The soil microbial communities were characterized using high-throughput sequencing of 16S and internal transcribed spacer (ITS) ribosomal RNA gene amplicons. The results showed that biochar combined with organic-organic fertilizer significantly improved soil properties  and soil enzymatic activities . Furthermore, soil bacterial relative abundance was higher in biochar and organic-inorganic treatments as compared to control plots and the most abundant phyla were Acidobacteria (40%), Proteobacteria (21%), Chloroflexi (17%), Planctomycetes (8%), Bacteroidetes (4%), Verrucomicrobia (2%), and Gemmatimonadetes (1%) among others. Among the treatments, Acidothermus, Acidibacter, Candidatus Solibacter and F473 bacterial genera were highest in combined biochar and organic-inorganic treatments. The lowest bacterial abundance and bacterial compositions were recorded in control plots. The correlation analysis showed that soil attributes, including soil enzymes, were positively correlated with Chloroflexi, Planctomycetes, verrucomicrobia, GAL15 and WPS-2 bacterial abundance. This study demonstrated that biochar with organic-inorganic fertilizer improves soil nutrients, enzymatic activities and bacterial abundance.Fertilizer management can influence soil microbes, soil properties, enzymatic activities, abundance and community structure. However, information on the effects of biochar in combination with organic-inorganic fertilizer after 3 years under pomelo orchard on soil bacterial abundance, soil properties and enzyme activities are not clear. Therefore, we conducted a field experiment with seven treatments, i.e., (1) Ck (control), (2) T1 (2 kg biochar plant Pomelo is a vital citrus fruit, and its farming has been steadily increasing worldwide in recent years . China iSoil microbes play a significant role in soil quality and agricultural production in the soil ecosystem , 2022. TBiochar is a carbon-rich product prepared by pyrolysis of biomass such as straw, sawdust, livestock and poultry manure, and edible fungus residues under anaerobic or low-oxygen conditions . StudiesTherefore, the effects of biochar application combined with organic-inorganic fertilizer under pomelo orchard after 3 years on soil bacterial abundance and community composition are scanty. This study aimed to evaluate the impacts of the combined application of biochar and organic-inorganic fertilizer on soil phytochemical properties, biochemical properties, soil bacterial abundance, and community structure under pomelo acidic soil.°C and the average annual precipitation is 1718.3 mm. The soil type of the experimental field was sandy loam and the primary soil properties of the experimental fields, i.e., pH, organic matter, available nitrogen, available phosphorus, available potassium and available magnesium, were 4.41, 15.4 g kg–1, 113.3 mg kg–1, 246.1 mg kg–1, 370.8 mg kg–1, and 30.2 mg kg–1, respectively. The tested pomelo trees were 20 years old, and the trees with more consistent growth with the same size were selected to carry out the test.A field experiment was conducted in Bilian Town , Yongjia County, Wenzhou City, Zhejiang Province, China. The experimental place’s climate is classified as a subtropical monsoon climate, in which the annual average temperature is 18.3°C. The pH, TN, TP, TK and OC of rice straw biochar was 8.68, 17.38 g kg–1, 3.67 g kg–1, 7.27 g kg–1, and 389.56 g kg–1. The organic-inorganic compound fertilizer is produced by Zhejiang Zhongci ecological fertilizer Co., Ltd. .The test biochar is produced from rice straw in a traditional kiln, where the temperature was between 500–600–1), (3) T2 (4 kg biochar plant–1), (4) T3 (2 kg organic-inorganic mixed fertilizer plant–1), (5) T4 (4 kg biochar + 1.7 kg organic-inorganic mixed fertilizer plant–1), (6) T5 (4 kg biochar + 1.4 kg organic-inorganic mixed fertilizer plant–1), and (7) T6 (4 kg biochar + 1.1 kg organic-inorganic mixed fertilizer plant–1). The experiment consisted of four replication and five plants in each replication. Biochar was applied in late February 2018. A 30 cm wide and 20 cm deep annular fertilization ditch was dug near the drip line. Biochar and organic-inorganic compound fertilizer were applied into the ditch and mixed with the soil. The annual fertilizer amount was nitrogen (N) 300 kg hm–2, P2O5 75 kg hm–2, K2O 112.5 kg hm–2, and organic-inorganic compound fertilizer was used for spring, summer, and autumn fertilization which account for 40, 30, and 30%, respectively of the annual total; biochar was only applied once during the experiment. Conventional orchard management was mainly based on artificial weeding, and disease-pest control with mancozeb, copper sulfate preparations, and abamectin.The experimental layout was a single-factor randomized block design with 7 treatments. The treatment combinations were as follows (1) Ck (control), (2) T1  °C. Then the mixture was measured for the produced glucose with a colorimetric method , and the quality of the DNA was checked by 1% agarose gel electrophoresis. Secondly, the bacteria DNA for soil sample was sent to Guangdong Meige Gene Technology Co., Ltd., for high-throughput sequencing. The analysis of soil bacteria high-throughput sequencing data is based on the cloud service of the Illumina MiSeq platform determined by Guangdong Meige Gene Technology Co., Ltd. The project number is MGWH20210317A1NZGQ-1N_A-20210610-9248.de novo technique  software . Under Uechnique . Followiechnique . At 97% echnique . The SILechnique . RDP assp-values was used to examine whether there were significant differences in the diversity indices or species richness among the plant rhizosphere soil samples. At a 97 percent level of OTU similarity, the rarefaction curves and rank abundance curves were generated. The similarity index of community structure among all the samples was determined using beta diversity analysis. Beta diversity was counted at the OTU level of genotypes using weighted UniFrac distances and visualized using principal coordinate analysis (PCoA). QIIME software (v1.8.0) was used to cluster and estimate the weighted UniFrac distance matrices, which revealed phylogenetic relationships among distinct communities as well as their abundance in all samples.An OTU-based analysis method was used to assess bacterial diversity in each sample from each plant . QIIME software (v1.8.0) was used to calculate OTU richness, Chao1, Simpson, and Shannon indices to measure the diversity index and species richness  among the genotypes for each sample, with a sequencing depth of 3%. ANOVA with 1 ; the sample hierarchical clustering and non-metric multi-dimensional analysis were performed using the UPGMA non-weighted group average method. SPSS venison 17 and EXCEL 2010 were used for statistical analysis of test data, and SigmaPlot 12.5  software was used to make charts.The experimental data analysis was carried out based on the cloud service provided by Guangdong Meige Gene Technology Co., Ltd. . Before data analysis, the data was leveled according to the minimum number of sample sequences. Among them, the diversity index  was calculated using Qime softwareBiochar combined with organic-inorganic fertilizer to soil significantly affected soil properties of pomelo orchard . ResultsThe application of biochar, organic-inorganic and mixed fertilizer significantly affected phosphate, urease, dehydrogenase (DHO) and nitrate reductase (NR) activities in pomelo orchard . ResultsCompared to Ck, biochar addition treatments T1, T2, T4, T5, and T6 significantly improved DHO activity by 27, 17, 24.90, 15.54, and 8.81%, respectively . SimilarAfter the screening, pre-clustering, and chimera removal, a total of 810,347 reads of good quality bacterial 16Sr RNA were recovered, with an average of 40517 reads per sample, resulting in 26 phyla, 49 classes, 92 orders, 111 families, and 204 genera. The unique numbers of OTUs in Ck, T1, T2, T3, T4, T5, and T6 were 840, 1055, 1036, 3807, 2234, 917, and 1236, respectively . Overall–1 (T3) decreased the relative abundance by 22% over the control treatment (Ck). The second most abundant bacteria were proteobacteria and were higher in organic-inorganic mixed fertilizer treatment (T3), ranging by 31.59% followed by Ck (21.35%) compared to other treatments. The lowest relative abundance of proteobacteria ranging by 17.38% was recorded in T4 (4 kg biochar + 1.7 kg organic-inorganic compound fertilizer) compared to all other treatments. Furthermore, Actinobacteria, Planctomycetes and Chloroflexi had maximum relative abundance, with range of 11.78, 11.78, and 4.38% in T4 and T3, respectively. Except for Acidobacteria and Proteobacteria, all other bacteria were higher in higher biochar rate than control.Acidothermus was the most abundant bacteria at genus level after others, not assigned and uncultured species, followed by Acidibacter, Candidatus Solibacter and FCPS473, the treatments showed that Acidothermus was highest in control treatment, followed by T4 and T5, while the lowest was recorded in biochar applied treatments (T2 and T3). In contrast, Acidobacteria species were increased in biochar applied soil compared to all other treatments. Furthermore, Candidatus Solibacter species were higher in control and T3, followed by T2 and T1, and the lowest was observed in T6.The bacterial species at the genus level as affected by different biochar and organic-inorganic compound fertilizers are presented in Alpha diversity indices were generated for each sample to examine treatment diversity . Alpha dFurthermore, the Shannon index was higher (5.74 ± 0.120) in T2 and was lowest (5.21 ± 0.539) in Ck. Simpson index was not significantly affected by biochar, organic-inorganic fertilizers treatments. Overall results showed that biochar application increased the alpha diversity indexes.Principal coordinate analysis was utilized to compare and contrast the similarities and differences in bacterial beta diversity across all treatments . AccordiCorrelation among soil properties, enzymatic activities and soil bacterial abundance were determined by R software using the core plot package . The resPomelo orchard’s soil is severely degraded due to many factors such as soil acidification, low organic matter content and poor soil management . FertiliSoil chemical properties such as pH, AK, AN, AP, and AM play an essential role in soil fertility and plant growth and development. In the present study, sole biochar and biochar combined with organic-inorganic fertilizer significantly improved soil pH compared to control and non-biochar applied treatments. The possible explanation for improving soil pH in biochar used soil might be due to biochar’s chemical properties; biochar itself has a high pH value, enhancing the pH of acidic soil. Similar results of improving soil pH by biochar application were reported by 2O emissions. The higher soil enzymatic activity in biochar treatments is supported by Soil enzymes such as phosphate, urease, DHO, NR and invertase activities are involved in several soil biochemical processes, for example, organic matter mineralization and other biogeochemical cycling of nutrients . In the –1 increased bacterial 16S rRNA gene copy numbers by 35–62%  were also positively correlated with soil bacteria and soil properties. These findings were intended to serve as a guide and foundation for improving soil in pomelo orchards using a combination of biochar and organic-inorganic fertilizer with good application potential.The original contributions presented in this study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.YS and DL designed the study and wrote the manuscript. QZ, XG, and IA performed the data analysis and revised the manuscript. QZ, IA, XG, FL, and SL performed the data curation. All authors listed have made a substantial, direct, and intellectual contribution to the work, and approved it for publication.


