uncharacterized Order) occurred in tailings at site C, at D = 25 and 30 cm with a relative abundance of 5 and 41% (Gaiella throughout the tailings profile (3–18%), Nitrospira within the top 20 cm’s (3–8%), Sulfurifustis (6–28%) and Thiobacillus (2–7.5%) at D = 25 and 30 cm, respectively, and Nitrosospira at D = 20 and 25 cm (∼3 and 7%)  .R-value of 0.742 with a p-Value of 0.001. An R-value closer to 1 implied a high dissimilarity between tested groupings, whereas a p-Value < 0.05 implied a high statistical significance. This indicated a strong, statistically significant difference in the microbial communities based on the site grouping  test was used to evaluate the relationships between the microbial community compositions, the environmental (or field parameters), and geochemical variables between each site, the data for which can be found in r2-Values for the correlations relating the environmental and geochemical variables to each other are listed in the In the following sections, environmental and geochemical variables ± mineralogical compositions and As valences were related to the overall  and siteD = 20, 25, and 30 cm) being very similar , with the bottom 15 centimeters of site A , including the Fe# and depth. The second included pH, Eh, the concentrations of As, Ca, Co and S(T), and the Co#, of which have slightly less significant r2 and p-Values   .The following observations from (1)The Fe# was highest in tailings of site A  and lowest in tailings from site B and could have a strong influence on the community dissimilarity;(2)(T), concentrations of As and Co), the concentration of Ca, the Co# and the amount of CO2(T) were highest at greater depths (D ≥ 15 cm) in the tailings from site C; andThe group 1 , group 2 D = 5 cm).Tailings from site C had the lowest pH values (lowest in (T) all of which have r2-Values > 0.75 and p-Values < 0.05 . At D = 5 cm, the highest concentration of group 3 ) and group 4  variables occurred  (5+ and As1–-species were highly correlated to depth (r2 > 0.65). The highest contact pH increase occurred within the first 15 cm’s of tailings from site B, with the lowest pH values at D = 25 and 30  variables  were inversely correlated to As1– (r2 > 0.9), and have the highest values at D = 10 and 15 cm  . Within 5 and 30 . The larnd 15 cm . The con(T), and the percent abundances of As5+, As3+, and As1–-species, all of which have r2 ≥ 0.75 and p-Values ≤ 0.06 (r2 > 0.8 or r2 <-−0.8). The Co#, along with the G1 and G2 variables, all increased with depth, with maxima for each variable or group of variables at D = 30 cm or D = 25 and 30 cm (r2 ≤-−0.65). The contact pH values were inversely proportional to the CO2(T) concentrations (r2 =-−0.9), with the highest and lowest values, respectively, at D = 5 cm. Maxima of the average As valence, percent abundance of As5+-species and the AVR occurred at D = 15 cm.The microbial community composition at site C was controlled dominantly by: pH, depth, Eh, concentrations of As and Co, Co#, S#, Ss ≤ 0.06 . Similarnd 30 cm . The G4 2(T), pH, Fe#, Co#, S#, and S(T) varied with depth.The significance of the depth profile on the microbial community composition at site A can be also recognized in the site-specific NDMS plot . This pl5+ with respect to total As; i.e., the proportion of arsenates to arsenides  when transitioning from D = 5 cm to D = 25 cm (5+ (5%) and the Fe# between D = 25 and 30 cm coincided not only with an increase in the S and Co# but also with a change in the microbial community composition.The microbial community composition at site C followed a continuous increase in the concentration of As, Co#, S#, and the amount of As = 25 cm , and corWe will first discuss environmental parameters controlling microbial community composition before addressing their relationships to the mineralogy and geochemistry of the tailings material at sites A, B, and C of the Cobalt Mining Camp.Microbial diversity in mine waste environments can be shaped by various environmental factors and geochemical variables. The characterization of their diversity is important in understanding the biological response to the environmental and geochemical conditions.The variables associated with governing the microbial structure in the studied tailings were different and more complex than those associated with trends in the mineralogical data . The following were the most important observations from the relationships between the overall and site-specific microbial communities to the environmental and geochemical variables described,(1)depth and the Fe# were the primary controllers for the CMC microbial communities;(2)depth was the only common variable in shaping the structure of the bulk and site-specific microbial communities;(3)(T) played a dominant role in determining the microbes present at specific sites;the concentrations of various elements , pH, Eh and S(4)5+ vs. As3+ and As1–) strongly correlated to different microbial communities present; andin site C, the abundances of different As-species although the Fe# did not play an important site-specific role in the microbial community structure, it was an important parameter for differentiating the microbial composition between each site.-30 cm) samples. Similar trends were observed in As and Sb contaminated soils as well as in slightly acidic mine tailings  and deeper  and Fe2+) to the reduction of a terminal electron acceptor (such as oxygen (O2) and sulfate (SO4)2–) under aerobic and anaerobic conditions. In legacy tailings sites, surrounding influence and inputs from soil and aquatic environmental can introduce micronutrients as well as a mix of metal-cycling and soil nutrient-cycling organisms .In a tailings system, lithotrophic organisms commonly occur as the most abundant metabolic groups. These organisms can couple the oxidation of reduced inorganic compounds  are known to grow under neutral pH, and are dependent on nitrate as either a chemical oxidant (under anaerobic conditions) or as an electron acceptor  and putative SRB (class Thermodesulfovibrionia) co-occurred in tailings at site C at 30cm suggesting the transition between sulfur oxidation and reduction were present at this depth. This coincided with the highest total S, As and Co concentrations in these materials suggesting these genera were likely involved in the biotic transformation of arsenides (discussed below).In general, the CMC tailings had a higher abundance of FeOB and SOB relative to IRB and SRB through the 30 cm profile. Many of the putatively identified FeOB and SOB  content decreased from 5 wt% at the top to 3 wt% at the bottom  and those that couple nitrate-reduction to Fe and/or S-oxidation . The high abundance of these microbes could be due to the implementation of cyanide during ore-processing  could also be cycled in the tailings and occurred in sediments with observed As3+-oxidation under oxic conditions at neutral pH, but in in the absence of known As-oxidizers , the reduction of arsenates or arsenites, and the oxidation of arsenides or arsenites .The most apparent and distinct change in Arsenic mineralogy and microbial structure occurred at site C where the mineralogical transition from the reduced (arsenides) to oxidized (arsenates) zone correlated with a decrease in the abundance of S and As-reducing bacteria.At site B, the higher abundance of arsenates and arsenides at shallower and greater depth, correlated well with a higher abundance of methanotrophic bacteria and SOB and FeOB, respectively. Similarly, the higher abundance of (sulf)arsenides at site A correlated well with increasing abundances of methanotrophic bacteria, SOB and FeOB at greater depths.These observations indicated that tailings material enriched or depleted in (sulf)arsenides or arsenates had distinct microbial community composition but they also suggested that the latter compositions were not solely controlled by the type of As-bearing minerals.Sulfurifustis, and Thiobacillus spp.), were able to couple S-oxidation and As-reduction, and Thiobacillus spp. using nitrate as the preferred electron donor  As-reducing bacteria were in higher abundance. However, it is possible that some of the SOB was able to reduce As. For example, two of the identified SOB (on donor . Additionditions . The putnditions . Kelly eIron-(hydr)oxides are the most thermodynamically stable Fe-phase under near-neutral to alkaline conditions, with their formation being inhibited by the presence of IRB . Courche2+-species may have also been promoted by putative IRB such as those of the genera Bacillus and Paenibacillus and Rhodoferax genera 2– as a terminal electron acceptor) or those found in As  contaminated sites. Furthermore, the occurrence of novel neutrophilic, As-cycling bacteria within the characterized CMC tailings were likely present, due to the ∼ 25–55% uncharacterized genera and >85% uncharacterized species in the amplicon data. Many of the identified microbial genera in the CMC tailings are known to have As resistant genes, encoded by the ars gene, such as genera Bacillus, Sphingomonas and Sulfuricella, as well as additional previously unmentioned genera such as Xylophilus Sites A and B in this study shared a similar geochemistry and mineralogy to that observed by (2)Site C was geochemically and mineralogically dissimilar to sites A and B, as site C had distinct reduced and oxidized zones, as observed in more acidic mine tailings environments;(3)The groupings observed of the Co# vs. Fe# and S# between each site, correlated well with site-specific microbial groupings;(4)The abundance of microbial communities involved in cycling Fe, S, N and C correlated with depth, i.e., the occurrence of SRB at greater depths coincided with a low average valence of As;(5)The presence of FeOB and SOB at greater depths within the CMC tailings could be due to the presence of (sulf-)arsenides;(6)Samples at site B with low Co#’s and high Fe and/or S#’s had a proportionally higher abundance of FeOB or SOB, due to the abundance of (sulf-)arsenides, whereas at site C, a low Fe# and high Co and S#’s showed an abundance of (sulf)-arsenides, sulfides, and SRB;(7)At individual sites, high Co#’s did not correlate with a higher abundance of potential As-cycling bacteria; and(8)The low abundance or absence of IRB and SRB at the bottom of site A and B suggested that the tailings remained largely oxic or at the oxic-anoxic transition up to 30cm.PRJNA700089.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: NCBI BioProject, accession no: BC, MS, and NM were involved in the design of the study. MS and NM supervised the study. MS conducted a portion of the scanning electron microscopy and X-ray diffraction work. BC wrote the first draft and finalized the manuscript. All authors contributed to the writing of the manuscript and approved submission.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.


