Paeonia suffruticosa Andr.) is a popular ornamental plant in China due to its showy and colorful flowers. However, yellow-colored flowers are rare in both wild species and domesticated cultivars. The molecular mechanisms underlying yellow pigmentation remain poorly understood. Here, petal tissues of two tree peony cultivars, “High Noon” (yellow flowers) and “Roufurong” (purple–red flowers), were sampled at five developmental stages (S1–S5) from early flower buds to full blooms. Five petal color indices  and the contents of ten different flavonoids were determined. Compared to “Roufurong,” which accumulated abundant anthocyanins at S3–S5, the yellow-colored “High Noon” displayed relatively higher contents of tetrahydroxychalcone (THC), flavones, and flavonols but no anthocyanin production. The contents of THC, flavones, and flavonols in “High Noon” peaked at S3 and dropped gradually as the flower bloomed, consistent with the color index patterns. Furthermore, RNA-seq analyses at S3 showed that structural genes such as PsC4Hs, PsDFRs, and PsUFGTs in the flavonoid biosynthesis pathway were downregulated in “High Noon,” whereas most PsFLSs, PsF3Hs, and PsF3’Hs were upregulated. Five transcription factor (TF) genes related to flavonoid biosynthesis were also upregulated in “High Noon.” One of these TFs, PsMYB111, was overexpressed in tobacco, which led to increased flavonols but decreased anthocyanins. Dual-luciferase assays further confirmed that PsMYB111 upregulated PsFLS. These results improve our understanding of yellow pigmentation in tree peony and provide a guide for future molecular-assisted breeding experiments in tree peony with novel flower colors.Tree peony ( Paeonia suffruticosa Andr.) belongs to the section Moutan in the genus Paeonia of the family Paeoniaceae and is a traditional horticultural plant in China1. Floral color is one of the crucial ornamental characteristics of tree peony, which has more than 2000 cultivars with 9 major colors worldwide3. The flower colors of domesticated tree peony are generally white, pink, purple, or red, but few flowers are pure yellow. Currently, new tree peony cultivars with novel pure yellow corollas have become a breeding focus with great economic potential4. “High Noon,” a hybrid of P. suffruticosa × Paeonialutea, is one of the most famous and popular tree peony cultivars. It possesses pure yellow, cup-shaped, and semidouble flowers. Due to its strong ecological adaptability, “High Noon” has been increasingly cultivated in the major production areas of tree peony around the world. However, the molecular basis of yellow coloration in tree peony remains elusive. Therefore, yellow-colored “High Noon” provided a good candidate for research on yellow coloration in tree peony flowers.Tree peony , luteolin (Lu), and chrysoeriol (Ch); and flavonols, including multiform glucosides of kaempferol (Km), quercetin (Qu), and isorhamnetin (Is)10. In eight different color series from white to red, to yellow of tree peony, a total of 39 flavonoids  have been identified12. Recently, 56 flavonoids were further characterized from 15 traditional Chinese tree peony cultivars with white, pink, and red color series13. In yellow-colored tree peony flowers, 26 flavonoid components have been detected, among which Km, Ap, Lu glucosides, and chalcones were dominant11. The main flavonoid components in yellow flowers of P. lutea were chalcones, flavones, and flavonols, including tetrahydroxychalcone (THC), isosalipurposide (ISP), Km, Qu, Is, Ch, and Ap5. The production of chalconaringenin 2’-O-glucoside  was presumed to be a leading reason for yellow pigmentation in petals of P. lutea14.The chemical compounds determining flower colors mainly include flavonoids, carotenoids, chlorophylls, and their derivativesO-glucosyltransferase (UFGT), and anthocyanin O-methyltransferase16. These structural genes are regulated by the MBW complex, which contains MYB, bHLH transcription factors (TFs), and WD40 proteins17. In the regulation of flavonoid production, MYBs serve as key TFs and bind directly to bHLH regulators or the promoters of structural genes to activate gene expression18. As chaperone proteins, WD40 plays a role in stabilizing the MBW complex. In addition, other TFs, such as SPLs, NACs, WRKY, HY5, and ERFs, are also involved in regulating flavonoid biosynthesis20. In particular, SPLs can disturb the formation of the MBW complex by competing for bHLHs or MYBs, thus inhibiting anthocyanin production21. Recently, many flavonoid synthesis-related genes have been identified in tree peony, including the structural genes PsCHS1, Ps-CHI1, PsF3H1, PsDFR1, and PsANS1, and the TF genes PlWDR3, PlWDR18, PlbHLH3, PoMYB2, PsMYB12, and PoSPL128. However, only a limited number of studies have reported candidate genes for yellow pigmentation in tree peony. For example, transgenic tobacco plants constitutively expressing Ps-CHI1 exhibit an up to threefold increase in total flavonol and flavone levels, and a significant reduction in anthocyanin content and floral color strength compared to wild-type (WT) controls24. High coexpression of THC2’GT, CHI, and FNS II in P. lutea flowers guarantees the accumulation of yellow pigments14. Notably, no TF gene regulating yellow pigmentation in tree peony has been identified.Flavonoid synthesis can be divided into three phases. The first phase includes the transformation of phenylalanine to coumaroyl-CoA, which is shared by many secondary metabolism pathways. The second phase corresponds to the conversion of coumaroyl-CoA to dihydroflavonols, including dihydrokaempferol, dihydroquercetin, and dihydromyricetin (DHM). This process is catalyzed by a battery of enzymes covering chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), flavonoid 3’-hydroxylase (F3’H), flavonoid 3’5’-hydroxylase, flavone synthase (FNS), and flavonol synthase (FLS). The second phase is critical for yellow coloration due to its accumulation of chalcones, flavones, and flavonols. Subsequently, the third phase is the synthesis of a series of stable anthocyanins catalyzed by dihydroflavonol 4-reductase (DFR), anthocyanidin synthase (ANS), UDP-glucose flavonoid 3-31. Recently, the third-generation sequencing (TGS) platform single-molecule real-time sequencer PacBio RS  has been developed for more advanced RNA sequencing (RNA-seq)33. Compared to SGS, TGS has longer read lengths, higher consensus accuracy, a lower degree of bias, and simultaneous capability for epigenetic characterization34. In turn, the sequence alignment errors of TGS can be algorithmically improved and corrected by SGS reads35. Therefore, higher quality transcriptome assemblies can be achieved by combining SGS and TGS technologies.Second-generation sequencing (SGS) technology has been widely applied to transcriptomic analysis in tree peonyIn this study, we performed metabolomics and full-length transcriptomics analyses of the petal tissues of two tree peony cultivars, “High Noon” (yellow flowers) and “Roufurong” (purple–red flowers), at different flowering stages. Differentially expressed genes (DEGs) and the metabolic profiles of the two cultivars were identified and characterized. Candidate genes responsible for yellow pigmentation were further investigated by quantitative real-time PCR (qRT-PCR) analysis, subcellular localization, overexpression in tobacco, promoter sequence analysis, and dual-luciferase assays. The molecular mechanisms of yellow pigmentation in tree peony flowers were discussed.L* (brightness) value increased gradually from S1 to S5, indicating the elevation of petal color brightness. C* (chroma) and b* (yellowness) peaked at S3 and subsequently declined at S4 and S5, demonstrating that S3 was the yellowest stage. In contrast to L*, C*, and b*, h (hue angle) decreased from S1 to S3, followed by a slight increase at S4 and S5. For “Roufurong”, no significant change in L* was observed throughout the flowering process. h dropped from S1 to S3 and then increased dramatically at S4 and S5. h-Values from S3 to S5 were ~0° (360°), consistent with the purple–red color. Similar to h, C* in “Roufurong” also decreased from S1 to S3 and increased at the later stages S4 and S5. Consistent with the flower color, b* in the purple–red-colored “Roufurong” was significantly lower than that in the yellow-colored “High Noon” from S1 to S5. Consistently, a* (redness) was much higher in “Roufurong” than in “High Noon”.To characterize flower color development in tree peony, the petal tissues of the yellow-flowered cultivar “High Noon” and purple–red-flowered cultivar “Roufurong” were sampled at five developmental stages from early flower buds to full blooms Fig. . The petO-glucoside (Pn3G) increased dramatically as the flower bloomed and reached the maximum level at S5, implying that Pn3G may contribute to purple–red flower pigmentation. Compared to “High Noon”, the THC, Is, and Ap contents showed a quick elevation during the first four periods and a moderate decline at S5. Their variation ranges were relatively smaller than that of pelargonidin 3-O-glucoside (Pg3G). In contrast, the contents of Lu, Km, Qu, cyanidin 3-O-glucoside (Cy3G), and Ch did not display a clear developmental pattern, and thus, these components may have no significant influence on the purple–red coloration.Significant variations in flavonoid contents were observed between the two tree peony cultivars at each developmental stage. As shown in Fig. L* and b* were positively correlated with THC levels, indicating that the increase in the THC content led to the accumulation of yellow pigments. In contrast, the h-value was negatively correlated with THC levels. Ch had a positive effect on C* but a negative effect on a*. Therefore, THC and Ch may be associated with the production of yellow flowers. In “Roufurong”, Pg3G had a negative impact on L* but a positive impact on h, suggesting that a high content of Pg3G may lead to a low brightness of the petals. In addition, C* was negatively correlated with the Lu content, which may affect petal saturation as a copigment. Moreover, a* was found to be positively and negatively associated with Pn3G and Ch, respectively, whereas b* had a negative correlation with Pn3G and Ap. These results indicated that a high content of Pn3G and a low content of Ch were critical for the purple–red color of petals. Considering that THC and Ch displayed the highest accumulation levels at S3 in “High Noon”, and C* and b* also peaked at S3, stage S3 was further selected for subsequent transcriptome analyses to identify the candidate genes for yellow pigmentation.To understand the relationship between flower color and flavonoid components, multiple linear regression (MLR) analysis was performed Table . In “HigVitis vinifera (27.40%) and more transcripts fell under biological process, of which metabolic process, cellular process, and single-organism process were mainly enriched with transcripts  were obtained, with an average read length of 1,883 bp  and 5 TF genes (PsMYB4 (F01_cb7851_c8), PsMYB111 (F01_cb8732_c11), PsTT8 (F01_cb3692_c7), PsEGL3 (F01_cb8475_c28561), and PsSPL9 (F01_cb9156_c0)). The expression profiles of these DEGs related to flavonoid biosynthesis are displayed in Fig. To identify the candidate genes for yellow pigmentation, DEGs between “High Noon” and “Roufurong” at S3 were identified. There were 16,017 upregulated DEGs and 13,527 downregulated DEGs in “High Noon” compared with “Roufurong” Fig. . Gene OnPsCHSs (F01_cb9709_c117 and F01_cb9709_c59) and two PsCHIs (F01_cb13354_c0 and F01_cb13354_c4). Based on gene annotation, PsCHS functions in the synthesis of THCs, whereas PsCHI plays a critical role in the production of flavanones, which contribute to yellow coloration. In addition, eight putative PsF3Hs , two PsF3’Hs (F01_cb6443_c24 and F01_cb6443_c9), and nine PsFLSs  also exhibited higher transcription in “High Noon”. Of these genes, PsFLS can divert the flavonoid pathway in favor of flavonol production. These expression variations between “High Noon” and “Roufurong” were consistent with the observed higher levels of THC, flavones, and flavonols in “High Noon”. In contrast to the structural genes at the second stage, genes at the third stage of flavonoid biosynthesis, including four PsDFRs  and three PsUFGTs , were significantly downregulated in “High Noon”. PsDFR and PsUFGT are responsible for the biosynthesis of anthocyanins such as Cy3G, Pn3G, and Pg3G. Therefore, the downregulation of PsDFRs and PsUFGTs in “High Noon” may explain the lack of anthocyanin production in this yellow-flowered cultivar.As shown in Fig. 17. Candidate MYB proteins related to flavonoid biosynthesis were identified by phylogenetic analyses with MYBs from Arabidopsis. The results showed that PsMYB4 belonged to subgroup 4 (S4) of Arabidopsis  and repress the transcriptional activities of MBW complexes, thereby leading to the inhibition of anthocyanin and phenylpropanoid syntheses36. PsMYB111 was clustered to S7 , PsCHS (F01_cb9709_c126), PsCHI (F01_cb13354_c0), PsF3H (F01_cb4853_c44), PsF3’H (F01_cb6443_c9), PsFLS (F01_cb4550_c24), and PsDFR (F01_cb12671_c6), as well as TF genes PsMYB4, PsMYB111, PsTT8, PsEGL3, and PsSPL9 at five flowering stages, S1–S5, in “High Noon” and “Roufurong” petals  was constructed and transiently transformed into Nicotiana benthamiana leaves. The subcellular localization of the protein was observed after 72 h. Fluorescence of the fusion protein PsMYB111-GFP was detected specifically in the nucleus . Two T2 lines (OE-1 and OE-2) were obtained. The flower color of OE-1 and OE-2 was light pink, in contrast to the rosy red color of the WT    Fig. . Corresp01) Fig. . These rNtCHS, NtCHI, and NtFLS increased significantly compared to WT, whereas NtC4H, NtF3H, NtF3’H, and NtDFR were only mildly upregulated. The upregulation of NtFLS was the highest among the analyzed structural genes. In addition, NtbHLH and NtSPL9 exhibited similar expression abundances to WT. Taken together, PsMYB111 may regulate the expression of structural genes alone rather than in a complex and may enhance the accumulation of flavonols such as Qu and Km.To investigate whether PsMYB111 can regulate the transcription of flavonoid biosynthetic structural genes, we analyzed the expression of selected structural genes in OE-1 petals using qRT-PCR Fig. . The resNtCHS and NtFLS. To verify its regulatory role on PsCHS and PsFLS, we sequenced the promoter sequences of PsCHS (750 bp) and PsFLS , and analyzed their cis-acting elements , bHLH-binding site (5′-CATGTG-3′), and G-box (5′-CACGTG-3′)  Fig. , supportPsCHS and PsFLS served as reporters. After coinfiltration of the TF and promoters in N. benthamiana leaves, the LUC/REN ratios were detected. As shown in Fig. PsCHS and PsMYB111-PsFLS constructs compared with the corresponding controls, suggesting that PsMYB111 can activate the promoters of both PsCHS and PsFLS. The activation intensity of PsMYB111 on PsFLS (2.59-fold) was stronger than that of PsCHS (1.58-fold), indicating that PsMYB111 may preferentially regulate the transcription of PsFLS.To further confirm this hypothesis, a dual-luciferase assay was performed. The pGreenII62-SK vector carrying PsMYB111 served as an effector and the pGreenII0800 LUC vector carrying the promoters of Pure yellow flowers in tree peony are extremely rare. The molecular mechanisms of yellow pigmentation in this species remain unclear. Here we selected the cultivar “High Noon” with yellow flowers as the research material, with the purple–red-flowered cultivar “Roufurong” as a control. The floral color phenotypes and flavonoid profiles of “High Noon” petals were characterized, together with combined full-length and comparative transcriptome analyses. Candidate genes underlying yellow pigmentation were further validated by functional analyses.P. lutea from Yunnan, which also contains abundant THC, ISP, Km, Qu, Is, Ch, and Ap5. Among these compounds, THC and Ch were the main pigments causing yellow color. The high amount of Chalcone 2’G has been speculated to be an important reason for the yellow-flowered phenotype in P. lutea14. In “Roufurong”, the accumulation of the dominant anthocyanin Pn3G is the most likely reason for its purple–red-colored petals. It has been reported that reductions in anthocyanins increased the L*-value and decreased the a*-value, causing flower colors of two herbaceous peony cultivars “Coral Sunset” and “Pink Hawaiian Coral”, to change from coral to pink and then to yellow41. In addition, the sharp decrease in anthocyanins during flowering of “Jinyi Hualian” and “Xiaguang” elicited colors from red to orange and then to yellow42. Therefore, the high contents of THC, flavones, and flavonols without anthocyanins may be essential for the yellow pigmentation of “High Noon” flowers.Flavonoids play a key role in the coloration of different plant organs. In this study, we showed that the contents of THC, flavones , and flavonols  in the flowers of “High Noon” were markedly higher than in those of “Roufurong”. No anthocyanin was detected in the yellow petal tissues of “High Noon”. Similar observations have been found for yellow-flowered 14. It was previously reported that a low expression level of PlCHI in the Paeonia lactiflora cultivar “Huangjinlun” induced chalcone accumulation and produced a yellow color43. In the present study, we assume that the high expression of PsCHS and low expression of PsCHI in “High Noon” during S3–S5 contributed to the large accumulation of THC, which also resulted in yellow coloring. In addition, the high expression of PsF3H, PsF3’H, and PsFLS from S3 to S5 in “High Noon” promoted metabolic flow to the synthesis of flavonols . In white Muscari armeniacum flowers, when FLS was upregulated, the substrates used for cyanidin synthesis were then available for the synthesis of kaempferol44. The downregulation of PsDFR and PsUFGT is consistent with the lack of anthocyanin accumulation in “High Noon”. Similarly, observations have been made in P. lactiflora, in which the lower expression levels of PlDFR, PlANS, Pl3GT, and Pl5GT in the inner petals inhibited anthocyanin production, resulting in yellow pigment formation45. Notably, the substrate competition mechanism between FLS and DFR may cause variations in anthocyanin and flavonol synthesis, as FLS strengthens dihydroflavonol flux toward flavonols and finally limits anthocyanin accumulation46. In the present study, we also observed competition between FLS and DFR, which may have also contributed to the yellow-flowered phenotype in “High Noon”. The upregulated expression of FLS transcripts in “High Noon” may consume dihydroflavonols as a substrate, thereby increasing the accumulation of flavonols and reducing the accumulation of anthocyanins at the DFR branch. Similar findings have been revealed in Paeonia ostii, which exhibited upregulated expression of PoFLS4 and a transition of dihydroflavonols into flavonols in nearly white flowers28. The competition between FLS and DFR in M. armeniacum for DHM might inhibit the synthesis of delphinidin, thereby altering the ratio of flavonol to anthocyanin and furthering the elimination of blue pigmentation44. The latest research on Brassica oleracea L. italica also showed that the difference in kaempferol accumulation was likely caused by the expression level of FLS47. In contrast, the increased transcription of PlDFR in pigmented flowers of Pleione limprichtii was accompanied by a decrease in PlFLS transcription, causing increased production of anthocyanins48. In addition, the competition mechanism between FLS and DFR has also been reported to underlie the lack of anthocyanins in white-fruited Chinese bayberry27.In the flavonoid biosynthetic pathway, CHS catalyzes the synthesis of a key intermediate THC, which can be further isomerized by CHI, leading to the production of flavones and flavonolsArabidopsis, the triple mutant Atmyb4/7/32 displayed elevated anthocyanin and phenylpropanoid accumulation compared to the WT plants, which is consistent with our results36. We also observed that PsDFR and PsC4H were obviously negatively correlated with PsSPL9. The high expression of PsSPL9 and low expression of PsDFR may inhibit anthocyanin production in “High Noon”. Similarly, the AtSPL9 TF in Arabidopsis was also proven to inhibit the expression of anthocyanin biosynthetic genes, particularly DFRs21. In addition, yellow coloration in the floral tissues of P. lactiflora is possibly under the regulation of miR156e-3p-targeted SPL1 by suppressing PlPAL, PlFLS, PlDFR, PlANS, Pl3GT, and Pl5GT49. For PsMYB111 and PsTT8, although they displayed a consistent expression pattern  could be generally upregulated in transgenic tobacco overexpressing flavonol-specific MYB TF genes54. The overexpression of GtMYBP3 and GtMYBP4 identified in Gentiana triflora promoted the expression of