Primary hyperoxaluria type I (PH1) is caused by AGXT gene mutations that decrease the functional activity of alanine:glyoxylate aminotransferase. A build-up of the enzyme’s substrate, glyoxylate, results in excessive deposition of calcium oxalate crystals in the renal tract, leading to debilitating renal failure. Oxidation of glycolate by glycolate oxidase  is a major cellular source of glyoxylate, and siRNA studies have shown phenotypic rescue of PH1 by the knockdown of HAO1, representing a promising inhibitor target. Here, we report the discovery and optimization of six low-molecular-weight fragments, identified by crystallography-based fragment screening, that bind to two different sites on the HAO1 structure: at the active site and an allosteric pocket above the active site. The active site fragments expand known scaffolds for substrate-mimetic inhibitors to include more chemically attractive molecules. The allosteric fragments represent the first report of non-orthosteric inhibition of any hydroxy acid oxidase and hold significant promise for improving inhibitor selectivity. The fragment hits were verified to bind and inhibit HAO1 in solution by fluorescence-based activity assay and surface plasmon resonance. Further optimization cycle by crystallography and biophysical assays have generated two hit compounds of micromolar (44 and 158 µM) potency that do not compete with the substrate and provide attractive starting points for the development of potent and selective HAO1 inhibitors. AGXT gene  has been proposed as a target for substrate reduction therapy. Accumulation of the substrate of HAO1 is benign as the accumulated glycolate is highly soluble and can be excreted freely. Similarly, there is no damaging deficit of metabolites downstream of HAO1 as sufficient levels of glycine and pyruvate can be obtained through other pathways. The safety of HAO1 inhibition is further supported by reports of asymptomatic loss-of-function n humans . Proof-on humans , RNAi knn humans , and sman humans . RNAi tan humans  and recen humans .2O2. Human HAO1 (hHAO1) exhibits a broad substrate range from the two-carbon glycolate to the 16-carbon 2-hydroxypalmitate, with glycolate being the preferred substrate (in vitro (HAO1 is a flavin mononucleotide (FMN)-dependent enzyme that oxidizes α-hydroxy acids to the corresponding α-keto acids, with the concomitant reduction of molecular oxygen to Hubstrate . The othin vitro , but itsin vitro . Two keyin vitro . This gain vitro   or MolPort (gating loop site compounds).E. coli BL21 (DE3) cells. hHAO1 was cultured in auto-induction Terrific Broth  buffer, pH 8.0 . For soain vitro was determined using the Amplex Red fluorescence hydrogen peroxide assay (Sigma Aldrich). The Amplex Red assay reagent contained horseradish peroxidase  and its substrate Amplex Red  in assay buffer. The assay buffer contained 50 mM sodium phosphate, pH 7.4, 200 mM KCl, 2 mM MgCl2, and 0.01% TritonX. Determination of suitable assay parameters is described in the ®). Following 10 min incubation at room temperature, 10 µl/well of Amplex Red reagent was added. Fluorescence emission was measured at 585 nm, with excitation at 570 nm, after a further incubation period of 10 min, using a PHERAstar plate reader with a FI 540 590 optics module. All reactions were performed in technical triplicates for two different preparations of hHAO1. Reaction rate, defined as total H2O2 produced by the hHAO1 reaction, was determined from the H2O2 standard curve. Data were plotted using GraphPad Prism software; curve fitting was performed with nonlinear least-squares regression fit to log (inhibitor) vs response (three parameters) equation (for IC50 determination) and to mixed model, competitive, non-competitive, and uncompetitive inhibition models from the GraphPad Prism Enzyme kinetics–Inhibition equations and the best fit was selected for each ligand by comparison of Akaike’s information criterion probability scores and extra sum-of-squares F test p values calculated by the software (for inhibition mode determination). An additional parameter calculated when fitting to enzyme kinetics–mixed model inhibition equation was the α value. This value is defined as the difference between the inhibition constant for the free enzyme (Ki) and the inhibition constant for the enzyme-substrate complex (αKi) and can be related to the initial velocity (V0), substrate concentration ([S]), inhibitor concentration ([I]), and other kinetic parameters  as follows: V0 = Vmax [S]/(Km (1 + [I]/Ki) + [S](1 + [I]/(α∗Ki))). When an inhibitor binds with equal affinity to both the enzyme alone and the enzyme-substrate complex, α = 1, indicating a non-competitive inhibition mode (Ki = αKi). When an inhibitor preferentially binds to the enzyme alone, α > 1, with a very large value indicating an inhibition mode close to competitive inhibition . When inhibitor preferentially binds to the enzyme-substrate complex, α < 1, with a very small value indicating an inhibition mode close to uncompetitive inhibition .hHAO1 activity Purified hHAO1 (30 μg/ml) was attached via the C-terminal His6-tag to a Ni-NTA chip to a density of 5000 RU. The assay buffer was 20 mM HEPES, pH 7.5, 0.05% TWEEN20, 200 mM NaCl, 0.5 mM TCEP, 5% DMSO. A serial dilution (11 concentrations) was prepared in the above buffer for each analyte  by 1:1 dilution from 100 to 0.05 µM and the subsequent solutions were passed over the chip at a flow rate of 30 μL/min. Data are from an n = 1 experiment.o-Fc) electron density maps, with background correction, performed using multi-crystal isomorphous difference density maps  and one fragment at the surface-exposed gating loop, ∼12 Å from the active site (3) (o-Fc maps before background (ground state) subtraction and 2mFo-Fc maps output from PanDDA are shown in 4, 5) and one gating loop fragment (6).We applied crystallography-based fragment screening to determine hHAO1 crystal structures with novel ligands bound . Among t1, 2, 4, and 5 occupy the glycolate binding site, stacking with the FMN co-factor sulfanyl]-1,2,3-thiadiazole-4-carboxylic acid (CCPST) determined in this study  makes few interactions but in fragment 2 it displaces Tyr208, further disrupting the hydrogen bonding network around the gating loop.Fragments omplexes . In bothCollectively these four fragments represent embellishment to known inhibitor scaffolds that have not been explored, and we next aimed to characterize their utility as starting points for inhibitor development by characterizing them in solution.3 and 6, bound above the active site where the gating loop would otherwise be, contain a phenylpiperazine scaffold  and maintains the hydrogen bond with the backbone nitrogen of Lys135 but the sulfanyl-triazole extension prevents movement of Tyr208 to the location as seen with 3 and instead the piperazine ring is twisted slightly to avoid clashing (Binding of clashing .We next asked whether this gating loop pocket had been observed indirectly in structures of HAO1 (spinach or human) bound to active site inhibitors. We inspected structures of HAO1 bound to active site inhibitors that contained a secondary moiety, away from the heteroaryl-carboxylic acid, that could theoretically reach the gating loop pocket described in this work. Of the seven compounds that could occupy the gating loop pocket , only onin crystallo, we next characterized their binding and inhibition of hHAO1 in solution. We first determined the binding affinity of the control inhibitor CCPST for hHAO1, which has not previously been reported for any HAO enzyme, using surface plasmon resonance (SPR). We immobilized His-tagged hHAO1 to an Ni-NTA coated SPR chip and passed increasing concentrations  of CCPST across it at a flow rate of 30 μl/min. Using this method, we measured a KD of 47.5 µM for CCPST (1–3) demonstrated measurable, specific binding to hHAO1 in solution at concentrations from 15 to 50 μM, though the binding was too weak to determine KD values  (50 3.6 µM), flavin dehydrogenase domain of yeast flavocytochrome b2 (IC50 6 µM), and hHAO1 (IC50 4.5 µM)  , rat LCH 4.5 µM) . However 4.5 µM) , which wof hHAO1 . In thisof hHAO1 , consist1 had an IC50 of 420 µM whereas the remaining active site fragments, 2, 4, and 5, demonstrated 10, 26, and 37% inhibition respectively at 1 mM. The loop site fragments 3 and 6 showed 2% and 12% inhibition respectively at 1 mM, with fragment 3 showing 19% inhibition at 10 mM.We then measured inhibition of hHAO1 activity by the fragments , showingThe above-observed binding and inhibitor effects prompted us to perform one round of optimization to improve fragment potency. Of the different approaches described to optimize fragment hits , we adop1 scaffold and substitution of the cyclohexane (that showed no interaction with the protein) for the fragment 2 scaffold. Compounds were screened for inhibition of hHAO1 in the Amplex red assay at 1 mM concentration. Half-maximal inhibitory concentration (IC50) and  values   or an amino-ethyl  to the carboxamide of fragment 1 provided a 5-fold increase in potency , 1,3-dimethyl , 1-methyl , or 6-methyl  substituents result in a more than 10-fold increase in potency  versus glycolate concentrations (0–500 µM) to determine the inhibition mode of these improved compounds. As expected, all compounds derived from active site fragments demonstrate competitive inhibition mode with respect to glycolate, entirely consistent with their binding in the substrate-binding pocket , or a non-orthosteric site . Glycolate-titration experiments with compound 13 showed a concentration-dependent decrease in Vmax, up to 2.8-fold reduction at 1 mM compound (p < 0.0001) and a concentration-dependent increase in Km, up to 4-fold increase at 1 mM compound (p < 0.0001), collectively indicating mixed model inhibition with respect to glycolate by compound 13, which is supported by fitting in GraphPad Prism  and a concentration-dependent increase in Km, up to 4.9-fold increase at 1 mM compound (p < 0.0001), supporting mixed model inhibition, as observed when fitting in GraphPad Prism (α) values  and binding to the enzyme-substrate complex (αKi), see materials and methods for details) of 13 and 18 for compounds 13 and 14, respectively, which indicates both compounds bind more readily in the absence of substrate , and in doing so reduce both substrate turnover (decreased Vmax) and hHAO1 affinity for glycolate (increased Km).Therefore, our competition data indicate that compounds Inhibition of hHAO1 is a promising and recently validated approach for the treatment of primary hyperoxaluria. There is also potential to inhibit hHAO1 in the treatment of multifactorial kidney stone formation disorders as approximately 12% of the world population will suffer from kidney stone disease (urolithiasis) within their lifetime and 76% of these stones contain oxalate .Currently, the only approved therapeutic modality for hHAO1 inhibition is RNAi. Existing small molecules in development include chemical series of pyrazole , triazolAside from the lack of diversity in these chemical series, their