Protein palmitoylation is a reversible post-translational modification by fatty acids (FA), mainly a palmitate (C16:0). Palmitoylation allows protein shuttling between the plasma membrane and cytosol to regulate protein stability, sorting and signaling activity and its deficiency leads to diseases. We aimed to characterize the palmitoyl-proteome of ovarian follicular cells and molecular machinery regulating protein palmitoylation within the follicle. For the first time, 84 palmitoylated proteins were identified from bovine granulosa cells (GC), cumulus cells (CC) and oocytes by acyl-biotin exchange proteomics. Of these, 32 were transmembrane proteins and 27 proteins were detected in bovine follicular fluid extracellular vesicles (ffEVs). Expression of palmitoylation and depalmitoylation enzymes as palmitoyltransferases (ZDHHCs), acylthioesterases (LYPLA1 and LYPLA2) and palmitoylthioesterases (PPT1 and PPT2) were analysed using transcriptome and proteome data in oocytes, CC and GC. By immunofluorescence, ZDHHC16, PPT1, PPT2 and LYPLA2 proteins were localized in GC, CC and oocyte. In oocyte and CC, abundance of palmitoylation-related enzymes significantly varied during oocyte maturation. These variations and the involvement of identified palmitoyl-proteins in oxidation-reduction processes, energy metabolism, protein localization, vesicle-mediated transport, response to stress, G-protein mediated and other signaling pathways suggests that protein palmitoylation may play important roles in oocyte maturation and ffEV-mediated communications within the follicle. Palmitic acid C16:0) is the most abundant fatty acid (FA) produced in mammalian cells, which represents 32% of total FA content in bovine oocyte 6:0 is th,5,6. Prohttps://swisspalm.org/, accessed on 12 March 2021) [Development of methodological approaches of large-scale protein palmitoylation profiling in mammalian cells, which are based on acyl-biotin exchanges (ABE) method ,16,17, ach 2021) . In the ch 2021) ,18,19. Mch 2021) , althougch 2021) ,24. In mch 2021) .ZDHHC genes is linked to cancer progression [ZDHHC genes are involved in pathogenesis of known neurological disorders as Huntington’s disease (DHHC17), schizophrenia (ZDHHC8) and X-linked mental retardation (ZDHHC9 and ZDHHC15) [The mechanism of protein palmitoylation are extensively studied in human cells. Although some proteins are able to autopalmitoylate spontaneously , the majgression , and mangression . MutatioZDHHC15) ,34. CLN1 gene) and its paralog PPT2 can remove thioester-linked fatty acyl groups from modified Cys residues in palmitoylated proteins or peptides during lysosomal degradation [Protein depalmitoylation is a part of palmitoylation dynamics and involves several enzymes, including two members of serine hydrolase family, acyl-protein thioesterases (APTs) LYPLA1 and LYPLA2 that cleaved palmitate from S-palmitoylated proteins . In addiradation . In addiradation ,35. Reperadation , and plaReversible protein palmitoylation allows shuttling the proteins between cell compartments, including subcellular vesicle transport . SignifiAs any biological fluid, ovarian follicular fluid (FF) contains EVs of different types, which differ by their genesis, size and biological properties. Large/medium size EVs  formed by outward budding and fission of the plasma membrane. Small 30–150 nm EVs, also defined as exosomes, are formed within the endosomal network, and released from multivesicular bodies upon their fusion with the plasma membrane ,41. In bThe objective of our study was to characterize, for the first time, protein palmitoylation machinery in bovine ovarian follicle. We have identified palmitoylated proteins from follicular GC and cumulus-oocyte complexes and looked for these proteins in ffEVs. Expression of specific ZDHHCs and depalmitoylation enzymes was analyzed in follicular GC, CC and oocytes, and then investigated in relation to oocyte maturation. p < 0.05), were considered potentially palmitoylated proteins. All these potentially palmitoyl-proteins correspond to 84 proteins with unique identifiers (IDs), which are reported in In order to identify palmitoylated proteins in bovine follicular cells, we performed purification of the proteins with thioester-bonded palmitate, using an acyl-biotinyl exchange (ABE) protocol from bovine GC and COCs  were identified from both COCs and GC samples, and six proteins were only identified from COCs: DCD, LDHC, PRR9, SELENBP1, S100A9 and TUBB4B , revealed only 10 proteins (11.9%) common with our list of 84 potentially palmitoylated proteins , other were for the first time reported as palmitoyl-proteins in bovine. However, when comparing with human SwissPalm dataset (4598 protein IDs), 76 out of 84 proteins were already detected in one or more human palmitoyl-proteomes  transmembrane proteins TMPs [Using Membrane Proteome database  were already identified as a part of human EV protein cargo . To compWe then compared the lists of the proteins identified from bovine follicular fluid exosome-like EVs (294 IDs) , cumulusIn summary, the comparative analyses of proteomic data suggests that a significant portion of here identified palmitoylated proteins are TMBs and could be a part of protein cargo of exosome-like EVs released and/or captured by GC, CC and the oocyte. g: (n = 606). To confirm the presence of potentially palmitoylated proteins in follicular fluid EVs, the small ffEVs were isolated from follicular fluid by differential centrifugation and precipitated at 100,000× g: a. ElectrWestern-blot analyses of total proteins from GC, CC and ffEVs were performed in order to detect several protein-candidates identified as palmitoylated in this study b. ProteiEnrichment analysis of cellular component GO terms of 84 palmitoyl-protein IDs is shown in In this case, 10 proteins were matched with GO term Cytoskeleton. The most enriched GO terms in cellular components were ER-Golgi intermediate compartment, Recycling endosome membrane, and Trans-Golgi network membrane . Analysis of Biological Process and Molecular Function GO enrichment of palmitoylated proteins from follicular cells using DAVID software is shown in In addition, detailed GO analysis of the list of 84 follicular palmitoyl-proteins revealed their particular association to GO term Protein Localization to Membrane, in accordance with numerous TMPs identified. Significant enrichment of palmitoyl-proteins to several GO terms related to biological processes and molecular functions was observed . Thus, GO:0055114 Oxidation-Reduction Processes regrouped the proteins HSD17B1, TMX4, TMX1, AIFL1, SELEBP1, PRDX6, FASN, PLOD1, DLAT, ACAT1, PGK1, ACAA2, ALDH6A1, LDHC, IDH2, GNAS, BLVRA and SCCPDH. Proteins S100A9, S100A8, SCAMP3, SCCPHD1, MLEC, CKAP4, DNAJC5, CD36, ERGIC3, LMAN1, ERP44, PI4K2A, CANX, TUBB4B, PRDX6, CD81, TMX3, CD58, SCAMP2, SORT1, VALP3, SCARB2 and M6PR are associated with GO:0016192 Vesicle Mediated Transport. Proteins VAMP3, SCARB2, TMX3, TMX4, TMX1, MAN1B, ERP44, AIFM1, CD58, TUBB4B, PRDX6, LMAN1, CD36, ACAT1, ACAA2, PGK1, SIGMAR1, SERPINH1, MMP14, GNAS, GN11, GNAI1, GNA13, S100A9 and S100A8 are related to GO:0006950 Response to Stress. Moreover, 40 out of 84 palmitoyl-proteins matched GO:0003824 Catalytic activity, notably Oxidoreductase activity , and GTPase Activity . 25 palmitoyl-proteins are related to GO:0036094 Small Molecular Protein Binding, notably GO:0005102 Signalling Receptor Binding , GO:0050662 Coenzyme Binding , and GO:1901567 Fatty Acid Derivative Binding . p < 0.05) enriched  between these cells  showed similar expression level in GC, CC, and oocytes. Heatmap representation of normalized gene expression values showed that 13 differential genes overexpressed in somatic cells (GC and CC) compared to the oocytes (CLN1 (coding PPT1), acyl-protein thioesterases LYPLA1 and LYPLA2, and palmitoyltransferases ZDHHC4 and ZDHHC5  were analyzed in bovine oocytes, follicular granulosa cells (GC), and cumulus cells (CC). From the global transcriptome dataset earlier obtained on bovine GC, CC, and oocytes (GEO accession GSE149151), we could retrieve the normalized expression values for 37 out of 41 genes coding for palmitoylating and depalmitoylating enzymes, and performed differential analysis of gene expression in GC, CC and oocyte . In thisse cells , and eigd ZDHHC5 b. Clusted ZDHHC5 a include ZDHHC16 b.Therefore, bovine oocytes, CC and GC express different enzymes, which are involved in protein palmitoylation and depalmitoylation, and their expression patterns differ between these cell types.To investigate expression of palmitoylation and depalmitoylation enzymes in bovine follicular cells and oocytes, we have compared the list of 41 known enzymes with the lists of proteins identified from bovine oocytes , cumulusThus, in silico analysis of follicular cells transcriptomes and proteomes revealed the most abundant enzymes, which can regulate protein palmitoylation in oocytes, GC, and CC.Using immunofluorescence (IF), we have analyzed the localization of ZDHHC16, PPT1, PPT2, and LYPLA2 in GC and cumulus-oocyte-complexes (COCs) . In granIn COCs, ZDHCC16 demonstrated strong heterogeneous labelling within the ooplasm and, at a lower level, in the cytoplasm of attached CC. Both PPT1 and PPT2 enzymes were also detected in the oocytes and surrounding CC. PPT1 was relatively homogenously localized through the ooplasm, but more significant abundance of PPT1 was observed in CC cytoplasm. In contrast, PPT2 was more abundant in the oocyte, where it was spread through the ooplasm and more concentrated to oocyte nucleus . In CC, PPT2 was observed not only in the cytoplasm by also in the nuclei. Therefore, intracellular localization of one potential palmitoyltransferase ZDHHC16, palmitoyl-protein thioesterases PPT1 and PPT2, and acyl-thioesterase LYPLA2 was confirmed at the protein level in bovine oocytes, CC and GC. We have analyzed transcriptomic data that were previously obtained on bovine CC, which surrounded either immature oocytes, or the oocytes matured in vivo, or the oocytes after 24 h of in vitro maturation (IVM) . DiffereZDHHC4, ZDHHC7, ZDHHC16, ZDHHC18), and depalmitoylation enzymes . Expression of ZDHHC4, ZDHHC7 and ZDHHC18 was significantly increased whereas expression of CLN1 and LYPLA2 significantly decreased during maturation (p < 0.05). Expression of many others ZDHHCs also increased in CC during oocyte maturation in vivo and/or in vitro ; in contrast, ZDHHC13, the only one PAT detected in CC, was significantly more abundant in in vivo matured CC compared to in vitro matured (p < 0.05); other enzymes did not show any significant difference. At the protein level, analysis of the bovine CC proteome from in vivo or in vitro matured COCs  demonstr matured . In addiBy analyzing quantitative proteomics data on bovine immature and in vitro mature oocytes from the recent study , we founBos taurus proteins were identified as palmitoyl-proteins [In the present study, we have isolated and identified for the first time 84 potentially palmitoylated proteins from ovarian follicular cells in bovines. According to recent data presented in SwissPalm, 19.86% of human proteins were detected or predicted to be palmitoylated; however only 0.54% of proteins . This isproteins , retina proteins , or pulmproteins . Taking proteins , and 28 The identified follicular palmitoyl-proteins are involved in different molecular functions and biological processes, similar to other palmitoyl-proteomes. Functions of reported here palmitoyl-proteins were related to protein processing and secretion, binding of