Gossypium hirsutum, specifically Gossypium purpurascens, has the potential to facilitate the restoration of genetic diversity that has been depleted due to selective breeding in modern cultivars. This investigation evaluated 45 G. purpurascens varieties and a salt-tolerant cotton variety based on 34 morphological, physiological, and biochemical indicators and comprehensive salt tolerance index values. This study effectively identified a total of 19 salt-tolerant and two salt-resistant varieties. Furthermore, transcriptome sequencing of a salt-tolerant genotype  and a salt-sensitive genotype  revealed 2776, 6680, 4660, and 4174 differentially expressed genes (DEGs) under 0.5, 3, 12, and 24 h of salt stress. Gene ontology enrichment analysis indicated that the DEGs exhibited significant enrichment in biological processes like metabolic (GO:0008152) and cellular (GO:0009987) processes. MAPK signaling, plant-pathogen interaction, starch and sucrose metabolism, plant hormone signaling, photosynthesis, and fatty acid metabolism were identified as key KEGG pathways involved in salinity stress. Among the DEGs, including NAC, MYB, WRKY, ERF, bHLH, and bZIP, transcription factors, receptor-like kinases, and carbohydrate-active enzymes were crucial in salinity tolerance. Weighted gene co-expression network analysis (WGCNA) unveiled associations of salt-tolerant genotypes with flavonoid metabolism, carbon metabolism, and MAPK signaling pathways. Identifying nine hub genes  across various intervals offered insights into the transcriptional regulation mechanism of salt tolerance in G. purpurascens. This study lays the groundwork for understanding the important pathways and gene networks in response to salt stress, thereby providing a foundation for enhancing salt tolerance in upland cotton.Elevated salinity significantly threatens cotton growth, particularly during the germination and seedling stages. The utilization of primitive species of  Salinizn uptake . The worn uptake .Plants primarily respond and adapt to salt stress through various hormones and biosynthetic and signaling pathways controlled by gene expression regulation ,6. TransWRKY11, TaFBA1, BES1, R2R3-MYB, and GhLOG3 were detected in soybean, tobacco, Arabidopsis, and cotton [Advancements in sequencing technology and decreasing costs have propelled the field of plant science into the omics era. Various omics methodologies, such as phenomics, proteomics, transcriptomics, metabolomics, and genomics, have gained prominence in research ,16. Amond cotton ,26,27,28d cotton ,30,31. Ed cotton , wheat [d cotton , maize [d cotton , rice [3d cotton , and cotd cotton . Gossypium hirsutum L.), a member of the Malvaceae family, is a globally significant crop accounting for 90% of textile fiber production. It is an allotetraploid species cultivated in over 80 countries, including semi-arid and tropical regions [G. purpurascens was identified as a perennial plant in various regions such as Brazil, Africa, China, Egypt, Congo, and India [G. hirsutum [G. hirsutum [G. purpurascens has been naturally occurring in the South China Sea Islands for centuries without human intervention [G. purpurascens [G. purpurascens [G. purpurascens subjected to salt stress and potential candidate genes were identified through WGCNA analysis. The findings of this study have the potential to provide valuable insights into the molecular mechanisms underlying salt stress responses in various cotton species and contribute to our understanding of salinity stress in other crop plants.Upland cotton  and data were recorded at various time points  after the onset of salt stress. Several parameters related to plant growth and ion concentrations were assessed, including fresh root weight, dry root weight, fresh shoot weight, dry shoot weight, root-shoot ratio, relative water content, and ion-selective coefficients for Na, K, and Mg ions. Statistical analyses were performed on the collected data, including standard deviation, coefficient of variation, principal component analysis, and correlation analysis  variety, K386 (NY2), and one salt-sensitive (SS) genotype, K396 (GP2), which were screened and selected based on biomass and ionome indexes. These varieties were subjected to a salt treatment of 0.4% NaCl for different durations  under the same growth conditions . The SS Total RNA was collected from leaf tissues of cotton seedlings subjected to different salinity stress durations . The RNA samples were then subjected to transcriptome sequencing to investigate the molecular mechanisms underlying salt tolerance in cotton seedlings. A total of 29 cDNA libraries were generated, and high-throughput sequencing generated 221.06 Gb of raw sequencing data, with a range of 39,604,862 to 58,894,078 raw reads per library. After quality filtering, the number of clean reads per library ranged from 19,802,431 to 29,447,039, with 50% having clean reads. The quality of the sequencing data was assessed based on Q20 and Q30 values, which ranged from 99.18% to 94.89%, indicating high-quality sequencing . The ranClear contrasts in gene expression were observed between SS and ST genotypes at 12 and 24 h, indicating significant transcriptomic shifts as GP2-24-1 and GP2-24-2 were enlisted in the fourth cluster while GP2-24-1, NY2-24-2, NY2-24-1, and NY2-24-3 were observed in the sixth cluster. GP2-12-2, GP2-12-1, GP2-12-3, NY2-12-3, NY2-12-1, and NY2-12-2 were assigned to the fifth cluster. Due to the significant distances between GP2-24-1 and GP2-24-2 and GP2-24-3, the sample was removed from subsequent differential expression analysis. The analysis indicated that salinity stress affects seedling growth differently in salt-tolerant and salt-sensitive cultivars, highlighting the significant differences between the two accessions. Pearson’s correlation analysis further indicated that the salt-tolerant cultivar may provide valuable insights into the mechanisms underlying salt tolerance. Differential expression analysis and identification of new genes were also performed using the filtered high-quality reads.p-value < 0.05) representing a greater than twofold change in transcriptional expression compared to the control (0 h time point) under salt treatment. The number of DEGs at different time intervals  in both GP2 and NY2 genotypes were compared to the control to understand the global transcriptional changes in response to salt treatment. In the GP2 genotype, the number of DEGs increased with increasing salt stress. At 0.5, 3, 12, and 24 h, 7028, 9590, 20,333, and 28,330 DEGs were detected, respectively. Similarly, in the NY2 genotype, 5192, 12199, 16125, and 17,343 DEGs were identified at 0.5, 3, 12, and 24 h, respectively  were identified based on a significant value  in both salt-sensitive (SS) genotype GP2 and salt-tolerant (ST) genotype NY2 E. The heUnder established conditions, we identified genotype-specific and salt-responsive differentially expressed genes (DEGs) related to salt tolerance in two cotton genotypes. A total of 2776, 6680, 4660, and 4174 salt-tolerant DEGs were identified at 0.5, 3, 12, and 24 h, respectively . Three cThis study employed GO enrichment analysis to categorize the functional roles of the DEGs observed in seedlings under salinity stress conditions in both genotypes . A totalPP2C (Gh_D03G014500), ERF1 (Gh_A02G045500) and ACS1 (Gh_D11G099900)) and downregulated genes (SPCH (Gh_D12G271000) and CHB (Gh_A03G238200)) associated with salt tolerance (KCS1 (Gh_D06G142800) was upregulated in all comparison groups except the GP2-00 vs. GP2-05 group in plant-pathogen interaction. AP1 (Gh_D05G091100), ABI1 (Gh_A13G222500), and ABI2 (Gh_D13G222500) were upregulated and PIF3 (Gh_D11G310300), AHK4 , and XTH22 (Gh_D05G318000) were downregulated in plant hormone signal transduction. Similarly, KCS1 (Gh_D08G142800) was upregulated in all the comparison groups except the GP2-00 vs. GP2-05 group. TPS (Gh_D08G093700) was upregulated in starch and sucrose metabolism, while INV (Gh_A04G061600) was downregulated. Overall, the analysis indicated that MAPK signaling, plant-pathogen interaction, starch and sucrose metabolism, and biosynthesis of amino acids pathways played crucial roles in regulating salt tolerance at the seedling stage of G. purpurascens.Furthermore, a KEGG pathway enrichment analysis was conducted to explore the roles of DEGs concerning salinity tolerance. A total of 2804 salt-tolerant DEGs were found to be involved in KEGG enrichment pathways. At different time intervals, various pathways showed enrichment, including MAPK signaling, plant-pathogen interaction, starch and sucrose metabolism, and biosynthesis of amino acids C–F. The olerance . KCS1 (GATHB-7) (Gh_A11G107200), NAC (NAC056) (Gh_D13G165700), ERF (ERF114) (Gh_D05G245400), R2R3-MYB (MYB102) (Gh_A06G146700) and C2H2 (ZAT10) (Gh_A12G020100), which were associated with salinity tolerance. Additionally, bZIP (bZIP1) (Gh_D09G076500) and HSF (HSFC1) (Gh_A06G070600) showed upregulation at multiple time intervals, while GRAS (Gh_A13G161600) demonstrated upregulation at 12 and 24 h.A total of 924 genes belonging to 25 transcription factor (TF) families were identified in the DE salt tolerance-related genes. These TF genes showed diverse expression patterns, indicating their various roles in response to salinity . WRKY, RSD-2b , L-LEC (Gh_D10G026800), DLSV (Gh_D01G031000), LysM (Gh_D11G049400), and CrRLK1L-1 (Gh_D07G198500) subfamilies was observed. Notably, RLCK (Gh_A11G195200) was identified as a novel gene, upregulated in the NY2 genotype but downregulated in the GP2 genotype. Carbohydrate-active enzymes (CAZymes), which are involved in carbohydrate metabolism, also showed distinct expression patterns. Genes such as GH36 (Gh_A05G362500), GT8 (Gh_D13G204000), GH18 (Gh_D13G213600), GT20 (Gh_D08G093700), and CBM18 (Gh_A10G141200) were consistently upregulated across different comparison groups (GT1 (Gh_D13G179300) and GT43 (Gh_D06G029900) were upregulated at 3, 12, and 24 h. Other gene families, such as cellulose synthase-like protein G2 (Gh_D02G048800) and EXP (Gh_A12G200600), exhibited specific expression patterns concerning salinity tolerance groups  revealed that GP2 and NY2 exhibited tolerance against salinity at 3, 12, and 24 h . Upreguln groups . GT1  explored the gene regulatory network associated with salt stress response in GP2 and NY2 genotypes. Initially, 216,006 genes exhibiting differential expression between adjacent developmental stages of GP2 and NY2 were identified through transcriptome analysis. These