therapeutic use is limited by the metabolic instability, poor cell permeability, and cellular toxicity associated with carboxylic acid-containing drugs , and con1 and its derivatives  are superficially similar to published pyrazole inhibitors in structure except that, instead of a carboxylic acid, they contain a carboxamide (1), methyl-carboxamide (4), hydroxy-carboxamide (7), or aminoethyl-carboxamide (8) group as part of the polar head group and the hydrophobic moiety is a phenyl ring fused to the pyrazole. Exploration of similar rLCHAO inhibitors showed no activity upon similar modification of the pyrazole ring and a 5-fold reduction in potency with a carboxamide  are somewhat similar to published diketone scaffolds  had a 6-methyl substituent and potency was 3-fold lower with a carboxylic acid, the traditional HAO1 binding functional group, at this position (9). Further structural work will be needed to unravel the interactions made by these very small fragments to guide their optimization.Fragment caffolds  but pipehe plant , pig , combined with the enzyme kinetics of follow-up compounds , indicate a persistent, true allosteric site at the gating loop that is a novel target for developing hHAO1 inhibitors to treat primary hyperoxaluria. A more specific and thorough investigation optimizing compounds 13 and 14 would likely yield potent, selective lead candidates for the development of therapeutic hHAO1 inhibitors for primary hyperoxaluria, and as such these early compounds present considerable utility for developing molecules distinct in both chemical scaffold and mechanism of action from all previously known inhibitors.Collectively the two fragment-bound structures (


Dear Editor,The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel emerging coronavirus that has spread worldwide since breaking out in late 2019 and has led to hundreds of millions of infections and millions of human deaths . The genHere, we try to map the phosphorylation sites in N protein of SARS-CoV-2 and investigate their functions. Flag-tagged N protein was expressed in HEK293T cells and the phosphorylation level of N protein was detected by immunoprecipitation followed by western blotting assay. As expected, the phosphorylation can be easily detected by anti-phospho-serine (S)/threonine (T) antibody , implyinWe integrated our results with the reported phosphorylation sites of N protein in different experimental settings . In totain vitro-transcribed 5′ untranslated region (UTR) of viral RNA. The in vitro RNA-binding assay showed that the phosphorylation mimic mutations, S51E and T54E, dramatically blocked the RNA-binding capacity of the NTD domain 268, respectively Figure . Intrigun Figure .We further evaluated the influence of phosphorylation in the NTD domain. The full-length WT or mutant N proteins were transfected into Caco-2 cells and the distribution was visualized by immunostaining. Similarly to the WT N protein, T49A, T49E, T54A, and T54E mutant proteins diffused in cytoplasm Figure . HoweverStress granules (SGs) are nonmembranous mRNA‒protein aggregates, which can be induced by viral infection and function to restrict viral proliferation . PreviouIn summary, our study demonstrate that N protein of SARS-CoV-2 is highly phosphorylated in host cells and finds several novel phosphorylation sites, among which the phosphorylation on S51 resides directly at the center of the RNA-binding pocket of the N protein NTD. The phosphorylation mimicry study suggests that S51 phosphorylation may inhibit the RNA-binding capacity of the NTD domain and further promote the phase separation of N protein and release its inhibition in the SG formation. Further efforts are needed to find out the kinase/phosphatase of this site and illustrate its detailed regulatory mechanism in the future. Also, it will be interesting to explore the influence of S51 phosphorylation on viral RNA replication or virion packaging with a live virus system in the high-level biosafety laboratory.mjac003_Supplemental_FileClick here for additional data file.


Cryopreservation of biological matter in microlitre scale volumes of liquid would be useful for a range of applications. At present, it is challenging because small volumes of water tend to supercool, and deep supercooling is known to lead to poor post-thaw cell viability. Here, we show that a mineral ice nucleator can almost eliminate supercooling in 100 µl liquid volumes during cryopreservation. This strategy of eliminating supercooling greatly enhances cell viability relative to cryopreservation protocols with uncontrolled ice nucleation. Using infrared thermography, we demonstrate a direct relationship between the extent of supercooling and post-thaw cell viability. Using a mineral nucleator delivery system, we open the door to the routine cryopreservation of mammalian cells in multiwell plates for applications such as high throughput toxicology testing of pharmaceutical products and regenerative medicine. ThereTnuc) is determined by the presence (or absence) of heterogeneous ice nucleating sites in the form of impurities within the sample media or features on walls of the vessel [in situ in a high-throughput multiwell plate format would be highly beneficial to the field of drug discovery and toxicity screening. Also, reliably controlled IN would be advantageous in fields such as reproductive medicine and the emerging fields of the banking of complex tissues for transplantation [In the absence of controlled IN, the temperature that IN occurs is an unpredictable variable with aqueous CPA solutions typically supercooling well below their equilibrium melting points –8. Ice ne vessel ,10, ofteantation ,15 and rantation . Howeverantation . CurrentCustomary practice for inducing IN during cryopreservation is ‘manual’ nucleation, where touching the outside of the vessel with a very cold object locally cools the contents enough to trigger IN ,18. WhilAn alternative method of controlling IN is introduction of ice nucleating materials to the cryopreservation medium in a manner that does not interfere with the specimen and which facilitates removal after thawing . Ice nucin situ in 96-well plate format. Mineral powders are emerging as materials that could be used for controlling IN in cryopreservation applications [We report here a formulation based on a ‘hyperactive’ variety of potassium (K)-feldspar, ‘LDH1’, as a novel passive ice nucleating material with biocompatibility potential for use with cryopreserving cultured cells ications ,36 and pications –39 due tications ,41. In pications ,37,39 anications ,42,43. Wications . These sications .in situ within multiwell plates using monolayer cultures of immortalized human hepatocytes under small aliquots of CPA. We do this firstly by directly linking Tnuc of CPA in a 96-well plate to the post-thaw cell recovery rate on an individual well by well basis using remote temperature measurements from infrared (IR) thermography. Secondly, we trial the high throughput cell freezing and recovery performance in several 96-well plates with mammalian cell monolayer cultures frozen with IN controlled by LDH1 delivered in IceStart arrays [In this study, we characterize the exceptional ice nucleating ability of LDH1 in sub-millilitre aliquots of both pure water and CPA solution in 96-well plates in order to quantitatively compare its ice nucleating activity with other ice nucleating substances applied to this format. We then demonstrate how LDH1 can aid cell cryopreservation t arrays ,36.. 2. 2.1et al. [An ice nucleating mineral-based material—LDH1—was sourced for this study along with four additional materials  that are already known as having highly active ice nucleating activities and also easily dispersible in water. LDH1 was obtained from a sample of potassium feldspar originating from the Mt Malosa locality, Malawi, a source that has produced samples with similarly exceptional ice nucleating activity ,44, the et al. , then gret al. –27.. 2.2To quantify the ice nucleating activity of LDH1 and other ice nucleators, we performed droplet freezing assays using a combination of two instruments ,46. The 2 gas flow to prevent frost formation on the coverslip. For each run, the droplets were cooled at a rate of 1°C min−1 until all droplets had frozen. Droplet freezing IN events were detected visually with a webcam and the corresponding temperature obtained from the plate temperature thermocouple in the cooling plate.For the µl-NIPI experiments, 30–50 µl sized droplets of IN material suspension were pipetted at room temperature onto a cleaned hydrophobic glass coverslip  resting on an aluminium cooling stage powered by a programmable Stirling engine freezer . The stage was then enclosed inside a Perspex cell with a dry N−1 until all droplets were frozen. An IR camera  mounted above the cooling plate collected images of the 96-well plate every 15 s during freezing runs with each pixel in the images comprising temperature data with the pixel at the centre of each well being defined as the droplet temperature. Harrison et al. [b). The latent heat released by a freezing droplet can cause significant warming of droplets in neighbouring wells, so for each run every other well was left empty and filled in ‘chessboard pattern’ so to prevent such artefacts [The larger volume droplet freezing assays were performed on the IR-NIPI instrument where 50 µl droplets of IN material suspended in water (or CPA as required) were pipetted into gamma-ray sterilized 96-well plates  and placed onto the cooling plate of a programmable Stirling engine freezer  then subjected to a cooling protocol of 1°C minn et al.  report trtefacts . Followirtefacts . This mefice(T)—for a given aqueous suspension:n(T) is the number of droplets frozen at temperature T and N is the total number of droplets in the assay. Combined with knowledge of the mass of ice nucleator per droplet (m), we can derive for any material the density of active sites per unit mass as a function of temperature, nm(T)—a description of the intrinsic ice nucleating activity of a material on a mass-by-mass basis—using the following equation [Both the µl-NIPI and IR-NIPI techniques produce data for fraction of droplets frozen as a function of temperature—equation ,48:2.2nmnm(T) allows us to compare the intrinsic ice nucleating activity of particulate samples such as ground mineral powders, normally measured by active sites per INP surface area, ns(T), with non-particulate ice nucleating materials such as Snomax and pollen washing water.This assumes that sites contained within the material activate at a characteristic temperature without any significant time-dependent component . Using n. 2.32 in T75 flasks with culture medium (CM) comprised of RPMI 1640 supplemented with 10% fetal bovine serum, 100 kIU l−1 penicillin and 0.1 µg l−1 streptomycin for 1 week before first passage. Upon each passage up to five (depending on cell yield), 96-well plates pre-coated with rat tail collagen  were seeded with cells at a density of 50 000 per well under 200 µl per well