Native chickens are vitally important livestock at the community level and represent food security and a source of savings, especially in developing countries where they are considered a valuable genetic resource for use in the development of high-yielding breeds. However, due to the constraints of their slow growth rate, there is an inadequate supply to meet consumer demand. Therefore, genetic improvement for growth traits is one sustainable way to solve these problems. In this study, a multiple trait animal model and selection index are developed as a solution for this problem to improve growth performance in smallholder farms.p > 0.05). The heritability estimates of body weight, average daily gain, and breast circumference in the chickens raised at the on-site farm and on-station were moderate to high, with on-station values slightly higher than on-site farm values. The heritability estimates of body weight were 0.236 to 0.499 for the on-site farm, and 0.291 to 0.499 for on-station. For average daily gain, the heritability estimates were 0.274 to 0.283 for the on-site farm and 0.298 to 0.313 for on-station; meanwhile, and for breast circumference, the heritability estimates were 0.204 to 0.268 for the on-site farm and 0.278 to 0.296 for on-station. Both phenotypic and genetic correlations among and between growth traits were positive and ranged from low to high values. The top 20% of the estimated breeding values and selection indices in the on-site farm and on-station experiments showed that the body weight at eight weeks of age (BW8), ADG from 4–8 weeks of age (ADG4–8), and breast circumference at eight weeks of age (BrC8) should be used as selection criteria for Thai native chicken breeding programs. In conclusion, the genetic parameters and breeding values in on-station experiments showed that the breeding program by selection index for improving growth performance is valid. Therefore, to implement such a breeding program in an on-site farm, an intensive or semi-intensive farm system should be considered to minimize the effect of genotype-environment interaction.To ensure that any new technology developed within an experimental station is appropriate to the community’s needs and compatible with the existing systems, on-site farm research is an important component in examining the effectiveness of agricultural research. The present study examined the growth performance and genetics of Thai native chickens under conditions typically experienced by farmers on smallholder farms (on-site farms) compared with at an experimental unit (on-station). There were 1694 Thai native chickens (Pradu Hang dum) used in this experiment, and they were divided into 613 chickens for the on-station and 1081 chickens for the on-site farm experiments. The individual chicken data included the birth weight (BW0) and body weight at 4, 8, 12, and 16 weeks of age , ADG from 0–4, 4–8, 8–12, 12–16 weeks of age , and breast circumference at 8, 12, and 16 weeks of age . A multiple traits animal model and a selection index were used to estimate the variance components, genetic parameters, and breeding values of growth traits. The results showed that the body weight, average daily gain, and breast circumference at 8, 12, and 16 weeks of age of Thai native chickens raised on-station were higher than those raised on-site at the farm among mixed-sex and sex-segregated chickens, while the birth weight and body weight at four weeks of age (BW0 and BW4) and ADG from 0–4 weeks of age (ADG0–4) were not significantly different ( Native chickens are vitally important livestock at the community level and represent food security and a source of savings, especially in developing countries ,2,3,4. TAlthough the benefits of native chickens are distinctive in many respects, they present a significant limitation that affects their production worldwide: their growth rate is slow compared to that of commercial broilers and crossbred native chickens. The slower growth rate significantly affects feed conversion as well. At the same time, most Thai indigenous chickens are raised in backyards with poor husbandry practices, inadequate nutrition, and hot environments. With this system, the farmers must spend a long time raising chickens to achieve the weight requirements of the market ,14. PrevTo solve this problem, a genetic method for improving growth performance should be one of the most effective and sustainable methods. Therefore, the Thailand Research Fund (TRF) and the Thai Department of Livestock Development (DLD) have cooperated in collecting a population of Thai native chicken breeds for conservation and future utilization since 2001. The Pradu Hang dum chicken breed was designated in the project and is now raised throughout Thailand. In 2010, the Research and Development Network Center on Animal Breeding (NCAB), Faculty of Agriculture, Khon Kaen University, was established to conduct research on genetic improvements of Thai native chicken breeds, including growth and carcass characteristics ,19,20, eTo ensure that “Pradu Hang dum KKU55” which has been genetically improved in its growth performance after seven generations within our experimental station is appropriate to the community’s needs, and compatible with existing systems, the on-site farm  is an important component in examining the effectiveness of breeding programs. In the past, several studies have compared the effects of farming systems (intensive and extensive) on genetics parameters ,24,25,26The study was carried out at the Research and Development Network Center of Animal Breeding and Omics, Khon Kaen University, Khon Kaen, Thailand. Meanwhile the farmers’ on-site experimental trials were performed in a rural community in Thung Pong Distinct, Khon Kaen Province (distance from on-station 50 km). These trials were supported by a career development initiative under the “Development of indigenous chicken farming for sustainability” project (reference no. 007/2562) through the collaboration between the Royal Initiative Discovery Foundation and the Research and Development Network Center of Animal Breeding and Omics, Khon Kaen University. This research project was reviewed and approved by the Institutional Animal Care and Use Committee of Khon Kaen University based on the Ethics of Animal Experimentation of the National Research Council of Thailand (No. IACUC-KKU-144/64).On-station farm research.: A total of 613 chickens were produced in two hatches, randomly selected, and mixed by sex. After hatching, the chicks were weighed individually, and an identification number was attached to the leg. Chicken management occurred up to four weeks of age, followed by wing banding to maintain body weight and growth records. All chickens received Newcastle vaccines and antibiotics according to the chicken vaccination program.2 per eight chickens. Therefore, each on-site farm received between 200–240 native chickens depending on the chicken house size. They were managed in a manner similar to that followed for the on-station research.On-site farm research. A total of 1081 chickens were produced in three hatches by farmers who had experienced raising native chickens before and had participated in training on raising native chickens with at Khon Kaen University. In addition, farmers must have had equipment for raising native chickens that included a chicken house with a concrete padded floor with rice husks foundation, while the four sides of the house were composed of wire mesh, with water tanks inside, feed tanks, and lighting. In front of the chicken house, there was a device to record body weight, feed intake, and other information. The chickens were raised within a house with 1 mThe chickens in both the on-site farm and on-station were fed with standard commercial broiler diets with two formulas according to the age of the chickens. From hatching to 4 weeks of age, 21% crude protein (CP) and 3000 Kcal/kg  were provided, followed by a grower feed with 19% crude protein (CP), and 2900 Kcal/kg  until the end of the experiment . They weIndividual chicken data consisted of the birth weight (BW0) (g), body weight at 4, 8, 12, and 16 weeks of age ), average daily gain (g/day) during 0–4, 4–8, 8–12, and 12–16 weeks of age , and breast circumference (cm) at 8, 12, and 16 (slaughter weight) weeks of age . Before using the data for statistical analysis, the Proc UNIVARIATE procedure by SAS v.9.0 software was used to examine data distribution, including assessing normality and checking data outliers.p < 0.05).The recorded data were validated and analyzed for the least squares mean (LSmean) value, and significant differences were compared by research site (on-site farm and on-station) between mixed-sex and separated-sex chickens using the generalized linear model for an unbalanced analysis of variance (GLM procedure) in the SAS package to investigate significant differences. If significant differences were detected, then multiple pairwise comparisons were conducted using Scheffe’s test (The average information-restricted maximum likelihood (AI-REML) method with the best linear unbiased prediction (BLUP) were used to estimate the variance components, genetic parameters , and estimated breeding values (EBVs) . The mulP = phenotypic variance-covariance matrix, The generalized linear model for an unbalanced analysis of variance (PROC GLM) using the SAS package was used to compare the EBV by research site. The accuracy of the selection index by research site was calculated based on the following equation: The selection index was calculated based on three traits: body weight, average daily gain, and breast circumference (used to represent the quantity of breast meat). The relative economic value (p < 0.05), while the birth weight and body weight at four weeks of age (BW0 and BW4) were not significantly different (p > 0.05). The results showed consistent trends with the sex-segregated analysis, as shown in p < 0.05). Only the ADG at 0–4 weeks of age was not significantly different between the on-site farm and on-station chickens (8.11 vs. 8.48 g/day) (p > 0.05). In terms of breast circumference (BrC) traits, we found significant differences at 8, 12, and 16 weeks of age  (p < 0.05) in both the mixed-sex and sex-segregated  chickens, as shown in A comparison of the least square means of the body weight, average daily gain, and breast circumference between Thai native chickens raised on-site farm and on-station is shown in The phenotypic correlations of body weight (BW), average daily gain (ADG), and breast circumference (BrC) in Thai native chickens raised on-site farm and on-station are presented in The genetic correlations of body weight (BW), average daily gain (ADG), and breast circumference (BrC) in Thai native chickens raised on-site farm and on-station are presented in The top 20% of the estimated breeding values and selection indices for the on-site farm and on-station Thai native chickens are presented in p > 0.05). However, from 8 weeks onward, the native chickens raised on-station had statistically significantly higher body weight than the native chickens raised on-site farm (p < 0.05). The differences in body weight between the native chickens raised on-station and on-site farm were 72, 192, and 424 g at 8, 12, and 16 weeks, respectively. The results of this study agree with several studies. For example, Lwelamira [This study used a multitrait animal model and selection index to compare the growth performance and genetic variations in Thai native chickens raised in different environments, namely, on-site farm and on-station environments. Differences were not observed in birth weight, body weight at four weeks of age or ADG from 0–4 weeks of age in the mixed-sex and sex-segregated chickens raised on-site farm and on-station  about their satisfaction. We received interesting information that some farmers had other activities besides raising chickens, such as rice planting and cassava farming. Therefore, it may be one reason farmers do not fully spend time raising native chickens ,43.The heritability estimates for the BW, ADG, and BrC traits in the present study were medium to high  between the growth traits were positive and ranged from 0.02 to 0.90 see . The pheThe EBV showed that the native chickens raised on-station presented high phenotype growth efficiency see  and highThe results of this study indicate that different environmental effects (on-site farm and on-station) affect both the growth characteristics and genetic expression of Thai native chickens. The phenotypic and genetic growth traits of native chickens raised on-station showed better correlations than those of native chickens raised in an on-site farm. In addition, the genetic parameters and breeding values in on-station showed that the breeding program by selection index for improving growth performance is valid. Therefore, to implement such a breeding program in an on-site farm, an intensive or semi-intensive farm system should be considered to minimize the effect of genotype-environment interaction.