Dipsaci Radix, is verified to exert therapeutic effect on alleviating recurrent spontaneous abortion (RSA). However, due to the lack of relevant research, its molecular mechanism is still unclear. We retrieved targets for AS6 and RSA, and then used their overlapped targets for PPI analysis. In addition, we used GO and KEGG enrichment analyses, and molecular docking to investigate the anti-RSA mechanisms of AS6. Furthermore, we conducted in vitro experiments to validate the predictions of network pharmacology. Results showed that a total of 103 AS6-associated targets and 2084 RSA-associated targets, with 49 targets overlapped. GO enrichment analysis showed 845 significant biological processes like decidualization, while KEGG pathway enrichment analysis revealed 76 significant entries including 18 signaling pathways, which were closely linked to PI3K-Akt, HIF-1, TNF, IL-17, and VEGF signaling pathways, etc. Molecular docking findings verified that AS6 had tight link with the key targets including JUN, CASP3, STAT3, SRC, and PTGS2. Notably, in vitro experiments revealed that AS6 treatment could exert lower expressions of JUN, pro-CASP3, CASP3, STAT3, SRC, and PTGS2 in decidual cells compared with progesterone despite the expressions of STAT3, SRC, and PTGS2 with no significant difference, and mifepristone could interfere with the effects. In general, numerous targets and multiple pathways involve during the process of AS6 treatment against RSA. Moreover, our in vitro research first reported that AS6 may regulate the expressions of key targets  in decidual cells to promote decidualization, thus treating RSA.Asperosaponin VI (AS6), as the quality marker of  Recurrent spontaneous abortion (RSA) is a common reproductive endocrine disorder, which refers to consecutive spontaneous abortions happening twice or more before 20 weeks of gestation , 2. AccoDipsaci Radix, which is an important drug in Shoutai pill. Pharmacological studies in recent decades have shown that Dipsaci Radix has a variety of biological activities, including antiuterine contraction, antiinflammatory, antiaging, antiarthritic, antiosteoporosis, fracture healing, neuroprotection, and it has been verified to benefit Chinese women from miscarriages, serving as the preferred herb in clinical treatment  098.5 cells/ml. After 30 min, we removed nonadherent cells and replaced the medium after 48 h.Fresh decidua tissues were taken aseptically, washed in PBS to remove blood, cut into pieces, digested with trypsin-EDTA (0.25%) for 5–10 min, and digestion was stopped by adding DMEM/F12 medium containing 10% FBS. The cell clusters in the final digestion were extracted with a 23-gauge needle, filtered through a 200-mesh sieve, centrifuged at 2000 rpm for 5 min, resuspended in DMEM/F12 medium containing 10% FBS, and incubated in flask at 5 × 10We treated the decidual cells with a concentration gradient of progesterone (P), mifepristone (M), and AS6 for 12, 24, and 48 h. According to the manufacturer's protocol, we performed a cell counting kit 8 (CCK-8) assay to detect cell proliferation abilities using an optical density (OD) setting of 450 nm in the microplate reader .μL RIPA lysis buffer prepared with phosphatase inhibitor and protease inhibitor per dish (Beyotime). The protein bands were transferred to polyvinylidene fluoride membranes  by electrophoresis and wet transfer steps, closed with QuickBlock™ Western closing solution (Beyotime) at room temperature for 30 min, added primary antibody and incubated overnight at 4°C in a shaker, then added the corresponding secondary antibody and incubated in shaker at 24°C for 1.5 h. The antibody reactivity level was subsequently detected by gel imaging system (Bio-Rad). Finally, the grayscale values were quantitated using ImageJ software.We cultured decidual cells in 6-well plates and treated them with the specific concentration of progesterone, Asperosaponin VI with or without mifepristone according to CCK-8 screening results. After the treatment, the protein was extracted by adding 200 t-tests were used to compare two separate samples. One-way ANOVA was used for comparison of univariate samples between multiple groups. p-value <0.05 indicates statistical significance.All results were expressed as mean ± standard deviation. Student's We obtained a total of 103 AS6-associated targets. After data standardization by the UniProt database, we obtained AS6-related target proteins called as Gene symbols. AS6-associated structure and target results were shown in supplementary Tables A total of 2084 RSA-associated target proteins were retrieved. We took the overlap of AS6- and RSA-associated targets as OTPs, which included 49 overlapped targets, as shown in OTPs were imported into the STRING platform with the targets having no link to others hidden. We imported the PPI data into Cytoscape to draw PPI network in p-value set to 0.001. We got 845 items of biological process (BP). The top 20 items were shown in We finally got totally 76 items including 18 key signaling pathways listed in Among 21 core targets, the top five target proteins in terms of degree were chosen for molecular docking, including JUN, CASP3, STAT3, SRC, and PTGS2, respectively, which were considered as the key targets in the process of AS6 treating RSA. To verify how AS6 binds to the key targets, we adopted molecular docking using Autodock Vina to predict their docking interactions. Based on Based on Based on Based on Based on in vitro research. The concentrations of progesterone used in the study were 0 (control group), 5, 10, and 20 μmol/L. The results revealed that the progesterone concentration at 20 μmol/L exerted proliferative effect on the proliferation of decidual cells, which was selected for subsequent experiments  exerts essential effects on the maintenance of pregnancy, the declining level of which in blood in early pregnancy leads to necrosis of the decidua, thereby causing miscarriage . MifepriAccording to PPI network topology analysis of OTPs, we noticed that these targets were characteristics of decidualization, autophagy, angiogenesis, oxidative stress, inflammation, and endocrine-related proteins. We identified five key targets including JUN, CASP3, STAT3, SRC, and PTGS2, which are in close conjunction with AS6 according to molecular docking findings, indicating that they may be the key targets of AS6 in treating RSA.β, TNF-α, and IFN-γ in FTDCs, which can be mediated by the activation of JUN-related signaling [JUN (Transcription factor AP-1 subunit Jun), which is the mediator of trophoblast invasion, plays a critical role in decidualization , 31. It ignaling . NotablyCaspase-3 (CASP3) is an apoptosis-related gene, whose expression has close correlation with placental separation . Some stSignal transducer and activator of transcription 3 (STAT3) phosphorylation has a close relationship with embryo implantation and decidualization . RSA resα, IL-6, IL-1B, and TNF-α in a dose-dependent manner [Similar to PPI analysis, GO enrichment results show consistent results as demonstrated in t manner .in vitro by upregulating HIF-1α/VEGF pathway and can effectively promote the angiogenesis of regenerative tissues and promote wound healing in vivo [KEGG enrichment results revealed that PI3K-Akt, HIF-1, TNF, IL-17, and VEGF signaling pathways may exert regulatory functions on AS6 against RSA. Some studies have verified that the inhibition of PI3K-Akt signaling pathway can reduce trophoblast cell proliferation and migration . Moreove in vivo . So HIF- in vivo . Abnorma in vivo .in vivo and in vitro experiments are needed to confirm our findings.In summary, our results predict some potential therapeutic targets and pathways, providing reference for future studies on AS6 treatment against RSA. However, one limitation of this study is that further in vitro study first reported that AS6 may regulate the expressions of key targets in decidual cells including JUN, CASP3, STAT3, SRC, and PTGS2 to promote decidualization, thus treating RSA.Collectively, our results revealed that AS6 may treat RSA possibly by regulating numerous signaling pathways and targets related with decidualization, autophagy, blood vessel endothelial cell migration, angiogenesis, inflammatory response, oxidative stress, and immune response, etc. Moreover, our


StDnaJs were identified from a genome-wide survey, and their classification, phylogenetic relationships, chromosomal locations, and gene duplication events were further analyzed. Together with 20 StHSP70s and 7 StHSP90s previously identified in the potato genome, the global expression patterns of the members in 3 HSP families were investigated in 2 potato cultivars during Potato virus Y (PVY) infection using RNA-seq data. Of them, 16 genes  were significantly up- or downregulated. Further analysis using qRT-PCR demonstrated that 7 of the 16 genes  were remarkably upregulated in the potato cultivar ‘Eshu 3’ after PVY infection, implying their potential roles in the potato-PVY compatible interaction. Subsequent virus-induced gene silencing (VIGS) assays showed that silencing of the homologous genes of StDnaJ17, StDnaJ21, StHSP70-6, and StHSP90.5 in Nicotiana. benthamiana plants dramatically reduced the accumulation of PVY, which indicated the four genes may function as susceptibility factors in PVY infection. This study provides candidate genes for exploring the mechanism of potato-PVY compatible interaction and benefits breeding work aiming to produce new cultivars with the ability to grow healthily under PVY infection.HSP40 , HSP70, and HSP90 are major heat shock protein (HSP) families that play critical roles in plant growth and development and stress adaption. Recently, several members of the three HSP families were reported to be widely involved in the plant host-virus interactions. However, their global expression profiles and core members recruited by viruses are largely unknown. In this study, a total of 89  Solanum tuberosum L.) is the third-largest food crop after rice and wheat in terms of human consumption. It has become a significant strategic crop for poverty eradication, health promotion, and food security in developing countries [Potato virus Y (PVY), one of the top 10 plant viruses recognized in the world, is considered to be the most harmful virus affecting potatoes [Potato  were first identified as proteins induced by elevated temperatures in both prokaryotes and eukaryotes. However, HSPs also contribute to responses to other environmental stresses, including drought, cold, and salinity, and offer protection against pathogens ,6. PlantNicotiana tabacum (NtMPIP1) was demonstrated to interact with the movement protein of tobacco mosaic virus (TMV), and the silencing of this gene remarkably hindered the movement of TMV [N. benthamiana (NbDnaJ) could interact with Potato virus X (PVX) stem-loop 1 RNA and capsid protein and played negative roles in PVX replication and movement [N. benthamiana promote Potato virus A (PVA) infection by regulating viral capsid protein (CP) functions [Red clover necrotic mosaic virus (RCNMV), host HSP70 and HSP90 interacting with p27, a virus-encoded component of the 480 kDa replication complex on the ER membrane, are required for viral RNA replication [N. benthamiana HSP90 interacts with Tm-22, encoding a coiled coil—nucleotide binding site—leucine-rich repeat type resistance protein, and the silencing of HSP90 reduced the steady-state levels of Tm-22 protein and Tm-22-mediated resistance to TMV [Although HSPs are ubiquitous constitutive proteins induced by heat and other abiotic stresses, increasing evidence indicates that HSP members are widely involved in plant-virus interactions. For example, PVY infection induced the HSP signaling pathway and modulated the HSP response triggered by heat stress in susceptible hosts ,16, and t of TMV . Similarmovement . HSP70 aunctions , and furunctions . In Red lication . Furthere to TMV .DnaJ gene family has not been identified yet, although the HSP70 and HSP90 gene families have been characterized in this crop previously [DnaJ family in the potato genome through bioinformatic methods. Subsequently, the gene expression patterns of the members in the DnaJ, HSP70, and HSP90 gene families were investigated under PVY invasion using RNA-seq data and the qRT-PCR method, respectively. At last, the core HSP members involved in potato-PVY interaction were identified by VIGS in N. benthamiana. Our findings provide an overview