Repressor protein period (PER) complexes play a central role in the molecular oscillator mechanism of the mammalian circadian clock. While the main role of nuclear PER complexes is transcriptional repression, much less is known about the functions of cytoplasmic PER complexes. We found with a biochemical screen for PER2-interacting proteins that the small GTPase regulator GTPase-activating protein and VPS9 domain-containing protein 1 (GAPVD1), which has been identified previously as a component of cytoplasmic PER complexes in mice, is also a bona fide component of human PER complexes. We show that in situ GAPVD1 is closely associated with casein kinase 1 delta (CSNK1D), a kinase that regulates PER2 levels through a phosphoswitch mechanism, and that CSNK1D regulates the phosphorylation of GAPVD1. Moreover, phosphorylation determines the kinetics of GAPVD1 degradation and is controlled by PER2 and a C-terminal autoinhibitory domain in CSNK1D, indicating that the regulation of GAPVD1 phosphorylation is a novel function of cytoplasmic PER complexes and might be part of the oscillator mechanism or an output function of the circadian clock. Circadian clocks are endogenous mechanisms that drive rhythmicity of physiology and behavior, enabling light-sensitive organisms to anticipate daily recurring changes in the course of the day–night cycle. The mammalian circadian system is synchronized to the environment by a light-responsive master clock in the hypothalamus that transmits timing information to peripheral oscillators via neuronal and endocrine pathways . RhythmiPER and CRY proteins function as integral components of PER complexes, highly dynamic protein assemblies that work in the core feedback loop of the molecular clock. Nuclear and cytoplasmic PER complexes differ in their protein compositions, which reflect diverging functions in different cellular compartments. Cytoplasmic PER complexes exist in two variants; a smaller complex that contains the core components PER1/2, CRY1/2, and casein kinase 1 delta (CSNK1D), and a bigger complex that additionally contains PER3 and GTPase-activating protein and VPS9 domain-containing protein 1 (GAPVD1) ,5,6. WhiFBXL3, thereby linking the circadian clock to cell cycle control [The circadian clock regulates a wide variety of output functions by controlling rate limiting steps of cellular pathways. For this purpose, clock proteins possess additional regulatory functions outside the oscillator mechanism that establish molecular links to cellular physiology, in general, via the transcriptional regulation of individual sets of target genes . However control . Such fi control ., but nothing is known about the consequences of the association of GAPVD1 with the PER complex for the regulation of GAPVD1. Our results show that the control of CSNK1D-dependent GAPVD1 phosphorylation is a novel function of the PER complex that is linked to GAPVD1 degradation and that PER2 regulates GAPVD1 phosphorylation through a C-terminal autoinhibitory domain of CSNK1D.In this study, we address the role of the PER complex in the posttranslational regulation of GAPVD1, a regulator of small GTPases that has been identified as a substoichiometric component of cytoplasmic PER complexes in mice . Knockdop < 0.0005)  A. This g 0.0005) B.p-value , CSNK1D-GFP (C), or PER2-GFP and CSNK1D-RFP (red fluorescent protein) (CP). In CP cell lines that overexpress both proteins the GAPVD1 protein band migrated considerably slower than in wild type cells or cells that overexpress only PER2 or CSNK1D A. The efIn combination, the knockdown and overexpression experiments strongly indicated a requirement for both CSNK1D and PER proteins for the establishment of GAPVD1 phosphorylation. At this point, two major questions seemed to arise. First, why is CSNK1D necessary but not sufficient for GAPVD1 phosphorylation? And second, which mechanistic role do PER proteins play in this process? It had been shown previously that the kinase activity of CSNK1D is inhibited by autophosphorylation of its C-terminal tail. More specifically, deletion of a region between Histidine 317 and Proline 342, which contains five serines and one threonine, creates a mutant enzyme with 10-fold increased activity . Stable These results suggest that PER2 stimulates the CSNK1D-dependent phosphorylation of GAPVD1 by counteracting the autoinhibitory activity of the C-terminal domain of CSNK1D, most likely via direct interaction with CSNK1D in the context of the PER complex. Indeed, deletion of an essential part of the casein kinase interaction domain in the PER2 mutant PER2(Δ489–618)  resultedS (underlined is the target site for phosphorylation) [Since GAPVD1 is phosphorylated by CSNK1D as part of the PER complex, and CSNK1D-dependent phosphorylation at two antagonistic sites regulates the degradation of PER2 in a mechanism called the phosphoswitch , we askeylation) , which mylation) . PrimaryFinally, we examined the degradation kinetics of GAPVD1 after blocking translation with cycloheximide under the same conditions that were used for the phosphopeptide analysis, i.e., in wild type, CP, and PF670462-treated cells, all of which have distinguishable GAPVD1 phosphorylation patterns A. TreatmGAPVD1 is linked to the molecular circadian oscillator as a component of cytoplasmic PER complexes, but little is known about the functional consequences of this interaction. It has been suggested that GAPVD1 might play a role in the assembly, accumulation, trafficking, or nuclear entry of PER complexes, since knockdown of GAPVD1 lengthens the circadian period of cultured cells and GAPVD1 must, therefore, interfere with the molecular clockwork . Our stuHow do PER proteins regulate the phosphorylation of GAPVD1? Our data indicates that PER2 can relieve CSNK1D from the effect of an autoinhibitory domain in its C-terminus. Unlike many other kinases, CSNK1D is not regulated by phosphorylation of its activation loop . Its actAnother question concerns the biological role of GAPVD1 phosphorylation. We show here that the phosphorylation state of GAPVD1 affects the speed of its degradation, which might affect the function of GAPVD1 as a regulator of the molecular oscillator, for example, by delaying or promoting the nuclear entry of the PER complex. However, our experiments do not allow any conclusions about the direct effects of phosphorylation by the PER complex on GAPVD1 degradation. Nevertheless, in analogy to the phosphoswitch model, we found