RAC875_c103017_302, Tdurum_contig42087_1199, and Tdurum_contig46877_488 on chromosomes 4B, 6B, and 7B respectively), two (BobWhite_c836_422 and BS00010616_51) and three  on chromosomes 5A, 1B, and 3D at the positions 243.59cM, 77.82cM and 292.51cM) showed pleiotropic effects in studied traits under normal, heat-stressed and both conditions respectively. The present study not only authenticated the numerous previously reported MTAs for examined attributes but also revealed novel MTAs under heat-stressed conditions. Identified SNPs will be beneficial in determining the novel genes in wheat to develop the heat tolerant and best yielded genotypes to fulfill the wheat requirement for the growing population.Dissecting the genetic basis of physiological and yield traits against tolerance to heat stress is an essential in wheat breeding programs to boost up the wheat yield for sustainable food security. Herein, a genome-wide association study (GWAS) was performed to reveal the genetic basis of heat tolerance using high-density Illumina 90K Infinium SNPs array through physiological and yield indices. These indices were phenotyped on a diverse panel of foreign and domestic genotypes of Pakistan, grown in normal and heat-stressed environments. Based on STRUCTURE analysis, the studied germplasm clustered into four sub-population. Highly significant variations with a range of moderate (58.3%) to high (77.8%) heritability was observed under both conditions. Strong positive correlation existed among physiological and yield related attributes. A total of 320 significant (-log10 P ≥ 3) marker-trait associations (MTAs) were identified for the observed characters. Out of them 169 and 151 MTAs were recorded in normal and heat stress environments, respectively. Among the MTA loci, three ( Wheat is one of the staple crop in the world, high and stable yield in this crop is an essential goal for growing wheat. Wheat has an optimum day-time growing temperature due to cool season crop during reproductive development of 15°C and for each degree Celsius more than optimal temperature a yield decrease of 3–4% were recorded . ClimateThe actual grain yield of wheat crop does not match to its potential due to several restricting factors, one of which is heat stress . SeveralHeat tolerance is a complex and polygenically controlled character that is influenced by numerous genes with a minor impact on the morphology of plant. Hence, the selection of tolerance to heat stress in field conditions is very difficult due to its genetically complex, meteorological variations and the effect of genotypes-environment interactions. In such a manner, the identification of quantitative trait loci (QTLs) and molecular marker linked with heat stress tolerance is essential to improve breeding efficacy through marker-assisted selection (MAS) , 7. HeatProducing of maximum yielded and heat stress tolerant genotype is difficult by numerous complications because yield and heat stress tolerance associated genes are polygenic and complex in nature. Several procedures were practiced for choosing the spring wheat genotypes for heat stress environments. Rehman et al  suggesteenzymes activity during grain filling that consequently leads to reduction in grain size, weight and ultimately grain yield. Grain filling duration and grain filling rate determines grain development. Longer grain filling duration facilitates the longer time to capture available resources and improve the grain weight. Days to heading (DH) is most important to determine life of plant and days taken for anthesis [The effect of stimulation to heat stress was studied with maximum crop canopy temperature. Canopy temperature (CT) at vegitative and reproductive stage is main character that is used by a breeder to select best wheat lines against tolerance to heat as well as drought stress . Grain yanthesis , 25.Numerous attempts were made using the conventional breeding strategies to improve wheat yield in heat stressed conditions, but these approaches have only contributed to an increase of more than only 1% in annual production , 14. In In this study, a miscellaneous panel of wheat genotypes was genotyped with 90K SNPs array as well as phenotyped in non-stressed and heat stressed environments during 2 crop seasons in 2018–19 and 2019–20. This study delivers valued evidence about the genetics of tolerance to heat stress in wheat. Novel SNPs were observed to be related with key heat stress tolerant attributes. The significantly associated SNPs with physiological and yield indices would be useful to identify and mapping the candidate genes for heat stress tolerance in wheat.2 in this study. Formula used for the calculation of flag leaf area described by Muller [Total 96 Pakistani and foreign  wheat genotypes were preserved at the Department of Plant Breeding and Genetics, University of Agriculture Faisalabad PBG-UAF that were used in this experiment. Complete data about the accessions code, their origin, the names and the pedigrees are mentioned in S1 Table in y Muller  as underFlag leaf area = length of flag leaf × width of flag leaf × 0.74. Cell membrane thermo-stability (CMT) was measured by using the previously described procedure . Two fulWhere, C and T represent the electrical conductivity of normal and heated sample while the subscript 1 and 2 indicate electrical conductivity readings before and after autoclaved, respectively.Days to heading (DH) was counted from the date of sowing up to the date when it has above 50% plant with the complete heading. At booting stage of the crop, a hand-held digital Infrared thermometer LT300 is used to calculate canopy temperature at vegetative stage (CTV) in centigrade. The distance from the canopy was kept 50cm and the slope was 45°, taking care that laser light should only focus on the canopy and data was recorded on a sunny day from 10:00 a.m. to 2:00 p.m. Data were recorded for canopy temperature at grain filling stage (CTG) at mid grain filling stage with the help of infrared thermometer with the same procedure and precautions. From selected plants, days taken to maturity (DM) were examined as the duration from sowing to maturity dates when plants were physiologically mature. Ten plants were randomly selected from all replications of each entry and bulked separately. TGW were taken from bulk seeds and were weighted by using and electric balance (Compax- Cx-600) to record TGW. Threshing of all spikes from the selected plants than with the use of electric balance, (Compax- Cx-600) weighted. Mean data of grain yield/plant (GYP) was calculated from replicationsThe noted data were applied for the combined analysis of variance (ANOVA) through the GenStat ® Ver.17  for examH2 is the broad sense heritability, ny number of years and nr number of replications”. Pearson’s correlation coefficients (r) noted to determine the association among studied traits through the SPSS ver.23 [Where S ver.23  under obSingle accession was seeded in a polythene bag. Fresh 15-day-old wheat seedling leaf used for DNA isolation follow to the CIMMYT Manual of Molecular Genetics procedures . DNA of 4 cycle and admixture modal selection have been used in this study. An ad-hoc web based method “Structure Harvester v0.6.93” was experienced obtaining high values or peak of ‘‘K” for the confirmation to identify the STRUCTURE results [Bayesian grouping practice was used with SNP to rank cluster of genetically similar accessions using STRUCTURE v.2.3 . Burn-in results . We sele−3 or more [P) value [The GAPIT packages were applied to perform association mapping of studied attributed in RStudio . This to or more  after Bo or more . To deteP) value , 40. OveP) value .2 = 61.6% respectively under heat stressed conditions. Descriptive statistics data of studied characters in normal and heat stressed condition base on averaged data overs year have given in S3 Table in 2 and 28 cm2 under normal and heat stressed conditions, respectively. The CMT had mean values 57% under normal conditions while in heat stress conditions as 48% in this study. The CTV and CTG exhibited mean performance as 35 and 28 under normal conditions, while, it was 27 and 30 under heat stressed condition, respectively. Day to headings and day to maturity was 90 days and 112 days under normal conditions while in heat stress conditions it changed as 81 days and 102 days respectively. Mean values of TGW was 73g and 31g in normal and heat stressed conditions, respectively. Mean values of GYP was 22g and 16g in normal and heat stressed conditions, respectively. Correlation coefficient of physiological and yield traits based on data average over year in normal and heat stressed conditions presented in In the current experiments, ANOVA of physiological and yield traits for 96 spring wheat genotypes exhibited significant effects on phenotype variability (P < 0.01). Studied attributes exhibited highly significant influence between examined genotypes as shown in S2 Table in Δk) was calculated from structure harvester, which exhibited maximum values when the given k  was four  explain the high values of trait variation (19.83%) on chromosome 1A at 341.14cM while the marker (Ra_c11906_1618) from chromosome 1D at 172.13cM explain the low value (12.68%) of trait variation of flag leaf area under normal conditions (S4 Table in Kukri_c55051_414) had maximum PVE (37.15%) on chromosome 5A at 68.2cM whereas the marker (RAC875_c1036_897) on the 6B chromosome  explain maximum phenotypic trait variation (32.35%) on chromosome 4A at 542.67cM while the marker (tplb0028p08_626) on chromosome 3D at 13.29cM explain a low value (24.13%). Under heat stress conditions, 27 MTAs explain phenotypic variation ranged from 19.21% to 41.09% in this attribute. These significantly associated SNPs were perceived at chromosomes 1A, 3A, 4A, 5A, 7A, 1B, 2B, 3B, 4B, 5B, 7B, 2D, 3D, 5D and 7D  explained highest phenotypic trait variation on chromosome 3A at 276.05cM while the marker (RFL_Contig1319_507) on chromosome 3B at 268.75cM explained low value  exhibited more phenotypic variation (34.98%) on chromosome 1A at the position of 312.46cM, while the marker (BS00079076_51) showed least phenotypic variation (18.12%) at chromosome 7B at the positions of 296.21cM in normal conditions. In heat stressed conditions, fourteen MTAs explained phenotypic variation ranged from 24.94% to 37.40% in this attribute. Three SNPs were detected at chromosome 6D, two at each of 1B, 3D, 4A, 5A, while on at 7B (BS00010616_51) explained more phenotypic trait variation (37.40%) on chromosome 7B at 186.24cM, while the marker (GENE-4153_101) on chromosome 6D at 180.23cM explain minimum value (24.94%) under heat stress conditions  was highly correlated with 30 MTAs, out of them, seven MTAs were situated at each chromosomes 2D and 7B, three MTAs on each chromosome 4B and 5A, two MTAs on each chromosomes 3B and 7A while one MTA associated on each of chromosome 1A, 1B, 2B, 6B, 5D and 7D . They exon at 7B . The marTdurum_contig48049_705) explain the highest value of trait variation (37.86%) on chromosome 4A at 160.42cM while the marker (Kukri_c78263_296) from chromosome 6D at 118.9cM explain the least value (20.58%) of trait variation of this character under normal conditions (S4 Table in BobWhite_c25703_160) on chromosome 7A at 687.47cM had more PVE (28.92%), while the marker (Kukri_rep_c69515_183) on the 5B chromosome explained the minimum proportion (22.32%) of the trait PVE at 199.86cM position  on chromosome 4A at position 160.42cM for this trait. The marker namely TA001877-0445 exhibited the minimum phenotypic variation which located at chromosome 3D with position 263.93cM in this studied trait. Associated MTAs dispersed on A, B and D genome, including five SNPs from the A-genome, eight from the B-genome and six from the D-genome. In heat stressed conditions, 24 significant SNPs were positively associated