flavonol biosynthesis genes in tobacco and Arabidopsis51. In addition, the expression of NtCHS, NtF3H, and NtFLS was strongly upregulated in GhMYB1a-overexpressing transgenic tobacco lines and GhMYB1a significantly activated the promoters of NtCHS and NtFLS over GhDFR and GhMYB10 in gerbera39. Similarly, we showed that PsMYB111 had a significant activation effect on PsCHS and PsFLS promoters, particularly PsFLS . Two cultivars, “High Noon” with pure yellow flowers and “Roufurong” with purple–red flowers, were used as the experimental materials. All selected plants were grown in fields with adequate light and moisture  of fresh petals at these five stages were measured by a tristimulus color meter . The materials for other tests were quickly frozen in liquid nitrogen and then stored at −80 °C until further use. Tobacco plants (N. benthamiana and Nicotiana tabacum) were cultivated in an incubator at 25 °C with a 16/8 h day/night photoperiod. The color-related values  of N. tabacum petals were measured according to standard methods for tree peony.11. Frozen petal samples of tree peony and tobacco were promptly ground to a powder in liquid nitrogen with mortars and pestles. Approximately 300 mg of tree peony petal powder and 100 mg of tobacco petal powder were divided into two parts and dissolved in 6 ml (for tree peony)/2 ml (for tobacco) of methanol-hydrochloric acid  solution for anthocyanin detection and 6 ml (for tree peony)/2 ml (for tobacco) of methanol solution for the detection of other flavonoids. Next, the samples were leached in the dark at 4 °C for 24 h and shaken and mixed once every 6 h. After 30 min of ultrasound-assisted extraction, the samples were centrifuged at 10,000 r.p.m. for 10 min to collect the liquid supernatants. Finally, each supernatant was filtered through a 0.22 μm nylon microporous membrane. Ten microliters of pure supernatant was quantified by HPLC  equipped with a diode array detector. A 4.6 × 250 mm C18 column  was used. Eluent A was a 0.04% formic acid aqueous solution and eluent B was acetonitrile. The following gradient elution conditions were used: 5% B at 0 min, 40% B at 40 min, 100% B at 45 min, 100% B at 55 min, and 5% B at 60 min. The flow rate was 0.5 ml/min at a 40 °C column temperature. Standards of THC, Ap, Lu, Ch, Km, Qu, Is, Cy3G, Pn3G, and Pg3G were purchased from Shanghai Yuanye Bio-Technology Co., Ltd . Mean values and SDs were calculated from three independent biological replicates.Flavonoids were detected using a previously reported high-performance liquid chromatography (HPLC) method with some modificationsL*, a*, b*, C*, and h were dependent variables.Furthermore, MLR analysis was conducted by SPSS (version 23.0) to explore the relationship between flower colors and flavonoid components. The contents of THC, Ap, Lu, Ch, Km, Qu, Is, Cy3G, Pn3G, and Pg3G were independent variables, whereas the values of Ten petal samples at five flowering stages of yellow- and purple–red-flowered tree peony cultivars were collected for isoform and RNA-seq. First, the total RNA of ten samples was extracted using the RNAprep Pure kit for plants . RNA quality and integrity were detected using an RNA 6000 Nano Assay Kit in an Agilent Bioanalyzer 2100 system . Subsequently, equal amounts of RNA from each sample were pooled together. A SMARTer PCR cDNA Synthesis Kit  was applied to reverse-transcribe total RNA into cDNA. The sequencing process was conducted on a Pacific Bioscience RS II platform. Second, two petal samples at S3 from two tree peony cultivars with three biological replicates were sequenced on an Illumina HiSeq 2500 platform for comparative analysis.https://github.com/TransDecoder/TransDecoder/releases). TFs were predicted by iTAK software (https://omictools.com/itak-tool)55. Genes were annotated based on the following databases: Nr (NCBI nonredundant protein sequences), Pfam (protein family)56, KOG/COG/eggNOG (Clusters of Orthologous Groups of proteins)58, Swiss-Prot 59, KEGG (Kyoto Encyclopedia of Genes and Genomes)60, and GO61.Raw reads were filtered for ROIs through the Iso-seq pipeline and those with completed 5′- and 3′-cDNA primers, as well as poly A tails, were identified as FLNC transcripts. Furthermore, Iterative Clustering for Error Correction was conducted to screen consensus isoforms. High-quality full-length transcripts were confirmed under the criteria of postcorrection accuracy above 99%. ORFs of the transcripts were predicted using TransDecoder 64. The false discovery rate (FDR) was corrected using the posterior probability values. FDR < 0.01 and |log2(fold change)| ≥ 2 were regarded as the thresholds for significant differential expression. GO enrichment analysis of DEGs was performed by the GOseq R package65. The statistical enrichment of DEGs in KEGG pathways was tested by KOBAS software66.DEGs between two samples based on the RNA-seq results were identified using the EBSeq R package−1, qRT-PCR was performed using TB Green TaKaRa Premix Ex Taq™ II  according to the manufacturer’s instructions. The reaction took place under the following conditions: denaturation at 95 °C for 15 s and 45 cycles of amplification . PsUbiquitin was used as an internal reference for the expression level normalization of DEGs. Relative expression levels were calculated by the 2−∆∆CT method67. The specific primers are listed in Supplementary Table DEGs putatively involved in the flavonoid biosynthesis pathway were selected for qRT-PCR analysis. Total RNA was extracted with the RNAprep Pure kit for plants  and first-strand cDNA was synthesized using the PrimeScript™ RT Master Mix reverse transcription kit . After diluting the cDNA template five times to 200 ng µLMYB, two bHLH, and one SPL genes implicated in the flavonoid biosynthesis pathway were selected. Phylogeny trees of their corresponding proteins along with 101 MYBs, 94 bHLHs, and 16 SPLs from Arabidopsis were constructed with MEGA 6.0 using the neighbor-joining clustering method. Sequence homology alignment was performed with DNAMAN software. Structural genes related to flavonoid synthesis were screened and clustered based on their expression profiles using TB tools.Two R environment (https://www.r-project.org/); correlations with a coefficient of R ≥ 0.5 or R ≤ −0.5 were retained. The coexpressed genes with strong interconnections were considered hub genes. The relationships between candidate genes, including TF genes and structural genes, and flavonoid components were visualized by Cytoscape (v.3.7.0).The interaction network was established on the basis of Pearson’s correlation coefficients, which were calculated in the PsMYB111 without the termination codon was cloned into the pCAMBIA1302-GFP vector. The primers are listed in Supplementary Table Agrobacterium strain GV3101 by the freeze–thaw method. The Agrobacterium containing the target plasmid was resuspended in infiltration buffer  to an OD600 of 0.4 and stationarily cultured for 2 h until infiltration. The Agrobacterium mixture was then injected into two young leaves of each N. benthamiana plant from the lower epidermis via a syringe without a needle. The infiltrated plants were grown in a growth chamber under the same conditions described above for 72 h, and GFP and 4′,6-diamidino-2-phenylindole fluorescence was observed under a Nikon C2-ER confocal laser scanning microscope . All transient expression assays were repeated three times.The full-length ORF of PsMYB111 construct was transferred into the Agrobacterium strain GV3101, sterilized N. tabacum leaf disc transformation was performed following previously described methods68. Hygromycin and kanamycin were used to screen the transgenic tobacco lines. Rooted transgenic plants were transferred to a soil mix and grown in a greenhouse until flowering. Flowers of T2 transgenic plants were used for the detection of color indices, quantification of flavonoid levels, and qRT-PCR verification as described above. Specific primers are shown in Supplementary Table After the pCAMBIA1302-PsMYB111 was cloned into a pGreenII62-SK vector (effector). The promoter sequences of PsCHS and PsFLS were isolated from the genomic DNA of “High Noon” using a genome walking kit  and then inserted into the pGreenII0800 LUC vector (reporter). All effector and reporter vectors were transformed into Agrobacterium strain GV3101 and isolated with kanamycin (50 mg L−1). Transient expression in N. benthamiana was conducted following the method described in subcellular localization assays. The enzyme activities of firefly luciferase (LUC) and Renilla luciferase (REN) were detected at 72 h after infiltration with a dual-luciferase assay system on GloMax® Discover . Only the promoter-LUC reporter construct with no effector was regarded as a blank control. Three independent experiments were conducted for each combination and all experiments were technically repeated three times. The specific primers used for genome walking and dual-luciferase assays are listed in Supplementary Table The full-length ORF of Supplementary TableSupplementary Figure


Palliative care knowledge and health literacy are frequently underestimated in American adults; for example, as measured by the Newest Vital Sign , 79.2% (n = 247) of participants within a Geriatrics Clinic sample displayed “adequate” functional health literacy, while 11.8% (n = 37) scored within the “possibly limited” range, and 9.0% (n = 28) scored within the “highly limited” range. There was additionally a significant association between health literacy and age  within this sample. The Palliative Care Knowledge Scale  was administered to participants, and higher scores indicated a greater knowledge of palliative care. This construct is particularly important to measure, as racial/ethnic disparities exist within this domain; for example, African Americans may have lower overall knowledge of palliative care services and advance care planning than non-Hispanic Whites . In the current study, knowledge of palliative care was measured using the PaCKS , and scores represented the widest possible range of 0 to 13 . There was a significant correlation between age and PaCKS score , as palliative care knowledge increased with age. Females scored significantly higher  than males , t(309) = 3.18, p < .001. There was no main effect of race on palliative care knowledge, and post-hoc analysis using Tukey HSD did not demonstrate significant differences between groups.