of CM and then cultured for 48 h by which time confluent monolayers were achieved. One plate out of each set was assigned as a non-freeze control ‘baseline’ plate while the remaining plates were prepared for cryopreservation. All wells were seeded apart from the edge wells in order to avoid edge effects that are a known source of variability in cell growth when culturing cells in 96-well plates [Tnuc and cryopreservation survival to be quantified, a single culture plate was cryopreserved using the IR-NIPI instrument. Here every other well in the plate was left empty of cells and CPA, consistent with the droplet freezing assay method described above.HepG2 cells, an immortalized human hepatocyte carcinoma cell line, were used in all cryopreservation experiments  and procl plates ,52. For For the high-throughput trials, four plates were cryopreserved—two plates for the immediate post-thaw trial and two plates for the extended 5-day post-thaw trial (see below). Prior to cryopreservation culture medium was replaced with 100 µl per well of a CPA formulation optimized for this cell type, consisting of 10% (v/v) dimethyl sulfoxide and 0.015 M trehalose in CM. The plates were then cooled with ice-bath-cooled aluminium blocks to minimize the toxic effects of the CPA.Tnuc values were needed. We achieved this by spiking a small number of wells with a few grains of LDH1 powder to nucleate ice close to the melting point and allowing the remaining wells to nucleate in an uncontrolled manner at much lower IN temperatures. For plates used in the high-throughput trials, the individual plates were divided into areas, with each area being subject to a different IN control: IceStart arrays, manual nucleation or uncontrolled nucleation.For the plate cooled on the IR-NIPI instrument and used in the correlation experiment, a wide range of b,c) are disposable plastic devices, containing a mineral nucleator (in this case LDH1), that can be inserted into a 96-well plate containing adherent cells prior to undergoing freezing and then removed after thawing without contacting the cells. They contain ‘feet’ that slot into each well of the 96-well plate, each of which is loaded with approximately 25 mg of ice nucleating material to trigger IN in the CPA in each well.IceStart arrays b,c are d−1 from +4° to −80°C. The manual nucleation procedure was performed when the plate temperature had dipped below the CPA melting point: the cover strips were removed from the section to be nucleated and then a deep cooled object was held above the plate with the falling ice mist triggering IN in the exposed wells. The technique is described in detail by Daily et al. [The manual nucleation treatment was unsuitable for evaluation in plates that were cultured on after thawing as this method of IN was incompatible with the maintenance of a sterile culture environment. The sections for manual nucleation (if used) and uncontrolled nucleation were covered with sterile plastic strips to prevent potential contamination from IceStart arrays triggering IN. IceStart arrays loaded with LDH1 powder pre-cut into suitably sized portions were wet sterilized by immersion in 70% (v/v) ethanol which was allowed to fully evaporate before the arrays were inserted into their section of the plate. The plates were then sealed and placed on a controlled rate freezer (Asymptote Via Freeze Duo) and cooled at 1°C miny et al. . The pla2 over 5 days with CM replenished after 48 or 72 h.For thawing, the cryopreserved culture plates were first removed from the −80°C freezer and transferred to a −20°C freezer and held for 20 min to minimize any thermal expansion experienced by the plate upon warming . The pla. 2.4−1 in CM and allowed to equilibrate at 37°C for 2 h, during which time the dye was taken up by viable cells only. The dye solution was then removed from the wells and replaced with a wash-fix solution of 10% (w/v) calcium chloride and 1% (v/v) formaldehyde solution followed by a releasing solution (50% (v/v) ethanol acidified with 1% (v/v) acetic acid). Dye concentration per well was quantified by optical absorbance at 540 nm using a microplate reader (Thermo Scientific Multiskan GO). The absorbance values were translated to cell numbers using a standard curve constructed from neutral red absorbance values for a series of known HepG2 cell concentrations  from each culture passage.Viable cell counts were performed using the neutral red dye uptake assay ,54,55 eia). Each of these experiments consisted of eight replicate plates frozen with sections of the plates divided and subject to the varying IN control treatments outlined above. This comprised three sections of 2 × 10 wells for the immediate thaw trial and two sections of 3 × 10 wells for the 5-day trial—we did not include the manual nucleation treatment due to the lack of sterility involved in this procedure. The differences in post-thaw viable cell counts between these groups were analysed by one-way ANOVA with a Games Howell post hoc test, required when comparing groups with unequal variances. As the 5-day trial included only two treatments, IceStart and uncontrolled nucleation, comparisons were conducted using a two-sample t-test. All statistical significance tests were performed using Minitab v17.20.2. We previously performed preliminary toxicity experiments which showed, using two-sample t-tests, neither treatment of the cells with CPA nor IceStart array installation had a significant effect on cell viability. These activities were not regarded as additional treatments. Viable cell numbers were converted to post-thaw recovery rates  by normalizing data to mean viable cell numbers of unfrozen (baseline) plate cultures from the same passage. This was averaged over eight replicate plates for both experimental series.A pair of high-throughput plated cryopreservation trials were performed which determined cryopreservation success in terms of post-thaw cell viability either immediately post-thaw or after 5 days of post-thaw culture a. Each o. 3. 3.1−1. First, we focus on the droplet freezing temperatures for 50 µl droplets of ice nucleating materials at various concentrations in 96-well plates, using IR-NIPI , shown as boxplots in figure 4a–i. In the pure water blank runs, most aliquots of water supercool by more than 15°C before nucleation occurred. The degree of supercooling is clearly much smaller when heterogeneous ice nucleating materials are present. Strikingly however, for wells with LDH1 at suspension concentrations above 1%, none of the well temperatures noticeably dip below 0°C in contrast to the next most active material, Snomax. Instead, they plateau at around 0°C before cooling again after crystallization was complete. This observation implies that no measurable supercooling occurred and IN induced by LDH1 took place close to the melting point,To assess the ability of LDH1 to control IN relative to other materials, we performed droplet freezing assays of both 1 µl  and 50 µ IR-NIPI a,b, shownm(T) (nm(T) fits for LDH1 and cholesterol plotted alongside existing literature nm(T) parameterizations for K-feldspar , quartz, birch pollen and Snomax to enable comparison of ice nucleating activity. We found that LDH1 is more ice-active on a mass-by-mass basis than any other mineral, cholesterol or AgI and is comparable to Snomax. Moreover, median freezing temperatures within 1.5°C of the melting point of ice were seen for less than 0.1% LDH1 (0.05 mg of LDH1 per well) suspension droplets in 96-well plates. If LDH1 was able to virtually eliminate supercooling in 50 µl droplets at concentrations as low as 0.1 wt%  it implies that it contains sites able to nucleate ice close to the equilibrium melting point. In principle, the IR-NIPI cannot reliably determine supercooling of less than its nominal uncertainty of ± 1°C [Using the 1 µl and 50 µl droplet freezing assay data from this study and from the literature, we plot the number of active IN sites per unit mass of material as a function of temperature: nm(T) . Figure of ± 1°C . Howeverof ± 1°C , was roud).The only non-biological materials other than LDH1 previously recorded to induce IN close to the melting point of water are long chain aliphatic alcohol monolayers . This hafigure 4. 3.2While it is known that ice formation at severe supercooling is likely to be hazardous to cells undergoing cryopreservation, it is not quantitatively clear from the literature whether inducing IN at the melting point has significant benefits . To examTnuc values ranging from −21.8°C to −3.4°C in the plate. These clearly correspond to the distribution of plate PTV values (a–c) and the scatter plot of well PTV and Tnuc, shown in figure 6a, shows that well IN temperature and PTV are strongly positively correlated (r2 = 0.89) and appears to show a linear dependence. The thermal histories for each well, shown in figure 6d, contrast the temperature fluctuations that occur in wells with IN controlled at warm temperature with those with IN occurring at colder temperature. Figure 6d illustrates how the maximum rate of cooling of wells after the ice crystallization phase is far higher for wells with low (uncontrolled) Tnuc (approx. 7°C min–1) than those with Tnuc near the melting point (3°C min−1). The rate of warming upon IN was far faster than the 15 s time interval we used for temperature measurements apart from when nucleation occurred close (within 1°C) to the melting point. For wells nucleating below −10°C the rise to melting point appeared instantaneous implying warming rates of hundreds of °C per minute. Overall, it is clear that warmer well IN temperatures and corresponding smaller rates of change of temperature are conducive to cell survival.Using a combination of uncontrolled IN of most wells and spiking a few with LDH1 powder, we were able to produce well V values a–c and t. 3.3We then tested our method of 96-well plate cell cryopreservation with controlled IN using IceStart arrays as vehicle for delivery of LDH1 ,61. ThisWe performed two 96-well plate freezing trials, each of which comprised eight replicate plates of HepG2 monolayer cultures cryopreserved with different methods of IN control. Post-thaw cell viability  was assessed immediately upon thawing and was compared to that of a non-frozen control 96-well plate culture to determine acute cell post-thaw survival rates . The first trial determined PTV immediately upon thawing, while for the second trial the cryopreserved plates were cultured for a further 5 days after thawing to determine longer term cell viability and performance. Plates were frozen with portions divided into varying methods of IN control: IceStart arrays loaded with LDH1 powder, uncontrolled nucleation as a negative control and manual nucleation as a positive control. The manual nucleation method used ice mist falling from a cold object and has been previously demonstrated to induce IN uniformly at moderately low supercooling (at most 5°C) during similar experiments with 96-well plates [t-test for the 5-day post-thaw trial) are shown in p < 0.05). Differences were seen