Saccharomyces cerevisiae. As with the echinocandins, such as caspofungin, PA targets the synthesis of cell wall β-1,3-glucan and has potential utility in the treatment of medically important fungi. However, the antifungal activity of PA against human pathogenic Candida species has not been explored and the precise mode of action of this compound is not understood. Here, we show that PA sensitivity is regulated by the calcineurin pathway and that susceptibility to PA varied significantly between Candida species, but did not correlate with in vitro β-glucan synthase activity, cell wall β-glucan content or the sensitivity of the species to caspofungin. Strains with point mutations (S645Y or S645P) in the hotspot1 region of the β-1,3-glucan synthase subunit Fks1, had decreased sensitivity to caspofungin but increased sensitivity to PA. C. guilliermondii, C. orthopsilosis, and C. parapsilosis were more sensitive to PA than C. albicans, C. dubliniensis, C. tropicalis, and C. glabrata. These observations suggest that there are significant differences in the mode of action of PA and caspofungin and that PA or PA analogues are not likely to have broad spectrum activity in the treatment of Candida infections.The rise of widespread antifungal resistance fuels the need to explore new classes of inhibitory molecules as potential novel inhibitors. Recently a plant natural product poacic acid (PA) was shown to inhibit β-1,3-glucan synthesis, and to have antifungal activity against a range of plant pathogens and against  They ar strains . These m strains . Howeverin vitro . Paradoxynthesis . Further species , includiesistant . These hSaccharomyces cerevisiae, Alternaria solani and the oomycete Sclerotinia sclerotiorum in vitro. Lesion development on soybean leaves by S. sclerotiorum was also inhibited by PA ex vivo. PA apparently acts by directly binding to β-1,3-glucan polysaccharide, and therefore differs from the mode of action of echinocandins which directly target the β-1,3-glucan synthase enzyme. In this study we contrast the modes of action of two β-glucan synthesis inhibitors, poacic acid (PA) and caspofungin and show that they have distinct mode of actions and different host range activities. We determined the activity of PA against a range of clinically important fungal pathogens and demonstrate that PA is differentially active against a range of Candida species and examine the relationship between species-specificity, β-1,3-glucan synthesis and its sensitivity to PA. Our findings demonstrate that PA and echinocandins inhibit β-1,3-glucan synthesis in different ways and that PA-based pharmacophores may not be suitable as pan-Candida inhibitors.Poacic acid , PA, is a natural plant metabolite found in the lignocellulosic hydrolysates of grasses and has been characterised as a promising antifungal agent that targets β-1,3-glucan synthesis . This coC. albicans and S. cerevisiae to PA. First we investigated the antifungal activity of PA against the C. albicans reference strain SC5314, and S. cerevisiae S288C (shown previously to be sensitive to PA) as well as the echinocandin resistant C. albicans NR3 strain. As shown in S. cerevisiae was 5- to 10-fold more sensitive  to PA than both C. albicans strains, NR3 (IC50 = 515 µg/ml) and SC5314 (IC50 > 1000 µg/ml). The measured sensitivity against S. cerevisiae corresponds with that cited previously  was determined for β-1,3-glucan synthases isolated from the total membrane fractions in SC5314, NR3, and S288C. The glucan synthase activity of all three stains was found to be inhibited to a similar extent by PA  and S288C (IC50GS = 206 μg/ml) suggesting that this hotspot mutation that confers echinocandin resistance is irrelevant for PA sensitivity. Therefore, in in vitro assays, PA inhibited C. albicans and S. cerevisiae β-1,3-glucan synthases to the same degree suggesting that the differential sensitivity of the C. albicans and S. cerevisiae is not due to differences in the direct action of PA on Fks1 activity.Kinetic analyses revealed a correlation between the inhibition of β-1,3-glucan synthase enzyme activity and echinocandin MIC . In addiecreased . Therefont by PA E. The PACandida infections. Piotrowski et al. demonstrated that PA and CSF acted synergistically against S. cerevisiae .Combinatorial synergy between PA and cell wall synthesis inhibitors. PA could potentially be useful as part of a combinatorial therapy against revisiae . Therefoor alone A. Notice>8 μg/ml A. This pae S288C B. HoweveS. cerevisiae  (a competitive inhibitor of chitin synthase) and CFW  . Similarrevisiae , we obsealbicans C. Howeven growth C. These C. albicans wild type cell wall. In fungi, β-glucan damage often leads to the induction of chitin synthesis  and inner (glucan + chitin) layers of S. cerevisiae and C. albicans cell walls after PA treatment      , 2) mutand CHS8) , 3) mannnd MNS1) , and 4) nd PGA5) . PA sensty to PA A. The ICes to PA . Therefofks1-S645S/P (a heterozygous mutant) and fks1-S645P (a homozygous mutation at 645 serine to proline), were generated using CRISPR-CAS9 to introduce this point mutation . A heterozygous FKS1/fks1Δ null mutant was compared displaying marginally reduced sensitivity to PA and increased sensitivity to CSF  and four strains of S. cerevisiae  , whereas M476/93/6, SCS192139P, AM2005/0181, and AM2005/0052 were somewhat less sensitive to PA (IC50 = 616 to > 1000 μg/ml). This species is also variable in its sensitivity to CSF  . The S. albicans . Comparey to CSF . InteresCandida species. PA has been reported to bind directly to β-1,3-glucan  did not impact cell growth of either C. albicans and S. cerevisiae (data not shown). In C. albicans the PA sensitivity was hardly affected by exogenous β-1,3-glucan at 0.25 mg/ml and 0.5 mg/ml  and S. cerevisiae S288C were comparable, but the IC50 values were 111 μg/ml and > 1000 μg/ml, respectively. Therefore, the PA sensitivity did not correlate simply with the basal β-1,3-glucan content of the cell wall of different yeast species.Next we hypothesized that varied basal levels of glucan in different  by FACS B. C. albC. albicans Fks1 were highly conserved in C. dubliniensis and C. tropicalis Fks1 but different in S. cerevisiae Fks1 and C. glabrata Fks1   . HoweverCandida. Despite of the demonstrated potency of PA against S. cerevisiae  by the Rho1-dependent β-1,3-glucan synthases Fks1, Fks2 and Gsc1 using UDP-glucose as the substrate  (We showed that point mutations in s (TMHs) . The cens (TMHs) . Computa2+/calcineurin pathways  and PTS1 (encoding a protein phosphatase) resulted in increased resistance to PA. Furthermore, S. cerevisiae Sur1 and Csg2, which are involved in mannosylinositol phosphorylceramide synthesis, activate the PKC pathway and are resistant to PA when the cognate genes are deleted. It is however possible that PA treatment could stimulate cell wall remodelling by via PKC/Mkc1 independent mechanisms . For overnight cultures, single colonies from the agar plates were inoculated in either YPD or NGY broth , and incubated at 30 °C with shaking at 200 rpm. Uridine (25 μg/ml) was supplemented in the medium for uridine auxotrophic mutants. CSF was obtained from the Aberdeen Royal Infirmary. Echinocandin B was generous gift from Osamu Kondo .The fungal strains used in this study are listed in Candida species and S. cerevisiae strains was determined according to CLSI M27-A3 guidelines in using modified RPMI-1640 , pH7.2) as described previously  was dissolved/gelatinised in DMSO, and used in some experiments in which β-glucan was provided exogenously. After 24 h incubation in microtitre plates, the optical density of wells was measured using a VERSAmax plate reader  at 405 nm. For the post-sensitivity assays of cell viability, 2.5 μl samples were taken from the sensitivity assay microtitre plates and were spotted on a fresh YPD and incubated at 30 °C overnight. The inhibitory concentration of MIC plates at 50% cell survival (IC50) or a 10% cell survival (IC90) was calculated from LOWESS curves using GraphPad Prims 5 (v5.04).The antifungal susceptibility of PA against various eviously . BrieflyE = A × B/C  is applied alone; B is the percentage growth when compound B (CSF) is applied alone; C is the percentage growth DMSO control. Sub-MIC or IC50 concentrations of PA or caspofungin were used for analysis of synergistic effects. Combinations of 1000, 500 and 62.5 µg/ml PA and 0.062, 2, and 0.25 µg/ml CSF for SC5314, NR3 and S288C respectively were used. A combination of 500 µg/ml PA and 25 µg/ml CFW or 40 µM NKZ were used for SC5314.Combinatorial inhibition assays were performed to investigate synergistic effect of PA with echinocandins. Growth conditions were identical to that used in antifungal susceptibility tests described above. A 12 × 8- or 8 × 8-dose-defined matrix was used in 96-well plates containing PA (0.98 to 1000 μg/ml) in combination with either CSF (0.032–16 μg/ml), NKZ (0.625–40 μM), or CFW (0.2–200 μg/ml). After incubating for 24 h at 37 °C, the final OD at 405 nm was determined. Results are presented as a heatmaps or a bar charts of the percentage of growth inhibition observed. Synergistic effects were calculated as described  using thCells treated with either PA or CSF were fixed with 10% neutral buffered formalin and stained with 25 µg/ml CFW to visualise cell wall chitin. Samples were observed by differential interference contrast (DIC) microscopy as described in the previous study  using a β-1,3-glucan was stained with Aniline Blue . Mannoprotein was stained with Alexa594-ConA . β-1,3-glucan and mannoprotein were stained as described previously  with sli6 cells/ml, 0.5 mM EDTA was added, and samples were sonicated in a water bath for up to 10 min to disrupt cell clumps. Aniline Blue (25 μg/ml) was added immediately before FACS analysis. Unstained controls were used to establish the basal level of fluorescence. A total of 10,000 cells per sample were analysed by FACS according to BD Fontesa manufacturer’s guidelines (BD Biosciences). Data obtained by the BD FACSDiva software were transferred and analysed by FlowJo (v10.02) to semi-quantify the fluorescence intensity representing β-glucan contents.For fluorescence-activated cell sorting, applied in flow cytometry (FACS) analysis, cells were first grown in YPD at 30 °C for 6 h. Cells were washed twice with PBS, and fixed in 3.7% formaldehyde. Fixed cells were diluted in PBS to reach 1 × 107 cells/ml. The following procedures were carried out at 4 °C. Cells were harvested, washed with 1 mM EDTA, and disrupted by vortexing 4 times for 2 min each with 5 ml of glass beads in 10 ml of breaking solution containing 0.5 M NaCl, 1 mM EDTA with 1 mM phenylmethylsulfonyl fluoride. After centrifugation at 1,200 × g for 5 min, the supernatant was collected and transferred to 33 PC tubes . Pellets were resuspended in breaking solution, and centrifuged at 1,200 × g for 1 min. The supernatant was also transferred to new 33 PC tubes. Membrane fractions in 33 PC tubes were collected by centrifugation at 100,000×g for 30 min in an RP70T-203 rotor (Hitachi) with Himac CP 65β (Hitachi). The resultant microsomal membrane pellet was suspended in membrane buffer containing 50 mM Tris-HCl, pH 7.5, 10 mM EDTA, 1 mM β-mercaptoethanol, and 33% glycerol, homogenized, and stored at − 80 °C to obtain membrane mixture.To prepare membrane microsomal fractions, cells were grown at 30 °C to a density of 2–4 × 1014C]; NEC403; PerkinElmer). Brij-35 was used for membrane solubilisation was included in the reaction solutions for assays of C. albicans β-1,3-glucan synthase activity (GS) was calculated after fitting with Gamlss beta in the R Gamlss package.