small molecules as ions, nucleotides or lipids, protein-protein binding, FA transport, mitochondrial activity and energy metabolism, various enzymatic activities, signal transduction and oxidative stress response. Many of them are transmembrane proteins, participating in signal transduction in response to hormonal stimulation through different pathways, such as PI3K signaling (S100A9), MAPK cascades (G-proteins), EGF-signaling , IGF1-signalling (ITGA6) or WNT-signaling  and others. Lipid and carbohydrate metabolism were among the most enriched functional pathway of identified palmitoyl-proteins. According to Swisspalm database, many proteins involved in FA metabolism, including FA transporters, enzymes of lipid synthesis and transformation are palmitoylated in human cells. In our study, CD36, a major lipid transporter of FAs to the cell, was identified in follicular cells in palmitoylated form. In mice adipocytes, only palmitoylated CD36 mediates FA uptake through caveolar endocytosis, and depalmitoyalation of CD36 initiates the formation of endocytic vesicles, which assure delivery of FAs inside the cell and target them to lipid droplets . TherefoGap-junction functional pathway and vesicle mediated transport were among the enriched GO terms of follicular palmitoyl-proteins. These functional activities and pathways largely imply bidirectional oocyte-somatic cells communications and are crucial for oocyte growth and maturation within the follicle. Cell interactions permanently occur between the oocyte and surrounding cells through cell-to-cell communications via gap-junctions ,63,64,65Protein palmitoylation has a role in targeting of the proteins not only to plasma membranes but also to the nucleus, spindle and cleavage furrow , which cMost of the palmitoyl-proteins identified here were matched to different membrane compartments: Golgi, recycling endosome membrane, lysosomes, exosomes and other membrane structures. This localization was expected, because the major functions of palmitoylation are stable membrane anchoring of soluble proteins and regulation of protein sorting by either partitioning of proteins into cholesterol-rich lipid rafts, or modulating protein-protein interactions, or regulation of ubiquitination status . In factRegulation of protein palmitoylation within the follicle may affect not only sorting, localization and activity of the modified proteins, but may also regulate stress response to lipotoxicity, which occurs when level of free saturated FAs, especially palmitate, is elevated and affects oocyte quality ,77,78. AThe tight relationship between protein palmitoylation and intracellualr lipid metabolism metabolism is also supported by high depalmitoylation activity in the mitochondria of mammalian cells, suggesting a role of palmitoylation in mitochondrial lipid homeostasis, and therefore cell functioning and viability . ZDHHC4 and ZDHHC14 were overexpressed in somatic follicular cells (GC and CC) in contrast to ZDHHC3, ZDHHC5, ZDHHC13 and ZDHHC16, which demonstrated higher expression in the oocytes. In concordance, although ZDHHC16 protein was detected by immunofluorescence in follicular somatic cells, the most intensive immunostaining was observed in the oocyte. Protein palmitoylation occurs mainly at the plasma membrane and at the Golgi apparatus, although palmitoylated proteins could be localized to various subcellular organelles, including mitochondria, nucleus, ER, and others . Granular aspect of ZDHHC16 immunofluorescence labeling corroborates with preferential location of several PATs to Golgi, ER and plasma membranes [In the present study, we characterized enzymatic machinery of protein palmitoylation in bovine ovarian GC, CC and enclosed oocytes, for the first time. According to transcriptomic data, most of the genes coding the enzymes, which either catalyze protein S-palmitoylation (PATs and associate proteins), or remove palmitate (APTs and associate proteins) demonstrated cell-specific expression pattern of protein palmitoylation in follicular cells and enclosed oocyte. In human, substrate-specific PAT activity was reported for 17 out of 23 known ZDHHC proteins . Accordiembranes . Xenopus egg, ZDHHC3 palmitoylates a protein Gs alpha (GNAS), and oocyte-specific knockdown of ZDHHC3 led to the acceleration of progesterone-induced meiosis resumption, demonstrating the importance of PAT-mediated palmitoylation in oocyte meiosis [Zdhhc13-deficient mice model [A protein can be palmitoylated specifically by single ZDHHC, or by several PATs. In mice, FA transporter CD36 is specifically palmitoylated by ZDHHC5 . ZDHHC7  meiosis . Transcr meiosis  and in C meiosis  indicatece model . Among tce model , and in ce model . Proteince model . Thus, pLYPLA1 and LYPLA2, palmitoyl-thioesterases PPT1 (gene CLN1) and PPT2 (PPT2), as well as two ABHD enzymes (genes ABHD12 and ABDH17C) strongly and cell-specifically expressed in follicular somatic cells and enclosed oocyte. CLN1 and ABHD12 significantly more expressed in GC and CC, whereas PPT2 and ABDH17C overexpressed in the oocytes. According to proteome analysis of bovine granulosa cells [Protein depalmitoylation is promoted by specific enzymes including APTs, PPTs and ABHD-containing proteins . In our sa cells , cumulussa cells ,56 and osa cells ,53, the sa cells . The onlsa cells . CLN1 (coding for PPT1) and LYPLA2 decreased during maturation of enclosed oocyte. In concordance, proteomic data revealed significantly lower abundance of PPT1 and LYPLA2 proteins in bovine CC surrounding in vitro matured oocytes compared to in vivo maturation [In CC, genes turation . In the turation . These dturation . TherefoGlobally, expression modulations of palmitoylation/depalmitoylation enzymes in CC accompanied oocyte maturation, and in vivo changes of protein palmitoylation machinery were more significant than after IVM. Indeed, oocyte competence to develop embryo was higher after in vivo oocyte maturation compared to IVM in cow ,86,87, tIn conclusion, it the present study we characterized molecular machinery of protein palmitoylation in bovine granulosa cells, cumulus cells and oocytes, and identified the most abundant palmitoyl-proteins from these cells. Palmitoylation is likely mediated by several highly expressed ZDHHCs, which increase their expression in CC during oocyte maturation, whereas expression of depalmitoylation enzymes decreased. In the oocyte, depalmitoylation seems to increase and thus may be involved in meiosis regulation. Follicular palmitoylated proteins are involved in different cell metabolism processes and signaling and can be transported by follicular fluid extracellular vesicles. However, the role of palmitoylation in functioning of each of these proteins in follicular cells or oocytes remains to be investigated.No experiments with living animals were performed. All chemicals were provided by Sigma-Aldrich  unless others stated. Bovine ovaries were collected at a commercial slaughterhouse. Bovine follicular cells, mainly granulosa (GC) and cumulus-oocyte complexes (COCs) were retrieved from the antral follicles 3–8 mm in diameter by vacuum aspiration. Immature COCs with compact cumulus layers were selected and washed in TCM199-HEPES medium complemented with 0.04% BSA, and 25 μg/mL gentamycin. g for 10 min and washed by pipetting with modified McCoy’s 5A serum-free medium containing 3 mM L-glutamine and 20 mM HEPES (pH-7.6). Suspended GC were dropped off on a Percoll density medium  and purified GC were collected after centrifugation . Selected COCs and GC samples were then washed twice in sterile PBS, centrifuged again and the pellets kept at −80 °C until protein isolation and proteome analysis. For proteomic studies, GC from the sediments of follicular fluid and TCM199-HEPES after COCs’ retrieval were centrifuged at 300× General workflow of analyses of protein palmitoylation in bovine follicular cells included proteomic approach to purify and identify palmitoyl-proteins and in silico analyses of their functions . In addiAcyl-biotinyl exchange chemistry (ABE) technology  was usedn = 500), collected from about 120 ovaries in tree experiments, using 10 mM N-ethylmaleimide (NEM) to block the free thiols. After removing the NEM, the subsequent treatments with hydroxylamine (HA) were performed in order to cleave the palmitoylation thioester linkages. Following treatment with HPDP-biotin biotinylated free thiols. Proteins were precipitated three times using chloroform-methanol, resuspended in 4% SDS and divided into two equal portions (+HA sample and −HA sample as control). The +HA sample was diluted five-fold by addition hydroxylamine-containing +HA buffer ; for the −HA samples, −HA buffer  was added. The +HA and −HA samples were incubated at room temperature for 1 h with end-over-end rotation, and then precipitated to remove chemicals. After serial precipitations, affinity purification of biotinylated proteins was performed per each −HA and +HA sample, then precipitated using 100% trichloroacetic acid (TCA), and stored −20 °C.Briefly, total proteins were extracted from frozen pellets of GC , and COCs  coupled to an Ultimate® 3000 RSLC Ultra High Pressure Liquid Chromatographer . For identification of palmitoylated proteins, each pellet from +HA and −HA samples was resuspended in SDS-PAGE sample buffer containing 4% SDS, 5% beta-mercaptoethanol, 125 mM Tris-HCl pH 6.8, 20% glycerol and bromophenol blue as the dye, and incubated for 5 min at 95  °C, then centrifuged at 10,000× Bos taurus protein databases was performed to obtain a species-specific identification of the protein. The abundance of identified proteins within one sample was estimated by calculating the Exponentially Modified Protein Abundance Index [For protein identification, mass spectrum ion searches were performed using Mascot search engine v2.3.2  via Proteome Discoverer 2.1 software . Research was performed against the mammalian non-redundant NCBI database (released July 2018). Peptides and proteins identified by MASCOT were subjected to Scaffold v4.8.4 software . Peptide and protein identifications were accepted if they could be established at greater than 95% probability by the Peptide Prophet algorithms ,89, respce Index . To characterize enriched palmitoylated proteins, +HA and −HA conditions analyzed in triplicate , with three technical replicates per band were compared using Scaffold Q+ software . Label-free quantitative approach was employed. Protein clusters were defined as enriched palmitoylated proteins if they met the following conditions: (1) detection once among the three replicates only in +HA and never in control condition −HA; (2) detection in both conditions but with an enrichment in +HA with a fold change > 3. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE  partner p-values. Multiple pair analysis was performed by Tukey test. Difference at p < 0.05 was considered significant. Microarray expression data were validated by real time PCR for 10 genes involved in lipid metabolism in our previous study [Microarray expression data (public repository Gene Expression Omnibus (GEO) accession number GSE149151) were used for analysis of gene expression in GC, CC and oocytes. Normalized expression values were retrieved for the genes coding the enzymes involved in palmitoylation (GO:0018345) and depalmitoylation (GO:0018345) and identified palmitoylated follicular proteins. The analysis of differential expression between the follicular cells  was performed on normalized expression values of four biological replicates using XLSTAT software  by applying ANOVA with Benjamini- Hochberg correction of us study ; in addius study .https://swisspalm.org/, accessed on 12 March 2021), which contains the proteins with experimentally detected palmitoylation [http://microvesicles.org/index.html, accessed on August 2018), which contains the information about molecular composition of extracellular vesicles in different species [https://membranome.org/, accessed on 22 March 2021) holding structural and functional data of more than 6000 single-helix (bitopic) transmembrane proteins [For functional annotation of palmitoyl-proteins, we have used SwissPalm database, released 3, 8 September 2019  and DAVID functional Annotation Bioinformatics microarray analysis . Gene Ontology analyses were performed with FunRich software v3.1.3. , STRING g 15 min, and the second one at 2000× g 15 min at room temperature. The samples of clarified follicular fluid were then centrifuged 30 min at 12,000× g at 4 °C to remove the large EVs. To sediment the small ffEVs, supernatants were ultra-centrifuged at 100,000× g for 90 min at 4 °C, and the pellets were washed with 4 mL PBS and centrifuged for 90 min at 100,000× g. Pellets were resuspended in 25 µL of PBS per 1 mL of original follicular fluid. The preparations of ffEVs were kept at −80 °C. Small extracellular vesicles (exosome-like ffEVs) were isolated from the 3 pools of follicular fluid aspirated from 3–8 mm follicles by differential centrifugation, as previously described . BrieflyOf each fresh preparation of ffEVs, 3 µL were fixed with 3 µL of 2% glutaraldehyde solution in PBS at room temperature. TEM was performed with 3 µL of fixed EVs as described earlier . Microgrg. Protein samples were resolved on ready 4–12% SDS-PAGE gels  in replicates and transferred onto nitrocellulose membrane . After blocking in 5% dry milk/Tris-buffered saline/0.1%Tween (TBST), membranes were incubated with primary antibodies raised against human ATP1A1 , CD63 (1/500 dilution), CD81 (1/1000 dilution), PTGFRN (1/500), VIM (1/1000 dilution) and HSPA8 (1/1000) at 4 °C overnight. Membranes were washed four times using TBST and incubated with corresponding secondary horseradish peroxidase (HRP)-conjugated secondary antibodies  in TBST for 1 h at room temperature. Specific signals were revealed by chemiluminescent reagent SuperSignal™ West Dura Extended Duration Substrate . Each membrane was used to reveal at least different size proteins, after stripping of previous antibody. Signals were captured using a GeneGnome camera  and Genesys 1.5.4 software (Syngene). Total protein samples of GC, CC and ffEVs were prepared as described elsewhere . Briefly® Nunc, Thermo-Fisher Scientific, Courtaboeuf, France) for 24 h at 38.8 °C, in humidified thermostat at 5% CO2 and 20% O2. Culture media was removed, GC rinsed with PBS and used for immunofluorescence analysis (n = 3).GC, collected and purified as described above, were diluted McCoy culture medium supplemented with 5% of foetus calf serum at a concentration 1 million cells per mL and incubated on an 8-well chamber slide  using 20× objective or an oil 40× objective with a numerical aperture of 1.3 and the appropriate filters. Images were captured using Zen 2012 software .Immature COCs  partially stripped from their cumulus and GC after 24 h of culture were washed in Tris-buffer saline (TBS), fixed in 4 in PBS containing 4% paraformaldehyde (PAF) for 20 min and then washed in TBS-0.1% BSA for 3 min at room temperature. After blocking in TBS containing inactivated sheep serum (5%) and 0.1% Triton X100 for 30 min (GC), or 1 h (COCs) at room temperature, cells were incubated overnight at 4 °C in TBS containing BSA (0.2%) with primary rabbit antibodies against human PPT2 or ZDHHC16, or LYPLA2, or mouse antibodies against human PPT1 all at 1:100 dilution. Control cells were incubated with similar concentration of mouse or rabbit was IgG (negative control). Three 5-min washes (GC), or four 30-min washes (COCs) in TBS containing 0.1% BSA and 0.1% Tween 20 were performed, then samples were incubated with 1:400 dilution of secondary Alexa Fluor 488-conjugated goat anti-rabbit IgG and/or Alexa Fluor 594-conjugated goat anti-mouse IgG , in TBS containing 0.2% BSA for 1 h (GC), or 2 h (COCs) at room temperature. Then, four 10 min washes in TBS containing BSA (0.1%) and incubated in TBS with 2.5 µg/mL Hoechst 33258 for 15 min at room temperature to stain nuclear chromatin. Sections were then mounted using MoviolOvarian follicular cells express a number of specific enzymes controlling protein palmitoylation and depalmitoylation, and their expression patterns are cell-specific. In bovine oocytes and surrounding cumulus and granulosa cells, reversible palmitoyation is mediated by different ZDHHCs and APTs, which are strongly expressed in these cells and varied their expression during oocyte maturation. The most abundant palmitoyl-proteins within the follicle were identified. They matched to different biological processes and pathways attributed to palmitoyl-proteins in other cells, and were enriched in the proteins regulating energy metabolism, cell signaling and extracellular transport. Palmitoylated proteins could be transported by follicular fluid extracellular vesicles and therefore might be intermediators of intra-follicular signaling pathways, involved in follicular growth and oocyte maturation. Protein palmitoylation seems to be crucial for progression of oocyte maturation in vivo and in vitro although the roles of palmitoylation for each protein involved in these processes remain to be investigated.


This review aims to describe the type 1 tyramine receptors (TAR1s) in insects with a multidisciplinary approach and might be an important tool for a wide scientific audience, including biochemists, molecular physiologists, ethologists, and neurobiologists with a biological entomology background. In fact, in the last years, TAR1 has received much attention due to its broad general interest. The review is composed of a general introduction about the tyraminergic and octopaminergic systems and the corresponding tyramine (TA) and octopamine (OA) receptors, including the recent classification as well as their brief structural and functional information. The four chapters then describe TAR1s: (1) Molecular and structural characterization, with the purpose to provide a clear biochemical overview of the receptor that ensures a well-defined TAR1 identity; (2) pharmacology, in which a clear TAR1-mediated intracellular signaling pathway is detailed; (3) physiology and behavior, focusing on the TAR1-controlled traits in insects; (4) insecticide target, in which the knowledge on TAR1 roles in insects is associated with the growing evidence about the pest management strategies based on this receptor. The conclusions summarize TAR1 features as well as future directions on which the receptor research should move.Tyramine is a neuroactive compound that acts as neurotransmitter, neuromodulator, and neurohormone in insects. Three G protein-coupled receptors, TAR1-3, are responsible for mediating the intracellular pathway in the complex tyraminergic network. TAR1, the prominent player in this system, was initially classified as an octopamine receptor which can also be activated by tyramine, while it later appeared to be a true tyramine receptor. Even though TAR1 is currently considered as a well-defined tyramine receptor and several insect TAR1s have been characterized, a defined nomenclature is still inconsistent. In the last years, our knowledge on the structural, biochemical, and functional properties of TAR1 has substantially increased. This review summarizes the available information on TAR1 from different insect species in terms of basic structure, its regulation and signal transduction mechanisms, and its distribution and functions in the brain and the periphery. A special focus is given to the TAR1-mediated intracellular signaling pathways as well as to their physiological role in regulating behavioral traits. Therefore, this work aims to correlate, for the first time, the physiological relevance of TAR1 functions with the tyraminergic system in insects. In addition, pharmacological studies have shed light on compounds with insecticidal properties having TAR1 as a target and on the emerging trend in the development of novel strategies for pest control. Drosophila [Drosophila melanogaster [Apis mellifera [Periplaneta americana [Bombyx mori [i and Gq proteins, is described in the next chapters. On the other hand, TAR2 is thought to be a receptor highly specific for TA, and its activation elicits a selective stimulation of Ca2+ release [2+ levels [Drosophila [In insects, the main biogenic amines are dopamine (DA), serotonin 5-HT), tyramine (TA), and octopamine (OA). Together, they control and modulate a broad range of biological functions essential for the life of the insects. Whereas DA and 5-HT functions and pathways are highly conserved in both vertebrates and invertebrates, TA and OA can be considered the invertebrate counterparts of the catecholamines epinephrine and norepinephrine of vertebrates . TA, OA,-HT, tyraosophila . Octα-Rsnogaster , is exprnogaster  and has ellifera , Periplamericana , and Bombyx mori . All α-abyx mori , reprodubyx mori , circadibyx mori . The othbyx mori , locomotbyx mori , and feebyx mori . The TAR release . TAR2 se release ,29,30,31+ levels ,15. The osophila , therewiosophila .TAR1s, like all GPCRs, consist of a single polypeptide chain containing seven hydrophobic transmembrane domains, connected by six hydrophilic loops, along with an extracellular N-terminal and an intracellular C-terminal region . To dateP. americana expresses a second type 1 tyramine receptor, named PeaTAR1B (accession number: LT900530), in addition to the PeaTYR1 or PeaTAR1A (accession number: AM990461) [D. melanogaster and called Tyr-dro [D. melanogaster TAR1, a putative eighth transmembrane domain was found close to the N-terminal region [Drosophila suzukii and Phormia regina TAR1 [D. melanogaster, D. suzukii, and P. regina, respectively [Sitophilus oryzae TAR1, the IL3 region is a very flexible element and is stabilized by TA binding, a key event for signaling with the G-protein [A. mellifera TAR1 has a relatively short IL3, composed of 110 amino acids. Blenau and colleagues linked this unusual aspect to the fact that this receptor couples only with Gi and not also with Gq [D. suzukii TAR1) to 19 (Rhodnius prolixus TAR1) [B. mori TAR1 [B. mori TAR1 was completely abolished. Furthermore, the double mutant S218A and S222A was also not able to attenuate cAMP levels after stimulation with TA. The authors suggested a binding scheme by which the carboxylic group of D134 residue forms an ion pair with the protonated amine of TA, while the S218 and S222 are involved in H-bond between the hydroxyl groups.Recently, it has been reported that M990461) ,43. PeaT Tyr-dro . The amil region . The samina TAR1 ,37 but iina TAR1 , this eiina TAR1 . Interesectively ,36,37. Iectively . Given t-protein . However with Gq . The GPC with Gq . In all us TAR1) . These sus TAR1) . Palmitous TAR1) . In TAR1us TAR1) . In factus TAR1) . A conseus TAR1) . Throughori TAR1 . In partS. oryzae TAR1 binds TA by forming H-bonds with D114 (in the third transmembrane domain) and with N427 (in the sixth transmembrane domain) [83, C118, W394 and S428, are involved in TA binding suggesting a more complex binding pocket for TA.Through molecular docking approaches, Braza and colleagues confirmed that  domain) . FurtherD. melanogaster TAR1 in Chinese Hamster Ovary (CHO)-K1 cells [i coupling activity. In particular, TA was able to reduce forskolin-stimulated cAMP levels in a dose-dependent manner with a pEC50 of 5.62  assay, an assay able to investigate both the Gi and Gq contribution to intracellular signaling simultaneously. In this study, DMR assays revealed that TA is able to evoke a positive concentration dependent signal in HEK 293 cells stably expressing the D. suzukii TAR1 (pEC50 of 6.87), while OA can elicit an intracellular Ca2+ release only at 10 µM [P. americana, as discussed above, is the only insect presenting two distinct TAR1 [i coupling, PeaTAR1B displayed a different response to antagonist compounds. In fact, several antagonists completely lacked inhibitory potential on the receptor, such as chlorpromazine [R. prolixus TAR1 was expressed in HEK 293, the rank order of antagonist potency was yohimbine > metoclopramide > phenoxybenzamine > phentolamine > cyproheptamide > gramine > mianserin > chlorpromazine [P. xylostella TAR1 expressed in HEK 293T cells [i and Gq proteins, whether TA preferably activates the Gi- or Gq-mediated transduction pathway, and how much OA, even though less potent, contributes to this, remain to be elucidated. Nevertheless, it is obvious that TAR1s are promiscuous GPCRs which are able to couple with both Gi and Gq proteins as summarized in Conversely, OA was less potent, with a pEC of 4.52 . Furtherproteins . TAR1 sive as OA . These vly to Gi . StudiesH. halys ,38,45,47ecessary . D. suzuat 10 µM . P. amernct TAR1 ,43. In f3T cells ,45,46,472+ and cAMP intracellular variations  [D. melanogaster TAR1 transcripts were found to be abundant in the pars intercerebralis, in the mushroom bodies, and in the antennal and olfactory lobes [TAR1 in nervous tissues compared to the periphery was also observed in D. suzukii, Chilo suppressalis, R. prolixus, P. xylostella, H. halys, Mamestra brassicae, and Agrotis ipsilon, suggesting a crucial role of the receptor in controlling a broad range of physiological functions and behaviors [TAR1 was also strongly expressed in the antennae of M. brassicae, A. ipsilon, and H. halys, where it could regulate olfactory-mediated behaviors [D. melanogaster TAR1-defective line, called honoka, whose behavioral responses to repellents were reduced in comparison to wild-type flies. Furthermore, using in situ hybridization, Brigaud and colleagues observed that TAR1 was expressed at the base of the olfactory sensilla trichodea, pheromone-sensitive sensilla, rather than in sensilla chaetica, which are mechano-sensitive in A. ipsilon [H. halys. In the antennae, TAR1 appeared to be highly expressed in flagellomeres, apical structures rich in sensilla trichoidea which are essential for pheromone perception. Furthermore, RNAi-mediated TAR1 silencing resulted in a lower sensitivity to the alarm pheromone (E) -2-decenal [A. mellifera. In two studies conducted in 2017 on the honeybee brain, the authors showed that TAR1 is mainly expressed at the presynaptic sites of olfactory receptor neurons (ORNs), innervating the antennal lobes and the mushroom bodies, which are essential structures for the olfactory system [TAR1 mRNA localization was observed by Mustard and colleagues via in situ hybridization in honeybees [TAR1 showed a higher expression in the antennae of pollen foragers in comparison to nurse ones. In contrast, OAR1 exhibited the opposite expression profile [L. migratoria and D. melanogaster. In locusts, the ratio between TAR1 and OAR1 expression levels influenced olfactory preferences during the solitary-gregarious phase transition. In fact, high levels of TAR1 promoted solitary behavior by inducing the perception of gregarious pheromones as repellent, while RNAi-mediated TAR1 downregulation in solitary locusts was able to mediate the transition to the gregarious-like behavior [tspo transport protein [A. mellifera, movement impairment could be attributed to TAR1 [TAR1 expression in leg muscles of D. melanogaster, P. americana, and A. ipsilon [Drosophila, the TA/OA ratio was modulated by nutritional state, such as satiation and starvation. In particular, satiation inhibits locomotion through the increase of TA levels. Moreover, this mechanism controlling locomotor behavior requires a functional TAR1 in D. melanogaster [TAR1 transcripts such as the central complex [D. melanogaster TAR1PL00408 defective line exhibited higher body fat accumulation, starvation resistance, and food intake in comparison to wild-type flies [D. melanogaster, where, like many other insects, lipids are mainly stored in the fat body. Their storage and release are mainly controlled by two hormones, the insulin-like peptides (mainly ILP2) and the adipokinetic hormone  [PL00408, as compared to control flies, might be related to a direct tyraminergic action on the ILP2 release. RNAi-mediated D. melanogaster TAR1 silencing, targeted to the fat body, indeed triggered a ILP2 reduction in insulin-producing cells, located in the pars intercerebralis, and an increased TG accumulation [PL00408 flies correlates well with other traits observed, such as enhanced food intake, lower movement propensity, and lower metabolic rate [honoka flies, a D. melanogaster TAR1-defective line, exhibited a higher starvation resistance but, in contrast to TAR1PL00408 flies, a reduced responsiveness to sugar stimuli compared with control flies [TAR1 is highly expressed in neurons located in the sub-esophageal ganglia that are presumably associated with the salivary glands and neck muscles control, thus linking TAR1 with feeding. In honeybees, the topical administration of TA induced an increased Gustatory Response Score (GRS) that was sensitive to yohimbine [TAR1 knockdown in D. melanogaster significantly reduced bitter sensitivity in starved flies, suggesting that TAR1 might be directly involved in the gustatory behaviors [TAR1 expression level in the fat body in comparison to nurses, suggesting a correlation between the receptor and sugar responsiveness [D. suzukii and R. prolixus, TAR1 is expressed in the reproductive organs [R. prolixus ovaries display higher TAR1 transcript levels in comparison to other reproductive tissues such as the lateral oviduct and common oviduct, suggesting its importance in modulating reproductive processes [D. suzukii male abdomens showed a significant difference in the TAR1 expression levels as compared to females, suggesting a possible role of TA in male reproductive system [TAR1 gene has been successfully downregulated through RNAi approaches in A. mellifera and H. halys to investigate the receptor physiological functions [em (CNS) . Throughry lobes . A higheehaviors ,47,48,50ehaviors . This sty system ,69. A sioneybees . Further profile . Therefo profile . Behaviobehavior . In a su protein . It is e to TAR1 , since t ipsilon ,42,50. F ipsilon . Furthernogaster . However complex . TAR1 hape flies ,77, thuslucagon) . Under alucagon) ,79. Therlic rate . It has ol flies . It is wohimbine . LeDue aehaviors . Furthersiveness . Last bue organs ,45. In pe system . Recentlunctions ,85. ThusC. suppressalis TAR1 was expressed in HEK 293 cells, 10 µM of amitraz was able to inhibit forskolin-stimulated intracellular cAMP, mimicking TA effects [Rhipicephalus microplus TAR1. When expressed in the CHO cell line, TAR1 was allosterically positively modulated by BTS-27271, an amitraz metabolite [R. microplus TAR1 (T8P and L22S) could be responsible for a lower susceptibility, or even resistance, to the amitraz insecticide action [D. melanogaster TAR1. However, the same monoterpenes exhibited a different pharmacological effect on D. suzukii and R. microplus TAR1 receptors. In fact, they appeared able to increase the TA potency acting as positive allosteric modulators and not as agonists [S. oryzae TAR1 might provide new insights and possibly new molecules for TAR1-related pest control [In addition to their role in the physiology and behavioral control of insects, TAR1s have proven to be interesting targets for insecticides. Amitraz is an acaricide and non-systemic insecticide that targets OA receptors . However effects . Amitraz effects ,88. Thro effects ,90. On ttabolite . Even ife action . These me action  was the agonists ,94. Thisagonists . Recent  control .Tyramine receptor 1 appears central in controlling physiological processes and defining specific behavioral traits in insects. In recent years, the number of molecular and physiological data have helped shed some light on the role of this receptor. However, despite its importance in controlling the different physiological mechanisms of insects, many questions about the interconnection between the tyraminergic and octopaminergic systems in the modulation of physiology and behavior remain. An intriguing hypothesis suggests that also the ratio between TA and OA receptors might influence behavioral decisions and physiological states. This aspect is evident in social insects, including honeybees, in which both the levels of TA and OA and the expression patterns of the TA and OA receptors seem to influence the caste identities, characterized by well-defined physiological and behavioral profiles ,66,96. M


Marek’s disease virus (MDV) is an oncogenic avian alphaherpesvirus whose genome consists of unique long (UL) and short (US) regions that are flanked by inverted repeat regions. More than 100 open reading frames (ORFs) have been annotated in the MDV genome, and are involved in various aspects of MDV biology and pathogenesis. Within UL and US regions of MDV, there are several unique ORFs, some of which have recently been shown to be important for MDV replication and pathogenesis. In this review, we will summarize the current knowledge on these ORFs and compare their location in different MDV strains. Gallid alphaherpesvirus 2 (GaHV-2), also known as Marek’s disease virus (MDV). Infection with MDV causes neurological disease, immunosuppression, paralysis, blindness, and lymphomas in chickens. MDV was originally thought to be a gammaherpesvirus due to its ability to induce rapid onset of T-cell lymphoma in chickens. In the early 1980s, an electron microscopy study revealed that the genome of MDV and its close relative, Meleagrid alphaherpesvirus 1 (MeHV-1) or turkey herpesvirus (HVT), consists of two unique regions, each flanked by inverted repeat regions [Mardivirus genus in the Alphaherpesvirinae subfamily, family Herpesviridae. In addition, MDV strains vary in their virulence and are classified into different pathotypes, including mild (m), virulent (v), very virulent (vv), and very virulent plus (vv+). It is generally accepted that infection of MDV causes four phases of pathogenesis in susceptible chickens, including early cytolytic phase, latent phase, late cytolytic/immunosuppressive phase, and proliferative phase [Marek’s disease (MD) is an infectious and neoplastic disease of chickens caused by an oncogenic alphaherpesvirus,  regions ,4. Accorve phase . Featherve phase .In 1969, a serial-passage attenuated MDV, HPRS-16/att, was first used as a vaccine to control MD ,8,9. In In 2000, genomes of GA (GenBank: AF147806.2) and Md5 (GenBank: AF243438.1) strains of MDV were fully sequenced and more than one hundred open reading frames (ORFs) were identified and annotated [meq, vIL8, and vTR, encoded within the repeat regions, are specific to MDV and have been shown to be important for MDV pathogenesis and/or oncogenesis, and have been summarized previously [When compared to other alphaherpesviruses, genes such as eviously ,15,16. Ieviously . AlthougProteins encoded by genes in the UL region of MDV are involved in various