that total phosphorylation levels seem to be less important for the degradation kinetics than phosphorylation of individual sites in GAVPD1. In particular, phosphorylation of site 5 markedly responded to the level of CSNK1D kinase activity, and degradation was strongly delayed when site 5 was fully dephosphorylated. Interestingly, a comprehensive study of the circadian phosphoproteome found site 5 to be rhythmically phosphorylated  and to bCaenorhabditis elegans, a function attributed to its C-terminal VPS9-domain, which acts as a GEF (Guanine nucleotide exchange factor) for small GTPases of the Ras-related in brain 5 (RAB5) family [There is also evidence that phosphorylation of GAPVD1 has a role in vesicle mediated transport processes . Initial) family . The fun) family . It is u) family ,37. Neve) family . Control) family , shown t) family  and to r) family  as well ) family . Future 2 environment.HT1080 and HeLa cells were purchased from the German Collection of Microorganisms and Cell Cultures (DSMZ). Stable cell lines were generated by Effectene  transfection of HT1080 cells. One microgram plasmid-DNA was used in a T25 cell culture flask; antibiotic containing selection medium was added after 48 h, and single clones were isolated after 10–30 days. The resulting cell lines were HT1080-PER2-GFP (P); HT1080-GAPVD1-GFP (G); HT1080-CSNK1D-GFP (C); HT1080-GFP; HT1080-CSNK1D-RFP-PER2-GFP (CP); HT1080-CSNK1D(D317-342)-RFP (C(DC)). All cell lines were cultured in Dulbecco’s modified Eagle’s medium  supplemented with 10% FCS, 100 U/mL penicillin, 100 µg/mL streptomycin and, depending on the cell line, 0.4 µg/mL puromycin or 300 µg/mL hygromycin, and maintained at 37 °C in a 5% COGAPVD1/RAP6 ; GAPVD1/RAP6 in Duolink ; PER2 ; CSNK1D ; CyTM3 Anti-Mouse IgG Antibody ; CyTM3 Anti-Rabbit IgG Antibody ; ECLTM Anti-Mouse IgG Antibody ; ECLTM Anti-Rabbit IgG Antibody .2MoO4, 1mM Na3VO4, 10 mM NaF, 2.5 mM Na4P2O7, 1x protease inhibitor cocktail (PI) ). Twenty-five milligrams of cleared whole cell protein extract was incubated with 40 µL equilibrated GFP-Trap® beads  with rotation at 4 °C overnight. Thereafter, the beads were washed three times with 1 mL CHAPS lysis buffer and then three times with high salt CHAPS lysis buffer ). The beads were suspended in 60 µL Buffer A  and then subjected to LC-MS or Western blot analysis. For affinity enrichment of GAPVD1 from HT1080 and HT1080-CSNK1D-RFP-PER2-GFP cell lines, 107 cells were lysed in Triton X-100 lysis buffer , 1X phosphatase inhibitor cocktail (Roche). Thereafter, the cleared cellular lysates were incubated with 120 µL Protein A microbeads  and 30 µL GAPVD1 antibody  with rotation at 4 °C overnight. Subsequently, the cell lysates were applied on equilibrated m Column (Miltenyi Biotec) that were placed in the magnetic field of the m MACS™ Separator (Miltenyi Biotec). The columns were washed 4 times with high salt lysis buffer , 1X phosphatase inhibitor cocktail (Roche)) and then one time with low salt lysis buffer , 1X phosphatase inhibitor cocktail (Roche)). Proteins were eluted with 40 µL preheated (95 °C) SDS gel loading buffer , 1X phosphatase inhibitors cocktail (Roche)) and then subjected to LC-MS. For CSNK1D inhibitor treatment, cells were incubated for 18h with 20 µM PF-670462 before lysis.For affinity enrichment of GFP from HT1080-PER2-GFP and HT1080-GFP cell lines, cells were lysed in CHAPS lysis buffer  connected to an Ultimate 3000 Rapid Separation liquid chromatography system (Dionex/Thermo Scientific) equipped with an Acclaim PepMap 100 C18 column  was used. The length of the LC gradient was 60 min. The mass spectrometer was operating in positive mode and coupled to a nanoelectrospray ionization source. The capillary temperature was set to 250 °C and source voltage to 1.4 kV. In the QExactive plus mass spectrometer for the survey scans, a mass range from 200 to 2000 m/z at a resolution of 140,000 was used. The automatic gain control was set to 3,000,000, and the maximum fill time was 50 ms. The 10 most intensive peptide ions were isolated and fragmented by high-energy collision dissociation (HCD).p ≤ 0.01) on the peptide level was set as the identification threshold.Proteome Discoverer  was used for peptide/protein identification with Mascot  as a search engine employing the human database . A false discovery rate of 1%  for 24 h at 37 °C. Cells were fixed and permeabilized with 4% formaldehyde for 10 min, followed by 0.1% Triton X-100 for 5 min at room temperature. Blocking was performed with 2 drops of Duolink blocking solution  per well for 60 min at 37 °C. Thereafter, slides were incubated with primary antibodies diluted in Duolink antibody diluent at 4 °C overnight on an orbital shaker. After washing four times with wash buffer A, PLA probes were added (70 µL/well) for 1 h at 37 °C in a humid chamber. Ligation and amplification were performed according to the manufacturer’s instructions . Slides were washed two times with wash Buffer B at room temperature for 10 min, then overnight at 4 °C. After washing, cells were stained with DAPI 1:1000 in PBS for 4 min, and slides were covered with slide slips. Pictures were taken with an Axiovert 100 microscope and analyzed with Image J.Whole cell extracts of HT1080 cells lysed in 1× Laemmli buffer without SDS were sonicated and subsequently incubated at 65 °C for five minutes. The samples were cooled on ice and then treated with 1U Calf Intestinal Phosphatase  per microgram of protein in CutSmart buffer (NEB) for 30 min at 37 °C. After the dephosphorylation reaction, SDS was added to a final concentration of 1%, and proteins were separated by SDS-PAGE.Ten to the five cells were seeded on 6-well plates one day before siRNA transfection. siRNAs targeting CSNK1D , CSNK1E , or non-target (nt) control siRNA  were incubated with DharmaFECT 1 Transfection Reagent  according to the manufacturer’s instructions. Transfection was carried out at a concentration of 100 nM for each siRNA. Cells were collected after 48 h for mRNA analysis by qPCR or after 72 h for protein analysis by Western blotting.Two point five times ten to the six U2OS cells were seeded on T175 flasks two days before dexamethasone shock. Cell synchronization was performed with 100 nM dexamethasone  for 30 min at 37 °C. Cells were collected after 26 h, 30 h, 34 h, 38 h, 42 h, and 46 h after dexamethasone treatment. Immunoprecipitations were performed from 4 mg whole cell protein extract with 4 µg CSNK1D  or 4 µg GAPVD1 antibody .