β-1,3-glucan synthase activity was measured as described previously  with sliactivity . MembranC. albicans SC5314 and S. cerevisiae S288C were grown in YPD at 30 °C overnight. The following day, overnight cultures were diluted in a fresh YPD medium to an OD600 of 0.2 and incubated at 30 °C for 1 h. Then cells were treated for further 6 h with DMSO (0.5% for SC5314 or 0.1% for S288C) or PA (500 μg/ml for SC5314 or 100 μg/ml for S288C). After incubation, cells were collected and washed with mQ-water. Cells were freeze-substituted by using a Leica EM AFS2 automatic freeze substitution system (Leica Micro-systems). Further steps were carried out according to the previous study , and S. cerevisiae were diluted in fresh YPD and then incubated for 4 h at 30 °C with shaking until they reached mid-log phase. Cells were then treated for 10 min with appropriate treatments, with equivalent non-treated cells used as a control. After treatments, cells were collected, and their total protein complement was extracted.Western analysis was performed using methods described previously with modifications . OvernigProteins (15 µg) were separated by SDS–PAGE (polyacrylamide gel) electrophoresis; NuPAGE®Novex Bis-Tris 4–12% Precast gels (Invitrogen). Blotting was performed according to the manufacturer’s instructions. A mix of the SeeBlue® Plus2 Pre-stained (Invitrogen) and MagicMarkTM standards (Invitrogen) were loaded as markers. After blotting, separated proteins were transferred onto PVDF membranes which were rinsed with PBS and blocked in PBS-T 10% BSA  for 30 min at RT. Membranes were then incubated overnight at 4 °C with Phospho-p44/42 MAP Kinase (Thr202/Tyr204) Antibody  in PBS-T 5% BSA . For secondary antibody staining, the membranes were incubated at RT for 60 min in PBS-T 5% BSA containing an anti-rabbit IgG, HRP-linked Antibody . Signals were enhanced by SuperSignal® West Femto Maximun Sensitivity Substrate (Thermo Scientific) according to the manufacturer’s instructions. Chemiluminescence signal detection was performed using the Fusion image acquisition system v15.15 .C. albicans, using the CRISPR-Cas9 system according to a previous study with modifications  for the Cas9 system was digested with KpnI and SacI. The repair template was created using oligos of FKS1rt_Fwd and S645P_Rev. Repair templates (10 μg) were purified and transformed along with linearized pV1200_FKS1sgRNA into C. albicans SC5314. The repair templates contained the desired point mutation (serine to proline at 645), and a silencing mutation at 635  to prevent the Cas9 system repeatedly cutting the NGG site. A positive colony was selected by plating on an YPD agar 200 μg/ml nourseothricin, and then grown on YPD agar containing 0.5 μg/ml caspofungin. All positive transformations were confirmed by the DNA sequencing analysis as previously described .Amino acid substitutions in Fks1 (orf19.2929/C1_02420C) were introduced in ications . A singlications . The sinescribed . To creaCandida genome database (http://www.candidagenome.org), the Saccharomyces genome database (http://www.yeastgenome.org) or from the NCBI (http://www.ncbi.nlm.nih.gov)  using S. cerevisiae Fks1 as the comparator sequence. The phylogenetic tree of Fks1 was created after MUSCLE analysis, and then re-generated as an unrooted phylogenetic tree, using TreeDyn 198.3 (http://www.phylogeny.fr/). The transmembrane topology prediction of homologue Fks1 was performed via the web-tool, Phobius (http://phobius.sbc.su.se/index.html).The amino acid sequences of Fks1 or homologues in all reference strains was obtained from the nih.gov) . The mulThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


The emergence in recent years of DNA editing technologies—Zinc finger nucleases (ZFNs), transcription activator-like effector (TALE) guided nucleases (TALENs), clustered regularly interspaced short palindromic repeats (CRISPR)/Cas family enzymes, and Base-Editors—have greatly increased our ability to generate hundreds of edited cells carrying an array of alleles, including single-nucleotide substitutions. However, the infrequency of homology-dependent repair (HDR) in generating these substitutions in general requires the screening of large numbers of edited cells to isolate the sequence change of interest. Here we present a high-throughput method for the amplification and barcoding of edited loci in a 96-well plate format. After barcoding, plates are indexed as pools which permits multiplexed sequencing of hundreds of clones simultaneously. This protocol works at high success rate with more than 94% of clones successfully genotyped following analysis. The past two decades have seen the explosion of available genomic editing tools for eukaryotic systems including Zinc finger nucleases (ZFNs) , transcrAlthough methods for both editing cells and isolating clones of edited cells have become much more accessible, techniques for high-throughput screening of large numbers of clones are still required. These techniques are particularly important for projects where exact nucleotide changes are required as HDR occurs much less frequently than NHEJ, requiring larger numbers of clones to be screened to isolate correctly repaired loci ,12,16. WConventional screening approaches use Sanger sequencing , which iThis protocol describes the genotyping of one or more loci of interest using next-generation sequencing. The protocol requires cells to have previously been edited, single-cell sorted and clonally expanded following a cell-type specific protocol Dimethyl sulphoxide, DMSO 96-well V-bottomed plates Parafilm 96-well PCR plate Tris, 1 M, pH 8.0 Ethylenediaminetetraacetic acid (EDTA), 0.5 mM, pH 8.0 Tween 20, 50% PCR grade water DNA low bind tubes Proteinase K Platinum PCR master mix Locus specific primers, 10 µM mix, see Agarose 10× Tris acetate-EDTA, TAE 100 bp ladder Escherichia coli Exonuclease I, Shrimp alkaline phospatase, rSAP Custom iR5 and iC7 barcoded primers, Agencourt Ampure XP SPRI Beads 100% Ethanol Qubit BR DNA assay kit NEB Ultra II PCR Tube NEB Next Multiplex Oligos for Illumina primer set 1 NEB Next Multiplex Oligos for Illumina primer set 2 Herculase II Fusion polymerase kit Tris-EDTA, TE D1000 reagents D1000 loading tips D1000 screen tape KAPA Library Quantification Complete Kit MiSeq Reagent Nano Kit, v2 500-Cycles PhiX Control v3 8- or 12-channel pipette Centrifuge with buckets for plates Thermocycler with 96-well plate capacity Electrophoresis gel tank and power packMagnetic rack Minifuge Qubit fluorometer Agilent 2200 TapeStation Real-time quantitative polymerase chain reaction (qPCR) thermocycler MiSeq Microcentrifuge The first PCR step of this protocol requires amplification of the edited locus using custom primers . PrimersAs the addition of the m13 linkers may alter the binding specificity of the primer, at least two primer pairs are ordered and tested on genomic DNA . The PCRLocus specific PCR products are barcoded with one of eight forward primers (iR5) and one of twelve reverse primers (iC7) in all 96 permutations to uniquely identify each well . These pThe custom plateScreen96 scripts are available on GitHub , with adHere we provide instructions for splitting non-adherent cells into two plates, one for freezing and storage (stock plate) and the other for lysis (genotype plate), amplification and indexing producing a next-generation sequence ready library . Cells s1.Using the appropriate growth media, clonally expand single-cell sorted colonies, splitting them as necessary until they occupy two to four wells of a 96-well plate at a high level of confluence (80–90%).2.Reduce media volume to <100 µL per well and mix by pipetting.3.Prepare two new 96-well V-bottomed plates (one for stock storage and one for genotyping) by combining two wells of highly confluent cells. Note: If cells have only grown to occupy two wells of a flat-bottomed 96-well plate, transfer a single well to each V-bottomed plate.CRITICAL STEP Clones should occupy identical wells in both the stock and genotyping plates.4.g, 5 min, room temperature).Set aside the genotyping plate and pellet the cells in the stock plate by centrifugation (250× 5.v/v) per stock plate.During this spin step, prepare 5 mL of sterile freezing media .Pellet cells in the genotyping plate from step 4 in 11.Using an 8- or 12-channel pipette remove supernatant and resuspend in 30 µL lysis buffer.12.Seal plate and incubate at 37 °C overnight.13.On the following day, heat to 95 °C for 10 min in thermocycler to deactivate proteinase K.14.Cool to 4 °CPAUSE STEP Lysed cells can be stored at −20 °C for up to one month or at −80 °C for one year.15.On ice, prepare 1150 µL of Locus PCR master mix per lysed plate by combining 1125 µL Platinum master mix and 25 µL of 10 µM locus primer mix .16.Aliquot 11.5 µL of Locus PCR master mix into each well of a 96-well PCR plate on ice.17.On ice, add 1 µL of cell lysate to the corresponding well of the PCR plate and mix by pipetting.18.Place in thermocycler and amplify using the Platinum PCR cycling settings in 19.Transfer 1 µL of PCR products to the corresponding well of a new 96-well PCR plate.20.Prepare 220 µL PCR clean-up master mix per plate by combining 16.5 µL exonuclease I, 16.5 µL shrimp alkaline phosphatase (SAP) and 187 µL PCR grade water.21.Add 2 µL of PCR clean-up master mix to each well, mix by pipetting.22.Incubate in a thermocycler at 37 °C for 30 min, 85 °C for 15 min, and then cool to 4 °C.PAUSE STEP After stopping the reaction, the mix can be stored at 4 °C overnight.23.To each well of cleaned-up PCR product add 11.5 µL Platinum master mix (perform on ice).24.Prepare a stock 96-well plate of barcoding primers by combining all unique pairs of row and column primers at 5 µM each by adding equal volumes (2–5 µL) of each iC7 and iR5 primer at 10 µM. This primer plate may be stored at −20 °C and used multiple times.25.Using the barcoding primers prepared in step 24 and a multichannel pipette add 0.5 µL of primers to the appropriate wells of the genotyping plate.26.Place in a thermocycler and amplify using the Platinum PCR cycling settings in 27.During amplification bring an aliquot of 875 µL Ampure XP beads to room temp in an Eppendorf tube.28.Pool 5 µL from each well of a single plate into a single 1.5 mL DNA low bind tube . Store excess PCR reaction at −20 °C.29.Add 864 µL Ampure XP beads (1.8 volumes) to the pooled PCR products. Mix by pipetting.30.Incubate at room temp for 5 min.31.During incubation prepare 80% ethanol .32.Place on magnetic stand. After the liquid has cleared ~5 min), remove and discard the liquid without disturbing the beads.33.Add 700 µL of fresh 80% ethanol again without disturbing beads. Incubate 30 s.34.Remove the ethanol and the repeat wash with another 700 µL of fresh 80% ethanol.35.Discard the ethanol, spin briefly on a bench-top centrifuge and replace on magnetic stand.36.Discard the residual ethanol and allow to air dry until beads appear matte in appearance ~5 min).CRITICAL STEP Do not over dry the beads as this will reduce yield. Beads should appear like damp mud, neither glossy wet nor dry. Cracks in the bead pellet are indicative of over-drying.37.Remove tube from magnet and resuspend beads in 55 µL of PCR grade water by pipetting 10 times.38.Incubate at room temp for 2 min.39.Replace on magnetic stand.40.Once clear (~4 min) recover 53 µL of eluted PCR product and transfer to a new 1.5 mL DNA low-bind tube.41.Use 2 µL of eluted PCR to quantify the DNA concentration using a Qubit BR DNA kit.42.Use 1 µL of eluted PCR to evaluate the product size on a D1000 TapeStation gel.PAUSE STEP After purification the products can be stored at 4 °C overnight or at −20 °C for several months.These steps follow the NEB Ultra II protocol with minor modifications.43.Combine 50 µL of DNA (0.5–2 µg), 7 µL 10× End Prep Buffer and 3 µL End Prep Enzyme. Mix by pipetting and incubate at 20 °C for 30 min in a thermocycler (lid open or un-heated).44.Increase temperature to 65 °C and incubate for 30 min (lid heated to 75 °C).45.Add 30 µL Ultra II Ligation Master Mix, 2.5 µL NEBNext Adaptor, 1 µL Ligation enhancer. Mix by pipetting and incubate at 20 °C for 15 min.46.TM enzyme. Mix by pipetting and incubate at 37 °C for 15 min.Add 3 µL USER47.TM enzyme reaction warm 90 µL of Ampure XP beads to room temp.During this USER48.Add 87 µL Ampure XP beads (0.9×) and mix by pipetting. Incubate at room temp for 5 min.49.During incubation prepare 80% ethanol .50.Place on magnetic stand. Without disturbing beads, discard liquid when clear (~5 min).51.Add 500 µL of fresh 80% ethanol without disturbing beads. Incubate 30 s.52.Remove ethanol and repeat wash with another 500 µL of fresh 80% ethanol.53.Discard ethanol, spin briefly on a bench-top centrifuge and replace on magnetic stand.54.Discard residual ethanol and allow to air dry until beads are matte in appearance (~5 min).CRITICAL STEP Do not over dry the beads as this will reduce yield. Beads should appear like damp mud, neither glossy wet nor dry. Cracks in the bead pellet are indicative of over-drying.55.Remove tube from magnet and resuspend beads in 30.5 µL of PCR grade water by pipetting 10 times.56.Incubate at room temperature for 2 min.57.Replace on magnetic stand.58.Once clear ~4 min) recover 28.5 µL of eluted PCR product and transfer to a new PCR tube.59.