genes were filtered based on their fold change (|log2FC| > 1) and false discovery rate (FDR < 0.05). Genes with FPKM values less than five were excluded from the analysis. The WGCNA package was used to compute weight values for network construction, and a “soft threshold” of 12 was established to ensure a scale-free network distribution.A gene clustering tree was constructed by utilizing the correlation between distinct gene expression levels. The dynamic cutting tree method was subsequently employed to partition the branches and produce distinct modules A. Sixteep < 0.01) with salt stress treatments. The ME coral3 module demonstrated the highest number of genes (2566), followed by MElightcyan (295), MEbisque4 (239), and MEdarkolivegreen (168) modules  expression levels were used to examine gene expression within co-expression modules, representing the overall expression levels of each module. The weights for each gene were computed by WGCNA using measures of gene importance, such as lower ectivity .Within the ME bisque3 module, MYB4 (Gh_A01G171400) was identified as the main differentially expressed gene (DEG) in the co-expression network. This module had 23 associated genes . In the G. purpurascens under salt stress conditions when exposed to 0.4% NaCl. These genes belonged to the cellular component “mitochondrion” according to GO annotation (GO:0005739), and PFAM annotation revealed their association with LEA5 (Late embryogenesis abundant protein).Real-time PCR analysis was performed on eighteen randomly selected genes identified as significantly differentially expressed genes (DEGs) to validate the gene expression profiles obtained from the transcriptome data. This analysis aimed to compare the results with the corresponding transcriptome data, as shown in The results of this study demonstrated a significant positive correlation between the gene expression levels obtained through qRT-PCR and RNA-seq approaches. This finding confirms the reliability of the RNA-seq data used in the research. Furthermore, the hub genes identified by WGCNA exhibited differential expression under salt stress conditions compared to the expression levels in control seedlings. This observation implies that the identified hub genes may have a significant impact on the cellular response to salt stress.Plants have developed an advanced mechanism for altering their physiological and metabolic states in response to environmental variations. Salinity stress significantly challenges crop productivity, affecting various important agricultural crops such as maize, barley, wheat, rice, and cotton ,51,52,53+ and K+ ions in shoot and root tissues. The results indicated that the GP2 genotype was significantly more affected by salt stress than the NY2 genotype. These findings validate the divergent interactions exhibited by specific cotton genotypes when subjected to saline conditions, as reported in previous studies [+ to K+ in their systems compared to the tolerant genotypes. This ion distribution pattern in plant tissues is significant because it is crucial in salt tolerance mechanisms [+ and K+ ions and their respective ratios are important parameters in understanding plant responses to salt stress. Under salt stress conditions, the NY2 genotype exhibited a preferential accumulation of K+ and reduced accumulation of Na+, thereby maintaining shoot Na+/K+ ratios. Due to the distinct nature of the wild species, it is reasonable to consider that a Na+ extrusion from the root cells, coupled with an increased salt exclusion from the shoots, could potentially support the hypothesis in favor of wild cotton. The salt tolerance exhibited by wild cotton can be understood within the framework of anticipated evolution. This characteristic potentially reinforces the proposed evolutionary strategy found in wild plants adapted to saline environments. These observations aligned with prior rice and chickpea research [In our study, we assessed the impact of salt stress on different cotton genotypes collected from the South China Sea Islands by evaluating various phenotypic and physio-biochemical attributes, including seedling growth parameters, relative water content, and the distribution of Na studies ,55. Ionochanisms . The conresearch ,58,59. Aresearch , salt-toresearch ,61. By aUnder salt stress conditions, the growth parameters of seedlings, including the fresh and dry weights of roots and shoots, showed a declining trend. Importantly, the salt-sensitive genotype GP2 exhibited a more significant growth reduction than the salt-tolerant genotype NY2. This observation can be attributed to the detrimental effects of oxidative stress, ion toxicity, disruption of metabolic processes, and osmotic imbalances triggered by salt-induced stress ,61. The Understanding the molecular mechanisms underlying salinity tolerance is critical for developing salt-tolerant cultivars through molecular breeding techniques in cotton. In recent years, transcriptome data and RNA sequencing analysis have provided valuable insights into the molecular and biological processes involved in plant stress responses. RNA-seq is a simple, fast, and cost-effective method that has enabled the identification and characterization of stress-responsive genes in various plant species, including wild barley, rice, eggplant, sweet potato, pearl millet, and soybean ,70,71,72Transcriptome analysis revealed several key pathways in the plant’s response to salt stress. Specifically, mitogen-activated protein kinase (MAPK), plant-pathogen interaction (PPI), starch and sucrose metabolism (SSM), plant hormone signaling pathway (PHST), photosynthesis, and fatty acid metabolism (FAM) pathways were found to be transcriptionally reprogrammed in response to salt stress. The significant transcriptional reprogramming observed in these pathways provides evidence of their involvement in the plant’s response to salt stress. Understanding the regulation and function of genes within these pathways can contribute to developing strategies for enhancing salt tolerance in plants ,75,76. STranscription factors (TFs) play a crucial role in plants’ responses to salt stress by regulating downstream gene expression ,83. We iG. purpurascens, associated with improved salt tolerance in Arabidopsis [Carbohydrate-active enzymes (CAZy Family), including glycosyltransferases (GTs) and glycoside hydrolases (GHs), also play a role in plants’ responses to salt stress. Expression patterns of carbohydrate-active enzymes highlighted that GH36 (Gh_A05G362500), GT8 (Gh_D13G204000), GH18 (Gh_D13G213600), GT20 (Gh_D08G093700), and CBM18 (Gh_A10G141200) were upregulated in each comparison group while GT1 (Gh_D13G179300) and GT43 (Gh_D06G029900) were upregulated at 3, 12, and 24 h. Our investigation revealed higher expression levels of GT and GH genes in bidopsis ,95. ThesA significant implication of the present study is establishing a direct correlation between a comprehensive RNA-seq dataset and qRT-PCR analysis. This correlation facilitated the identification of key genes linked to salt tolerance in cotton and established a relationship between differentially expressed genes (DEGs) and salt stress tolerance. The upregulation of the Gh_A10G166600 gene in salt-tolerant and salt-sensitive genotypes is attributed to late embryogenesis abundant (LEA) proteins and is a possible candidate gene for salt tolerance in cotton. LEA proteins are low-molecular-weight proteins (10–30 kDa) that protect higher plants from environmental stresses . LEA proAdditionally, LEA proteins may be involved in ion sequestration. Furthermore, it is hypothesized that the adoption of an amphipathic α-helical conformation in the desiccated state aids in the incorporation of certain LEA proteins into the lipid bilayer, thereby serving to inhibit membrane fusion . SimilarThe mechanisms of plant salt stress responses are regulated by intricate signaling and transduction pathways that involve cross-talk between various components ,104. OurGossypium purpurascens genotypes were collected from Sansha Island in Hainan province , Naozhou Island , and Techeng Island  of Zhanjiang in Guangdong province, China (Gossypium hirsutum (Zhong 9807) known for its high salt tolerance [Forty-five e, China . As a coolerance  was obtaolerance ,107,108.To initiate the experiment, cotton seeds were subjected to a treatment process. Firstly, the seeds were immersed in a 15% sodium hypochlorite solution for 15 min. Subsequently, they were thoroughly rinsed five times with double-distilled water (ddH2O) to remove residual chemicals. The treated seeds were then grown in cups containing a sand and water mixture with a ratio of 8:1. This growth phase took place over three weeks in a thermostatically controlled greenhouse. The greenhouse maintained a relative humidity of 70–75% and followed a 14 h light and 10 h dark cycle at Zhengzhou Research Base, State Key Laboratory of Cotton Biology, Zhengzhou University, located in Zhengzhou, China.At the three-true-leaf stage (3TLS), the plants were subjected to two different concentrations of sodium chloride (NaCl): 0% (control) and 0.4% (treatment). To minimize the immediate impact of salt on the plants, the salt was gradually added with the final concentration reaching 0.4% NaCl. Each experimental group consisted of three replicates to ensure reliable results. Based on the salt resistance evaluation of all varieties, we selected one salt-tolerant (ST) variety K386 (NY2) and one salt-sensitive (SS) variety K396 (GP2) for RNA sequencing. Leaf samples were collected at various time points following the onset of salt treatment, including 0 h, 0.5 h, 3 h, 12 h, and 24 h. The collected samples were immediately stored at −80 °C to preserve their RNA integrity for subsequent experiments.A subset of ten seedlings from both the control and treatment groups were randomly selected to assess plant growth and physiological parameters. Samples of roots and shoots were obtained from each of these selected seedlings. The initial step involved the measurement of the fresh weight (FW) of both the roots and shoots of each seedling. Subsequently, the seedlings were placed in individual paper bags, ensuring proper labelling for identification. The bags containing the seedlings were then transferred to an oven set at 105 °C and subjected to drying for 30 min. This initial drying step helped to remove surface moisture from the plant tissues. The seedlings were then dried at a constant temperature of 80 °C until a stable weight was achieved. Following