To evaluate the efficiency of the ball tip technique for S2AI screw placement and introduce this technique.Sixty‐three patients who underwent pelvic fixation with S2AI screws were retrospectively reviewed. They were 29 males and 34 females with an average age of 59.6 ± 12.5 years. Among these patients, 35 patients  received ball tip technique and 28 patients  received conventional freehand technique. Ball tip technique was used in ball tip technique group. After a pedicle probe just penetrated the sacroiliac joint, a ball‐tipped probe consisting of a ball shaped metal tip with a flexible shaft was malleted to make a guide track within ilium. This ball‐tipped probe could bend automatically away from the cortex and forward through the cancellous bone when the tip met the cortical lamina of ilium, which can avoid penetration. After repeating the procedures, a guide hole was gradually formed. S2AI screw was inserted along the guide hole after tapping. In the conventional freehand group, S2AI screw was placed according to the conventional method. Postoperative computed tomography (CT) was used to assess the accuracy of screws. The time cost of screw insertion and screw‐related complications were recorded. Independent t‐test was used to compare the time cost between ball tip group and conventional freehand group. A chi‐square test was used to compare the accuracies of the ball tip group with the conventional group.P < 0.05). Four screws penetrated iliac cortex in the ball tip group vs 10 screws in conventional freehand group (5.7% vs 17.9%) (P < 0.05).There were 35 patients (70 S2AI screws) in ball tip group and 28 patients (56 S2AI screws) in conventional freehand group. No screw‐related complication occurred in all patients. Time costs were 9.8 ± 4.5 mins in ball tip group and 20.2.0 + 8.6 mins in conventional freehand group, respectively (The ball tip technique enhances the accuracy of screw placement and has less time cost compared with conventional freehand technique. This work introduced a novel technique named ball tip technique for S2AI screws placement. Ball tip technique enhances the accuracy of S2AI screw placement. To achieve solid osseous fusion across the lumbosacral junction has historically been, and continues to be, a challenge in spine deformity surgery involved in sacropelvic unitThis is a retrospective study, and it was approved by Ethic Committee of The First Affiliated Hospital of Nanchang University.Sixty‐three consecutive patients who underwent spinopelvic fixation using S2AI screws by a senior surgeon over 40‐month period (April 2015–August 2018) were reviewed retrospectively. The inclusion criteria: adult patients underwent sacropelvic fixation with S2AI. The exclusion criteria: S2AI screws were inserted with other techniques such as 3D template guided technique (TGT). The patients comprised 29 males and 34 females with an average age of 59.6 ± 12.5 years. Among these patients, 35 patients  received ball tip technique  and 28 patients  received conventional freehand technique   joint through S2 alar by aiming the point two‐finger width proximal to the ipsilateral greater trochanter. When the pedicle probe penetrated the SI joint and just broke through the iliac joint surface, a special ball‐tipped probe was used instead of pedicle probe and inserted along the primary track hole. The probe was then gently malleted to advance it through the cancellous channel to make a guiding track in ilium. The ball tip probe consisted of a ball‐shaped metal tip with a semi‐flexible metal shaft. It had the characteristics of metal's semi‐flexibility and could bend automatically when meeting the lateral or medial cortical lamina of ilium, which could make a track hole within ilium ultimately Fig. . After rWhen the trajectory was confirmed, the S2AI screw was inserted through the track hole after surrounding and tapping  CT is used to assess the accuracy of screw placement after surgery. The screw contained in the iliac cancellous bone was defined as being accurately placed. By contrast, screw that violated or broke medial or lateral cortex, or the cortex of sciatic notch, was defined as inaccurate position Fig. . The scrTime cost of unilateral screw placement was recorded and compared between the two groups. Timing started at the time of finding the starting point and finished at the time the screw was inserted completely without a revision.Any cortex violation‐related and screw‐related complication was recorded and compared between two groups to evaluate safety of screw placement, including major vessel injury, nerve injury, screw broken, and screw loosened. The complications occurred in follow‐up were considered to be no relation with accuracy of screws and were not evaluated.P < 0.05.Statistical analysis was performed using SPSS 17.0 . Data are presented as the mean ± standard deviation. Independent t‐test was used to compare the time cost between ball tip group and conventional freehand group. A chi‐square test was used to compare the accuracies of the ball tip group with the conventional free hand group. Statistical significance was defined as vs 17.9%)   were placed with mild cortical breaches and no screw was placed with moderate to severe cortical breaches. No breach occurred anterior, while four were posterior. In conventional freehand group, a total of 10 screw (in nine patients) breaches were noted, including four screws were placed with mild cortical breaches and six screws were placed with moderate to severe cortical breaches. Two breaches occurred anterior, while eight were posterior. A significant difference in the accuracy of screws placement was observed between two groups (5.7% P < 0.01).Time cost of unilateral S2AI screw placement were 9.8 ± 4.5 mins in ball tip group and 20.2 + 8.6 mins in conventional freehand group, respectively. There is a significant difference in the time cost between two groups. Ball tip technique for S2AI screw placement has less time cost compared with conventional freehand technique (t = −8.738, No cortex violation‐related or screw‐related complications occurred in any of the patients.Cortex violation of the ilium would occur when S2AI screws were placed by free hand technique. The cortical breach of an S2AI screw may result not only in decrease fixation strength but also potentially injury of the major vessels, particularly the internal iliac artery with an anterior breach and the superior gluteal artery with a caudal breachFurthermore, compared with conventional freehand technique, ball tip technique decreased the frequency of using C‐arm, which would significantly reduce radiation exposure to the patient and surgeon. In the conventional freehand technique, the surgeon had to find the teardrop and ensured the guided probe and the inserted screw inside the teardrop under repeated intraoperative fluoroscopy. On the contrary, in procedure of ball tip technique, the surgeon took the special probe with characteristics of semi‐flexible metal shaft to find the track hole in the cancellous channel automatically, rather than having to continue watching the probe inside the teardrop, which obviously reduces the frequency of using C‐arm. Consequently, the efficacy of S2AI screw insertion was significantly improved. Especially for some patients with severe lumbosacral deformity and rotational pelvis, ball tip technique significantly decreased frequency of intraoperative fluoroscopy and simplified surgical procedure Figs , 5, 6.Some surgeons who used conventional freehand technique reported that only 5% of the S2AI screws demonstrated moderate to severe cortical breaches, all of which perforated the pelvis posteriorly and all only involved the distal part of the screw tip depending on the horizontal and caudal anglesIn this study, the time cost between ball tip technique and conventional freehand technique were also compared, our result demonstrated that ball tip technique could statistically decrease the time cost of screw placement from 20.2 ± 8.6 min to 9.8 ± 4.5 min. Less surgical time means less intraoperative blood loss and lower rate of wound infection. Less operation time may also decrease surgeon fatigue, improve concentration, and potentially correlate with positive patient outcomesvs conventional freehand technique. Additionally, although our result demonstrated that the ball technique is useful and reliable for S2AI screw placement in this study, it still needs to be emphasized that it may not be helpful to a surgeon who has no experience in placing S2AI screws, since a mistake in the identification of starting point could lead to missed screw placement even if the ball tip technique is used.There were limitations to this study. The ball tip technique was not generalized in pediatric patients, whether this technique could be used in pediatric patients needs to be further investigated. This study is a small series in a single center and the patients were not randomized for both techniques in this retrospective study. Limited results were obtained from this study and future studies may benefit from randomizing patients divided into ball tip Ball tip technique for S2AI screw placement is not only a reliable but also a practical technique. This technique significantly enhances the accuracy of S2AI screw placement and improves the efficacy by reducing the time cost for S2AI screw placement in spine surgery involved in sacropelvic unit compared with conventional freehand technique.This study was approved by the Ethics Committee of The First Affiliated Hospital of Nanchang University.Written informed consent was acquired from each patient to authorize treatment, imageology findings, and photographic documentation. The patients consented to the publication of their pictures as well as their anonymous and clustered data.