Ovalbumin (OVA) is one of major allergens of hen egg white with excellent nutritional and processing properties. Previous research exhibits that pulsed electric field (PEF) treatment could partially unfold OVA. This may contribute to the improvement of OVA phosphorylation. In this study, the effect of PEF pretreatment combined with phosphorylation on the structure and immunoglobulin (Ig) G/IgE-binding ability of OVA was investigated. The structural changes were measured by circular dichroism (CD), ultraviolet absorption, and fluorescence spectroscopy. The IgG- and IgE-binding abilities were determined by inhibition enzyme-linked immunosorbent assay (ELISA) using rabbit polyclonal antibodies and egg-allergy patients’ sera, respectively. The results showed that PEF pretreatment combined with phosphorylation markedly reduced the IgG- and IgE-binding abilities. It was attributed to the changes in secondary and tertiary structure, which was reflected in the increase of ultraviolet (UV) absorbance, α-helix content, and the increase the molecular weight. Moreover, it suggested PEF pretreatment improved the phosphorylation of OVA and enhanced the reduction of IgG/IgE-binding capacity of phosphorylated OVA. Therefore, PEF pretreatment combined with phosphorylation has the potential for developing a method for OVA desensitization. Hen’s egg is one of the most important daily nutrition resources and food ingredients. It has been widely applied into the food industry because of its outstanding nutritional and functional properties, such as excellent absorption, gelation, and emulsification . HoweverOvalbumin (OVA), named as Gal d 2, which constitutes about 54% of egg white proteins, is a 45-kDa glycoprotein with 385 amino acids. It becomes an ideal experimental model protein of food allergy because of its strong allergenicity and available quantity , pulsed Phosphorylation is a common protein modification that realized by chemical or enzymatic method . It is gCompared with the enzymatic phosphorylation, chemical phosphorylation has some advantages, such as low cost and significant phosphorylation effect. It could not only improve some of OVA functional properties, but also could promote calcium absorption by using as a dietary supplement because of the increase in the solubility of calcium phosphate through introduction of phosphate groups . RecentlPulsed electric field (PEF) is an emerging promising non-thermal technology for food processing and preservation . CompareIn the present study, phosphorylation combined with PEFs pretreatment was used to treat OVA, aiming to evaluate the effect of phosphorylation after PEFs pretreatment on IgG/IgE-binding ability and structural properties of OVA, and also to expound upon their relationships. It will enrich the relevant theory of chemical phosphorylation combined with the PEFs for OVA desensitization.Goat anti-rabbit IgG–HRP conjugate, goat anti-human IgE–HRP conjugate, STPP and 1-anilinonaphthalene-8-sulfonate (ANS) were purchased from Sigma-Aldrich . Egg allergy patients’ sera were bought from PlasmaLab International  and frozen (–80°C) until analysis. Their specific IgE levels ranged from 10.8 to 64.6 kU/L . The polt) was calculated as follows , f is the pulse repetition rate , W is the pulse width (μs), and v the flow rate (ml/s). In this study, the pulse repetition rate and pulse width were 20 Hz and 5 μs, respectively. The flow rate of OVA sample was 0.33 ml/s.where 0) × 100%, where B and B0 are the absorbance values of the well with and without the inhibitor, respectively. IC50 is the concentration of inhibitors that causes a 50% inhibition of antibody binding (μg/ml) (The IgG- and IgE-binding capacities of OVA were determined by inhibition enzyme-linked immunosorbent assay (ELISA) with rabbit polyclonal anti-OVA-sera and egg allergy patients’ sera, respectively . At firs (μg/ml) .According to the method of Zhang et al. , with soThe average number of phosphate molecules attached to each OVA molecule, evaluated as incorporation ratio (IR), was used to assess the degree of phosphorylation . Based oPA, and MWNA are the molecular weight of phosphorylated OVA and native OVA, respectively.where 79.9799 is the molecular weight of phosphates attached to OVA, MW2/dmol], and the contents of different secondary structures were analyzed with DichroWeb.1The secondary structure of OVA was defined by far-ultraviolet (UV) (190–240 nm) circular dichroism (CD) spectroscopy. The MOS-450 spectropolarimeter  was used to collect the CD spectra of OVA (0.1 mg/ml) at scan speed of 100 nm/min. The path length, bandwidth and step resolution were all 0.1 cm. The data were showed as molar residue ellipticity [(θ), deg cmThe UV-2910 Hitachi spectrophotometer  was applied to collect the UV absorption spectra. The OVA samples (0.5 mg/ml) were scanned from 240 to 320 nm at a scan speed of 800 nm/min. The F-7000 Hitachi spectrofluorometer  was used to measure the intrinsic fluorescence of OVA (0.2 mg/ml). The emission spectra were scanned from 300 to 420 nm at a speed of 1,200 nm/min with the excitation wavelength of 280 nm. The bandwidth, excitation, and emission slits were all 5.0 nm.0) was the initial slope of the fluorescence intensity vs. protein concentration plot, which was calculated by linear regression analysis.According to the method of Yang et al. , with moAll the experiments were carried out in triplicate, and all results were presented as mean value ± SD. The statistical analysis was performed by ANOVA with SPSS 19.0  and OriginPro 2020 .50 value is the inhibitor concentration that causes a 50% inhibition of the antibody-binding capacity, which reflecting that the higher the IC50 value, the lower the binding capacity. 50 values of OVA treated under different conditions. As shown in 50 value of OVA-H was almost the same as that of OVA-N. However, after OVA was phosphorylated with phosphates, it markedly increased from 2.02 mg/ml (OVA-N) to 5.64 mg/ml (OVA-P). Moreover, when phosphorylated with PEF pretreatment, OVA showed further higher IC50 values, with the highest value (14.77 mg/ml) observed at PEF pretreatment at 25 kV/cm. Similar results were shown in p < 0.05). However, OVA-P had higher IC50 value than OVA-N. Furthermore, phosphorylated OVA with PEF pretreatment had further higher IC50 values and OVA-P-25 had the lowest IgE-binding ability (15.31 mg/ml).In this work, the IgG- and IgE-binding abilities of OVA samples were determined by inhibition ELISA with rabbit polyclonal anti-OVA-sera and egg-allergy patients’ sera, respectively. The ICPhosphorylation is the result of the typically covalent binding of the phosphates to proteins, without participation of enzyme. It will result in the increase of the molecular weight of protein. p < 0.05) in CD spectra of OVA after dry-heating without phosphates. However, the α-helix content of OVA phosphorylated with STPP increased from 24.1% (OVA-N) to 28.5% (OVA-P) while the β-sheet content decreased from 25.5% (OVA-N) to 19.8% (OVA-P). Furthermore, compared with OVA-P, the phosphorylated OVA with PEF pretreatment had more α-helix content and less β-sheet content. Besides, the unordered content of phosphorylated OVA increased by up to 10.1% when it is compared with that of OVA-N.The changes of the far-UV CD spectra of OVA samples were depicted in max) intensity of OVA slightly increased from 843 (OVA-N) to 881 (OVA-H) after dry-heated without phosphates. The result was consistent with UV absorption analysis, suggesting that dry-heating at 55°C for 4 h had limited influence on the OVA tertiary structure. However, after phosphorylation, the λmax intensity of OVA-P, OVA-P-20, OVA-P-25, and OVA-P-30 decreased to 551, 485, 433, and 460, respectively.The changes in the intrinsic fluorescence spectra of OVA samples were shown in p < 0.05) in surface hydrophobicity of OVA after dry-heating without phosphates, implying that dry-heating had little effect on the surface hydrophobicity of OVA. However, after phosphorylation with phosphates, the surface hydrophobicity of OVA dramatically increased from 59.6 (OVA-N) to 118.8 (OVA-P). Moreover, when OVA was phosphorylated after PEF pretreatment, its surface hydrophobicity was further decreased, with the highest value of 179.5 (OVA-P-25).via a covalent bond between proteins and phosphates. According to the ways of phosphorylation, it is divided into enzymatic phosphorylation and chemical phosphorylation. The latter has more application potential than the former due to its low cost and convenience. Phosphorylation has been used to improve protein functional properties among them allergenicity  and double-blind placebo-controlled (DBPC) trials, should be implied to prove the decreased allergenicity by PEF pretreatment combined with phosphorylation.In conclusion, it was shown that phosphorylation combined with PEF pretreatment dramatically decreased the IgG- and IgE-binding abilities of OVA, which was closely correlated with the structural changes. Moreover, PEF pretreatment promoted the reduction of IgG- and IgE-binding capacities by improving the phosphorylated of OVA. The results imply that PEF pretreatment combined with phosphorylated may be a promising method for OVA desensitization. However, in the future, the phosphorylated sites of OVA treated under the condition should be revealed by mass spectrometry to explore the mechanism of PEF pretreatment combined with phosphorylated reduces the IgG and IgE-binding of OVA at the molecular level. Besides, other experiments The original contributions presented in this study are included in the article/supplementary material, further inquiries can be directed to the corresponding authors.WY and QW designed and wrote the manuscript. WY and DD analyzed the data. QL and WD provided the data and revised the manuscript. All authors contributed to the article and approved the submitted version.