-5 µL NEB Universal Primer-5 µL NEB Index Primer-10 µL Herculase II 5× Buffer-0.5 µL deoxynucleotide triphosphate (dNTP)-1 µL Herculase II PolymeraseTo the adaptor ligated library add:60.Mix by pipetting and amplify using the Herculase PCR cycling settings in 61.During the PCR warm 40 µL of Ampure XP beads to room temp.62.Add 40 µL Ampure XP beads (0.8×) to the PCR and mix by pipetting. Incubate at room temperature for 5 min.63.During incubation prepare 80% ethanol .64.Place on magnetic stand. Without disturbing beads, discard liquid when clear ~5 min).65.Add 500 µL of fresh 80% ethanol without disturbing beads. Incubate 30 s.66.Remove ethanol and repeat wash with another 500 µL of fresh 80% ethanol.67.Discard ethanol, spin briefly on a bench-top centrifuge and replace on magnetic stand.68.Discard residual ethanol and allow to air dry until beads are matte in appearance (~5 min).CRITICAL STEP Do not over dry the beads as this will reduce yield. Beads should appear like damp mud, neither glossy wet nor dry. Cracks in the bead pellet are indicative of over-drying.69.Remove tube from magnet and resuspend beads in 30 µL of 0.1× Tris-EDTA (TE) by pipetting 10 times.70.Incubate at room temperature for 2 min.71.Replace on magnetic stand. Once clear (~4 min) recover 28 µL of eluted PCR product and transfer to a new DNA low bind tube.72.Confirm PCR product size using either a 2% agarose gel or D1000 TapeStation.PAUSE STEP After this step the indexed library can be stored at 4 °C overnight or at −20 °C for several months.73.Make a 1:10,000 and 1:20,000 dilution of indexed PCR products and quantify by real-time qPCR using the KAPA Illumina quantification kit.74.If sequencing in-house prepare 8 nM dilutions of each PCR plate pool and combine differently indexed plates in equal volumes.75.If sequencing in a core facility prepare DNA pools to their specifications.76.Sequence on a MiSeq using a 500-cycle Nano kit  with 10% PhiX.CRITICAL STEP Locus-specific PCR products will have near identical sequences, PhiX is essential to avoid MiSeq run failure from low complexity. If sequencing a single locus specific PCR product, increase PhiX to 30%.77.Download fastq files from either BaseSpace or core facility provider.78.PIPE_fastqPasths.txt: Containing names and paths to fastq files.PIPE_spacerBarcodePrimer_FWD.txt: Containing forward primer sequences.PIPE_spacerBarcodePrimer_REV.txt: Containing reverse primer sequences.PIPE_targetLocus_xxX.bed: Locus PCR amplification coordinates and additional region(s) of interest highlighted In a single UNIX directory prepare the following metadata plain text files:All scripts as well as sample files and fastq files  are available via GitHub as a user tutorial.CRITICAL STEP Plain text files are essential as rich text files will not be read correctly and cause analysis failure.79.Run analysis with the following minimal command : > ./plateScreen96.sh-g hg19.80.Analyze pipe output and select clones to recover (see Expected Results).81.Prepare one 1.5 mL microcentrifuge tube per clone by adding 1 mL sterile phosphate-buffered saline (PBS).82.Recover plate from −80 °C storage and remove parafilm.83.Working quickly, add 150 µL of sterile PBS to wells containing clones to be recovered.84.As each clone thaws, pipette mix to quickly combine with PBS, transfer melted clones to microcentrifuge tubes containing PBS.85.g, 5 min).Centrifuge to pellet cells (250× 86.Discard supernatant and resuspend in appropriate amount of growth media and plate for expansion.During amplification and indexing of the edited locus, quality-control checks that are performed ensure libraries are successfully prepared for sequencing . AnalysiLysis Buffer  can be m


These results may ultimately find applications in the development of potential inhibitors against amyloid fibril formation and its biologically adverse effects.Deposition of soluble proteins as insoluble amyloid fibrils is associated with a number of pathological states. There is a growing interest in the identification of small molecules that can prevent proteins from undergoing amyloid fibril formation. In the present study, a series of small aromatic compounds with different substitutions of 1,3,5-triphenylbenzene have been synthesized and their possible effects on amyloid fibril formation by hen egg white lysozyme (HEWL), a model protein for amyloid formation, and of their resulting toxicity were examined. The inhibitory effect of the compounds against HEWL amyloid formation was analyzed using thioflavin T and Congo red binding assays, atomic force microscopy, Fourier-transform infrared spectroscopy, and cytotoxicity assays, such as the 3--2,5-Diphenyltetrazolium Bromide (MTT) reduction assay and caspase-3 activity measurements. We found that all compounds in our screen were efficient inhibitors of HEWL fibril formation and their associated toxicity. We showed that electron-withdrawing substituents such as –F and –NO The conversion of peptides or proteins from a soluble state into insoluble, β-sheet rich, fibrillar aggregates, generally called amyloid fibrils, is a key event in a large family of human pathologies, including neurodegenerative and non-neuropathic systemic amyloidosis ,2. AmongFibril formation is a polymerization process which can be simply described by a sigmoid curve with three phases, originally suggested to be a three-stage process consisting of nucleation, fibril elongation, and the final plateau , althougThe propensity of proteins and peptides to form amyloid fibrillar structures is correlated to their physicochemical properties such as net charge, secondary structure tendency, hydrophobicity, and aromatic interactions ,19. Regi1–6). These pharmaceutical important compounds are made by the condensation of aryl methyl ketones catalyzed by HPA/NCP under solvent-free conditions. Nanoclinoptilolite (NCP) refers to a natural zeolite composed of a microporous arrangement of silica and alumina and also HPA (H6P2W18O62) refers to the Wells–Dawson heteropolyacid. The heteropolyacid catalyst activates the carbonyl group toward the electrophilic substitution. Using this procedure, six compounds belonging to the category of para-substituted 1,3,5-triarylbenzenes have been synthesized and purified as previously described [1); 1,3,5-tris(4-fluorophenyl)benzene (Compound 2); 1,3,5-tris(4-nitrophenyl)benzene (Compound 3); 1,3,5-tris(4-methylphenyl)benzene (Compound 4); 1,3,5-tris(4-methoxyphenyl)benzene (Compound 5); and 1,3,5-tris(4-hydroxyphenyl)benzene (Compound 6).escribed ,44. ThesThe names and chemical formulas of the six compounds are reported in 1–6. All compounds clearly showed a pattern of dose-dependent inhibition, as suggested by the observed decrease of ThT fluorescence intensity  in 50 mM glycine buffer, pH 2.5, and 57 °C, while stirred gently by magnetic bars (rpm 250), in the absence or presence of 0.03–2.6 µM compounds ntensity . The IC5termined . The relA control experiment was also designed to rule out the possibility that the low ThT fluorescence intensities measured in the presence of these compounds might result from the quenching of ThT fluorescence by the compounds themselves. These compounds were separately added at final concentrations of 2.6 μM to pre-formed HEWL aggregates and the ThT fluorescence assay was performed immediately before and after compound addition. The ThT fluorescence values were found to be similar in each case, before and after compound addition, indicating the lack of any significant quenching effect .1, 2, 3, 4, and 5 were found to be lower than that measured in the absence of the compounds  DMSO used here has a minimal effect on the aggregation of HEWL  for dissolving our compounds, and, thus, a control experiment was carried out to determine whether DMSO itself had an effect on the HEWL aggregation. However, 2%  in 50 mM glycine buffer, pH 2.5, and 57 °C, under stirring (250 rpm), in the absence or presence of 0.32 µM of compounds d by AFM . HEWL ag1–6, according to the aggregation protocol described for the ThT experiments. HEWL aggregates were collected by centrifugation, dried under N2, dissolved in cell culture medium, and added to SH-SY5Y human neuroblastoma cells at a concentration of 2 µM . The ability of the resulting aggregates to cause cellular dysfunction was assessed by performing the MTT reduction inhibition assay, a widely used indicator of mitochondrial dysfunction [1 (to 92.7 ± 3.2%), 2 (to 96.2 ± 3.2%) and 5 (to 93.9 ± 2.9%). By contrast, incubation with compounds 3, 4, and 6 resulted in a partial rescue of the cell viability. Very similar results were obtained by measuring the activity of caspase-3, a well-recognized apoptotic marker [1, 2, 4, and 5 during HEWL aggregation significantly prevented the apoptotic response induced by aggregates phenylmethane, i.e., 12.3 ± 1 µM [In this work, a series of novel 1,3,5-triarylbenzenes derivatives having four aromatic rings and different electron-donor or -acceptor substitutions were synthesized and evaluated for their potential to prevent HEWL amyloid aggregation. These compounds, similar to bis(indolyl)phenylmethanes studied previously, were shown to inhibit HEWL amyloid fibril formation under conditions in which the protein is initially partially unfolded, but with different magnitude. In particular, considering the various techniques used here, including the ThT and CR assays, AFM, and FTIR, compounds e effect . These f3 ± 1 µM .The amorphous and unstructured HEWL aggregates formed in the presence of all compounds were less toxic than the HEWL fibrils formed in their absence, as shown by MTT cell viability and caspase activity assays . Moreove2, –F, or –CN [2 group. On the other hand, when an electron-donating group, such as a –OH, –CH3, or –OCH3, was added, amyloid inhibition is decreased due to a repulsion between the electron-rich aromatic ring of the inhibitor and the electron-rich aromatic residues of HEWL, which does not allow the establishment of a stable complex between the inhibitor and the protein undergoing aggregation. In this context, the –OH group is the least effective in inhibiting HEWL aggregation.It has been proposed that π–π stacking of aromatic rings can be enhanced when two rings are electron poor and rich, respectively . Our wor, or –CN  on the aInhibitory effects of bis(indolyl)phenylmethanes may be discussed from different points of view, including different factors, such as the importance of inter- and intra-molecular hydrogen bonds, electronic effects and steric hindrances, the intrinsic steric congestion originating from the structural framework of bis-indolylarylmethanes, all affected by the substituents. In the case of 1,3,5-triarylbenzenes, a fully planar formula presents three aryl substituents in the same plane as the central benzene ring. In this case, only the aryl substituents may exert a little steric congestion around the aryl rings. Therefore, the net structure should have a planar identity and we can assume an effective diffusion of these types of compounds into amyloid fibrils. This can explain both the most effective inhibitory potential of 1,3,5-triarylbenzenes relative to bis-indolylarylmethanes and the critical role played by the electron releasing/donating substituents on the aryl rings in determining such an inhibition.Hen egg white lysozyme (HEWL), thioflavin T (ThT), and Congo red (CR) were purchased from Sigma-Aldrich . Other reagents were purchased from Merck , unless stated otherwise. All synthesized molecules were compared, in terms of their spectral and physical data, with those reported previously .−1 using KBr pellets. 1H and 13C NMR spectra were recorded on a Bruker AVANCE 300 MHz instrument  using TMS as internal reference. All products were identified by comparison of their spectral and physical data with those previously reported. Wells–Dawson diphosphooctadecatungstic acid H6P2W18O62·24H2O was prepared according to the literature method [All reagents and starting materials were commercially available and were used as received. Natural clinoptilolite-rich tuffs were obtained from the Sabzevar region in the north-east of Iran. Fourier-transform infrared (FT-IR) spectra were recorded on a 8700 Shimadzu Fourier-transform spectrophotometer  in the region of 400–4000 cme method .2O (3:1) as confirmed by an intense single spot in TLC. The pure products were specified based on the spectral data and determination of their melting points.In a typical reaction, acetophenone (1 mmoL) and the surface-modified HPA/NCP (15 mg) were added to a small test tube and the reaction mixture was stirred for 2.5 h at 100 °C. After completion of the reaction, as indicated by thin layer chromatography (TLC), the reaction mass was cooled to 25 °C; then, hot ethanol was added to the reaction mixture and the mixture was stirred for 5 min. The insoluble catalyst was isolated via simple filtration. The filtrate containing water as the only byproduct of the cyclotrimerization was concentrated under reduced pressure, and finally the obtained crude product was purified through re-crystallization in EtOH:Hmax = 3053, 1651, 1494, 1431, 1125, 758, 700 cm−1. 