A. suum. In our study, GABA (3 μM) produced significant increase of contractile EC50 value for pyrantel, and nonsignificant for bephenium and morantel, from 8.44 to 28.11 nM, 0.62 to 0.96 µM, and 3.72 to 5.69 nM, respectively. On the other hand, the maximal contractile effect (Rmax) did not change in the presence of GABA. However, when A. summ muscle flaps were incubated with GABA 3 μM and carvacrol 100 μM, the EC50 value of pyrantel, bephenium, and morantel was increased significantly to 44.62 nM, 1.40 μM, and nonsignificantly to 7.94 nM, respectively. Furthermore, Rmax decreased by 70, 60, and 65%. Presented results indicate that the combined use of GABA receptor agonists and nicotinic receptor antagonists can effectively inhibit the neuromuscular system of nematodes, even when one of the nicotinic receptor subtypes is dysfunctional, due to the potential development of resistance.Resistance of parasitic nematodes to anthelmintic drugs is a growing problem in human and veterinary medicine. The molecular mechanisms by which nematodes become resistant are different, but certainly one of the possible processes involves changing the drug binding site on the specific receptor. The significance of changes in individual subtypes of nicotinic acetylcholine receptors (nAChRs) for the development of resistance has not been clarified in detail. This study investigates the interaction of antinematodal drugs, agonist of different types of nAChRs and carvacrol with gamma aminobutyric acid (GABA) on the contractions of parasitic nematode  A number of excitatory and inhibitory neurotransmitters are involved in the regulation of somatic muscle contractions in nematodes. It is well known that excitatory and inhibitory neurons, whose coordinated activity ensures sinusoidal movement have an equivalent role in nematode locomotion. The excitation of somatic muscles is provided by acetylcholine (ACh), which leads to contraction. Cholinergic transmission occurs both between neurons and at the neuromuscular junction. Furthermore, the contraction of the ventral wall causes the dorsal wall to relax on the other side of the body. The relaxation of the somatic muscles takes place through GABA, and in this way the two neurotransmitters coordinate the movement of the nematode. Acetylcholine (ACh) is a neurotransmitter in the excitatory synapses, while GABA is the neurotransmitter in the inhibitory synapses between interneurons and motor neurons. These interneurons could be related evolutionarily to mammalian Renshaw cells, and are potentially a very attractive drug target .Ascaris suum, there are at least three types of nAChR located on the muscle, with distinguishable pharmacological and single-channel properties. Each of the three subtypes was named based on its preferred agonist, N (prefers nicotine and oxantel), L (prefers levamisole and pyrantel), and B (prefers bephenium) , tetrahydropyrimidines , quaternary/tertiary amines (bephenium and tribendimidine), pyridines (methyridine), and amino-acetonitrile derivatives . This group of drugs produces spastic paralysis of the nematode by selectively gating acetylcholine receptor ion-channels at the synapses of motor neurons and interneurons, or at somatic muscle cells. The result of opening of the sodium ion channel is depolarization, followed by a contraction . Pharmacphenium) .Another large group of antinematodal drugs are avermectins. Avermectins interact with the gamma aminobutyric acid (GABA) receptor in somatic muscle, and the glutamate-gated chloride channel, which exists only in the pharynx of nematodes. This interaction leads to increased permeability of the postsynaptic membrane to chloride ions, causing hyperpolarization of the membrane and muscle relaxation, or atonic paralysis and death of parasites .Carvacrol is a phenolic monoterpenoid, present as a secondary metabolite in many plant essential oils, such as thyme oil and oregano oil. Evidence of antinematodal effects of carvacrol has been published both in vivo and in vitro experiments. Our previous studies have shown that carvacrol is most likely a non-competitive antagonist of nAChRs in parasitic nematodes . FurtherA. suum. This study could indicate the possibility of using two or more antinematodal drugs with different mechanisms of action in order to neutralize the resistance of the parasite.We considered it important to examine the effect of antinematodal drugs, agonists of different types of nAChRs and carvacrol with GABA on the contractions of the parasitic nematode Ascaris suum were collected at the Ambar slaughterhouse in Surčin, Serbia and maintained in Locker solution (mM): NaCl 155, KCl 5, CaCl2 2, NaHCO3 1.5, and glucose 5, at a temperature of 32°C. The locker solution was changed twice a day. After collection, the worms were used for contractions assay for the next four days. Ascaris muscle flaps for contractions were prepared as previously described . Each flap  was monitored isometrically by attaching a force transducer in an experimental bath maintained at 37°C, containing 20 ml Ascaris Perienteric Fluid Ringer/APF-Ringer (mM): NaCl, 23; Na-acetate, 110; KCl, 24; CaCl2, 6; MgCl, 5; glucose, 11; HEPES, 5; pH 7.6, and bubbled with room air. After dissection, the preparations were allowed to equilibrate for 15 min under the initial tension of 0.5 g. Different concentrations of pyrantel, morantel, and bephenium were then added to the preparation, and the maximum contraction was observed before the washing and subsequent application of the next concentration of the drug. The responses for each concentration were expressed in grams (g), produced by each individual flap preparation. The effects of GABA (3 μM), and GABA + carvacrol 100 μM on control dose-response plots were determined. Contractions were monitored on a PC computer, using a BioSmart interface, and eLAB 44 software . Sigmoidal concentration-response curves for each agonist in the absence or presence of antagonist were described by the Hill equation.Adult females of Carvacrol, morantel, pyrantel, bephenium, and GABA were obtained from Sigma-Aldrich Co . Morantel, pyrantel, bephenium, and GABA were dissolved in the APF-Ringer, while carvacrol was dissolved in ethanol, with the final concentration of ethanol in the APF-Ringer of 0.1%v/v. When tested, 0.1% of ethanol did not alter the resting activity of preparations and did not alter the drug responses.50) is the concentration of the agonist (Xa) producing 50% of the maximum response and nH is the Hill coefficient (slope). Prism 6.0  was used to estimate the constants EC50 and nH, by non-linear regression for each preparation. We determined mean contraction responses to each concentration of ACh (control dose-response: CR[Ach]) and mean responses to each of the concentrations of ACh in the presence of GABA. One-way analysis of variance (ANOVA) was applied for the analysis of the differences between the EC50 value and the Rmax . Differences were considered significant when the p value was < 0.05. The statistical analysis was done using Graphpad Prism software, while all values are expressed as mean ± standard error (S.E.).In the studies presented here, sigmoid concentration dose–responses were described by the equation: % response = 1/1 + [EC50/Xa]nH, where the median effective concentration  of pyrantel has not changed in the presence of GABA 3 μM, but decreased significantly after incubation with combination of GABA and carvacrol , because it did not change in the presence of GABA. However, the combination of GABA and carvacrol significantly reduced the Rmax value from 2.2 to 0.89 g   . Moreovee effect . Figure morantel  and sigmmorantel .The applied concentration of GABA (3 μM) did not cause relaxation of the tested muscle strips.We believe that the concept of simultaneous use of compounds with multiple sites of antiparasitic action is able to prevent the development of resistance in parasites. We have shown in previous studies that carvacrol is most likely to act as a non-competitive inhibitor of nAChRs of nematodes . It is k50 ~ 10 µM) on levamisole receptor subtypes (L-subtype) of nematode nAChRs. Our results are in accordance with the presented data. In our study, the contractile EC50 of pyrantel for A. suum muscle flaps was 8.44 μM, and it increased more than threefold in the presence of GABA, without altering the Rmax. Such an inhibition is manifested by competitive antagonists, but GABA certainly is not a competitive antagonist of nAChRs. We previously obtained a similar result with ACh and hypothesized that this phenomenon occurs as a consequence of the binding of GABA, in low concentrations, to the GABA receptor in the synapses between inhibitory interneurons and motoneurons (synaptic effect). This process immediately activates the competitive