Inhibitory oligodeoxynucleotides (ODNs) are short single-stranded DNA, which capable of folding into complex structures, enabling them to bind to a large variety of targets. With appropriate modifications, the inhibitory oligodeoxynucleotides exhibited many features of long half-life time, simple production, low toxicity and immunogenicity. In recent years, inhibitory oligodeoxynucleotides have received considerable attention for their potential therapeutic applications in immune-mediated inflammatory diseases (IMIDs). Inhibitory oligodeoxynucleotides could be divided into three categories according to its mechanisms and targets, including antisense ODNs (AS-ODNs), DNA aptamers and immunosuppressive ODNs (iSup ODNs). As a synthetic tool with immunomodulatory activity, it can target RNAs or proteins in a specific way, resulting in the reduction, increase or recovery of protein expression, and then regulate the state of immune activation. More importantly, inhibitory oligodeoxynucleotides have been used to treat immune-mediated inflammatory diseases, including inflammatory disorders and autoimmune diseases. Several inhibitory oligodeoxynucleotide drugs have been developed and approved on the market already. These drugs vary in their chemical structures, action mechanisms and cellular targets, but all of them could be capable of inhibiting excessive inflammatory responses. This review summarized their chemical modifications, action mechanisms and applications of the three kinds of inhibitory oligodeoxynucleotidesin the precise treatment of immune-mediated inflammatory diseases. Nucleic acids derived from microorganisms can trigger powerful immune responses through a variety of signaling sensors, including Toll-like receptors (TLRs), cyclic GMP-AMP Synthase (c-GAS), melanoma differentiation-associated protein 5 (MDA5), retinoid-induced gene-1 (RIG-I) and oligomerization domain (NOD)-like receptors (NLRs) . UnmethyIn the past few decades, inhibitory ODNs have attracted great attention because their potential therapeutic applications in immune-mediated inflammatory diseases (IMIDs) . A bunchAntisense oligodeoxynucleotides (AS-ODNs) are short, synthetic, single-stranded oligodeoxynucleotides that are complementary to specific sequences of target RNA . They alDNA aptamers are oligodeoxynucleotide compounds synthesized from single-stranded DNA molecules, usually consisting of 15–100 nucleotides, with the ability to form secondary and tertiary structures that bind specifically to target molecules . AptamerImmunosuppressive oligodeoxynucleotide (iSup ODN) refers to a class of artificially synthesized single-stranded DNA molecules with a length of tens of nucleotide sequence to function the negative regulation of immune response . CompareSingle-stranded ODNs face several challenges that complicate drug development. The unfavorable properties of ODNs include: 1) degradation by nucleases when introduced into biological systems, 2) poor uptake through cell membranes, 3) unfavorable biodistribution and pharmacokinetic properties and 4) suboptimal binding affinity for complementary sequences . TherebyFirst-generation chemistries include the widely used phosphate backbone modifications, e.g., phosphorothioate (PS). PS modification, replacement of a non-bridging phosphodiester oxygen by sulfur, is the most widely used single alteration in nucleic acid drug development . PS linkIn addition, other types of chemical modifications have been developed, such AS locked nucleic acid (LNA), peptide nucleic acid (PNA), and phosphorodiamidate morpholino oligonucleotides (PMO) . PNA canAS-ODNs prevent protein translation of certain mRNA strands by binding to them, in a process called hybridization. By contrast, DNA aptamers can directly bind to specific target molecules especially proteins with high affinity and specificity. Unlike the above mentioned inhibitory ODNs, iSup ODNs play their role mainly by competitively binding receptors or inhibiting signal transduction of important factors in inflammatory pathways. Relatively, the mechanism of AS-ODN is clearer due to its widely used in basic research and clinical application.RNase H is responsible for the degradation of RNA-DNA hybrids synthesized along the chain in the nucleus. RNase H is also present in the cytoplasm and degrades mature mRNA . The DNAAS-ODNs can block the binding of some functional groups to the functional sequences involved in gene splicing in pre-mRNA, regulate or change the gene-splicing mode of the pre-mRNA, and induce the production of new functional protein heteroform  Figure . During Triple helix-forming oligonucleotides could bind to the major groove of double-stranded purine-rich chain to form Triple-stranded DNA through Hoogsteen or reverse Hoogsteen hydrogen bonds . Thus, tin vivo , hepatitis B and C viruses (HBV and HCV), herpes viruses or any new virus . Fomivirin vivo, and increases the level of surfactant in experimental models of allergic asthma (via receptors with a common beta subunit (betac) that transduces cell signaling. TPI ASM8 is an AS-ODN targeting betac with PS modification. It has been confirmed that TPI ASM8 downregulated the biologic activities of GM-CSF, IL-3, and IL-5 simultaneously by inhibiting betac mRNA expression with antisense technology. Moreover, TPI ASM8 has been shown to be safe and well tolerated in human trials , but also associated with NF-κB signaling (+ (sTLR9) neutrophils to play a protective role in the development of systemic inflammatory response syndrome (SIRS) in locally inflammatory areas  . Interesignaling . In addiignaling . A closeignaling . Anotherignaling . Meanwhiignaling . In addiry areas . YW002 cry areas .in vitro  is an autoimmune disease with glomerulonephritis and multifocal terminal organ damage caused by many pathogenic autoantibodies and immune complexes . The mamin vitro . Rheumatin vitro . In genein vitro .In the past two decades, a great deal of research has been done on the mechanism of inhibitory ODN in cells and its application in therapy, and great progress has been made in this field, providing potential therapeutic options for many medical problems that cannot be solved at present, and it is the most promising tool for gene targeted therapy at present. However, it is not difficult to find that there are still some problems in the research and development of inhibitory ODN drugs, such as easy to be degraded by ribozyme in the blood, and small molecular weight leads to fast renal clearance. Of course, appropriate modifications and efficient encapsulation are necessary for inhibitory ODNs to enhance the transport across membranes. In addition, off-target problem is also a non-negligible problem in drug development of inhibitory ODNs. Sometimes, inhibitory ODNs cannot accurately combine its corresponding target to achieve therapeutic effect due to the potential poor specificity and low affinity with the target. To solve the problem, it is necessary to modify inhibitory ODNs with longer length and more stable three-dimensional structure to enhance its binding specificity and affinity with the target.Inhibitory ODN is a multi-faceted tool capable of delicate basic research and discovery that can later be molded into a therapeutic agent for clinical application. Reliability and reproducibility will be the pillars of future inhibitory ODN research as they are critical to successful clinical translation. With the increasing maturity of molecular genetics, pharmacology and chemical synthesis, as well as the rapid development of bioinformatics, we look forward to the discovery of more new mechanisms of action and the application of genomic information to ODN design, so further promote the application of inhibitory ODNs in the treatment of immune-mediated inflammatory diseases.