of the core members in three major HSP families that possibly participate in PVY infection, which helps to explore the mechanism of potato-PVY compatible interaction, and benefits breeding work aiming to produce new cultivars with the capacity to grow healthily under PVY infection.The above findings indicate that HSP members should be conservative components recruited by viruses to facilitate their infection cycle. However, their global expression profile and the core members recruited by viruses are largely unknown in potato. Moreover, the potato eviously ,25. In thttp://solanaceae.plantbiology.msu.edu/pgsc_download.shtml, accessed on 2 January 2022) were used to construct the local database using BioEdit7.0 software. The Hidden Markov Model (HMM) profile of the J-domain (PF00226) was acquired from Pfam  and set up as queries to blast against putative potato DnaJ genes with e-value < 10−5. Additionally, the keywords “HSP40”, “J-protein”, and “DnaJ” were applied to blast against the PGSC database. After redundant sequences were removed, putative DnaJ sequences were analyzed by Pfam and Smart  to verify the conserved domains. All the non-redundant genes with high confidence were named S. tuberosum DnaJ (StDnaJ) according to their location on the chromosome.Potato genomic sequences obtained from Potato Genome Sequencing Consortium  for computation of the isoelectric points (PI), molecular weights (MW), and amino acid numbers. The intron numbers and chromosome locations of the StDnaJs were determined using the PGSC database. The potato DnaJ proteins were classified based on the composition of the domains predicted by Pfam and Smart.Newly identified StDnaJ amino acid sequences were submitted to EXPASY PROTOPARAM  at least 2 StDnaJs were identified within a size range of 100 kb; (2) the alignments of these DnaJs were observed to have a high coverage rate of the longer gene (≥70%); and (3) the identity of the aligned region was also greater than 70% [http://chibba.agtec.uga.edu/duplication/index/locus, accessed on 20 January 2022).The mapping of than 70% . The detN. benthamiana plants preserved in our laboratory were used in this experiment. ‘Eshu 3’ is a cultivar from China that is susceptible to PVYO while ‘Exploits’ from Canada possesses a temperature-dependent hypersensitive resistance to PVYO, and is susceptible to PVYO at high temperatures (30 °C) [N. benthamiana plants, which is the time when the cotyledons and the first 2–4 true leaves emerged, were used for virus-induced gene silencing [The potato cultivar ‘Eshu 3’ and ‘Exploits’ together with  (30 °C) . The potilencing .O-FL strain was used for inoculation in this study [O-FL isolate according to the method described previously [The PVYis study . The oneeviously . The plaN. benthamiana plants, PVYO-FL tagged with the green fluorescent protein (GFP) was cloned into the vector pCB301-2μ-HDV  to construct the recombinant PVYO-full-length cDNA clone . The recombinant binary plasmids were transformed into the Agrobacterium. tumefaciens strain GV3101 by electroporation. The agrobacteria were grown overnight at 28 °C with shaking and then resuspended with the MMA buffer  to OD600 = 0.5. In total, 3 middle-upper leaves of each plant were selected and injected with 0.5 mL of agrobacterium solution. The accumulation of PVY in the upper uninoculated systematic leaves was investigated at 8 dpi.For gene-silenced To fully understand the expression patterns of the 3 potato HSP families under PVY infection, the upper leaf samples from 2 potato varieties (‘Eshu 3’ and ‘Exploits’) during PVY infection were harvested with 3 biological replicates after the plantlets were treated for 15 days. Then, the expression patterns of the three heat shock protein families were investigated from RNA-seq data, which has been not published yet . TPM , and cloned into the TRV (tobacco rattle virus) RNA2 vector. The empty vector TRV RNA2 was set as the control. The pTRV1, pTRV2, and 7 new pTRV2 vectors were transformed into the A. tumefaciens strain GV3101 by electroporation, respectively. The mixture of Agrobacterium cultures containing pTRV1 and pTRV2  and pTRV1 and pTRV2-T-target genes  at OD600 = 0.5 was incubated for 3 h in the darkness at room temperature before inoculation. The gene silencing efficiency was tested by qRT-PCR (the primers are shown in Virus-induced gene silencing (VIGS) has been applied routinely in ignaling . To consStef1α (AB061263) and Ntactin (XM_016658880) genes were set up as internal controls. Quantitative real-time polymerase chain reaction (qRT-PCR) was carried out in an optical 96-well plate with a CFX Connect TM Real-Time System q-PCR machine . The reaction volume consisted of 5 μL of EVAGreen Express 2xqPCR MasterMix , 1 μL of cDNA, 0.3 μL of each primer (10 μM), and 3.4 μL of ddH2O. The PCR program was set as follows: 95 °C for 3 min, 40 cycles at 95 °C for 10 s, 55 °C for 15 s, and 72 °C for 20 s. Three technical replications were performed for each sample. The obtained data were dealt with the 2−ΔΔct method [The total RNA was isolated by the Plant Total RNA Kit  following the manufacturer’s protocol. The cDNA was synthesized by HiScript II Reverse Transcriptase . The gene-specific primers were designed by Primer Premier 5.0 . The Stet method . The enzt method .DnaJ genes, HMM and keywords searches were performed using the PGSC database. After the removal of redundant sequences, the maintained sequences were subjected to analysis by Pfam and SMART to verify the J-domain and other conserved domains. Finally, 89 sequences were assigned as S. tuberosumDnaJ (StDnaJ) genes and named StDnaJ01 to StDnaJ89 according to their location on the chromosome. The biological information of each StDnaJ is shown in To achieve an overview of the potato Based on the presence/absence of 3 characterized domains identified in StDnaJ proteins, the J-domain, zinc-finger domain (CXXCXGXG), and C-terminal domain, the StDnaJ proteins were divided into 4 groups  with 9, 8, 67, and 5 members, respectively . Group IStDnaJ genes were segmented into 6 subfamilies   . Sub-famStDnaJs were randomly mapped on 12 chromosomes, and most of them were identified on the proximate or distal ends of the chromosomes. Among them, in the order from more to less, 16 StDnaJs were located on chromosome 1; 11 on chromosomes 3; 10 on chromosome 4 and 5; 8 on chromosome 7; 7 on chromosomes 9 and 11; 5 on chromosomes 2; 4 on chromosomes 6, 8, and 12; and 3 on chromosome 10 (The 89 osome 10 .StDnaJ62 and StDnaJ63) were found on chromosome 7. This pair of StDnaJ genes was mapped proximally in distance and separated by less than one gene. Six segmentally duplicated genes were found. StDnaJ30 on chromosome 3 exhibited synteny with StDnaJ53 on chromosome 6. Similar duplication events were also discovered for StDnaJ50 on chromosome 5 and StDnaJ75 on chromosome 9, and StDnaJ55 on chromosome 6 and StDnaJ85 on chromosome 11  [StDnaJ, StHSP70, and StHSP90) during PVYO infection, the upper systematic leaves were collected from ‘Eshu 3’ and ‘Exploits’ in compatible interactions with PVY at 15 dpi and used for RNA-seq analysis (The potato cultivar ‘Eshu 3’ is susceptible to PVY (30 °C) ,35. To vanalysis .StDnaJs, 20 StHSP90s, and 7 StHSP90s were identified in this study and previous studies [StDnaJs, 12 StHSP90s, and 7 StHSP90s exhibiting the minimum expression level (TPM > 5) were included in the further analysis  in 2 potato cultivars. A few members were downregulated in one or two potato cultivars, for example, StDnaJ19 and StDnaJ61 were only downregulated in ‘Eshu 3’ while StDnaJ56 and StDnaJ72 were inhibited (>2-fold) in both cultivars  were significantly upregulated (>2-fold) in both potato cultivars  in both cultivars  were remarkably upregulated (>2-fold) or downregulated (>2-fold) under PVY infection. Subsequently, these genes were selected and their expression levels in response to PVY infection further confirmed in ‘Eshu 3’ using qRT-PCR  were significantly upregulated compared to the control (mocked). Therefore, we assumed that these seven genes possibly played a role in the potato-PVY interaction process.Based on the RNA-seq data, a total of 16 candidate genes  were associated with the accumulation of PVY, we performed VIGS assays by silencing their homologous genes in the model plant N. benthamiana, a method frequently used to verify the functions of candidate genes in Solanaceae, due to its difficulty in silencing genes by VIGS in potato crop. The homologous genes in N. benthamiana were identified based on the highest sequence similarity to the corresponding potato genes (N. benthamiana were used to generate the TRV2 VIGS vector.To identify whether the 7 significantly upregulated genes (to genes . Fragmenphytoene desaturase (PDS) gene led to plants being bleached at 10 days , NbDnaJ21-silenced (TRV: NbDnaJ21), NbHSP70-6-silenced (TRV: NbHSP70-6), and NbHSP90.5-silenced (TRV: NbHSP90.5) plants compared with the control (TRV: 00)  A, althouTRV: 00) B, and thTRV: 00) C. These DnaJ gene family has been identified in many species from bacteria to human, although the number of DnaJs varies in different species. However, this family had not previously been studied in potato. Therefore, a genome-wide analysis of the DnaJ/HSP40 gene family was performed in potato in this study, which characterized the protein structure, classification, chromosome location, and gene duplication events of a total of 89 DnaJ genes in potato  [S.cerevisiae, H. sapiens, and A. thaliana possess four groups of DnaJ genes (I to IV) [DnaJ genes were also classified into 4 groups (I to IV) (DnaJ genes in C. annuum were categorized into five groups (I to V) [DnaJ genes to perform various functions. In addition, group III DnaJ genes containing only a J-domain are the most abundant in all species, indicating that they may play more complex roles or participate in multi-biological processes.Besides the difference in the total number, the classification of  to III) ,38. S.cI to IV) ,39,40. AI to IV) . However(I to V) . The resStDnaJ genes were mapped randomly across 12 chromosomes, with most of them on the terminal regions of chromosomes. Despite the fact that the potato genome size was nearly 7-fold larger than that of A. thaliana, there were fewer DnaJ genes found in potato (89 genes) than in A. thaliana (120 genes) [A. thaliana. A total of four pairs of StDnaJ genes were involved in gene duplication events, including one tandem duplication (StDnaJ62 and StDnaJ63) and three segmental duplication events   [HSP70 gene family, three tandem duplication events and two segmental duplication events were found [It was reported that the gene family expansions, and genome evolutionary mechanisms, primarily depended on gene duplication events , which m0 genes) . The postDnaJ75) . Thereforonments . Our resectively . Moreovere found . The aboS. tuberosum L.), belonging to Solanaceae, is a well-known “friendly” host for PVY. This suggests that a series of host factors (susceptibility genes) in potato can facilitate the infection of this virus, which are largely unknown. Increasing studies have reported that members of the plant DnaJ, HSP70, and HSP90 gene families play important roles in the host-virus interaction process [StDnaJ06, StDnaJ17, StDnaJ21, StDnaJ56, StDnaJ63, StDnaJ69, StDnaJ71, StDnaJ73, StHSP70-1, StHSP70-6, StHSP70-10, StHSP70-17, StHSP70-18, StHSP70-19, StHSP90.2, and StHSP90.5) were differently expressed genes (DEGs) in 2 potato cultivars during PVY infection  belonged to group II and IV, respectively (StDnaJ69 shares 92% and 96% identity with IPKN. benthamiana P58 (IPKNbP58) and tomato IPKP58 (IPKLeP58) at the nucleic acid level, respectively. P58IPK contains nine tetratricopeptide repeats (TPRs) arranged in tandem at the N terminus and a J-domain at the C terminus, which is conservative in plants and animals [IPK can interact with the TMV-P50 and TEV-helicase and functions as a susceptibility gene required for virus virulence [IPK is recruited by the influenza virus and Hantaviruses to combat the protein kinase R (PKR)-mediated innate antiviral response [Pepino mosaic virus (PepMV) [NbHSP70C was reported to be inhibited by a natural antiviral agent (quercetin) in tobacco to disturb the replication of TMV [Potato  , and recn of TMV .StDnaJ06, StDnaJ17, StDnaJ21, StDnaJ63, StHSP70-6, StHSP70-19, and StHSP90.5) were significantly induced in ‘Eshu 3’ under PVY infection  dramatically reduced the accumulation