We find that high CSPG4 expression is independently associated with disease-free survival, and with an immune landscape favorable to induce strong cytotoxic immune response after NK cell stimulation. Our results suggest the potential value of CSPG4-specific chimeric antigen receptor-redirected cytokine-induced killer lymphocytes treatment in GIST, notably “CSPG4-high” tumors, and calls for preclinical validation, drug testing in vivo, then in clinical trials.Gastrointestinal stromal tumors (GIST) are the most frequent sarcomas of the gastrointestinal tract. Identification of novel prognostic and/or therapeutic targets is a major issue to overcome tyrosine kinase inhibitors resistances. CSPG4, a cell surface proteoglycan, emerged as a potential therapeutic target for immune therapy in different cancers, including sarcomas. CSPG4 expression has never been studied in GIST. In this work we analyzed CSPG4 mRNA expression in a large series of clinical GIST samples given the scarcity of disease  must be improved through the development of more reliable prognostic factors and of therapies able to overcome imatinib resistance. The immune system represents an attractive tool. CSPG4, a cell surface proteoglycan, emerged as a potential therapeutic target for immune therapy in different cancers, including cell therapy based on CSPG4-specific chimeric antigen receptor (CAR)-redirected cytokine-induced killer lymphocytes (CSPG4-CAR.CIKs) in sarcomas. CSPG4 expression has never been studied in GIST. We analyzed CSPG4 mRNA expression data of 309 clinical GIST samples profiled using DNA microarrays and searched for correlations with clinicopathological and immune features. CSPG4 expression, higher in tumors than normal digestive tissues, was heterogeneous across tumors. High expression was associated with AFIP low-risk, gastric site, and localized stage, and independently with longer postoperative disease-free survival (DFS) in localized stage. The correlations between CSPG4 expression and immune signatures highlighted a higher anti-tumor immune response in “CSPG4-high” tumors, relying on both the adaptive and innate immune system, in which the boost of NK cells by CSPG4-CAR.CIKs might be instrumental, eventually combined with immune checkpoint inhibitors. In conclusion, high CSPG4 expression in GIST is associated with better DFS and offers an immune environment favorable to a vulnerability to CAR.CIKs. However, primary or secondary resistance occurs in all cases and a critical goal is to develop new therapies able to overcome it. Adjuvant imatinib results in lower relapse rates [Gastrointestinal stromal tumors (GIST) are the most frequent sarcomas of the gastrointestinal tract ,2. They se rates ,8 and inse rates . Neverthse rates ,12,13. IPDL1 expression, immune-related parameters, and prognosis in imatinib-naïve patients with localized GIST [The role of immunotherapy in sarcomas is growing fast with no current standard of care. If our current understanding of the immune response in GIST remains limited, several data suggested that the exploitation of immune system may be clinically interesting . The prezed GIST . Altogetzed GIST . During zed GIST , and morzed GIST .CSPG4, a cell surface proteoglycan, displays overexpression in certain human cancers, low expression in normal tissues, and roles in tumor growth and dissemination . AlthougTo our knowledge, CSPG4 expression has not been studied in GIST clinical samples. Here, we analyzed CSPG4 expression in 309 GIST samples and searched for correlations with both the clinicopathological features including clinical outcome and the tumor immune landscape. We show that expression is heterogeneous, high GSPG4 is a favorable independent prognostic factor and is associated with an immune profile suggesting potential vulnerability to cellular immunotherapy.https://depmap.org/portal; accessed on 14 December 2021) to compare the RPPA-based protein vs. RNA-seq-based mRNA expression of CSPG4.Our data set included clinicopathological and gene expression data of clinical GIST samples from 15 public data sets that we collected from the National Center for Biotechnology Information (NCBI)/Genbank GEO, ArrayExpress databases, and authors’ websites . The samA pre-analytic processing of data was done. In a first step, we normalized each data set separately: a quantile normalization was applied to the already processed non-Affymetrix data, and Robust Multichip Average (RMA) with non-parametric quantile algorithm to the raw Affymetrix data. That was done in R using Bioconductor and associated packages. In a second step, we mapped hybridization probes represented across the different technological platforms, and when multiple probes mapped to the same GeneID, we kept the most variant probe in a given data set.CSPG4 expression value in each study separately, the value was transformed by subtracting the mean of the gene in that dataset divided by its standard deviation in the GIST samples. Analysis was done by using binary values using the median expression level of the whole series as a cut-off. Because CSPG4 is the target of cellular immunotherapy, we searched for correlations of its expression in tumors with several immune variables. In each data set separately, several immunity-related multigene classifiers were applied to each tumor: the 24 Bindea’s innate and adaptative immune cell subpopulations [Next, we corrected the 15 studies for batch effects using z-score normalization. Briefly, for each ulations , the Immulations , and metulations , of tertulations , of cytoulations , of IFNαulations , the antulations , and ESTulations .CSPG4 expression-based groups and clinicopathological variables were calculated with the Student’s t-test or Fisher’s exact test when appropriate. Since the delay of relapse and follow-up were not available in seven data sets, we used as primary endpoint the occurrence of disease relapse or death during follow-up. The prognostic analyses for disease-free survival (DFS) were done using a logistic regression based on the lm function , the significance of which was estimated by specifying a binomial family for model with a logit link. Several variables were tested in univariate analysis: the CSPG4 expression status , the patients’ age and sex , the tumor location  and mutational status , and finally the AFIP risk . All variables with a p-value inferior to 5% in univariate analysis were included in the multivariate analysis. When the time to follow-up was available, the 5-year DFS was estimated using the Kaplan-Meier curves; and curves were compared using the log-rank test. A logistic regression analysis using the lm function tested the correlations of molecular variables with “CSPG4-high” vs. “CSPG4-low” groups (R’s package). All statistical tests were two-sided and the significance threshold was 5%. All analyses were done with the survival package (version 2.43) from R software (version 3.5.2).The continuous variables are presented using median and range, whereas the discrete values are presented using number and percentage. The correlations between KIT, mainly in exon 11, then PDGFRA, mainly in exon 18. A wild-type KIT and PDGFRA status (WT) was found in 16% of cases (WT). The disease extension stage was localized in most of cases (83%) and advanced  in 17%. The relapse risk, defined according to the AFIP classification, was low-risk in 53% of cases, intermediate-risk in 19%, and high-risk in 28%.A total of 309 GIST samples was available for analysis. Their characteristics are summarized in CSPG4 mRNA expression varied among the 309 tumors with a range of intensities over 8 units in log2 scale  showed higher mRNA CSPG4 expression in GIST samples (N = 77) than in digestive tract normal tissues (N = 144)  .CSPG4 expression (high vs. low) and clinicopathological features (p = 0.063), with more PDGFRA mutations in “CSPG4-high” tumors and more WT status in “CSPG4-low” tumors. Significant correlations were found with prognostic features: AFIP classification, with more low-risk in “CSPG4-high” tumors and more intermediate- and high-risk in “CSPG4-low” tumors (p = 3.83 × 10−4); tumor site, with more gastric site in “CSPG4-high” tumors and more small intestine and other sites in “CSPG4-low” tumors (p = 4 × 10−6); and disease stage, with more localized diseases in “CSPG4-high” tumors and more advanced disease in “CSPG4-low” tumors (p = 2.83 × 10−3).We searched for correlations between features . There wp = 3.79 × 10−6). In univariate analysis (p = 2.82 × 10−10) class was associated with a higher risk of relapse when compared with low/intermediate-risk class, whereas patients’ age, sex, and mutational status were not significant.A total of 161 out of 309 patients fulfilled the following criteria—localized tumors, treated with primary complete surgery, without adjuvant imatinib, and with available information regarding an eventual disease relapse—and were thus included in the prognostic analysis. Their characteristics are summarized in analysis , AFIP hip = 5.12 × 10−4; logit test), and AFIP high-risk (p = 6.93 × 10−9) class remained significant, suggesting independent prognostic value  for relapse of 0.85 (95%CI 0.78–0.92) in CSPG4-high vs. CSPG4-low patients. Similarly, among the AFIP low/intermediate-risk patients, the Odds Ratio (OR) for relapse was 0.74 (95%CI 0.56–0.99) in CSPG4-high vs. CSPG4-low patients.In multivariate analysis, CSPG4 expression-based group  in the whole series A and wasN = 17) than CSPG4-high (N = 11). We found more “Responders” in the “CSPG4-low” group than in the “CSPG4-high” (71% vs. 55%), but the difference was not significant, likely because of the small number of patients ). As a continuous value, the tumor shrinkage was greater in the “CSPG4-low” group than the “CSPG4-high” group , while “CSPG4-low” tumors showed higher tumor purity (p = 3.65 × 10−3). This identified “CSPG4-high” tumors as more infiltrated by immune cells than “CSPG4-low” tumors , in T lymphocytes  and more specifically with cytotoxic cells subsets .We next looked at the composition and functional orientation of this immune infiltrate in “CSPG4-high” vs. “CSPG4-low” tumors. The analysis of the 24 immune cell types defined as the immunome  revealedp = 2.86 × 10−3) [p = 1.56 × 10−3) [p = 7.37 × 10−4) [p = 1.60 × 10−3) [p = 6.46 × 10−3 and p = 3.23 × 10−3). Antigen processing/presentation machinery (APM) score [p = 4.12 × 10−2).Additional immune functional signatures reinforced this observation, showing higher Immunologic Constant of Rejection (ICR) score ( × 10−3)  in “CSPG × 10−3)  and tert × 10−4) , coheren × 10−3)  from bot × 10−3) , in “CSPM) score  was alsoCSPG4 expression in 309 GIST clinical samples. We report that high expression is an independent, favorable prognostic factor for disease relapse and is associated with cytotoxic immune response. To our knowledge, this is the first study analyzing the expression of this new potential target for immune therapy in GIST.We analyzed CSPG4 mRNA expression. Such an approach allowed us to avoid the limitations of