Despite several advances in the field, pharmacodynamic outcome measures reflective of LRRK2 kinase activity in clinical biofluids remain urgently needed. A variety of targets and approaches have been utilized including assessments of LRRK2 itself , or its substrates (e.g. Rab10 or other Rab GTPases). We have previously shown that intrinsic kinase activity of LRRK2 isolated from PBMCs of G2019S carriers is elevated, irrespective of disease status. In the present study we find that phosphorylation of Rab10 is also elevated in G2019S carriers, but only those with PD. Additionally, phosphorylation of this substrate is also elevated in two separate idiopathic PD cohorts, but not in carriers of the A53T mutation in α-synuclein. In contrast, Rab29 phosphorylation was specifically reduced in urinary exosomes from A53T and idiopathic PD patients. Taken together, our findings highlight the need for the assessment of multiple complimentary targets for a more comprehensive picture of the disease. Similarly, early biomarker studies, assessing LRRK2 auto-phosphorylation (pS1292-LRRK2) in urinary exosomes, have indicated that LRRK2 activity may be elevated during the clinical progression of LRRK2-associated, as well as idiopathic, PD (iPD)3. In addition to auto-phosphorylation of LRRK2 and its intrinsic kinase activity, which are measures of distinct aspects of its kinase function, other readouts of the activation state of LRRK2 include phosphorylation of endogenous substrates, such as members of the Rab family of small GTPases4. Extending these findings, the group of Greenamyre and colleagues5 applied a novel proximity ligation assay to detect pS1292-LRRK2 and pT73-Rab10 in ventral midbrain of post mortem tissue from iPD patients. In subsequent studies, we have reported that the in vitro kinase activity of purified LRRK2 from peripheral blood mononuclear cells (PBMCs) of G1029S-LRRK2 mutation carriers is elevated6. Alternatively, while phosphorylation at the S935 site within the N-terminal region of LRRK2 is a robust measure of target engagement (e.g. refs. 8), it does not completely track with kinase activity of LRRK29.The kinase activity of leucine-rich repeat kinase 2 (LRRK2) has emerged as the primary target for both therapeutic development and biomarker design in Parkinson’s disease (PD). Both cellular and animal models have demonstrated that neurodegeneration induced by mutant forms of LRRK2 is kinase activity-dependent  in neutrophils was not significantly changed in carriers of the G2019S LRRK2 mutationLRRK2 carriers only, whereas levels in healthy carriers and A53T-SNCA PD patients are not changed from healthy controls. Additionally, phosphorylation of Rab29 is decreased in urinary exosomes from SNCA mutation carriers and iPD patients. Thus, it appears that alterations in peripheral LRRK2-dependent Rab phosphorylation are not uniformly linked to either disease status or genotype.In the present study, we find increased Rab10 phosphorylation in iPD patients and affected G2019S-6, expression of Rab10  was not significantly altered in either iPD cohort. As an alternative approach, similar to what has been reported by Wang and colleagues8, we normalized pRab10 to total LRRK2 expression; however, while the same trends were retained (i.e. G2019S+ PD+ and iPD levels were slightly increased) the differences were not statistically significant . Finally, in PBMCs from affected carriers of the A53T mutation in α-synuclein (ASYN), phosphorylation of Rab10 was unchanged in PBMCs compared to healthy controls , even in the presence of a signal boost reagent , suggesting that within PBMCs, Rab29 is not a particularly prominent LRRK2 substrate, either in the healthy control population or in any of the PD cohorts; or that specific Rab29 phosphatases are particularly active in these cells.10, and similarly it is not possible, in the preparations we employed, to determine the cellular source of the EVs recovered from the patient urine samples. A more targeted study of specific blood cell types and immuno-isolated EVs  would be better suited to reveal such a correlation.In extracts of urinary extracellular vesicles (EVs), the detection of LRRK2 was highly variable; in many samples, total and pS1292-LRRK2 was un-detectable  from the CUMC-Movement Disorders center. In PBMCs from these individuals, we found a significant elevation in phosphorylation of the LRRKtide peptide substrate in both G2019S carrier groups, compared to healthy controls and idiopathic PD patients6. This is not surprising given the conserved motif that this mutation lies within in the LRRK2 kinase domain (e.g. ref. 14). In the same report, we could not detect changes in the in vitro kinase activity of LRRK2 isolated from iPD PBMCs6.In the present study, we assessed Rab GTPase phosphorylation in PBMCs and urinary exosomes from multiple PD cohorts collected at different clinical centers. We recently reported several LRRK2-based outcome measures in a cohort of G2019S-only in affected carriers of this mutation, healthy G2019S carriers exhibited pT73-Rab10 levels similar to healthy controls. Thus, the isolated enzyme bearing the Gly to Ser substitution in the critical DYG kinase domain motif exhibits elevated intrinsic kinase activity, regardless of disease status6; however, in a cellular context the disease status clearly interacts with the genotype to modulate Rab10 phosphorylation, at least in peripheral immune cells. In other words, in vitro kinase activity is a good predictor of LRRK2 mutation status, but not of disease status. In another recent study, combining western immunoblotting and targeted mass-spectrometry measures of phosphorylated Rab10, it was reported that T73-Rab10 levels in a small sample of carriers of the R1441G were significantly elevated in neutrophils compared to healthy control subjects12. Unlike the present study, phosphorylated Rab10 levels from carriers of the G2019S-LRRK2 mutation were not different from healthy controls, regardless of disease status12; however in an earlier study also using an MS-based approach, pT73-Rab10 levels in neutrophils were approximately 2-fold higher in a set of G2019S carriers15. We did not detect changes in Rab10 phosphorylation in CD14+ monocytes in either the iPD or the A53T-ASYN patient groups, suggesting that the cellular source of increased pRab10 in the iPD and G2019S+PD+ PBMCs is not this subset of monocytes, but perhaps neutrophils (e.g.12), or other monocyte subsets, such as CD16+ monocytes which have been shown to exhibit increased LRRK2 expression in PD16. Additionally, using a novel phospho-specific Rab10 antibody in an ELISA-based outcome measure, Wang and colleagues reported slightly elevated pRab10, normalized to LRRK2 levels, in PBMCs of both healthy and affected G2019S carriers8. It should be noted that in this study, the authors reported a slight but significant decrease in LRRK2 expression in both G2019S-LRRK2 groups, which may account for the elevated pRab10 levels when normalized to LRRK2 instead of total Rab10. Nevertheless, while the current study remains semi-quantitative in its approach, the study by Wang and colleagues represents an important advance in the field in terms of more quantitative assessments of changes in LRRK2 pathway signaling in PD.In PBMCs from G2019S carriers, we report here increased phosphorylation of Rab10 (T73) 6; and manuscript in preparation); whereas in two independent cohorts (CUMC and Athens), we find increased phosphorylation of Rab10 in PBMCs from iPD patients. Not surprisingly, there is considerable variation in phosphorylated Rab10 levels in these clinical samples, which is reflective of course of LRRK2 kinase activity but also potentially other kinases, as well as the activity of PPM1H, a phosphatase implicated in Rab10 de-phosphorylation17. Further, deeper analyses should be performed to determine if specific subsets of patients exhibit increased Rab10 phosphorylation, which would likely be correlated with other biochemical outcomes or other specific clinical measures. In this study, however, we did not observe any correlations with clinical outcomes (e.g. UPDRS-III and others). Previous reports have not revealed differences in Rab10 phosphorylation in this patient group (e.g. ref. 11); the reasons for this discrepancy are unclear, however may be related to the normalization methods taken in each study. Rab10 phosphorylation is reported to be increased in post-mortem brain from iPD patients5; thus, taken together with our present findings, it suggests that increased phosphorylation of this key substrate, and by extension LRRK2 kinase activity, is a systemic phenomenon in this disease cohort. The lack of significant increases in Rab10 phosphorylation in A53T-PD patients suggests that there are multiple PD progression pathways, depending on the form of the disease. Alternatively, in this specific patient group, with an earlier age of disease onset . While we could not detect changes in phosphorylation of Rab10 in this cohort, the reduction in Rab29 phosphorylation is the first indication of possible changes in LRRK2 activation in clinical samples from PD patients carrying the A53T-SNCA mutation; and bolsters the notion that LRRK2 function plays an important role in PD in general. A summary of our findings in the current study, in the context of our previously reported findings from one of the two cohorts (from CUMC) is presented in Table 20.A second key finding we report here is the loss of phosphorylation of Rab29 (Thr71) in urinary exosomes from iPD and A53T-PD patients. Recently, it was reported by two independent groups that over-expression of Rab29 can trigger the recruitment of LRRK2 to intracellular membranes and the trans-Golgi network (TGN) leading to its activation-LRRK2/PD+ & G2019S-LRRK2/PD−) followed at Columbia University Medical Center  have been reported in detail in a separate manuscript6, and are summarized in Table SNCA PD) was derived from patients followed at the Departments of Neurology at Henry Dunant Hospital Center  and Aiginiteio University Hospital . All samples were collected, processed and coded at the time of collection so that the analyses of the samples were performed by researchers blinded to the PD and mutation status. The study was approved by the institutional ethical review boards of both Columbia University (RNA) as well as Biomedical Research Foundation of the Academy of Athens , and all participants signed written informed consent. Thus, in this study we have included two independent groups of healthy controls and iPD patients. Demographics and key clinical features of the Athens cohort are summarized in Table The samples that we assessed were collected as part of two separate biomarker studies. The demographics of a subset of the clinical cohort  was collected from participants in the morning of their clinic visit and tested for urinary infections.PBMCs were isolated from whole blood collected in Heparin-coated Vacutainer collection tubes (BD), using protocols established in our recent work3, and lysed under the same conditions as PBMCs. Briefly, an initial centrifugation took place for 30 min, at 4500 g, at 4 °C for debris clearance and the supernatant was stored at −80 °C in 50 ml falcon tubes. After quickly thawing the urine in a 42 °C waterbath, the EVs were isolated by ultracentrifugation  at 100,000 g, for 1 h at 4 °C. EV pellets were lysed with 50 μl lysis buffer , for 1 h on ice. Lysates were then centrifuged at 13,000 rpm, for 15 min at 4 °C and the supernatant was collected and stored at −80 °C. Parallel EV pellets from sample preps were resuspended in PBS after ultracentrifugation for TEM imaging  and 15 ml was transferred to a 50 ml falcon and 750 μl ‘’Isolation Cocktail” from the EasySepTM Direct Human Monocyte Isolation Kit (StemCell Technologies) was added. ‘’RapidSphere Magnetic Beads” from kit were vortexed for 30 s before use and added to the blood sample. Blood was mixed gently by tube inversion and incubated for 5 min, at RT. The volume was increased to 50 ml with 1 mM EDTA-PBS Ca2+/Mg2+ free (Gibco), and the solution was gently mixed. The tube was placed into a magnet (StemCell Technologies) without the lid, for 10 min, to immobilize the magnetic beads along the wall of the centrifuge tube. The enriched monocyte cell suspension was carefully removed (leaving ~10 ml behind) into a new 50 ml falcon, where 750 μl of fresh magnetic beads were added and tube was gently mixed followed by 5 min RT incubation. The cell suspension was again removed (leaving ~5 ml behind) and to ensure complete removal of magnetic beads, the tube was placed in the magnet for another 10 min, at RT. Purified CD14+ monocytes were then transferred to a new falcon (leaving ~5 ml behind) and the 30 ml suspension was increased with 1 mM EDTA-PBS Ca2+/Mg2+ to a final volume of 40 ml. Centrifugation for 5 min, at 335 g, at RT followed and the cell pellet was washed once with 1X PBS and stored at −80 °C. Prior to biochemical analysis, pellets were lysed with 50 μl lysis buffer  added fresh), for 30 min on ice. Lysates were then centrifuged at 45,000 rpm, for 1 h, at 4 oC and S/N was collected and stored at −80 °C.Where possible, in the Athens cohort, we collected CD14+ monocytes in addition to PBMCs and urine. Blood was collected in KFor western immunoblotting, protein samples were separated by SDS-PAGE, and the membranes blocked in either 5% Milk/TBST  or in 5% BSA/TBST , for 1 h at RT. The primary antibodies used were as follows: Rab10 , Rab29 , Flotilin-1 , LRRK2 , pT73-Rab10  and pT71 Rab29 , and pS1292-LRRK2 , shaking overnight at 4 °C. Initially, full-length membranes were probed with a primary antibody , and band intensities were obtained by an independent researcher, before the sample ID’s were decoded. Band intensities of phosphorylated Rab10 were first normalized to total Rab10 expression; and, alternatively to total levels of LRRK2 determined by ELISAt-tests where indicated.For statistical comparison of the different study groups, we employed ANOVA with Tukey’s post-hoc multiple comparisons, or if required non-parametric Kruskal–Wallis tests & Dunn’s post-hoc test; or two-tailed Supplementary MaterialSupplementary Figure 1Supplementary Figure 2Supplementary Figure 3