1H NMR : δ 7.88 , 7.87–7.86 , 7.52–7.49 , 7.43–7.40 .13C NMR : δ 141.6, 140.1, 128.9, 127.7, 127.2, 124.4.Light yellow solid; m. p. 171–173 °C. IR (KBr): vmax = 3059, 1604, 1508, 1449, 1224, 1159, 825, 771 cm−1. 1H NMR : δ 7.67 , 7.61 , 7.59 . 13C NMR : 161.7, 137.6, 132.4, 129.0 , 125.3, 115.9 .White solid; m. p. 238–240 °C. IR (KBr): vmax = 3043, 1595, 1489, 1379, 1085, 1007, 849 cm−1. 1H NMR (CDCl3): δ = 7.56 , 7.62 , 7.70 . 13C NMR : δ 122.3, 125.0, 128.9, 132.1, 139.7, 141.6.White solid; m. p. 151–152 °C. IR (KBr): vmax = 3017, 2922, 1612, 1511, 1491, 1109, 812 cm−1. 1H NMR : δ 7.74 , 7.62 , 7.30 , 2.44 . 13C NMR : 142.2, 138.3, 137.4, 129.4, 127.3, 124.7, 21.1.White solid; m. p. 178–179 °C. IR (KBr): vmax = 2953, 2918, 2848, 1598, 1460, 1378, 1263, 1097, 802 cm−1. 1H NMR : δ 7.59 , 7.37 , 6.99 , 3.86 . 13C NMR : 159.7, 141.7, 134.0, 129.3, 122.7, 114.2, 55.9.White solid; m. p. 142–143 °C. IR(KBr): vmax = 2953, 2918, 2848, 1606, 1460, 1378, 1263, 1097, 802 cm−1. 1H NMR (CDCl3): δ = 3.81 , 6.94–7.41 , 7.79 . 13C NMR : δ 55.4, 114.3, 123.8, 128.3, 133.9, 141.8, 159.3.White solid; m. p. 237–239 °C. IR (KBr): v280 = 2.65 L·g−1·cm−1) [1–6, under gentle stirring at 250 rpm by a Teflon magnetic bar [HEWL concentration was determined by optical absorption at 280 nm (ε−1·cm−1) . HEWL waetic bar .A mother solution of 2.5 mM ThT was prepared in 10 mM sodium phosphate buffer, 150 mM NaCl, pH 7.0. It was then passed through a 0.45 μm filter paper and stored at 4 °C until it was placed at room temperature immediately before use. At regular time intervals, a 10 µL aliquot of protein solution was mixed with 990 μL of ThT solution in a 10 × 10 mm quartz cuvette and the fluorescence emission spectra were recorded at room temperature using a Cary Eclipse VARIAN fluorescence spectrophotometer  and an excitation wavelength of 440 nm .1–6 have optical absorption spectra partially overlapped to that of CR, all protein samples were centrifuged at 10,000 rpm and the pellets resuspended in 50 mM glycine buffer, pH 2.5, at room temperature. A mother solution of 20 mM CR was prepared in 5 mM sodium phosphate buffer, 150 mM NaCl, pH 7.4, it was then filtered using a center-glass N4 filter and stored at 4 °C, until it was placed at room temperature immediately before use. At different time points during protein aggregation, a 60 μL aliquot of resuspended protein solution was mixed with 440 μL of CR solution. Optical absorption spectra were acquired after 2–3 min equilibration at 25 °C, using a Cecil 7200 UV–visible spectrophotometer  and a 1 cm path-length cell.Since compounds 1–6, centrifuged at 13,000× g for 10 min and resuspended in D2O to a final volume of 72.8 μL, to reach a final concentration of 27.8 mg/mL. For each sample, 40 μL was deposited onto an Omni-Cell transmission cell with a 25 μm spacer , and Fourier-transform infrared (FTIR) spectra were recorded using a FTS-40 FTIR spectrometer  purged with a continuous flow of dry air. Sixty-four interferograms were accumulated in each case at a spectral resolution of 1 cm−1. The FTIR absorbance spectra were blank-subtracted and baseline-corrected to reveal the amide I region (1600–1700 cm−1). For each spectrum, the sharp water peaks in the amide I and flanking regions (1580–1800 cm−1) were eliminated by using a multiple Gaussian function to fit only the experimental data that were devoid of such peaks in this region and replotting the spectrum as the best fitted function. All spectra were normalized to the same amide I region area. Additional smoothing was not carried out.Samples were incubated for 48 h to promote aggregation as described above in the presence of 0.32 µM compounds 1–6 were diluted 100 times in filtered deionized water. A 5 µL aliquot of each of the resulting samples was placed on freshly cleaved mica at room temperature. After a few minutes, the mica was slowly washed with 100 μL of deionized water and dried with N2. Each image was acquired in a non-contact mode at a scan speed of 30 μm/s, loop filter of 3 Hz and force of 200 nN with a Dual Scope Probe Scanner  with an area of 5 × 5 μm2. Conical shape silicon tips (MikroMasch NSC16) with a resonance frequency of 150 kHz and a nominal constant of 40 N/m were used. Fifteen images were collected for each compound.HEWL samples pre-incubated for 48 h under aggregating conditions with or without 0.32 µM compounds 3 (1:1) supplemented with 10% fetal bovine serum (FBS), 1.0 mM glutamine and 1.0% penicillin and streptomycin solution. Cells were maintained in a 5.0% CO2 humidified atmosphere at 37 °C and grown until 80% confluence for a maximum of 20 passages.Authenticated human neuroblastoma SH-SY5Y cells were purchased from American Type Culture Collection (ATCC)  and they tested negative for mycoplasma contaminations. SH-SY5Y cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM), F-12 Ham with 25 mM HEPES and NaHCO1–6 were centrifuged and the pellets were dried under N2 to remove any compounds and DMSO, dissolved in cell culture medium at a final HEWL concentration of 2 μM , and added to SH-SY5Y cells seeded in 96-well plates for 24 h at 37 °C. The cell medium was then removed and the MTT solution (0.5 mg/mL in RPMI) was added to the cells for 4 h. The medium was then aspirated, and the formazan product was solubilized with cell lysis buffer  for 1 h, as previously described [HEWL samples pre-incubated for 48 h under aggregating conditions in the absence or presence of 0.32 µM compounds escribed .1–6 as described in the previous section and then added to the cell culture medium of SH-SY5Y cells seeded on glass coverslips for 24 h at 2 μM . Caspase-3 activity was then analyzed by using FAM FLICATM caspases 3 & 7 solution . Cell fluorescence was analyzed by the TCS SP8 scanning confocal microscopy system  equipped with an argon laser source, as previously reported [HEWL aggregates were formed in the absence or in the presence of 0.32 µM of compounds reported . A serieAll data were expressed as means ± standard error of mean (S.E.M). ANOVA followed by Bonferroni’s post comparison test was used to compare different values.3, and –CH3 groups as electron-donor groups positioned on aromatic rings have a low efficacy, whereas –F and –NO2 as electron-withdrawing groups have great efficacy in inhibiting amyloid formation by HEWL. Based on the results of this study and others, it is apparent that small aromatic compounds and functional groups appended on these aromatic molecules can have a profound effect on their ability to inhibit amyloid formation. These results may ultimately find applications in the development of potential amyloid inhibitors and, thus, possible use of these compounds as a therapeutic approach in the treatment of amyloid deposition diseases.In conclusion, we have provided experimental evidence of different effects of various substituents of the architecture of the 1,3,5-triarylbenzene molecule on the inhibition of the self-assembly of HEWL. We showed that –OH, –OCH


Benefiting from the unique tubular sphere architecture, the rich inner defects and the enhanced electron interactions between Fe, Ni and S, this electrocatalyst shows low overpotential of 48 mV for HER at 10 mA cm−2 in 1.0 mol L−1 KOH solution, which is one of the lowest value of non-previous electrocatalyts for HER in alkaline electrolyte. Furthermore, assembled this versatile electrode as an alkaline electrolyzer for overall water splitting, a current density of 10 mA cm−2 is achieved at a low cell voltage of 1.56 V, and reach up to 30 mA cm−2 only at an operating cell voltage of 1.65 V.Earth-abundant and efficient bifunctional electrocatalysts for both hydrogen evolution reaction (HER) and oxygen evolution reaction (OER) are highly significant for renewable energy systems. However, the performance of existing electrocatalysts is usually restricted by the low electroic conductivity and the limited amount of exposed active sites. In this work, (Fe Electrochemical water splitting including the cathodic hydrogen evolution reaction (HER) and the anodic oxygen evolution reaction (OER), which provides us with a feasible method for sustainable hydrogen production4. However, both the HER and OER are greatly limited by the high overpotential and low electrocatalytic efficiency. It is thus imperative to explore efficient electrocatalysts. At present, the most active electrocatalysts for HER and OER are Pt and RuO2/IrO2, respectively, but the wide-spread utilization of these catalysts is restricted by their high cost and scarcity. Therefore, enormous efforts have been made to design alternative electrocatalysts, such as transition-metal chalcogenides7, phosphides8, alloys9, nitrides10, carbides11 for HER and transition-metal oxides/hydroxides13, phosphates14, borates15 for OER. Nevertheless, most of these researches concentrate on the perfection of either HER or OER activity. In consideration of simplifying the fabrication procedures and decreasing the overall cost for water splitting system, the exploitation of efficient and low-cost bifunctional electrocatalysts for both HER and OER is highly attractive and desirable21.Hydrogen energy has been regarded as the most promising alternative to replace those conventional fossil fuels due to its renewability, sustainability, high calorific value and great energy conversion with zero CO24. Interestingly, we have noticed that the enhanced electronic conductivity and electrocatalytic performance have been achieved after converting the LDHs to transition-metal sulfides (TMSs)27. Recently, it has been confirmed that the charged states of sulfur and metals in TMSs are similar to those in hydrogenase and its analogs, which can severally as the proton-acceptors and hydride-acceptors in the water splitting processes and thereby result in efficiently catalytic activity25. Also, the inner defect-rich structure of TMSs has been attracting great attention because it can expose more active sites, and thus lead to outstanding improvement of electrocatalytic performance28.Transition-metal layered double hydroxides (LDHs) have exhibited efficient performance for both HER and OER, while their electrocatalytic activity is still limited by the low electronic conductivity30. Comparing to the solid structures of materials , hollow micro/nanostructures have a kinetically favorable open structure, more exposed active sites and even a shortened ion diffusion length, and thereby possess high activity for HER and OER33. Guan et al.32 had fabricated CoS2 nanotube arrays on a carbon cloth as a bifunctional electrocatalyst for overall water splitting, achieving a current density of 10 mA cm−2 at a cell voltage of 1.67 V. Zhang et al.30 had reported the alkaline electrolyzer assembled by carbon paper/carbon tubes/cobalt-sulfide sheets electrode needed a cell voltage of 1.743 V to reach 10 mA cm−2. Moreover, Chao et al.33 had synthesized Co9S8 hollow microspheres exhibited efficiently electrocatalytic activity for OER, HER, and even the oxygen reduction reaction. In spite of these crucial advances have been done, the rational design and construction of TMSs electrocatalysts with uniquely hollow nanostructure and high performance for both HER and OER still needs more efforts.Besides, the nanostructure modification is an effective way to optimize the electrocatalytic activity0.2Ni0.8)0.96S tubular spheres on Ni foam ((Fe0.2Ni0.8)0.96S TSs/Ni) via vulcanizing FeNi-LDH spheres grown on Ni foam (FeNi-LDH Ss/Ni). As expected, owing to the unique tubular sphere architecture to facilitate the release of gaseous products, the rich inner defects to expose more active sites, and the strong electron interactions between the Fe, Ni and S to improve the charge-transfer kinetics, the (Fe0.2Ni0.8)0.96S TSs/Ni possesses improved performance for both HER and OER in basic electrolytes, compared with the FeNi-LDH Ss/Ni. It just needs overpotentials of 48 mV for HER and 233 mV for OER to drive a current density of 10 mA cm−2. Especially, an alkaline electrolyzer assembled by the (Fe0.2Ni0.8)0.96S TSs/Ni electrode can be driven by a single 1.5 V AA battery, demonstrating greatly practical prospect and feasibility for water splitting.Herein, we have prepared (Fe3)3·9H2O and Ni(NO3)2·6H2O were purchased from Tianjin Yongda Chemical. CO(NH2)2 (urea), NH4F, and CH3CSNH2 (thioacetamide) were obtained from Aladdin. Ni foam was acquired from Kunshan Electronic Limited Corporation. All chemicals were directly used as received without any purification.Fe(NO−1 HCl solution and absolute ethanol with an ultrasound for 10 min each. 484.8 mg Fe(NO3)3·9H2O, 872.4 mg Ni(NO3)2·6H2O, 303.0 mg urea and 18.5 mg NH4F were dissolved in a solution comprised of 30 mL distilled water and 20 mL absolute ethanol under vigorous stirring for 10 min. Then, the above solution was transferred