in individual plates between IceStart and manual nucleation wells; however, overall neither nucleation method resulted in constantly higher or lower results. The overall immediate post-thaw recovery rates of the eight replicates were 46.2%, 45.3% and 16.6% using IceStart, manual nucleation and uncontrolled nucleation respectively, meaning both IceStart and manual nucleation resulted in significantly higher PTS than uncontrolled nucleation. There was no significant difference in outcome between the IceStart and manual nucleation methods . For the 5-day post-thaw trial, where only IceStart and uncontrolled nucleation methods of IN were used, all 8 replicate plates showed significantly higher PTV cell number with IceStart deployed. Overall PTS over all plates averaged: 80.5% versus 53.1%, respectively . In contrast to the immediate post-thaw viability trial, when the cryopreserved plates were cultured for a further 5 days after thawing the data were far more variable. For example, the uncontrolled nucleation datasets included clusters of ‘dead’ wells and ‘recovered’ wells within plates: this is clearest in plate#10, #14 and #15 (e). In contrast, the LDH1 nucleated wells have a much greater and less variable PTV.The results of the freezing trials for both immediate post-thaw and 5-day post-thaw, along with statistical significance testing . In the cases where the cell layers had detached, there were cells still visibly stained by the dye and therefore still viable. Closer inspection of the monolayer (where still in situ) at the immediate post-thaw stage revealed a network of spherical viable cells that had lost their epithelial morphology and were clumped together in places together with dead (unstained) cells (figure 8d). The ratio of viable cells to dead cells was qualitatively lower where IN was uncontrolled compared to controlled, either by IceStart or manual nucleation. Re-examination after 5 days of culture post-thaw  showed the surviving cells had re-attached and started proliferating once again, consistent with the general higher cell viability seen for all treatment groups compared with immediate post-thaw. The manner of proliferation in wells frozen with controlled IN versus uncontrolled IN was, however, distinct and reflected the variable amount of initial post-thaw monolayer detachment where well cultures grew as confluent monolayers compared with irregular masses.Using an inverted light microscope, we visually inspected the cell monolayers in the 96-well plates of non-frozen control cultures compared with those after thawing at the point where neutral red dye had stained all the viable cells. Lower magnification images detailing the condition of the monolayers and higher magnification images resolving individual cells are shown in figure 8. 4. 4.1Our observations quantitatively support the generally accepted principle that for slow-freezing cryopreservation of cells, avoidance of severe supercooling is required for good cell post-thaw recovery and functionality ,32,62,63−1 compared with up to 7°C min−1 in the most deeply supercooled wells. This resulted in an effective cooling rate much higher than the nominal 1°C min−1 defined by the cooling programme on the controlled rate freezer. Not only do higher than optimum cooling rates after extracellular ice formation mean that cells cannot dehydrate fast enough to avoid IIF [et al. [et al. [Tnuc is far more extreme than that of the cooling rates. In summary, severe temperature fluctuations following IN events are a plausible reason for the cell monolayer detachments and were minimized by controlling IN at the melting point with IceStart arrays. This may negate the need for using thermally matched substrate materials for cell monolayer freezing.Detachment of the HepG2 cell monolayer from the surface of culture plate appeared to result in low post-thaw viability and mostly only occurred when IN was left uncontrolled . Monolayvoid IIF ,67. Eska [et al.  and Rutt. 5Tnuc is difficult to control in a repeatable way [Overall, we have demonstrated how the survival of cell monolayers frozen within multiwell plates is directly related to the temperature of IN and how it can be significantly improved with the aid of a mineral-based ice nucleating agent. A sterilizable device inserted into the 96-well plate passively and reliably induces IN at close to the melting point of the host cryoprotectant, thus avoiding severe thermal stresses caused by rapid latent heat release associated with ice crystal growth from deeply supercooled liquid. able way , and it able way . Hence, Tnuc and cell viability described in this work could be adapted to other cell or tissue types and biological markers as well as other multiwell plate formats (e.g. 384-well plates). This will enable examination of the importance of IN control for cryopreservation of other cell types and biological entities. This might enable existing cryopreservation protocols to be suitably tailored. Further research is needed to identify the nature of the hyperactive ice nucleating sites found in mineral specimens such as LDH1 to enable further sources of this material to be identified or manufactured. More detailed studies are required to determine improved delivery methods for LDH1 , such as encapsulation or integration into vessel surfaces [Our method for quantifying the relationship between surfaces . Moreovesurfaces ,70. This


An orthogonal, noncovalentapproach to direct the assembly of higher-orderDNA origami nanostructures is described. By incorporating perfluorinatedtags into the edges of DNA origami tiles we control their hierarchicalassembly via fluorous-directed recognition. When we combine this approachwith Watson–Crick base-pairing we form discrete dimeric constructsin significantly higher yield (8x) than when either molecular recognitionmethod is used in isolation. This integrated “catch-and-latch”approach, which combines the strength and mobility of the fluorouseffect with the specificity of base-pairing, provides an additionaltoolset for DNA nanotechnology, one that enables increased assemblyefficiency while requiring significantly fewer DNA sequences. As aresult, our integration of fluorous-directed assembly into origamisystems represents a cheap, atom-efficient means to produce discretesuperstructures. Furthermore, the incorporation of recognition elementsin ODN staples permits the spatial arrangement of components withsubnanometer precision, providing additional functionality for applicationsspanning biosensing, drug delivery, and the fabrication of photonicdevices.9DNA self-assembly is the pre-eminent strategyto construct multidimensionalfunctional nanostructures with angstrom-level precision.11 An alternative strategy to address these size limitations is thehierarchical assembly of individual DNA origami nanostructures intohigher-order superstructures  M13mp18 phage DNA is themost commonly used, confining the dimensions of folded origami structuresto ∼100 nm. To overcome this limitation, a methodology is nowrequired to enable the assembly of origami nanostructures into discretemicron-scale ensembles. Although increasing the length of the scaffoldstrand is one strategy, this method requires an increase in the numberof staples required to fold the resultant structure, complicatingthe design and increasing the cost of singular origami motifs.ructures 1a.12−1418 Theoretically, these highly programmable pure DNAapproaches should enable architecture scaling to any desired size;however, in practice they have distinct limitations. Blunt end stackingis restricted by the number of helical edges within an origami structureand requires stringent alignment of these edges, making it sensitiveto global origami distortions. Both sticky- and blunt-ends methodshave the disadvantage that the binding strength of individual recognitionelements is sequence-dependent, coming with the associated cost oflarge sets of unique DNA sequences. They are further hampered by thelow yield of final products, which drops precipitously as the assemblysizes increase (for structures approaching 1 μm in size yieldscan be as low as 2–3%).19Incorporating ssDNA overhangs(sticky ends) onto the edges of nanostructuresthat base-pair with complementary sequences or the use of blunt-endedbase-stacking interactions are the most common of these strategies,and they have been used to great effect to promote the assembly ofextended two-dimensional assemblies and complex, three-dimensionalorigami superstructures.22 has the potential to address the limitations of thesepure DNA methods and represents a step toward creating a library ofnucleic acid recognition elements analogous to the more diverse rangeof elements employed in protein–protein assembly.23 The incorporation of perfluorinated tags atprecise locations within a DNA sequence is one such molecular recognitionmodality. These fluorine-rich groups preferentially associate withone another while excluding other forms of hydrophilic and hydrophobicinteractions, a phenomenon known as the “fluorous effect”.24 The fluorous effect has been utilized extensivelyin the preparation of microarrays28 in which small molecules or biomacromoleculesincorporating perfluorinated tags [-(CF2)nCF3 where n ≥ 3] are immobilizedonto fluorous-micropatterned surfaces. The affinity of self-associationof perfluorinated tags (RF) is far stronger than equivalenthydrophobic interactions, rendering the fluorous effect an atom-efficientyet reversible noncovalent interaction.Blending orthogonal modes of molecular recognition that can functionco-operativelyWe surmised that thestrategic placement of perfluorinated tagsat the interface between origami nanostructures would provide an orthogonalrecognition mode for their hierarchical assembly; a strong yet mobilebinding solution (the fluorous pony-tails can slide over one anotherwithout separating) to deliver added stability when mixed with ssDNAsticky ends. In this paper, we show that blending DNA base-pairingwith fluorous-directed recognition results in a “catch-and-latch”system; the fluorous effect provides strength and stability, whilethe DNA provides specificity, locking the individual origami intothe correct positions 1. The reF-ODNs) tosticky ends of an identical sequence, providing a flexible systemin which the tile itself is assembled first and is later modifiedby incubation with the appropriate RF-ODN, allowing fora maximum of 14 tags. Assessment of the origami assembly was doneusing a combination of agarose gel electrophoresis (AGE) and atomicforce microscopy (AFM). RF-ODN tags were prepared by asolid-phase synthesis using phosphoramidite chemistry , with thelengths of RF including RF4, RF6,and RF8 variants. In addition, branched designs 2, and (RF8)4, respectively) were preparedusing a branching phosphoramidite installed at the 5′ end tomodulate the density of perfluorinated groups 2, and (RF8)4. It was anticipated thathigher fluorous content would lead to stronger interactions betweenorigami and thus higher dimerization yields.The first phase of this study focused