Influenza viruses are still a serious threat to human health. Cytokines are essential for cell-to-cell communication and viral clearance in the immune system, but excessive cytokines can cause serious immune pathology. Deaths caused by severe influenza are usually related to cytokine storms. The recent literature has described the mechanism behind the cytokine–storm network and how it can exacerbate host pathological damage. Biological factors such as sex, age, and obesity may cause biological differences between different individuals, which affects cytokine storms induced by the influenza virus. In this review, we summarize the mechanism behind influenza virus cytokine storms and the differences in cytokine storms of different ages and sexes, and in obesity. The COVID-19 pandemic showed the catastrophic impact of a new type of virus on human health. Since the H1N1 Spanish influenza outbreak in 1918, there have been many influenza pandemics, such as the H2N2 Asian influenza in 1957, the H3N2 Hong Kong influenza in 1968, and the H1N1 influenza pandemic in 2009 ,5.A mature influenza virus is wrapped by a protein membrane that contains two antigenic determinants: Hemagglutinin and neuraminidase (HA and NA) ,7,8,9,10Although it is easy to associate the severe consequences of influenza-virus infection with viral load, the host’s inflammatory response to the influenza virus is more related to the lung injury induced by the influenza virus ,19,20,21A severe cytokine storm can cause acute respiratory distress syndrome (ARDS) . ClinicaAlthough the influenza B virus (IBV) is similar to the influenza A (IAV) virus in morphology, the influenza A virus has a variety of hosts, prone to antigenic drift and antigenic shift, and a variety of epidemic strains. The influenza B virus, on the other hand, has only two hosts, humans and seals, and there are only two epidemic strains, Victoria and Yamagata. The influenza B virus is more likely to infect children ,42. The The center of the cytokine storm is the cytokine. Cytokines are small proteins secreted by cells that are essential for the communication between cells in the immune system and the resolution of infectious diseases. Since the influenza virus is a single-stranded negative-sense RNA virus, viral replication requires viral RNA polymerase to produce sense messenger RNA from the viral genome . The preThere are three types of interferons (Types I–III), defined by their receptor specificity . Type I After the influenza virus infects the lungs, Type I IFNs are rapidly expressed in various myeloid and parenchymal cells ,59. Althλs or Type III IFNs share homology, expression patterns, signaling cascades, and antiviral functions with Type I IFNs [Staphylococcus aureus infection, and significantly improve the proliferation of lung epithelial cells [IFNe I IFNs ,65. Simie I IFNs . Excessial cells . Higher al cells . In clin+ and CD11b+ DCs carrying viral antigens [+ T cells can produce cytokines, including IFN-γ and TNF-α, through a variety of antigen-dependent pathways [+ T cells are important for clearing the influenza virus, if the CD8+ T-cell response is unregulated, it can cause considerable lung disease. IFN-γ produced by CD8+ T cells promotes the release of lung epithelial chemokines, leading to inflammatory cell infiltration, and aggravating lung pathology and thymocyte apoptosis [NK cells produce Type II IFN, IFN-γ, in the early stage of influenza-virus infection. In the subsequent immune response, T cells become the main producer of IFN-γ . T cellsantigens . Once acantigens . Influenpathways . Althougpoptosis ,72. Antipoptosis . IFN-γ cpoptosis .TNF-α is almost everywhere in the body. Macrophages, monocytes, NK cells, endothelial cells, epithelial cells, and T and B lymphocytes can all express and produce TNF-α . TNF-α cThe name interleukin originally referred to the cytokine secreted by leukocytes, but many kinds of cells can produce interleukin. Inactive precursor cytokines pro-IL-1β and pro-IL-18 require nucleotide-binding domain, leucine-rich-containing family, and pyrin domain-containing-3 (NLRP3) inflammasomes to mature into biologically active IL-1β and IL-18 . In the IL-6 as a marker of inflammation is produced by macrophages, DCs, mast cells, and other innate immune cells. Clinical studies revealed that excessive IL-6 is closely related to the poor prognosis of influenza patients ,94,95. TThe IL-17 family includes at least six cytokines: IL-17A (IL-17), IL-17B, IL-17C, IL-17D, IL-17E (IL-25), and IL-17F. IL-17A and IL-17F have the highest homology in the IL-17 family, and both can transmit signals through IL-17RA . Both arNumerous biological factors lead to biological differences between different individuals, which are very important in