antagonistic excitatory mechanism through excitatory interneurons . Furthermore, the addition of carvacrol led to a significant increase in bephenium EC50 but also a decreased the Rmax. This is obviously mostly non-competitive antagonism and can be explained by the fact that carvacrol binds to a different site on the receptor relative to the binding site of bephenium, and that the B-subtype of nAChRs are predominantly located postsynaptically.Bephenium is an anthelmintic compound used against human and dog hookworms and gastrointestinal parasitic nematodes in sheep . Bepheni50 of morantel, nor the Rmax value. There was no significant change in EC50 of morantel even after the incubation with the combination of GABA + carvacrol, but the Rmax was significantly reduced. Similar to bephenium, we can state that the inhibitory effect of GABA is almost absent, while the combination of GABA and carvacrol exhibits mostly non-competitive antagonism. As before, we could assume that the receptors to which morantel binds are predominantly localized postsynaptically, which causes the absence of activation of inhibitory interneurons, and consequent competitive response. Parascaris equorum expressed in Xenopus oocyte, are able to form a functional nAChR highly sensitive to pyrantel and morantel.The results obtained with morantel are especially interesting. Morantel also belongs to tetrahydropyrimidines, but it is still not clear which subtype of nAChRs it predominantly prefers. The incubation of the muscle strips with GABA did not significantly change the EC50 to the right, producing a dose-ratio (EC50control/EC50GABA) of 2.39, while the Rmax was reduced by 30%. Now, after the incubation with 3 μM of GABA the dose ratios were 3.33 for pyrantel, 1.54 for bephenium, and 1.60 for morantel. On the other hand, Rmax for pyrantel, bephenium, and morantel was reduced by 9, 0.1, and 14.6%, respectively. It is obvious that GABA most effectively inhibits the effect of pyrantel and that the majority of this effect is competitive. We assume that this is the result of the synaptic effect in the synapses between inhibitory interneurons and motoneurons. However, when GABA was combined with carvacrol the dose-ratio (EC50control/EC50GABA + carvacrol) was 6.93, 2.25, and 2.13 for pyrantel, bephenium, and morantel, respectively, while Rmax decreased by 70, 60, and 65%, respectively. Their effect is more dominantly non-competitive, but also indicates that the combined use of GABA receptor agonists and nicotinic receptor antagonists can effectively inhibit the neuromuscular system of nematodes, even when one of the nicotinic receptor subtypes is dysfunctional due to the development of resistance. This is useful information for the formulation of commercial drugs that could prevent and overcome resistance.In our previous study , GABA 3


Children’s Hospitals’ Solutions for Patient Safety (SPS) is a network of over 140 children’s hospitals who share the vision of working together to eliminate serious harm across all pediatric hospitals. The SPS network is built on the fundamental belief that by sharing successes and failures transparently and learning from one another, children’s hospitals can achieve their goals more effectively and quickly than working alone. Each year, SPS hosts National Learning Sessions to which members are invited to submit abstracts describing relevant safety research or improvement work. The following abstracts were among the top submitted for the SPS Spring 2021 National Learning Session. Higher volumes of patients with behavioral crises admitted to medical units have led to patient and staff injuries. Staff felt ill-equipped to predict and respond to behaviors to support patients and ensure safety for all involved.To implement a standardized, multidisciplinary team response to behavioral escalation on the medical inpatient units.A multidisciplinary committee performed a Failure Modes & Effects Analysis, which yielded improvement opportunities, notably the need for a team to prevent and respond to crises. Key drivers related to development of a Behavior Response Team (BRT) included: identification of roles-responsibilities, behavior de-escalation, BRT members trained in de-escalation techniques collaborating with the bedside team and medical providers, and notification process development. Two pathways were designed: (1) BRT Huddle for early signs of escalation to develop a behavioral/safety plan and (2) BRT STAT for urgent response to patients at immediate risk of harm to self or others. The BRT leader trained in de-escalation techniques utilizes a scripted process to facilitate the response and post-event debriefing.The BRT was piloted on one medical unit and subsequently implemented on 14 inpatient units and the pediatric intensive care unit. BRTs have been utilized with frequency across the system since implementation. In the 7 months since implementation, 91 BRTs have been called, with 3 non-OSHA injuries. During this same time, 3 OSHA-recordable injuries occurred due to aggressive interactions, none during a BRT event. It has been 151 days since our last OSHA-recordable event (Fig. BRT implementation streamlined response efficiency and increased staff support during complex behavioral crises. Next steps include spread to other noninpatient areas.


Inhibition of ERK1 also downregulated AKT phosphorylation. To test if DEPTOR phosphorylation mediated this crosstalk, MM cells were transfected with WT or phosphomimetic DEPTOR and exposed to ERK inhibitors. Although WT DEPTOR had no effect on the inhibition of AKT phosphorylation, the phosphomimetic DEPTOR prevented inhibition. These results indicate that ERK1 maintains AKT activity in MM cells via phosphorylation of DEPTOR. We propose that DEPTOR-dependent crosstalk provides MM cells with a viability-promoting signal (through AKT) when proliferation is stimulated (through ERK).DEPTOR is a 48 kDa protein upregulated in multiple myeloma (MM) cells. DEPTOR inhibits mTOR and, by repressing a negative feedback loop, promotes AKT activation. We previously identified a compound that binds to DEPTOR in MM cells and induces its proteasomal degradation. To identify the mechanism of degradation, here, we screened for drug-induced posttranslational modifications and identified reduced phosphorylation of DEPTOR on serine 235 (S235). We show that an S235 phosphomimetic DEPTOR mutant was resistant to degradation, confirming the importance of this posttranslational modification. In addition, a DEPTOR mutant with a serine-to-alanine substitution at S235 could only be expressed upon concurrent proteasome inhibition. Thus, S235 phosphorylation regulates DEPTOR stability. Screening the DEPTOR interactome identified that the association of USP-7 deubiquitinase with DEPTOR was dependent upon S235 phosphorylation. Inhibition of USP-7 activity resulted in DEPTOR polyubiquitination and degradation. A scansite search suggested that ERK1 may be responsible for S235 phosphorylation, which was confirmed through the use of inhibitors, ERK1 knockdown, and an  By repressing a negative feedback loop, DEPTOR-induced inhibition of TORC1 results in activation of the PI3K–AKT (a serine/threonine-specific protein kinase) pathway  domain–containing mechanistic target of rapamycin (mTOR)–interacting protein (DEPTOR) is a 48 kDa protein that binds to the mTOR and downregulates its kinase activity within TOR kinase complex 1 (TORC1) and TOR kinase complex 2 (via phosphorylated residues in a conserved degron sequence between amino acids 286 and 291. Phosphorylation events seem to be mediated by a synergistic interaction between the mTOR kinase and casein kinase 1α (CK1α), although there may also be a role for the S6 kinase 1, downstream of mTOR, and the ribosomal protein S6 kinase 1  and v-maf avian MAF oncogene homolog B . Furtherkinase 1 . These dIn previous work , 8, 9, wThe aforementioned data indicated a novel mechanism of DEPTOR degradation, which is the subject of this report. Our results show that drug 3g induces a dephosphorylation of DEPTOR on serine 235 (S235), which results in release of the ubiquitin-specific protease 7 (USP-7) deubiquitinase. Polyubiquitination ensues with proteasomal degradation. The critical phosphorylation state of S235 is mediated by the extracellular signal–regulated kinase 1 (ERK1). Furthermore, our results suggest that the level of S235 phosphorylation is also a physiologic regulator of DEPTOR turnover in the absence of drug 3g and that DEPTOR S235 phosphorylation plays a key role in the crosstalk between ERK and AKT in MM cells.A). Gao et al. . S235 is located between the C-terminal DEP domain and the degron . Spectra of S235 phosphorylation specifically are shown in The 8226 MM cell line contains high levels of DEPTOR because of