on determiningwhich, if any, of the fluorous tags could promote dimerization ofthe origami. The tags, in order of increasing fluorous content, wereRF4 and RF6 tags were used.Using the RF8 tag resulted in the formation of some higher-orderstructures, including dimers and larger aggregates (visible on theAFM and observed in the gel as bands with lower mobility). When usingthe (RF8)2 tag there was a clear shift towarddimers as the predominant formation . This tendency toward dimerization did not appear forthe (RF8)4 tags, which produced a mixture offormations. Larger aggregates were particularly prominent for thisvariant in AFM analysis, although monomers appear to dominate in theAGE analysis. No origami assembly was observed when unmodified structureswere used, and, significantly, no assembly was observed for the alkyltag, which is directly comparable in size to the fluorous RF8 tag, confirming that origami-to-origami assembly is specificallydriven by the fluorous effect rather than a general hydrophobic effect.First, the origamistructure was modified with one of the fluorous tag variants, at all14 positions along its short edge, F-ODNs included ineach structure. Having obtained the highest dimer yield using the(RF8)2 tag, we systematically varied the numberof (RF8)2-ODNs incorporated into the origami2 tags are used .While these results demonstrate that the fluorous effect can promoteorigami assembly when all 14 modification sites are used, we nextwanted to explore how the strength of the fluorous effect could bemodulated by changing the number of Rare used 2e,f. As (a)ii), reducing conformational flexibility,improving the likelihood of successful contact between elements (particularlyin systems that include both RF-tags and DNA sticky ends),and decreasing the likelihood of unwanted aggregation between multipleorigami structures. A series of origami nanostructures was preparedusing this technique, with a systematic increase in (RF8)2-staples from 2 to 14. An equivalent series of nanostructureswas also prepared, which incorporated sticky ends at the equivalentsites.Havingdemonstrated fluorous-directed dimerization and established guidelinesfor the type and number of fluorous tags necessary to achieve assembly,we sought to refine the design by integrating the fluorous tags atthe origami folding stage (as fluorous-modified staple strands), ratherthan add them to preformed nanostructures, as we did for the resultsshown in Figure F stapleand sticky endsystems i,ii, respectively) shows that, for all modificationnumbers, the fluorous effect is significantly more efficient thanDNA hybridization at promoting dimer assembly. The largest yield improvementobserved was for the 10-tag systems, where the fluorous-directed approachproduced a 7.4x increase in yield. Generally, the percentage of dimerformations increases with the number of RF staples used,until we reach 12 and 14 RF staples, at which point trimers,tetramers, pentamers, and higher-order aggregates begin appearingin larger numbers. However, as can also be seen in both the AFM 2 staples in both origami motifs, folding them separately before mixingboth designs  becomes too high, while alsostill leaving room along the origami edge for the addition of ssDNAsticky ends. To do this, we created a rectangular origami design thatcould easily be distinguished from the motifs used thus far 4a. We in designs 4b.F8)2 staples integrated into the designs. The smallnumber of heterodimers may indicate that the fluorous interactionsare relatively stable, with little dissociation and recombinationoccurring. This suggests that, if the fluorous-driven dimerizationis too stable (because too may fluorous tags have been used), theinclusion of ssDNA sticky ends may be insufficient to dissociate thehomodimers and promote heterodimer formation. Since 8, 10, and 12RF tags result in a high degree of dimerization and a smallnumber of multimers (2.5% for 12 RF tags) , where the fluorous-drivendimerization is less pronounced, the number of remaining attachmentsites is high, and no multimers are formed, would offer a flexibleplatform for integration with Watson–Crick base-pairing, i.e.,integrating fluorous-driven association with sequence-selective molecularrecognition of DNA sticky ends.AFM analysis revealed that homodimerspredominate over heterodimerswith the overall dimerization rate increasing with the number of (RRF tags) 4c, they F8)2 tags, Our studies show that the strategic placementof perfluorinated tags at the interface of origami tiles is an effectivestrategy for the assembly of higher-order origami nanostructures.Although discrete dimeric species are the major products of our fluorous-directedassembly experiments, the formation of slipped dimers, multimers,and larger aggregates is inevitable given the nonspecific, mobilenature of the fluorous effect. We therefore explored the potentialto enhance the formation of discrete origami heterodimers by combiningfluorous and Watson–Crick modes of molecular recognition. Tothis end, a series of single-stranded, five-nucleotide sticky endswere integrated into the origami structures in positions flankinga central fluorous core comprising four and characterized by matrix-assisted laser desorption/ionization massspectrometry (MALDI-MS).Supporting Information) were purchased unpurified from Integrated DNATechnologies (IDT) in 96-well plates, suspended in water, and normalizedto 100 μM. DNA origami were folded in a buffer solution containing1 × TAE  with 12.5mM magnesium acetate. M13mp18 concentrations ranged from 4 to 10 nM,with staple strands being at a 10x molar excess. Solutions were heatedto 95 °C and cooled to 25 °C at a rate of 1 °C/min.Single-strandedM13mp18 DNAwas purchased from Tilibit nanosystems . Staple strands .Excess staple strandswere removed using Amicon Ultra-0.5 mL 100 kDa. 500 μL of 1xTAE-Mg2+buffer was added and spun at 10 0004 μL of loading dye was added to 20μL of annealed and filtered samples before being loaded into the wells. Four microliters of DirectLoadT1 kb ladder  was used as a reference. Thegel consisted of 1% agarose containing 1xMg-TAE buffer stained withSYBR safe . The electrophoresisvoltage was set to 80 V and run in an ice bath for a total of 90 min.2 for 10 s. Samples wereimaged under ambient conditions using Tapping Mode on a DimensionIcon (Bruker) with FESPA-V2 probes. For counts of origami assembliesmade from AFM images, n > 275 for each dimer type.Typical scan parameters were scan rate: 1 Hz, resolution: 3072 ×1024, area: 10 μm, amplitude set point: 250 mV, drive frequency:75 kHz, drive amplitude: 1000 mV.Ten microliters of sample containing1 nM of origami were deposited onto freshly cleaved mica and leftto adsorb for 2 min. Samples were then rinsed with DI water beforebeing dried under a weak flux of N


MAR2014). Their structures were verified utilizing varied spectral analyses. The isolated metabolites (1–3), alongside the earlier reported integracides G (4), H (5), and J (6), were inspected for 5-LOX inhibition capacity. Interestingly, 1–6 possessed marked 5-LOX inhibition potentials with IC50s ranging from 1.18 to 3.97 μM compared to zileuton (IC50 1.17 µM). Additionally, molecular docking was executed to examine the interaction among these metabolites and 5-LOX, as well as to validate the in vitro findings. The docking study revealed their inhibitory activity interactions in the binding pocket. These findings highlighted the potential of integracides as lead metabolites for anti-inflammation drug discovery.Inflammation is a complicated disorder that is produced as a result of consecutive processes. 5-LOX (5-lipoxygenase) is accountable for various inflammation mediators and leukotrienes synthesis, and its inhibition is the target of anti-inflammation therapeutics. Fungi have acquired enormous attentiveness because of their capability to biosynthesize novel bio-metabolites that reveal diversified bio-activities. A new tetracyclic triterpenoid, integracide L ( Inflammation is a complicated reaction that is controlled by an interaction between the cells and inflammatory mediators . Among tFusarium species, a widespread cosmopolitan group of fungi, have been separated from both marine and terrestrial sources +  in HRESIMS, indicating eight DBE. It showed a UV band at λmax 235 nm. The IR displayed absorptions at 3439, 1729, 1663, and 879 cm−1, corresponding to hydroxyl, ester carbonyl, and the exocyclic di-substituted double bond, respectively. Compound 1 was one degree of unsaturation and 18 mass units more than integracide F (3), indicating the loss of one double bond and the presence of an extra OH group in 1. It exhibited characteristic HRESIMS fragments at m/z 532.3758 [M + H-OAc]+ and 490.3658 [M + H-2OAc]. It’s NMR data of 1 were similar to that of 3 D +19.3  was examined as priorly stated, utilizing zileuton as the positive control [5-LOI of integracides . The % 5-LOX inhibition of and IC50s are listed as mean-values ±SD. Statistical significance was analyzed among the samples by one-way-ANOVA and subsequently by Tukey–Kramer test (p < 0.005).IC2+ coordinates with His residues were in the active site. The protein-Fe2+ bonds were deleted, and finally, the minimization of the protein was applied using the OPLS4 force field. All ligands were prepared before docking using the “LigPrep” tool [The docking study was performed with the Schrodinger program . The crystal structure of stable 5-LOX complexed with the NDGA inhibitor was downloaded from the protein data bank . The protein was prepared using the “Protein Preparation Wizard” tool in Maestro software, where the missing hydrogens were added to the residues, the metal ionization state was corrected, and the water molecules > 5 Å from protein residues were deleted. The protein contained two chains: A and B. Chain B was complexed with the inhibitor; therefore, it was chosen to perform the docking study. The protein was then refined by predicting the pKa of the ionizable residues using PROPKA and water molecules > 3 Å (not involved in the water bridge), which were removed . Additioep” tool . The 2D ep” tool .To perform the docking, a grid box was generated around the active site of stable 5-LOX (PDB: 6N2W) containing the co-crystallized inhibitor NDGA and using Glide’s “Receptor-Grid-Generation” tool in Schrödinger suite . All stu1) and two metabolites (2 and 3), were purified from Fusarium sp. (FS No. MAR2014). Their specification was achieved with the assistance of extensive spectral analyses. Compounds 1–6 demonstrated a noticeable 5-LOX inhibition potential. The docking study of integracides illustrated their binding mode in the active site with possible amino acid interactions, which could explain their inhibitory activity.A new tetracyclic triterpenoid, integracide L (These findings may foster more inspection of the possible usage of integracides as 5-LOX inhibitors and draw further interest to the synthesis of structure-similar analogs with enhanced 5-LOX inhibition capacity. Moreover, additional in vivo and mechanistic investigations are needed.