immunohistochemistry  while providing opportunities to work on a relatively large series of samples and to search for associations with the expression of other multigene signatures.Our preliminary analysis of more than 350 cancer cell lines revealed a strong correlation between mRNA and protein expression levels, allowing us to base our study on CSPG4 expression in GIST was heterogeneous, making possible to search for correlations with clinicopathological variables. High expression was associated with favorable prognostic variables: low-risk according to AFIP classification, gastric site, and localized extension stage at diagnosis. In the literature, other immune tumor features have been found to be associated with a more favorable AFIP class, such as high NK cells infiltration, low T-regs infiltration [CSPG4 expression was higher in samples from patients without disease relapse than in samples from patients with relapse. This favorable prognostic value was conserved in multivariate analysis, as was conserved the prognostic value of AFIP classification. In addition, we showed that “CSPG4-low” patients seemed more sensitive to neoadjuvant imatinib than “CSPG4-high” patients; however, the results were non-significant, likely because of the small number of patients, which calls for an analysis of larger series of patients. The prognostic value of high CSPG4 expression has already been reported in melanoma [As already observed in many cancers, we confirmed higher expression in GIST tumor samples than in normal digestive tissues. ltration , and higltration , suggestmelanoma , glioblamelanoma , breast melanoma , head anmelanoma , and hepmelanoma ; howevermelanoma . In sarcmelanoma  and softmelanoma ; althougmelanoma . To our dim cells; (iii) higher expression of signatures evoking higher immune cytolytic activity. Altogether, these results suggested a higher anti-tumor immune response in “CSPG4-high” tumors than in “CSPG4-low” tumors that relies on both the adaptive and innate immune system. Of note, the Th1 immune cell type was not significantly different between the two tumor groups, suggesting that, even though the immune infiltrate was higher in “CSPG4-high” tumors, there is still room to increase anti-tumor immune response efficiency. The actual cytotoxic response seems to rely on innate NK-cell subset activation. This also has been suggested by several reports [bright NK cells that accumulated in tumor foci after imatinib treatment [CSPG4, a transmembrane proteoglycan, had been originally identified as a highly immunogenic tumor antigen on the surface of melanoma cells , and dur reports ,50. Of nreatment , and thereatment .In this fertile immune environment, complementing the boost of NK cells with immune checkpoint inhibitors, which will fully unleash CD8 T cells cytotoxic potential, might be extremely powerful. Novel immunotherapies, such as anti-NKG2A or anti-KIR, that target both NK and other cytotoxic T cells subsets, might be promising as well. In this line, “CSPG4-high” GIST, as compared to “CSPG4-low” GIST, might be more sensitive to CSPG4-CAR.CIKs, not only because of higher target expression level, but also thanks to a more favorable anti-tumor cytokine microenvironment with a synergic action of resident cytotoxic T and NK cells. Such analysis has never been reported in GIST.CSPG4 expression. It also includes a few limitations: (i) its retrospective nature and (ii) the mRNA, rather than protein, analysis on bulk tissue samples. Of course, analysis of larger patient series is warranted to confirm our observation, as well as functional analyses of GIST preclinical models. However, our results suggest the potential value of CSPG4-CAR.CIKs treatment in GIST, notably “CSPG4-high” tumors, that calls for drug testing in vivo, and then in clinical trials.We showed that expression of CSPG4, a new potential target for immune therapy, notably specific CAR-CIKs, is heterogeneous in GIST clinical samples and that high expression is associated independently with DFS and with an immune landscape favorable to induce strong cytotoxic immune response after NK cell stimulation. Our study displays several strengths: (i) its originality, (ii) a relatively large size of series given the rarity of GIST, and (iii) the biological and clinical relevance of


Subgroup analysis showed that SMD was reduced as the subjects’ CRF level increased, with a 6–8% CHO solution composed of GL:FRU improving performance (exercise: 1–4 h); administration during the event led to a superior performance compared to administration before the exercise, with a 6–8% single-source CHO solution increasing performance in intermittent and ‘stop and start’ sports and an ~6% CHO solution appearing beneficial for 45–60 min exercises, but there were no significant differences between subjects’ gender and age groups, varied CHO concentrations, doses, or types in the effect measurement. The evidence found was sound enough to support the hypothesis that CHO solutions, when ingested during endurance exercise, have ergogenic action and a possible crossover interaction with the subject’s CRF.Carbohydrate (CHO) supplementation during prolonged exercise postpones fatigue. However, the optimum administration timing, dosage, type of CHO intake, and possible interaction of the ergogenic effect with athletes’ cardiorespiratory fitness (CRF) are not clear. Ninety-six studies (from relevant databases based on predefined eligibility criteria) were selected for meta-analysis to investigate the acute effect of ≤20% CHO solutions on prolonged exercise performance. The between-subject standardized mean difference [SMD = ] was assessed. Overall, SMD [95% CI] of 0.43  was significant ( Interest in the role and contribution of carbohydrates (CHOs) as an energy fuel, particularly during endurance exercise, dates back to the beginning of the 20th century . Christe−1 for exercise lasting up to 2.5 h and up to 90 g·h−1 when the duration of exercise exceeds 2.5 h ). The alpha level for statistical significance was set at An overview, along with the subjects’ characteristics, descriptive characteristics of the protocols used, and the effect (in SMD [95% CI]) of experimental CHO supplementation as compared to the control on an exercise task of the reviewed articles included in this meta-analysis, is reported in alphabetical order, by first author surname, in p < 0.001). All studies used similar methods (pre-post design). The sample population of the control groups used was usually identical to that of the experimental groups in a crossover design, with the exception of three studies, which used a different sample population as control groups in a parallel design  involving 1560 subjects in experimental groups (1534 in control groups) satisfied the inclusion criteria and were approved for further analysis. So, 142 SMDs were assessed and the total SMD [95% CI] estimate of 0.43  was significant,  in comparison to dose rates of 81–100 g·h−1 ,  solution compared with control (placebo) on prolonged exercise performance in subjects over 18 years old found that regardless of the CHO supplementation time administration, form, and concentration, and exercise type, mode, or protocol, consumption of CHO solution has positive effects on performance. However, a crossover interaction between SMD and the subjects’ CRF level could exist. Carbohydrate solution seems to have a tendency to favor cyclists’ performance more than that of runners. As the exercise duration increases from 1 up to 4 h, the effect size of CHO intake on performance is also increased. A 6–8% CHO solution composed of two CHOs (GL:FRU) than more components appeared sufficient to increase the chances of a better performance. Interval frequency administration during the event proved superior to administration before the exercise. Moreover, though the effect size on performance seems to be unaffected by the use of different CHO doses schedules, it was found that a CHO dose of 80–100 g·hn = 679 subjects) and no effect in the remaining 18% [n = 1560).In 2011, two meta-analyses reported a significant acute effect of CHO supplementation on endurance exercise performance  ,144. In ning 18% . Anotherning 18% . SimilarOn the other hand, high concentrated CHO sport solutions (>8% CHO) may in some cases impede the process of fluid absorption, hinder performance, ,149,150,CHO supplementation supposedly has different effects on performance in different modes of exercise. This may be due to different muscle groups at work  or to possible differences in the CHO absorption rate, which may be aided by small stomach movements during different exercise modes . Undoubtedly, the athletes’ weight is one of the determinant factors of energy expenditure, running economy, and, consequently, endurance exercise performance . As runnPrevious studies have demonstrated that fatigue during prolonged exercise in subjects fed CHO supplement occurs at approximately the same time as muscle glycogen depletion ,165,166.−1 [−1 as an acute fueling strategy is generally recommended [−1 using a 7% solution, [−1, no positive result was found [Originally, most of the studies investigated the role of CHO supplements during continuous (rather than intermittent) endurance exercise and showed an increase in endurance efficiency. During the prolonged intermittent and intermittent shuttle exercise (periods of intermittent bursts of high or lower intensity exercise (punctuated by rest or lower intensity activities)), energy is derived conjointly from anaerobic and aerobic metabolism ,168. So,−1 . Based oommended ,169. Simommended ,49,63,79as found ,108.−1) than the guidelines suggest [Theoretically, CHO availability via the blood is not limited during exercise in individuals taking CHO supplements. Our findings show that: (a) as exercise duration increases up to 4 h, so does the effect size of CHO intake on performance ; and (b) suggest  in endur suggest ,177,178. suggest ,127,128) suggest , (b) mus suggest , and (c) suggest ,145.Given that (a) GL expenditure is closely correlated with exercise intensity , (b) traIn terms of sex, the already existing guidelines often assumed a similarly acute exercise effect of CHO supplementation in males and females. A study with a mixed-gender group  revealed−1) [−1 (60 g·h−1) [−1). A series of studies also demonstrated even higher oxidation rates, up to 75%, using MTC (GL:FRU in ratio of 2:1) [−1) benefits performance more than the consumption of sport solutions containing GL-only (or MD-only) and minimizes the chances of GI discomfort [The energy-dependent sodium-glucose link transporter (SGLT1) actively transports GL  and GAL through the intestinal mucosa ,193,194.−1) , exogeno0 g·h−1) ,204,205.0 g·h−1) ,209,210.0 g·h−1) ) speeds  of 2:1) ,212. Morscomfort ,215,216.