with DH counting 7 markers situated on chromosome 4B, 5 markers situated on chromosome 3B, 4 markers situated on chromosome 3D, 3 markers situated on chromosome 6A and 2 markers situated on chromosome 2B, while 1 SNPs on each chromosomes 1B, 5A and 6B , while the marker Kukri_rep_c105589_73 on chromosome 1A at 261.32cM explain the least values (18.35%) under normal conditions. Significant MTAs for this trait were dispersed on A, B and D genomes, including 10 SNPs from each A and B-genomes while seven from the D-genome under normal conditions. Under heat stressed conditions, twenty significant SNPs were strongly linked with DM as well as four markers detected on chromosome 7B, 2 SNPs on each chromosomes 1A, 1B, 3D, 5A, and one on each 4A, 7A, 2B, 3B, 1D, 2D, 5D and 7D as mentioned in Tdurum_contig48049_705 explain the maximum trait variation (35.71%) on chromosome 4A at 160.42cM, while the marker Tdurum_contig42214_2997 on chromosome 2B at 285.18cM explained the least values (20.00%) under heat stress conditions. Six associated markers were observed from the A genome, 8 SNPs from B-genome and 6 from the D-genome under heat stress conditions. These markers explained 20.00% to 35.71% of the phenotypic variation in days to maturity under heat stress conditions (S5 Table in Days to maturity (DM) was highly linked with twenty-seven markers in non-stressed environments. Significantly associated MTAs were perceived on chromosome 1A, 3A, 4A, 5A, 7A,1B, 2B, 3B, 4B, 7B, 1D, 2D, 3D, 5D, 6D and 7D  for thisExcalibur_c53131_187 explain the maximum phenotypic variation (31.26%) on chromosome 3A at 276.05cM, while the marker Kukri_c56014_275 on chromosome 4A at 425.2cM explain least values (17.47%) under normal conditions  on chromosome 2D at 110.64cM explained less phenotypic variation under heat stress conditions  showed the least phenotypic variation on chromosome 2B at the position of 351.23cM in non-stressed conditions  followed by DM (27), CTG (23), FLA (22), TGW (22), DH (19) GYP (17) and CMT (9) under normal conditions. Under heat stressed conditions, high number of MTAs was perceived for CMT (27) followed by DM (20), FLA (19), CTG (19), CTV (14), TGW (16) and GYP (12) in the present study . ChromosBS00065292_51 and Excalibur_c13242_1178) showed the least phenotypic variability 13.99% and 17.27% on chromosomes 5A and 3A having the position of 400.13cM and 279.61cM were considerably linked with flag leaf area under non-stressed and heat stressed condition, respectively  and 6B (386.86cM) explain the minimum phenotypic traits (13.08% and 17.10%) variation in normal and heat stressed conditions, respectively. In genome B the significant SNPs (BS00013824_51 and BS00010616_51) observed the maximum trait variation 32.73% and 37.40% from chromosomes 1B and 7B with the position of 208.49cM and 186.24cM for CTV under both examined conditions  and 3D (379.04cM) were linked with DH in D genome exhibiting maximum phenotypic variation 36.51% and 39.13% in non-stressed and heat stressed conditions, respectively  correlation among SNPs and the studied traits , 3A , 4A , 5A , 7A , 1B , 2B , 3B , 4B , 6B , 7B , 1D , 2D , 3D , 5D , 7D  in non-stressed condition. The Q gene located on chromosome 5A established the easy threshing of spike, plant height, spike length and emergence time of spike, and the pleiotropic effects of several yield related attributes like  PleiotroPSY-1) gene, the existence of this gene show the pleiotropic effect at 7B . The TaSnRK2 gene resolute sucrose non-fermenting 1-related protein kinase located at chromosomes 4A and 4B. It has played an important role in responding against numerous environmental conditions and depict significance correlation with grain yield . Wheat y TaGW2-A1 at 6A TaTGW6-A1 at 3A, TaCwi at 2A, TaGS5-A1/TaGS-D1 at 4A, TaSus1 at 7A and TaSus2 at 7B described by many scientists , 63.A pleiotropic locus modified the appearance of numerous phenotypic traits, in this study, several pleiotropic loci have been documented. In normal conditions, pleiotropic locus BobWhite_rep_c61300_88 on chromosome 7B at 206.22cM were significantly linked with yield and heat stress related attributes such as DH, CTG and DM. Another pleiotropic locus Excalibur_c20796_395 at chromosome 7A on the positions 372.34cM was also associated with physiological and yield related traits in this study. The examined characters like DH, CTG, DM and GYP was influenced by a pleiotropic locus Excalibur_c27873_266 on chromosome 1D at 268.67cM under normal conditions. The markers Tdurum_contig42087_1199, Tdurum_contig46877_488 and RAC875_c103017_302 on chromosomes 6B, 7B and 4B at 229.64cM, 178.15cM and 263.93cM respectively, showed pleiotropic effects for DH, CTG, DM and GYP under normal conditions as shown in S4 Table in BobWhite_c836_422 and BS00010616_51 were associated with CTV and DM, situated at chromosomes 5D and 7B with the position of 162.12cM and 186.24cM, respectively, in heat stress condition. Other pleiotropic loci D_GA8KES401BNLTU_253, Kukri_rep_c87210_361 and Tdurum_contig1015_131 on chromosomes 1B, 5A and 3D at the position of 243.59cM, 77.82cM and 292.51cM, respectively, showing the significance behavior with examined attributes in non-stressed and heat stressed environments. Herein, MTAs for physiological and yield related characters identified on chromosome 4A, 5A, 1B, 7B, 2D and 3D in both environments as given in RAC875_c103017_302, Tdurum_contig42087_1199, and Tdurum_contig46877_488 and on chromosomes 4B, 6B and 7B at 263.93cM, 229.64cM, and 178.15cM respectively, were not only significantly associated with physiological and yield traits but also showed pleiotropic effects for DH, CTG, DM and GYP. The Markers BobWhite_c836_422 and BS00010616_51 on chromosomes 5D and 7B also showed pleiotropic effects for CTV and DM in non-stressed and heat stressed conditions. The outcomes of the current experiment are timely in relation to developing efficient molecular markers and determining the function of related genes. The significantly associated SNP markers and genomic regions recognized would be useful for marker-assisted selection for heat tolerant wheat breeding scheme.Heat stress showed adverse effects and declined grain yield in wheat. Positive association exists among physiological and yield attributes like FLA, CMT, CTV, CTG, DH, DM, TGW, and GYP in heat stressed environments. Therefore, the germplasm selection based on these traits would be fruitful in breeding for high temperature tolerant wheat genotypes. Under normal conditions, the markers S1 File(DOC)Click here for additional data file.


Highly pathogenic avian influenza viruses (HPAIVs) of H5 subtype pose a great threat to the poultry industry and human health. In recent years, H5N6 subtype has rapidly replaced H5N1 as the most predominate HPAIV subtype circulating in domestic poultry in China. In this study, we describe the genetic and phylogenetic characteristics of a prevalent H5N6 strain in Guangdong, China.Nucleotide sequencing identified a H5N6 subtype HPAIV, designated as A/chicken/Dongguan/1101/2019 (DG/19), with a multibasic cleavage site in the hemagglutinin (HA). Phylogenetic analysis revealed DG/19 was a reassortant of H5N1, H5N2, H5N8, and H6N6 subtypes of avian influenza viruses. A number of mammalian adaptive markers such as D36N in the HA were identified.Our results showed that HPAIV H5N6 strains still emerge in well-managed groups of chicken farms. Considering the increasing prevalence of H5N6 HPAIV, and the fact that H5N6 HPAIVs are well adapted to migratory birds, an enhanced surveillance for the East Asian-Australasian flyway should be undertaken to prevent potential threats to the poultry industry and human health.The online version contains supplementary material available at 10.1186/s12917-021-02903-z. Avian influenza viruses (AIVs) are a group of enveloped viruses characterized with an eight-segmented, single-stranded, negative-sense RNA genome . AIVs cawww.who.int/influenza/human_animal_interface). During the past several years, the HPAIV H5N6 subtype has continuously evolved and has replaced H5N1 as one of the main HPAIV subtypes in domestic poultry in China [In 1996, the first HPAIVs of H5N1 subtype (A/goose/Guangdong/1/1996 (H5N1)) was isolated from an outbreak affecting domestic geese in Foshan city, Guangdong province, China. A year later, the first human clinical respiratory case of H5N1 was reported in Hong Kong, China . Since tin China –16. A moin China . TherefoIn this study, we report the genetic and phylogenetic characteristics of a HPAIV H5N6 strain isolated (October 2019) from chicken farms in Dongguan, Guangdong province, China.Primers targeting the conserved region of M segment were used to detect the influenza A virus in the liver and spleen of chickens  and GeneBank by ClustalW. Subsequently, the phylogenetic trees for HA GlcNAcβ) were shown on the respiratory track of terrestrial birds but not on the intestinal tract of ducks [Southern China has been considered as a hot spot for the generation of novel AIVs. In this study, we reported the genetic characterization of a recent H5N6 HPAIV strain isolated from chickens in southern China. In addition, novel mutations such as D36N, which is one of the six common amino acid substitutions of H2 subtype AIV from mallards to swine , are emeof ducks , 22. Theof ducks , 16.In addition, a number of novel mutations in the HA of H5N6 HPAIV were identified, including Q142H, N89S, and D193N. It has been suggested that the D193N mutation of H10N7 virus promoted virus binding to α2,6-linked sialic acid receptors without an impair of binding to α2,3-linked sialic acid receptors . HoweverThe NA protein of DG/19 has a 11-aa stalk deletion at residues 59–69, which has been shown to enhance virulence in mammals . MoreoveIn this study, we report the genetic characteristics of a recent HPAI H5N6 (DG/19) strain from chickens in southern China. Our results revealed HPAIV H5N6 strain circulating in south China is potentially a reassortment between H5N1, H5N2, H5N8, and H6N6 AIVs, highlighting the importance of continuous surveillance of AIVs through the East Asian-Australasian flyway. Further studies are needed to investigate the biological role of those novel mutations identified in this study.The routine surveillance of AIV was performed in domestic poultry in Guangdong, China. In October 2019, an outbreak of AIV was reported in a chicken farm in Dongguan, Guangdong province. The liver and spleen samples collected from chickens were homogenized 3 cycles by Precellys Evolution Super Homogenizer (France) at 6000 rpm for 15 s. The viral RNA extraction and reverse transcription was performed as previously described , 26. BriAll 8 gene segments of DG/19 were successfully amplified by Phusion Hot Start II High-Fidelity PCR Master Mix  with a set of universal primers described by Hoffmann et al. . The temwww.gisaid.org) or Influenza research database  were selected for molecular evolutionary analyses. The full length of HA (1701 bp) and NA (1380 bp) genes were aligned by ClustalW and phylogenetically clustered by maximum likelihood method with 1000 bootstrap replicates in MEGA7.A total of 95 representative H5N6 strains from Global Initiative on Sharing Avian Influenza Data  [The 3D structure model of HA of DG/19 was analyzed by the SWISS-MODEL server (sy.org/) . In briesy.org/) .Additional file 1: Figure S1. Clinical pictures of chickens in this study.Additional file 2: Figure S2. The amplification of conserved M gene segments (A) and full genome of A/chicken/Dongguan/1101/2019 (DG/19).Additional file 3: Figure S3. The maximum likelihood phylogenetic trees for the PB2(a), PB1(b), PA(c), NP(d), M(e), and NS(f) gene segments of A/chicken/Dongguan/1101/2019 (DG/19). The virus detected in this study was indicated in red star. The scale bars represent the number of substitutions per nucleotide.