of PVY in N. benthamiana plants , a chloroplast-replicating viroid inducing clear bleaching-type chlorosis [HSP90 knockdown on the resistance or susceptibility to the viroid in the transgenic plants. Our results of the VIGS assays indicated that NbHSP90.5, together with NbDnaJ17, NbDnaJ21, and NbHSP70-6, might be recruited as susceptibility factors to facilitate PVY infection, which partially supported our above hypothesis. Although NbDnaJ06, NbDnaJ63, and NbHSP70-19 were markedly induced by PVY infection, the silencing of these genes did not significantly suppress or promote PVY accumulation in the VIGS assays. Our data indicate that these genes may not be indispensable for PVY accumulation. Nevertheless, this result cannot exclude the possibility of these genes being involved in PVY infection since knockdown of these genes by RNA interference is incomplete compared with knockout by the CRISPR/Cas9 systems. Clearly, the functions and underlying mechanisms of these HSPs involved in the host-virus interactions need to be clarified by further study.Further qRT-PCR analysis on the above differently expressed genes revealed that seven HSP genes (nfection , and thea plants , suggestaperones . In Arabaperones ,50,51,52aperones ,49, althGS assay . In the hlorosis . Togethehlorosis . UnfortuStDnaJ genes were identified in the potato genome in this study. A series of analyses of this gene family, including their classification, phylogenetic relationships, chromosomal locations, and gene duplication events, were carried out using bioinformatics methods. Moreover, the RNA-seq data and qRT-PCR analysis demonstrated that StDnaJ06, StDnaJ17, StDnaJ21, StDnaJ63, StHSP70-6, StHSP70-19, and StHSP90.5 were significantly upregulated during the PVY infection process, implying their potential roles in the potato-PVY compatible interaction process. Further, the knockdown of the homologous genes of StDnaJ17, StDnaJ21, StHSP70-6, and StHSP90.5 in N. benthamiana by VIGS dramatically reduced the accumulation of PVY, which indicates that the four genes may function as susceptibility factors in the PVY infection process. Our work provides candidate genes that possibly participate in the plant-virus interactions in the three major HSP families, which helps to understand the mechanism of potato-PVY compatible interaction, and benefits breeding work aiming to produce new cultivars with the capacity to grow healthily under PVY infection.In summary, a total of 89


In the current study, we characterized how acute myeloid leukemia (AML) promotes the generation of senescent-like CD8We analyzed NanoString, bulk RNA-Seq and single-cell RNA-Seq data from independent clinical cohorts comprising 1,896 patients treated with chemotherapy and/or immune checkpoint blockade (ICB).+ T cells were impaired in killing autologous AML blasts and that their proportion negatively correlated with overall survival (OS). We defined what we believe to be new immune effector dysfunction (IED) signatures using 2 gene expression profiling platforms and reported that IED scores correlated with adverse-risk molecular lesions, stemness, and poor outcomes; these scores were a more powerful predictor of OS than 2017-ELN risk or leukemia stem cell (LSC17) scores. IED expression signatures also identified an ICB-unresponsive tumor microenvironment and predicted significantly shorter OS.We show that senescent-like bone marrow CD8The IED scores provided improved AML-risk stratification and could facilitate the delivery of personalized immunotherapies to patients who are most likely to benefit.ClinicalTrials.gov; NCT02845297.John and Lucille van Geest Foundation, Nottingham Trent University’s Health & Wellbeing Strategic Research Theme, NIH/NCI P01CA225618, Genentech-imCORE ML40354, Qatar National Research Fund (NPRP8-2297-3-494).  Acute myeloid leukemia (AML) is a molecularly and clinically heterogeneous disease . We rece+ T cell infiltration has been shown to correlate inversely with OS in select tumor types, including AML, because of the establishment of highly dysfunctional T cell states , CTLA4, HAVCR2 (encoding TIM3), CD160, and 2B4 (encoding CD244), and display an impaired ability to secrete effector cytokines and to exert cytotoxic functions. Senescent T cells downregulate costimulatory molecules CD27 and CD28, express senescence-associated surface markers B3GAT1 and KLRG1, as well as MAPK p38 and γ-H2AX intracellular molecules, remain metabolically active and continue to secrete proinflammatory cytokines  in combination with immune checkpoint blockade (ICB) with pembrolizumab  (designated as AZA+Pembro). We integrated these with publicly available gene expression data from multiple cohorts of children and adults with AML to validate our RNA metric of immune effector dysfunction (IED), and we analyzed BM samples collected longitudinally at the time of AML onset and response assessment (https://doi.org/10.1172/JCI159579DS1). The derived gene signatures of IED correlated with molecular features of leukemia stemness and with distinct clinical characteristics. IED gene sets served as a reliable biomarker to stratify OS after standard-of-care therapy and ICB, both in AML and in melanoma   , 17.+ T cells in both experimental conditions. Consistent with previous observations  and nonsenescent (CD3+CD8+CD57–KLRG1–) T cells against autologous AML blasts using an anti–CD3/CD33 bispecific T cell engaging (BiTE) antibody construct (+CD8+CD57+KLRG1+) T cells in baseline BM samples was associated with significantly worse OS (P = 0.004) after treatment with standard chemotherapy (+CD8+CD57+KLRG1+ T cells = 31.9%). Senescent-like T cells measured at time of response assessment in 22 patients from the JHU1 cohort who achieved a CR also correlated with shorter OS  previously shown to be expressed by circulating CD8+ T cells from patients with AML  and correlated the expression of genes in the immunosenescence signature with markers of immune cells and leukemia blasts.Transcriptional profiling of the tumor microenvironment (TME) has been used to identify immunological signatures, characterize biological processes, and develop predictors of protective immunity , 26. We with AML  and to bwith AML  and on dwith AML . We usedCD34, CD38, IL3RA, KIT), or with markers of accessory cells of the monocyte/macrophage lineage  gene sets within the BM microenvironment might reveal core biological processes involved in antitumor immune responses and in therapeutic outcomes. To this end, both TCGA-AML and Beat-AML cases were split into quartiles based on average expression levels of the 7 T cell senescence-associated genes. Gene set enrichment analysis (GSEA) was used to identify core gene sets accounting for the enrichment signal in immunosenescencehi (highest quartile) versus immunosenescencelo cases (lowest quartile). Among the 4,872 curated gene sets from the ImmuneSigDB, only gene sets with a FDR of less than 0.05 and a normalized enrichment score of more than 2.0  were carried forward for leading-edge analysis. We reasoned that those genes contained in the leading edge would represent biologically related genes enriched for a phenotype of interest , dysfunction and/or exhaustion    , and sennd ETS1) . We herend ETS1) , solid tnd ETS1)  34, 35), 35CXCR3nd ETS1)  2). The. TheCXCRnd ETS1) . Furthernd ETS1) , as wellnd ETS1) –39 (Suppnd ETS1) , B and Cnd ETS1) –43, we fnd PD-L1 . A princnd PD-L1 .https://www.leukemia-net.org/home/) risk category, or mutation count  revealed significantly higher IED172 scores at baseline in patients with primary induction failure  compared with those achieving complete remission . We. We46). on count , A and Bon count  or in thutations . These fated AML , 48 and ated AML . The anaTP53 and RUNX1 mutational status in de novo AML  score has previously been associated with poor clinical outcomes and with novo AML , 52. Thenovo AML , A and Bnovo AML , immune novo AML  53), an, anTP53 novo AML . This finovo AML  54). Wh. WhTP53 edict OS .FLT3-ITD and NPM1-mutational status at diagnosis  statistical method to fit an L1-regularized linear model  that reviagnosis . On straividuals , 30 wereividuals . High PIAs shown in + and NK cells, characterized by the expression of inhibitory molecules, and inversely correlated with leukemia burden  and the same weights as those provided in the original publication , A and Bhi cases , C–F. SpS of 11% , were fuy of 55% . Furtherectively , G–J.2 statistics (P < 0.001) in this modeling framework than the LSC17 score (P = 0.001). In addition, the interaction between the 2 continuous variables was statistically significant (P = 0.013), indicating that a higher PI20 value will increase the association between the LSC17 score and OS. Taken together, these analyses suggest that the PI20 score and its integration with the LSC17 score could provide accurate prognostic risk stratification.We formally tested the interaction between senescence- and stemness-related pathways by a multiplication term in the Cox PH model. As shown by the Wald χatistics , the PI2n = 31 paired BM samples; P = 0.0044) and OS   . In lineE134589)  and in BE134589) . FinallyE134589) . These dP = 0.011; P = 0.0015; To further examine the effect of induction chemotherapy on IED scores, we generated nCounter gene expression data using serial BM samples from a large number of donors with newly diagnosed AML (the Studien Allianz Leukämie [SAL] and Johns Hopkins University 2 [JHU2] cohorts), totaling 90 patients and 183 BM specimens that were longitudinally collected at time of diagnosis and response assessment) . The IEDCD3G, CD8A, CD8B, CD28, GZMK, and GZMB), cosignaling molecules , myeloid  and dendritic cell differentiation , and in chemoattraction  after chemotherapy , leukemia stem cells , and candidate genes overexpressed in AML  were downregulated after chemotherapy, consistent with recent scRNA-Seq and immunohistochemistry data  in the PIlo group, prompting us to examine the ability of a published IFN signature to predict OS . The analysis of scRNA-Seq data from 8 patients with relapsed/refractory AML treated with AZA + nivolumab ; ivolumab  confirmeecursors . In lineecursors . Compare8 scores . Taken tGZMA, GZMB, PRF1, KLRD1, and NCR1), T cell and NK cell cosignaling molecules , cytokine receptors , IFN responsiveness (ISG20), and T cell signaling  . We identified 43 genes that were significantly differentially expressed in both postchemotherapy and post-AZA+Pembro BM samples (We sought to identify genes at the intersection of responses to chemotherapy and AZA+Pembro. We examined DEGs between matched posttreatment (available in 31 patients after cycle 2) and pretreatment BM samples in the immunotherapy cohort. Treatment with AZA+Pembro resulted in upregulation genes associated with immune effectors (d ZAP70) . As withd ZAP70) , AZA+Pem samples , and we  samples . This pr samples .lo cases, patients in the PI24hi group had lower numbers of lymphocyte clusters and tumor infiltrating lymphocyte (TIL) patches and higher myeloid/macrophage RNA scores  . The PI2A scores . Further subtype ; P = 0.0n = 41) or combination anti-PD-1 + anti-CTLA-4 , and had significantly higher PFS and OS rates  from pretherapy lesions of nonresponders, i.e., patients with progressive or stable disease, compared with responders  and were found to be associated with leukemia stemness and with poor response to induction chemotherapy. OS prediction afforded by validated clinical cytogenetic categories and experimental LSC17 signatures  and associated clinical data were obtained with written informed consent from the donors in accordance with the Declaration of Helsinki on research protocols approved by the Ethics Committee of TU Dresden and Studienallianz Leukämie, Germany (EK98032010), and by the Institutional Review Boards of the Children’s Hospital of Philadelphia (no. 10-007767) and Johns Hopkins University.S Rutella and LL conceptualized and designed the study. S Rutella, JV, FM, TY, RM, S Reeder, VR, and LL developed the methodology. RM, HA, MK, HAK, JFZ, AA, BW, HAA, MDM, SKT, MB, IG, and LL acquired, obtained consent from, and managed patients, as well as processing patient samples. S Rutella, JV, FM, S Reeder, TY, RM, BD, BRB, VR, BW, HAA, MDM, SKT, MB, IG, and LL analyzed and interpreted data. S Rutella wrote the manuscript. S Reeder, JV, FM, RM, BD, BRB, JFZ, SKT, MB, IG, LL reviewed and/or revised the manuscript. S Rutella and LL supervised the study.