−1), and consequently ensure better performance during prolonged exercise [The main goal of the intake of CHO sport solution during exercise is euhydration and euglycemia maintenance, and the minimum GL concentration required to boost water absorption is 0.9% . So, in exercise ,220,221.exercise . This isexercise ,169.Regarding CHO administration time , there seems to be a lack of relevant comparative studies and only a few reviews have addressed this topic, albeit not extensively ,160. Bri−1), probably because of the large variance of SMD among different trials that used different experimental methods . On the other hand, some reviews consider that CHO intake of up to 60 g·h−1 for exercise lasting up to 2.5 h and up to 90 g·h−1 when the exercise duration exceeds 2.5 h should be recommended [N = 8) did not actually test the dose–response in terms of CHO g·h−1 intake but tested the CHO ingestion at a rate of 1.8 g·min−1 during 120 min of exercise where the significant effect was confirmed [N = 9) compared the effect of two different iso-caloric beverages, containing GL or GL:FRU (no placebo/control group), at an ingestion rate of 2.4 g·min−1 ~144 g·h−1 on exercise lasting more than 3 h [N = 12) compared ingestion of a placebo or CHO at doses of 15, 30, and 60 g·h−1  on exercise lasting more than 2 h [N = 51) suggested an CHO ingestion dose between 60 and 80 g·h−1 for an optimum performance enhancement when exercise lasts 2 h [−1 for exercise lasting up to 2.5 h in the trained male cyclists population only, rather than recommend a CHO intake up to 60 g·h−1 for exercise lasting up to 2.5 h and up to 90 g·h−1 when the duration of exercise exceeds 2.5 h for the general population in various exercise modes.Concerning the CHO dose–response, our results show that the effect size on performance is unaffected by the use of different CHO dose schedules , relevant research studies are still limited at the present time [−1 during an endurance event lasting up to 2.5 h, and up to 90 g·h−1 when the duration of exercise exceeds 2.5 h [−1 has also been suggested for an optimum performance benefit when exercise lasts ~2 h [−1 of MTC , theoretically, the chance of an optimum CHO oxidation rate [−1 GL-only (or MD-only) ingestion [−1 (instead of 60 g·h−1) composed of MTC for endurance events lasting 1–2.5 h [In 2013, Jekendrup (one of the leading researchers in the field) argued that, with a few exceptions, decent dose–response research was notably absent and many of the published reports had substantial methodological flaws, which made it difficult to establish a valid relationship between the quantity of CHO consumption and the efficiency of the dose–response . In the ent time . We onlyds 2.5 h ,220. Nevds 2.5 h ,172,173.ds 2.5 h . A CHO ists ~2 h . So, in ion rate  will be ion rate , thus inngestion . In thisngestion ,228, the 1–2.5 h ,146.−1 (instead of 60 g·h−1) when exercise duration exceeds 2.5 h is to compensate for the metabolic demands due to the increased glycogen depletion rates [−1 in race durations ~4.28 h [−1·h−1 in race durations ~24 h [−1 in single-stage and multistage ultramarathon events [−1 in race durations of 24 h [The main reason for recommending a CHO dose up to 90 g·hon rates ,220. Howon rates ,172,173. ~4.28 h ; (b) mosns ~24 h  or 20–40n events ,177,178; of 24 h , which i−1 units and not, correspondingly, in individual BM, as proposed [−1 depends on the athlete’s size among other factors [−1 values.However, another question arises regarding dose: Why are the doses recommended so far expressed mainly in g·hproposed , given t factors . On the  factors , a concl factors ,143,220. factors ,232,233. factors ,232,233. factors ,234,235)Though meta-analysis has been widely applied to human performance research ,238,239,Gut trainability derived from ‘nutritional training’ in order to train the GI tract by improving the rate of emptying and fluid absorption and thus favor exercise performance in endurance events is a known practice among endurance athletes ,235. How−1) response in endurance performance, and various studies recommend 30–60 g·h−1 to 90 or even a surprising 120 g·h−1 [−1·kg−1 of BM, as suggested by the American College of Sports Medicine. This will enable global scope organizations to update their recommendations and make them more suitable and accurate for a wider range of athletes’ body sizes [Most of the review studies were conducted on cycling , in laboratory environments 133 trials) and in a male population , making it hard to generalize to other natural sporting environments , elite athletes, and the female population. Perhaps more applied research is needed to investigate the effect of CHO supplements on endurance exercise of different types, durations, and modes at different phases of a female’s menstrual cycle  and in e3 trials 20 g·h−1 ,9,242. A20 g·h−1 . Since nIn this article, we selected and analyzed relevant papers from a specific period (1975–2021) and verified the hypothesis that CHO supplementation during prolonged exercise enhances performance or delay fatigue progression. Maintaining blood glucose concentrations and increasing CHO oxidation rates could be the main reasons behind such performance improvement. Fatigue is not solely located at the peripheral level, as explained by the appearance of a “metabolic endpoint”, in which muscle glycogen levels are exhausted and plasma GL levels are lowered  ,244. The−1 for the first 2.5 h and then be increased to 90 g·h−1, or the dose should be 90 g·h−1 from the beginning of an endurance event when the exercise lasts more than 2.5 h . There are also many who are misled by retailers, possibly due to the difficulty of interpreting the scientific data, the existence of a plethora of different guidelines, and various misconceptions [In the last 45 years, the undiminished interest in research on CHO solutions (sport drinks) has promoted our insights and sport supplementation practices. Gastrointestinal absorption, splanchnic and muscle blood flow, muscle energy uptake, and substrate oxidation have all been established as possible barriers to the potential ergogenic effects of CHO solutions . Howeverceptions ,257,258.−1 of two transportable CHOs (GL:FRU in a ratio of 2:1) in the form of a 6–8% CHO solution during exercise events (intermittent and continuous) lasting from 1 to 4 h  in prolutrition ,145,146.utrition .−1 has a greater effect size on performance in comparison to 60–80 g·h−1 and is significantly more beneficial for endurance performance in comparison to a CHO dose >100 g·h−1. However, all included studies investigating the efficacy of CHO supplements during prolonged exercise have shown some evidence of risk-of-bias, an issue that should be addressed in future research. More research is needed to investigate the impact of CHO supplementation and CHO dose, preferably expressed per kg of BM as suggested [We examined the efficacy of CHO (≤ 20%) solution compared to controls on prolonged exercise performance in subjects over 18 years old in 96 studies . We found that CHO solution favors performance in prolonged exercise, continuous and intermittent, and cyclists tend to have a greater effect size in comparison to runners. As the exercise duration increases from 1 to up to 4 h, so does the effect size of CHO intake on performance. A crossover interaction between SMD and the subjects’ CRF level was also considered, although not between gender classes; however, the number of studies in the female population are limited and the athlete’s gender may be a worthy variable for future consideration. A 6–8% CHO concentrated solution composed of two transportable CHOs  appears to be sufficient to increase the chance of a better performance. An administration schedule during the event seems superior for performance in comparison to administration prior to exercise on its own, although CHO supplementation prior to exercise should not neglected. Additionally, although the effect size on performance seems to be unaffected by different CHO dose schedules, it appears that a CHO dose of 80–100 g·h


Heat sinks are commonly used for cooling electronic devices and high-power electrical systems. The ever-increasing performance of electronic systems together with miniaturization calls for better heat dissipation. Therefore, the heat sink materials should not only have high thermal conductivities, low densities, and cost, but also have coefficients of thermal expansion matching to those of semiconductor chips and ceramic substrates. As traditional materials fail to meet these requirements, new composite materials have been developed with a major focus on metal matrix composites (MMCs). MMCs can be tailored to obtain the desired combination of properties by selecting proper metallic matrix and optimizing the size and type, volume fraction, and distribution pattern of the reinforcements. Hence, the current review comprehensively summarizes different studies on enhancing the thermal performance of metallic matrices using several types of reinforcements and their combinations to produce composites. Special attention is paid to the types of commonly used metallic matrices and reinforcements, processing techniques adopted, and the effects of each of these reinforcements (and their combinations) on the thermal properties of the developed composite. Focus is also placed on highlighting the significance of interfacial bonding in achieving optimum thermal performance and the techniques to improve interfacial bonding. Heat sinks are commonly used for cooling electronic devices and high-power electrical systems . ChingulMicroelectronics systems find wide application in today’s world, ranging from digital watches to supercomputers . The inc−6 K−1 and 7 × 10−6 K−1) [The electronic systems have been continuously evolving following two important trends, namely ‘enhanced performance’ and ‘miniaturization’ ,22. The 0−6 K−1) , heat si0−6 K−1) ,21. Trad0−6 K−1) ,25. Al aMetal matrix composites (MMCs) are a type of composite in which, typically, the ceramic reinforcements are embedded in a metallic or alloy matrices ,26,27,28−1K−1), high melting temperature, and good weldability [2O3 and WO3 particles [Copper is the most widely used matrix material for heat sink applications due to its high thermal conductivity 400 Wm0 Wm−1K−1dability ,43,44,45dability ,65,66,67dability ,69,70, cdability ,71,72,73dability ,75, SiC dability ,71,76,77dability ,79, molydability , and a harticles .The achievement of desired thermal properties from the copper matrix composite is determined by the interface between the matrix and the reinforcement ,82,83,84−1K−1) [−6 K−1 [−1K−1 at 1 wt% Zr. Li et al. [−1K−1 at 0.5 wt% Zr. Bai et al. [−1K−1 at 5 wt% B. Weber et al. [−1K−1 at 0.005 at% and 2.5 at% Cr and B, respectively. Che et al. [−1K−1 by alloying the matrix with 3 vol% Ti. It was observed that the concentration of alloying elements should be optimized to achieve the maximum value of thermal conductivity as shown in Diamond-reinforced copper matrix composites have attracted the most interest of researchers due to their high thermal conductivity (up to 2200 Wm−1K−1) ,41) and  [−6 K−1  reportedi et al.  and Wangi et al.  reportedi et al.  found thr et al.  reportede et al.  obtained−1K−1 for copper/diamond composite reinforced with (1.9 µm