C4) confers genetic risk for schizophrenia. The variation includes numbers of the increased C4A copy number, which predicts increased C4A mRNA expression. C4-anaphylatoxin (C4-ana) is a C4 protein fragment released upon C4 protein activation that has the potential to change the blood–brain barrier (BBB). We hypothesized that elevated plasma levels of C4-ana occur in individuals with schizophrenia (iSCZ). Blood was collected from 15 iSCZ with illness duration < 5 years and from 14 healthy controls (HC). Plasma C4-ana was measured by radioimmunoassay. Other complement activation products C3-ana, C5-ana, and terminal complement complex (TCC) were also measured. Digital-droplet PCR was used to determine C4 gene structural variation state. Recombinant C4-ana was added to primary brain endothelial cells (BEC) and permeability was measured in vitro. C4-ana concentration was elevated in plasma from iSCZ compared to HC . The patients also carried more copies of the C4AL gene and demonstrated a positive correlation between plasma C4-ana concentrations and C4A gene copy number. Furthermore, C4-ana increased the permeability of a monolayer of BEC in vitro. Our findings are consistent with a specific role for C4A protein in schizophrenia and raise the possibility that its activation product, C4-ana, increases BBB permeability. Exploratory analyses suggest the novel hypothesis that the relationship between C4-ana levels and C4A gene copy number could also be altered in iSCZ, suggesting an interaction with unknown genetic and/or environmental risk factors.Structural variation in the complement 4 gene ( The complement system, a part of the innate immune system, contributes to the first line of defense against pathogens Fig. . In addiCSMD1 gene = 2.90, p-value = 0.09). C5-ana and TCC concentrations did not differ between groups.Results are shown in Fig. Only 5 patients and 6 controls completed lumbar puncture. Procedures are discussed and preliminary data are shown in Supplementary Fig. p = 0.03) in individuals with schizophrenia and a trend for all C4A (p = 0.06)  Fig. .Fig. 3Thρ = −0.37, p = 0.05). We then analyzed individuals with schizophrenia and controls separately. In individuals with schizophrenia, plasma C4-ana was positively correlated with C4A GCN ; in controls, there was no correlation . The correlations in individuals with schizophrenia and in controls were significantly different based on the modified Fisher’s Z-transformation test for rho  . The group-specific correlations between C4-ana and C4B GCN were in the opposite directions and were not significantly different from each other . A negative correlation between C4A and C4B GCNs is expected, based on population allele frequencies [Figure 18) Fig. . This diWe hypothesized that increased C4-ana could have a pathological effect by binding to PAR1 and PAR4  on brainCSMD1, is also associated with schizophrenia [Elevated C4 gene copy number has been implicated in schizophrenia etiology by GWAS  and by mophrenia , 11, 12.ophrenia . Based oophrenia .We found significantly higher plasma C4-ana levels, but not C3-ana or C5-ana levels, in individuals with schizophrenia compared to healthy controls Fig. . A previC4AL GNC and a trend towards increased total C4A GCN in individuals with schizophrenia compared to controls , including BEC . Indeed,We found that the addition of C4-ana increases the permeability of a BEC monolayer  the effect of medication and (ii) BMI on complement activation products. (i) One previous study reported an increase in C3-ana after medication treatment , howeverFurther work is needed to test hypotheses arising from the current findings: that a peripheral mechanism of C4 protein activation in patients with schizophrenia interacts with increased C4 GNC and structural variants, leading to increased C4-ana. Given the small sample size of our study, larger cohorts are needed in FEP and individuals with chronic schizophrenia.Supplementary Material


Furthermore, plenty of achievements have been announced regarding H2S working in combination with other signal molecules to mitigate environmental damage, such as nitric oxide (NO), abscisic acid (ABA), calcium ion (Ca2+), hydrogen peroxide (H2O2), salicylic acid (SA), ethylene (ETH), jasmonic acid (JA), proline (Pro), and melatonin (MT). This review summarizes the current knowledge within the mechanism of H2S and the above signal compounds in response to abiotic stresses in plants, including maintaining cellular redox homeostasis, exchanging metal ion transport, regulating stomatal aperture, and altering gene expression and enzyme activities. The potential relationship between H2S and other signal transmitters is also proposed and discussed.Hydrogen sulfide (H Further, under normal circumstances, the reactive oxygen species (ROS) are tightly controlled in plants, because plants have a reactive oxygen scavenging system, which keeps the production and removal of reactive oxygen species in a dynamic balance. This ROS includes hydrogen peroxide (H2O2), superoxide anion (O2·-), singlet oxygen (·O2), and hydroxyl radical (·OH)   in white clover (Trifolium repens) under dehydration conditions [2S scavengers could not reduce the generation of H2O2 induced by Spd, but H2O2 scavengers could effectively inhibit the increase of NO and H2S induced by Spd. The H2S signal induced by Spd was also significantly inhibited by NO scavenger [2S may be the downstream signaling molecule to interact with NO and H2O2 (Drought stress is one of the most serious abiotic stresses in the world. Treatment by spermidine (Spd) remarkably enhanced Hnditions . Furthercavenger . Hence, and H2O2 .2S, H2O2, and putrescence (Put) could alleviate the negative effects of UV-B stress. The protective role of Put in UV-B radiation damage was reduced by the inhibitors of H2S, H2O2, and Put [2O2 was increased by exogenous H2S, and the enhanced H2O2 promoted the accumulation of UV absorbing compounds in hulless barley  seedlings, thus preserving the steady state of oxidation-reduction under UV-B stress and improving its UV-B tolerance [UV-B is a common stress in practical agricultural production. When plants encounter the UV-B stress, the levels of electrolyte leakage, MDA, and ultraviolet absorbing compounds decreased, and the activities of antioxidant enzymes, GSH, and AsA also declined . However and Put . Moreoveolerance .2S, NO, and H2O2 had a significant impact in response to low temperature (10 °C) by modulating the plasma membrane proton pump in cucumber roots [2O2 treatment could improve the heat resistance in maize  seedlings, and this effect could be strengthened by NO and H2S donors but abolished by NO and H2S scavengers or synthesis inhibitors [2S act downstream of H2O2 in the acquisition of heat resistance in plants  under toxic environment in plants has also been explored to some extent.In recent years, many kinds of signal transmitters have emerged to regulate plant growth and development, and to acclimate to environment changes. The protective role of H2S enhanced heat tolerance by strengthening the activities of antioxidant enzymes and increasing osmolyte content in maize seedlings [2S generation by enhancing the activity of H2S synthesis enzyme DES [2S, this downstream role of H2S in SA-induced stress responses was also similarly reported in Cd tolerance in A. thaliana [2S. Pb stress accelerated endogenous H2S production [2S content, which was further reinforced by H2S donor NaHS. It seems that SA triggers endogenous H2S accumulation, which further regulates the AsA-GSH cycle to resist Pb toxicity  and benzoic-acid-2-hydroxylase (BA2H) ,88,89. Seedlings . Furtherzyme DES . While tthaliana . Thus, toduction . Moreoveoduction . In additoxicity .2S biosynthesis in guard cells in tomatoes under osmotic stress [2S scavenger HT or H2S synthetic inhibitor PAG, suggesting a downstream component of H2S in ethylene-triggered stomatal closure under osmotic stress. Further, H2S induced the persulfidation of 1-aminocyclopropane-1-carboxylic acid oxidase1 (ACO1) and ACO2, and restrained their expressions. As a result, H2S negatively regulated ethylene generation in response to osmotic stress [Prunus persica L. Batsch) seedlings [2S restrained over-synthesis of ethylene as well as inhibited oxidative damage under waterlogging stress  and proline dehydrogenase (PDH) in foxtail millet, whereas H2S scavenger or inhibitor reduced the above effects [2S and Pro resulted in preferable growth status, stomatal movement, and oxidative remission under stress conditions. These results indicate a cooperation of Pro and H2S under adverse environments (Pro is a kind of organic osmolyte with a wide distribution in plant cells. Previous studies have demonstrated the increase of Pro after the application of signal transmitter agents in defense of abiotic stresses ,103,104. effects . Moreoveronments .2S can reinforce plant tolerance to these stresses through constructing a luxuriant crosstalk with other signal molecules, such as NO, ABA, Ca2+, H2O2, SA, ETH, JA, Pro, and MT. The genes regulated by H2S and other molecules under abiotic stress conditions are displayed in 2S generation and improve the activities of H2S synthesis enzymes under the stress condition. Meanwhile, H2S represents a feedback manner to enhance the signal cascades in inducing the accumulation of some signal messengers, especially NO, ABA, and Ca2+. In addition, the existence of DES1-related auto-persulfidation and persulfidation may be the reason for the extensive inspiration of its enzyme activity in different stress conditions. In summary, H2S acts as a downstream signal member in cooperation with ABA, H2O2, SA, ETH, JA, and MT, but an upstream signal member of Pro under stress condition. Nevertheless, the crosstalk between H2S, NO, and Ca2+ represents a two-side signal cascades manner, whereas relationships between H2S and other signal molecules vary on account of the specific stress pattern.The disadvantageous environment conditions cause oxidative damage, ionic imbalance, and osmotic stress to plants, resulting in a weakened growth and development status. H2S and one single signal transducer under abiotic stress conditions. Firstly, the feedback molecular mechanism of H2S and NO, and the interactions within protein persulfidation, S-sulfhydration, and S-nitrosylation, remain unclear. Next, more post-translational modification proteins need to be discovered and identified that are triggered by H2S in ABA- or NO-dependent signal pathways under stress condition. Finally, new signal messengers related to H2S activity are waiting to be discovered.Multiple types of research need to be done to explore the point-to-point mechanism within the crosstalk between H


Francisella novicida Cas9, which is a CRISPR-Cas9 ortholog. Using FnCas9(H969A) nickase, the targeting limitation of existing Streptococcus pyogenes Cas9 nickase [SpCas9(H840A)]-based prime editing is dramatically extended, and accurate prime editing is induced specifically for the target genes in human cell lines.Prime editing can induce a desired base substitution, insertion, or deletion in a target gene using reverse transcriptase after nick formation by CRISPR nickase. In this study, we develop a technology that can be used to insert or replace external bases in the target DNA sequence by linking reverse transcriptase to the The online version contains supplementary material available at 10.1186/s13059-022-02644-8. F. novicida)  compared to SpCas9(H840A)-RT on average  according to the location of the nicking guide RNA   by using FnCas9(H969A)-RT in PE4 manner, the averaged efficiency increased 2,94 fold compared to the conventional PE3 method  was indicated according to the point at which nicks were formed (from − 6 to + 3) by both nickase modules -RT shows a precise, expanded range of prime editing and versatile editing properties. These advantages of prime editor are expected to have a big ripple effect on the bio/medical field by enabling target-specific and various types of gene editing in the future. In particular, further engineering of Cas9 protein or AAV based delivery of the FnCas9(H969A)-RT is possibly applied to most of the existing human disease-causing mutations, and it is thought that FnCas9 based prime editing will increase the possibility of application as a therapeutic agent by compensating the role of SpCas9 based one.Escherichia coli BL21(DE3) cells, respectively, and colonies were grown at 37 °C. After growing to an optical density of 0.6 in a 500-ml culture flask, induction was performed at 18 °C for 48 h by isopropyl β-d-1-thiogalactopyranoside treatment. The cells were then precipitated by centrifugation and resuspended in lysis buffer . The cells were disrupted by sonication on ice for 3 min, and the cell lysates were separated by centrifugation at 20,000×g for 10 min. The further purification process was carried out as previously described , and nicking sgRNA (60 pmol) via electroporation using an Amaxa electroporation kit . In parallel with the SpCas9 prime editor, the FnCas9 prime editor was transfected targeting the same nucleotide sequence in HEK293FT cells. Transfected cells were transferred to a 24-well plate containing DMEM (500 μl/well), pre-incubated at 37 °C in the presence of 5% CO2 for 30 min, and incubated under the same conditions for subculture. All the cell lines used in this study have been authenticated.Human-derived cell lines  were purchased from Invitrogen (R70007), and American Type Culture Collection , respectively. The HEK293FT, U2OS, and HeLa human cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (both from Gibco) at 37 °C in the presence of 5% COGenomic DNA (gDNA) was extracted from the cultured cells 72 h after genome editing. The gDNA was isolated using DNeasy Blood and Tissue Kit (Qiagen). Target amplicons were obtained through PCR . Reverse complementary sequences of PAM such as CCN for NGG are also included for the analyses, and Y chromosome and mitochondrial sequences were excluded. The analyses process was conducted using R software (version 4.1.1).For analyses of effective windows of FnCas9 and SpCas9, position information of pathogenic single point mutations was extracted from ClinVar (NCBI) data. Next, the position information was compared with the PAM sequences of FnCas9 and SpCas9 within human genome -RT and FnCas9(H969A)-RT. Figure S3. Various methods of prime editor delivery by using SpCas9(H840A)-RT or FnCas9(H969A)-RT expression plasmids. Figure S4. The target sequence and NGS analysis results commonly edited by SpCas9(H840A)-RT or FnCas9(H969A)-RT. Figure S5. Cell diversity experiment of prime editing using FnCas9(H969A)-RT. Figure S6. Comparative analysis of nickase activity of SpCas9(H840A)-RT and FnCas9(H969A)-RT. Figure S7. Targeting of multiple loci for direct comparison between SpCas9(H840A)-RT and FnCas9(H969A)-RT based prime editing. Figure S8. Comparative analysis of off-target editing of SpCas9(H840A)-RT and FnCas9(H969A)-RT based on in-silico predicted sites. Figure S9. Multiplexed prime editing with SpCas9(H840A)-RT and FnCas9(H969A)-RT system. Figure S10. Expansion of the range of prime editing in EMX1 locus by FnCas9(H969A)-RT. Figure S11. Result of the prime editing in target sequence induced by FnCas9(H969A)-RT. Figure S12. Expansion of the targetable range by prime editing with FnCas9(H969A)-RT or RHA-FnCas9(H969A)-RT.Additional file 2. Review history.