Substance use disorders (SUDs) are chronic, lifelong disorders that have serious consequences. Repeated substance use alters brain function. G-protein-activated inwardly rectifying potassium (GIRK) channels are expressed widely in the brain, including the reward system, and regulate neuronal excitability. Functional GIRK channels are identified as heterotetramers of GIRK subunits (GIRK1–4). The GIRK1, GIRK2, and GIRK3 subunits are mainly expressed in rodent brain regions, and various addictive substances act on the brain through GIRK channels. Studies with animals  and humans have demonstrated the involvement of GIRK channels in the effects of addictive substances. Additionally, GIRK channel blockers affect behavioral responses to addictive substances. Thus, GIRK channels play a key role in SUDs, and GIRK channel modulators may be candidate medications. Ifenprodil is a GIRK channel blocker that does not have serious side effects. Two clinical trials were conducted to investigate the effects of ifenprodil in patients with alcohol or methamphetamine use disorder. Although the number of participants was relatively low, evidence of its safety and efficacy was found. The present review discusses the potential of GIRK channel modulators as possible medications for addiction. Therapeutic agents that target GIRK channels may be promising for the treatment of SUDs. Substance use disorders (SUDs) are characterized by compulsive drug use despite clinically significant distress and other negative consequences in life. Substance use disorders alter how the brain and body respond to addictive substances, and patients with SUDs suffer from an inability to quit addictive substances. Addictive substances comprise natural, semi-synthetic, and synthetic substances, such as amphetamine/methamphetamine, cocaine, opioids, cannabinoids, alcohol, hypnotics/anxiolytics, inhalants, nicotine, and caffeine. Addictive-substance misuse and problems that are associated with SUDs have been a serious societal concern worldwide. In 2022, the United Nations Office on Drugs and Crime reported on opioid-related overdose deaths in North America and Canada, the distribution of quantities of heroin and morphine seized in the Balkan route , expansion of methamphetamine production in Afghanistan, distribution of quantities of cannabis resin seized, and trafficking in North Africa, Western and Central Europe, and South-West Asia . HoweverKcnj gene and assembled in homotetrameric or heterotetrameric units comprising four subunits: GIRK1 (Kir3.1/Kcnj3), GIRK2 (Kir3.2/Kcnj6), GIRK3 (Kir3.3/Kcnj9), and GIRK4 (Kir3.4/Kcnj5) [GIRK channels are encoded by the GIRK1 Kir.1/Kcnj3,4/Kcnj5) ,8. Three+ selectivity, inward rectification, and G-protein-dependent gating [+ currents through GIRK channels inhibit cellular excitability [N-methyl-D-aspartate (NMDA) receptors, nicotinic acetylcholine receptors, adenosine receptors, and opioid receptors, as well as dopamine transporters [i/o family of G-proteins to those receptors and gated by G-protein βγ subunits that are released from the G-protein α subunit [GIRK2 subunits can uniquely form functional homomers in the brain . These vt gating . Outwardtability . The cortability . Dopamintability . Dopamintability ,6. Addictability . Activatsporters ,17,18,19 subunit ,21  [50 values: 7.01 ± 0.92 μM at GIRK1/GIRK2, 8.76 ± 1.26 μM at GIRK2, and 2.83 ± 0.69 μM at GIRK1/GIRK4 [1-adrenergic receptors, GluN2B subunit-containing NMDA receptors [50 than GIRK channels  [Although GIRK channels are activated in a G-protein-dependent or -independent manner by addictive substances, antipsychotic compounds, psychoactive compounds , and a cerebral circulation/metabolism ameliorator were shown to block brain-type GIRK1/GIRK2 channels and cardiac-type GIRK1/GIRK4 channels in the on assay ,45,46,47on assay ,45,46,47oxapine) ,45,46,47K1/GIRK4 ,49. Howeeceptors ,52, and eceptors , and alseceptors . Ifenproeceptors , and preeceptors . Ifenproeceptors . Pretreaeceptors . Notablys above) . A recens above) . These ss above) . GIRK chIfenprodil has been used as a treatment for dizziness after brain ischemia <60 mg/day) in a few countries, including Japan and France. A high dose of ifenprodil  is used as an analgesic in patients with cancer in Japan . Ifenpro mg/day iGoto reported that ifenprodil diminished pain in the extremities, headache, and tremors in the fingers in patients with alcohol use disorder . In a re®, cough medicine, and alcohol) [n = 10), 60 mg/day ifenprodil group (n = 11), and 120 mg/day ifenprodil group (n = 11). The primary outcome was the use or nonuse of methamphetamine during the drug administration period in the placebo group vs. the 120 mg/day ifenprodil group (see our previous studies for secondary outcomes). In this clinical trial, we did not find effects of ifenprodil on the primary or secondary outcomes [B receptor, was shown to modulate the kinetics of GIRK channels, resulting in rapid desensitization [In the second clinical trial, the effects of ifenprodil were examined in patients with methamphetamine use disorder ,67. Methalcohol) . The admoutcomes ,67. The outcomes ,67. Impooutcomes . ML297 (outcomes . V052933outcomes . Crystaloutcomes . In 2019tization . These sSubstance use disorders are chronic diseases. Patients with SUDs and associated societal problems exist worldwide, including in Japan . The FDA1-adrenergic receptors, GluN2B-containing NMDA receptors, and sigma-1/2 receptors. Future studies should clarify the mechanisms by which ifenprodil acts to treat SUDs. Understanding the molecular and structural bases by which addictive drugs act on GIRK channels and how they change their gating and conformation properties will provide new insights into the development of novel treatments for SUDs.GIRK channels are essential mediators of cellular excitability in the central nervous system and are involved in the rewarding effects of addictive substances. Previous studies with GIRK channel knockout and mutant mice confirmed that GIRK channels are involved in regulating behavioral responses to addictive substances. Our two clinical trials suggest that ifenprodil might be effective for patients with alcohol use disorder and methamphetamine use disorder, without causing severe adverse events. Ifenprodil is not a selective GIRK channel blocker; it also inhibits α


Triple negative breast cancer (TNBC) is the most aggressive subtype of breast cancer, with a higher mortality rate when compared to the other subtypes. Because of the absence of a molecular target and the heterogeneity of TNBC molecular subtypes, the development of targeted therapies is limited. Protein tyrosine kinases (TKs), in particular, have emerged as important molecular targets and biomarkers in cancer. Thus, we identified the signature TK of individual TNBC subtypes by analyzing RNA-seq-based transcriptome data of TNBC patients from The Cancer Genome Atlas database and validated this finding against other TNBC patients and cell line datasets. Our discovery of the signature TK for TNBC subtypes can be used as actionable targets for anti-cancer therapies, as well as subtype-specific biomarkers for TNBC.LYN, CSF1R, FGRF2, and SRMS, respectively. These findings could serve as a potential biomarker of specific TNBC subtypes, which could lead to an effective treatment for TNBC patients.Triple negative breast cancer (TNBC) shows impediment to the development of targeted therapies due to the absence of specific molecular targets. The high heterogeneity across TNBC subtypes, which can be classified to be at least four subtypes, including two basal-like , a mesenchymal (M), and a luminal androgen receptor (LAR) subtype, limits the response to cancer therapies. Despite many attempts to identify TNBC biomarkers, there are currently no effective targeted therapies against this malignancy. In this study, thus, we identified the potential tyrosine kinase (TK) genes that are uniquely expressed in each TNBC subtype, since TKs have been typically used as drug targets. Differentially expressed TK genes were analyzed from The Cancer Genome Atlas (TCGA) database and were confirmed with the other datasets of both TNBC patients and cell lines. The results revealed that each TNBC subtype expressed distinct TK genes that were specific to the TNBC subtype. The identified subtype-specific TK genes of BL1, BL2, M, and LAR are  Breast cancer is a heterogeneous group of diseases. Expression of hormone receptors, such as estrogen receptor (ER) and progesterone receptor (PR), or human epidermal growth factor receptor 2 (HER2), can indicate the probability of responsiveness to therapies targeting these receptor proteins. Triple negative breast cancer (TNBC), which is approximately 15–20% of all breast cancer cases, shows an impediment to the development of targeted therapies due to the absence of molecular targets . Each suA potential target that is generally used for the prognostic marker, as well as cancer therapy, is a protein kinase because of its association with human cancer initiation and progression ,10. AmonOver the past three decades, the FDA has approved at least 24 new therapeutic drugs for breast cancer, including 18 small molecules, three monoclonal antibodies (mAbs), and three antibody-drug conjugates (ADCs) ,22,23. EAlthough TKs are commonly activated in cancers and serve as important targets for anti-cancer therapies, as well as predictive markers for therapeutic response , there ahttps://xenabrowser.net/, accessed on 17 July 2022). The transcripts per million (TPM) RNA-seq data of TNBC patients were selected based on the TCGA-BRCA project [http://cbc.mc.vanderbilt.edu/tnbc/, accessed on 17 July 2022), based on the highest positive centroid correlation and p-value on normalized mRNA expression of TNBC samples. In total, 183 TNBC samples were assigned to either BL1 (64 samples), BL2 (37 samples), M (54 samples), or LAR (28 samples) subtypes. We performed analysis on publicly available transcriptomic data from TCGA  . The criteria to select the DE gene specific to each TNBC subtype was set as |log2FC| ≥ 1, where upregulated and downregulated genes were defined as the genes with log2FC ≥ 1 and log2FC ≤ −1, respectively. We also employed Venn, the online tool , to generate a Venn diagram to show the relation of DE genes among TNBC subtypes.Differential gene expression analysis of RNA-seq data, excluding long noncoding RNAs (lncRNAs), was performed using R package DESeq2 (version 1.30.1) . To idenp-value < 0.05 and log2FC ≥ 1) were analyzed using the functional annotation tool, that is, the Database for Annotation, Visualization, and Integrated Discovery  [p-values < 0.05 were selected for further analysis.To further investigate DE genes in each TNBC subtype, GO enrichment analysis and KEGG pathway analyses were performed. Upregulated DE genes from each TNBC subtype , which is a web server application that infers overrepresentation of upstream kinases of which putative substrates are in a user-inputted list of proteins [http://www.kinhub.org/kinmap/, accessed on 13 August 2022) [Upregulated DE genes in each TNBC subtype were converted into protein. Then, these proteins were used as the input to Kinase Enrichment Analysis 3, KEA3 (proteins . The kinst 2022) .Other publicly available transcriptomic data of TNBC patients and cell lines from the following datasets were analyzed to validate the TCGA results. The Clinical Proteomic Tumor Analysis Consortium (CPTAC) dataset  includesy-axis) and 1–specificity (x-axis). In addition, the area under the ROC curve (AUC), which ranges from zero to one (one indicating perfect performance), was