experimental studies of immunology and virology. Biological factors such as sex, age, and obesity may affect the host’s susceptibility to the influenza virus and their autoimmune response.Among hospitalized patients with influenza, those younger than 5 years old and older than 65 years old are more likely to develop acute pneumonia ,121,122.During influenza-virus infection, the individual’s gender and hormonal status can regulate the process of inflammation and immunopathology ,136. AltApproximately 1.9 billion people in the world are overweight, and there are more than 670 million obese adults and more than 100 million obese children ,148. So The innate and adaptive immunity of obese individuals changes ,153. ObeObese individuals have higher long-term concentrations of leptin (a proinflammatory adipokine), while adiponectin (an anti-inflammatory adipokine) is at a lower concentration . Leptin The influenza pandemic poses a huge threat to human health. Many severe influenza patients die from serious complications caused by cytokine storms, such as ARDS. Endothelial dysfunction can also cause inflammation, which potentially impacts the cytokine storm caused by influenza viruses . In this


SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE (SPL) gene family affects plant architecture, panicle structure, and grain development, representing key genes for crop improvements. The objective of the present study is to utilize the well characterized SPLs’ functions in rice to facilitate the functional genomics of TaSPL genes. To achieve these goals, we combined several approaches, including genome-wide analysis of TaSPLs, comparative genomic analysis, expression profiling, and functional study of TaSPL3 in rice. We established the orthologous relationships of 56 TaSPL genes with the corresponding OsSPLs, laying a foundation for the comparison of known SPL functions between wheat and rice. Some TaSPLs exhibited different spatial–temporal expression patterns when compared to their rice orthologs, thus implicating functional divergence. TaSPL2/6/8/10 were identified to respond to different abiotic stresses through the combination of RNA-seq and qPCR expression analysis. Additionally, ectopic expression of TaSPL3 in rice promotes heading dates, affects leaf and stem development, and leads to smaller panicles and decreased yields per panicle. In conclusion, our work provides useful information toward cataloging of the functions of TaSPLs, emphasized the conservation and divergence between TaSPLs and OsSPLs, and identified the important SPL genes for wheat improvement.The  Wheat is one of the most important crops worldwide, providing a food supply for about 28% of the global population . HoweverTranscription factors (TFs) represent a particular type of DNA-binding proteins encoded by certain gene families, which bind to their target genes in a sequence-specific manner and activate or inhibit the transcription of target genes. Thus, TF families are involved in various aspects of plant growth and development, and often contain master regulators and key genes for crop improvement.2+ binding sites—Cys-Cys-His-Cys (CCHC) and Cys-Cys-Cys-His (CCCH)—and a nuclear localization signal (NLS) . A . A SPL ge-miR156 , all of  , based on their RNA-seq expression patterns  are those with tissue-specific expression patterns, while Cluster 5 (TaSPL2/13/16/18) contains TaSPLs preferentially expressed in some tissues or developmental stages. The remaining TaSPLs , shoots and developing roots, stems, inflorescences, and seeds, indicating that these TaSPLs might play pleiotropic roles in the regulation of plant development. Meanwhile, we validated the expression profiles of several TaSPLs, including TaSPL2/3/4/6/8/10/17/18, in wheat cv. Chinese Spring at multiple development stages and in various tissues . For example, TaSPL18 was expressed in the leaf sheath across multiple stages, while TaSPL16 was not; furthermore, TaSPL18 showed stronger expression levels in flower organs  than TaSPL16. In contrast, the other two duplicated pairs of TaSPLs (TaSPL5/10 and TaSPL14/17) showed relatively similar expression profiles.These expression results also indicated potential functional divergence between the paleoduplicated TaSPLs using RNA-seq data. Statistical analysis of the expression ranges between the A, B, and D sub-genome TaSPL homeologous copies revealed 11 TaSPL genes with SEBs. Particularly, many TaSPLs (TaSPL1/3/4/5/9/15/18) from the B sub-genome had relatively lower expression levels, when compared with the corresponding copies from A and D sub-genomes . We analyzed the SEBs of -genomes B. TypicaTaSPLs, we questioned whether the wheat SPL orthologs retained similar expression preferences after rice–wheat divergence. To