In vitro studies, decreased P4HA1 significantly inhibits proliferation and cell cycle, by regulating cyclin-dependent kinases (CDKs), cyclins and CDK inhibitor (CKI). Moreover, via inhibiting epithelial-mesenchymal transition (EMT) and matrix metalloprotease (MMPs), dysregulation of P4HA1 could restrain the tumor cell invasion and metastasis of lung adenocarcinoma. In addition, we found that P4HA1 could enhance cell stemness and cisplatin-resistance in lung adenocarcinoma. In summary, P4HA1 plays a crucial role in the development of NSCLC and may provide a brand-new target for lung cancer treatment.Prolyl 4-hydroxylase subunit alpha 1 (P4HA1) is the core active catalytic portion of prolyl 4-hydroxylase, and has contributed to tumorigenesis in several cancers. In this study, we identified that P4HA1 mRNA and protein are both up-regulated in non-small cell lung cancer (NSCLC). Besides, overexpressed P4HA1 is correlated with poor clinical outcomes and serve as an independent prognosis biomarker in lung adenocarcinoma (LUAD), but not lung squamous cell carcinoma (LUSC).  Lung cancer, as a type of prevailing malignant tumor, led to considerable mortality and morbidity worldwide 2β2 tetrameric, and the α subunit owns peptide binding and catalytic activity. In mammalian there have found three P4HA subtypes including of P4HA1/2/3. P4HA1 being the major isoform of P4H exists many human tissues, and contributes a lot to the prolyl 4-hydroxylase activity The prolyl 4-hydroxylase subunit alpha 1 (P4HA1) is a member of the prolyl 4-hydroxylase (P4Hs), which catalyzes the post-translational modification and modulates protein folding and stability https://xenabrowser.net) https://www.ncbi.nlm.nih.gov/geo/) The Cancer Genome Atlas (TCGA) - The Genotype-Tissue Expression (GTEx) Lung Cancer data were downloaded from the University of California Santa Cruz Xena Browser  and all research was performed in accordance with relevant guidelines/regulations. All research samples were obtained with written informed consent. If the patient is juvenile, a written consent will be signed by caretakers, or guardian on behalf of the juvenile participating in this study.Between 2002 and 2012, we collected 500 NSCLC patients, which diagnosed with NSCLC and underwent surgery and 103 cases of non-cancerous control lung tissues from The Second Xiangya Hospital, Central South University's. All tumors were assessed by expert pathologists using the WHO lung cancer histological classification and the Eighth Edition Lung Cancer to establish tumor stage. At the time of the original procedure, no patients had received radiation or chemotherapy, and none of them had received therapy targeting PD-1/PD-L1 throughout the follow-up period. The time from diagnosis to death, or the final known moment of survival, was used to calculate overall survival time. The Ethics Committee of Central South University's Second Xiangya Hospital accepted this study (No: S039/2011), and all patients with written informed consent had access to comprehensive clinical and follow-up data. In this study, we used the TMA technology to construct high-throughput NSCLC TMAs according to rules previously described Eligible patients included in this article for TMAs are in accordance with the following inclusion criteria: (1) NSCLC; (2) Complete follow-up data and clinicopathological data. The detailed clinic parameters of enrolled patients were presented in Exclusion criteria included the following: (1) Other treatments were used after the operation; (2) Missing follow-up or clinic parameters.The immunohistochemistry experiment was conducted following the protocol our former study 2.The non-neoplastic lung epithelial cell 16HBE, BEAS-A2 and six NSCLC cell lines  were obtained from the ATCC (American type culture collection). Cell lines were cultured in RPMI-1640 medium  containing 10% fetal bovine serum . The culture dishes were placed in the incubator at 37 °C, 5% COThe lentiviral vectors were designed and synthesized by landbiology . The target sequence of shRNA is as follows: P4HA1: 5′-CTAGTACAGCGACAAAAGA-3′. The infection process was performed according to the manufacturer's instructions. A549 and SPC-A1 cells were infected with a multiplicity of infection (MOI) of 2 and incubated for 8 hours. Three days after infection, cells were cultured in 10% FBS medium with 2.5 μg/ml puromycin (Sigma-Aldrich).Total RNA was extracted using Trizol RNA reagent . Reverse transcription was performed using the PrimeScript™ RT reagent Kit with gDNA Eraser . qRT-PCR was conducted using SYBR Green™ Premix Ex Taq™ II  in Applied Biosystems 7500 . The primers were as follows: GAPDH, forward ACAACTTTGGTATCGTGGAAGG; reverse GCCATCACGCCACAGTTTC; P4HA1 forward GGAACAAGCCCTAAGG CAACT; reverse TGCTGATATACCGCATAGCTCAA.Western blotting was performed according to the standard protocol 2. Colonies were washed triple with PBS and stained with crystal violet for 15min.Cells were planted into 96-well plates with 1000 cells per well. CCK-8 reagents  were added at every at 24h for five days. The optical density was estimated at 450 nm wavelength. Cells were planted in in 6-well plates (500/well) and incubation for 2 weeks at 37 °C, 5% COCell for cell cycle analysis were harvested after 48 h incubation and washed with PBS. The cells were further fixed with 70% ice-cold ethanol at 4 °C overnight. The samples were sent to Sun Yat-Sen hospital for further experiments.5 cells) were seeded with serum-free medium onto the top chamber, and the bottom chamber was filled with 10% FBS medium. For invasion assays, BD Matrigel were first added in the top chambers for 2h, and then cells (2×105 /well) were planted. After 18h, all the chambers collected and fixated with methanol for 30 min, stained with 0.01% crystal violet for 15 min.Migration and invasion assays were performed using Transwell chambers (Corning USA). For migration assays, cells (2×103 cells/well) were plated in 24-well plates with ultra-low adherence  and cultured in DMEM/F12 medium, supplemented with B27, 20 ng/ml EGF and 20 ng/ml bFGF for 14 days to form spheres.Cells  https://david.ncifcrf.gov/summary.jsp) Genes co-expression with P4HA1 were extracted using Pearson's correlation analysis (|r| ≥ 0.3) in cBioPortal  or R version 3.6.2 were used for data analysis. Unless specifically mentioned, all values are presented as the mean ± SD. Two groups had equal variances, two‑group independent sample comparisons Student's t‑test (two‑tailed) were performed, while Welch's t‑test was used for unequal variances. Multi‑group samples statistics were analyzed via one‑way ANOVA if the variances were equal; if not, Welch's ANOVA was performed. Bonferroni post hoc tests were performed for all ANOVAs. Samples from TCGA Lung Cancer Cohort were divided into high and low expression groups according to median values. DSS, PFI, OS, DFI curves were plotted using the Kaplan‑Meier method, log‑rank test was used to assessed differences. Cox proportional hazard regression model was hired to estimate the prognostic role of P4HA1 in the patients with NSCLC. GTEx-TCGA lung cancer cohort having the largest samples were first selected to assessed the mRNA expression of P4HA1 in our study. In the cohort, P4HA1 showed distinct differential expression in both lung adenocarcinoma and lung squamous cell carcinoma compared with normal lung tissue Figure . NeverthNext, we explored the P4HA1 protein expression in our NSCLC tissue arrays by Immunohistochemistry. We found that P4HA1 protein increased in lung cancer tissues compared to non-cancerous lung tissues in both lung adenocarcinoma Figure B.We also analyzed with TCGA lung samples with follow-up data that the prognostic value of increased expression of P4HA1 in lung adenocarcinoma and lung squamous cancer, separately. Kaplan-Meier analysis revealed that P4HA1 was negatively correlated to overall survival (OS), disease-specific survival (DSS), and progression free interval (PFI), but not with disease-free interval (DFI) in lung adenocarcinoma Figure A. The reMoreover, we investigated with the Cox regression, both univariate Figure D and mulTaken together, these results indicated that P4HA1 might serve as an independent prognostic biomarker for lung adenocarcinoma patients. Since P4HA1 displayed greater prognostic value in lung adenocarcinoma, we will focus on lung adenocarcinoma in our further study.To investigate the role of P4HA1 expression in lung adenocarcinoma progression, we assessed the association of clinicopathological features with P4HA1 mRNA and protein expression (Table To get insight into the biological functions and potential molecular pathways of P4HA1 implicated in the tumorigenesis of lung adenocarcinoma, we carried on the Gene enrichment analysis with TCGA LUAD Cohort. Firstly, we analyzed the co-expression genes of P4HA1 in cBioPortal. Genes with |r| ≥ 0.3 were further used to Gene ontology analysis and KEGG pathways analysis in DAVID. GO analysis revealed that P4HA1 involved in multiple biological processes, including cell proliferation, hypoxia response and cell cycle A. KEGG pWe investigated P4HA1 expression in six different lung cancer cells and human bronchial epithelial cell line with real-time PCR and Western blotting. The results indicated P4HA1 was highly expressed in six lung cancer lines in both mRNA and protein level, in which A549 and SPC-A1 were the two most high expressed adenocarcinoma cell lines C-D. qPCRTranswell migration assays demonstrated that knockdown P4HA1 inhibited the ability of tumor cell migration in lung adenocarcinoma. Moreover, Matrigel invasion assay indicated that knockdown of P4HA1 significantly inhibited tumor cell invasion of lung adenocarcinoma (Figure To detect the function of P4HA1 in the stemness of lung adenocarcinoma, we applied cancer cell sphere formation analysis. From the results, we found that knockdown P4HA1 formed smaller and fewer stem spheres compared to the corresponding groups Figure A-B. BesiPrevious studies have shown that inhibiting P4HA1 could make triple-negative breast cancer sensitive to chemotherapy. We detected P4HA1 expression in cisplatin-resistant cell lines to demonstrate whether P4HA1 is associated with chemoresistance in lung adenocarcinoma. The expression of P4HA1 in cisplatin-resistant lung adenocarcinoma remarkably increased compared with control groups Figure D.P4HA1 is the key component of the rate-limiting enzyme P4H, which plays a vital role in synthesis of various collagens Many studies have reported that P4HA1 overexpresses and serves as a satisfying prognostic biomarker. In mammary cancer, P4HA1 showed a dramatical high-expression on both mRNA and protein level, and P4HA1 mRNA expression was correlated with shorter overall survival times in vitro study validated that decreased P4HA1 expression inhibited the proliferation, migration and invasion of lung adenocarcinoma cells. According to the gene enrichment analysis, P4HA1 may regulate the proliferation of lung adenocarcinoma by regulating cell cycle. Flow cytometry cell cycle analysis further proved that knockdown P4HA1 inhibited G1/S transition of cell cycle. MMPs and EMT are the most common mechanism for cancer metastasis. In prostate cancer, P4HA1 could promote prostate cancer metastasis via regulating MMP1 expression After analyzing the relationship between P4HA1 and clinical pathological parameters of lung adenocarcinoma patients, we found P4HA1 mRNA expression relating to tumor diameters and distant metastasis, while not relating to age, gender, smoking history and clinical stage. However, P4HA1 protein expression was associated with stage, which was different from mRNA level. Additionally, Gene Ontology and KEGG pathways enrichment analyses suggested that P4HA1 could regulate proliferation and metabolism. Previous studies reported that P4HA1 promoted proliferation, metastasis in breast cancer, prostate cancer, and glioma Our study firstly proves the functional of P4HA1 in the progression of NSCLC disease. These results suggest that P4HA1 may act as a potential drug target and prognostic indicator for lung adenocarcinoma. Consequently, the development of highly specific and potent P4HA1 inhibitors may be the key to overcome chemotherapy resistance and improve lung cancer treatment.Supplementary figures and tables.Click here for additional data file.