MAF dysregulation , 7 and iFig. 1o et al.  reportedo et al. , demonsto et al. , and thuC), and simultaneous blocking of proteasome function with MG-132 allowed normal S–A protein expression (D). Thus, the absence of S235 phosphorylation destabilizes DEPTOR protein resulting in proteasomal degradation. As expected, when the S–A mutant was protected by MG-132, exposure to drug 3g had no effect on expression (A). To test if decreased phosphorylation at S235 mediates drug-induced DEPTOR degradation, 8226 cells were transfected with either tagged WT or the phosphomimetic S–Asp mutant and treated in the presence or the absence of drug 3g for 3, 6, or 12 h. Drug 3g (1 μM) induced WT DEPTOR degradation rapidly by 3 h (E). This rapid kinetics is comparable to drug-induced degradation of endogenous DEPTOR in sensitive MM cells (E). These results confirm the relevance of S235 dephosphorylation in drug-induced DEPTOR fragmentation. In addition, the results with the S–A mutant shown in Figure 1D also indicate S235 phosphorylation physiologically regulates DEPTOR stability in the absence of drug 3g. There was only a slight prolongation of the T1/2 of the aspartate mutant versus WT DEPTOR in the absence of drug 3g (B). This was probably explained by the finding that, in the 8226 cell line, WT DEPTOR is constitutively phosphorylated at S235 (see later) so there is little difference in T1/2versus the phosphomimetic mutant.To test the relevance of diminished S235 phosphorylation, we generated DEPTOR mutants that would resist S235 phosphorylation  or present a phosphomimetic signal (serine-to-aspartate [S–Asp]) at residue 235. FLAG-tagged constructs were transfected into MM cells, and immunoblot was performed for tagged DEPTOR expression. Although both mutants were normally transcribed, there was no protein expression of the S–A mutant identified C, and sipression D. Thus, pression A. To tesy by 3 h E. This rMM cells . In contMM cells E. These  drug 3g B. This wi.e., no drug 3g) with those identified after drug 3g treatment yielded a dataset of 301 proteins. From these 301 separate proteins, the DEPTOR binding of 49 proteins significantly decreased  + MG-132 and compared with cells treated with MG-132 alone. Nonspecific binders were identified and discarded by use of IgG instead of anti-DEPTOR immunoprecipitating antibody. In the absence of drug 3g, 242 distinct binding proteins were identified by MS. Following exposure to drug 3g, there were 238 distinct DEPTOR binders identified. Combining the proteins bound to DEPTOR in the basal condition . This inhibitor has shown promise as an anti-MM drug, and one of its proposed mechanisms is that USP-7 paralysis results in an enhanced ubiquitination and fragmentation of human and/or murine double minute-2 protein (HDM2) with subsequent p53 activation, which is thought to mediate MM cell death . Also, targeting USP-7 resulted in loss of DEPTOR expression in both MM cell lines . This was associated with enhanced ubiquitination of DEPTOR (B). Comparable results were seen when USP-7 was targeted genetically by two separate shRNAs in 8226 or OPM-2 cells (C and D). As shown, the silencing of USP-7 was associated with decreased expression of HDM2 and DEPTOR (C) in both MM cell lines. A second independent shRNA was used in 8226 cells (D) to further support the negative effect on DEPTOR expression when USP-7 is silenced. In addition, Figure 3D demonstrates that this negative effect is reversed by MG-132. Thus, targeting USP-7 pharmacologically and genetically results in DEPTOR ubiquitination and degradation and further supports the notion that loss of DEPTOR–USP-7 binding is the key molecular alteration that mediates drug 3g-induced DEPTOR degradation.To test if USP-7 binding regulated DEPTOR stability, we first utilized a novel USP-7 inhibitor, P5091 A. This ill death . We, thu 12.5 μM . At our f DEPTOR B. Compar-2 cells , C and Dd DEPTOR C in both26 cells D to furtversus a FLAG-tagged empty vector [EV]) followed by challenge with P5091. Results shown in Figure 3E suggest that DEPTOR overexpression affords some protection against P5091, specifically identified at the 12 μM dose. Although P5091, by inhibiting USP-7, would be expected to induce some degradation of the S–Asp–transfected DEPTOR protein, there may have been a sufficient level still remaining to protect against cytotoxicity. An immunoblot assay (F) performed after 48 h exposure to 12 μM of P5091 suggests this possible explanation. As shown, the P5091 USP-7 inhibitor induced degradation of the FLAG-tagged DEPTOR while, as expected, having no effect on expression of the FLAG-tagged EV. However, following exposure to P5091, there was still some DEPTOR expression remaining in DEPTOR-transfected cells compared with EV-transfected cells (the latter cells demonstrating complete loss of expression of endogenous DEPTOR).As described previously, the P5091 USP-7 inhibitor is selectively cytotoxic to MM cells , possiblot assay F performA confirms the specificity of the antibody. WT or S–A mutant DEPTORs were expressed and immunoprecipitated. As shown, the WT DEPTOR was immunodetected with the antibody, whereas the S–A mutant, incapable of S235 phosphorylation, was not.A scansite search for kinase consensus sites identified ERK1 as a potential mediator of S235 phosphorylation. In addition, a previous study  used in the 00 of MG-132 (to prevent DEPTOR degradation) markedly decreased phosphorylation of S235 after a 4 h incubation in 8226 and OPM-2 MM cells. When the PD98059 ERK inhibitor was applied in the absence of MG-132, DEPTOR expression was lost (C). Similar results were obtained with the U0126 ERK inhibitor (D), further demonstrating that ERK inhibition phenocopies the effects of drug 3g on DEPTOR. In addition, silencing of ERK1 utilizing two separate shRNAs in either cell line in the presence of MG-132 also inhibited S235 phosphorylation (E). In the absence of MG-132, ERK1 knockdown also resulted in loss of DEPTOR expression . In addition, the PD98059 ERK inhibitor induced dephosphorylation of S235 and loss of DEPTOR expression in primary MM cells from patients . An in vitro kinase assay confirmed the ability of recombinant activated ERK1 to directly phosphorylate DEPTOR on S235 .To test the role of the ERK, we first used ERK inhibitors. As shown in Figure 4was lost C. Similanhibitor . The PD9nhibitor D, furtherylation E. In theA and B) also decreased S235 phosphorylation in a concentration-dependent fashion. A similar inhibition of DEPTOR S235 phosphorylation and DEPTOR protein expression was identified in an additional primary MM specimen (C). Notably, while preventing DEPTOR S235 phosphorylation, drug 3g had little effect on phosphorylation of ERK or the ERK1 substrate, ELK-1 (A–C). Thus, the inhibited ERK-induced phosphorylation of S235 in drug 3g-treated MM cells is not because of a general inhibition of the ERK mitogen-activated protein kinase cascade but, more likely, occurs secondary to a structural alteration of DEPTOR after drug binding.As expected, exposure of 8226 cells to drug 3g in the presence or the absence of MG-132 , A and Bspecimen C. Notable, ELK-1 , A–C. ThD (for MM1.S) and right side in Fig. 5D) demonstrates that, following reactivation with serum add-back, DEPTOR phosphorylation is reconstituted. Importantly, the rescued effects on ERK reactivation and DEPTOR phosphorylation with serum add-back are prevented by coincubation with the ERK inhibitor, PD98059. Similar results were seen with the OPM-2 cell line  gene, its ERK activation is not serum dependent. We, thus, performed these experiments in OPM-2 cells and an additional MM cell line with upregulated DEPTOR expression, MM1.S. Figure 5ell line . Thus, uA demonstrates this effect where drug 3g inhibited AKT phosphorylation in a concentration-dependent fashion in both 8226 and OPM-2 MM cells. We, thus, tested if ERK inhibitors similarly regulated AKT phosphorylation. Lysates from PD98059-treated MM cells  and U0126-treated cells , consistent with the expected effects of DEPTOR degradation on the negative feedback loop. In similar fashion, shRNA knockdown of ERK1 also inhibited AKT phosphorylation in association with DEPTOR loss . In these experiments, the targeting of DEPTOR by drug 3g or ERK inhibition concurrently enhanced phosphorylation of p70S6K and IRS-1 (A–C), supporting the notion that AKT downregulation was mediated by the negative feedback loop. To confirm that DEPTOR degradation mediated the negative effects of ERK inhibitors on AKT phosphorylation, 8226 MM cells were either transfected with EV, WT DEPTOR, or the phosphomimetic DEPTOR mutant. Transfected cells were then exposed to the PD98059 ERK inhibitor, and effects on AKT were assessed. As shown in Figure 6D, PD98059 inhibited AKT phosphorylation in MM