Stress in poultry can lead to changes in body metabolism and immunity, which can increase susceptibility to infectious diseases. However, knowledge regarding chicken responses to viral infection under stress is limited. Dexamethasone (Dex) is a synthetic glucocorticoid similar to that secreted by animals under stress conditions, and has been widely used to induce stress in chickens. Herein, we established a stress model in 7-day-old chickens injected with Dex to elucidate the effects of stress on IBV replication in the kidneys. The metabolic changes, immune status and growth of the chickens under stress conditions were comprehensively evaluated. Furthermore, the metabolic profile, weight gain, viral load, serum cholesterol levels, cytokines and peripheral blood lymphocyte ratio were compared in chickens treated with Dex and infected with IBV. An LC-MS/MS-based metabolomics method was used to examine differentially enriched metabolites in the kidneys. A total of 113 metabolites whose abundance was altered after Dex treatment were identified, most of which were lipids and lipid-like molecules. The principal metabolic alterations in chicken kidneys caused by IBV infection included fatty acid, valine, leucine and isoleucine metabolism. Dex treatment before and after IBV infection mainly affected the host’s tryptophan, phenylalanine, amino sugar and nucleotide sugar metabolism. In addition, Dex led to up-regulation of serum cholesterol levels and renal viral load in chickens, and to the inhibition of weight gain, peripheral blood lymphocytes and IL-6 production. We also confirmed that the exogenous cholesterol in DF-1 cells promoted the replication of IBV. However, whether the increase in viral load in kidney tissue is associated with the up-regulation of cholesterol levels induced by Dex must be demonstrated in future experiments. In conclusion, chick growth and immune function were significantly inhibited by Dex. Host cholesterol metabolism and the response to IBV infection are regulated by Dex. This study provides valuable insights into the molecular regulatory mechanisms in poultry stress, and should support further research on the intrinsic link between cholesterol metabolism and IBV replication under stress conditions. Animal stress is a complex, multidimensional and universal phenomenon that is difficult to evaluate and has important biological significance . Poultryin vivo and in vitro  axis is an important system for integrating and regulating stress responses in vitro . In genein vitro . Activatin vitro . GCs arein vitro . Excessiin vitro . Throughin vitro . Additioin vitro . GCs alsin vitro . The secin vitro  that is in vitro . In poulin vitro . CORT isin vitro . Therefoin vitro . Dex is in vitro , which hin vitro . In partin vitro . TherefoHerein, we established a chicken stress model by using Dex and found that Dex-induced chicken stress increased the IBV viral load in the kidneys. Untargeted metabolomics was used to detect differentially enriched metabolites in chicken kidney tissue to evaluate the correlations between IBV replication and metabolic changes induced by Dex. Serum cholesterol levels significantly increased in chickens under stress. Many metabolic pathways and metabolites may be associated with the replication and pathogenicity of IBV. This finding provides insight into the intrinsic link between host metabolism and IBV replication under stress conditions.’s laboratory   fetal calf serum. The above cells were cultured at 37 °C under 5% CO2.DF-1 , Vero and H1299 cell lines were obtained from the American Type Culture Collection (ATCC) . The IBVversity) . The culversity) , In brie d in a multi-stage incubator  at a constant machine temperature of 37.6°C and relative humidity of 53%. Eggs were placed with the air cell up and turned hourly at an angle of 90°. From E17 to E21.5, the eggs were not turned, the air temperature remained constant at 35.0°C, and the air speed was lower than 0.2 m/s, which is considered still air.Anti-IBV N antibody was obtained by immunization of a rabbit with IBV N antigen in our laboratory as previously described . CD4-FITDF-1 cells, Vero cells and H1299 cells were harvested at the indicated infection time points and lysed with 2× SDS loading buffer in the presence of 100 mM dithiothreitol, then denatured at 100°C for 10 min. The obtained protein samples were separated with SDS-PAGE to detect intracellular IBV protein levels according to a previously described method .via an eye dropper with 200 μl of 105 EID50 of IBV-QX or PBS as the negative control. Chickens were observed daily, and their clinical symptoms and mortality were recorded. At 7 dpi (14 days old), the chickens’ responses to IBV infection under stress induced by Dex were comprehensively assessed through measurement of viral load, histopathology, metabolomics, serum cholesterol concentration, peripheral blood lymphocyte ratio, survival rate and growth performance , Dex (N=52), IBV (N=52), IBV-P-Dex  and IBV-A-Dex . The feeding and care of chickens was performed in accordance with the Institutional Animal Care and Use Committee guidelines. The approval number was SHVRI-chicken-2022021802. According to previous studies , a chickThe trachea, heart, lung, liver, kidney and spleen tissues were isolated and soaked in 10% neutral formalin at room temperature for more than 48 h, then subjected to routine processing. H&E staining was performed, and the slices of organs for each chicken were observed under an optical microscope.Samples were thawed on ice, and 50 mg tissue fragments were taken from each sample and mixed with 500 μl ice-cold methanol/water . After homogenization at 30 Hz for 2 min, the mixture was vibrated for 5 min and incubated on ice for 15 min. After centrifugation at 12,000 rpm at 4°C for 10 min, 400 μl supernatant was transferred into another centrifuge tube; subsequently, 500 μl of ethyl acetate/methanol  was added into the original centrifuge tube. The mixture was oscillated for 5 min, incubated on ice for 15 min and centrifuged at 12,000 rpm at 4°C for 10 min, and 400 μl of supernatant was collected. The two supernatants were combined and concentrated. Then 100 µl of 70% methanol water was added to the dried product, and ultrasonic treatment was performed for 3 min. Finally, samples were centrifuged at 12,000 rpm at 4°C for 3 min, and 60 µl of supernatant was aspirated for LC-MS/MS analysis.Untargeted metabolomics profiling was performed using a UPLC-Q-TOF/MS . The chromatographic seperation system was equipped with a Waters ACQUITY UPLC HSS T3 column . UPLC conditions were as follow: column temperature, 40°C; flow rate, 0.40 mL/min; injection valume, 1ul. The mobile phase consisted 0.1% formic acid inwater (phase A), and 0.1% formic acid in acetonitrile (phase B). The linear gradient programm was as follows: 95% to 10% phase A over 11 min, and holding 10% for 1 min, and 10% to 95% in 0.1 min, and holding 95% phase A for 1.9 min. The mass spectrometer was operated in positive/negative polarity mode with the following settings: Nitrogen was used as the drying gas, nebulizer gas and sheath gas, and the flow was maintained at 8 L/min, while sheath flow was at 11 L/min. The drying gas and sheath gas temperature was maintained at 325°C. The ESI+ and ESI- voltages were set at 2500 V and 1500 V respectively. Mass range was set at m/z 50-1700 and the resolution was 30,000 (FWHM). The mass spectrometer was calibrated daily in the mass range m/z 100-1700 before starting the sample analysis by using Agilent tune mix (Part no G1969-85000). The mass accuracy values were good in full scan range (mass error < 5 ppm).Total RNA from kidneys was used for quantitative real-time PCR (qPCR) analysis to confirm the propagation of the IBV in chicks, and the mock group was used as a negative control. The primer pairs used in the PCR assay for the IBV-N gene were IBV Fw-5′-CAG AAG AAG GGC TCT CGC ATT AC-3′ and IBV Re-5′-AGG TTG AGC ATT GCC GTA ACA C-3′. Positive and negative strands of IBV genomic RNA were detected according to our previous report . The qPCR program ” method was used to correct the peak area . The pea program .in vivo, we assessed the effect of Dex on IBV replication, on the basis of chicken mortality and IBV viral load in kidney tissue , we used the principle component analysis (PCA) to compare the metabolite composition , P value (<0.05) and VIP (≥1) in the OPLS-DA model was used to screen for differentially enriched metabolites, and a total of 147 differentially enriched metabolites were obtained in the Dex group. The lists of differentially enriched metabolites are shown in via a volcano plot , National Natural Science Foundation of China (grant No. 32030108), Shanghai Agriculture Applied Technology Development Program, China (2022-02-08-00-12-F01154) and Natural Science Foundation of Shanghai (grant Nos. 21ZR1476800 and 20ZR1469400).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The purpose of this study was to compare outcomes between orthopaedic trainees using various preoperative training platforms  in a slipped capital femoral epiphysis model.Participants were randomly assigned to one of the three groups: (1) reading/video control group (n = 7), (2) VR group (n = 7), or (3) PS group (n = 7). Participants in the VR group completed a VR slipped capital femoral epiphysis module while participants in the PS group practiced the placement of a screw in the physical module before evaluation of percutaneous screw placement in the PS model. Outcomes evaluated included overall surgical time, amount of fluoroscopy, Global Rating Scale score, radiographic screw position, physical screw accuracy, presence of breeching of the articular surface or femoral neck, and overall platform rating (0 to 10).P = 0.001).No difference was observed in surgical time, Global Rating Scale score, radiographic or physical accuracy of screw position, or articular surface breaching between the groups. Subjectively, there was a difference in utility of platform rating between the groups (PS: 10 ± 0, VR: 7 ± 2, and control: 6 ± 1, Training with VR was subjectively rated higher in value compared with reading/video methods and had similar performance outcomes compared with training with PS. Although not widely adopted, both traditional virtual reality (VR) surgical platforms  and more modern VR systems (simulators with head-mounted display and generic hand controllers) may have some benefit in orthopaedic training. Previous literature reports that VR simulator platforms may improve resident skill level in the operating room or on cadaver models for knee arthroscopy or total hip arthroplasty.Slipped capital femoral epiphysis (SCFE) screw fixation is a routine pediatric orthopaedic procedure which demands advanced three-dimensional skills. The utility of VR as a surgical learning platform to train novice orthopaedic trainees on how to perform in situ screw fixation has not yet been evaluated. The primary purpose of this study was to compare screw placement accuracy and surgical technique among orthopaedic trainees trained with either VR, isolated text, video, and literature review or physical simulation (PS) training in a SCFE sawbones model.Twenty-one orthopaedic trainees, consisting of 9 junior orthopaedic residents and 12 senior medical students, were recruited from a single institution to participate in this study. A customized phantom limb model (Sawbones) encasing a SCFE radiopaque Sawbones femur was used to simulate a pediatric patient  SCFE Sawbones model and placed on a radiolucent table. Bone models were replaced with a new sample between every trial. Before beginning a trial, a felt sticker was used to cover previous incisions sites on the reusable model. Surgical instruments included a scalpel, 2.8-mm Kirschner wire with power, a 4.5-mm drill bit with power, and a 7.0-mm cannulated screw (OrthoPediatrics). Operating room setup and instrument tray are shown in Figure 10111213 on how to perform in situ screw fixation , (2) group 2 (PS group): PS training on the SCFE Sawbones model, or (3) group 3 (VR group): VR-based SCFE simulation training module using the Osso VR platform  group 1 (control group): standardized reading and video materialm Figure . All groTrainees were given the option to either maintain the C-arm in a single position and manually maneuver the limb to obtain an AP and frog-leg lateral view or position the C-arm while keeping the phantom model static and obtaining an AP and lateral view. An imaging technology was available for all C-arm trials to assist in intraoperative positioning of the C-arm. Total number of AP and lateral images were recorded for each