S100A9 gene expression in HCC; we found that S100A9 expression was associated with HCC prognosis. In addition, S100A9 protein expression was assessed by immunohistochemistry analysis of tissues from 382 HCC patients. We found that the infiltration of S100A9+ cells in both tumor and nontumor tissues could predict poor overall survival  and a high recurrence risk  in our tissue microarray analysis. Furthermore, immunofluorescence double staining revealed that the primary S100A9-expressing cells in adjacent nontumoral tissue were CD15+ neutrophils, and both CD68+ macrophages and CD15+ neutrophils expressed S100A9 in HCC tumor tissues. Taken together, the results suggest that high S100A9+ cell density predicts a poor prognosis in HCC patients, and S100A9 expression could potentially serve as an independent prognostic marker for HCC.S100A9 is differentially expressed in various cell types and is associated with the development, progression and metastasis of various cancers. However, the expression, distribution, and clinical significance of S100A9 in hepatocellular carcinoma (HCC) remain unclear. In the present study, The Cancer Genome Atlas (TCGA) database was used to examine  Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related death worldwide , 2. CurrAccumulating evidence has highlighted the significance of inflammation, which dramatically affects the progression and prognosis of HCC patients. S100A9 is a calcium-binding protein that plays an indispensable role as a mediator in inflammatory processes . Evidenc+ cell density can serve as an independent predictor of poor prognosis in patients with HCC.Solid tumors comprise not only cancer cells but also many other nonmalignant cell types, which produce a unique microenvironment that regulates tumor progression. S100A9 is expressed mainly by neutrophils, monocytes, and activated macrophages , 8. ReceS100A9 gene expression was significantly downregulated in tumor tissues compared to adjacent nontumor tissues . As shown in + cell density was significantly higher in the nontumoral tissue than in the tumoral tissue (P < 0.0001).We performed IHC to assess the protein expression level of S100A9 in the HCC microarray chip. First, we applied automated measurements with computerized image analysis to count S100A9y assays . To asse+ cells in the tumoral region and nontumoral region. Kaplan-Meier analysis revealed a negative association between overall survival (OS) and the density of S100A9+ cells in the nontumoral region .We next investigated the prognostic role of S100A9 expression in HCC. A total of 382 treatment-naïve HCC patients who had long-term follow-up data were divided into two groups according to the median counts of S100A9+ cells in tumor and nontumor regions. Using the median value as a cutoff, patients were classified into four groups: I, N-S100A9low T-S100A9low (n = 110); II, N-S100A9low T-S100A9hi (n = 81); III, N-S100A9hi T-S100A9low (n = 81); and IV, N-S100A9hi T-S100A9hi (n = 110). Significant differences in OS (P < 0.0001), group II , and group III . In addition, patients in group IV exhibited the highest recurrence rate (5-year recurrence rate: 76.07%) among all patients, including those in group I , group II , and group III . The above data suggest that S100A9 might represent a potential target for HCC therapy.We also evaluated the combined influence of S100A9es in OS  and recues in OS  were fou+ cell density and patient clinicopathological features. + cell number did not significantly correlate with patient sex, age, HBsAg, AFP, ALT, cirrhosis, differentiation, tumor multiplicity, tumor size, Barcelona Clinic Liver Cancer (BCLC) stage or TNM stage. However, nontumoral S100A9+ cell density, but not tumoral S100A9+ cell density, was associated with sex, tumor size, BCLC stage and TNM stage.We also analyzed the correlation between S100A9S100A9 gene expression and tumor immune cell infiltration using the TCGA-LIHC data set. Only 7 nontumoral and 49 tumoral samples with a CIBERSORT P-value < 0.05 were retained for further study. The abundances of twenty-two immune cell types in each HCC sample are presented in the boxplot in S100A9 gene (data not shown). However, in terms of the component of cells, macrophages (M0 + M1 + M2 Mφ) were the main cell populations in the microenvironment of HCC tumor tissue. Hence, we investigated the prognostic significance of Mφs in 49 patients in the TCGA-LIHC dataset. As shown in + cells, CD15+ neutrophils and CD68+ Mφ infiltration in our tissue microarray was analyzed  in nontumor areas, but this proportion was significantly reduced in tumor areas (29.01 ± 24.20%)  –6C. In a8+ cells –6F. Coll+ cells in HCC tissue. A high S100A9+ cell density in either tumoral or nontumoral tissue was found to be a predictor of unfavorable prognosis and could serve as an independent risk factor in patients with HCC. Furthermore, we demonstrated that Mφs and neutrophils accounted for most S100A9+ cells in HCC tumoral tissue and that neutrophils were the dominant S100A9+ cells distributed in nontumoral tissue.S100 proteins are involved in a variety of biological processes and expressed in various cell types in tissues. We delineated the distribution and prognostic value of S100A9+ cells. Few HCC cells themselves express S100A9. These results were consistent with those of previous studies demonstrating high S100A9 expression in infiltrating immune cells in various cancer types, including colorectal cancer and pancreatic cancer [S100A9, a member of the S100 family, is expressed in granulocytes, monocytes, macrophages, MDSCs and tumor cells in various cancers , 19, 20.c cancer . In othec cancer , prostatc cancer , nasophac cancer , and brec cancer , S100A9 c cancer . However+ cell number was an independent and significant prognostic factor in HCC. Consistent with our results, previous studies showed that S100A9+ cell density was associated with a poor prognosis in patients with other cancers, such as clear cell renal cell carcinoma [The relationship between S100A9 expression in HCC and disease progression remains unclear. Here, we observed that high tumoral S100A9 expression at both the RNA  and protein (IHC detection) levels was associated with a poor prognosis. Furthermore, multivariate analyses revealed that S100A9arcinoma , invasivarcinoma , and nonarcinoma .S100A9 plays an important role in malignant development and cancer progression by triggering protumor immune responses. Previous studies have shown that S100A9 acts as a chemotactic molecule to recruit inflammatory cells or immunocytes, such as MDSCs and neutrophils , 28, to Interestingly, S100A9 staining of the nontumor tissue of HCC patients provided prognostic information. Current evidence has demonstrated that tumor-adjacent, morphologically normal tissue is not completely normal –43. S100+ cells in nontumoral and tumoral regions are still largely unclear, further studies are needed. Nevertheless, our current study identified that high S100A9 expression was associated with poor OS and a high recurrence risk in patients with HCC. Consequently, S100A9 expression could be considered a significant prognostic marker in patients with HCC.However, this study has certain limitations. The underlying molecular mechanisms regulating distinct cell sources of S100A9This study was approved by the Ethics Committee of Sun Yat-sen University Cancer Center (SYSUCC). A total of 382 patients with pathologically confirmed HCC who underwent surgery at the SYSUCC from June 21, 2006 to September 17, 2010 were included in this study. The patients provided informed consent for participation in the present study. The patients did not receive any immunotherapy or neoadjuvant therapy before the operation. The clinical data of the patients were extracted from their electronic medical records and are listed in The clinical stage of tumors was determined according to the tumor-node-metastasis (TNM) classification system of the American Joint Committee on Cancer . The follow-up period ended in July 2014, and the median survival time was 63 months . Recurrence was diagnosed pathologically via surgical biopsy and/or radiologically via computed tomography or positron emission tomography. OS was defined as the length of time between surgery and death or the last follow-up examination.S100A9 gene in human HCC were obtained from TCGA up to April 17, 2020. Samples included only those that were annotated as “untreated, primary HCC”. A total of 365 HCC tissues and 50 normal tissues were selected based on the official TCGA-liver hepatocellular carcinoma (LIHC) dataset. S100A9 gene expression (as transcripts per kilobase million (TPM) values) and corresponding clinical and pathological data of these samples were downloaded and processed as previously described. The gene expression TPM data of S100A9 are shown in Data on the expression levels of the n = 183; high, n = 182).According to the expression level of S100A9 in the tumor tissues of 365 patients in the TCGA-LIHC database, the patients were divided into high and low groups , and 399 genes were highly expressed in the S100A9low group (the fold change of high group/low group < 0.5). These differentially expressed genes are shown in S100A9high group were analyzed using GO enrichment tools (http://enrich.shbio.com/index/ga.asp).Under the condition of S100A9low and 182 S100A9high patients were subjected to 50 hallmark gene analyses of sets by using GSEA software . The detailed results are shown in All transcriptome expression results of A tissue microarray (TMA) was constructed, and the slides were incubated with a rabbit monoclonal primary antibody against S100A9  at 4°C overnight. The sections were stained using an Envision system  as previously described [Double immunofluorescent staining was performed as previously described . Briefly2) in the immunohistochemical staining of the tissue microarray is shown in Immunohistochemically and fluorescently stained sections were scanned at a magnification of ×20 using the Aperio Digital Pathology Scanner  to capture a digital whole slide image. Digital image analysis for quantification of the staining for S100A9, CD34, CD56, CD20, CD3, CD15 and CD68 was measured using Aperio ScanScope AT Turbo, eSlide Manager and ImageScope software  in accordance with the manufacturer’s recommendations. The 3 most representative high-power fields were captured for each tumor and nontumor region in all specimens. All measurements were performed by the same researcher, who was blinded to the histologic and patient survival data, to prevent interoperator variability. Moreover, the protein expression level of S100A9  and GraphPad Prism (version 8). The Kolmogorov-Smirnov test and Shapiro-Wilk test were used to analyze normality. The significance of differences between groups was determined by the Mann-Whitney test and Wilcoxon matched-pairs signed-rank test. Pearson’s correlation was used to evaluate the correlation between investigator count and computer count, and to evaluate the correlation of variables with immune cell infiltration. Survival curves were assessed by Kaplan-Meier analysis with the log-rank test. The Cox proportional hazards model was used to identify prognostic factors through univariate and multivariate analyses. The statistical significance of differences between groups was determined using the two-tailed Student’s All patients’ samples were anonymously coded in accordance with local ethical guidelines (as stipulated by the Declaration of Helsinki), and written informed consent was provided. The Review Board of Sun Yat-sen University Cancer Center approved the study protocol.Supplementary Table 1Supplementary Table 2Supplementary Table 3Supplementary Table 4Supplementary Table 5