the cooling process, the shoots’ and roots’ dry weights (DW) were determined and soon after, the root-shoot ratio and water content were computed (Formulas (1) and (2)).v/v) nitric acid, dried samples were examined for Na, Mg, and K contents using inductively coupled plasma-optical emission spectroscopy  [Later, each dried sample was weighed (about 0.2 g), ground, and made into ashes by heating the samples at 550 °C for 12 h. Following microwave digestion in 70%  ,110. TheSTIi designates the ith salt tolerance index for a genotype, and STImin and STImax indicate the minimum and maximum values among i salt tolerance indexes, respectively.iW indicates the weight of component i’s contribution to the total variance percentage.To evaluate the salt tolerance of the 46 cotton varieties, the comprehensive salt tolerance index (CSTI) of each variety was obtained using associated functions and principal component analysis (PCA) . In shoriW indicates the weight of component i’s contribution to the total variance percentage.Finally, the CSTI values were generated using the following equations:To investigate the plants’ transcriptional response to salt stress, we conducted transcriptome profiling using the Illumina HiSeq platform. Leaf samples were collected from two selected genotypes, with three replicates for each genotype and treatment combination. Total RNA was extracted from the samples using TRIzol reagent, and the quality and integrity of the RNA were assessed using 1% agarose gel electrophoresis and the Agilent 2100 Bioanalyzer system . We generated paired-end reads for sequencing and performed quality control to remove low-quality and short reads using FastQC (Version 0.11.9) and a Perl program. Gene expression levels were quantified using featureCounts, and the expected number of fragments per kilobase of transcript per million mapped reads (FPKM) was calculated for each gene based on the read counts.Differential expression analysis was conducted using the DESeq R package (Version 1.34.1), applying a significance threshold of FDR  ≤ 0.05. Genes were considered differentially expressed if they exhibited at least a 2-fold difference in FPKM values between the compared samples. This analysis allowed us to identify genes that showed significant changes in expression levels in response to salt stress. Transcriptome profiling was employed to comprehensively understand the alterations in gene expression linked to salt stress in the examined genotypes. This approach enabled us to uncover potential candidate genes and pathways involved in the plants’ response to salt stress and to compare the transcriptional profiles between salt-tolerant and salt-sensitive genotypes.® Ultra™ RNA Library Prep Kit  was utilized to create libraries under the manufacturer’s guidelines. The library fragments were purified using the AMPure XP system . Subsequently, polymerase chain reaction (PCR) was executed utilizing 3 μL of uracil-specific excision reagent (USER) enzyme procured from New England Biolabs (NEB), United States of America (USA), in conjunction with the complementary DNA (cDNA) at a temperature of 37 °C for 15 min, followed by 95 °C for 5 min. Subsequently, the PCR products underwent purification via the AMPure XP system, and the quality of the library was assessed utilizing the Agilent Bioanalyzer 2100 system. The sequencing of libraries was conducted using an Illumina Novaseq 6000 platform provided by Beijing Allwegene Technology Company Limited in China. The sequencing adapters were removed from the raw data and low-quality reads were eliminated to obtain clean reads. The reference genome employed in this study was Gossypium hirsutum, as reported by [Following the completion of quality inspection, a quantity of 1.5 μg RNA was extracted from each sample, and three replicates were established for library construction. The NEBNextorted by . The refhttps://www.omicshare.com/tools/ (accessed on 1 April 2023). To further analyze the specific genes related to salt tolerance between the two genotypes, we established the following strict screening parameters: (1) at four salt treatment time intervals, only genes will be selected with differential expression found between NY2 and GP2 ; (2) only differentially expressed genes in salt-tolerant varieties will be selected ; (3) in both salt-tolerant and salt-sensitive varieties, there are differentially upregulated genes, but the difference in log2 fold change will be greater than 0.6 ; (4) in both salt-tolerant and salt-sensitive varieties, there are differentially downregulated genes, but the difference in log2 fold change will be less than −0.6 . The specific screening process, gene count statistics, and final results are all included in the DESeq2 was utilized to conduct differential expression analysis (DEGs) between two treatments . The crip-value were determined between the module eigenvectors of each module and various samples. Positive and negative correlations were shown by correlation coefficients with r > 0 and r < 0, respectively. The specificity modules in this study were further examined as |r| > 0.6 and p modules < 0.05. The genes corresponding to the top five weight values of their expression correlation were retrieved after the target genes with the strongest correlation coefficient among modules were sorted out. The WGCNA graph was constructed using the Gephi-0.91 program [R programming was used to analyze weighted gene co-expression networks with the help of the WGCNA package . FPKM >  program . Utilizi−ΔΔCt approach [−ΔΔCt method, the relative values of the stress samples at different time points can be obtained.Quantitative real-time PCR (qRT-PCR) analysis was conducted to measure the expression levels of 18 genes associated with salt tolerance in NY2 and GP2 at 0.5, 3, 12, and 24 h. The first set of qRT-PCR cDNA was produced in a 20-mL solution, which included 1 mg of RNA as the template. The First-strand cDNA Synthesis SuperMix kit  was utilized for this purpose, and the manufacturer’s instructions were followed. Subsequently, the Novostar R SYBR qPCR SuperMix Plus  was utilized to conduct qRT-PCR on a LightCycler 480 . The gene used as a housekeeping gene in this study was identified as GhHistone. The Primer-BLAST online tool2 was utilized to design gene-specific primers . The qRTapproach . Here, wMicrosoft Excel 2021 was utilized to statistically analyze various phenotypic markers and determine their salt tolerance value. IBM SPSS 25.0 was employed to conduct morphological, physiological, and biochemical data analyses . Principal component analysis and cluster analysis were performed using R programming. Two-way ANOVA determined the significance of the phenotypic trait data between the two cotton genotypes, and qRT-PCR results were presented with GraphPad Prism 9.0 . Each experiment was repeated at least three times, and the values were expressed as mean ± SE.G. purpurascens cultivars at the seedling stage. This study identified one salt-tolerant variety and one salt-sensitive variety based on 34 traits and CSTI values in wild cotton species. Transcriptome analysis revealed 2776, 6680, 4660, and 4174 salt-tolerant differentially expressed genes at 0.5, 3, 12, and 24 h, respectively, and pathways associated with salt resistance by comparing the transcriptome data of salt-tolerant and salt-sensitive genotypes. GO and KEGG pathway enrichment analysis revealed the involvement of various biological processes, including the MAPK signaling pathway, plant-pathogen interaction, starch and sucrose metabolism, the plant hormone signaling pathway, photosynthesis, and fatty acid metabolism, in response to salt stress. ERF, bHLH, and WRKY were key transcription factors in salt tolerance. WGCNA revealed that metabolic pathways and MYB4, MYB36, MYB43, MYB105, bZIP16, and bZIP43 played significant roles in salinity tolerance. Furthermore, qRT-PCR highlighted the sensitivity of Gh_A10G166600 to salt stress and its potential synergistic relationship with LEA proteins in mitigating the harmful effects of salinity. The findings contribute to our understanding of the genetic basis of salt tolerance in cotton and provide valuable genetic resources for future salt-resistant breeding efforts. This pioneering research expands our knowledge of salt stress responses in Gossypium purpurascens and lays the foundation for developing improved salt-tolerant cotton varieties.In conclusion, this study utilized RNA-seq analysis to explore the molecular mechanisms underlying salt tolerance in two


Podocytes are highly specialized cells that play a pivotal role in the blood filtration process in the glomeruli of the kidney, and their dysfunction leads to renal diseases. For this reason, the study and application of this cell type is of great importance in the field of regenerative medicine. Hypertension is mainly regulated by the renin–angiotensin–aldosterone system (RAAS), with its main mediator being angiotensin II (ANG II). Elevated ANG II levels lead to a pro-fibrotic, inflammatory, and hypertrophic milieu that induces apoptosis in podocytes. The activation of RAAS is critical for the pathogenesis of podocyte injury; as such, to prevent podocyte damage, patients with hypertension are administered drugs that modulate RAAS signaling. A prime example is the orally active, non-peptide, selective angiotensin-II-type I receptor (AGTR1) blocker losartan. Here, we demonstrate that SIX2-positive urine-derived renal progenitor cells (UdRPCs) and their immortalized counterpart (UM51-hTERT) can be directly differentiated into mature podocytes. These podocytes show activation of RAAS after stimulation with ANG II, resulting in ANG II-dependent upregulation of the expression of the angiotensin-II-type I receptor, AGTR1, and the downregulated expression of the angiotensin-II-type II receptor 2 (AGTR2). The stimulation of podocytes with losartan counteracts ANG II-dependent changes, resulting in a dependent favoring of the specific receptor from AGTR1 to AGTR2. Transcriptome analysis revealed 94 losartan-induced genes associated with diverse biological processes and pathways such as vascular smooth muscle contraction, the oxytocin signaling pathway, renin secretion, and ECM-receptor interaction. Co-stimulation with losartan and ANG II induced the exclusive expression of 106 genes associated with DNA methylation or demethylation, cell differentiation, the developmental process, response to muscle stretch, and calcium ion transmembrane transport. These findings highlight