used to represent the overall performance of identified TK genes as signature genes to classify TNBC subtypes.To validate the results, the mRNA expression z-score of TK genes of interest in each dataset was used to compare the expression across all TNBC subtypes. Furthermore, the receiver operating characteristic (ROC) curve of those TK genes was used to illustrate the performance of identified TK genes in classifying the TNBC subtype from TNBC patient and cell line datasets. The ROC curve is made up of sensitivity . The statistical details of all experiments were reported within the text, in the figure legends, and in figures, including statistical analysis performed and statistical significance.The TCGA RNA-seq dataset of TNBC patients was analyzed with DESeq2 to identify DE genes ,29. Usinp-value < 0.05 in BL1, BL2, M, and LAR, respectively. Among these, there were 138 (BL1), 341 (BL2), 344 (M), and 966 (LAR) upregulated genes, and 970 (BL1), 341 (BL2), 1020 (M), and 1043 (LAR) downregulated genes  and the Janus family . In LAR, the kinases of the PTK6 family, including SRMS and PTK6 , were found to be upregulated, followed by BMX from the Tec family. On the other hand, upregulated kinase genes of the SRC family, such as LYN and SRC, were detected in BL1 and M, respectively.The log2FC distribution of NRTK genes revealed differences among TNBC subtypes B. EighteEPHA4 overlapping between BL2 and M. In BL1, only EPHA10, which is a class of Eph receptor RTKs, was found to be upregulated. In BL2, the top-upregulated kinase gene with the highest FC was CSF1R. Several RTKs of Eph family dominantly involved in BL2 in both upregulation  and downregulation . Furthermore, EGFR was found to be significant in BL2. A variety of RTKs from different families, such as the Eph, DDR, FGFR, and NTRK families, were positively or negatively involved in the M subtype. In addition, EPHA7, EPHB1, EPHB3, FGFR1, FGFR2, FGFR3, IGF1R, and NTRK3 were found to be upregulated, especially EPHA7 and NTRK3, which showed the highest FC in M but the lowest in the BL2 and LAR subtypes. In LAR, seven RTKs, e.g., ERBB2, ERBB4, FGFR4, FLT3, LMTK3, RET, and TEK, were upregulated where ERBB4 and FGFR4 had the highest FC compared to the rest of TNBC subtypes. We also found that the expression of ERBB2 (or HER2), which is typically used to classify one of the major types of breast cancer, was found to be upregulated in LAR.Similar to NRTK, changes in RTK gene expression levels in each TNBC subtype were also unique C. ThirtyEPHA10, LYN, and PTK2 for BL1; CSF1R, EPHB2, and TXK for BL2; EPHA1, FGFR2, and NTRK3 for M; ERBB4, FGFR4, and SRMS for LAR. These identified TKs could potentially be subtype-specific signature TK genes of the particular TNBC subtype.Combining TK gene expression results from both NRTKs and RTKs, the top three TKs with the most significant and the highest FC in each subtype were To infer the potential upstream kinases in accordance with the upregulated DE genes in each TNBC subtype, we performed kinase enrichment analysis using KEA3 . SearchiTo further investigate the relation of TKs on different subtypes of TNBC, only the top 10 TKs were investigated A,B. We fThe most enriched kinases in each TNBC subtype were relatively unique with a small number of TKs overlapping between any two subtypes C. In BL1Comparing the results of predicted upstream kinases and subtype-specific TK genes of the TNBC subtype, we found that 10%, 40%, 60%, and 50% of TKs inferred by KEA3 were identity to the subtype-specific TK genes of the BL1, BL2, M, and LAR subtype, respectively. These overlapping TKs could also be used as the subtype-specific signature TKs of each TNBC subtype.p-values < 0.05 were selected for further analysis. The top 10 pathways for KEGG and GO analyses were displayed in To investigate the unique biological functions of DE genes, functional enrichment analyses were performed using KEGG and GO analysis on unique and upregulated DE genes A. Only tKEGG pathway analysis of DE genes revealed differences between TNBC subtypes B. The baGO analysis, including biological process (BP), cellular component (CC) and molecular function (MF) analysis, revealed distinct characteristics of each TNBC subtype . BP analTo validate the potential signature TKs for each TNBC subtype, we compared our findings to the RNA expression of TKs in other TNBC patient and cell line datasets. Three datasets are from TNBC patients, including CPTAC (27 patients), MET500 (39 patients), and METABRIC (325 patients). Another dataset is from CCLE, including 20 TNBC cell lines. These datasets were then subcategorized into BL1, BL2, M, and LAR, according to previous reports ,41. Two EPHA10, LYN, and PTK2, could possibly be involved in this subtype since their RNA expression was the highest in BL1. Furthermore, the z-score of the top three kinases, including TXK, EPHB2, and CSF1R for BL2, NTRK3, EPHA7, and FGFR2 for M, and ERBB4, FGFR4, and SRMS for LAR, were consistently the highest value in BL2, M, and LAR, respectively. We found that the RNA-expression of TKs from TCGA, CPTAC, MET500, METABRIC, and CCLE were mostly consistent, except for certain kinases, including EPHA10, LYN, TXK, and FGFR2, of which the z-score from METABRIC and CCLE did not agree well with the rest. The differences could be possibly due to the heterogenicity of TNBC, as well as experimental methods, such as TNBC microarray from METABRIC or small sample space of TNBC cell lines. Interestingly, signature TK genes in the LAR subtype showed distinguished RNA expression than other subtypes, indicating the promising unique signature of this specific subtype.The mRNA expression z-score of the top three subtype-specific signature TK genes of each TNBC subtype was used to compare the changes in the expression level of selected kinases within the same dataset, as well as to compare the trend across datasets . We founEPHA10, LYN, and PTK2 were 0.633 ± 0.129, 0.747 ± 0.031, and 0.702 ± 0.063, respectively, where the individual AUC values of LYN and PTK2 were similar across all four datasets. In BL2, the average AUC values of TXK, EPHB2, and CSF1R were 0.500 ± 0.167, 0.705 ± 0.076, and 0.708 ± 0.029, respectively. The individual AUC values of EPHB2 and CSF1R were consistent across all datasets, while TXK performed poorly in CCLE, CPTAC, and METABRIC datasets. In M, the average AUC values of EPHA7, FGFR2, and NRTK3 were 0.637 ± 0.128, 0.675 ± 0.237, and 0.599 ± 0.075, respectively. These M subtype-specific TK genes performed better in CPTAC and MET500 compared to METABRIC and CCLE datasets. Particularly, the AUC of FGFR2 from CPTAC and MET500 datasets were 0.849 and 0.875, respectively. This indicated that FGFR2 could be a potential biomarker for the M subtype. TK genes in LAR had the highest AUC values, especially SRMS and FGRF4, with average AUC values of 0.822 ± 0.095 and 0.813 ± 0.122, respectively, whereas ERBB4 had a lower AUC value of 0.641 ± 0.135 due to inconsistent performance in CCLE and METABRIC datasets. In summary, eight out of 12 candidate subtype-specific TK genes could be potential biomarkers to classify TNBC subtypes, including LYN and PTK2 for BL1, CSF1R for BL2, EPHA7 and FGFR2 for M, and SRMS, ERBB4, and FGFR4 for LAR (average AUC > 0.700 from RNA-seq data of TNBC patients (CPTAC and MET500)). Among these TK genes, FGFR2 for M and SRMS for LAR achieved the highest average AUC at 0.862 and 0.874, respectively.In addition, the sensitivity and specificity of our TNBC subtype-specific signature TK genes were evaluated using ROC analysis A–D. The Tyrosine kinases are commonly involved in cancers and serve as important targets for anti-cancer therapies, as well as predictive markers for therapeutic response . BecauseEPHA10, LYN, and PTK2 (BL1 subtype-specific TK genes) is linked to the severity of breast cancer and can be used as the biomarker for TNBC [CSF1R and EPHB2 (BL2 subtype-specific TK genes) were reported as having similar expression in different types of breast cancer, including TNBC [TXK has the highest FC of RNA-expression in BL2 among TNBC subtypes, the ROC analysis showed the opposite. Thus, the activity of TXK on breast cancer requires further investigation [For the DE TK genes, we found that the expression of these genes was unique in each subtype; therefore, they could be used as the predictive marker to distinguish the TNBC subtype. Although several evidences supported the high expression of our identified TKs in TNBC, and some of them have been under the clinical study , the sigfor TNBC ,43,44,45for TNBC ,46. Intefor TNBC ,47. Howeing TNBC ,49. Eventigation . NTRK3, EPHA7, and FGFR2 (M subtype-specific TK genes) has been linked to a worse prognosis in breast cancer and could promote TNBC formation [ERBB4, FGRF4, and SRMS (LAR subtype-specific TK genes), overexpression of ERBB4 and FGRF4 was reported to be associated with poor prognosis in TNBC [In addition, overexpression of ormation ,52,53,54 in TNBC ,56. Apar in TNBC . Moreove in TNBC ,59. On t in TNBC . OverallUpregulated DE genes in each TNBC subtype were also used to predict the upstream kinases responsible for the observed differential phosphorylation. Out of more than 500 protein kinases, we found that TKs were dominant in the top-ranked predicted kinases of the TNBC subtype. This suggests that TKs could play an important role in the phosphorylation activities that contribute to signal transduction in TNBC. The basal-like subtype of TNBC showed the similarity to the enriched TKs. About half of them in BL1 and BL2 were overlapping, such as LYN, JAK2, SYK, and EGFR, which were reported to be associated with the aggressiveness of breast cancer, particularly in TNBC ,38,39,40For M and LAR, all unique TKs generally acted as cell–surface receptors with different functions. Three major types of enriched RTKs in M were FGFR, DDR, and LMTK3. The amplification of FGFR1, FGFR2, and FGFR3 was found in TNBC with variations associated with poor prognosis and overall survival ,71. DDR1FGFR2 for M and SRMS for LAR, with the highest average AUC > 0.860. However, the results from CCLE were particularly inconsistent. This is likely due to the difference in the microtumor environment surrounding TNBC cells between the cell line and the tumor cells within the patient’s body. Besides the heterogenicity of TNBC patients, the differences in experimental methodologies, such as RNA-seq  and microarray (METABRIC), cause inconsistency in gene expression where we found that the results from TCGA, CPTAC, MET500 datasets were mainly consistent. Although TKs appear as potential candidates for personalized medicine, the plasticity and redundancy of the kinome present key challenges for drug development. Targeting this TK may result in the upregulation and system-wide changes in multiple TK expressions and activities. More investigation is still required to better verify these identified TKs prior clinical practice.Despite about 40% correlation between expression levels of mRNA and protein, genes with differentially expressed mRNA have significantly higher correlations between mRNA and protein than those with non-differentially expressed mRNA ,76. Our Analyzing transcriptomic TK profiles of TNBC from TCGA datasets and validating the findings with the other TNBC patient datasets reveals potential signature TKs of TNBC subtypes, which are uniquely and highly expressed, for future investigation. These subtype-specific TKs could serve as novel targets for clinical exploration and therapeutic application. Moreover, these identified TKs could be adapted as an additional staining procedure to distinguish the TNBC subtype.