aspects of MDV biology, such as DNA replication, gene expression, virion packaging, and morphogenesis. In the UL region of Md5 strain, a total of 12 unique ORFs were identified A, and thMDV009/LORF1  is located at the TRL/UL junction region of Md5 genome, and encodes a 333 amino acids (aa) long protein, with a predicted molecular weight (MW) of 37.9 kDa. It is in opposite orientation to MDV008/R-LORF14  which encodes phosphoprotein pp24. MDV009/LORF1 is unique to MDV-1 and has no homologue in MDV-2, HVT, and other non-avian herpesviruses , its funMDV010/LORF2  is located at the left terminus of the UL region (where UL1 is located), and is a spliced gene that encodes a 756-aa long viral lipase (vLIP) protein. MDV010/LORF2 homologues are present in MDV-1, MDV-2, and HVT . LipasesMDV vLIP is the first reported herpesvirus lipase and aa 229–369 shows significant homology to the critical structure of pancreatic lipase α/β hydrolase fold, a protein-folding structure that is important for the enzymatic activity of pancreatic lipase ,27. vLIPIn a more recent Md5 genome annotation (GenBank: NC_002229.3), two originally annotated ORFs MDV011*  and MDV012*  were re-annotated as a single spliced ORF, named MDV012 , which has been shown to encode protein p012 , suggestSchippers et al. studied the importance of MDV012 in regulating MDV replication and characterized the properties of p012 protein . They foLORF4  encodes a 142-aa long protein with a predicted MW of 16 kDa. It is antisense to MDV013/UL1  which encodes glycoprotein L (gL). LORF4 is unique to MDV-1 and is not present in MDV-2 and HVT. Using a two-hybrid screening method and in vitro binding assays, Niikura et al. identified an interaction between the protein encoded by LORF4 and chicken MHC class II beta chain . Later, LORF5  encodes a 119-aa long protein with a predicted MW of 13.1 kDa. A total of 295 nucleotides at the 3′ end of LORF5 overlap with MDV040/UL27 , which encodes gB, a major membrane glycoprotein. LORF5 is unique to MDV-1 and is not present in MDV-2 and HVT genomes. In 2000, Schumacher et al. characterized a gB mutant MDV where 2 kb nucleotides of gB, including 49 nucleotides at 3′ end of LORF5, were deleted . Their sLORF6  encodes a 155-aa long protein with a predicted MW of 17.2 kDa. It is antisense to the 5′ end of MDV049/UL36  which encodes UL36 major tegument protein. LORF6 is unique to MDV-1 and is not present in MDV-2 and HVT. The importance of LORF6 in MDV pathogenesis remains unknown.LORF7  encodes a 120-aa long protein with a predicted MW of 12.9 kDa. It is antisense to 5′ end of MDV050/UL37  which encodes UL37 tegument protein. LORF7 is unique to MDV-1 and is not present in MDV-2 and HVT genomes. The importance of LORF7 in MDV pathogenesis remains unknown.LORF8  encodes a 208-aa long protein with a predicted MW of 23.0 kDa. It is antisense to MDV058/UL45 , which encodes UL45 envelope transmembrane protein. LORF8 is unique to MDV-1 and is not present in MDV-2 and HVT. Both LORF8 and MDV058/UL45 are located downstream of MDV057/UL44, which encodes gC. Previously, Tischer et al. showed that both deletion of the entire gC coding region and mutation of the start codon of gC resulted in larger MDV plaque size; in addition, plaque size of the gC deletion virus was larger than that of the gC start codon mutant virus, suggesting that plaque size differences might be due to downstream LORF8 or MDV058/UL45 genes, since deletion of the entire gC coding sequence may affect their expression .MDV069/LORF9  encodes a 269-aa long protein with a predicted MW of 29.7 kDa, and is located at the right terminus of the UL region. MDV069/LORF9 homologues are present in MDV-1, MDV-2, and HVT . MDV069/meq deleted did not cause tumors in infected chickens and provided superior protection than CVI988/Rispens does against MD caused by highly virulent MDV [meq deletion virus retains the ability to cause lymphoid organ atrophy in chickens [meq deletion virus [vIL8 and meq resulted in the development of a safe and efficacious MDV vaccine [meq double deletion virus.Meq is the major oncogene of MDV, which is essential for MDV tumorigenicity . Mutant lent MDV ,38,39. Hchickens . By deleon virus . Recentl vaccine . Thus, iMDV071/LORF10  encodes a 194-aa long protein with a predicted MW of 21.4 kDa, and is located at the right terminus of the UL region. MDV071/LORF10 homologues are present in MDV-1 and HVT, but not in MDV-2 genomes . We haveMDV072/LORF11  encodes a 903-aa long protein with a predicted MW of 103.2 kDa, and is located at the right terminus of the UL region. MDV072/LORF11 homologues are present in MDV-1, MDV-2, and HVT genomes . MDV LORLORF12 homologs of Md5, RB-1B, 648a, and CVI988 encode a 126-aa long protein with a predicted MW of 14.1 kDa; while LORF12 of GA  encodes a 148-aa long protein with a predicted MW of 16.4 kDa . LORF12 S3 protein kinase and ICP22) involved in DNA replication and gene expression [Ten ORFs were annotated in the US region of Md5 strain ,46, inclpression ,50,51,52SORF1 homologues of GA, RB-1B, 648a, and CVI988 are located in the US region, which encodes protein ranging from 76 to 114-aa long . HoweverSORF2 homologues of GA, RB-1B, 648a, and CVI988 are located at the left terminus of the US region  and encoMDV090/SORF3  encodes a 351-aa long protein with a predicted MW of 40.1 kDa. MDV090/SORF3 homologues are present in MDV-1, MDV-2, and HVT . A deletMDV093/SORF4  encodes a 147-aa long protein with a predicted MW of 16.8 kDa, and its importance in MDV pathogenesis remains unknown. MDV093/SORF4 is unique to MDV-1 and is not present in MDV-2 and HVT.In this review, we summarized our current knowledge of unique ORFs encoded within the UL and US regions of MDV. There are also some ORFs in the repeat regions of MDV that remain to be studied. Some of these ORFs partially or completely overlap with other MDV genes; thus, partial or single amino acid mutations, such as those disrupting the start or stop codon, can without affecting the protein sequence of the overlapping gene be used to elucidate specific function of these ORFs.meq, RLORF4, and RLORF5a, have been shown to splice with exons II and III of vIL8, and the Meq-vIL8 splice variant was demonstrated to be an important virulence factor of MDV [Other than these genes, taking advantage of RNA sequencing and reverse transcription PCR analyses, some novel ORFs were identified in MDV. For example, two additional splicing variants of gC were identified and have been shown to be required for efficient horizontal transmission of MDV . In addir of MDV ,58. Usinr of MDV , the rol


DPEP1, KRT80, FABP6, NKD2, FOXQ1, CEMIP, ETV4, TESC, FUT1, and GAS2) are observed in CRC cell lines and public CRC datasets. Moreover, we find that a high mRNA expression of DPEP1, NKD2, CEMIP, ETV4, TESC, or FUT1 is significantly correlated with a worse prognosis in CRC patients. Further investigation reveals that CTNNB1 is the key factor in the interaction of the canonical Wnt signaling pathway with 10 upregulated CRC-associated genes. In particular, we identify NKD2, FOXQ1, and CEMIP as three CTNNB1-regulated genes. Moreover, individual inhibition of the expression of three CTNNB1-regulated genes can cause the growth inhibition of CRC cells. This study reveals efficient biomarkers for the prognosis of CRC and provides a new molecular interaction network for CRC.Colorectal cancer (CRC) is a heterogeneous disease with changes in the genetic and epigenetic levels of various genes. The molecular assessment of CRC is gaining increasing attention, and furthermore, there is an increase in biomarker use for disease prognostication. Therefore, the identification of different gene biomarkers through messenger RNA (mRNA) abundance levels may be useful for capturing the complex effects of CRC. In this study, we demonstrate that the high mRNA levels of 10 upregulated genes ( Colorectal cancer (CRC) is one of the most malignant cancers and the third most prevalent cancer worldwide . By 2030Gene expression profiles have been broadly used to identify novel biomarkers through high-throughput technology . HoweverDPEP1, KRT80, FABP6, NKD2, FOXQ1, CEMIP, ETV4, TESC, FUT1, and GAS2) exhibit simultaneous upregulation in CRC cell lines and public CRC datasets. In addition, we find that the mRNA expression levels of 10 upregulated CRC-associated genes are significantly and positively related to the CRC stage. We further investigate the prognostic significance of these genes in CRC outcomes. Further investigations reveal that the key gene involved in the canonical Wnt signaling pathway that interacts with the 10 upregulated CRC-associated genes is CTNNB1. We observe NKD2, FOXQ1, and CEMIP to be CTNNB1-regulated genes, and individual inhibition of the expression of three CTNNB1-regulated genes can cause growth inhibition of CRC cells.Thus, in the present study, we demonstrate that the mRNA levels of 10 upregulated CRC-associated genes . The cell line was cultured in RPMI-1640, supplemented with 10% fetal bovine serum, and 1% penicillin–streptomycin  , and were maintained at 37 °C in a humidified atmosphere containing 5% COCTNNB1, NKD2, FOXQ1, CEMIP, and scrambled negative control small interfering (si)RNAs were purchased from Invitrogen , and transfected into cells using the jetPRIME transfection reagent  according to the manufacturer’s instructions. Sequences of the siRNAs are described in the 5 dissociated cells by the jetPRIME transfection reagent according to the manufacturer’s instructions. Next, cells were plated in 96-well plates at 3000 cells/mL after transfection with control siRNA or siRNA of NKD2, FOXQ1, and CEMIP for 48 h. After cells had grown for 48 h, cells were stained with 0.5% crystal violet for 10 min at room temperature. Next, the plates were washed with tap water three times. After drying, cells were lysed with a 0.1 M sodium citrate solution , and the absorbance was measured at 550 nm on a microplate reader.Cell viability was determined with the crystal violet-staining method. In brief, the oligonucleotide (50 or 100 nM) was introduced into 5 × 10NKD2, FOXQ1, and CEMIP for 48 h, respectively. The medium was changed every 3 days. After 7 days, the cells were fixed and stained with 0.5% crystal violet. Foci of >5 mm in size were counted, and average focal counts and standard deviations (SDs) were calculated.HCT116 cells were seeded at 1000 cells/well in six-well dishes and grown overnight after transfection with control siRNA or siRNA of Total RNA was extracted from CRC cell line by using a Qiagen RNeasy kit  and QIAshredder columns according to the manufacturer’s instructions . One microgram of total RNA was reverse-transcribed to complementary DNA (cDNA) by using a reaction-ready First Strand cDNA Synthesis Kit . To detect the mRNA expression levels of NKD2, FOXQ1, and CEMIP genes, as well as GAPDH, specific products were amplified and detected using the cycle profile of the Qiagen SYBR green reagent . The sequences of primers used in this study were shown in the http://discover.nci.nih.gov/cellminer, accessed on 30 March 2021) was used to compare and plot the relative baseline expression of DPEP1, KRT80, FABP6, NKD2, FOXQ1, CEMIP, ETV4, TESC, FUT1, and GAS2 mRNAs in the National Cancer Institute (NCI)-60 cell line panel. The tool enables the retrieval and integrated analyses of baseline and experimental data compiled from the 60 cell lines included in the panel. We selected gene transcript level z scores for the analysis of the 10 genes as gene identifier inputs.The CellMiner tool  is useful for the effective