into an 80 mL Teflon-lined stainless-steel autoclave, in which contained a piece of cleaned Ni foam. The autoclave was heated at 120 °C for 6 h and allowed to cool to room temperature naturally. The product was taken out, washed with distilled water, and dried at 60 °C.The Ni foam (3 cm × 3 cm) was cleaned with 3.0 mol L0.2Ni0.8)0.96S TSs/Ni was obtained after drying at 60 °C overnight. The mass loading is 2.5 mg cm−2.Typically, 300.5 mg thioacetamide (TAA) was dissolved in a solution consisted of 40 mL distilled water and 10 mL absolute ethanol. The above solution was transferred into an 80 mL autoclave, containing a piece of as-prepared FeNi-LDH Ss/Ni. The autoclave was maintained at 140 °C for 4 h. After cooling down to room temperature, the product was taken out and severally washed with distilled water and absolute ethanol for four times. Finally, the (Fe3)3·9H2O, and altering the amount of Ni(NO3)2·6H2O to 1.2 g, followed by the same sulfuration process. For FeS, the similar procedures without adding the Ni foam and replacing Ni(NO3)2·6H2O with Fe(NO3)3·9H2O (1.7 g) were employed. Then the FeS were bonded to a Ni foam to form FeS/Ni  analysis was recorded by a MSAL-XD2 X-ray diffractometer using Cu Kα radiation (λ = 1.5406 Å). The inductively coupled plasma optical emission spectrometer (ICP-OES) was tested by Perkin Elmer Optima 2000DV. The scanning electron microscopy (SEM) observations were performed on Philips SEM-XL30S microscope operated at 15 kV. Transmission electron microscopy (TEM), high-resolution transmission electron microscope (HRTEM) and energy dispersive X-ray spectroscopy (EDS) were characterized using a JEOL JEM-2100F instrument at 200 kV. Nitrogen adsorption isotherms were recorded on a Micromeritics TriStar 3000 Analyzer at −196 °C. The Brunauer-Emmett-Teller (BET) surface area was determined by adsorption data. The X-ray photoelectron spectroscopy (XPS) measurements were carried out by using a model of ESCALab250 with an Alumina Ka (1486.6 eV) source.−1. All potentials in this work were converted to the reversible hydrogen electrode (RHE) according to the Nernst equation:The electrochemical measurements were performed in a conventional three-electrode setup controlled by a CHI 660D electrochemical workstation . The as-prepared samples, Hg/HgO electrode and platinum foil were used as the working, reference, and counter electrodes, respectively. Linear sweep voltammetry (LSV) was conducted with a scan rate of 1 mV svia the Tafel equation:η is the overpotential, b is the Tafel slope and j is the current density. Electrochemical impedance spectroscopy (EIS) experiments were performed in the frequency range from 105 to 0.01 Hz with an amplitude potential of 5 mV. Chronoamperometry tests were implemented at certain potentials. The double layer capacitance (Cdl) is proportional to the electrochemical surface area (ECSA), tested by cyclic voltammograms (CV) cycles with scanning rates of 4, 6, 8, 10, 12 and 14 mV s−1. The linear slope of capacitive currents versus scan rates is equal to 2Cdl34.Tafel plots were recorded 0.2Ni0.8)0.96S TSs/Ni has been prepared via two-step hydrothermal treatments. In step I, the FeNi-LDH Ss/Ni was prepared by the reactions between the metals ions and products released by the hydrolysis after heating at 120 °C for 6 h. The XRD pattern in Fig. ο, 23.0ο, 34.4ο, 39.0ο, 46.0ο, 59.9ο, 61.3ο, corresponding to the (003), (006), (012), (015), (110) and (113) planes of rhombohedral Ni0.75Fe0.25(CO3)0.125(OH)2·0.38H2O (JCPDS no. 40–0215), respectively, matching well with the typical profile of LDH materials, and the other two peaks at 44.5ο and 51.8ο are indexed as the (111) and (200) planes of Ni foam (JCPDS no. 65–2865). In step II, the as-prepared FeNi-LDH Ss/Ni underwent a conversion via a sulfur treatment at 140 °C for 4 h, using TAA as the sulfur source to synthesize (Fe0.2Ni0.8)0.96S TSs/Ni. It can be seen that the diffraction peaks of FeNi-LDH are disappeared and the emerging peaks at 30.1°, 34.4°, 44.8° and 53.2° are attributed to the (100), (101), (102) and (110) planes of the hexagonal (Fe0.2Ni0.8)0.96S (JCPDS no. 50–1790). ICP-OES results indicate that the atomic ratio of Fe and Ni is about 0.2: 0.8 in Table 0.2Ni0.8)0.96S. Moreover, the EDS spectrum suggests the coexistence of Fe, Ni, and S, while the Cu elemental comes from the copper mesh 0.96S TSs/Ni retains the sphere characteristics of FeNi-LDH Ss/Ni, except for the enhanced surface roughness in Fig. 2 sorption measurement 0.96S TSs/Ni exhibits a BET surface area of 73 m2 g−1, which is larger than that of FeNi-LDH Ss/Ni (53 m2 g−1). And the pore size distribution curve illustrates that both the mesopores and macropores exist in the (Fe0.2Ni0.8)0.96S TSs/Ni 0.96S TSs/Ni was further elucidated by TEM. Figure 0.2Ni0.8)0.96S spheres are comprised of the nanotubes with a pore diameter of 84 nm. Notably, the formation of (Fe0.2Ni0.8)0.96S tubular spheres can be explained by the Kirkendall diffusion effect37. The specific illustration is described as follows: when the solution is heated at 140 °C, it will release the S2− ions from TAA. The S2− will react with Fe and Ni ions to form a thin layer of (Fe0.2Ni0.8)0.96S on the surface of FeNi-LDH 0.96S39. In addition, the HRTEM image shows lattice fringe spacing of 0.172 and 0.202 nm in Fig. 0.2Ni0.8)0.96S, respectively. Significantly, compared with the FeNi-LDH Ss/Ni 0.96S, which may provide more enrich active sites40, and further improve the electrocatalytic activity. The elemental mappings in Fig. 0.2Ni0.8)0.96S TSs/Ni.The SEM image in Fig. ent Fig. , the 0.96S TSs. As shown in Fig. 3/2 and Fe 2p1/2 peaks at 713.5 eV and 723.5 eV, respectively, indicating the Fe3+ oxidation states of (Fe0.2Ni0.8)0.96S TSs, while another peak at 707.6 eV is assigned to Fe-S bond42. The Ni 2p XPS spectrum in Fig. 3/2) and 874.1 eV (Ni 2p1/2), as well as their satellite peaks, are assigned to Ni2+, and the peak at 857.3 eV is attributed to Ni3+ 43. It is worth noting that the Fe 2p and Ni 2p peaks of (Fe0.2Ni0.8)0.96S TSs show a little positive shift after vulcanizing the FeNi-LDH Ss 0.96S TSs are mainly attributed to the influence of electron transfer between FeNi LDH Ss and (Fe0.2Ni0.8)0.96S TSs, suggesting the enhanced electron transfer from Fe, Ni to S45. Correspondingly, the relative interaction between Fe, Ni and S are enhanced after the sulfuration, which is beneficial to the charge transfer in the electrocatalysis process and hence favors the electrocatalytic activity.XPS analysis was employed to investigate the surface chemical states on the 0.96S TSs/Ni for HER was assessed by LSV curves in a standard three-electrode system. For comparison, the FeNi-LDH Ss/Ni, 50 wt% Pt/C loading on Ni foam  and the bare Ni foam were also tested. Figure −1. Unquestionably, 50 wt% Pt/C/Ni exhibits the best performance with an onset potential close to zero, while bare Ni foam shows almost no HER performance. The as-prepared (Fe0.2Ni0.8)0.96S TSs/Ni exhibits excellent HER activity with sharp increase of current density, which requires low overpotentials of 48 mV and 198 mV to deliver the current density of 10 mA cm−2 and 100 mA cm−2 , respectively, outperforming that of ƞ10 = 153 mV and ƞ100 = 380 mV for FeNi-LDH Ss/Ni. This result indicates the enhanced HER activity of (Fe0.2Ni0.8)0.96S TSs/Ni after the sulfuration. Such excellent HER activity of (Fe0.2Ni0.8)0.96S TSs/Ni is also superior to the reported noble-metal-free materials listed in Table Electrocatalytic activity of the 0.96S TSs/Ni is 52.9 mV dec−1, much smaller than that of the FeNi-LDH Ss/Ni (85.7 mV dec−1), implying more favorable kinetics on (Fe0.2Ni0.8)0.96S TSs/Ni. To further evaluate the charge-transfer kinetics, the EIS of (Fe0.2Ni0.8)0.96S TSs/Ni and FeNi-LDH Ss/Ni was tested. As displayed in Fig. Rct) of 0.65 Ω for (Fe0.2Ni0.8)0.96S TSs/Ni is obviously lower than that of the FeNi-LDH Ss/Ni (2.79 Ω), suggesting better charge-transfer kinetics in the HER electrochemical processes. The lower charge-transfer resistance of (Fe0.2Ni0.8)0.96S TSs/Ni comparing to FeNi-LDH Ss/Ni probably arises from its unique tubular sphere architecture with a shortened ion diffusion length and the optimization of electron interactions between Fe, Ni and S after the sulfuration.The corresponding Tafel plots were carried out to estimate the catalytic kinetics for HER. Figure 0.2Ni0.8)0.96S TSs/Ni was further measured by the chronoamperometric method. As shown in Fig. 0.2Ni0.8)0.96S TSs/Ni maintains the electrocatalytic activity after continuing hydrogen-release for 12 h. The polarization curve of (Fe0.2Ni0.8)0.96S TSs/Ni after 1000 cycles also reveals a negligible difference to the initial one 0.96S TSs/Ni after stability measurements demonstrate that this electrocatalyst is still retained their tubular sphere structure with just a little aggregation 0.96S TSs/Ni, the HER activities of Ni3S2/Ni and FeS/Ni were also investigated as shown in Fig. 0.2Ni0.8)0.96S TSs/Ni exhibits improved HER activity comparing to the Ni3S2/Ni and FeS/Ni. In addition, this electrocatalyst gives a smaller Tafel slope (52.9 mV dec−1) than Ni3S2/Ni (76.3 mV dec−1) and FeS/Ni (89.7 mV dec−1) 0.96S TSs/Ni.To get insight into the inherent electrocatalytic activity of (Fe−1) Fig. . These r0.2Ni0.8)0.96S TSs/Ni for OER in 1.0 mol L−1 KOH was further evaluated by LSV measurement at a scan rate of 1 mV s−1. Figure 0.2Ni0.8)0.96S TSs/Ni, FeNi-LDH Ss/Ni, RuO2 loading on Ni foam  and the bare Ni foam. Interestingly, the anodic peak around 1.4 V vs RHE before OER can be observed for FeNi-LDH Ss/Ni, which is due to Fe and Ni species change to higher oxidation states47. However, the anodic peak of (Fe0.2Ni0.8)0.96S TSs/Ni shifted negatively to 1.38 V vs RHE, which may be attributed to the improved charge transfer. Specifically, the (Fe0.2Ni0.8)0.96S TSs/Ni exhibits a low ƞ10 of 233 mV and an ƞ100 of 310 mV for OER, superior to those of the FeNi-LDH Ss/Ni (ƞ10 = 263 mV), Ni foam (ƞ10 = 405 mV) and even the RuO2/Ni (ƞ10 = 253 mV). In addition, a smaller Tafel slope of (Fe0.2Ni0.8)0.96S TSs/Ni (46.2 mV dec−1) compares to those of FeNi-LDH Ss/Ni (63.0 mV dec−1) and RuO2/Ni (82.2 mV dec−1), suggesting a more favorable OER catalytic kinetics in Fig. 0.2Ni0.8)0.96S TSs/Ni, which makes this uniquely tubular sphere as one of the best noble-metal-free electrocatalysts in Table 3S2/Ni and FeS/Ni 0.96S TSs/Ni to reach a current density of 100 mA cm−2 with respect to Ni3S2/Ni and FeS/Ni 0.96S TSs/Ni.The electrocatalytic activity of  is herein employed in Fig. Cdl of 100.1 mF cm−2 for (Fe0.2Ni0.8)0.96S TSs/Ni is considerably larger than that of the FeNi-LDH Ss/Ni (54.4 mF cm−2), revealing the existence of enriched active sites on (Fe0.2Ni0.8)0.96S TSs/Ni. This is ascribed to the inner defect-rich crystal structure of (Fe0.2Ni0.8)0.96S TSs/Ni. The long-term stability of (Fe0.2Ni0.8)0.96S TSs/Ni for OER was tested at static potentials of 1.46, 1.50, and 1.52 V vs. RHE. Figure 0.2Ni0.8)0.96S TSs/Ni is also tested by using continuous CV sweeps in 1.0 M KOH at a scan rate of 100 mV s−1. The polarization curve reveals negligible degradation after 2000 cycles of CV scanning, confirming a satisfactory stability of (Fe0.2Ni0.8)0.96S TSs/Ni. Similar to HER, the SEM images, TEM image, N2 sorption isotherm and the corresponding pore-size distribution indicate the structure of (Fe0.2Ni0.8)0.96S TSs/Ni is insignificant change after OER durability testing, as displayed in Fig. Besides, the electrochemically active surface area (ECSA) is an important parameter to understand the intrinsic activity of electrocatalysts, the double-layer capacitances (0.2Ni0.8)0.96S after OER performance, XPS is characterized. As shown in Fig. 0.2Ni0.8)0.96S after OER tests. Moreover, the peaks located at 711.6 eV and 721.2 eV belonged to Fe-OOH bonds in the Fe 2p region are observed and the binding energy in the Ni 2p region shows a little positive shift, as shown in Fig. 0.2Ni0.8)0.96S could be attributable to the Ni-Fe oxo/hydroxyl species, consistent with the previous reports48.To further investigate the nature of (Fe0.2Ni0.8)0.96S TSs/Ni was further served as anode and cathode in an electrolyzer for overall water splitting. As shown in Fig. −2 and 30 mA cm−2, respectively. Although the cell voltage of (Fe0.2Ni0.8)0.96S TSs/Ni couple to generate 10 mA cm−2 is larger than that of the NixCo3-xS4/Ni3S2/NF (1.53 V)49, it is superior to those of FeNi-LDH Ss/Ni (1.66 V), RuO2–50 wt% Pt/C couple (1.58 V), NiCo2S4@NiFe LDH/NF (1.60 V)24, Ni0.7Fe0.3S2/Ni (1.625 V)26, NiCo2S4/NF (1.68 V)27, CoS2 NTA/CC (1.60 V)33, FeSe2/NF (1.73 V)50, FeB2-NF (1.57 V)51, Zn0.76Co0.24S/CoS2/TM (1.66 V)52 and even most of the reported works exhibited in Table 2 and O2 with a predicted ratio of 2: 1 are obtained, and the amount of experimentally quantified gas is in good accordance with theoretically calculated gas, indicating that the (Fe0.2Ni0.8)0.96S TSs/Ni affords a Faradaic efficiency of ~100% for both HER and OER 0.96S TSs/Ni can be attributed to the following aspects: (1) the inner defect-rich crystal structure of (Fe0.2Ni0.8)0.96S TSs/Ni can expose more effectively active sites. Meanwhile, the outer unique tubular sphere architecture of (Fe0.2Ni0.8)0.96S TSs/Ni is beneficial to intimate contact between the material and electrolyte, and the release of gaseous products; (2) the change in composition between FeNi-LDH Ss/Ni and (Fe0.2Ni0.8)0.96S TSs/Ni leads to strong electron interactions between Fe, Ni and S, and further optimizes charge transfer; (3) the in-situ growth of (Fe0.2Ni0.8)0.96S TSs on Ni foam avoids the use of a binder, which can enhance the conductivity.The excellent electrocatalysis performance of (Fe0.2Ni0.8)0.96S TSs/Ni has been successfully synthesized via the hydrothermal sulfuration treatment of FeNi-LDH SAs/Ni. As expected, by taking advantage of the unique tubular sphere architecture, the rich inner defects and the enhanced electron interactions between Fe, Ni and S, the as-synthesized (Fe0.2Ni0.8)0.96S TSs/Ni possesses higher HER and OER performance with respect to FeNi-LDH Ss/Ni. Furthermore, the alkaline electrolyzer with (Fe0.2Ni0.8)0.96S TSs/Ni as the anode and cathode just needs cell voltages of 1.56 V and 1.65 V to achieve 10 mA cm−2 and 30 mA cm−2, respectively, suggesting the great value for the practical application. More importantly, this study will encourage new opportunities to design versatile electroactive materials with a uniquely hollow structure and high performance for water splitting, fuel cells, supercapacitors, and even the batteries.In summary, (FeSupplementary Information