Inhibition, associated with self-efficacy, enables people to control thought and action and inhibit disturbing stimulus and impulsion and has certain evolutionary significance. This study analyzed the neural correlates of inhibition modulated by self-efficacy. Self-efficacy was assessed by using the survey adapted from the Motivated Strategies for Learning Questionnaire. Fifty college students divided into low and high self-efficacy groups participated in the experiments. Their ability to conduct inhibitory control was studied through Go/No-Go tasks. During the tasks, we recorded students’ brain activity, focusing on N2 and P3 components in the event-related potential (ERP). Larger No-Go N2 amplitudes for the high self-efficacy group were found compared with the low self-efficacy group. Conflict detection as represented by N2 was modulated by self-efficacy, whereas conflict inhibition as represented by P3 was not modulated by self-efficacy. The highly self-efficacious students were more capable of detecting conflicts but not necessarily more capable of inhibiting action given that conflict was detected. Taken together, these findings offer neurophysiological evidence of the important regulatory role of self-efficacy in inhibitory control ability development. Inhibition or inhibitory control, referred to as response inhibition, is an executive function defined as the ability to deliberately withhold or override a dominant, prepotent , or automatic response to resist distraction or temptation and to achieve the desired goal . InhibitThe common way to examine the neural correlates of inhibition is using Go/No-Go tasks . In a tyThe ability to conduct inhibitory control is studied through Go/No-Go tasks. The experimental indicators are No-Go N2 and No-Go P3.This study was conducted at a public university of science and engineering in the city of Beijing, China. We used convenience sampling for this study. There were 61 students who had previously been tested for self-efficacy and appropriate samples were selected based on their pretest scores. Twenty-four college students with high self-efficacy were selected, and 26 college students with low self-efficacy were selected. There were 12 females and 12 males, 11 undergraduate students and 13 graduate students in the high self-efficacy group; and 14 females and 12 males, 12 undergraduate students and 14 graduate students in the low self-efficacy group. The experimental participants were right-handed, with normal or corrected vision, and they had no history of mental illness. At the request of the Academic Ethics Committee of the university, they signed informed consent to participate in this study. Self-efficacy was assessed before Go/No-Go tasks by using the survey adapted from the Motivated Strategies for Learning Questionnaire (MSLQ) . The MSLBefore the experiment, the experimental procedure was described and the students participated in the experiment in a relaxed state. Pictures including single triangle and double triangle were selected as the stimulus. The participants were instructed to respond by pressing the button “/” (right hand) or “z” (left hand) as quickly as they could whenever the “Go” stimulus (double triangle) was presented and not to press the button when the “No-Go” stimulus (single triangle) was presented. A fixation cross to orient attention to the middle of the screen was presented for 500 ms. Stimuli were presented for 50 ms, and participants could respond anytime within the onset of the stimulus and the interstimulus interval (950 ms). A practice phase of 20 trials with feedback was given. Each condition consisted of 150 test trials. Go stimuli were presented for most of the trials (80%). All experimental tasks were presented using the E-prime software 3.0.Electroencephalogram (EEG) signals were continuously recorded with the NeuroLab digital amplifier system , using NeuCap with Ag/AgCl electrodes at 32 sites according to the extended international 10–20 system. The reference electrode was placed on the nose tip. The nose-tip reference was converted into bilateral mastoid for reference in offline data analysis. Vertical and horizontal electrooculography (EOG) signals were recorded with two electrodes placed above and below the right eye and with two electrodes at the right and left outer canthi of the eyes, respectively. Data were recorded continuously at a sampling rate of 1,000 Hz and filtered offline with a bandpass of 0.01–100 Hz. Electrode impedance was maintained below 5 kΩ throughout the experiment.1 was used to analyze EEG data. Blinks were corrected using an ICA procedure. The plotted average, condition-specific activation had to account for the N2/P3 time course. The EOG components were identified and selected according to the topographical maps that had to show a fronto-central scalp distribution, which is usually seen with the N2 and P3. One of the EOG components was removed on average. They were performed on all subjects. Remaining artifacts exceeding ± 100μV in amplitude or containing a change of over 100μV within a period of 50 ms were rejected. After performing EOG correction and visual inspection, only artifact-free trials were considered .The EEGLab softwareThe EEG was segmented in epochs of 700 ms, time-locked to picture onset, and included a 100 ms pre-stimulus baseline. Trials contaminated by amplifier clipping, bursts of EMG activity, or peak-to-peak deflection exceeding ± 100 μV were excluded from averaging (excluded 5%). The averaged ERP waveforms were low-pass filtered at 30 Hz. Filter frequencies correspond to the half-amplitude cut-off (24 dB/octave rolloff).For response times, only Go trials with correct responses were included. There were no response times for correct response No-Go trials, as no response was made if participants inhibited the response successfully. Means for all the conditions are presented in post hoc comparisons were performed with Bonferroni correction. Analyses were conducted using the SPSS software .The ERP literature has examined Fz, Cz, and Pz to analyze the inhibition process or self-efficacy effects e.g., . The Fz,F = 4.48, p = 0.04, partial η2 = 0.09) and the main effect of the site  = 6.21, p = 0.003, partial η2 = 0.11), with the Go trials (−2.59 μV) exhibiting smaller amplitude than the No-Go trials (−2.83 μV), and the N2 amplitude at the Cz (- 3.45 μV) significantly larger than those at the Fz (- 1.19 μV) and Pz (- 2.77 μV), there was no interaction among self-efficacy level, Go/No-Go trials, and electrode sites, between Go/No-Go trials and electrode sites, and between self-efficacy and electrode sites. But the main effect of self-efficacy was significant  = 7.12, p = 0.01, partial η2 = 0.13). There is interaction between self-efficacy and Go/No-Go trials  = 4.76, p = 0.03, partial η2 = 0.09). The amplitudes of No-Go N2 were significantly larger in the high self-efficacy group (- 3.42 μV) than in the low self-efficacy group .While repeated-measures ANOVA see  revealedF = 6.74, p = 0.002, partial η2 = 0.12), with the P3 amplitude at the Pz (0.76 μV) significantly larger than those at the Cz (- 0.47 μV) and Fz (- 0.70 μV). A significant interaction effect was found between Go/No-Go trials and electrode sites  = 18.77, p < 0.001, partial η2 = 0.28), with no other interaction effect observed. When we explored each site, self-efficacy level and Go/No-Go trials significantly interacted at only Pz  = 5.02, p = 0.01, partial η2 = 0.12), and an enhanced P3 amplitude on No-Go trials in high self-efficacy group (- 0.30 μV) compared with low self-efficacy group (- 0.61 μV) was identified, but this difference was not significant (p > 0.1).Repeated-measures ANOVA see  revealedThis study investigated the neural correlates of inhibition modulated by self-efficacy. Two groups (low self-efficacy vs. high self-efficacy) of 50 college students participated in the experiments. We adopted Go/No-Go tasks and selected two ERP components associated with inhibitions—N2 and P3. The amplitudes of No-Go N2 were significantly larger in the high self-efficacy group compared with the low self-efficacy group. But there was no significant difference in P3 amplitudes on No-Go trials between the high self-efficacy group and the low self-efficacy group.The finding of larger No-Go N2 amplitudes in the high self-efficacy group indicates that a person with a higher self-efficacy level is more likely to have a stronger ability to detect a conflict. According to In contrast with previous studies, this study found there was no significant difference in No-Go P3 amplitudes between the high self-efficacy group and the low self-efficacy group. The P3 component has been used to ensure that stimulus analyses are appropriately linked with the correct behavioral actions in the monitoring processes; inhibition of the action operation is associated with the No-Go P3 . The finThis study analyzed neural correlates of inhibition modulated by self-efficacy. It was found that students with a higher self-efficacy level tended to have a stronger ability to detect a conflict. Conflict detection was modulated by self-efficacy, whereas conflict inhibition was not modulated by self-efficacy. The high self-efficacious students were more likely to be capable of detecting conflicts but not necessarily followed by inhibition of the action operation.In classrooms, teachers could design meaningful or student-relevant activities to increase their self-efficacy to regulate their behaviors in response to conflicts or challenges, and thus enhance motivational aspects. Teachers also could emphasize the significance of positive reinforcement and a supportive environment, and teach students problem-solving and information processing skills to encourage students to persist longer in learning tasks and engage students actively in class work.This study explored neural correlates of inhibition modulated by self-efficacy based on Go/No-Go task monitoring and processing, but it did not reflect the whole process of inhibition and also focused on a small sample of students. Future research could implement multi-task or more complex measures to assess inhibition modulated by self-efficacy with a larger sample size. We also need to know more about how other predictors contribute further to understanding inhibition behavior change.To sum up, the findings of this study support previous reports that inhibition in response to a stimulus is associated with self-efficacy from the perspective of cognitive neurology, and provide evidence that inhibition, indexed by N2 amplitude, may be one mechanism through which self-efficacy improves task performance. The analysis of ERP components serves to yield a more complete picture of the cognitive mechanism underlying inhibition modulated by self-efficacy. This study provides a new perspective for studies on inhibition and self-efficacy, and it will contribute to our understanding of cognitive ability development.The original contributions presented in this study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.The studies involving human participants were reviewed and approved by China University of Petroleum-Beijing. The patients/participants provided their written informed consent to participate in this study.The author confirms being the sole contributor of this work and has approved it for publication.