cells transfected with an EV or WT DEPTOR, but AKT inhibition was prevented in cells transfected with the phosphomimetic (Asp) version of DEPTOR. Thus, the ERK–AKT crosstalk is mediated by ERK-induced effects on DEPTOR that are regulated by the phosphorylation status of S235. To test if this has ramifications for MM cell viability, MM cells, transfected as in Figure 6D, were challenged with the ERK inhibitor. As shown in Figure 6E, the cytotoxicity induced by ERK inhibition was prevented specifically by transfection of the phosphomimetic DEPTOR, which remains expressed even when ERK is inhibited. In contrast, MM cells transfected with WT DEPTOR were not protected.ERK activity in MM cells is crucial for tumor cell proliferation either induced by myeloma growth factors , 12 or mFigure 6Our investigation on the mechanism by which a small-molecule inhibitor induces DEPTOR degradation has uncovered a novel ERK1-dependent regulation of DEPTOR protein stability. Our results indicate that ERK1-induced phosphorylation of S235 on DEPTOR recruits and/or maintains an interaction with the USP-7 deubiquitinase. USP-7 protects DEPTOR against polyubiquitination and proteasomal degradation. When exposed to the anti-DEPTOR drug 3g, S235 becomes dephosphorylated, USP-7 is released, and polyubiquitination and degradation ensues. Several previous detailed studies , 5 eluciet al. , it is not surprising that ERK inhibition also phenocopies the other effects of drug 3g, namely loss of USP-7 binding and DEPTOR degradation. However, the interaction between DEPTOR and ERK may be complex. As mentioned previously, DEPTOR associates with ERK1/2 in pull-down assays . Furthermore, our results in Figure 6D, demonstrating that only the S235 phosphomimetic DEPTOR version prevented AKT downregulation during ERK targeting, underscores the critical role of S235 DEPTOR phosphorylation in the crosstalk. Indeed, the cytotoxic effect of an ERK inhibitor was significantly protected by ectopic expression of the phosphomimetic DEPTOR (E). Thus, myeloma-specific upregulation of DEPTOR expression provides a key step to maintaining cell viability during tumor clonal expansion.Previous work , 13, 14 c DEPTOR E. Thus, versus nonphosphorylated peptide when used at dilutions of 1:100 to 1:10,000. It was used in Western blot experiments with dilution at 1:1000. The mutant DEPTOR constructs were generated by cloning full-length complementary DNA of the WT human DEPTOR into plenti6 lentivirus vector as previously described. Mutants with its S235 residue replaced by an alanine or an aspartic acid residue, respectively, were created using Quick-change II Site-directed mutagenesis kit (Agilent Technologies). Lentiviral transduction of MM cells was as described . The anti-USP-7 drug, P5091, was obtained from Abcam. The ERK inhibitors U0126 and PD98059 were obtained from Millipore Sigma–Aldrich. The phosphospecific antibody recognizing phosphorylated S235 in DEPTOR was generated in rabbits immunized with the phosphorylated peptide Cys-Glu-Leu-Leu-Asn-Glu-Lys-(p-Ser)-Pro-Ser-Ser-Gln-Glu-NH2 (where pSer represents phosphoserine) and subsequently affinity purified. It was specific for phosphorylated peptide escribed .Bone marrow aspirate from newly diagnosed patients with MM was separated as previously described . IsolateBinding of endogenous DEPTOR or FLAG-tagged DEPTOR constructs to USP-7 was assayed by coimmunoprecipitation as previously described . Real-ti°C. Cell lysates were immunoprecipitated with FLAG antibody . Ubiquitination levels of FLAG-DEPTOR were measured with an antiubiquitin antibody used at 1:1000 dilution .FLAG-DEPTOR-transfected 8226 or OPM-2 MM cells were treated with drug 3g (2 μM) or P5091  at 37 °C overnight. Peptides were extracted from gel bands by 50 μl of 50% ACN/5% formic acid solution for three times, before being dried in speed vac.In-gel digestion was performed as follows: 100 μl of 100 mM ammonium bicarbonate/50% acetonitrile (ACN) was added to gel slices and incubated at 37 °C for 30 min to destain the gel slices. The destaining step was repeated twice until all stain was removed. Next, 50 μl of ACN was added to gel pieces and incubated for 15 min at room temperature to shrink the gel pieces. Gel pieces were dried under speed vac for 5 min, and 50 μl 0.01 mg/ml trypsin solution (dissolved in 100 mM ammonium bicarbonate) was added to gel pieces for digestion at 37 °C for 1 h and then alkylating them by adding 40 mM iodoacetamide at room temperature for 30 min in the dark. Two hundred microliter of cold acetone was then added to the protein solution and stored in −20 °C for 1 h before centrifugation at 13,000g for 10 min at 4 °C. The supernatant was then removed, and protein pellets were air dried for 10 min in room temperature. Trypsin dissolved in 50 mM ammonium bicarbonate was added to protein pellets for digestion at 37 °C overnight. Protein digests were desalted by Empore stage-tip the following day. Elution from the stage-tip was dried by speed vac and resuspended in 3% ACN with 0.1% formic acid.In-solution digestion was performed by first reducing proteins in solution (50 μl) by adding 5 mM Tris(2-carboxyethyl) phosphine at 56 2O with 0.1% formic acid; B = ACN with 0.1% formic acid) over 40 min and from 35% to 85% solvent B in 5 min at a flow rate of 300 nl/min. The nano-LC was coupled with a Q Exactive Plus orbitrap mass spectrometer (Thermo Fisher Scientific). The electrospray ionization voltage was set at 1.9 kV, and the capillary temperature was set at 275 °C. Full spectra (m/z 350–2000) were acquired in profile mode with resolution of 70,000 at m/z 200 with an automated gain control target of 3 × 106. The most abundance 15 ions were subjected to fragmentation by higher-energy collisional dissociation with normalized collisional energy of 25. MS/MS spectra were acquired in centroid mode with resolution of 17,500 at m/z 200. The automated gain control target for fragment ions are set at 2 × 104 with maximum injection time of 50 ms. Charge states 1, 7, 8, and unassigned were excluded from tandem MS experiments. Dynamic exclusion was set at 45.0 s.A total of 1.0 μg protein was injected to an ultimate 3000 nano–liquid chromatography (LC), which was equipped with a 75 μm × 2 cm trap column packed with C18 3 μm bulk resins , and a 75 μm × 15 cm analytical column with C18 2 μm resins . The nano-LC gradient was 3–35% solvent B  by Proteome Discoverer, version 1.4 (ThermoFisher Scientific) for binding protein identification and label-free quantification. UniProt human DEPTOR sequence  was used for PTM analysis. “Precursor ions area detector” node is used within Proteome Discoverer, version 1.4, for label-free quantification. Following parameters were set: precursor mass tolerance ±10 ppm, fragment mass tolerance ±0.02 Th for higher-energy collisional dissociation, up to two miscleavages by semitrypsin. Variable modification includes methionine oxidation, lysine methylation, and alkylation; serine, threonine, and tyrosine phosphorylation. Cysteine carbamidomethylation was set as fixed modification. False discovery rate (FDR) was at 1.0% calculated by Proteome Discoverer, version 1.4, percolator node, which searches Decoy database to determine FDR. Minimum of one peptide was required for protein identification.t test was performed to determine the p values.The phosphorylation percentage was calculated by normalizing the peak area of the phosphorylated peptide to the total peak area of the same peptide . The quantification information was determined based on two biological replicates, each of which was repeated twice as technical replicates. Student's p Values were calculated based on three biological replicates.Label-free quantification of DEPTOR-binding proteins was performed based on the average peak areas of top three peptides of each protein. Three biological replicates were used for average to determine the relative abundance of DEPTOR-binding proteins in control  and treated (3g + MG-132) samples. https://massive.ucsd.edu/ProteoSAFe/dataset.jsp?task=31938adc56e843aba65dd52304d25c74). Project ID number: MSV000088145. Search engine and release version: “Proteome Discoverer, version 1.4.” Release version/date of sequence database searched: DEPTOR sequence, version 2, released July 7, 2009, human database released, July 2, 2015. Number of entries in the database actually searched human database: number of entries is 23,292; DEPTOR database: number of entries is 1. How FDR was calculated: FDR was calculated by Proteome Discoverer, version 1.4, percolator node, which searches Decoy database to determine FDR.Raw MS data and search results were placed in the MassIVE public repository (This article contains The authors declare they have no conflicts of interest with the contents of this article.