the usefulness of UdRPC-derived podocytes in studying the RAAS pathway and nephrotoxicity in various kidney diseases. Kidneys perform numerous essential tasks in the human body; these are filtration of the urine to dispose of urinary components such as urea, uric acid, and creatine into the urine . They reNPHS2), Nephrin (NPHS1), KIRREL1 (NEPH1), and CD2-associated protein (CD2AP) through which podocytes are connected to cell–cell contacts and control podocyte function [Podocytes are highly differentiated and specialized cells located on the outside of the glomerular capillaries that project into the urinary cavity of Bowman’s capsule. They have a large cell body from which numerous foot processes taper around the glomerular capillaries. Adjacent podocytes thus form lamellipodia-like foot processes, between which an interdigitating cell–cell contact is formed to ensure the size selectivity of glomerular filtration via the slit membrane. Numerous podocyte-specific genes have been identified whose products play important roles in maintaining the filtration barrier. There is a highly conserved complex consisting of Podocin NPHS, Nephrinfunction ,4. The mThe renin–angiotensin–aldosterone system (RAAS) is a cascade that contributes to various systematic and localized processes. Among the systematic processes is the regulation of blood pressure. The octapeptide angiotensin II (ANG II) is the biological effector of the renin–angiotensin system and regulates blood pressure and extracellular volume. Angiotensin II acts on the two subtypes of angiotensin II receptors AT1 (AGTR1) and AT2 (AGTR2). Based on this, AGTRs were shown to be expressed in various segments of the nephron in the distal tubule, collecting duct, and renal vasculature ,7,8,9,10ACTN4 and decreased expression of focal adhesion in podocytes [Persistent activation of the RAAS is critical for the pathogenesis of podocyte injury and causes proteinuria. ANG II stimulation triggered the downregulation of odocytes . In summodocytes ,19. The odocytes ,21. ACE odocytes . In contodocytes ,23.We previously reported human urine as a non-invasive source of renal stem cells with regenerative potential. These urine-derived renal progenitor cells (UdRPCs) express, among others, the renal stem cell markers sine oculis homeobox homolog 2 (SIX2), Cbp/P300-interacting transactivator with Glu/Asp rich carboxy-terminal domain 1 (CITED1), Wilms tumor 1 (WT1), CD133, CD24, and CD106 . FurtherIn the current study, we demonstrate that losartan counteracts ANG II-dependent molecular changes by elevating the expression of podocyte-specific markers such as NPHS1. Additionally, cells treated simultaneously with ANG II and losartan undergo receptor-usage switching from AGTR1 to AGTR2. We carried out a transcriptome analysis of human urine-derived podocytes stimulated with 100 μM ANG II with and without 1 μM losartan. To date, transcriptome data derived from podocytes treated with losartan are restricted to rodents ,26,27. OIn a previous manuscript, we reported the existence of urine-derived renal progenitor cells (UdRPCs) inherently capable of differentiating into podocytes ,24,28. UFor the investigation, the optimal concentration of losartan was tested with and without ANG II. Based on these findings, a losartan concentration of 1 μM and a cell density of 60–70% were used in all further experiments. Therefore, the urine-derived podocytes were pre-treated with 1 μM losartan for 24 h, thereafter 100 μM ANG II was added for another 24 h. We differentiated primary SIX2-positive UdRPC UM51 and the transformed counterpart UM51-hTERT into podocytes . The expp = 0.002). In contrast, the addition of 1 μM losartan induced a slight significant downregulation of AGTR1 (p < 0.05) compared to ANG II treatment (p = 0.007) while in UM51, hTERT podocyte mRNA expression was slightly increased  (p < 0.05) expression in both cell lines (p = 0.004 and p = 0.01) expression compared with ANG II treatment (p = 0.003 and p = 0.004)  C. As hypll lines C. To furll lines D. This ereatment D. In par= 0.004) D. We prop < 0.05), 1219 are exclusively expressed in the untreated control podocytes and 94 in the podocytes treated with losartan alone  derived from somatic cells are available and serve to study nephrogenesis and various kidney associated diseases such as hypertension ,35,36,37The use of losartan allowed us to determine whether the cytoskeletal change was mediated by ANGII induced signaling through AGTR1. The present data indicate that the addition of losartan is sufficient to counteract the cytoskeletal restructuring induced by ANG II by rescuing NPHS1 expression. It is interesting to note that losartan causes a redistribution of the expression of AGTR1 and AGTR2. From this, it can be concluded that losartan can efficiently block binding to AGTR1, causing ANG II to bind with a higher preference, presumably to AGTR2. Furthermore, this indicates that activated angiotensin receptors trigger a signaling cascade into the nucleus to augment their own transcriptional and translational expression. AGTR2 appears to reintroduce cells into differentiation, again upregulating the expression of podocyte-specific genes. The 1243 genes that were upregulated in the podocytes treated with ANG II are annotated with GO-BP terms such as renal system development, cell division, metal ion transport, calcium signaling pathway, and regulation of the cell cycle process, suggesting that these cells might dedifferentiate and lose their cell type-specific properties by signaling through AGTR1. Podocytes are terminally differentiated cells that do not undergo cell division in vivo. Therefore, a process of dedifferentiation is needed when kidney damage occurs. This process has been described for many renal pathologies, such as diabetes and lupus nephritis, and is even discussed as a potential biomarker [ADRA1A) and shroom family member 2 (SHROOM2). Like AGTR1, the alpha-adrenergic receptor ADRA1A mediates its action through association with G-proteins that activate a phosphatidylinositol calcium second messenger system. These in turn activate mitogenic responses and regulate the growth and proliferation mediated by Klf15 in the kidney [SHROOM2, which is involved in endothelial cell morphology changes during cell spreading [In comparison, GO-BP terms for losartan treatment included cell–cell communication, vascular smooth muscle contraction, oxytocin signaling pathways, and renin secretion. These include the calmodulin-like 4 (CALML4), natriuretic peptide A (NPPA), and protein phosphatase 1 regulatory subunit 12B (PPP1R12B) genes. The blockade of AGTR1 and the resulting signaling through AGTR2 seem to lead to vasodilation and reduction in vascular tone ,45,46. Ie kidney . Additiopreading . LCK, NPPA, CCL3, SLC10A1, SLC12A3, CACNA1H, CATSPER1, LRRC38, HEPHL1, CFTR, TMC8, CSRP3, DMD, and MMP9. These genes have been reported to contribute to reduction in inflammation and oxidative stress [NPPA, for example, is responsible for extracellular fluid volume and electrolyte homeostasis. This signaling cascade can apparently be activated by the addition of losartan and seems to play a major role in hypertension [LCK results in a reduction in oxidative stress as described by Alqarni [Some of the losartan-induced 94 genes are associated with the GO-BP term metal ion transport, which includes e stress . ARBs pre stress . NPPA, frtension ,52,53. T Alqarni . Interes2+ dependent. As early as 1978, Kerjaschik was the first to propose that the Ca2+ increase in podocytes is one of the first events leading to podocyte injury [2+ influx via TRPC5/TRPC6. This cascade enables the downstream transcription of Calcineurin. AGTR1, TRPC, Calcineurin, and the actin cytoskeleton form a linear signaling cascade in which ANG II activates Calcineurin in a TRPC5-dependent manner [The 106 genes associated with the co-stimulation of losartan and ANG II are annotated with GO-BP terms such as extracellular matrix organization, metal ion transport, DNA methylation or demethylation, cell differentiation, developmental process, response to muscle stretch, regulation of cytokine production involved in inflammatory response, and calcium ion transmembrane transport. It has already been reported that podocytes expresse the transient receptor proteins TRPC3, TRPC4, TRPC5, and TRPC6 ,56. Undee injury . AGTR1 se injury . It was e injury ,60. Upstt manner . Changedt manner . This dot manner . The efft manner . Here, tt manner . It is kt manner . A well-t manner ,46.Transcriptome analysis revealed 94 losartan-induced genes associated with diverse biological processes and pathways such as vascular smooth muscle contraction, the oxytocin signaling pathway, renin secretion, and ECM-receptor interaction. Co-stimulation with losartan and ANG II induced exclusive expression of 106 genes associated with extracellular matrix organization, metal ion transport, DNA methylation or demethylation, cell differentiation, the developmental process, response to muscle stretch, calcium ion transmembrane transport, and the regulation of cytokine production involved in the inflammatory response.These findings highlight the usefulness of human urine-derived podocytes in studying nephrogenesis, kidney-associated diseases such as hypertension, the RAAS pathway, nephrotoxicity, and drug screening. Urine-derived renal progenitor cells (UdRPCs) of a 51-year-old male (UM51) were isolated as described in Rahman et al. . FurtherFor the immunocytochemistry, the podocytes were fixed with 4% paraformaldehyde (PFA)  followed by three wash steps with PBS for 5 min each. To block unspecific binding sites, the fixed cells were incubated with blocking buffer containing 10% normal goat or donkey serum, 1% BSA, 0.5% Triton X-100 (Sigma-Aldrich Chemistry) and 0.05% Tween-20 (Sigma-Aldrich Chemistry) for 2 h at room temperature. The primary antibodies were diluted in blocking buffer according to −∆∆CT method and specified by fold change expression. The samples were run in independent experiments three times. The mean value of these experiments was used for the calculations. The statistical significance was calculated using the Two-Sample test assuming unequal variances [p = 0.05.Quantitative PCR was performed using podocyte-specific genes and angiotensin II receptors of type I and II. The samples were run in triplicate on a 384-well reaction plate and Power Sybr Green PCR Master Mix  using Step One Plus Real-Time PCR systems. The amplification conditions were denaturation at 95 °C for 13 min followed by 37 cycles of 95 °C for 50 s, 60 °C for 45 s, and 72 °C for 30 s. To normalize the quantitative real-time PCR, the ribosomal encoding gene-RPL0 was used. The primer sequences are listed in ariances  with a sTM ECL Western Blotting Substrate solutions from Thermofisher  were used. The quantification was carried out with ImageJ. Detailed information on the antibodies used can be found in The podocytes were lysed in RIPA buffer (Sigma-Aldrich Chemistry) containing 5 M NaCl, 1% NP-40, 0.5% DOC, 0.1% SDS, 1 mM EDTA, 50 mM Tris, pH 8.0, and freshly added 10 μL/mL protease and phosphatase inhibitor (Sigma-Aldrich). We dissolved 20 μg of each obtained protein lysate in a 10% sodium dodecyl sulfate PAGE gel and then transferred it to an Immobilon-P membrane . The membranes were incubated with the primary antibody overnight at 4 °C, then washed three times with 0.1% Tween-20 in Tris-buffered saline followed by 1 h of incubation of the secondary antibodies at room temperature. For visualization of the blotted proteins, PierceFor the microarray experiments, 1 μg RNA preparations were hybridized on the Human Clariom S Gene Expression Array  at the core facility Biomedizinisches Forschungszentrum (BMFZ) of the Heinrich Heine University Düsseldorf. The raw data were imported into the R/Bioconductor environment  and furtGene expression data will be available online in the National Centre of Biotechnology Information (NCBI) Gene Expression Omnibus. In this study, urine samples were collected with the informed consent of the donors and the written approval  of the ethical review board of the medical faculty of Heinrich Heine University, DuȠsseldorf, Germany. All methods were carried out in accordance with the approved guidelines. The medical faculty of Heinrich Heine University approved all experimental protocols. t-test with a significance threshold p = 0.05.Data are presented as arithmetic means + standard error. In total, three independent experiments were performed and used for the calculation of mean values. The statistical significance was calculated using the two-sample Student’s


Increased exposure to environmental heavy metals and metalloids and their associated toxicities has become a major threat to human health. Hence, the association of these metals and metalloids with chronic, age-related metabolic disorders has gained much interest. The underlying molecular mechanisms that mediate these effects are often complex and incompletely understood. In this review, we summarize the currently known disease-associated metabolic and signaling pathways that are altered following different heavy metals and metalloids exposure, alongside a brief summary of the mechanisms of their impacts. The main focus of this study is to explore how these affected pathways are associated with chronic multifactorial diseases including diabetes, cardiovascular diseases, cancer, neurodegeneration, inflammation, and allergic responses upon exposure to arsenic (As), cadmium (Cd), chromium (Cr), iron (Fe), mercury (Hg), nickel (Ni), and vanadium (V). Although there is considerable overlap among the different heavy metals and metalloids-affected cellular pathways, these affect distinct metabolic pathways as well. The common pathways may be explored further to find common targets for treatment of the associated pathologic conditions.  •Heavy metal-induced oxidative stress is damaging for biomolecules such as DNA, proteins, and lipids.•Exposure to heavy metals affects cellular pathways associated with various disease conditions.•Some of the metals may play a protective role against pathological conditions like diabetes and neurodegeneration. Enhanced recombination frequency may be related to inhibition of DNA replication and subsequent strand breaks Arsenic-induced carcinogenesis involves members of the MAPK families. Low-level (1 to 2 µM) arsenic trioxide (ATO) exposure activates extracellular signal-regulated kinases (ERK1/2), causing cell transformation 3.1.22 but attenuation at (10 and 20 µM) VEGF (induced by 5 µM CdCl2) overexpression via ERK and PI3K/AKT pathways  and ROS generation c-fos and c-myc early genes and NF-κB signaling-dependent genes, and promotes macrophage proliferation 2 for 9 to 15 weeks) cadmium exposure can cause lung cancer via activating the Notch1 signaling system. HIF-1α, AKT, and ERK may activate Notch1 in this mechanism as found in cell culture studies The half-life of cadmium is 25 to 30 years 3.1.3Hexavalent chromium Cr[VI] compounds are highly toxic and cause most chromium toxicity. Cr[VI] has been classified as a human pulmonary carcinogen Several factors are involved in chromium-induced carcinogenicity, including tissue and cell type, Cr[VI] concentration, exposure period, free radical formation, and Cr[V] and Cr[IV] reactivity. The hexavalent Cr[VI] does not bind to DNA or other macromolecules itself; rather, it is reduced to Cr[V], Cr[IV], and Cr[III] by reacting with cellular reductants, triggering Fenton-type reactions. This generates hydroxyl radicals in the presence of hydrogen peroxide and induces oxidative stress. Chromium cross-linking and adduct formation with cellular antioxidants cause oxidative stress and ROS generation leading to DNA damage, including DNA–chromium–protein crosslinks, DNA inter- and/or intra-strand crosslinks, SSBs and DSBs, p53 point mutations, and lipid peroxidation 3.1.4+ lymphocytes crucial for anti-tumor activity Iron is found in the earth’s crust and coexists with manganese in groundwater 3.1.5Vanadium is a metalloid with different oxidation states that can generate free radicals Vanadium complexes have anti-cancer properties, too, including generation of ROS, inhibition of tyrosine phosphatases, induction of apoptosis, DNA cleavage, cell cycle arrest, and lipoperoxidation 3.1.6Nickel, a group 1 carcinogen, depletes cellular GSH and increases ROS 3.23.2.1Insulin resistance causes type 2 diabetes (T2D) 4 groups in glucose-metabolizing enzymes and biomolecules with high affinity. Pentavalent As[V] interacts with ATP phosphate binding sites and inhibits ATP-dependent processes like pentose phosphate pathway (PPP) and insulin secretion ., pyruvate dehydrogenase and α-ketoglutarate dehydrogenase Arsenic compounds bind to —SH and—PO3.2.2Cadmium affects insulin secretion from pancreatic β-cells. While low cadmium levels enhance insulin release, high levels diminish the rate 3.2.3Chromium, unlike arsenic and cadmium, may help patients with diabetes and MetS by improving insulin sensitivity and glucose metabolism 3.2.4High levels of nonpathological iron, such as dietary iron, have been found to be associated with an increased risk of diabetes. Elevated serum ferritin levels are associated with insulin resistance, T2D , obesity, MetS, and CVDs 3.2.5Studies link mercury to the pathogenesis of MetS. Mercury-induced oxidative stress causes insulin resistance, hypertension, dyslipidemia, and obesity, however direct association with diabetes is inconclusive Similar to chromium, vanadium compounds can enhance insulin response in T2D 3.33.3.1Arsenic has an enormous impact on lipid and glycolipid metabolism. Long-term arsenic exposure can deteriorate the structural integrity and functions of the cardiovascular system, leading to CVDs, hypertension, dyslipidemia, obesity, and fatty liver disease Atherogenesis is a pathophysiological condition characterized by inflammation and proliferation of smooth muscle cells, followed by thrombosis and vascular wall damage Arsenic reduces cyclic guanosine monophosphate (cGMP)—a NO surrogate, and inhibits endothelial nitric oxide synthase (eNOS) in endothelial cells 3.3.2., lysoPCs 2) Cadmium exposure is associated with intracellular lipid accumulation and elevated levels of pro-inflammatory lipids, e.g3.3.3+ conductance, modulate chlorine channels, deactivate Na+/K+-ATPase, inhibit phospholipid turnover, and activate phospholipase C (PLC). However, lower concentrations (0.5 to 5 µM) is enough to cause DNA damage Mercury, especially MeHg, has a high affinity for —SH groups and selenium, which reduces antioxidant defense by disrupting GSH, GPX, and catalase and promotes free radical-mediated oxidative stress and lipid peroxidation 3.3.42) through AKT, ERK, and NF-κB activation may be analogous to cadmium-associated VEGF production and atherosclerosis as shown in cell culture studies Nickel increases the fluidity of membranes containing phosphatidylinositol--bisphosphate [PIP2] and the lipid clustering of phosphatidylinositol-3-phosphate (PI3P) systems, interfering with the development of signaling lipid domains and leading to nickel toxicity 3.3.5. ceramide and lysoPC, derived from lipid peroxidation, notably of PUFAs Ferroptosis is an iron-dependent cell death process resulting from iron accumulation and lethal lipid species, e.g2+-ATPase and activation of potassium channels. These cellular events are involved in the regulation of angiogenesis and vasorelaxation Vanadium can act as a cardioprotective agent. The activation of vanadium-induced PI3K/AKT signaling pathway results in the phosphorylation of eNOS and production of NO. NO, in turn, activates cGMP which leads to the subsequent inhibition of Ca3.43.4.12+ imbalance. These are linked to central and peripheral nervous system damage and may explain arsenic-related neurological symptoms Arsenic-induced neurotoxicity can occur through a number of mechanisms, including ROS-induced oxidative stress, decreased activities of mitochondrial complex I, II-III, and IV, lipid peroxidation followed by DNA damage and neuronal cell death, apoptosis by caspase-9, p38, and JNK activation, decreased acetylcholinesterase activity, and Ca3.4.2c-fos gene. The Fos transcription regulator forms a complex, AP-1, with another protein named Jun. The AP-1 site is in the promoter region of several neuronal activity or degeneration genes. Iron-induced oxidative stress may activate early genes, which may explain the link between iron toxicity and neurological disorders such as epilepsy, stroke, Alzheimer’s disease, Parkinson's disease, Huntington’s disease, and amyotrophic lateral sclerosis (ALS) Since the brain consumes 20% of body oxygen, the central nervous system (CNS) is especially vulnerable to oxidative stress. Iron overexposure causes oxidative stress and ROS, which upregulate the 3.4.3The CNS is the primary target of MeHg toxicity. Mercury-induced alterations in protein synthesis, a key factor in cellular degeneration, may cause nervous system changes 3.4.4Vanadium-induced