thick) Cu-0.5B-coated diamond particles was reported [−1K−1 [−1K−1 at 500-nm-thick tungsten coating on diamond reinforcement to 900 Wm−1K−1 with the reduction in the coating thickness to 100 nm. Kang et al. [−1K−1 by applying 1-μm-thick WC coating on the diamond particulate reinforcement. Some researchers have reported thermal conductivities of 562 [−1K−1 [−6 K−1 and 7.15 × 10−6 K−1 with Cr7C3- and Mo2C-coated diamond particulate-reinforced copper matrix composites. Zhu et al. [−1K−1. Cho et al. [−1K−1. Chang et al. [2·K). On the other hand, an increase in the coating deposition temperature (10 nm thickness) to 873 K increased the interfacial thermal conductance from 29 to 85 MW/(m2·K) due to the transformation of the intermediate TiC layer from an amorphous to crystalline state.The secondary approach to improve the interfacial bond between diamond particles and the copper matrix is the diamond surface modification. The carbide-forming elements, such as B, Ti, W, Cr, Mo, or Si, are deposited on the surface of diamond by magnetron sputtering ,49,60,63reported . Maximumd [−1K−1  were repd [−1K−1 ,58,59 obg et al.  synthesis of 562  and 596 2 [−1K−1  accompanu et al.  reinforco et al.  reinforc−1K−1. In a similar study involving alloying matrix and coating diamond with Cr, a thermal conductivity of 810 Wm−1K−1 was achieved [−1K−1. Some researchers have adopted a new technique of applying a dual layer coating on diamond particles with W [−1K−1, respectively. The advantages of this technique are a uniform distribution of diamond particles, lowering of the sintering temperature, and a very strong interfacial bond.Alternatively, some studies  have comachieved . In an aachieved , but thes with W ,64 or Mos with W  as the i−1K−1), negative coefficient of thermal expansion (−1.45 × 10−6 K−1), and low cost have made graphite fibers a potential reinforcement. The enhanced machinability of the composite is an additional advantage [−1K−1 and 6.1–9.4 × 10−6 K−1 [−1K−1 perpendicular to the pressing direction. To obtain isotropic properties, W-coated graphite particles were used as reinforcement, but the resulting thermal conductivity reported was just 158 Wm−1K−1 [−1K−1 and 7.4 × 10−6 K−1, respectively) suitable for heat sink application was obtained by infiltrating liquid copper into graphite foam [High thermal conductivity  along the fiber orientation. They were used as reinforcements to synthesize copper matrix composites for thermal management applications. Thermal conductivities of 220 and 120 Wm−1K−1 of unidirectional composites in the longitudinal and transverse direction, respectively, were reported by Korab et al. [−1K−1 in the longitudinal and transverse directions, respectively, were reported by incorporating 35% volume fraction pitch-type carbon fibers (K6371T) in the copper matrix. In order to overcome anisotropy in properties, cross-ply composites were fabricated, but the thermal conductivities obtained in the in-plane and transverse directions  were below 300 Wm−1K−1 [−1K−1 with 40% volume fraction of copper-coated carbon fibers [Carbon fibers have a thermal conductivity as high as 900 W/mK and a negative coefficient of thermal expansion  with an extraordinarily low coefficient of thermal expansion (0 × 10−6 K−1), which can be exploited to achieve enhanced thermal performance. Unfortunately, retaining the thermal conductivity of matrix metal while incorporating CNTs has been a great challenge due to the inhomogeneous dispersion of CNTs. The formation of such clusters deteriorates the thermal conductivity of the composite [−1K−1 and 5 × 10−6 K−1, respectively, was successfully fabricated by filling copper in the pores of the macroscopic CNT [CNTs possess outstanding thermal conductivity  when added to Cu/WC-TiC-Co composite improved its thermal conductivity from 190 Wm−1K−1 to 350 Wm−1K−1 [Graphene possesses outstanding in-plane thermal conductivity in the range of 1000–5300 Wm10−6 K−1 ,97. Thoutallurgy . Chu et tallurgy ,97 devel10−6 K−1 , 35 vol% Wm−1K−1 . The pow−1K−1and 4.5 × 10−6 K−1, respectively. Only a few researchers have focused on investigating the appropriateness of using SiC as a heat sink material. A maximum achieved thermal conductivity of 400 Wm−1K−1was reported for a composite with 40% volume fraction SiC reinforcements [−1K−1 with a coefficient of thermal expansion of 11 × 10−6 K−1 was reported with Mo-coated SiC particle inclusion in copper matrix [SiC possess a thermal conductivity and coefficient of thermal expansion of 200–300 Wmrcements . Since Srcements , and Mo rcements . A thermr matrix .−1K−1, respectively, over a temperature range of 300 to 600 °C [−6 K−1, and 9.7 to 10.3 x10−6 K−1 with 30% and 50% volume fraction W particle reinforcements, respectively, over a temperature range of 150 to 550 °C. In another study, a composite with 60 wt.%W–40 wt.% Cu showed stable thermal conductivity (260–240 Wm−1K−1) over a temperature ranging from room temperature to 1000 °C [Tungsten (W) is specifically used as a reinforcement in Cu ,43,78,79o 600 °C . The coe−1K−1 with 55% volume fraction Mo/Cu matrix composite.Molybdenum (Mo) as a reinforcement has not received much research attention. Chen et al.  reported2O3 and WO3 to synthesize Y2W3O12 hybrid powders. The thermal conductivities of all the composites with 40% to 70% volume fraction Y2W3O12 reinforcements were below 300 Wm−1K−1.Metal oxides as reinforcements have also been the least explored. Das et al.  used Y2O−6 K−1) of aluminum is compensated by reinforcement with carbides , oxides3N4 [2O3 ), diamon3N4 [2O3 ,125,126,3N4 [2O3 ,128,129,3N4 [2O3 ,132,133.−6 K−1) along with a low thermal conductivity (160 Wm−1K−1). Zhang et al. [−6 K−1) with 73 vol% SiC particle-reinforced Al-Si alloy matrix. However, the resulting AMC suffered profound thermal fatigue damage as a result of thermal cycling. Schobel et al. [9Si and Al3.21 Si0.47), which contributes to the increment of the thermal conductivity. The maximum thermal conductivity (235 Wm−1K−1) was reported at 45 vol% of finer SiC particles .SiC is the most commonly used carbide reinforcement for aluminum matrix composites. Saraswati et al.  achievedg et al.  achievedl et al.  focused l et al.  demonstrl et al. . The inc2O3. A comparison of their thermal performance discovered that the CBN reinforcement yielded enhanced thermal conductivity, although the value remained relatively low for most applications. In another study, Manivannan et al. [4C and AlN-reinforced aluminum composites have not attracted much research focus. Tayebi et al. [−6 K−1 with Al/25%B4C composite. Zhang et al. [−1K−1 and 11.2 x10−6 K−1 with Al/50% AlN composite.Manivannan et al.  developen et al.  reinforci et al.  reportedg et al.  reported3N4 nanocomposite through the powder metallurgy route with subsequent microwave sintering and hot extrusion. A coefficient of thermal expansion of 19.3 × 10−6 K−1 (reduction by 17.2%) was reported. Reddy et al. [−6 K−1 (reduction by 17.6%) was reported. In both instances, the resulting coefficient of thermal expansion was above the acceptable range to meet market requirements.The efforts to develop aluminum nanocomposites for heat sink application is scarce. Matli et al.  fabricaty et al.  fabricat−1K−1 and a coefficient of thermal expansion of 7 × 10−6 K−1 was achieved with AlSi/diamond (60 vol%) composite through the gas pressure infiltration route (GPI), where SiC was not formed at the interface [4C3 instead of SiC was considered to serve as a thermal barrier. To prevent the formation of Al4C3 and to improve interfacial bonding, the diamond particles were coated with SiC [−1K−1 in combination with a low coefficient of thermal expansion of 5.69 × 10−6 K−1 was reported with 60 vol% TiC-coated diamond particle reinforcement [−6 K−1 and 9.27 × 10−6 K−1 with 50 vol% diamond particle reinforcement [−1K−1 with gas pressure-infiltrated W-coated diamond-reinforced AMC.Diamond forms weak interfacial bonds with Al matrix. Alloying matrix material with a small amount of Si was found to form SiC at the matrix–diamond interface. A thermal conductivity of 375 Wmnterface . The forwith SiC , TiC [11with SiC , Ti [123with SiC . A thermorcement . Yang etorcement . Che et orcement  reported−1K−1 of squeeze-casted Al/diamond composite shot up to 670 W/m−1K−1 when fabricated by GPI. The characteristic long exposure time of diamond crystals to aluminum melt involved in this process promotes interfacial bonding and thermal conductance. Monje et al. [−1K−1 was reported at a contact time of 15 min and infiltration temperature of 760 °C. At the higher infiltration temperature of 850 °C, a maximum thermal conductivity of 676 Wm−1K−1 was achieved at a contact time of 1 min [4C3 formation. After exceeding the solubility limit (43 atomic%) of carbon in Al, the Al4C3 phase precipitates on the diamond surface either in a particle form or as a continuous layer. For enhanced interfacial bonding and thermal conductance, the Al/diffusion layer/diamond system is preferred [−1K−1 was recorded at an optimum infiltration temperature and pressure of 800 °C and 0.8 MPa [−1K−1. The enhanced thermal conductivity was attributed to the activation of the diffusion reaction on the {111} face of diamond, resulting in good interfacial bonding and interfacial thermal conductance. Further studies are required to confirm the mechanism of improvement of thermal conductivity in Al/diamond composite and to explore the effect of Al4C3 on the thermal conductivity.Significant progress was realized when Ruch et al.  establise et al.  further of 1 min . The enhreferred . Zhang ereferred  demonstr 0.8 MPa . Later,  0.8 MPa  also dem−1K−1 [−1K−1 at merely 4.6 vol% of diamond [Some new approaches were adopted to reduce the usage of high-volume fraction of diamond reinforcement. Diamond film (6.5 vol%) was coated on spiral W wire and was reinforced in the Al matrix, resulting in a thermal conductivity of 294 Wm−1K−1 . A novel diamond .