analysis of cancer data in the Cancer Genome Atlas (TCGA) database [The UALCAN website  by using colorectal tumors and normal colorectal tissues. In this study, the mRNA expression levels of the top 10 upregulated CRC-associated genes were compared in CRC and normal control samples using the Oncomine database.Gene expression changes were analyzed using the TCGA microarray dataset of the Oncomine website  [http://string-db.org/, accessed on 30 March 2021) was used for protein–protein interaction (PPI) predictions [The WEB-based Gene SeT AnaLysis Toolkit   was useddictions . Functiodictions .cBioPortal, an open-access online tool for the multidimensional analysis of cancer genomics, enables the visualization and analysis of genes, including DNA copy number analysis, mRNA, nonsynonymous mutation analysis, and DNA methylation analysis . The colp < 0.05 considered statistically significant.The public Gene Expression Profiling Interactive Analysis (GEPIA) database can be used to analyze gene expression profiles . Furtherhttps://www.colonomics.org/, accessed on 30 March 2021); Jorissen et al. [https://cancergenome.nih.gov/, accessed on 30 March 2021).In silico analysis was performed using the CANCERTOOL . Genes wn et al. ; Laibe en et al. , and TCGp values and fold-changes for the differential expression analysis of genes generated from the CRC tissues in the Oncomine database, UALCAN database, and survival analyses were calculated using a one-sided Student’s t-test. p values of <0.05 were considered significant.Our recent study  identifiDPEP1 , compared with normal colon tissues (n = 19). In addition, to verify the specificity of 10 genes in CRC tissues, we increased the number of samples and integrated 716 samples from six studies including, Hong, Kaiser, Ki, Sabates-Bellver, Skrzypczak, and TCGA databases in ONCOMINE by meta-analyses. As shown in the To confirm the mRNA expression levels of the aforementioned 10 genes in numerous CRC tissues, we analyzed the mRNA expression profiles of 10 genes by using the TCGA dataset of the Oncomine database. Compared with normal samples, the mRNA expression of the 10 genes significantly increased in colorectal tumor tissues. Significant increases were found in the mRNA expression of n = 466) was analyzed using a SurvExpress survival analysis [DPEP1   to perfoAPC gene are detected in approximately 70%–80% of human colon tumors, indicating that the activation of the Wnt/beta-catenin (CTNNB1) pathway plays a crucial role in human colon tumorigenesis [APC, CTNNB1, and the top 10 upregulated CRC-associated genes through the cBioPortal database [APC, CTNNB1, and the top 10 upregulated CRC-associated genes in CRC were 69% for APC, 13% for CTNNB1, 8% for DPEP1, 7% for KRT80, 6% for FABP6, 7% for NKD2, 6% for FOXQ1, 8% for CEMIP, 8% for ETV4, 7% for TESC, 6% for FUT1, and 8% for GAS2, respectively . We observed that the CTNNB1 transcript was downregulated by approximately 1.5 times in siCTNNB1-transfected SW480 cells and the expression trend of NKD2, FOXQ1, and CEMIP transcripts was also downregulated A. Next, DPEP1, KRT80, FABP6, NKD2, FOXQ1, CEMIP, ETV4, TESC, FUT1, and GAS2) transcripts were upregulated in CRC cell lines and public CRC datasets. Secondly, we found that the top 10 upregulated CRC-associated genes affected the prognosis of CRC patients, and six of the genes had high expression in the CRC subgroup with more severe malignancy. Thirdly, we found that a high expression of DPEP1, NKD2, CEMIP, ETV4, TESC, or FUT1 was significantly correlated with worse prognosis in CRC patients. Fourthly, the PPI network demonstrated that CTNNB1 is the key factor involved in the interaction of the canonical Wnt signaling pathway with the 10 upregulated CRC-associated genes. Fifthly, we demonstrated NKD2, FOXQ1, and CEMIP as three CTNNB1-regulated genes, and individual inhibition of the expression of the three CTNNB1-regulated genes can cause growth inhibition of CRC cells. Collectively, this study identifies three putative CTNNB1-regulated genes from the top 10 upregulated CRC-associated genes and provides a new molecular interaction network for CRC.An increase in both the number of biomarkers and their uses helps with prognostication and treatment decision-making for CRC . In addiNKD2, FOXQ1, and CEMIP are three CTNNB1-regulated genes. In addition, through cellular component analysis, we found that these three genes were located in the nucleus. Furthermore, NKD2 [The aberrant Wnt/beta-catenin (CTNNB1) signaling pathway plays a crucial role in CRC development . In the re, NKD2  and FOXQre, NKD2  proteinsre, NKD2 . Based op < 0.01). Subsequently, the multivariate stepwise Cox regression analysis of the top 10 upregulated CRC-associated genes was applied and three genes are eventually proved to be correlated with overall survival  relate with CRC prognosis, and six of the genes  are highly expressed in the CRC patients in advanced stages. CTNNB1 is the key factor involved in the interaction of the canonical Wnt signaling pathway with 10 upregulated CRC-associated genes. We identified NKD2, FOXQ1, and CEMIP as three CTNNB1-regulated genes and and individual inhibition of the expression of the three CTNNB1-regulated genes can cause growth inhibition of CRC cells. However, further experiments should be conducted to verify the regulatory mechanism between CTNNB1 and the three aforementioned CTNNB1-regulated genes.


Cx3cr1-positive MCs were isolated by fluorescence-activated cell sorting from six-week (young) and two-year-old (old) GFPCx3cr1/+ mice, both during physiological aging and laser-induced CNV development. High-throughput RNA-sequencing was performed to define the age-dependent transcriptional differences in MCs during physiological aging and CNV development, complemented by immunohistochemical characterization and the quantification of MCs, as well as CNV size measurements. These analyses revealed that myeloid cells change their transcriptional profile during both aging and CNV development. In the steady state, senescent MCs demonstrated an upregulation of factors contributing to cell proliferation and chemotaxis, such as Cxcl13 and Cxcl14, as well as the downregulation of microglial signature genes. During CNV formation, aged myeloid cells revealed a significant upregulation of angiogenic factors such as Arg1 and Lrg1 concomitant with significantly enlarged CNV and an increased accumulation of MCs in aged mice in comparison to young mice. Future studies need to clarify whether this observation is an epiphenomenon or a causal relationship to determine the role of immunosenescence in CNV formation.Immunosenescence is considered a possible factor in the development of age-related macular degeneration and choroidal neovascularization (CNV). However, age-related changes of myeloid cells (MCs), such as microglia and macrophages, in the healthy retina or during CNV formation are ill-defined. In this study,  Age-related macular degeneration (AMD) is the most common cause of blindness in the elderly and can progress in its advanced stages to an atrophic or a neovascular form (nAMD) ,2,3,4,5.CFH, ARMS2/HTRA1 and C3, belong to the established risk factors for AMD [The pathogenesis of AMD and CNV formation is incompletely understood and involves a complex interplay of demographic, genetic, and environmental risk factors . Since a for AMD . Further for AMD ,13,14,15 for AMD ,17,18,19 for AMD ,21, they for AMD ,23. HoweIn this study, we analyzed the transcriptional profile of myeloid cells in the healthy retina and at sites of laser-induced CNV by using young and old myeloid-cell-specific reporter mice and high-throughput RNA-sequencing. Our data revealed that myeloid cells significantly alter their transcriptional profile during physiological aging and that senescent CNV-associated myeloid cells are characterized by the increased expression of cell proliferation and angiogenesis factors, which is associated with increased myeloid cell accumulation and CNV severity.GFP/+Cx3cr1 mice with and without laser-induced CNV, we isolated myeloid cells, defined as CD45lowCD11b+Cx3cr1-GFP+Ly6C−Ly6G−, using fluorescence-activated cell sorting. Following RNA extraction, we performed bulk RNA-sequencing to obtain the quantitative measurements of a total of 29,852 RNA transcripts , with higher discrepancies between the laser and control groups than between the young and old groups , a total of 422 differentially expressed genes (DEGs) were identified, of which 335 were upregulated and 87 were downregulated in myeloid cells of old compared to young mice. The visualization of DEGs is shown as a readplot in Cxcl13, Spp1, Vcam1, Lgals3, and Cxcl14  and Cxcl14  were strongly upregulated in myeloid cells isolated from old mice, thus suggesting the activation of migration and recruitment processes in aged myeloid cells. The enrichment of proliferation- and chemotaxis-related genes in senescent myeloid cells was found to be associated with an increase in Cx3cr1-GFP+-positive cells in the outer and inner plexiform layers of the retina , fibronectin 1 , and secreted phosphoprotein 1 , also known as osteopontin. Subsequent Gene Ontology enrichment analysis revealed that the DEGs between these two groups contributed to biological processes associated with the proliferation of cells, such as the mitotic cell cycle , cell cycle process , and chromosome segregation  , a total of 1135 differentially expressed genes (DEGs) were identified, 771 of which were upregulated following laser CNV induction and 364 of which were downregulated compared with myeloid cells of unlasered young mice . The top× 10−30) . The topCxcl13 , Ccl8 , and Cxcl2  (n = 13 CNV lesions) compared to the young group   and mononuclear cell proliferation  B. The to = 0.01) C. In lin 0.0001) D,E.p = 0.02) (Lrg1) and arginase 1 (Arg1), which were significantly more upregulated in senescent compared to young myeloid cells associated with CNV  without showing an increase in expression during physiological aging (Il6) after CNV induction in old mice . Finally, we determined the CNV size 7 days after laser induction in old and young mice via the quantification of immunohistochemical collagen IV staining on RPE/choroidal flat mounts. In line with the increase of pro-angiogenic factors in myeloid cells of old mice, we found a 5-fold increased CNV lesion size compared to CNV in young mice  B. The moal aging F. FurtheAging is the main risk factor for age-related macular degeneration and associated CNV formation, which is linked to an activation of the innate immune system ,25,26. ICxcl13, Alpl, and Chic1, could be confirmed by our RNA-sequencing analysis to be differentially expressed in senescent microglia. Possible explanations for the different results between our and Ma’s study could include the different ages and strains of the mice studied, the different myeloid cell isolation protocol, and the use of microarray technology by Ma et al., which may be limited by technical issues such as limited probe coverage and inconsistent probe hybridization efficiency. The most significant difference, however, is the different cutoff criteria used in the two studies. While we used a log2FC of >2 or <−2 and an adjusted p-value of <0.05, Ma et al. chose to only work with fold-change values and no p-value adjustment. Nevertheless, for the sake of completeness, we conducted a reanalysis of the data from Ma et al., comprising the comparison of 3-month-old vs. 24-month-old mice, which were the time points closest to the ones chosen in our analysis. Applying the cutoff criteria