CSNK2A1 gene, which encodes the alpha subunit of protein kinase CK2. The most frequently occurring mutation is lysine 198 to arginine (K198R). To investigate the impact of this mutation, we first generated a high-resolution phosphorylation motif of CK2WT, including the first characterization of specificity for tyrosine phosphorylation activity. A second high resolution motif representing CK2K198R substrate specificity was also generated. Here we report the impact of the OCNDS associated CK2K198R mutation. Contrary to prior speculation, the mutation does not result in a complete loss of function, but rather shifts the substrate specificity of the kinase. Broadly speaking the mutation leads to 1) a decreased preference for acidic residues in the +1 position, 2) a decreased preference for threonine phosphorylation, 3) an increased preference for tyrosine phosphorylation, and 4) an alteration of the tyrosine phosphorylation specificity motif. To further investigate the result of this mutation we have developed a probability-based scoring method, allowing us to predict shifts in phosphorylation in the K198R mutant relative to the wild type kinase. As an initial step we have applied the methodology to the set of axonally localized ion channels in an effort to uncover potential alterations of the phosphoproteome associated with the OCNDS disease condition.Okur-Chung Neurodevelopmental Syndrome (OCNDS) is caused by heterozygous mutations to the  CSNK2A1 gene, which encodes the alpha subunit of protein kinase CK2, a well-characterized and conserved serine/threonine protein kinase  is broadly characterized by delayed psychomotor development and intellectual disability . The disn kinase . Of the n kinase , and thin kinase . Interesn kinase . Furthern kinase . Taken tL method  to generK198R mutant was done using the Q5® Site-Directed Mutagenesis Kit (New England BioLabs).For bacterial expression, a plasmid containing the human CSNK2A1 gene in an Invitrogen Gateway donor vector (pDONR223) was provided by The Broad Institute and was transferred to the pDEST17 backbone following the standard Gateway Protocol (Life Technologies). Generation of the CK2Escherichia coli and phosphorylates the endogenous proteome according to its native substrate preferences. These phosphorylated proteins can then be identified by LC-MS/MS to create a list of phosphorylation sites to be used to visualize substrate specificity. ProPeL experiments were carried out as previously described  cells (Lucigen) by IPTG induction. Optimal expression conditions were determined to be mid-log induction followed by expression for 24 h at 37°C in TB media (data not shown).The Proteomic Peptide Library (ProPeL) approach is a mass spectrometry-based method to experimentally determine high-resolution kinase specificity motifs. In this method, an exogenous kinase of interest is expressed in escribed  with theWT and CK2K198R was confirmed by western blotting using the primary antibody Anti-CSNK2A1 antibody (Abcam ab10466) at a 1/5,000 dilution and the secondary antibody IRDye® 680RD Donkey anti-Rabbit IgG at a 1/5,000 dilution.Comparable expression of CK22O) was injected onto a Waters nanoEase M/Z Peptide BEH C18 analytical column  and separated by reversed-phase UPLC using a gradient of 4–30% Solvent B (0.1% formic acid in acetonitrile) over a 100- min gradient at 300 nL/min flow. Peptides were eluted directly into the Q Exactive HF using positive mode nanoflow electrospray ionization and 1.5 kV capillary voltage. MS scan acquisition parameters included 60,000 resolution, 1e6 AGC target, maximum inject time of 60 ms, and a 300–1800 m/z mass range. Data-dependent MS/MS scan acquisition parameters included 15,000 resolution, 1e5 AGC target, maximum ion time of 40 ms, loop count of 15, isolation window of 2.0 m/z, dynamic exclusion window of 30 s, normalized collision energy of 27, and charge exclusion “on” for all unassigned, +1, and >+8 charged species.Peptide samples were subjected to mass analysis using a Thermo Scientific Ultimate 3000 RSLCnano ultra-high performance liquid chromatography (UPLC) system coupled to a high-resolution Thermo Scientific Q Exactive HF mass spectrometer. An aliquot of each peptide preparation in Solvent A , and the MaxQuant contaminants database. Variable modifications were oxidation of Met, acetylation of protein N- termini, deamidation of Asn/Gln, and for enriched samples, phosphorylation of Ser/Thr/Tyr. Carbamidomethylation of Cys was set as a fixed modification. Protease specificity was set to trypsin, allowing a maximum of two missed cleavages. LFQ quantification was enabled. All results were filtered to a 1% false discovery rate at the peptide spectrum match and protein levels; all other parameters were kept at default values. MaxQuant-derived output was further analyzed in accordance with the ProPeL method (Peptides were identified using MaxQuant (v1.6.10.43) and the embedded Andromeda search engine . The rawL method .E. coli expressed proteome) the scoring of phosphorylated and unphosphorylated serine, threonine, and tyrosine residues in E. coli could be used to convert motif scores to probabilities of modification. Specifically, phosphorylated 15mers (for either the wild type or K198R data set) were mapped back onto the UniProt reference K12 E. coli proteome to generate a list of expressed proteins phosphorylated by CK2 in E. coli. These proteins were then subjected to a full trypsin digestion in silico and resultant peptides with a length up to 50 residues were retained to generate a list of all potential phosphorylation sites that could have been hypothetically observed in our experiments. Seven residues upstream and downstream from each serine, threonine, and tyrosine residue in these peptides was extracted to create a complete list of serine, threonine, and tyrosine-centered 15mers. Each 15mer was labeled as either positive or negative depending on whether it was observed to be phosphorylated in our ProPeL experiments, resulting in 1,350 positive (phosphorylated) and 26,331 negative (not observed to be phosphorylated) 15mers in the CK2WT data set, and 1,024 positive and 20,083 negative 15mers in the CK2K198R data set. Every peptide was scored using the appropriate wild type or K198R serine, threonine, or tyrosine-centered position weight matrix derived from the log-odds binomial probabilities observed in the corresponding serine, threonine, or tyrosine-centered pLogo, as we have shown previously  the overall kinase activity of CK2K198R can be approximated at 85% of CK2WT, thus suggesting that the OCNDS CK2K198R mutation results in only a slight decrease in kinase activity relative to wild type CK2. This observed activity of CK2K198R stands in contrast to previous results indicating that the mutation results in a loss of activity  residues in the +1 position, as well as an increased preference for both glycine and alanine, with glycine superseding aspartate to become the most statistically significant residue at the +1 position , its shift is significant as it represents a near doubling of the proportion of phosphorylated tyrosine residues in the K198R data set (15%) relative to wild type (8.1%).Further comparison of the CK2acceptor , while CK198R pLogo was consistent among each individual phosphoacceptor. To investigate this question, we fixed each central residue  to generate phosphoacceptor specific pLogos as shown in WT pLogos indicated a preference for acidic residues at the +1 and +3 positions  in the sites unique to CK2K198R. The +3 acidic residues were similarly attenuated in the mutant kinase data set. Finally, with regard to the phosphoacceptor residue, the phosphorylation sites unique to CK2WT showed a highly significant disfavoring of tyrosine residues relative to serine residues that was lost in the CK2K198R mutant .To further explore motif differences between the three subsets noted in the Venn diagram, pLogos were generated for each subset . The pLoTaken together these results point to a significant shift at the substrate level for the K198R mutant kinase, whereby the most canonical wild type CK2 phosphorylation sites are either lost or phosphorylated with decreased efficiency, coupled with a large increase in previously unphosphorylated sites.K198R is the most frequently occurring mutation in patients with OCNDS  with the greatest change in probability between CK2WT and CK2K198R are listed in K198R and the remaining eight are less likely to be phosphorylated by CK2K198R. Additionally, four of the ten sites included in https://okurchung.com that provides users with an interactive view of our predictive results.CK2th OCNDS . Substraredictor , but witK198R mutation does not simply cause a loss of function, or an inability to recognize substrates  and tyrosine, which added additional preferences for acidic residues upstream of the modification site in the mutant.While wild type CK2 has previously been shown to be capable of phosphorylating tyrosine residues  the studE. coli, thus allowing researchers to assess shifts in phosphorylation at the