−1K−1) with zero or negative through-plane CTE was reported. However, a drastic reduction in hardness and tensile strength with an increasing volume fraction of graphite flakes was noticed. An increase in thermal conductivity (from 324 to 783 Wm−1K−1) was reported with an increase in the volume percentage of graphite flakes (from 10 to 80%) while the coefficient of thermal expansion reduced in the parallel (from 16.9 to −2.5 × 10−6 K−1) and perpendicular (from 15.2 to 10.1 × 10−6 K−1) direction to the basal plane [−1K−1 with the increase in the size of the graphite flakes from 150 to 500 µm [4C3, which reduces the thermal conductivity and corrosion resistance.Graphite flake-reinforced Al matrix composites features excellent machinability in combination with high specific thermal conductivity. Oddone et al.  developeal plane . Additioo 500 µm . These c4C3. A thermal conductivity of 540.8 W/mK in the longitudinal direction was reported. The coefficient of thermal expansion in the longitudinal direction was found to range from −1 × 10−6 K−1 to −1.9 × 10−6 K−1 with an increase in the temperature from 100 to 300 °C. The enhanced thermal performance was attributed to the amorphous layer formed at the fiber–matrix interface in the presence of oxygen, which probably was absorbed during the infiltration. Lee et al. [−1K−1 with AMC reinforced with unidirectional carbon fiber fabricated by the low-pressure infiltration process. It was demonstrated that the growth rate of Al4C3 was more profound during the cooling than the infiltration process. The increase in time from 10 to 60 min to cool from 849 to 500 °C resulted in a decrease in the thermal conductivity from 273.2 to 230 Wm−1K−1, respectively. Silvain et al. [−1K−1 and 7.09 × 10−6 K−1, respectively, were reported. Tokunaga et al. [−1K−1 with 40 vol% reinforcement of carbon fibers in aluminum matrix with Al-12.2 mass% Si alloy (10 vol%). Pei et al. [−1K−1 along the fiber direction with a very low coefficient of thermal expansion between −0.26 × 10−6 K−1 and 0.26 × 10−6 K−1 was reported.Another carbon-based reinforcement is carbon fiber. Beronska et al.  fabricate et al.  reportedn et al.  and Kurin et al.  reinforca et al.  achievedi et al.  reinforc−1K−1 with 0.3 wt% graphene-reinforced AMC fabricated by powder metallurgy.Studies investigating the thermal performance of CNT-reinforced AMCs are scarce ,137. The4C to form TiCx-TiB2/Al composites. The resulting thermal conductivity was just 160 Wm−1K−1. Molina et al. [−1K−1 by reinforcing Al-12wt%Si alloy matrix with graphite flakes and SiC particles. Chamroune et al. [−1K−1 with in-plane and through plane coefficients of thermal expansion of 15 × 10−6 K−1 and 2.4 × 10−6 K−1, respectively, using solid-liquid sintering. In another study, Peng et al. [−1K−1. Graphite flakes were coated with copper while the carbon fibers were doped with nitrogen to improve their wettability in Al matrix and the composite was fabricated through the optimized vacuum gas pressure infiltration method. Xue et al. [−1K−1 and a coefficient of thermal conductivity of 7.8 × 10−6 K−1 with diamond/SiC/ Al-7Si-0.3Mg hybrid AMC. Han et al. [−1K−1 and a coefficient of thermal of 2.5 × 10−6 K−1 by reinforcing aluminum matrix with 70 vol% graphite flakes, and 3-D copper networks coated with 5 vol% graphene. The enhanced thermal performance was attributed to the effective heat transfer path provided by copper networks and to the better distribution of graphite reinforcements.Recently, hybridizing AMCs with secondary reinforcements to mitigate the side effects of adding primary reinforcements ,141,142 a et al.  reportede et al.  hybridizn et al.  reported2O3, SiC, BN, and AlN reinforcements. However, a minimal improvement was observed with reinforcement with graphene and carbon fibers.−1K−1) among all the matrix materials considered for thermal management. Due to its scarcity and high cost, it has been rarely investigated. Zhao et al. [−1K−1 by reinforcing silver matrix with Cr-coated diamond particles. Pal et al. [−1K−1 due to lower interfacial resistance.Silver has the highest thermal conductivity  of magnesium, it has also rarely been explored for thermal applications. Molina et al. [−1K−1 with TiC-coated bimodal diamond particle reinforcement. The enhancement in their thermal conductivity was attributed to the nano-scale coating thickness, and to the bimodal mixture of thee reinforcing particles. Hou et al. [−1K−1 with Ni-coated short carbon fiber (1 vol%) reinforcement.Given the low thermal conductivity . The preheat temperature depends on the type of reinforcement. Meanwhile, the matrix metal  is melted separately. The preform is placed inside a die preheated to a relatively much lower temperature. The molten metal is immediately poured with simultaneous application of vacuum to the preform and a predetermined vertical pressure to the melt. The infiltration of molten metal into the preform is caused by the downward pressure applied by a hydraulic or pneumatic piston. The applied pressure is determined such that the piston displacement rate completes the infiltration in a few seconds before the actual commencement of solidification. The relatively cold die aids in rapid solidification of the composite under the applied pressure. The casted samples are either air cooled or annealed followed by furnace cooling to room temperature [The reinforcing particles are first pressed in a mold to make a preform. The preform is preheated in a forming gas atmosphere . No graphitization of carbon fibers was observed in the fabrication of AMC by this method [4C3 nor any reaction between diamond reinforcements and Si when present in the AMC matrix were observed [4C3 during fabrication of AMC reinforced with monocrystalline diamond, a transformation of diamond particles into the amorphous carbon phase was reported. Pingping wang et al. [The low-temperature processing ability along with rapid infiltration and solidification have made this technique more attractive for the fabrication of AMCs ,120,132.observed . Rather,observed . Though g et al.  optimizeIn this process, a pressurized inert gas or vacuum forces the molten metal/alloy in to the preform ,86,152. The gas pressure infiltration technique requires a longer processing time, which has been exploited to improve the interfacial bonding between the reinforcement and metal matrix  along with matrix alloying and/or reinforcement coating with carbide-forming elements. Mostly copper matrix composites ,153,154 The success of the liquid infiltration process (squeeze casting and gas pressure infiltration) depends on the porosity and the strength of the preform. The open pore structure allows the metal or alloys to flow. The strength enables the preform to withstand pressures applied during the infiltration process without becoming deformed ,156. SinThe powder metallurgy process has emerged as an effective means for synthesizing near net shape composite products. The process not only offers a homogeneous dispersion of reinforcements in the matrix phase but can also prevent undesirable interaction between them ,108,157.4C (25 vol%) [Hot press sintering is the conventional sintering method for MMCs. The copper matrix composites reinforced with carbon fibers (30–40 vol%) , SiC par25 vol%)  and grap25 vol%)  were con25 vol%)  and carb25 vol%) ,131, res25 vol%) . The ext25 vol%) ,108.In this method, the sample is heated by the applied electric field. This process involves simultaneous application of a pulsed direct current and uniaxial pressure on the powder or the compact. The current passing through the die and the compact (if conductive) is transformed into heat by a Joule heating mechanism ,129,158.This is a novel technique of sintering, which generates heat by transforming electromagnetic radiations into heat in the compact being sintered. Microwave sintering offers a high volumetric heating rate, shorter processing time, finer homogenous microstructure, improved mechanical properties, enhanced densification, reduced energy consumption, and environment friendliness over other methods ,108,157.−1K−1 [−6 K−1 to 7 × 10−6 K−1 [The heat sink materials should not only have high thermal conductivities above 300 Wm−1K−1 ,12,22, b−1K−1 ,7,11,21,2C [The thermal performance of the composites can be observed to be affected not only by the type of reinforcements, but also by the techniques to improve the interfacial bonding and processing routes. The diamond particles are observed to be the most promising reinforcement in copper matrix when the interfacial bonding was improved. Alloying copper matrix by carbide-forming elements like zirconium  or boron2C  are obse2C . FurtherThe other carbon-based reinforcements like graphite, carbon fiber, and CNTs are also featured in AMCs with acceptable ,141 and/The incorporation of metal particles like Mo  and W 7 in coppe−1K−1 and coefficients of thermal expansion typically in the range of 3 × 10−6 K−1 to 7 × 10−6 K−1. The current review summarizes the efforts of different researchers in enhancing the thermal performance of metal matrix using a combination of several types of reinforcements to produce composites. Based upon this extensive literature review, the main findings can be summarized as the following:Copper and aluminum are the most commonly used matrix materials for heat sink composites. Diamond particles were observed to be the most promising reinforcement when the interfacial bonding was improved either by alloying matrix or by surface metallization of diamond particles by a carbide-forming element.4C3 that serves as a thermal barrier.Another important technique to improve interfacial bonding and thereby the thermal properties in Al/diamond composites is to promote the formation of an Al-C diffusion layer. The formation of a diffusion layer precedes the precipitation of a brittle, hydrophilic interfacial phase of AlThe MMCs with other lone carbon-based reinforcements like graphite, carbon fiber, CNTs, and graphene are generally reported to demonstrate unacceptable and anisotropic thermal performances.A trend setting approach was noticed when a diamond film was coated on another reinforcement in the fabrication of AMCs. A significantly high ratio of thermal conductivity to diamond loading was observed with merely a small volume percentage of diamond. This technique needs to be further explored to establish the feasibility of this technique in providing MMCs with an acceptable thermal performance. This technique may not only reduce diamond loading but also reduce the overall cost.Using graphite foam or metallic foam coated with carbon-based reinforcements is another step towards obtaining MMCs with isotropic properties. Further, it can provide an effective heat transfer path for better heat conduction.The AMCs with acceptable and/or close to acceptable thermal performances possess a very high specific thermal conductivity, which makes them more attractive for heat sink applications in portable electronic devices.Among the liquid state processing techniques, the gas pressure infiltration technique has emerged as being effective. This technique offers control over the infiltration temperature, pressure, and reinforcement-matrix melt contact time. This feature can be exploited to improve the interfacial bonding between the reinforcement and the matrix material.The SPS and hot press sintering has emerged to be the most common sintering methods when the powder metallurgy route is adopted for the fabrication of MMCs. Though microwave sintering is hailed to offer significant advantages over other methods, it has rarely been used, and it needs to be further explored.In view of the above findings, it can be concluded that the thermal performance of the composites is affected not only by the type of reinforcements, but also by the techniques to improve the interfacial bonding and processing routes. The novel techniques should be further explored to meet the ever-increasing thermal management challenges.MMCs as heat sink materials were reviewed in this paper. The heat sink materials should demonstrate an acceptable thermal performance, i.e., thermal conductivities above 300 Wm