However, the H3N2 influenza virus component of the vaccine, A/Perth/16/2009 (Perth/09), back-boosted and elicited antibodies with HAI activity and Bmem response to historical H3N2 influenza virus strains. Following stratification of the pre-existing antibody with HAI against the CA/09 H1N1, there was a negative correlation with HAI seroconversion to other vaccine strains. Overall, strong immune responses against CA/09 H1N1 influenza virus negatively influenced the induction of novel humoral responses.In this study, we hypothesized that the humoral response to trivalent seasonal influenza virus vaccines was influenced by rapid antigenic switching of H1 HA. We tested archived sera and peripheral blood mononuclear cells (PBMC) collected at prior to vaccination at day 0, as well as days 30 and 90 after vaccination during the 2009/2010 and 2010/2011 influenza virus seasons. During the 2009/2010 season, vaccination successfully induced antibodies with hemagglutinin inhibition (HAI) activity against both H1N1 and H3N2 vaccine components. For the 2010/2011 season, the A/California/04/2009 (CA/09) H1N1 elicited seroconversion (HAI titer = 1:40) and novel memory B cell (B A novel H1N1 influenza virus (pH1N1) emerged in North America in March 2009 [Since the novel pH1N1 influenza virus shared common epitopes with previously circulating H1N1 strains, pre-existing memory B or T cells can be recalled by exposure to the novel H1 HA antigen. As a result, a single 15 μg dose of monovalent pH1N1 influenza virus vaccine elicited antibodies with hemagglutinin inhibition (HAI) activity at a titer (1:40 or higher) for seroconversion in healthy young adults that had not been previously exposed to the pH1N1 influenza virus influenza virus .The effectiveness of seasonal influenza virus vaccines has fluctuated from 19% to 64% since the introduction of the pH1N1 influenza virus into the human population. Of note, the highest vaccine effectiveness recorded in the past ten years coincided with the use of the monovalent pH1N1 influenza virus vaccine deployed during the 2009/2010 influenza virus season, as well as the trivalent seasonal influenza virus vaccine used during the 2010/2011 influenza virus season , 4. Two The study procedures, informed consent, and data collection documents were reviewed and approved by the IRB of the H-13000 and the University of Messachusetts (MA). The funding source had no role in sample collection, nor decision to submit the manuscript for publication. Subjects were recruited in MA, USA, and enrolled with written, informed consent. Exclusion criteria included documented contraindications to hyper sensitivity to vaccine components, previous life threatening reaction to influenza vaccine, altered immune competence state, neurological disorder; pregnancy; acute febrile illness; history of anemia or bleeding disorders; and donation of 60 cc or higher blood within the past 30 days.®, Sanofi Pasteur, Swiftwater, PA, USA). Blood samples were processed for sera and PBMC. For PBMC isolation, blood was collected in Vacutainer CPT tubes (BD Biosciences) at D0, D7, and D21. These samples were processed within 6 hours of collection and stored at liquid nitrogen (vapor phase) for future analysis. Serum was collected in Vacutainer SST tubes (BD Biosciences) and processed within 24–48 hours, stored at 4°C until separated, and aliquoted for long-term storage at –20°C. Overall workflow for each sample and time point is summarized in During the 2009/2010 and 2010/2011 northern hemisphere influenza seasons, 30 eligible participants were consented and enrolled in the study. Demographic information was only available for the 2009/2010 chorot . MonovalHAI assay was conducted in accordance with the protocols from the WHO laboratory influenza surveillance manual. As a testing antigen, influenza viruses were obtained through the Influenza Reagents Resource (IRR), BEI Resources, or the Centers for Disease Control and Prevention (CDC) and propagated in 10-day-old embryonated, specific pathogen-free (SPF) chicken eggs. Tested serum was treated with receptor-destroying enzyme (RDE) (Denka Seiken Co.) prior to being tested in accordance with manufacture’s recommendation. RDE-treated sera was serially diluted in PBS 2-fold across v-bottom microtiter plates (50 μL/well) and an equal volume of each influenza virus (8 HAu/50 μL l) was added. The plates were covered and incubated at RT for 20 minutes, and then 0.8% turkey erythrocytes  were added to the virus-serum mixture and incubated for 30 minute at room temperature. The HAI titer was determined by the reciprocal dilution which inhibited agglutination of TRBCs. People were considered seronegative with a titer less than 1:40.2O2 for 40 min at 37°C. The reaction was terminated with 1% (w/v) sodium dodecyl sulfate (SDS). Colorimetric absorbance at 414 nm was measured using a PowerWaveXS  plate reader. Background was subtracted from negative wells. Linear regression standard curve analysis was performed using the known concentrations of recombinant standard antigen to estimate the HA content in VLP lots.HA binding IgG level was measured by ELISA as previously described . Briefly6 viable cells/mL) with complete media containing RPMI 1640 medium (MilliporeSigma) with 10% FBS , 23.8 mM sodium bicarbonate (Thermo Fisher Scientific), 7.5 mM HEPES (Amresco), 170 μM Penicillin G , 137 μM Streptomycin (MilliporeSigma), 50 μM 2-mercaptoethanol (MilliporeSigma), 1 mM sodium pyruvate (Thermo Fisher Scientific), essential amino acid solution (Thermo Fisher Scientific), nonessential amino acid solution (Thermo Fisher Scientific), 500 ng/mL R848 (Invivogen), and 5 ng/mL rIL-2 (R&D Systems) for 7–9 days at 37°C in 5% CO2 [PBMCs were plated on 12-well cell culture plate  . In contParticipants vaccinated during the 2010/2011 influenza virus season had a higher pre-existing HAI titer against the CA/09 H1N1 than during the prior season . ApproxiDuring the 2009/2010 influenza season, 16 out of the 29 participants had pre-existing antibodies with HAI activity (median HAI titer = 20) against Uru/07 . AmongstPre-existing serum antibodies with HAI activity against the CA/09 H1N1 influenza virus may indicate that individuals were exposed to the virus by natural infection or monovalent vaccine deployed during between June and December, 2009. To evaluate the impact of prior exposure to the CA/09 H1N1 antigen, participants were stratified by pre-existing serum HAI titer at D0 prior to vaccination. Among subjects who received CA/09 H1N1 virus vaccination during the 2010/2011 influenza season, only 8 out of the 17 subjects with pre-existing CA/09 H1N1 HAI titers seroconverted to the CA/09 H1N1 virus . In contDuring the 2009/2010 influenza season, the median IgG titer to the Bris/07 H1 rHA and Bris/07 H3 rHA were 232.71 and 250.68 μg/ml prior to vaccination at D0, respectively . The medmem response. The HA-specific Bmem response was quantified by measuring the IgG antibody titers produced by ex-vivo stimulated peripheral blood mononuclear cells (PBMC) collected on days 0, 30, and 90 (mem-derived IgG (Bmem-IgG) from each three time point was controlled to be at the similar level  from D0 levels by five-fold at day 90 post-vaccination  t antigen . The ser strains , 9.mem secreted IgG [The HAI antibodies to Perth/09 is known is poorly reactive to the Uru/07 H3N2 or Bris/07 H3N2-like viruses . It is deted IgG . Insteadeted IgG . These ieted IgG . RecommeStratification of the HAI titers indicated that individuals with pre-existing antibodies with HAI activity against the CA/09 H1N1 virus had fewer immune responses against the H1N1 and H3N2 HA antigens in the influenza vaccine . Previouet al. cautiously suggested that one vaccine component could dominate others during the 2017/2018 influenza season [The negative interference by co-administrated vaccines has been widely investigated in pediatric immunizations since these vaccine schedules administer multiple vaccines simultaneously , 17. Onea season . The immmem response. Also, since we could verify subject information only for the 2009/2010 cohort, we could not verify compounding factors, as immunization history, age, or health conditions. Particularly, we could not evaluate the influence of monovalent vaccine or H1N1 infection due to the lack of history on 2010/2011 cohort. Third, the current study was conducted on samples collected from one locality in United States and two-consecutive influenza seasons. Follow-up longitudinal studies are needed to clarify the underlying mechanism and provide better insight. Also, our ex-vivo analysis of memory B cell response is limited to non-speicific stimulation only by IL-2. Future studies can extend to explore more antigen specific Bmem analysis and more optimal B cell stimulating condition.The limitations to this study including the use of archived serum and PBMC samples collected ~10 years ago. Since the serum and PBMCs were processed separately and assigned independent identification numbers, we could not correlate the individual serum antibody titer with the BOverall, the trivalent seasonal influenza virus vaccination during the 2009 pandemic was effective in inducing antibodies with HAI activity to HA antigens in the vaccine. However, dominant immunogenicity of one vaccine component could negatively influence immune responsiveness to other vaccine components. Seasonal influenza virus vaccinations may be expanded after the COVID-19 pandemic, the impact of sudden antigenic changes and vaccine interference needs to be thoroughly investigated. However, these findings should not be misinterpreted to discourage annual vaccinations as numerous reports still emphasize the importance of yearly immunization [S1 FigLower detection limit of the HAI assay was 1:10. The whiskers go down to the smallest value and up to the largest.*p<0.05, **p<0.01.(PPTX)Click here for additional data file.S2 Figmem-IgG was confirmed to be the same over three PBMC collection time points. It showed that the change of the Bmem-IgG level was a result of differentiation of the B cells, not the expansion of total B cells.Before analyzing HA-specific Bmem response, the total B(PPTX)Click here for additional data file.