trial.To gauge physical accuracy, the center of the femoral head was marked preoperatively to indicate optimal screw trajectory. After placement of the screw, a 2.8-mm Kirschner wire was used to breech the cortex of the femoral head to assess postoperative screw trajectory. Screw placement accuracy was measured as the shortest distance from the preoperative marked site on the femoral head to actual postoperative trajectory Figure .14 As described by Pring et al, “grade A” accuracy of pinning implied that the pin was placed within the central 50% of the physeal width, with the screw tip 5 to 10 mm across the physis, at least 5 mm from the subchondral bone, and at an angle of 70° to 90° to the capital physis. “Grade B” accuracy of pinning was reported when the pin was placed outside the “A” area, but not in the “C” area. This was considered to be an area containing the central 75% minus the central 50% of the physeal width, with the screw tip less than 5 mm across the physis or less than 5 mm from the subchondral bone, and at an angle of 50° to 90° to the physis. “Grade C” accuracy of pinning described screws placed outside the central 75% of the physeal width, less than 2.5 mm of screw tip across the physis, less than 2.5 mm from the subchondral bone, or at an angle less than 50° to the physis  to generate an overall performance rating (range: 6 to 30 in our study). GRSs have been used in multiple surgical specialties in both simulated and clinical settings.Rates of screw articular breaching and femoral neck in-out-in screw placement were recorded for each group. Articular breeching was defined as screw penetration through the articular surface of the femoral head. In-out-in was defined as screw penetration outside the femoral neck and re-entry into bone at a more proximal location within the femoral neck or head. Examples of screw breach and in-out-in screw placement are shown in Figure P value of 0.05 or less. Statistical analyses were done with Stata, version 15 (StataCorp LP).We used standard analysis of variance testing to compare continuous variables and chi-square test to compare categorical variables. Statistical significance was defined as a We recruited 21 junior orthopaedic residents and medical students from a single institution to participate in this study. Participants were equally divided into each of the three study platforms, with each group consisting of four medical students and three junior residents. All study participants completed the standardized reading and video study material before participation and verified that no additional learning tools were used.P = 0.69).No significant differences were observed in mean (±SD) physical accuracy of the screw placement between all three study platforms . Compared with the control and PS groups, participants in the VR group took significantly greater numbers of AP, frog-leg/lateral, and total radiographs (Table P = 0.31), frog-leg/lateral (P = 0.83), and total radiographs (P = 0.62) between the PS or control groups.No significant differences were observed in mean (±SD) total surgical time between all three study platforms . No differences were also observed in the number of screws that breeched the femoral neck before entering the femoral head .Across study platforms, no significant differences were observed in the number of participants who breached the articular surface of femoral head , there were no significant differences in AP Table  or laterP = 0.11, reviewer 2: P = 0.06; Table t-test analysis for each participant determined no significant differences in GRS scores between the two reviewers for a given participant .Across all three study platforms, no significant differences were observed in GRS scores (reviewer 1: P = 0.71).When participants were asked to evaluate how life-like  their study platform was, no significant differences were observed in mean (±SD) between the three groups .Participants ranked PS as the most useful study platform compared with the control group and VR group , VR headsets with hand controllers provided improved procedural in intramedullary nailing among first-year and second-year medical students. However, educational benefits of modern VR surgical training platforms and the transferability of skills to a physical model have never been evaluated in pediatric orthopaedic procedures.VR simulation has been previously described to improve resident skill level in the operating room or on cadaver models for knee arthroscopy, shoulder arthroscopy, and total hip arthroplasty.16 reported a complication rate of 26%, with joint penetration being the most common complication. Therefore, the inherent difficulty of performing this procedure may have masked any short-term gains in surgical performance from VR training in an extremely novice cohort. Moreover, the learning curve for successfully performing a SCFE procedure likely requires more than one trial. Although the minimum number of procedures to gain proficiency in SCFE fixation has not been previously quantified, the recommended number of supervised practice procedures for pediatric orthopaedic fellows for other procedures, such as supracondylar humerus pin fixation, has been reported to be approximately 15.17 Therefore, in our novice cohort, increased exposure to the basics of SCFE fixation and multiple trials may have resulted in greater differences among various educational platforms.Unlike the performance benefits previously reported in intramedullary nailing procedures among novices, our results indicated that no notable differences were observed in most objective parameters  between a modern VR headset and practice with a physical model. These results are likely secondary to (1) the relative technical difficulty of in situ SCFE fixation and (2) the lack of notable prior experience with SCFE fixation in our cohort of participants. SCFE fixation with in situ screws or pins has been previously associated with high rates of complications, even when done by experienced providers. For example, in a cohort of 202 pediatric patients with SCFE treated with screw fixation by trained orthopaedic surgeons, Riley et al18 Often times, these include more didactic sessions and video/text-based learning modules to decrease resident exposure to the virus.18 VR training modules may offer excellent supplementary content to didactic learning because it enables residents to directly visualize important surgical concepts and repeat procedures multiple times from the comfort and safety of their home while avoiding skill decay. However, despite the convenience of this system, program directors must consider the cost of the Osso VR platform. The hardware alone needed to run the Osso VR module is approximately 300 dollars for a single unit; residency leadership may consider bulk ordering the hardware and software implants from Osso for resident access as part of institutional educational funds. Although training with cadaver models may offer a more cost-effective method of instruction, because these specimens can be bulk ordered and used for multiple procedures across many residents, access to these models during times of social distancing and mandatory remote learning may preclude residents from participating in such learning opportunities. In this scenario specifically, surgical education with VR technology may be particularly useful and have a justified upfront cost.Although our results indicated that VR training offered similar performance as a phantom limb or a didactic teaching model, the mobility of modern VR platforms may offer extra convenience benefits. The novel coronavirus pandemic in 2020 forced orthopaedic residency curriculums to develop and adopt more remote-based learning modalities.Although the portability of the VR headset system offers notable convenience and advantages during a pandemic, participants offered some suggestions for VR training improvement to better suit their needs. First, the controllers with the VR system are not modular. As such, the same hand controller must represent a variety of different surgical instruments, decreasing the realism of the virtual simulation and potentially limiting gains in actual surgical technique. This could be rectified with addition of secondary controllers or instruments in the same VR kit. Second, we did not use the available test mode in the VR module, which would allow the user to critically think about pin placement rather than following outlined trajectories in the training module. Users may have gained further improvements in performance if both training and testing modules were completed before surgical simulation. Finally, the VR headset module for SCFE fixation does not have any modality to artificially palpate anatomic landmarks on the extremity to mark the incision site or feel differences in resistance while placing the guide wire. Although tactile components of anatomy are primarily limited by the portability of the system, ability to identify the incision site should be tested in the VR modules. These missing attributes in VR learning are key to mastering SCFE fixation and highlight the benefits of using a physical or cadaver model. Moreover, these limitations may explain why the VR group required greater radiograph images during the evaluation process.There are limitations to our study. First, our study had a small sample size, reducing the external validity of our results. Owing to the low sample size, outliers in the data set may have more notable effects on the mean. For example, one participant in the VR group took a total 185 radiographs during surgical simulation, compared with the next highest participant in the group with 132 radiographs. Notably, although there were no statistically significant differences in outcomes between all three platforms, our findings seem to trend toward favoring PS training over VR. Perhaps with a greater study sample size, these differences may be further pronounced. Moreover, individuals in the PS group were allowed to train and subsequently evaluated on the same phantom limb, which likely introduced some bias. Finally, using a sample of participants with limited orthopaedic experience may result in diminished gains in improvement with any educational platform. Participants may require prolonged exposure to the physical model and fundamentals of SCFE fixation to realize benefits from any particular surgical training methodology.http://links.lww.com/JG9/A203 and http://links.lww.com/JG9/A204).VR training holds promise for the future of orthopaedic training, and no statistically significant differences were observed in in situ screw placement accuracy, radiographic grades, and surgical technique compared with practice with a physical model in novice orthopaedic trainees. However, those trained with physical models performed nominally better across most of the outcomes and subjectively evaluated this method of training as the most life-like and useful. Future work may focus on improving current VR technology to make controllers more modular and realistic while simultaneously expanding training modules to be more detailed-oriented and rigorous (