site level between kinases bearing different characteristics and/or mutations. Utilizing this strategy here we have shown that there exists overlap between CK2WT to CK2K198R at the substrate level, as well as considerable extension to new atypical CK2 substrates by the mutant kinase. Another advantage of ProPeL in the present study is that the closed and finite system  allowed us to readily associate scan-x phosphorylation scores . Most kinases have multiple substrates throughout the cell, only a few of which will directly relate to a phenotype of interest. To investigate signaling changes that may underlie neurodevelopmental phenotypes associated with OCNDS, we focused on neuronal signaling in axonally localized ion channels, as CK2 has been shown to be highly enriched in the axon  binding motif, and is a known CK2 phosphorylation site  residue is present in the +1 position). The additional effects of the CK2K198R mutation, namely increased tyrosine phosphorylation and increased promiscuity towards the +1 position, could contribute in part to the heterogeneity observed in symptoms of OCNDS patients.It is worth noting that while the work presented here demonstrates the impact of the CK2variants  and nonsvariants , many ofL205R results in altered substrate specificity (Finally, as our group and others previously demonstrated that the Cushing’s Syndrome mutation PKAcificity , the pre


Sr2+-to-Ca2+ substitution did not lead to oxygen-vacancy ordering–disordering transition but an interesting Ca4-to-Sr4 type structure transition. Rietveld refinements revealed that the two-type structures exhibit similar oxygen-vacancy ordering and identical 1:1:1 triple-cation B-site ordering. Close inspection of the two-type structures revealed the subtle structure difference lies in the orientations of GaO4 tetrahedra, which is the origin of the formation of the narrow two-phase region (0.3 ≤ x < 0.65) in Cax4−SrxGaNbO8. More importantly, the A- and B-site cavities with large differences in size for both structures resulted in a site-selective doping behaviour for Sr2+ in Cax4−SrxGaNbO8. These structural changes found in Cax4−SrxGaNbO8 will provide a broad route approaching new oxygen-deficient phases with oxide ionic conductivities.Oxygen-deficient perovskites are a family of important materials that may exhibit oxide ionic conductivities. We attempted to introduce oxygen-vacancy disordering in perovskite Ca 2+-to-Ca2+ substitution resulted in new 1:1:1 triple-cation B-site ordered perovskites, where the structure difference lies in the orientations of GaO4-tetrahedra.Sr In contrast, the layered ordered and columnar ordered perovskites are relatively rare, and only a few specific phases have been reported up to now.12,13Among the various perovskites, 1:1 double-cation B-site ordering perovskites usually exhibit physical properties different from the disordered ones distinctively. For example, B-site ordered perovskites Srx3Lax2/3−TiO3, in which the pre-existing A-site vacancies allow for easy Li+ diffusion.14–16 In contrast to A-site ionic conductivity, oxygen ionic conductivity or mixed ionic and electronic conductivities for oxygen deficient perovskites are much more widely investigated due to their potential applications in solid oxide fuel cells (SOFCs) as electrolyte or cathode. For example, Sr2+ and Mg2+ co-doped LaGaO3 (LSGM) is the one of the best oxygen ionic conducting electrolytes.17–19 Much efforts have been devoted to improve the oxygen ionic conductivities of various perovskites by increasing the number of oxygen vacancies. However, increasing the number of oxygen vacancies may result in completely ordering of oxygen vacancies, which in turn lead to a significant decrease of oxide ionic conductivity. For example, oxygen-vacancy ordered perovskite Ba2In2O5 exhibits poor ionic conductivity in low temperature range (<900 °C), though there exists a large number of oxygen vacancies in the structure.20–22 Oxygen-vacancy ordering/disordering in perovskite is closely related to the A-site cationic size . For example, with an increase of the Sr2+-content (increase of tolerance factor) in Cax2−SrxFeCoOδ6− resulted in an oxygen-vacancy ordering to disordering transition.23 Therefore, the strategy of increasing the A-site cationic size of anion-ordered perovskites might be utilized to improve the oxide ionic conductivity.Owing to the structural flexibility, perovskite can accommodate various defects including A-site and oxygen vacancies, which can result in ionic diffusion at high temperature. A-site ionic diffusion was observed indeed in Li4GaNbO8, where the complex 1:1:1 triple-cation B-site ordering is coupled with the oxygen-deficient ordering.24 We attempted to incorporate larger Sr2+ cations into Ca4GaNbO8 to bring in oxygen-vacancy disordering, so as to obtain new oxide ionic conductors. Substitution of Ca2+ with Sr2+ led to a Ca4-type to the Sr4-type structure transition as we expected. Unfortunately, Rietveld refinements manifested that the oxygen vacancies are also ordered in the Sr4-type structure, which was further confirmed by AC impedance spectroscopy measurements on selected compositions due to the absence of oxide ionic conductivity. The subtle structural differences between the Ca4-and Sr4-type structures lie in the orientations of GaO4 tetrahedra, which is the origin of the coexistence of two phases in a narrow region 0.3 ≤ x < 0.65. Moreover, a site-selective doping behaviour was observed for Sr2+ in Cax4−SrxGaNbO8, however the A-site cationic ordering is only in short-range for all compositions.Herein this contribution, our attention is turned to the newly discovered oxygen-deficient perovskite Cax4−SrxGaNbO8 (x = 0–4) were prepared by high temperature solid-state reactions using calcium carbonate , strontium carbonate , gallium oxide , niobium oxide  as raw materials. The starting materials were preheated at 500 °C for 10 h to remove the absorbed moisture. Stoichiometric starting materials with a total weight of 1.2 g were weighted and mixed in an agate mortar by hands and then preheated at 950 °C to decompose the carbonates. After this calcination, the resultant white powders were further grinded and pressed into pellet with 13 mm diameter and then calcinated between 1200 °C and 1300 °C for 60 hours with intermediate grinding and pressing.The polycrystalline samples Ca25 were collected with a setting of 200 s/0.0131°.The phase purity of the samples was investigated by powder X-ray diffraction (PXRD). PXRD were performed on the PANalytical Empyrean diffractometer equipped with a PXIcel 1D detector (Cu Kα radiation). The operation voltage and current were 40 kV and 40 mA, respectively. The PXRD data for phase identification were collected with the setting of 30 s/0.0262°. High quality PXRD data, which were used for Rietveld refinements with the TOPAS-Academic V6 software,x4−SrxGaNbO8  were carried out over temperature range from room temperature to 800 °C using a Solartron 1260A impedance phase analyzer with the frequency from 10−1 to 107 Hz. Before the measurements, the pellets were coated with platinum pasts and then heated at 800 °C for 1 h to form electrodes.AC impedance spectroscopy measurements for Ca4GaNbO8 and Sr4GaNbO8 were measured over the range of 100–4000 cm−1 using the 325 nm line and with the light focus on the sample trough an optical lens. The spectral resolution was about 1–2 cm−1.The Raman spectroscopy measurements were performed on the LabRAM HR Evolution spectrometer. The room temperature Raman spectra for Cax4−SrxGaNbO8 (x = 0–4) are elucidated in 2+-content. This observation indicates the cell volume expansion of Cax4−SrxGaNbO8 upon substituting Ca2+ with Sr2+, which is consistent with the larger cationic radii of Sr2+ (1.44 Å in 12-fold coordination) in comparison with Ca2+ (1.34 Å in 12-fold coordination).26PXRD data for Ca4GaNbO8 revealed that this compound adopts a triple-cation B-site ordered perovskite-type structure and crystallizes in P21/c with lattice parameters a ≈ 11.18 Å, b ≈ 5.59 Å, c ≈ 14.07 Å, and β ≈ 121.55°.24 Our preliminary Le-bail fitting performed on Cax4−SrxGaNbO8 manifested that the XRD data for compositions with x < 0.65 could be indexed by this monoclinic cell. However, a group of weak reflections for compositions with x ≥ 0.65, which is not ascribe to impurity phases, could not be fitted with this monoclinic cell any more, indicating Sr2+-doping induced a change of lattice parameters. Indexing the XRD data of Ca3SrGaNbO8 yield a monoclinic cell with lattice parameters a ≈ c1/2, b ≈ b1, c ≈ √3a1, β ≈ 97°, where a1, b1, and c1 represent the cell dimension of Ca4GaNbO8 (denoted as Ca4-type phase). Similar unit cell was also observed for Sr4AlNbO8 (P21/c), which also adopt a triple-cation B-site ordered perovskite-type structure with anionic vacancy ordering, suggesting Cax4−SrxGaNbO8 (x ≥ 0.65) (denoted as Sr4-type phase) is isostructural to Sr4AlNbO8.27Previous study on Cax4−SrxGaNbO8 (x = 0.5) using Ca4GaNbO8 as the initial structure model resulted in unreasonable structural parameters, i.e. too