neuropathology, including neurobehavioral, neurochemical, and neurocellular changes, is known Interestingly, protective effects of vanadium compounds on cognitive impairments and Alzheimer's are also known. A link exists between T2D and Alzheimer's disease. Amyloid β binds to insulin receptor and alters downstream AKT and GSK3β activity, impairing brain insulin signaling 3.4.52+ accumulation, caspase-3 upregulation, B-cell lymphoma 2 (BCL-2) downregulation, and p53 deficiency Cadmium-induced apoptosis is linked to ROS generation, CaLimited literature exists on chromium’s neurotoxicity. The role of chromium-generated ROS in the brain is unknown. One study reported that Cr[VI] elevated ICAM-1 and VCAM-1 expression and activated NF-κB–caspase-1–IL-1β cascade which may associate chromium with neurodegenerative diseases Nickel increases the expression of caspases, cytochrome c, BCL-2-associated X protein (BAX), and BH3-interacting domain death agonist (BID) proteins, whereby lowering the expression of BCL-2. BAX and BID proteins are proapoptotic, whereas BCL-2 is antiapoptotic 3.53.5.1+ and CD8+ T cells are apoptosed by arsenic-induced ROS + T cells Arsenic exposure alters macrophage, dendritic, and T lymphocyte development, activation, and/or proliferation Inflammasome complexes are composed of NOD-like receptor proteins (NLRs) that specifically control caspase-1-dependent cleavage of pro-IL-1β and pro-IL-18. Arsenite and ATO suppress pro-IL-1β cleavage by inhibiting NLRP1, NLRP3, and NAIP5/NLRC4 3.5.2+/CD8+ ratio, downregulates cytokine production in Th1  and Th2  lymphocytes, suppresses the expression of class I and class II major histocompatibility complex (MHC) molecules in B lymphocytes, alters signaling through them, and inhibits B-lymphocytic activity and immunoglobulin IgE synthesis Cadmium impairs both innate and adaptive immunity. As reviewed, cadmium exposure impairs macrophage surface FcγRII expression and phagocytic capacity, inhibits macrophage activity in response to lipopolysaccharides and TLRs via decreased production of pro-inflammatory cytokines such as TNF-α and IL-1, induces immunosuppressive reactions and apoptosis of neutrophils and dendritic cells, and reduces the number of natural killer (NK) cells in the field of innate immunity. In case of adaptive immunity, cadmium decreases the CD4Cadmium causes respiratory disorders. Cadmium-induced oxidative stress enhances influenza virus proliferation. Cadmium-induced redox imbalance and subsequent activation of redox-sensitive cascades, including protein kinase C (PKC) and p38, JNK, and ERK MAPK pathways, may contribute to the process 3.5.3Both organic and inorganic forms of mercury are immunotoxins 3.5.4Immune response to Cr[VI] is dose-dependent. Lower concentrations of Cr[VI] stimulate lymphocyte blastogenesis, whereas higher concentrations inhibit it Iron causes hepatic inflammation. Iron overload causes oxidative stress, which activates apoptotic pathways in hepatocytes via Fas-FasL or TNF-TNF receptors, in individuals with chronic hepatitis C 2O5) affects IL-2-dependent PI3K/AKT/mTOR and MAPK pathways. It has also been reported to dysregulate or inhibit humoral responses in human and mice Vanadium compounds are both inflammatory and immunosuppressors 3.6+ efflux and NLRP3 activation Metal hypersensitivity is a rising concern with around 10-15% of human population being afflicted by contact hypersensitivity + T cells, and activation and proliferation of T cells following re-exposure Detailed nickel allergy mechanism is unknown, however the skin inflammation reaction involves production of cytokines and chemokines, activation of antigen presenting cells that present nickel allergen to naive CD4Mercury exposure is known to cause both type I (anaphylactic type) and type IV (delayed-type) hypersensitivity 4Heavy metal pollution is a global health and environmental concern. Sufficient evidence has supported the designation of ambient metal pollutants to be regarded as risk factors for cardiovascular, respiratory, metabolic, and neurological disorders. Rapid, uncontrolled urbanization and industrialization have exacerbated the release and exposure of heavy metals. Essential trace metals are needed for normal cellular and enzyme activity, including cellular metabolism, protein folding, DNA replication, redox reactions, immune reactions, electron transport and other metabolite transport, signal transduction, and neurotransmitter synthesis. However, high levels of these and other non-essential heavy metals and metalloids can damage biomolecules, impair cellular redox status, alter signal transduction, and cause protein misfolding, apoptosis, and malignant transformation.We reviewed the association between arsenic, cadmium, chromium, iron, mercury, nickel, and vanadium exposure and disruptions in common metabolic signaling pathways and pathophysiological conditions such as diabetes, carcinogenesis, cardiovascular, neurodegenerative, allergic, and inflammatory diseases. Among these are the MAPK, AMPK, PI3K/AKT, NF-κβ, HIF-1, TNF-α, TLR, NLR, WNT/β-catenin, Notch, TGF-β/BMP, ferroptosis, and caspase signaling pathways associated with such toxicities. Almost all the heavy metals lead to an imbalance in the homeostasis of cellular antioxidants .Some of these metals actually play a protective role by targeting those intracellular metabolic pathways. Arsenic and iron have anti-cancer properties, whilst chromium and vanadium supplements are now being investigated and trialled for the treatment of type 2 diabetes. Despite its possible involvement in carcinogenicity and neurotoxicity, vanadium is a pretty interesting metal since its complexes display potent insulin-mimetic effects that have promoted its use as an adjuvant in treating type 2 diabetes, and the ability to ameliorate the pathological conditions that are altered in neurodegenerative Alzheimer’s disease.The association between pathological states and alterations in metabolic pathways is complex, intricate, and dynamic in nature. This is a brief summary of the As, Cd, Cr, Fe, Hg, Ni, and V affected pathways and some associated diseases that are still being studied. Details and other inconclusive correlations between these metals and other diseases, such as renal toxicity, osteoporosis, gastrointestinal problems, etc., are not mentioned here.GARE) from the Ministry of Education, Bangladesh.Grant for Advanced Research in Education (The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Myeloid-derived suppressor cells (MDSCs), major components maintaining the immune suppressive microenvironment in lung cancer, are relevant to the invasion, metastasis, and poor prognosis of lung cancer, through the regulation of epithelial-mesenchymal transition, remodeling of the immune microenvironment, and regulation of angiogenesis. MDSCs regulate T-cell immune functions by maintaining a strong immunosuppressive microenvironment and promoting tumor invasion. This raises the question of whether reversing the immunosuppressive effect of MDSCs on T cells can improve lung cancer treatment. To understand this further, this review explores the interactions and specific mechanisms of different MDSCs subsets, including regulatory T cells, T helper cells, CD8 + T cells, natural killer T cells, and exhausted T cells, as part of the lung cancer immune microenvironment. Second, it focuses on the guiding significance confirmed via clinical liquid biopsy and tissue biopsy that different MDSC subsets improve the prognosis of lung cancer. Finally, we conclude that targeting MDSCs through action targets or signaling pathways can help regulate T-cell immune functions and suppress T-cell exhaustion. In addition, immune checkpoint inhibitors targeting MDSCs may serve as a new approach for enhancing the efficiency of immunotherapy and targeted therapy for lung cancer in the future, providing better comprehensive options for lung cancer treatment.  These T cells act as the main immune cells against cancer [+ T cells, through multiple pathways. MDSCs can encourage the depletion of CD8+, CD4+, and memory T cells (T-cell exhaustion), thus affecting the immune function of patients with tumors. These findings raise the question of whether reversing the immunosuppressive effect of MDSCs on T cells can lead to improved lung cancer treatment. To understand this further, this study reviews recent research on the interactions and specific mechanisms of different MDSCs subsets, including Treg, Th, CD8+ T, NKT, and exhausted T cells, as part of the lung cancer immune microenvironment. The review also focuses on the guiding significance confirmed via clinical liquid biopsy and tissue biopsy that different MDSC subsets improve the prognosis of lung cancer.Various types of immune responses, including those in cancer, involve T cells, such as CD8t cancer . The inct cancer , and a lt cancer , 6. OtheMDSCs are heterogeneous immature bone marrow cell (IMC) populations at different stages of differentiation. Normally, IMCs differentiate into mature granulocytes, monocytes, and dendritic cells (DCs), after they migrate to peripheral organs. However, within the tumor microenvironment (TME), tumor cells and their targets such as STAT3, IRF8, C/EBPβ, and Notch can inhibit the differentiation of IMCs. Specific factors produced by activated T cells, tumor, and tumor stromal cells, such as VEGF, CSF, IL-6, IL-10, MMP-9, TGF-β, NF-kB, and other immune-suppressive mediators, can stimulate the proliferation and activation of MDSCs , 8. Acti+Ly6G+Ly6Clow and CD11b+Ly6G−Ly6Chigh [+CD14−CD15+ or CD11b+CD14−CD66b+, whereas, M-MDSCs are defined as CD11b+ CD14+ CD15− HLA− DR−/low. Bronte et al. proposed naming a mixed group of MDSCs consisting of a greater number of immature progenitor cells with the phenotype of early stage MDSCs (e-MDSCs) [Animal experiments have shown that MDSCs express different surface antigens in animals and humans. In 2010, Dolcetti proposed that MDSCs can be divided into two subgroups—monocytic MDSCs (M-MDSCs) and polymorphonuclear MDSCs (PMN-MDSCs)—and confirmed that MDSCs are a heterogenous population of cells that can be dissolved using Ly6C and Ly6G in mice . MDSCs eLy6Chigh . HaverkaLy6Chigh . In humae-MDSCs)   in patients with lung cancer. PMN-MDSCs in such patients resemble activated PMN-MDSCs in mice in terms of genetic characteristics and have negative prognostic outcomes in the patients . With th+ [+ [MDSCs and their specific molecules, such as TIE2+ , S100A9++ [+ , PD-L1, + [+ , can be