Plasmodium falciparum pre-erythrocytic-stage antigens using whole-genome sequence data from 1010 field isolates. Of these, 699 were collected in Africa , 69 in South America , 59 in Oceania (Papua New Guinea), and 183 in Asia . Antigens surveyed include cell-traversal protein for ookinetes and sporozoites, circumsporozoite protein, liver-stage antigens 1 and 3, sporozoite surface proteins P36 and P52, sporozoite asparagine-rich protein-1, sporozoite microneme protein essential for cell traversal-2, and upregulated-in-infectious-sporozoite 3 and 4 proteins. The analyses showed that a limited number of these protein variants, when combined, would be representative of worldwide parasite populations. Moreover, predicted T-cell epitopes were identified that could be further explored for immunogenicity and protective efficacy. Findings can inform the rational design of a multivalent malaria vaccine.Failure to account for genetic diversity of antigens during vaccine design may lead to vaccine escape. To evaluate the vaccine escape potential of antigens used in vaccines currently in development or clinical testing, we surveyed the genetic diversity, measured population differentiation, and performed in silico prediction and analysis of T-cell epitopes of ten such  Plasmodium falciparum apical membrane antigen 1 (AMA1)-based malaria vaccine [Malaria eradication will require powerful new tools that interrupt transmission ,2,3. Vac vaccine ,8, and a vaccine  have sho vaccine ,11,12 an vaccine ,14.Plasmodium falciparum antigens [Over the past decade, genetic and genomic studies have been conducted to assess the diversity of antigens . Most ofantigens ,17,18, rantigens ,20, or cantigens . Moreoveantigens , the cirantigens , and theantigens ,25 have antigens , P52  for each sample were introduced, using GATK’s v3.7 FastaAlternateReferenceMaker [n = 22), Burkina Faso (n = 56), Cambodia (n = 129), Colombia (n = 16), Cameroon (n = 130), French Guiana (n = 36), Guinea (n = 123), Kenya (n = 57), Malawi (n = 150), Mali (n = 99), Myanmar (n = 18), Papua New Guinea (n = 59), Peru (n = 11), Tanzania (n = 68), and Thailand (n = 36), as reported in [www.malariagen.net/data/pf3k-5 (accessed on 9 March 2019)) ) , were usπS) and nonsynonymous (πN) site, overall nucleotide diversity (π), and the number of unique haplotypes for each given antigen.To assess if the candidate vaccine antigens would evolve under a neutral model, genetic diversity statistics were assessed separately for each sample set, and at the worldwide level, when warranted, using DnaSP . The par®. The mean haplotype diversity was compared between countries and regions using the non-parametric, 2-sided Mann–Whitney test. Antigens with three haplotypes representing more than 80% of a country’s parasite population were considered “priority” vaccine candidates. Immunogenic amino acid fragments that are conserved across countries could be ideal cross-protective vaccine candidates. Thus, for each antigen, conserved fragments, defined as a stretch of at least 20 amino acids without polymorphic residues in the 3D7 reference strain, were identified from the worldwide diversity dataset, and variant proportions were compared using a chi-square test in SAS 9.4®. This metric could identify short amino acid sequences that could be used in a multifragment malaria vaccine candidate.Haplotypes were based on the whole coding sequence for each gene without their untranslated regions. The frequencies of the three major haplotypes of each antigen were compared between countries and regions using a chi-square test in SAS 9.4®).Haplotype networks are commonly used to visualize the relationship between haplotypes from different sampling sites. Haplotype networks were generated with sequences both within sampling locations  and for the global dataset by median-joining network analysis using PopART  software . The datST (gene fasta files), as implemented in DnaSP v6 [To assess the null hypothesis that allele frequencies do not vary between countries, regions, or continents, we computed FDnaSP v6 , with sihttp://www.cbs.dtu.dk/services/NetMHCIIpan-3.0 (accessed on 28 October 2021)) and common human leukocyte antigen (HLA) frequencies in Mali, a country in West Africa with high malaria incidence  [To assess if polymorphisms in candidate genes were located in putative epitopes, we used the T-cell epitope prediction algorithm  ,44 to scB1_0120) ,46. A puB1_0120) . All epi®.Structural epitopes are groups of amino acid residues that are antibody targets. Most B-cell epitopes are discontinuous, comprising from 15 to 25 nonsequential residues , and theWe used the PROVEAN  software  to identhttps://services.healthtech.dtu.dk/service.php?NetSurfP-2.0 (accessed on 30 October 2021)) was used to predict each candidate protein’s structural features, including their secondary predicted structure and surface accessibility, using nucleotide sequences in fasta format. The sequence of each gene in the reference 3D7 strain was used in COACH (Consensus Approach) and COACH-D (Consensus Approach 3D) to predict proteins’ structures [The NetSurfP 2.0 server ) were obtained from the public domain (n = 721) or generated in-house (n = 289). Each of these vaccine candidates was analyzed separately to select potential infection-blocking vaccine candidates that could enter a vaccine development pipeline.To identify malaria vaccine candidate variants, or, more specifically, epitopes and protein fragments, that will not be the target of allele-specific efficacy, we surveyed the genetic diversity in the genes encoding ten datasets  (ParasitHd) and nucleotide diversity (π), following the rationale that the proteins with the fewest variants are the least likely to lead to vaccine evasion. While Hd estimates the probability that two randomly sampled alleles are different, π estimates the average number of nucleotide differences per site between two randomly sampled alleles. The analyses showed that Hd estimates ranged from 0.21 for UIS3, the least diverse protein, to 0.99 for the most diverse protein (SAP1), and the π statistic for the ten candidate proteins screened ranged from 0.038% for UIS3 to 1.530% for CelTOS  between regions. Moreover, our analyses showed that for each antigen, FST values ranged from a lack of clustering to a complete subdivision at the country level .To determine if the genetic diversity observed was similarly represented across geographic regions, we estimated  regions . FST stary level , with UITo determine the diversity of each protein, we started by estimating haplotype diversity. Most proteins except P36 and UIS3 showed considerable polymorphism suggestive of an excess of rare alleles .p = 0.02) with only 1 fragment identified in UIS4 and 58 fragments in SAP1  obtained from each antigen  and higher substructuring  of variants were observed for most antigens, UIS3, UIS4, and P36 had the same predominant haplotype in all continents . While P36 had the highest average number of CD4+ and CD8+ linear T-cell epitopes (9.3), few CD4+ and CD8+ (1.4) epitopes were predicted in LSA1  frequencies in Mali ,64, a ce in LSA1 . CelTOS Overall, for each antigen, we detected significant differences in the number of epitopes that could be used as components of a multiepitope malaria vaccine, with CelTOS and P36 having both CD4+ and CD8+ conserved T-cell epitopes.p values greater than 0.05). Although eight out of ten of these antigens had relatively conserved B-cell epitope-containing fragments, reported polymorphic residues were scattered across antigen-encoding genes with polymorphisms located in binding sites of LSA1, LSA3, P52, CelTOS, SAP1, and CSP  server for conformational epitope prediction and epitope sequence analysis tools, we identified conformational epitopes of all ten candidates and generated a concatenated fragment of individual nucleotides/epitopes. The resulting major epitope-containing fragment of CelTOS represented only 7.03% of the whole dataset sequences, while two and three major epitope-containing fragments of SPECT2 and CSP represented, respectively, 87% and 61.4% of the dataset’s epitope-containing fragments. LSA1 (90%), LSA3 (97%), P36 (99.8%), P52 (99.9%), and SAP1 (90.4%) each had one major epitope-containing fragment, representing at least 85% of the dataset variability. Furthermore, an assessment between countries and continents showed no differences in epitope-containing fragment distribution at the country and continent level  ,52. NoneDisordered regions in proteins/peptides , as well as their surface accessibility to the solvent and secondary structure, were predicted using NetSurfP. Except for P36 and P52, disordered regions were distributed across all proteins, while, excepting a few protein fragments, all protein surfaces were accessible to the solvent . Our resP. falciparum antigens that are currently included in the formulation of malaria vaccines in clinical development. Thanks to the public availability of whole-genome data for thousands of P. falciparum isolates from around the world [This study characterized the genetic diversity in ten pre-erythrocytic he world , it is nhe world .P. falciparum liver stage is an ideal parasite developmental stage for the investigation of potential vaccine targets as efficacy directed against this stage can provide protection against both infection and disease [The  disease . While t disease  is based disease  may have disease ,70. Whil disease ,72,73, m disease ,75,76.We have identified the specific variants of UIS3, UIS4, and P36 that cover worldwide diversity and, if appropriately immunogenic, could be used to formulate a cross-protective malaria vaccine. Furthermore, the lack of polymorphisms in binding and structurally relevant sites within these antigens suggests that potential mutations are highly detrimental to parasite fitness, which may prevent emergence of vaccine-resistant strains ,78.P. falciparum [Molecular epidemiology and population genetic analyses from a total of 1010 allelic sequences indicated that UIS3 with one variant, P36 with one variant, and UIS4 with three variants represented more than 90% of the worldwide diversity and were the most conserved of the ten proteins analyzed. Importantly, the allele in the 3D7 reference strain, on which in-development vaccines incorporating these antigens are based ,75,79, wlciparum , and sellciparum ,85, the FST, haplotype geographic distribution, supported a low-to-moderate subdivision of these three antigens by country and continent. Parallel to our findings, previous studies [Additional analyses based on  studies ,81 that Although P36 showed significant differentiation between continents, this substructuring at the continent level was clearly explained by the high prevalence of the 3D7 variant of P36 in Africa compared to Asia and the Americas. However, in Asia and the Americas, the 3D7 variant of P36 was the second most frequent variant, which may allow the design of a bivalent P36-based vaccine that would cover at least 80% of the worldwide parasite population. As studies have shown that polymorphic regions of proteins may be involved in immune escape ,86,87, tP. falciparum proteins. The lack of post-translational modifications in addition to its inefficiency in expressing higher-molecular-weight proteins has limited the use of Escherichia coli in malaria vaccinology [As the expression of full-length malaria antigens has been challenging , the concinology . Althougcinology ,91, thesCD8+ T cells play a critical role in an effective immune response against pre-erythrocytic-stage malaria parasites . This prAlthough ex vivo assays are ideal to evaluate the phenotype and function of T cells in peripheral blood along with antibody response, in silico analyses could be used as screening techniques to rule out peptides with a low binding affinity for their targeted ligand and those with polymorphic residues that are detrimental to the receptor and ligand interaction. Hence, binding residues were predicted and polymorphisms that may have an impact on protein binding to its receptors were identified. These analyses indicate P36 was the single antigen with the strongest binding peptides, and the residues of these peptides were conserved across samples from all continents surveyed.www.malariagen.net/pf7k (accessed on 9 March 2019)), which includes more sequences from these countries, may help improve haplotype frequency estimates. Hence, additional analyses are being conducted to validate P36 findings. Major histocompatibility complex (MHC) and HLA [Although a large dataset was used to assess the worldwide diversity of these ten liver-stage candidates, the recent release of Pf7k ( and HLA  polymorp and HLA . Thus, a and HLA .Overall, our findings suggest that among all ten antigens tested, P36, UIS3, and UIS4 were the top candidates for a successful strain-transcending vaccine based on protein residue conservation and the existence of many B- and/or T-cell epitopes. However, P36 stands out as the best candidate for the conservation of its epitopes and the lack of polymorphisms at functionally relevant residue positions. The essential role the protein plays during hepatocyte invasion  makes itWe have presented herein findings from in silico analyses for the screening and selection of malaria vaccine candidates. These results indicate that instead of CelTOS, CSP, LSA1, LSA3, SPECT2, or SAP1, a few variants of P36, UIS3, or UIS4 could be used to cover the worldwide diversity of these proteins and, were they to induce a protective response, prevent vaccine escape when the vaccine is deployed in the field. Moreover, epitope prediction and analysis, in addition to ongoing functional assays, will validate the use of these approaches in epitope-based vaccine design. These approaches applied systematically may accelerate malaria vaccine discovery.