used by Ma et al.  for their data and subsequently comparing the resulting DEGs with the DEGs in our analysis resulted in an overlap of 34 genes  could contribute to the recruitment of further immune cells in the aged retina [Itgax, Lgals3, Axl, Cst7, Clec7a, and Spp1  in aged myeloid cells, which was in accordance with studies exploring the expression profile of senescent brain microglia [On the other hand, our study identified 422 age-dependent genes, such as d retina . In addiicroglia ,35,36,37Lgals3, further proliferation-associated genes, such as Vcam1, Spn, and Gpnmb, are strongly enriched in aging retinal microglia cells, which may contribute to the increased myeloid cell density in the retina of aged mice [Adam8 and Dpp4, are enriched in aged myeloid cells. This could reflect the vertical migration of myeloid cells in the retina towards the subretinal space with age, which can be observed in both humans and mice [Spp1 emerged as one of the most highly expressed genes in senescent retinal microglial cells and in challenged MCs associated with CNV from young mice. SPP1 binds to multiple integrin receptors that are important for cell adhesion, migration, and survival [Cngb1 (cyclic nucleotide gated channel beta 1), Rp1l1 (retinitis pigmentosa 1 like 1), and Atp1b2 (ATPase Na+/K+ transporting subunit beta 2) were observed to be solely upregulated in the myeloid cells of old mice, which may be explained by increased photoreceptor phagocytosis, as previously described [Our study indicates that in addition to ged mice . Althougged mice , the resand mice ,13,14,15survival ,39 and msurvival ,41,42,43escribed . Taken tP2ry12 in aged compared to young MCs in the steady state .The laser-induced CNV model was used as described previously ,60,61,62® 647, Thermo Fisher Scientific, Waltham, MA, USA) overnight at 4 °C. Images of whole flatmounts were taken using a Hamamatsu NanoZoomer S60 . To quantify myeloid cell numbers, confocal images were taken using a Leica SP8 confocal microscope .After trans-cardial perfusion with phosphate-buffered saline (PBS) and 4% paraformaldehyde (PFA), eyes were fixed in 4% PFA for 1 h on ice and processed for flatmounts, as previously described ,63. AntilowCD11b+Cx3cr1+Ly6C-Ly6G-cells. The gating strategy is visualized in 3CR1-GFP-positive cells; third gate: exclusion of monocytes (Ly6c) and granulocytes (Ly6g); fourth gate: gating for CD45low myeloid cells). For one final sample, two retinae from one mouse were pooled. Following trans-cardial perfusion with 1× PBS and enucleation, eyes were dissected in ice-cold 1× PBS to isolate the retinae. Only central parts (50%) of the retinae were used for FACS, and the peripheral parts were omitted in order to enrich disease-associated myeloid cells. After tissue homogenization and filtration through a 50 µm cell strainer , dead cell exclusion was performed by incubation with fixable viability dye 780 . Anti-CD16/CD32 (Fc) receptor  was used to avoid unspecific binding. Following staining with anti-CD45 , anti-CD11b , anti-Ly6C , and anti-Ly6G  for 20 min at 4 °C, retinal myeloid cells were sorted into an RNA-stabilization reagent  using a MoFlo Astrios EQ High Speed Cell Sorter . Retinal myeloid cells were characterized as CD45RNA extraction, RNA library preparation, and RNA-sequencing were performed in collaboration with the Genomics Core Facility “KFB-Center of Excellence for Fluorescent Bioanalytics” , as previously described . In brieFirst-strand cDNA was generated using SMARTer Ultra Low Input RNA Kit for Sequencing v4 . Double-stranded cDNA was amplified with LD PCR and purified with AMPure XP beads. Library preparation was constructed by conforming to the Illumina Nextera XT Sample Preparation Guide . In brief, 150 pg of input cDNA were tagmented via Nextera XT transposome. The products were purified and amplified with a limited-cycle PCR program to construct sequencing libraries. The libraries were quantified with the KAPA SYBR FAST ABI Prism Library Quantification Kit . Equimolar amounts of each library were pooled for cluster generation on the cBot using the Illumina TruSeq SR Cluster Kit v3. The sequencing run was performed on a HiSeq1000 instrument with TruSeq SBS Kit v3 according to the Illumina HiSeq 1000 System User Guide. Illumina image analysis and base calling were recorded in library base call format (.bcl) before being further converted into Fastq files via the CASAVA1.8.2 software.http://www.bioinformatics.babraham.ac.uk/projects/fastqc last accessed on 30 July 2020). Reads were mapped to the mouse reference genome , as previously described ,65. Qual. For identification of CNV-specific changes in the transcriptional profile during aging, DEG between young and old control mice were defined at first. In a next step, the same was done for the young CNV and old CNV groups. For bioinformatic analysis, the intersections of these genes were determined and excluded before further analysis. This step was crucial to separate pure aging effects from CNV-associated changes in the expression level of DEGs. Gene enrichment analysis was performed using the R package clusterProfiler 3.10.1 [The output of featureCounts was imported to RStudio . Gene symbols were determined based on ENSEMBL  release r 3.10.1 . Heatmapr 3.10.1  and Vennr 3.10.1 , respectr 3.10.1 . Genes ir 3.10.1 .t test was applied. If the data were not normally distributed, the Mann–Whitney test was applied. Differences were considered significant at a p-value of <0.05.For statistical analysis, GraphPad Prism  was used. Data were tested for normality applying the D’Agostino–Pearson Omnibus test. If normality was proven, an unpaired


The research was performed under a triplicate randomized complete block design including a split-plot arrangement. Data were recorded for agronomic, biochemical, and fiber quality traits. Mean values of agronomic traits were significantly reduced under heat stress conditions, while hydrogen peroxide, peroxidase, total soluble protein, superoxide dismutase, catalase (CAT), carotenoids, and fiber strength displayed higher mean values under heat stress conditions. Under both conditions, high genetic advance and high heritability were observed for seed cotton yield (SCY), CAT, micronaire value, plant height, and chlorophyll-a and b content, indicating that an additive type of gene action controls these traits under both the conditions. For more insights into variation, Pearson correlation analysis and principal component analysis (PCA) were performed. Significant positive associations were observed among agronomic, biochemical, and fiber quality-related traits. The multivariate analyses involving hierarchical clustering and PCA classified the 23 experimental genotypes into four groups under normal and high-temperature stress conditions. Under both conditions, the F1 hybrid genotype FB-SHAHEEN × JSQ WHITE GOLD followed by Ghuari-1, CCRI-24, Eagle-2 × FB-Falcon, Ghuari-1 × JSQ White Gold, and Eagle-2 exhibited better performance in response to high-temperature stress regarding the agronomic and fiber quality-related traits. The mentioned genotypes could be utilized in future cotton breeding programs to enhance heat tolerance and improve cotton yield and productivity through resistance to environmental stressors.The ever-changing global environment currently includes an increasing ambient temperature that can be a devastating stress for organisms. Plants, being sessile, are adversely affected by heat stress in their physiology, development, growth, and ultimately yield. Since little is known about the response of biochemical traits to high-temperature ambiance, we evaluated eight parental lines (five lines and three testers) and their 15 F Cotton is the world’s most important natural fiber and oil crop . Climate°C, root development 30°C, and seedling development for boll development 25.5–29.5°C , superoxide radical (O2–), singlet oxygen (1O2), and hydroxyl radicals are produced in higher amounts  following a split-plot arrangement under a randomized complete block design (RCBD) with two replications. The plant × plant and row × row distance was 30 and 75 cm, respectively, with a 6 m row length for each genotype under each replication. The temperature was raised by constructing a tunnel using polythene sheets for 12 days at 50% flowering and it was removed at night. After screening, eight cotton genotypes were selected as parents regarding their SCY under heat stress. The selected parental material was crossed in line × tester mating fashion in the following season to obtain the subsequent F1 hybrids and their eight parents are listed in The 15 FDuring September, when all genotypes were at the 50% flowering stage, heat stress was implemented for 12 days during both the years. The covering of the polythene tunnel enhanced the temperature (5–6°C) during the daytime and the tunnel was removed during the night . The min2O2, CAT, peroxidase (POD), total soluble proteins (TSP), chlorophyll contents (Chl), and carotenoids (Car) in the leaves was performed to assess the effect of stress on biochemical attributes of the plants. For this purpose, the fourth fully expanded top leaf was considered for sampling from each genotype for biochemical analyses following the sampling method used by via UV spectrophotometer at different wavelengths . CAT activity was recorded at the wavelength of 240 nm (Enzyme extract (0.1 mL) was mixed with 3 mL of the reaction mixture, containing 5.9 mM Hf 240 nm  using a 2O2, 20 mM guaiacol, and 0.1 mL of enzyme extract according to Liu’s protocol, after certain amendments (The POD solution contained 50 mM phosphate buffer (pH = 5), 40 mM Hendments . At 470 The Bradford reagent method was used for the determination of protein content. Aliquots of 100 μL of the sample were blended with 5 mL of Bradford reagent. At 595 nm wavelength, the absorbance was recorded  using a The Arnon method  with spewhere,W = weight of leaf sample, V = volume of sample, Em = 2,500At crop agronomic maturity, data from five plants of each genotype regarding yield-related traits were recorded. The yield-related traits included plant height (PH), the number of bolls (TNB), boll weight (BW), SCY, and lint percentage (lint%). A representative sample from seed cotton obtained from the experimental genotypes was taken and weighed. The ginning of seed cotton samples was accomplished with the help of a single roller ginning machine  to separate seed and lint, and the ginning outturn was estimated by dividing the weight of lint in a sample by the seed cotton weight of the sample, expressed in percentage. Lint was further subjected to fiber quality analysis for the estimation of fiber strength (g/tex) (STR), short fiber (SF), micronaire value (MIC), reflectance (%) (RD), upper half mean length (mm) (UHML), and uniformity index ratio (%) (UI) with a high-volume instrument , following ASTM protocol, publication D5867-05 for HVI analysis .The preliminary screening data comprising of 50 upland cotton accessions based on yield performance across heat and normal conditions were subjected to a linear mixed model using ANOVA, followed by the construction of an ANOM-decision chart to graphically represent the genotypic behavior for selection of parents for crossing through analysis of mean methods as described by 1s across normal and heat stress for agronomic, biochemical, and fiber traits has been subjected to analysis of variance (2b). For the categorization of H2b, a method based on a range of values was used, as follows: low H2b had a < 30% value, medium H2b