The presence of fluorine atoms in many drugs opens new possibilities for developing radioligands labelled with fluorine-18. The trifluoromethyl group (CF3) represents a versatile structural motif in medicinal and pharmaceutical chemistry to design and synthesize drug molecules with favourable pharmacological properties. This fact also makes CF3 groups an exciting synthesis target from a PET tracer discovery perspective. Early attempts to synthesize [18F]CF3-containing radiotracers were mainly hampered by low radiochemical yields and additional challenges such as low radiochemical purity and molar activity. However, recent innovations in [18F]trifluoromethylation chemistry have significantly expanded the chemical toolbox to synthesize fluorine-18-labelled radiotracers. This review presents the development of significant [18F]trifluoromethylation chemistry strategies to apply [18F]CF3-containing radiotracers in preclinical and clinical PET imaging studies. The continuous growth of PET as a crucial functional imaging technique in biomedical and clinical research and the increasing number of CF3-containing drugs will be the primary drivers for developing novel [18F]trifluoromethylation chemistry strategies in the future.Positron emission tomography (PET) is a preclinical and clinical imaging technique extensively used to study and visualize biological and physiological processes in vivo. Fluorine-18 ( CF3 groups are incorporated into drugs to improve potency, binding selectivity, lipophilicity and facilitate their administration trifluoromethylation chemistry strategies.The rapidly increasing number of novel trifluoromethylation chemistry techniques and their impact on drug development and agrochemistry were summarised in detail in several recent review articles ,11,12,13PET is an important molecular imaging technique with unique diagnostic applications for the quantitative assessment of biochemical and physiological processes in vivo with specific radiotracers. PET is also used to assess the pharmacokinetics and pharmacodynamics of novel drug candidates ,17,18,1918F) is among the most commonly used radioisotopes for PET, as demonstrated by hundreds of fluorine-18-labelled radiotracers [18F]fluoro-2-deoxy-D-glucose ([18F]FDG). [18F]FDG is particularly useful in measuring enhanced glucose metabolism due to altered energy metabolism, also referred to as the Warburg effect, in many cancers [H-1-(3-[18F]Fluoro-2-hydroxypropyl)-2-nitroimidazole ([18F]FMISO) is another example of a fluorine-18-labelled radiotracer used in the clinic for the visualization of tissue or organ hypoxia in cancer and other diseases [18F-labeled radiotracers for clinical PET was recently demonstrated by the FDA approval of fluorine-18-labelled amino acid [18F]Fdopa for clinical PET imaging of Parkinson’s disease CF3 groups as part of the synthesis of hypoxia imaging agent [18F]TFMISO [18F]TFMISO in reasonable radiochemical yields (60%) but at a low molar activity (0.003 GBq/μmol). Riss et al. reported a nucleophilic alkene [18F]fluorination method resulting in products at a high molar activity (86 GBq/μmol) [18F]trifluoroethyl groups, but the operational simplicity of the chemistry and the obtained high molar activities are remarkable. While the molar activity of the method is impressive, the radiochemical yield was low [18F]CF3 groups. The process used a nucleophilic aliphatic radiofluorination reaction of no-carrier-added (n.c.a.) [18F]fluoride with difluoroiodomethane to form [18F]fluoroform. [18F]fluoroform was deprotonated and added to an aldehyde yielding respective [18F]trifluoromethyl carbinols in good yields. The experimental design of the reaction was relatively complex as it involves low-temperature distillation and trapping of highly volatile [18F]fluoroform at −80 °C in a secondary reaction vessel [18F]Ruppert-Prakash reagent ([18F]Trifluoromethyltrimethylsilane) [18F]Ruppert-Prakash reagent was achieved by the deprotonation of [18F]fluoroform with KHMDS and subsequent addition of this nucleophile to trimethylsilyl chloride to generate the [18F]trifluoromethyl transfer reagent. The synthetic utility of this method was demonstrated by the reaction of [18F]Ruppert-Prakash reagent with aldehydes, where TBAT (Tetrabutylammonium difluorotriphenylsilicate) was used as the initiator. The reaction with 4-nitrobenzaldehyde generated the radiolabelled compound in 11% RCY and 13 GBq/μmol molar activity  . The met was low . Van dern vessel ,36. In 2lsilane) . The synactivity .18F]CF3 groups. However, the method was limited to the synthesis of [18F]trifluoroacetoaryl ketones [18F]F2 resulting in products with diminished molar activity in the range of 0.02 GBq/μmol. Gomez et al. reported a method for the synthesis of fluorine-18-labelled trifluoroacetamides [18F]fluoride was not efficient. The addition of metal salts was also not successful, whereas the addition of base DBU proved to be crucial, and radiochemical conversion yields of 71% were observed. Further exploration of other nitrogen-based nucleophilic activators such as DBU confirmed the beneficial effects of nitrogen bases. The use of guanidine derivatives TBD and MTBD further improved the radiochemical conversions to 81% and 76%, respectively. The authors reported a moderate molar activity of 8.4 GBq/μmol for the reaction. Meyer and co-workers reported a nucleophilic halex reaction to synthesize α-CF3 ketones [Prakash et al. demonstrated the radiolabelling of protected enols as a strategy for synthesizing [ ketones . The stretamides . The dir ketones . The rea18F]trifluoroacetate during the synthesis of 2,2,2-[18F]trifluoroethyl triflate [18F]CF3 compound to hydride reduction to obtain their target molecule. The final product  was obtained at a molar activity of 0.037 GBq/μmol. In 2020, Szpera and co-workers reported an improved synthesis of 2,2,2-[18F]trifluoroethanol [Johnstrom et al. reported the formation of ethyl [triflate . The autoethanol . The ove18F]CF3 labelling. Here, the CF3 group is coordinated to an Au(III) complex. The Au(III) complex facilitates the C-F bond cleavage and migratory insertion to generate an isolable R-CF2-metal complex, which in turn provides easy access to the [18F]CF3 moiety [3-containing radiotracers in 6% radiochemical yield and with a low molar activity in the range of 0.3 GBq/μmol. The feasibility of using bis(trifluoromethyl)Au(III) complexes for the isotopic exchange with [18F]fluoride was successfully demonstrated with the synthesis of cannabinoid agonist [18F]BAY 59-3074  and its subsequent analysis as a potential radiotracer for quantifying aggregated tau protein levels in Alzheimer’s disease (AD) and progressive supramolecular palsy (PSP) [18F]fluoride on a difluoromethylidene to generate the [18F]CF3 group  . The autF3 group . They obN-methyl lanosoprazole ([18F]NML), an analogue of lansoprazole iodonium triflates [18F]CF3 group.Additional examples of aliphatic [riflates . The nuc18F]pentafluoroethyl groups from hypervalent iodanes via an electrophilic radiofluorination reaction with [18F]XeF2 [18F]XeF2. The reaction of [18F]XeF2 with a cyclic perfluorinated iodane afforded a [18F]CF3CF2-containing compound with a radiochemical yield of 9%. A carrier-added radiosynthesis reported by Josse et al. achieved a molar activity of 2.7 GBq/μmol [18F]CF3 group. Cheguillaume et al. reported a strategy for synthesizing perfluorinated alkyl motifs to develop novel nitroimidazole-based hypoxia PET markers [18F]HF-pyridine followed by an additional DBH-activated fluorination step with an excess of HF-pyridine to yield [18F]CF3-containing compounds.Gruber et al. discussed the synthesis of [18F]XeF2 . This waGBq/μmol . The pro markers . In this18F]CF3 groups by a Lewis acid-mediated dechlororadiofluorination using [18F]fluoride and Sb2O3 [. This study resulted in the generation of [18F]CF3 groups at low molar activities and moderate radiochemical yields. Das et al. utilized the high reactivity of the benzylic position in various bromo-difluoromethyl aryl compounds for nucleophilic substitution reactions with n.c.a. [18F]fluoride. The resulting [18F]CF3-containing compounds were obtained in low radiochemical yields of 2–4% at a low molar activity (1.5 GBq/μmol) [18F]trifluoromethylation reaction via Ag(I)-mediated halogen exchange with aryl difluorobromides [18F]CF3 groups with a molar activity of 0.04 GBq/μmol. The examples of these halex reactions are summarized in Angelini et al. attempted the synthesis of [nd Sb2O3 . This stBq/μmol) . Authorsbromides . The rea18F]fluoride on difluoroarylbromide to yield the desired [18F]CF3 compound [18F]CF3 chemistry was part of a multi-step synthesis of a fluorine-18-labelled GABA uptake inhibitor. Hammadi et al. reported the nucleophilic displacement of a benzylic bromide to generate 4-chloro-α,α,α-[18F]trifluoromethyl toluene in moderate radiochemical yields (~30%) but low molar activities in the range of 1 GBq/μmol [18F]-(S)-fluoxetine. The authors observed that achieving higher molar activity depends on a delicate balance between temperature and precursor concentration. Prabhakaran et al. described the synthesis of [18F]celecoxib via direct nucleophilic substitution of the corresponding bromide [18F]celecoxib at moderate molar activities of 4.5 GBq/μmol, which was tested for PET imaging in baboons and rats  containing radiotracer [18F]fluoride was purchased from an external source, and it underwent significant decay  before the experiment. Authors hypothesized this as a reason for the overall low molar activity.The authors used a direct nucleophilic radiofluorination reaction with n.c.a. [compound . The desGBq/μmol . The flu bromide . The reaand rats . A compeiotracer . They sy18F and tested in PET imaging experiments  specific antagonist was labelled with eriments . The stueriments . The rad18F]CF3 groups 2], which in turn facilitates the cross-coupling reaction with aryl iodides or aryl boronic acids.Ivashkin et al. described a novel approach for the generation of [3 groups . The autium salt . [18F]fl18F]fluoroform via a two-step strategy involving [18F]fluoromethane  [18F]trifluoromethyl-l-tryptophan using Cu(I)-mediated [18F]trifluoromethylation to image the serotonergic system [18F]trifluridine in radiochemical yields of 3% and a molar activity of 0.4 GBq/μmol. Radiotracer [18F]trifluridine was used for subsequent PET imaging describing the biodistribution and clearance profile of the compound in a tumour-bearing mouse  . The chec system . The Cu-c system . The difng mouse .18F]trifluoromethylation reaction for the synthesis of [18F]PTTP, a radiotracer that targets purinergic receptor P2X7 (P2X7R). P2X7R is an emerging molecular target for tracking and visualizing inflammation [Fu et al. reported a [ammation .2CO2Me was used as the difluorocarbene source. The radiosynthesis afforded [18F]PTTP in 7% radiochemical yield and a molar activity of 0.35 GBq/μmol suitable for PET imaging , [18F]fluoride and an SO2 source. The ammonium salt of [18F]trifluoromethane