short interatomic distances. Close inspection of the XRD data revealed that some reflections from the Sr4-type phase were observed for compositions x = 0.3, 0.4 and 0.5. To clarify this clearly, the diffraction components from Cu Kα2 were striped for comparison. As shown 4-type phases, of some characteristic reflections were visually observed. Moreover, a representative Le-bail fitting pattern for x = 0.5 presented in Fig. S1x = 0.3, 0.4 and 0.5 comprise two phases. More importantly, single-phase for compositions x = 0.3, 0.4 and 0.5 was not attainable by neither calcination at elevate temperature nor prolongation of calcination time  can be divided into three regions: (i) a single Ca4-type phase region (x < 0.3), (ii) a narrow two-phase region contains both Ca4-type and Sr4-type phases (0.3 ≤ x < 0.65), and (iii) a single Sr4-type phase region (x ≥ 0.65). We should note that the change of the relative content for the two phases within the two-phase region was slight (e.g. the two-phase region observed in Bax3−SrxZnSb2O9 (0.3 ≤ x ≤ 1.0).28 Such a difference is attribute to the subtle distinction in crystal structures between Ca4-type and Sr4-type structures, which will be discussed later.Preliminary Rietveld refinements performed on Cae Fig. S2, suggests slight , which i2+-content in Cax4−SrxGaNbO8 is presented in 4-type and Sr4-type phases also showed a linear increase within the two-phase region, which opposite to commonly observed constant lattice parameters for the two phases in the two-phase region. This uncommon phenomenon further corroborated that the co-existence of two phases in Cax4−SrxGaNbO8 is thermodynamically favorable.The evolution of normalized lattice parameters against Sr4-type and Sr4-type phases adopt oxygen-deficient perovskite-type structure with 1:1:1 triple-cation B-site ordering. Owing to their low structure symmetry (P21/c), both structures exhibit three distinctly different A-sites , which could be readily discerned through their surrounding environments. As shown in 6 octahedra , four NbO6 octahedra, and one GaO4 tetrahedron; A2 is surrounded by three B1O6 octahedra, one NbO6 octahedron, and four GaO4 tetrahedra; A3 is surrounded by two B1O6 octahedra, three NbO6 octahedra, and three GaO4 tetrahedra. Such difference in surrounding environments for A-site cations results in the A1-site is much larger than A2-and A3-site, suggesting Sr2+ is prone to occupy the A1-site at first.As described above, both Cax4−SrxGaNbO8  to get an insight picture of the site occupancy preference for Sr2+. The refined structure parameters for Ca4GaNbO8 is in good agreement with that reported by Yang et al. using combined refinements against neutron and synchrotron data  and structural parameters. However, some peaks with large discrepancies between the observed and calculated were observed, suggesting the A-site cations are not completely ordering in Ca3SrGaNbO8. Consequently, the occupancy factor for Ca2+ and Sr2+ cations at B1-site and three A-sites were refined freely during the subsequent refinements. It turned out that A1-site was dominantly by Sr2+, and A2, A3, and B1 sites were mainly occupied by Ca2+. Despite the occupancies for Ca2+ and Sr2+ at four independent sites were refined freely, the refined composition Ca2.92Sr1.08GaNbO8 agrees well with the nominal formula Ca3SrGaNbO8. Moreover, the reliable factors were improved significantly to Rwp = 5.543% and Rp = 4.074%. These results manifest that the Ca2+ and Sr2+ are partially ordered in Ca3SrGaNbO8. Rietveld refinements performed on other compositions further demonstrated that all the compositions with mixed A-site cations were partially ordered. We should note that the inversion between A- and B-sites, which is commonly observed in spines,29 was not considered in Cax4−SrxGaNbO8 because of the large differences in cationic size and coordination environment preference between  and . The final crystallographic parameters and selected bond lengths for Cax4−SrxGaNbO8 are summarized in Table S1 and S2.†Careful Rietveld refinements against laboratory XRD data were further performed on selected compositions Ca Table S1. For com4GaNbO8 and Sr4GaNbO8 are presented in p. Ca4GaNbO8 and Sr4GaNbO8 share the similar structure features with 1:2 layer-ordered hexagonal perovskites i.e. Ca4Nb2O9, however the structure for Ca4GaNbO8 and Sr4GaNbO8 are more complex because there is an another B-site cation (Ga3+) and ordered anionic deficiency.30 As shown in 2 layers creates tetrahedral cavities that could only accommodate smaller Ga3+ cations, which resulted in layered stacking sequence of (Ca/Sr)–(Ga1/2Nb1/2)–(Ga1/2Nb1/2)–(Ca/Sr) for B-site cations along the closed-packing direction [111]p. Owing to the ordered removal of every other oxygen-only O2 columns within the AO2 layers, Ga3+ and Nb5+ within (Ga1/2Nb1/2)–(Ga1/2Nb1/2) layers are column ordered along [110]p.The refined crystal structures for Ca4GaNbO8 and Sr4GaNbO8 seem identical at first glance. Close inspecting the structures revealed that the structural difference between Ca4GaNbO8 and Sr4GaNbO8 stems from the distinct orientations of GaO4 tetrahedra along-side of (Ca/Sr)O6 octahedra. As highlighted in 4 tetrahedra in Ca4GaNbO8 point to the same direction, however, the GaO4 tetrahedra in Sr4GaNbO8 point to opposite directions. Such a difference resulted in a doubled cell dimension along [111]p for Ca4GaNbO8 in comparison with Sr4GaNbO8  for Sr2+ at A1-site when x ≤ 1.5, whereas the increase of occupancy factors at A2 and A3 sites are relatively slow. The occupancy factors for Sr2+ at A2 and A3 sites show a synchronized increase behaviour in the range of 1.5 ≤ x ≤ 3.0, where the Sr2+-occupancy at A1 site manifests a slight increase. Further incorporation of Sr2+ into Cax4−SrxGaNbO8 lead to a sharp increase of occupancy factor for Sr2+ at B1 site when x ≥ 3.0, whereas a slight increase is observed for x ≤ 3.0. Such a sharp increase of occupancy of Sr2+ at B1-site lead to a significant deviation of the lattice parameters, especially for a, from the Vegard's law .31–33 Detailed inspection of the Nb–O bonds revealed that the Nb5+ exhibits a distorted coordination environment with three Nb–O atomic distances shorter than 2.0 Å and the remaining three bond lengths longer than 2.0 Å  effect.34,35 Though both Ga3+ and Nb5+ cations can adopt four- and six-fold coordinations, incorporation of Sr2+ into A- and B-sites did not lead to anti-site disordering between Ga- and Nb-sites for all compositions, which should unambiguous attribute to their large differences in charge and cationic size.As described above, Sr Table S2, indicat36 To gain an insight of structural change induced by Sr2+-doping, Raman spectra for Ca4GaNbO8 and Sr4GaNbO8 were measured. Raman spectra of Ca4GaNbO8 and Sr4GaNbO8 show similar features  are presented in Fig. S5R) and capacity (C) element. This large semicircle is associated with a capacity close to 1 × 10−12 F cm−1, implying the contribution of bulk response. The absence of inclined-like spike in low frequency range indicates the absence of oxide ionic conductivity. This observation is consistent with the structural features that there are no terminal oxygens, which is capable of immigration at high temperature, boned to only one B-site cation in both Ca4-and Sr4-type structures. Moreover, the oxygen-vacancy ordering is coupled with the B-site cation ordering, which could not be disrupted upon warming. Therefore, the absence of oxide ionic conductivity for Cax4−SrxGaNbO8 is understandable. The plots of conductivities for Cax4−SrxGaNbO8  against temperature is elucidated in −8–10−4 S cm−1) in the measured temperature range. Fitting the σ–T curves with Arrhenius equation gives active energy of ∼ 0.85 eV for all compounds, which in turn corroborated that the B-site cationic ordering is not disrupted by Sr2+-to-Ca2+ replacement.Typical AC impedance spectra at different temperatures for Ca2+ in Cax4−SrxGaNbO8 with larger Sr2+ did not lead to oxygen-vacancy and B-site cation ordering–disordering transition but a Ca4-type (x < 0.3) to Sr4-type (x ≥ 0.65) structure transition across a narrow two-phase region (0.3 ≤ x < 0.65). Rietveld refinements revealed that two-type structures possess similar anionic ordering and identical B-site ordering. The structural difference only lies in different orientations of GaO4 tetrahedra, which is responsible for the formation of the narrow two-phase region. In the process of changing the Ca4-phase to Sr4-phase, the Sr2+ cations were mainly incorporated into A1-site for x < 1.5, and then doped into A2/A3-site for x < 3, and finally doped into B1-site (x ≥ 3). Such site-selective doping behaviour observed in Cax4−SrxGaNbO8 was driven by the distinctly large difference in size for A1, A2/A3, and B1 sites. Incorporation of Sr2+ cations into both A and B sites had no influence on the arrangement of Ga3+/Nb5+ cations and oxygen defects, thus no significant change in electronic properties of Cax4−SrxGaNbO8 was detected.Substitution of CaThere are no conflicts to declare.RA-010-C9RA09970K-s001