Whole body exposure to ionizing radiation damages tissues leading to physical symptoms which contribute to acute radiation syndrome. Radiation biodosimetry aims to determine characteristic early biomarkers indicative of radiation exposure and is necessary for effective triage after an unanticipated radiological incident. Radiation metabolomics can address this aim by assessing metabolic perturbations following exposure. Gas chromatography–mass spectrometry (GC-MS) is a standardized platform ideal for compound identification. We performed GC time-of-flight MS for the global profiling of nonhuman primate urine and serum samples up to 60 d after a single 4 Gy γ-ray total body exposure. Multivariate statistical analysis showed higher group separation in urine vs. serum. We identified biofluid markers involved in amino acid, lipid, purine, and serotonin metabolism, some of which may indicate host microbiome dysbiosis. Sex differences were observed for amino acid fold changes in serum samples. Additionally, we explored mitochondrial dysfunction by tricarboxylic acid intermediate analysis in the first week with a GC tandem quadrupole MS platform. By adding this temporal component to our previous work exploring dose effects at 7 d, we observed the highest fold changes occurring at 3 d, returning closer to basal levels by 7 d. These results emphasize the utility of both MS-based metabolomics for biodosimetry and complementary analytical platforms for increased metabolome coverage. Fisher Optima2 to ~100 µL, transferred to a GC vial with a 250 µL glass insert, and evaporated to dryness in a speedvac without heat. Samples were derivatized inline with a Gerstel  multipurpose sampler for 1 h at 60 °C with rigorous shaking with MOX (10 µL) and an additional 1 h at 60 °C with MSTFA/1% TMCS (90 µL). For TCA intermediate analysis, urine (40 µL) was prepared as above, however, samples were deproteinated with 1 mL cold methanol  without urease pretreatment and batch derivatized per day. For global metabolomics, urine (60 µL) was aliquoted and treated with 5 µL (160 mg/mL) urease for 1 h at 37 °C with gentle agitation and serum (30 µL) was aliquoted without pretreatment. Quality control (QC) samples were prepared by aliquoting an equal volume from each sample. Samples were deproteinated with 1 mL 100% cold methanol with internal standards , incubated on ice for 10 min, and centrifuged at 13,000 rpm for 10 min at 4 °C. The supernatant was evaporated under N®-5 (G27) column ) mounted to a Leco  Pegasus HT TOF-MS. The GC settings were as previously described [m/z 40–600), ion source (200 °C), and 30 spectra/s acquisition rate. Samples were randomized and blanks, QCs, and liner exchange were performed every 10 samples.For global profiling, samples (1.5 µL) were injected into an Agilent  7890B GC system  mode for pyruvic, citric, isocitric, cis-aconitic, α-ketoglutaric (oxoglutaric), malic, succinic, and fumaric acid  Xevoric acid .® v. 4.72.0.0  and the statistical compare function as previously described [4, d-sorbitol-13C1, or l-leucine-d3 according to chemical class) and urine was further normalized to creatinine [For global analysis, data preprocessing, deconvolution, and alignment were performed with the software ChromaTOFescribed . Compounescribed . Pre-expeatinine . For muleatinine . The QC eatinine . For uniR2 = 0.99, Q2 = 0.80; performance measure: Q2P = 0.002; R2 = 0.98, Q2 = 0.65; performance measure: Q2P < 0.001; R2 = 0.95, Q2 = 0.57; performance measure: Q2P = 0.007; R2 = 0.92, Q2 = 0.08; performance measure: Q2P = 0.009; The pre-processed data matrices from our untargeted approach consisted of 408 compounds for urine and 112 compounds for serum . CreatinP < 0.001) from 5–60 d and higher levels of 5-hydroxyindoleacetic acid (P = 0.003) at 60 d in urine , isoleucine (P = 0.011), phenylalanine (P = 0.004), threonine (P = 0.003), serine (P = 0.029), and valine (P = 0.003) in the irradiated group , threonine , serine , and valine  at 3 d  and isoleucine  were similar between males and females. Leucine is a ketogenic branched chain amino acid (BCAA) that plays important roles in multiple conditions  including cachexia (muscle wasting), a known effect from radiation exposure [In serum, perturbed levels of several amino acids were evident at 3 d with significantly higher levels of leucine  at 3 d . Fold chexposure , and theexposure ,46 and hexposure , it mustP = 0.002) decreased from 15–60 d and oleic acid (P < 0.001) increased from 5–12 d  [In our previous work, we showed significant decreases in urinary TCA metabolites at 7 d after multiple doses of IR in a separate independent cohort . Citric tabolism  we used reported . In the reported . UrinaryC57Bl/6) . PyruvicC57Bl/6) . 50/60 in this NHPs model is ~6.6 Gy, which is consistent with other studies conducted without blood transfusion and antibiotics as supportive care [50/60 increase to ~7.5 Gy [50/60 may range from ~2.5–6.0 Gy depending on multiple factors including combined injury, pre-existing conditions, radiosensitivity, and time to supportive care [Radiation biodosimetry requires determining biomarkers to serve as a proxy for ARS severity and assignment of appropriate medical treatments. Here, a 4 Gy γ-ray TBI exposure was chosen to limit mortality and allow animal assessment over a 1–60 d time course. The LDive care  and with ~7.5 Gy . A 4 Gy ive care ,66. Geneive care . In the The majority of studies defining biophysiological responses to radiation exposures have focused on LC-MS platforms , however


Escherichia coli, and the enzyme was purified by means of standard chromatographic techniques. High-resolution NMR experiments revealed that HoLaMa is properly folded at pH 8.0 and 20°C. In addition, urea induced a cooperative folding to unfolding transition of HoLaMa, possessing an overall thermodynamic stability and a transition midpoint featuring ΔG and CM equal to (15.7 ± 1.9) kJ/mol and (3.5 ± 0.6) M, respectively. When the catalytic performances of HoLaMa were compared to those featured by the Klenow enzyme, we did observe a 10-fold lower catalytic efficiency by the HoLaMa enzyme. Surprisingly, HoLaMa and Klenow DNA polymerases possess markedly different sensitivities in competitive inhibition assays performed to test the effect of single dNTPs.We report here on the stability and catalytic properties of the HoLaMa DNA polymerase, a Klenow sub-fragment lacking the 3’-5’ exonuclease domain. HoLaMa was overexpressed in  First, a Mg2+-dNTP complex is bound and paired to the template DNA strand by the fingers domain, and the binding of a second divalent magnesium triggers the so-called fingers closure, i.e. a consistent conformational change leading to DNA elongation  + n[L0], with n representing the number of DNA molecules bound per enzyme molecule.Similar fluorescence experiments were performed to determine the dissociation constant, tal data :Q=Qmax*1H) NMR spectra were recorded both in 50 mM sodium phosphate, 50 mM NaCl (pH 8.0) and in 50 mM Tris-HCl, 50 mM NaCl (pH 8.0). The samples were prepared in a final volume of 500 μL containing 5% (v/v) D2O as field lock.High-resolution NMR spectroscopy was performed using a Bruker Advance III 600 MHz spectrometer equipped with a TCI-H/C/N triple resonance cryoprobe. All experiments were performed at 25°C. Water suppression was achieved by the Watergate sequence. Spectra were processed using TopSpin 2.1 software. One-dimensional proton  kJ/mol and the cooperativity of unfolding to m = (4.5 ± 0.5) kJ/mol*M. Accordingly, the midpoint for the chemical unfolding of HoLaMa is equal to CM = (3.5 ± 0.6) M of urea. Remarkably, this value of CM is very close to the value determined for the Klenow enzyme, i.e. CM = (3.6 ± 0.1) M [ΔG, and the cooperativity, m, of unfolding for Klenow, i.e. (20.9 ± 1.3) kJ/mol and (6.3±0.4) kJ/mol*M, respectively [ΔG and m determined for Klenow enzyme are most likely related to the higher molecular mass of this enzyme in comparison with that of its smaller derivative, HoLaMa. The ratio between the molecular mass of the two enzymes  is indeed equal to 0.69, and the ratio between the two m values equals 0.71.To further analyze the overall thermodynamic stability of HoLaMa, we also evaluated its sensitivity to denaturation as induced by increasing concentrations of urea. To this aim, we took advantage of the presence of two tryptophanes in HoLaMa, namely W866 and W924 (coordinates of DNA polymerase I primary structure). The fluorescence of protein tryptophanes is known to be affected by a number of factors , among w± 0.1) M . Howeverectively , were sin, as n = 1.24 ± 0.03, and the dissociation constant, KD, of the enzyme-DNA complex as equal to KD = (35.1 ± 6.1) nM, in reasonable agreement with the 67 ± 19 nM value we previously reported [Next, we tested whether the association with DNA does affect or not the thermodynamic parameters diagnostic of HoLaMa stability. First, we excited at 280 nm and detected the emission at 328 nm of samples containing 500 nM HoLaMa and increasing concentrations of the 40mer dsDNA  describing the folding to unfolding transition of HoLaMa was significantly affected by the binding of dsDNA to the enzyme. Indeed, we determined an overall thermodynamic stability of ΔG = (10.9 ± 3.5) kJ/mol, a cooperativity of unfolding of m = (3.3 ± 0.9) kJ/mol*M, and a transition midpoint of CM = (3.4 ± 1.4) M for the HoLaMa-DNA complex  s-1 (kobs = (64 ± 10) s-1 [In particular, we determined the fluorescence of W924 when an excess of enzyme was rapidly mixed to the 40mer polyA DNA see . The flu± 7) s-1 . This va 10) s-1 . However 10) s-1 , in agre-). In particular, we used for these assays three different 30mer DNAs featuring a template overhang of 15 bases  inactivating the proofreading activity  we determined v as equal to 13.08 ± 0.05, 11.04 ± 0.03, 17.98 ± 0.07 nM/s the activity of HoLaMa, Klenow, and Klenow exo-, respectively  was significantly higher when compared to that detected with 100 μM dTTP (v = 5.12 ± 0.02 nM/s). When the assay was repeated by using 1 μM 30mer 7AC DNA, 100 μM dTTP, and 100 μM dGTP the extension of the DNA substrate was expected to progress beyond the seventh position (v = (2.963 ± 0.003) nM/s, which is slower when compared to that detected in the presence of dTTP only (v = (1.880 ± 0.001) nM/s (v = (0.270 ± 0.005) nM/s which can be ascribed to the background, by unspecific reactions, of this series of assays  nM/s . TherefoWe used the 30mer 3AC DNA  to furth-,  the seventh position of the template overhang  Solutions containing 500 nM HoLaMa  and supplemented (blue line) or not (red line) with 7.6 M urea were excited at 280 nm. The emission spectra were recorded over the 290–400 nm interval and are reported in arbitrary units (a.u.). (B) Difference emission spectrum between native and denatured HoLaMa.(TIF)Click here for additional data file.