values range between 30 and 70%, and high H2b values above 70%. The H2b was classified following the procedure given by Johnson , geometric mean performance (GMP), and stress tolerance index (STI) for normal and heat stress with the help of the freely available online software package iPASTIC developed by The results from the preliminary screening experiment across normal and heat stress conditions, represented in 1 hybrids under heat stress during both years; this pointed toward the existence of genetic divergence , POD (105.29), TSP (16.03), SOD (98.01), Car (0.63), CAT (243.21), and STR (32.05) displayed higher mean values under high-temperature conditions (2O2 had moderate to high coefficient of variation (CV%) values (2b and genetic advance mean per percent (GAM) was observed in CAT, MIC, SCY, Chl a and b contents, and PH under both conditions. Moderate H2b and GAM were exhibited by TNB, H2O2, Car, SF, and STR under normal and stress conditions , TNB (20.63), BW (3.01), UHML (26.89), SCY (50.31), Lint% (50.55), RD (75.38), UI (86.20), MIC (5.12), Chl-a contents (1.12), and Chl-b contents (0.37) exhibited higher mean values under normal conditions. In contrast, Hnditions . The coe) values . The gennditions .2O2, POD, and Chl a and b under both the conditions. BW displayed a highly significant positive association with H2O2. The biochemical traits showed a significant positive correlation with POD, CAT, Chl a and b, and Car under both conditions  analysis was performed for the estimation of the degree of dissimilarity among experimental genotypes based on morphological, physiological, and biochemical traits measured under normal and high-temperature stress conditions. The cluster tree was shaped using the agglomerative hierarchical approach based on a “bottom-up” technique. The technique uses every single observation at the initial level as an individual cluster. These individual observations move forward to the next level, forming a hierarchy after pairing up successively until the final distinct cluster. The Euclidean distance method was used to calculate the distances between genotype pairs. Subsequently, all the genotypes were clustered together to create a full-fledged dendrogram This analysis divided all 23 experimental genotypes into four groups under both normal and heat stress conditions. Under normal conditions, Group-1, Group-2, Group-3, and Group-4 enclosed seven, one, 12, and three genotypes, respectively . Under hPrincipal component analysis is a multivariate statistical approach to studying and simplifying complicated and huge datasets. Based on the correlation among studied characters and extracted clusters, the variation patterns in cotton genotypes were also investigated using PCA to assess the genetic diversity of the genotypes and their relationship with the studied traits. Under both conditions, the total variation was divided into 19 principal components (PCs), out of which the first four PCs displayed > 1 eigenvalue. In contrast, the remaining PCs exhibited lower eigenvalues . The fir2O2, POD, TSP, and SOD had long vectors and revealed higher variation, whereas lint%, PH, MIC, STR, and UI exhibited the least variability. The SF, UI, and RD did not follow a desirable direction. PCA results displayed clear discrimination among all studied genotypes across normal and high-temperature stress conditions among the four PCs contributing to the maximum. The elaborated distribution details of studied traits under both normal and stress conditions among the four PCs are displayed in a scatterplot matrix in 2O2, STR, and BW show a positive correlation between PC2 and PC3 biplot, validating the correlation results.The summary biplot of studied traits along with their magnitudes of variation is displayed in 2O2 were closed and positively correlated in the biplot of PC-1 and PC-3. The biplot of PC-4 had the least variation compared with PC-1, with PC-2, and PC-3 being independent. In this biplot, SF and UI were more discriminating traits and had a strong positive correlation. The biplot of PC-1 with PC-4 contributed lower variation as compared with PC-2 and PC-3. In this biplot, RD, UHML, PH, and TNB lay close to each other and exhibited positive associations among themselves.The traits of POD, SOD, and H1 hybrids; four groups were observed (We have estimated stress tolerance indices (STI) based on mean performance (MP), geometric mean performance (GMP), and STI for test genotypes, considering yield as the most critical indicator for screening regarding heat-tolerant genotypes. In this method, genotypes were ranked according to their MP, GMP, and STI. For MP and GMP genotypes were with higher values, whereas STI ≥ 1 have been considered for heat tolerance . Out of observed . The genobserved . Based oobserved .Of all abiotic stressors, high-temperature stress is a major constraint in improving cotton yield and production, affecting numerous attributes and physiological and metabolic processes . The dev1 hybrids were obtained subsequently. Using CV for the studied traits, the variation assists with enhancing crop yields by assembling beneficial genes from genetically divergent genotypes. CV also assists in depiction of the precision regarding the experiment conducted. Genetic variation is highly prone to fluctuations that take place in a plants’ environment. As the genome of a plant tries to adapt according to the vagaries of its environment, internal modifications occur to produce desirable, modified, and flexible phenotypes. Those traits exhibiting high GCV and PCV with low adverse environmental effects are advantageous for selection. Cultivars with such characters should be selected to develop desirable and adaptable genotypes (Five lines and three testers were crossed in Line × Tester fashion (5 × 3), and 15 Fenotypes . In thisenotypes . Our finenotypes .2b, coupled with high GAM, may contribute to genetic gain owing to the selection process. Such a trend was observed in this work with CAT, MIC, SCY, PH, and Chl a and b content, indicating additive gene action. The mentioned traits can prove helpful in the selection of genotypes at early stages to be used further in improvement-based breeding programs. Interestingly, similar results were observed in earlier studies (2O2, Car, SF, and STR, had moderate H2b and GAM under both normal and stress conditions (2b, and GAM favor stabilized selection regarding the accumulation of alleles owing to the predominance of additive genes (Genetic improvement of crop plants relies on the magnitude of heritability of economic traits . Traits  studies . The folnditions . Higher ve genes . Some stve genes , 2011.2O2 production was observed to increase; however, owing to the scavenging activity of CAT and POD, its damaging impacts were prevented (2O2 into water and oxygen (2O2 to maintain its optimum level (2O2 level and were declared as heat-tolerant genotypes. Similar results have been observed previously in cotton (Previous findings on high mean values are incongruent with our findings for PH , Chl conrevented . CAT andd oxygen . An incrum level . The genn cotton , wheat (n cotton , chickpen cotton , and motn cotton . Carotenn cotton .2O2. Similar positive correlations among traits were also reported in earlier studies (A correlation matrix is used to study the dependency of variables upon each other for better phenotypes to give improved yields . The pos studies .1 hybrid genotype FB-SHAHEEN × JSQ WHITE GOLD with high SCY, BW, TNB, CAT, SOD, POD, and Chl content under both normal and stress conditions. This F1 hybrid genotype was also observed to be superior in terms of fiber quality traits. The other F1 hybrids, CCRI-24 × JSQ WHITE GOLD, and EAGLE-2 × JSQ WHITE GOLD showed maximum lint%, whereas the minimum lint% was recorded for GHUARI-1 × FB-FALCON under both conditions. The parental genotypes Eagle-2 and CCRI-24 were superior in yield and fiber quality parameters under both conditions.In some cotton cultivar leaves, the antioxidant enzymes become upregulated under heat stress but remain unable to safeguard cells from oxidative injury . This stTo select the best genotypes for agronomic, fiber-related, and biochemical traits, their discrimination from remaining low-performing ones was attained using hierarchical cluster analysis, indicating that they be utilized further in breeding programs . The gen1 hybrid FB-SHAHEEN × JSQ WHITE GOLD followed by Ghuari-1, CCRI-24, Eagle-2 × FB-Falcon, Ghuari-1 × JSQ White Gold, and Eagle-2 were identified as more heat tolerant as compared with the remaining experimental genotypes.Among the various stress tolerance indices, MP, GMP, and STI have been extensively used in various studies and are suitable selection criteria, as these parameters enable us to identify individuals with high performance regarding stress-tolerance potential under unfavorable conditions . In the Several previous studies have documented that species with higher heat tolerance show an increasing trend in antioxidant enzyme activity in response to high-temperature stress, but susceptible species fail to do so. Thus, the evidence accumulated from current data indicates that intrinsic antioxidant resistance mechanisms of plants may exhibit a strategy for the enhancement of tolerance against heat stress. However, to perform selection efficiently for genetically transformed heat-tolerant plants, the effects of underlying mechanisms under heat stress on plant morphology, physiology, growth, and antioxidative responses must first be identified.1 hybrid genotype: FB-SHAHEEN × JSQ WHITE GOLD, followed by Ghuari-1, CCRI-24, Eagle-2 × FB-Falcon, Ghuari-1 × JSQ White Gold, and Eagle-2 were the best performers under stress and normal conditions as they were not adversely affected. The adverse effects of heat stress usually include disruption of routine morphological, physiological, biochemical, and fiber characters in cotton and ultimately affect yield. Potential genotypes can be efficiently employed in future cotton breeding programs to improve cotton crop yield and productivity by enhancing their heat tolerance to withstand the changing climate.The continuously warming global climate drives plant genotypes to adapt through the modification of specific phenotypes. With this scenario of escalating temperature, the development of cultivars that may endure abrupt fluctuations without adversely affecting yield is necessary. The first solution is to screen the available cotton germplasm for its potential against high-temperature stress. Most plants exhibit high antioxidant enzyme activities as an important step involving the heat tolerance mechanism. This work identified that the FThe original contributions presented in the study are included in the article/MZ: experimentation, data collection, and drafting the manuscript. XJ and HM: visualization, validation, review, and editing manuscript. AS: conceptualization, resources, supervision, experimentation, review, and editing. ZS: formal analysis, visualization, validation, review, and editing. AM, AI, and AR: experimentation, data acquisition, review, and editing. AA: data acquisition, experimentation, review, and editing. YY: resources, visualization, validation, review, and editing. MI: formal analysis, software, visualization, validation, review, and editing. MR: conceptualization, funding, supervision, validation, review, and editing. All authors have reviewed the manuscript critically and approved the final draft for publication in Frontiers in Plant Science.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.