DBPrs4588 and DBP-rs7041) and cytochrome P450-24A1 (CYP24A1-rs6013897) in patients with cirrhosis with or without hepatocellular carcinoma (HCC), including demographic/clinical/biochemical profiles.To evaluate the association of genetic polymorphisms of vitamin D transporter protein (N = 158) with or without HCC, distributed into Group 1 (G1): cirrhosis and HCC; Group 2 (G2): isolated cirrhosis; and 225 individuals without hepatopathies (G3). Polymorphisms were analysed by real-time polymerase chain reaction. An alpha error of 5% was admitted.A total of 383 individuals were studied, considering the total group  of patients with cirrhosis  in G1 (98.3%) versus G2 . There was a moderate positive correlation between vitamin D and parathyroid hormone in patients . Smoking, alcoholism and diabetes mellitus (DM) stood out as independent factors for cirrhosis, as well as for cirrhosis with HCC, except for smoking, adding, in this case, advanced age, male gender, polymorphic allele of CYP24A1-rs6013897, viral hepatitis and high levels of serum gamma-glutamyl transferase (GGT), alpha-fetoprotein (AFP) and creatinine. An increase in survival was observed in the presence of the polymorphic allele of DBP-rs7041 (p = 0.0282).CYP24A1-rs6013897 is associated with cirrhosis and HCC as a predictor, while DBP-rs4588 is associated with reduced vitamin D, and DBP-rs7041 provides increased survival, suggesting a protective characteristic. Advanced age, alcoholism, DM, viral hepatitis and high levels of GGT, AFP and creatinine are also confirmed as predictors of HCC and cirrhosis, while smoking, alcoholism and DM for isolated cirrhosis only. Cirrhosis, the tenth leading cause of death in the world , is an iCirrhosis is directly associated with the development of hepatocellular carcinoma (HCC) , presentAdmittedly, biomolecular events that trigger hepatic carcinogenesis may result from alterations that interfere with the synthesis of deoxyribonucleic acid (DNA) . In addiDBP) participates and functions in metabolic and biological pathways, such as the regulation of bone development, including actin capture and modulation of immune responses [DBP-rs4588 and DBP-rs7041 are associated with reduced serum levels of 25-hydroxy-vitamin D [25(OH)D] [The vitamin D transporter protein gene (esponses . In this25(OH)D] , which m25(OH)D] , which nCYP24A1) encodes 25-hydroxyvitamin D3-24-hydroxylase. This enzyme catalyses the conversion of 25(OH)D3 and 1.25(OH)2D3 into hydroxylated products, which constitute the degradation of the vitamin D  can present satisfactory levels under the mediation of PTH . In thisDBP, which actively participate in the vitamin D metabolic pathway, could influence its serum level, with compensatory PTH secretion in case of vitamin D deficiency [DBP-rs4588 which in this series was associated with reduced levels of vitamin D.In this study, patients and controls showed similarity in PTH levels, corroborating a study in breast cancer . On the ficiency , which mCYP24A1-rs6013897 polymorphism is associated with cirrhosis and HCC, standing out as a predictor of the disease, while the DBP-rs4588 polymorphism can influence in the reduction of vitamin D level in patients and, on the other hand, the variant DBP-rs7041 confers increased survival, suggesting a protective character. Age, alcoholism, DM, viral hepatitis and high levels of GGT, AFP and creatinine are also confirmed as predictors of both diseases, while smoking, alcoholism and DM are predictors of isolated cirrhosis.In conclusion, the There are no conflicts of interest to declare.


Background: While magnetic resonance imaging (MRI) is the imaging modality of choice for the evaluation of patients with brain tumors, it may still be challenging to differentiate glioblastoma multiforme (GBM) from solitary brain metastasis (SBM) due to their similar imaging features. This study aimed to evaluate the features extracted of dual-tree complex wavelet transform (DTCWT) from routine MRI protocol for preoperative differentiation of glioblastoma (GBM) and solitary brain metastasis (SBM). Methods: A total of 51 patients were recruited, including 27 GBM and 24 SBM patients. Their contrast-enhanced T1-weighted images (CET1WIs), T2 fluid-attenuated inversion recovery (T2FLAIR) images, diffusion-weighted images (DWIs), and apparent diffusion coefficient (ADC) images were employed in this study. The statistical features of the pre-transformed images and the decomposed images of the wavelet transform and DTCWT were utilized to distinguish between GBM and SBM. Results: The support vector machine (SVM) showed that DTCWT images have a better accuracy (82.35%), sensitivity (77.78%), specificity (87.50%), and the area under the curve of the receiver operating characteristic curve (AUC) (89.20%) than the pre-transformed and conventional wavelet transform images. By incorporating DTCWT and pre-transformed images, the accuracy (86.27%), sensitivity (81.48%), specificity (91.67%), and AUC (93.06%) were further improved. Conclusions: Our studies suggest that the features extracted from the DTCWT images can potentially improve the differentiation between GBM and SBM. Glioblastoma multiforme (GBM) and brain metastasis are the most commonly identified brain neoplasms in the adult population ,2. The dGBM typically presents with central necrosis surrounded by an enhancing rim of tumor cells with peritumoral edema . Brain mThe radiomics-based extraction of mineable high-dimensional data from MRI for texture analysis can provide the underlying information related to pathophysiology, which is arduous to detect by visual perception ,12. The As mentioned, the boundaries of GBM and SBM would have different characteristics in the MR images, suggesting that algorithms sensitive to the edges would benefit from the distinction, such as the wavelet transforms mentioned in the online (version 5) image biomarker standardization initiative (IBSI) reference manual . However0 field. However, these techniques are not conducted routinely or universally owing to long acquisition times, inconsistent accuracy, or high cost. Conventional MRI techniques at a 1.5 T system are still primarily used in clinical practice. Herein, we attempted to differentiate GBM and SBM from the MRI images with routine protocols acquired at 1.5 T. We focused on the features obtained from the wavelet transformations, especially DTCWT, which could reveal more structure information.Several advanced MRI techniques, such as diffusion imaging, perfusion imaging, spectroscopy, and diffusion tensor imaging, have been used to discriminate between the two entities ,31,32. LWe collected the cases of GBM and brain metastasis from 1995 to 2020. There were 62 pathology-proven GBM cases found in the GBM database. Of the 62 GBM cases, 27 cases were recruited and retrospectively entered into this study. On the other hand, 85 pathology-proven cases of brain metastases were found in the brain metastasis database. Of the 85 cases with brain metastases, 24 cases with SBM were recruited and retrospectively entered into this study. In total, 51 patients were recruited between 2007 and 2020 (27 GBM and 24 SBM) in this retrospective analysis which the local Institutional Review Board approved . For the 24 patients with SBM, histopathological analysis revealed 13 cases that originated from the lung, five cases from the breast, two from the liver, one from the kidney, two from the colon, and one from the ovary. Inclusion criteria were as follows: (1) patients of single GBM or SBM with pathologically confirmed according to the WHO criteria ; (2) avaThe preoperative MRI was collected from the picture archiving and communication systems (PACS) at Ditmanson Medical Foundation Chia-Yi Christian Hospital. In detail, 19 GBM and 14 SBM cases were performed on a 1.5 T Signa™ HDxt scanner  with an eight-channel neurovascular array GE coil and fast spin echo (FSE) sequence. Eight GBM and 10 SBM cases were performed on a 1.5 T Optima™ MR450w  scanner with a 16-channel GE head-and-neck unit coil and the PROPELLER  sequence. The duration of each complete examination was about 20 min with ear plugs.2 and 2 for b value 1000 s/mm2; matrix, 128 × 128; FOV, 240 mm × 240 mm; b values, 0 and 1000 s/mm2), and contrast-enhanced T1-weighted image (CET1WI). We routinely apply FSE and fat suppression (FS) techniques on CET1WI. After a check-up of the patients’ renal function, CET1WI was obtained with the T1WI sequence about 30 s after intravenous administration of a standard dose of gadobutrol . The array spatial sensitivity encoding technique (ASSET) was applied for DWI sequences to accelerate image acquisitions. The apparent diffusion coefficient (ADC) maps were generated automatically using the GE built-in algorithm. All the images acquired were two-dimensional with static MRI scans.The acquisition protocols of the MRI examinations included FSE T1-weighted image (T1WI) (repetition time/echo time (TR/TE), 2600/24 ms; rephasing radiofrequency (RF) pulse, 160°; section thickness, 6 mm, intersection gap, 0.6 mm; number of acquired signals, 1; matrix, 320 × 224; field of view (FOV), 220 mm × 220 mm), FSE T2 fluid-attenuated inversion recovery (T2FLAIR) : 2200 ms; rephasing RF pulse, 160°; section thickness, 6 mm, intersection gap, 0.6 mm; number of acquired signals, 1; matrix, 320 × 224; FOV, 220 mm × 220 mm), single-shot echo planar imaging (SS-EPI) diffusion-weighted image (DWI)  to distinguish GBM and SBM on the basis of the possible combination of the selected features [® 2021a .The operational flow of the imaging processing and analysis is shown in features ,20, in wThe representative CET1WI and T2FLAIR images and the selected ROIs are shown in In contrast, these details are depicted in DWT’s horizontal, vertical, and diagonal components and six orientations in DTCWT. The corresponding wavelet-transformed DWI and ADC images are shown in t-test to select 21 features with a p-value smaller than 0.001 between GBM and SBM for further investigation , DWT (63 combinations), and DTCWT (511 combinations) images. We also considered all the combinations (221−1) of the selected features from the pre-transformed, DWT, and DTCWT images.However, by examining the box plots of these selected features , the disThe best feature combination from each group is shown in Several features observed on conventional MRI  , such asThe radiomics analysis quantifies the information of texture features extracted from MRI, enhancing clinicians’ existing data. Currently, radiomics-based analysis forces a shift from the visual perception of medical images, which is highly variable, to the extraction of highly dimensional meaningful data to support clinical decision making for precision medicine ,15,16,17An image can be transformed into frequency domains by employing a wavelet series. These wavelets are the images’ high- or low-pass filters and result in the approximation, vertical, horizontal, and diagonal components. The approximation component can be further transformed, resulting in a hierarchical multiresolution image ,46,47. Wt-test with a p-value <0.001  diagnostic accuracy compared with the performances of DWT and pre-transformed images. Further study with a more significant amount of sample data and inclusion of the peritumoral region for texture analysis is required to improve diagnostic accuracy and expand generalizability.