This study is the first to validate and compare the STATSports Apex 10 and 18 Hz. Between-analysis (t-test) for total distance and Vpeak reported non-significant differences. Apex units reported a small error of around 1–2% compared to the criterion distances during 400-m, 128.5-m circuit, 20-m trials, and Vpeak. In conclusion, both units could be used with confidence to measure these variables during training and match play.The aims of this study were (i) to investigate the criterion validity (vs. gold standard measurements) of the 10 and 18 Hz STATSports Apex units for measuring distances and peak speed (Vpeak) outcomes and (ii) to investigate the between-unit variability. Twenty university students were enrolled in the study . The criterion validity was tested by comparing the distances recorded by the units with ground truth reference . Vpeak values were compared with those determined by a gold standard criterion device  during a linear 20-m sprint. The distance biases for the Apex 10 Hz in the 400-m trial, 128.5-m circuit, and 20-m trial were 1.05 ± 0.87%, 2.3 ± 1.1%, and 1.11 ± 0.99%, respectively, while for the Apex 18 Hz the biases were 1.17 ± 0.73%, 2.11 ± 1.06%, and 1.15 ± 1.23%, respectively. Vpeak measured by the Apex 10 and 18 Hz were 26.5 ± 2.3 km h In team sport context, the common technologies utilized to quantify external load parameters are: global navigation satellite systems (GNSS), video tracking systems (VTS), and local positioning systems (LPS/radar) . PreviouGNSS devices present large variability in accuracy among different manufacturers’ models . TherefoGreater accuracy in the data collected may help sport scientists through understanding the players’ performances over a given training session, as well as small variations worth of interest in the players’ physical loads and efforts load among sessions. This is particularly relevant at professional and elite levels, where small differences can have a meaningful impact on performance outcomes throughout the season. By increasing the sampling frequency of the GNSS devices have resulted in several improvements in terms of data quality, however, some limitations continue to exist. Previous studies, employing 10 Hz GNSS devices, have shown low validity and reliability when tested in sport-specific circuits, as well as during high intensity short shuttle runs and change-of-direction maneuvers . NeverthThe STATSports Apex unit is an athlete-tracking system released in August 2017, and it is widely used in professional clubs . A previous validation of the STATSports Viper GNSS unit was performed in 2016, and it reported a distance bias of 2.5% during 20-m running activity and a small to moderate bias in speed (3–9%) during 5–20-m short shuttle runs . HoweverTo date, no validation and comparison studies of the Apex 10 and 18 Hz exist; therefore, it would be acknowledgeable to report accuracy data of such units and existing differences between the models, which could offer important practical applications for practitioners working in sport contexts. This need is further required when considering that GNSS-derived data is utilized to manage players’ training loads, recovery strategies, workload implementations, and subsequent training periodization . The valTwenty physical active male and female university students were enrolled  in this descriptive study (data recorded in 2018). The experimental protocol was in accordance with the Declaration of Helsinki for study on human subjects. The Institutional Ethics Board of the University of Suffolk  approved the experimental protocol. A written informed consent was obtained from the participants of this study.Figure 1A), and a 20-m trial (linear running) . ATS II radar uses high frequency radio waves and measures changes of speed of a moving object (Doppler radar). Radar gun and laser devices are considered to be gold standard instruments for evaluating Vpeak  data were collected outdoor on an athletic track, in absence of high and large buildings in the surrounding area to enhance satellites’ reception . Both Aprunning) . The parng Vpeak . The radng Vpeak . The speng Vpeak . Stalkerng Vpeak .Participants completed a 400-m trial at a self-selected speed (jogging pace), a 128.5-m trial, a 20-m trial (jogging pace), and a 20-m sprint at maximum speed. Each participant was verbally instructed before each trial to perform the correct procedure. Participants performed a familiarization trial (week 1) before the beginning of the experimental trial. A 400-m trial, 128.5-m circuit, 20-m trial, and 20-m sprint were performed by the participants of this study . The experimental session was performed during a sunny day without rain or clouds.Figure 2). Apex 10 Hz is a multi-GNSS augmented unit, which is capable of acquiring and tracking multiple satellite systems  concurrently to provide the best possible positional information. In contrast, Apex 18 Hz units have a higher sampling frequency than Apex 10 Hz, but its acquisition system is based only on GPS. GNSS data (speed and distance) recorded by the units were downloaded and further analyzed by the STATSports Apex Software .The Apex units (10 and 18 Hz) were turned on about 10–15 min before the beginning of the test, while the subjects were familiarized with the equipment as well as the protocol procedures. Apex units present the following characteristics: dimension 30 mm (wide) × 80 mm (high), weight 48 g, 100 Hz gyroscope, 100 Hz tri-axial accelerometer, and 10 Hz magnetometer. Prior to the experiments, both Apex unit models  were placed on the back of the participant, midway between the scapulas   of the statistical distribution. Validity was assessed by calculating the bias (%) between the known distance and the unit (absolute error). Bias was interpreted as poor (>10%), moderate (5–10%), or good (<5%) . A paire(CI 90%) . Effect (CI 90%) . Intracl(CI 90%) . The bet-1 and criterion was 26.3 ± 2.4 km h-1. Mean difference (90% CI) was 0.17 , t = 1.012, p = 0.32, ES (90% CI) = 0.08 , trivial. The absolute error of the Apex 10 Hz was 0.62 ± 0.45 km h-1 and the bias was 2.36 ± 1.67% (good). ICC between Apex 10 Hz and radar gun Vpeak was nearly perfect .The Shapiro–Wilk test confirmed the assumption of normality of the statistical distribution. Apex 10 Hz GNSS distance covered in the 400-m trial, 128.5-m circuit, and 20-m trial was 398.7 ± 7.6 m, 131.4 ± 1.4 m, 20.07 ± 0.29 m, respectively, with an absolute error of 4.19 ± 3.48 m, 2.85 ± 1.4 m, and 0.22 ± 0.20 m, respectively. The bias in each trial was 1.05 ± 0.87%, 2.3 ± 1.1%, and 1.11 ± 0.99%, respectively. Vpeak measured by the Apex 10 Hz was 26.5 ± 2.3 km h-1 and criterion was 26.3 ± 2.4 km h-1. Mean difference (90% CI) was 0.24 , t = 1.973, p = 0.064, ES (90% CI) = 0.08 , trivial. The absolute error of the Apex 18 Hz was 0.52 ± 0.30 km h-1 and the bias was 2.02 ± 1.24% (good). ICC between Apex 18 Hz and radar gun Vpeak was nearly perfect .Apex 18 Hz distance covered in the 400-m trial, 128.5-m circuit, and 20-m trial was 396.8 ± 11.6 m, 131.2 ± 1.3 m, and 20.19 ± 0.28 m, respectively, with an absolute error of 4.7 ± 2.9 m, 2.7 ± 1.4 m, and 0.23 ± 0.25 m, respectively. The bias in each trial was 1.17 ± 0.73%, 2.11 ± 1.06%, and 1.15 ± 1.23%, respectively. Vpeak measured by the Apex 18 Hz was 26.5 ± 2.6 km ht = 0.653, p = 0.522; in the 128.5-m circuit of 0.15 , t = 0.319, p = 0.753; in the 20-m trial of -0.12 , t = 1.45, p = 0.163; or in the 20 m sprint Vpeak of -0.04 , t = 0.403, p = 0.691, ICC (CI 90%) = 0.96 .Between-analysis (Apex 10 Hz vs. Apex 18 Hz) did not find any meaningful differences in the 400-m trial  1.78 , Table 1).The aims of this study were to validate and to compare the STATSports units (Apex 10 and 18 Hz) when measuring distance and Vpeak outcomes during sports-specific activities. The main findings of this research were that the STATSports Apex (10 and 18 Hz) reported a small bias (<5%) for distance measures  and Vpeak (20 m sprint), thus supporting the validity of both Apex models. Trivial (ES) differences were found between the Apex 10 and 18 Hz in the parameters analyzed   in team sports . PreviouApex reported lower or equal bias than previous wearable devices analyzed, such as 9.6–32.4% in TD (20 m sprint) MinimaxX team (1 Hz), 1.7–6.7% in TD (30 m sprint) MinimaxX v4.0 (10 Hz), and 2.9–7.7% in TD (30–40 m print) SPI-Pro (5 Hz) . A recenIn the current study, both Apex 10 and 18 Hz units resulted in small bias in distance measures during the 400-m, 128.5-m, and 20-m trials, as well as in Vpeak during the 20-m sprint. These scores are smaller than the bias reported for TD and Vpeak of previous Viper models. A previous study, employing the STATSports Viper (10 Hz) units, found a bias ranging from of 8.7 to 3.4%  in speed outcomes during 5–20-m running activity associated to this model . SeveralAn innovative finding of the current study is the comparison between Apex 10 and 18 Hz on TD and Vpeak. The between-unit analysis (Apex 10 Hz vs. Apex 18 Hz) found trivial to small differences in the 400-m trial (ES = 0.18), 128.5-m circuit (ES = 0.11), 20-m trial (ES = 0.42), and in 20-m sprint Vpeak (ES = 0.03). Following these results, it may be concluded that these two Apex models do not present meaningful differences in the parameters evaluated, therefore sport scientist and coaches could use both models interchangeably in their practice (even if consistency in the model utilization should be recommended). This is the first study to evaluate and compare such units, thus it is not possible to make any comparisons with other research work published. A future study could replicate the current study, analyzing Apex units.This study presents three main limitations: firstly, we evaluated sports-specific movements by linear running activity and circuits involving human participants. It is well known that this approach (largely used in the literature) might present errors, and therefore the results reported in the current study should be considered carefully. For instance, during the 400-m trial and the 128.5-m circuit, the variability due to the human movement inconsistency could have affected the findings. Sports scientists and coaches should consider that research studies involving human subjects might show some inconsistency between the designs. The application of mechanical devices collecting at higher frequency than the human movement could be used to test distance and speed. Secondly, the Vicon motion analysis system has been recently proposed as the new gold standard technology for evaluating sports-specific movements . Such teThe evaluation of GNSS Apex 10 and 18 Hz validity is a critical step for its application in team sports and for research purposes. Apex units are largely utilized in team sports . This study provides innovative findings and offers important implications for sports scientists and researchers involved with such technologies for practical and research purposes. Apex units (10 and 18 Hz) showed good levels of accuracy (bias < 5%) in sport specific metrics. Practitioners can be mindful that the units analyzed in this study can be used to evaluate distance covered during linear running and sports-specific activity. Moreover, Apex units can be used to evaluate Vpeak in sports, since non-significant and trivial differences were found compared to criterion Vpeak (radar gun). External load interpretation and the associated decision making processes can only be made when the validity of GNSS technology is well known. Coaches and sport scientists can use the metrics derived from STATSports Apex units, which have been analyzed in this study, to quantify players’ workload during training sessions and to optimize the overall training periodization.MB was the main investigator. GC was involved in the data analysis and statistics. AS had a critical role for data recording. AI was the supervisor of the project.AI was employed at the time of the study by Maccabi Tel Aviv FC. 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. The handling Editor declared a past co-authorship with one of the authors AI.