sulfinate was used for radiolabelling with n.c.a. [18F]fluoride in the presence of a Fe(III) salt and TBHP as a stoichiometric oxidant. The chemistry tolerates water, which enables its direct use for the radiolabelling of peptides under aqueous conditions. The described method was particularly effective for the introduction of [18F]CF3 groups into tyrosine and tryptophan residues of the peptide backbone. A successful application of the methodology yielded octreotide[Trp(2-CF218F)] with a molar activity of 0.3 GBq/μmol , 0.15 GBq/μmol with a radiochemical yield of 20%.Verhoog et al. utilized [residues . The grolsulfide . The mol18F]Trifluoromethylation reactions with thiophenols and phenols were reported by Khotavivattana et al. [18F]fluoride on bromodifluoromethylphenylsulfide and bromodifluoromethoxybenzene, respectively. The reaction required highly active AgOTf. The authors reported a significant reduction in the reactivity of AgOTf that was stored at room temperature and performed the addition of AgOTf inside a glovebox. The process yielded [18F]trifluoromethylated arenes with low molar activities in the range of 0.5 GBq/μmol  [Liang et al. discussed an elegant synthesis route to access [e (PDFA) . The des18F]trifluoromethylthiolation of α-bromo carbonyl compounds [Liang group followed up with a modified method for using PDFA in the context of transition metal-mediated [ompounds . These n18F]Trifluoromethyl cysteine as an amino acid mimicking radiotracer for imaging glioma was explored by Tang et al. [18F]trifluoromethylthiolation reaction was achieved by employing PDFA and S8, on cyclic sulfamidates. The one-step assembly of [18F]trifluoromethyl cysteine represents an interesting method for the synthesis of the fluorine-18-labelled amino acid mimics that was tested for PET imaging in C6 glioma-bearing mice  and a base. This reaction provided the corresponding [18F]trifluoromethyl-sulfur/selenium compounds in low molar activity of 0.38 GBq/μmol.Jubault et al. reported a method for [uoroform  68]. [1. [118F]tme group . [18F]fl18F]fluoride in the presence of AgOTf gave compounds containing a [18F]CF3-S group in good radiochemical yields but a low molar activity in the range of 0.1 GBq/μmol.A novel reagent for bromodifluoromethylthiolation reactions was reported by Wu et al. . α-Cumyl18F]Trifluoromethylation chemistry is a rapidly evolving area in radiopharmaceutical sciences for PET imaging. Considered a challenging and exotic subset of 18F radiofluorination reactions in the past, [18F]trifluoromethylations are now becoming a more established radiofluorination technique in radiopharmaceutical chemistry. The increasing presence of CF3 groups in many drug molecules and the continuous growth of PET as a cutting-edge functional imaging technique in biomedical and clinical research are the primary drivers for developing novel [18F]trifluoromethylation chemistry strategies. In comparison with traditional radiofluorinations, there are unique challenges associated with [18F]trifluoromethylations. A major challenge is the low molar activity of <5 GBq/μmol of many [18F]CF3 group-containing PET radiotracers. Traditional radiofluorination methodologies with n.c.a. [18F]fluoride typically achieve high molar activities >40 GBq/μmol. There is only a handful of reported [18F]trifluoromethylation methods that can achieve that. The low molar activity challenge of many [18F]CF3 group-containing PET radiotracers is observed with the majority of the reported methods. However, some of the discussed four major synthesis strategies for the installation of a [18F]CF3 group could offer opportunities to obtain radiotracers at a high molar activity.[18F]trifluoromethylation chemistry starting from n.c.a [18F]fluoride is the most widely explored and successful method for [18F]trifluoromethylation reactions. However, the synthesis of difluoromethyl groups with a suitable leaving group can pose a special synthetic challenge associated with this strategy. Both aliphatic and aromatic nucleophilic [18F]trifluoromethylation reactions provide radiotracers in reasonable radiochemical yields and high molar activities in the range of 30–120 GBq/μmol. The obtained favourable radiochemical yields and high molar activities predestine nucleophilic [18F]trifluoromethylation reactions as a method of choice for the preparation of [18F]CF3 group-containing radiotracers suitable for PET imaging experiments.One-pot isotopic exchange reactions are quite limited when it comes to producing radiotracers at higher molar activities due to the inherent challenge of removing the chemically and physically identical labelling precursor. A two-step isotopic exchange radiofluorination involving an isolable metal complex yielded the radiotracer at a low molar activity of 0.3 GBq/μmol. Nucleophilic [18F]trifluoromethylation strategies have also rapidly evolved due to recent advances in designing and synthesising suitable difluorocarbene precursors. [18F]Trifluoromethylation reactions with difluorocarbenes always require the presence of a transition metal, mainly copper. Undesired side reactions such as defluorination or elimination of [18F]fluoroform are also possible with this method. Radiolabelling strategies with [18F]fluoroform as a [18F]trifluoro-methylating agent was also explored. Despite the operational complexity of [18F]fluoroform-based synthesis strategies, the resulting radiotracers were generally obtained in good radiochemical yields and moderate to high molar activities. Electrophilic [18F]fluorinating reagents are not as widely used and tested as to their nucleophilic counterparts for [18F]trifluoromethylations. Recent utilization of reagents such as [18F]Selectfluor that are capable of n.c.a. radiofluorinations offers an opportunity for improving the molar activity of the obtained radiotracers. Several [18F]trifluoromethyl transfer reagents such as [18F]Umemoto reagents are also significant compounds used for electrophilic [18F]trifluoromethylations. Alkyl trifluoromethyl labelling strategies with electrophilic [18F]fluorinating reagents proceed with moderate radiochemical yields and low molar activities. The increasing number of drugs containing trifluoromethoxy (CF3O-) and trifluoromethylthio (CF3S-) groups also require innovative radiochemistry for the synthesis of respective fluorine-18-labelled radiotracers. The increasing number of innovative [18F]trifluoromethylation chemistries is becoming a significant driving force for the development of novel fluorine-18-labelled imaging agents. Although the reports discussed in this review demonstrate the considerable progress in [18F]trifluoromethylation chemistry over the last decade, fluorine-18 radiochemistry is still heavily dominated by synthesis strategies yielding mono-radiofluorinated radiotracers  by using traditional nucleophilic and electrophilic radiofluorinations. Thus, further investments in the development of novel [18F]trifluoromethylation chemistry are required. The development of innovative [18F]trifluoromethylation chemistry will significantly impact future radiopharmaceutical chemistry and preclinical and clinical molecular imaging.Difluorocarbene-based [


We discuss possible reasons for the lack of efficacy of the three plant growth regulators under field conditions and comment on future approaches to manipulating yield in the light of the strong homeostatic mechanisms controlling endogenous cytokinin levels.Using plant growth regulators to alter cytokinin homeostasis with the aim of enhancing endogenous cytokinin levels has been proposed as a strategy to increase yields in wheat and barley. The plant growth regulators INCYDE and CPPU inhibit the cytokinin degrading enzyme cytokinin oxidase/dehydrogenase (CKX), while TD-K inhibits the process of senescence. We report that the application of these plant growth regulators in wheat and barley field trials failed to enhance yields, or change the components of yields. Analyses of the endogenous cytokinin content showed a high concentration of  Food producers face a range of challenges in addressing global food security in the 21st century. These include continuing growth in food consumption in developing nations ,4. IncreThe cytokinins are a plant hormone group involved in many aspects of growth and development, including root and shoot growth ,18,19, fN6 isoprenoid side chain or an aromatic side chain; and the synthetic diphenyl ureas. Briefly, isopentenyl transferase (IPT) catalyses the first committed step towards the formation of the isoprenoid cytokinins. The first formed cytokinins are the nucleotides that are converted by LOG (LONELY GUY) to the active free base forms, trans-zeatin (tZ), N6-isopentenyladenine (iP), cis-zeatin (cZ), and dihydrozeatin (DHZ), which are detected by a two-component signaling system. Cytokinin levels are controlled through destruction by cytokinin oxidase/dehydrogenase (CKX) or inactivation by cytokinin glucosyl transferase to O- or N-glucosides cZ, [13C5]tZ, [2H5]tZR, [2H5]tZ7G, [2H5]tZ9G, [2H5]tZOG, [2H5]tZROG, [2H5]tZMP, [2H3]DHZ, [2H3]DHZR, [2H3]DHZ9G, [2H7]DHZOG, [2H3]DHZMP, [2H6]iP, [2H6]iPR, [2H6]iP7G, [2H6]iP9G, [2H6]iPMP stable isotope-labelled standards  were added to each sample to validate phytohormone determination. Sample purification was carried out with mixed-mode cation-exchange (MCX) cartridges . Analytes were eluted by two-step elution using a 0.35 M NH4OH aqueous solution and 0.35 M NH4OH in 60% (v/v) methanol solution. The resulting eluate was subsequently evaporated to dryness and then dissolved in the mobile phase (15 mM ammonium formate pH 4.0 in 5% (v/v) methanol). LC–MS/MS analyses were carried out using a Acquity UPLC® System (Waters) and a triple quadrupole mass spectrometer XevoTM TQ MS (Waters). The mass spectrometry data was then processed utilizing MassLynx™ Mass Spectrometry Software with TargetLynx™ (Waters).Grains were ground under liquid nitrogen and freeze dried with a Savant™ SPD131DDA SpeedVac™ Concentrator (Thermo Fisher Scientific) to produce samples weighing between 8 to 22 mg. For each treatment, three replicates were prepared. Samples were then analyzed according to . Sample p ≤ 0.05, were determined between PGR treatments and the respective DMSO control using a two-way ANOVA. A logit transformation was made to protein composition data prior to ANOVA analysis. Similarly, statistically significant differences for LC–MS/MS data were determined between PGR treatments and the control using two-way ANOVA (significance level: 0.05), with a post hoc two-sided Dunnett test . To ensure the assumptions of the ANOVA were met, an examination of Q-Q plots of standardized residuals was made, and where necessary the equality of variances ensured through a Levene’s test and plot of standardized residuals and predicted values.For yield and protein composition from the field trials, the mean was generated using four replicates for each treatment and the data presented with standard errors. Statistically significant differences, where