UTu1, a uniquely shaped (9/3) tRNA exploited for site-specific selenocysteine insertion, using single-molecule fluorescence. With this technique we identified ribosomal disassembly occurring from translocation of allo-tRNAUTu1 from the A to the P site. Using cryo-EM to capture the tRNA on the ribosome, we pinpointed a distinct tertiary interaction preventing fluid translocation. Through a single nucleotide mutation, we disrupted this tertiary interaction and relieved the translation roadblock. With the continued diversification of genetic code expansion, our work highlights a targeted approach to optimize translation by distinct tRNAs as they move through the ribosome.Ribosomes are remarkable in their malleability to accept diverse aminoacyl-tRNA substrates from both the same organism and other organisms or domains of life. This is a critical feature of the ribosome that allows the use of orthogonal translation systems for genetic code expansion. Optimization of these orthogonal translation systems generally involves focusing on the compatibility of the tRNA, aminoacyl-tRNA synthetase, and a non-canonical amino acid with each other. As we expand the diversity of tRNAs used to include non-canonical structures, the question arises as to the tRNA suitability on the ribosome. Specifically, we investigated the ribosomal translation of allo-tRNA  Escherichia coli (Protein synthesis is a highly conserved process performed by the ribosome. Aminoacyl-tRNA synthetases (aaRSs) acylate the 3′-terminal CCA sequence with the cognate amino acid to produce aminoacyl-tRNAs (aa-tRNAs) that are carried to the ribosome by EF-Tu. With 46 aa-tRNA substrates in hia coli ,2, the rhia coli ,6. Genethia coli .Methanosarcina PylRS , following the translation pathway of the other 20 canonical amino acids. Initial attempts to create a hybrid tRNA between tRNASec and tRNASer for recognition by EF-Tu resulted in low protein yields and minimal Sec insertion  containing recognition elements that resembled tRNASec but with a 12 bp acceptor domain (acceptor and T-stem combined) for proper recognition by EF-Tu (UTu1 was found to be a serine isoacceptor with a 9/3 (acceptor/T-stem bp) structure that allowed EF-Tu driven Sec incorporation  we visualized movement of tRNAUTu1 through the ribosome and compared it to natural tRNAs (tRNASer and tRNAsupD). From this, we were able to establish the roadblocks associated with tRNAUTu1 translation through the ribosome. Implementing cryo-EM, we visualized tRNAUTu1 in complex with the E. coli ribosome to pinpoint the structural feature in tRNAUTu1 hindering translation. With this information, we engineered a single-base mutation in tRNAUTu1 that removed the rigidity of the variable arm, enhancing translational processivity on the ribosome. This was verified through in vivo and in vitro assays and led to an increase in recombinant protein production. Our results demonstrate the importance of ensuring that synthetic tRNAs used for genetic code expansion are suitable substrates for ribosomal translation and show the power of combined dynamics and structural analyses for improved tRNA engineering.Here, we investigated the suitability of an allo-tRNA, tRNAfMet, biotinylated mRNA and 4 mM GTP. Prior to immobilizing the 30S PIC, we prepared the SMRT Cell v3 from Pacific Biosciences (Menlo Park), a ZMW chip, with Neutravidin. The 30S PIC was then diluted to 15 nM and loaded into the SMRT cell  was prepared from Cy3B-labeled 30S subunit, ribosomal protein S1, initiation factor IF2, fMet-tRNAMRT cell .Phe and Ser-tRNAs were formed with EF-Tu (GTP) using a previously described protocol . Before starting an experiment, the delivery mix was diluted into polymix mixture in the SMRT Cell and loaded into a modified PacBio RSII sequencer  of Phe(Cy5)-tRNAprotocol . After fequencer . At the 2. The micrographs were acquired as dose-fractionated image stacks.Details on preparation of grids and data processing are described in the Supplemental Information. Data was collected on a Titan Krios transmission electron microscope (Thermo Fisher Scientific) equipped with a K3 Summit direct electron detection camera (Gatan) in counting mode. Micrographs were accrued at a calibrated pixel size of 1.037 Å and with a nominal defocus range of –1 to –2 μm. Each micrograph consisted of 40 frames collected over a two second exposure at a dose rate of roughly 20 electrons per pixel per second for a total dose of roughly 40 electrons/ÅsupD, tRNAUTu1 or tRNAUTu1A) and B-95.ΔAΔfabR E. coli cells  were transformed into B-95.ΔAΔfabR E. coli cells  for intact mass spectrometry to quantify Sec incorporation. Spectra were analysed using Thermo BioPharma Finder™ 4.1 (ThermoFisher Scientific).Expression and purification of human GPx1 was performed as previously described ,11. Brieli cells . CultureUTu1 was identified in a metagenomic search for use in site-specific, EF-Tu driven, Sec insertion -based instrumentation  and compared it to a canonical serylated tRNA capable of UAG suppression (Ser-tRNAsupD).tRNAnsertion ,26. Sincentation . We obseentation . In addin Figure . We applPhe, signaled by a burst in Cy5 fluorescence. Successful peptide bond formation is visualized by medium Cy3B intensity from a FRET transition due to the ribosome intersubunit rotation into the rotated state. This successful decoding event is then followed by EF-G-driven translocation of Cy5-tRNAPhe from the A site to the P site, placing the UAG stop codon in the A site and driving intersubunit rotation back to the non-rotated state (low Cy3B intensity). Each Cy5-tRNAPhe occupancy lasts for two cycles of elongation as it translocates from A to P and then P to E sites. Therefore, elongation of consecutive Phe codons downstream of the stop codon results in cycles of double- and single-occupancy states, making the signal for translation downstream of stop codon unequivocal  of 83%. Similar suppression efficiency was measured when translating in the presence of Ser-tRNAsupD (72%)  provides us with information on the suppression efficiency of a natural substrate for the ribosome. Since decoding of UAG is known to be slower than a sense codon, we directly compared the two suppressor tRNAs (tRNAUTu1 to tRNAsupD) but also measured tRNASer as a control using single-molecule fluorescence. The decoding time is calculated from the dwell time of the non-rotated state prior to ribosome rotation at which point the tRNA is successfully incorporated and peptidyl transfer occurs , decoding times are not equivalent to those observed during cellular translation.Translation with Ser-tRNA) Figure . tRNAsupr occurs . Decodinodon UCG . Due to UTu1 matching the natural suppressor tRNAsupD, we next investigated what occurs after translocation of the suppressor tRNA from the A to the P site. After UAG suppression by tRNAUTu1, we found a significant drop in the ribosome processivity at the third codon. At low concentrations (50 nM) of Cy5-TCPhe, only 21% of ribosomes continued translating following tRNAUTu1 incorporation, compared to 84% of ribosomes that continued translating after tRNAsupD insertion  and tRNASec (PDB: 5LZE)  . The distRNAUTu1 .UTu1 is stabilized by canonical T-loop to D-loop pairing of G18:U55 and G19:C56. In addition, tRNAUTu1 forms a base pair between U16:U59 which is similar to C16:C59 of tRNASec but not observed in tRNAPhe between U16 and U59  and tRNASec (PDB:5LZE) central loop nucleotides interact with the D-arm forming base triples and do not form strong interactions with each other. Specifically, the corresponding residues in tRNASec (U45 and U9) point in opposite directions with G49 of the variable arm protruding into the D-arm to stabilize the tertiary structure  and UGTT . No significant difference was observed between the tRNAs with UKGE, however, a significant decrease in fluorescence was found for tRNAUTu1 compared to tRNAsupD with UGTT . We found that tRNAUTu1A expressed roughly three times more protein (0.036 ± 0.004 mg/g) compared to tRNAUTu1 (0.010 ± 0.001 mg/g)  Figure  with no ) Figure . MoreoveUTu1A processivity on the ribosome P site increased significantly (75%) relative to tRNAUTu1 (21%)  which was drastically decreased for tRNAUTu1A (15%)  Figure  and D, eD Figure . Results1A (15%) .UTu1A processivity on the ribosome P site, we generated a cryo-EM structure using the same strategy described above (i.e. capturing A and P site tRNAUTu1A on the ribosome). Data processing and particle sorting resulted with two maps, each with either A site tRNAUTu1A or P site tRNAUTu1A alone. A site tRNAUTu1A has a similar density to tRNAUTu1 and was modelled with a similar global fold . Highly flexible RNA chains are often poorly defined in cryo-EM, which in turn can point to higher flexibility of the variable loop of tRNAUTu1A in accordance with our single-molecule FRET data presented above  by the E. coli ribosome. Together, our approach allowed us to identify a structural element in tRNAUTu1 that hinders processivity in the ribosome P site. A single nucleotide mutation (C8A) removed the translational roadblock, increasing protein expression to match that of a natural suppressor tRNA (tRNAsupD)  Figure .Ser, tRNALeu and tRNATyr in E. coli). These additional variable arm contacts are suggested to be the driving force which move the ASF for the tRNA to translocate between the A and P site  C8 was chosen for mutagenesis over A45 to minimize any effect that a change of sequence in the variable arm would have on serylation. Although the chance of this would be small considering that SerRS specifically recognizes the structure of the long variable arm, rather than the sequence, this was avoided . MoreoveA Figure  suggest A Figure . It folle P site . This moUTu1 -dependent translation with SelB was overcome by rewiring translation to be EF-Tu compatible. This breakthrough allowed Sec to be site-specifically incorporated into any protein . EffortsUTu1 , which sUTu1 . Howevernsertion . SpecifiE. coli. tRNAPyl and M. jannaschii tRNATyr, which are both highly used for genetic code expansion applications, have small variable arms, similar in size to tRNAPhe. The movement required of the ASF is minimal for the tRNA to reach the A/P hybrid state, interacting only with the T-loop of the tRNA (E. coli ribosome is imperative to ensure efficient A to P site translocation. Moving forward with designing synthetic tRNAs, care should be taken when considering the tRNA scaffold to maximize protein yield and prevent translational roadblocks.This knowledge can apply to other synthetic tRNAs that are used for translation in the tRNA ,39. Howethe tRNA ,42–44 ThAtomic coordinates and structure factors for the reported cryo-EM structures have been deposited with the Protein Data Bank under accession numbers 7UR5, 7URI and 7URM and the Electron Microscopy Data Bank under accession numbers EMD-26705, EMD-26713 and EMD-26714.gkac576_Supplemental_FileClick here for additional data file.


The regulation of mammalian reproductive activity is tightly dependent on the HPG axis crosstalk, in which several reproductive hormones play important roles. Among them, the physiological functions of gonadotropins are gradually being uncovered. However, the mechanisms by which GnRH regulates FSH synthesis and secretion still need to be more extensively and deeply explored. With the gradual completion of the human genome project, proteomes have become extremely important in the fields of human disease and biological process research. To explore the changes of protein and protein phosphorylation modifications in the adenohypophysis after GnRH stimulation, proteomics and phosphoproteomics analyses of rat adenohypophysis after GnRH treatment were performed by using TMT markers, HPLC classification, LC/MS, and bioinformatics analysis in this study. A total of 6762 proteins and 15,379 phosphorylation sites contained quantitative information. Twenty-eight upregulated proteins and fifty-three downregulated proteins were obtained in the rat adenohypophysis after GnRH treatment. The 323 upregulated phosphorylation sites and 677 downregulated phosphorylation sites found in the phosphoproteomics implied that a large number of phosphorylation modifications were regulated by GnRH and were involved in FSH synthesis and secretion. These data constitute a protein–protein phosphorylation map in the regulatory mechanism of “GnRH-FSH,” which provides a basis for future studies on the complex molecular mechanisms of FSH synthesis and secretion. The results will be helpful for understanding the role of GnRH in the development and reproduction regulated by the pituitary proteome in mammals. As an intermediary factor in the hypothalamic–pituitary–gonadal (HPG) axis, follicle-stimulating hormone (FSH) is actively engaged in the regulation of hormonal secretion in the gonads, follicular maturation, spermatogenesis, and other reproductive activities in a variety of mammals . AdditioFshβ transcription; Lhβ is more preferentially synthesized under the stimulation of intermediate frequency GnRH pulses , and high-frequency GnRH pulses  tend to induce Cga transcription , and the maximum number of missed sites was set to 0. The peptide length is set to 7–25 amino acid residues, and cysteine alkylation is set as a fixed modification. Transition parameters: The parent ion charge is set to 2 and 3, the daughter ion charge is set to 1, and the ion type is set to b and y. The fragment ion selection starts from the third to the last, and the mass error tolerance of ion matching is set to 0.02 Da.p < 0.05 was considered to indicate statistical significance.SPSS 19.0 software  was used for statistical analysis. ANOVAs were used to analyze the data and student’s t tests were used to analyze the significance of differences. It was found that GnRH treatment did not significantly affect the morphological structure of the rat adenohypophysis, but was able to significantly promote the secretion of FSH. In addition, 81 significantly differentially expressed proteins and 621 proteins with significantly different phosphorylation modifications were identified in the GnRH-treated adenohypophysis by proteomics and phosphoproteomics techniques.