Moreover, their total phenolic contents (TPC) were detected by Folin-Ciocalteu assay and were in the range of 24.77 ± 2.02 to 252.65 ± 4.74 mg gallic acid equivalent (GAE)/g DW. Generally, Dianqing Tea, Lushan Yunwu Tea, and Xihu Longjing Tea showed the strongest antioxidant activities among 30 teas. Furthermore, the phenolic compounds in tea infusions were identified and quantified, with catechins most commonly detected, especially in green tea infusions, which were main contributors to their antioxidant activities. Besides tea polyphenols, considerable content of caffeine also presented in 30 tea infusions.Tea is among the most consumed drink worldwide, and its strong antioxidant activity is considered as the main contributor to several health benefits, such as cardiovascular protection and anticancer effect. In this study, the antioxidant activities of 30 tea infusions, which were obtained by the mimic of drinking tea of the public, from green, black, oolong, white, yellow and dark teas, were evaluated using ferric-reducing antioxidant power (FRAP) and Trolox equivalent antioxidant capacity (TEAC) assays, ranging from 504.80 ± 17.44 to 4647.47 ± 57.87 µmol Fe Camellia sinensis) is the most consumed drink in the world except for water, owing to its pleasant sensory properties, broad health benefits and unique sociocultural characteristics [Tea  of 30 well-known teas from China, including 4 black teas, 5 dark teas, 9 green teas, 4 oolong teas, 3 white teas, and 5 yellow teas were evaluated using ferric-reducing antioxidant power (FRAP) and Trolox equivalent antioxidant capacity (TEAC) assays, and their total phenolic contents (TPC) were determined by the Folin–Ciocalteu method. Moreover, polyphenols and caffeine in 30 tea infusions were detected using high-performance liquid chromatography coupled with photodiode array detector (HPLC-PDAD). Tea infusions could have different types and contents of antioxidants compared to the fat-soluble, water-soluble, and insoluble-bound fractions of tea. The results obtained can provide guidance for the public to select tea with strong antioxidant capacity. The results are also very helpful for the nutritionist and epidemic experts to calculate/assess the intake amounts of antioxidants from tea.The 2,4,6-tri(2-pyridyl)-s-triazine (TPTZ), 2,2′-azino-bis(3-ethylbenothiazoline-6-sulphonic acid) diammonium salt (ABTS), 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox) and Folin and Ciocalteu’s phenol were produced by Sigma-Aldrich . The standard chemicals, i.e., gallocatechin (GC), epigallocatechin (EGC), epigallocatechin gallate (EGCG), catechin (C), epicatechin (EC), epicatechin gallate (ECG), catechin gallate (CG), gallocatechin gallate (GCG), gallic acid, chlorogenic acid, ellagic acid, myricetin, quercitrin, kaempferol, astragalin, quercetin, theaflavin and caffeine, were obtained from Derick Biotechnology Co., Ltd. . The methanol and formic acid were of chromatography-grade and purchased from Kermel Chemical Factory . All the other reagents (such as sodium carbonate anhydrous) were of analytical grade and obtained from Damao Reagent Factory .The 30 teas  were pur4 was used as a standard, and the results were expressed as µmol Fe2+/g dry weight (DW).The FRAP assay was utilized to determine the reducing ability of tea infusions based on literature previously published with minor modification . In brie+ working solution with absorbance controlled at 0.710 ± 0.050. After the reaction at room temperature for 6 min, the absorbance was measured at the wavelength of 734 nm. Trolox was used as a standard, and the results were presented as µmol Trolox/g DW.The TEAC assay was used to measure the free radical-scavenging ability of tea infusions based on previously published literature that was slightly modified . BrieflyThe TPC was measured by the Folin–Ciocalteu method . The 0.5v/v), respectively, and the temperature was 35 °C with the flow rate of 1.0 mL/min. The elution gradient was set as follows: 5% A (0 min), 20% A (10 min), 22% A (15 min), 25% A (20 min), 40% A (40 min), 42% A (50 min), 50% A (60 min), 95% A (70 min), 5% A (70.1 min) and 5% A (75 min). Phenolic compounds and caffeine in tea infusions were identified via comparing their retention time and ultraviolet-visible (UV-vis) spectra with those of standard compounds. Quantitative analysis was performed according to the peak area under the maximal absorbance wavelength, and the contents were expressed as mg/g DW. The limit of detection (LOD) and limit of quantity (LOQ) of this method were 2 and 5 µg/mL, respectively.Tea infusions were subjected to HPLC coupled with a PDAD  and an Agilent Zorbax Extend-C18 column  for determination of phenolic compounds and caffeine. The HPLC method referred to previous literature with minor modification . In briep < 0.05.All data obtained after 3 repetitions were analyzed using SPSS 20.0.  and expressed as mean ± standard deviation (SD). Significant differences among groups were manifested using one-way analysis of variance (ANOVA) followed by an LSD post hoc test, with a significant level set at 0.05. For potential correlation among measurements, the linear regression analysis was performed. Statistical significance was defined at 2+/g DW , yellow tea (3582.93 ± 433.94 µmol Fe2+/g DW), oolong tea (1539.13 ± 351.86 µmol Fe2+/g DW), dark tea (1472.27 ± 691.91 µmol Fe2+/g DW), black tea (1283.47 ± 858.62 µmol Fe2+/g DW) and white tea (1160.80 ± 190.32 µmol Fe2+/g DW).The FRAP values of different tea infusions were quite different and varied from 504.80 ± 17.44 to 4647.47 ± 57.87 µmol Fe2+/g DW . Among tThe TEAC values of different tea infusions varied greatly in a range of 166.29 ± 24.48 to 2532.41 ± 50.18 µmol Trolox/g DW . Among 3The total phenolic contents of 30 tea infusions were quite differed ranging from 24.77 ± 2.02 to 252.65 ± 4.74 mg GAE/g DW . The phe2R of 0.840 (p < 0.001), which indicated that components in tea infusions possessed both good reducing ability and free radicals cleaning capacity. Moreover, the antioxidant activities of 30 teas were strongly correlated with their total phenolic contents . Thus, the phenolic compounds could play key roles in the strong antioxidant activities of teas.The correlations between values of FRAP, TEAC, and TPC of 30 teas are presented in The phenolic compounds in different tea infusions were quite diverse . The chrThe highest contents of catechins were found in green tea infusions among 6 tea categories, and all green tea infusions possessed such 6 principal catechins as follows (with contents in a decreasing order): EGCG (48.48 ± 9.16 mg/g DW), EGC (45.25 ± 25.87 mg/g DW), ECG (20.47 ± 8.38 mg/g DW), EC (7.97 ± 1.46 mg/g DW), GC (6.19 ± 2.02 mg/g DW), GCG (6.09 ± 1.47 mg/g DW). Thus, EGCG was the richest catechins in green tea infusions.The gallic acid was found in all 30 tea infusions in low contents varying from 0.29 ± 0.02 to 3.77 ± 0.32 mg/g DW. The top 5 teas with the highest contents of gallic acid were Yichang Congou Black Tea (3.77 ± 0.32 mg/g DW), Fuzhuan Brick Tea (3.22 ± 0.22 mg/g DW), Huoshan Large Yellow Tea (3.16 ± 0.26 mg/g DW), Fenghuang Shuixian Tea (3.05 ± 0.14 mg/g DW) and Keemun Black Tea (2.87 ± 0.15 mg/g DW). Furthermore, many tea infusions contained certain amounts of ellagic acid, quercitrin, astragalin, and chlorogenic acid, but the quercetin and kaempferol could only be found in 2 or 3 tea infusions. Additionally, theaflavin was only detected in Dianhong Congou Black Tea and Yichang Congou Black Tea, with contents of 0.66 ± 0.06 and 0.70 ± 0.08 mg/g DW, respectively.Aside from phenolic compounds, caffeine was also quantified in this study . High co3+ to Fe2+) and TEAC , were combined to evaluate comprehensively the antioxidant activities of tea infusions [It is difficult to fully evaluate the antioxidant activities of natural antioxidants just using a single determination method, so employing methods involved in different determination principles are essential ,25,26. Infusions ,28.2+/g DW; TEAC, 166.29 ± 24.48 to 2532.41 ± 50.18 µmol Trolox/g DW) and rich phenolic compounds (24.77 ± 2.02 to 252.65 ± 4.74 mg GAE/g DW), which were higher than many other natural products, like 223 medicinal plants  [2+/g DW; TEAC, 4.718–85.719 µmol Trolox/g DW; TPC, 2.440–44.844 mg GAE/g DW) [The 30 teas possessed potent antioxidant activities  oxidation [In this study, polyphenols in 30 teas were determined with catechins being the dominant phenolic compounds. Moreover, green tea was a richer source of catechins among 6 categories of teas, in which catechins comprised 11.08%–16.21% of the dry weight. EGCG was the richest catechins. It has been reported that EGCG possessing the most number of phenolic hydroxyl groups manifests the strongest antioxidant activity in catechins, which is even stronger than vitamins C and E . It has xidation . TherefoApart from phenolic compounds, habitual tea infusion intake could also introduce high contents of caffeine making up 1.24%–4.75% of the dry weight of tea leaves. A clinical trial has demonstrated that high caffeine intake could accelerate weight loss via relatively higher thermogenesis and fat oxidation ,44. HoweIn this study, the infusions of 30 selected teas were prepared mimicking daily practices, and their antioxidant activities were evaluated using different determination methods. The results suggested that 30 tea infusions possessed strong antioxidant activities and rich phenolic compounds, which varied greatly in specific varieties. Additionally, phenolic compounds were the main contributors to the antioxidant capacities of teas. Thus, tea is a good dietary source of natural antioxidants, especially phenolic compounds, which have a good potential to be developed into a functional drink or dietary supplements. Furthermore, the antioxidant activities and the profiles of specific bioactive substances  in tea varieties were quite different. Generally speaking, green tea possessed the highest antioxidant capacity and total phenolic content, which was also a richer source of polyphenols, especially catechins. Among 30 teas, Dianqing Tea, Lushan Yunwu Tea, and Xihu Longjing Tea are good sources of natural antioxidants. Thus, consumers should take into account the characteristics of different teas comprehensively for the selection of teas that are best suited to their needs. Moreover, the results are also very helpful for the nutritionist and epidemic experts to calculate or assess the intake amounts of antioxidants from tea.