follows: (1) The immune function of T cells is inhibited by MDSCs by affecting the amino acid metabolism. MDSCs have been shown to affect amino acid metabolism in T cells; they consume arginine, cysteine, tryptophan, and other essential amino acids necessary for T-cell proliferation and activation; and they affect T-cell immune function. MDSCs deplete L( +)-arginine, which plays a critical role in T-cell activation and metabolism in the immune microenvironment, T-cell receptor (TCR) CD3ζ chain downregulation and signaling is due to a high expression of arginase 1 (ARG1) by the MDSCs, and T-cell dysfunction. In addition, L ( +)-arginine depletion prevents T cells from entering the G1 phase by inhibiting cyclin D3 expression, which induces T-cell cycle arrest [(2) MDSCs inhibit T cells immune functions by secreting a variety of reactive oxygen species and immunosuppressive factors. MDSCs express various enzymes involved in the production of reactive oxygen species (ROS) and nitric oxide (NO). The production of MDSCs induces the upregulation of Arg-1 activity and ROS production in a pathway dependent on Signal Transducers and Activators of Transcription 3 (STAT3). Indicating that the STAT3 activator signal plays a critical role in MDSC functions. Peroxynitrite (the product of ROS reacting with NO) alters TCR and CD8 molecules, and deprives cytotoxic T lymphocytes (CTLs) of the ability to specifically bind to the major histocompatibility complex (MHC) class I molecules, leading to further loss of their ability to kill tumor cells [(3) MDSCs inhibit T-cell immune functions by elevating PD-L1 receptor expression. It is well known that the tumor immune microenvironment often presents an oxygen-deficient state, and in this hypoxic TME, hypoxia-inducible factor-1α (HIF-1α) is highly expressed. HIF-1α can induce programmed death ligand-1 (PD-L1) expression on the surface of MDSCs by binding to the programmed death factor-1 (PD-1) on the T-cell surface, which causes T-cell immune dysfunction and induces T-cell depletion and apoptosis [Activation of MDSCs is the major cause of lung cancer immune suppression. The mechanism of MDSC-mediated immunosuppression is closely related to the dysregulation of T-cell immune functions, and the mechanism by which MDSCs inhibit T-cell immune functions can be summarized as e arrest , 27. Cyse arrest . MDSCs de arrest , which ior cells –33. In aor cells , 34. (3)poptosis , 36.+ T lymphocytes cells that express CD25 and Foxp3 (CD4+CD25+ FoxP3+) [+ T cells to express Foxp3 and increase the number of Tregs [Tregs are a subgroup of CD4 FoxP3+) , and are FoxP3+) . Previou FoxP3+) , 40. Tre FoxP3+) , 42. At  FoxP3+) . Mark et FoxP3+) . MDSCs I FoxP3+) , and the FoxP3+) , 45. Ado FoxP3+) , In a st FoxP3+) . The app FoxP3+) . IL-10 a FoxP3+) . IL-10 cof Tregs , 51. It of Tregs . TGF-β rof Tregs , 54.+ T cells. Immunohistochemical tests among lung cancer patients showed that a large amount of immunosuppressive CD4+ T cells, including Th2 cells and Tregs, accumulated in the epithelium and stroma, whereas only a small number of Th1, Tfh, and Th17 cells were observed [Th cells are different subsets formed from naive CD4 + cells upon activation. They produce cytokines and play important roles in immune regulation via the secreted cytokines. They can be divided into Th1, Th2, and Th17 cells, depending on the specific cytokines that they produce. Th1 cells regulate viruses, bacterial infections, and the development of specific autoimmunity and secrete vital cytokines such as INF-α, IFN-γ, and IL-12. Their immune action is mainly achieved through the activation of immune cells such as macrophages, CD8 + T, and NK cells, to mediate protective immune responses within the TME , 56. Theobserved . Howeverobserved . Peng etobserved . In anotobserved .+ Th1 cells at the time of initiation and recall [Promotive and inhibitory regulatory relationships exist among MDSCs and Th cells within the lung cancer TME. A mutual inhibitory relationship between MDSCs and Th1 cells has been reported. Coculture experiments have found that Th1 produces cytokines such as IL-2 and IFN-γ, which are capable of reducing the number of MDSCs and inhibiting their function by inducing the production of chemokines like CXCL9 and CXCL10, and reducing the levels of immune factors like IL-6 and IL-1β that promote MDSC proliferation and activation . Yu et ad recall , which td recall . Additiod recall . In vivod recall , 71. Cocd recall .+ T cells, after activation by APC and recognition by the TCR of peptide-specific MHC-I complexes, activate and differentiate into effector CTLs, which are then capable of targeting and killing cells (such as infected cells and cancer cells) and secreting protective cytokines [+ T cells: migration and differentiation [+ T cells differentiates into effector CD8 + T cells, which are then activated and further differentiated into cytotoxic and memory CD8+ T cells [Naive CD8ytokines . In the ntiation , 75. Upo T cells , During  T cells .+ T cells, and the highly expressed iNOS and L-arginase I (Arg-1) of MDSCs promote the downregulation of CD3ζ chain expression of CD8+ T cells and inhibit the immune function of CD8+ T cells [+ T cells in vitro [+ T cells and inhibit their secretion of cytokines, which in turn prevents CD8+ T cells from exerting their immune response, promotes resistance to ICIs, and promotes tumor progression. The oversecretion of IL-10 by MDSCs may inhibit the production of protective factors such as IL-2, IFN-γ, and IL-12 by CD8+ T cells, thereby inhibiting their proliferation, leading to impaired antitumor immunity, and inducing apoptosis of CD8+ T cells [+ T cells. CTLS may produce IFN-γ, which in turn may inhibit the immune function of CTLS [+ T cells were promoted [+ T-cell activation, reducing T-cell proliferation, and decreasing the expression of cytotoxic molecules [Increased numbers of MDSCs in patients with lung cancer may inhibit the generation of protective immune CD8 T cells . MDSCs g T cells , 80. The T cells , and inh T cells . Combini T cells . Furtherin vitro . PD-1 re T cells , 80. The of CTLS , Howeverpromoted , It has promoted . Schouppolecules .+ T cells and induce apoptosis of CD8 + T cells by increasing ROS secretion and activating the Fas/Fas-L pathway [+ T-cell DNA damage and activating the p53 pathway [+ T cells through transcription factors: Marigo's experimental study found that the immunomodulatory activity of MDSC was dependent on C/EBP-β transcription factors, and the immune tolerance of CD8+ T cells was reversed after C/EBP-β was cleared from the bone marrow of tumor mice [Exosomes secreted by MDSCs deplete CD8 + T cells in mice. In vitro experiments have revealed that exosomes secreted by MDSCs can inhibit the proliferation of CD8 pathway . This me pathway . In addimor mice .+ T cells, which are involved in cancer progression [NKT cells are a subpopulation of T cells that express both the TCR and NK cell receptor NKR-P1. NKT cells produce large numbers of cytokines and, like NK cells, NKT cells can exert cytotoxic effects. They play immunomodulatory roles in cancer, autoimmunity, allergies, infections, and other diseases. Similar to Th1/Th2 cells, NKT cells can be divided into type I NKT cells for protective immunity, type II NKT cells for suppressing immunity, and invariant NKT (iNKT) cells. The glycolipid ligand galactose ceramide  activates iNKT cells in a cd1-dependent manner , 90, andgression . Type IIgression –99, and gression  + effector T cells. Under normal immune conditions, immunosuppressive molecule receptors can be temporarily expressed on the surface of activated effector T cells, but are quickly downregulated as T cells are activated. However, these inhibitory molecules cannot be downregulated during T-cell exhaustion and continue being strongly expressed on the effector T-cell surface. T-cell exhaustion is one of the major factors affecting the efficacy of immunotherapy; it not only causes effector T cells to lose their antitumor immune function but also causes ICIs and other immunotherapy methods to lose their roles and targets [T-cell exhaustion refers to the excessive activation of T cells in response to persistent antigenic stimulation, such as viral or tumor stimulation, the continuous co-expressing multiple inhibitory receptors on their surfaces, the inactivation of cellular effector effects, and the inability to differentiate into memory T cells and perform immune functions, such as tumor cell killing or virus clearance. The most significant features are the loss of effector cytotoxic functions , leading to changes in the expression of key transcriptional factors, and the upregulation or coexpression of multiple inhibitory molecular receptors  , 101, an targets . Studies targets .General control nonderepressible (GCN) is a serine/threonine kinase found in eukaryotic cells that may influence bone marrow function by regulating metabolism or protein production. It is a key driver of macrophage and MDSC polarization within the TME. Halaby found that the ability of GCN2 cKO tumors to express the T-cell exhaustion ligands CD206 and PD-L1 was significantly decreased. Simultaneously, the expression levels of PD-1 in CD4 + and CD8 + T cells decreased significantly. Flow cytometry showed that the expression of PD-1, LAG3, TIM3, and TIGIT on the surface of CD8 + T cells also decreased significantly. Thus, GCN, which promotes the polarization of MDSCs, can induce T-cell exhaustion .+ T-cell expression in lung cancer patients, and MDSCs can directly induce CD8+ T cells to express T-cell exhaustion inhibitory receptors such as PD-1, TIGIT, LAG3, CTLA4, and TIM3 [+ T cells, deprives effector T cells of glucose utilization, reduces the expression on CD8+ T cells, and inhibits antitumor functions [MDSCs negatively correlate with CD8and TIM3 , 106. MDand TIM3 . CD155, unctions .+ T cells and in the production of antitumor cytokines, both of which are evidence of CD8+ T-cell exhaustion [+ Tex cells, thereby enhancing viral control [MDSCs can cause T-cell cycle arrest by secreting IDO. However, the application of IDO1-shRNA to inhibit the expression of IDO1 in lung cancer mice downregulates the expression of the inhibitory receptors PD-1 and BTLA on T cells, positively regulates the secretion of cytokines such as IL-2 and TNF-α, reverses T-cell exhaustion, delays the onset of tumors, and inhibits tumor growth . Cytokinhaustion , 111 The control .+ T cells to proliferate and differentiate into Tregs and release immunosuppressive factors like TGF-β, IL-10 and IDO [MDSCs can also induce T cells exhaustion through an immunosuppressive microenvironment. MDSCs can induce primary CD4 and IDO . Transcr and IDO , or inhi+ or CD8+ T cellsare activated upon exposure to antigens and can differentiate into effector and memory T cells. Effector T cells bind specifically to antigens and memory T cells, which persist as a heterogeneous population at multiple sites, and can coordinate protective immune responses to re-exposure. There are two subgroups of memory T cells: central memory T cells (Tcm) and effector memory T cells (Tem). Tcm is primarily located in secondary lymphoid organs and can rapidly proliferate and differentiate into effector T-cells in response to antigens. Tem cells are mainly located in the peripheral tissues and can rapidly produce effector cytokines once stimulated by antigens [+ and CD8+ memory T cells in lung cancer patients contributes to long-term immunity [+ and CD4+ T-cell responses, thus promoting the metastasis and the progression of lung cancer [+ TEM PD-1 expression, which promotes CD4+ TEM exhaustion [+ T cells increase, and the STAT-3 pathway can also be activated to maintain long-term memory and improve antitumor immune mechanisms [Memory T-cell exhaustion is a new direction in tumor immunotherapy research. The degree of exhaustion of early memory T cells and T cells has been reported to correlate with the therapeutic efficacy of chimeric antigen receptor (CAR) T cells . Reversiantigens . Both Teantigens . They haantigens . TSCM isantigens . The metimmunity . Howeverg cancer , 122. Inhaustion . When MDchanisms  EMT refers to the morphological transformation of epithelial cells to a fibroblast or mesenchymal phenotype in response to physiological or pathological stimuli. Loss of cell polarity, rearrangement of the cytoskeleton, increased migratory motility, and resistance to apoptosis are important processes that mediate tumor invasion and metastasis . When MD+ and CD8+ T cells in the TME, recruitment of Tregs, and the exhaustion of T cells [+ and CD8+ T cells and promote CD4+FOXP3+ Treg differentiation, inhibiting the function of Th1 cells [MDSCs can inhibit T-cell immune functions in various ways, and the EMT process mediated by MDSCs is linked to the reduction of CD4 T cells , 132. Th T cells , and the T cells . EMT-act T cells . EMT is  T cells . High leh1 cells –138, andh1 cells . In addih1 cells , 141 (Fi+ and CD8+ T cells, memory T cells, and other immune cells were increased, and the secreted antitumor factors were also increased, which could promote the antitumor immune response [+ T cells, and improve antitumor immunity [+ T-cell infiltration, reduces Treg infiltration, alleviates chronic inflammation, and inhibits tumor progression and metastasis [+ T cells in the bronchoalveolar lavage fluid, and increased the antitumor effect of GalCer in a model of lung metastasis [Koh et al. used anti-Gr1 or anti-Ly6G to deplete MDSCs in mice with lung cancer; the activity and number of CD4response , 6. Studimmunity , 143. Intastasis . L-name,tastasis .+ and CD4+ T-cell exhaustion, and improve the quantity and quality of memory T cells, to further promote the T-cell-mediated antitumor immune response and enhance the therapeutic efficacy of chemotherapy [MDSCs immunosuppressive effect in the TME affects the long-term efficacy of lung cancer chemotherapy . Gemcitaotherapy , 147.+ T cells in multiple immune organs in mice, thereby enhancing the antitumor effect of anti-PD-1 treatment [+ and CD8+ T-cell infiltration and inhibiting Treg cell expression (Table ICIs have achieved some efficacy in NSCLC, and inhibitors targeting PD-L1 and/or PD-1 have been approved for use in NSCLC. Targeting PD-L1 and PD-1 increases tumor-specific T-cell immunity. PD-1/PD-L1 checkpoint inhibitors have demonstrated efficacy in advanced and refractory NSCLC in several clinical studies . MDSCs creatment . MEKi, Con Table  [150–152+ T cell activity, activate the antitumor immune response, and inhibit metastasis [+ T cells, promote antitumor immune responses, and enhance the effect of chemotherapy [Products that target MDSCs have also been shown to have inhibitory effects on the number and function of MDSCs. One of the most prominent features of MDSCs is ARG1. In recent years, vaccination for antibodies against ARG1, coupled with inhibitors of the immune checkpoint, have been found to increase T-cell infiltration, restore the function of CD8 + T cells, and promote antitumor immune responses , 154. Cltastasis . There atastasis . Clinicaotherapy . CurrentIn general, MDSCs in the peripheral blood and tumor tissues of lung cancer patients are often higher than those of healthy people. MDSCs inhibit the T-cell immune functions by upregulating the immunosuppressive pathway and secreting immunosuppressive molecules and chemokines, which promote the immunosuppression and treatment resistance of lung cancer patients, which then leads to tumor progression and metastasis. Therefore, MDSCs have been identified as a major impediment in treating patients with lung cancer, and the depletion of MDSCs in the lung cancer microenvironment has certain clinical implications for the treatment of lung cancer. Drugs targeting MDSCs have been developed, but there are still many open questions about their mode and mechanism. The mechanism of relevant drugs should be clarified in future experiments to ensure positive results in all lung cancer patient populations.


We report the developmentof an isothiouronium salt as a reagentfor the operationally simple synthesis of cyanomethyl thioesters withhigh functional group tolerance and avoiding the use of thiols. Additionally,we show that the products can be engaged in amide synthesis in eithera two-step or one-pot fashion. Perhaps the keythioester in this context is acetyl CoA, which plays a crucial rolein the citric acid cycle and thus in ATP production.3 Moreover, thioesters function as intermediates in the ubiquitinationof proteins,5 leading to protein degradation,6 DNA repair,7 or cellsignaling.8 To date, several thioester-containingdrugs have been developed, including fluticasone propionate (asthmatreatment),9 spironolactone (heart failureand hypertension),10 and derivatized GW870086(another fluticasone derivative with anti-inflammatory properties)11 (Thioesters are a privilegedclass of organosulfur compounds widely found in a variety of relevantsubstances for humankind.rties)11 1A.12 or anhydrides13). Recently, thioestersyntheses employing photoredox catalysis have also been developed.20 Although transition-metal-catalyzed approaches have been described,functional group tolerance remains a challenge in these variants.22 Thiocarboxylic acids also feature as suitable starting materialsfor thioester synthesis, either by reaction with an alkyl halide27 or coupling to a Michael acceptor.29 Nevertheless,thiols and thiocarboxylic acids suffer from limited commercial availabilityand are prone to oxidative dimerization . Interestingly, among these, only 2-(cyanomethyl)-1,1,3,3-tetramethylisothiouroniumbromide (2a) was found to productively react with benzoicacid to yield the corresponding thioester (Supporting Information for full optimization). Initially, reactions wereperformed in chloroform employing triethylamine as the base. However,under these conditions erratic behavior was observed, likely dependingon the quality of the chloroform employed as solvent, with aciditypresumably being a contributing factor (3 (entry 6). Finally,when 1.5 equiv. of isothiouronium salt 2a was employed,the desired thioester 3a was formed in 95% yield (NMR)(entry 7).Several isothiouronium salts were prepared quantitatively usingthis strategy , ortho-substitution led to lower efficiency(3d). Electron-withdrawing substituents were also tolerated(3e), as were heteroaromatic carboxylic acids , which gave moderate yields, while acidsbearing purely aliphatic substituents (3h) performedwell in this reaction. Olefinic substituents  were well tolerated. Notably, an unprotected hydroxylgroup did not interfere with thioesterification (3k)—aninteresting observation, given our recent work on alcohol-to-thioetherexchange promoted by isothiouronium salts.37 To assess the occurrence of racemization, (S)-(+)-2-phenylpropionicacid and Cbz-protected proline were subjected to the reaction conditions,providing products 3l  and 3m  with no observable epimerization. In lightof this finding, we further applied our procedure successfully toa diastereomerically pure cyclopropane (3n), which alsoreacted smoothly. Dehydroabietic acid, a particularly hindered substrate,also smoothly underwent conversion to the cyanomethyl thioester derivative 3o. Moreover, we were pleased to see that the reaction iseasily scalable, affording 98% yield of 3a on a 5 mmolscale. Thus, isothiouronium salt 2a offers a versatilealternative to procedures described in literature for the synthesisof cyanomethyl thioesters.With optimized conditions inhand, we studied the impact of varyingthe carboxylic acid coupling partner 3, focusi5a, 5b), and we once more found that hydroxylsubstituents were well tolerated (5c). Additionally,allylamine (5d), an α-secondary benzylic amine(5e), and pyridin-2-ylmethanamine (5f) wereamenable to amide coupling. Secondary amines are also suitable substrates,as demonstrated by the use of morpholine (5g). Additionally,reaction of 3j with cyclohexylamine gave the desiredamide (5i) in good yield. Notably, amide 5c could also be synthesized when water was used as a solvent insteadof acetonitrile.We then investigated cyanomethyl thioesters as precursors to amides.In acetonitrile at slightly elevated temperatures, these thioestersreadily reacted with a range of amines to provide the correspondingamides. Amines with aliphatic substituents gave excellent yields (Supporting Information) in moderate to good yields for this two-step,one-pot process.Next, we moved to the development of a one-potprocedure. Herein,after formation of the thioester as described previously, the correspondingamine was directly added to the reaction mixture. We found that, underthe same conditions as shown above, the desired amide products werereadily obtained are tolerated in a procedure which does not rely on reactants withlimited stability such as thiocarboxylic acids and thiols. Notably,both thioester formation as well as amide coupling can be performedwithout exclusion of air, enabling a particularly facile set up.Supporting Information.See thissection in the