address this, we retrieved both the microarray- and RNA-seq-based expression profiles of OsSPLs , in order to simplify the comparison between rice and wheat . In rice, OsSPL1/3/4/6/9/11/12/15 are ubiquitously expressed, OsSPL2/8/14/16/18 are expressed preferentially in some tissues, and OsSPL5/7/10/13/17 are expressed specifically in particular tissues  exhibited similar expression abundance at the organ level.In rice, the two r organs A. In conSPL triads showed similar organ-level expression preferences  were up- or downregulated, with different stress-response patterns. TaSPL6 was downregulated throughout the whole process of drought stress, while TaSPL10 was dramatically upregulated (by over seven-fold) after 3 h of treatment. After PEG treatment, the expression of TaSPL2/4/6/8/18 peaked at 1 h, TaSPL10 peaked at 12 h, while TaSPL3 was downregulated. Different from PEG treatment, NaCl treatment induced the expression of TaSPL2/6/10 but repressed the expression of TaSPL3/4/17. Under cold treatment, TaSPL6/10 responded rapidly at 1 h, while TaSPL2/3/18 had two upregulation peaks in response.Our qPCR analysis demonstrated that eatments . The expstresses . Under dSPL genes respond to drought [TaSPLs expression under several phytohormone treatments, including ABA, IAA, GA, GR24, MeJA, and BR . IAA mediated strong upregulation of TaSPL2/8/10/17 at 12 h, while TaSPL4/6/18 responded to IAA treatment earlier: at 1 to 3 h. For ABA treatment, all of the eight TaSPL genes showed a similar up-and-down expression pattern, with the majority of TaSPL expression induced in 3–6 h. The expression patterns of TaSPLs in response to GA were complex. TaSPL3/6 were downregulated by GA; TaSPL2/17 were first downregulated at early stages and then upregulated by GA at late stages; while TaSPL8/10/18 expression was significantly induced by GA treatment. Unlike the complex responses to GA treatment, a few TaSPL genes (TaSPL3/6/10/17/18) were upregulated at late stages (12 to 24 h) under GR24 treatment. As the only TaSPL-repressing phytohormone, MeJA induced downregulation of several TaSPLs, including TaSPL2/4/6/8/10/18. TaSPL17 was the only TaSPL gene significantly upregulated after MeJA treatment. For the BR treatment, several TaSPL genes  exhibited two upregulation peaks—one peak at 3 h and the other at 12 h—while TaSPL18 was downregulated by BR. These results suggest that the functions of some TaSPLs are (directly or indirectly) related to abiotic stress tolerance mechanisms and other biological processes, such as phytohormone biosynthesis, degradation, and signaling, thus regulating the plant growth and development of wheat. Previous studies have shown that  drought , heat [6 drought , auxin , TaSPL3-B (TraesCS6B02G138400.1), and TaSPL3-D (TraesCS6D02G098500.1), with over 96% identity for both the nucleotide and amino acid sequences. According to the public RNA-seq results of different tissues across wheat developmental stages, TaSPL3 triads exhibit particularly high expression in developing spikes, moderate expression in stems and roots, low expression in leaves, and almost no expression in seeds  were obtained, together with one line transforming the empty vector  as a negative control. PCR results confirmed that the six TaSPL3-OE lines were TaSPL3-positive, with various expression levels of TaSPL3, as determined by qPCR  of the T2 generation were grown in the experimental field  using a complete random block design. Meanwhile, another batch of transgenic and control lines were grown in pots and placed beside the experimental field for ease of observation.To investigate the function of etection . Six tra by qPCR . To inveTaSPL3 in rice promotes heading. The period from sowing to mature ranged from ~91 to ~96 days for the six TaSPL3-OE lines, while the same period for the control lines was 97 to 99 days . All five OsSPL pairs are retained and exhibit partially redundant functions and evidence for neofunctionalization [OsSPL3 knockout altered the heading date, whereas OsSPL12 knockout did not; OsSPL4 knockout altered the heading date, whereas OsSPL11 knockout did not; OsSPL5 knockout changed the tiller number, whereas OsSPL10 knockout did not [TaSPL11 and TaSPL12, suggesting that their functions may have been replaced by other SPL members.First, the paleoduplicated on wheat . The prelization ,37,50,69 did not . In wheaTaSPLs differ from OsSPLs in terms of gene duplication patterns. During the allohexaploidization process of the wheat genome, the number of TaSPL genes was not only tripled by polyploidization, but also increased by tandem duplications, such as the case of TaSPL10a, 10b, 10c, and 10d , tandem duplications and segmental duplications [OsSPLs and drove subfunctionalization within the OsSPL pairs [TaSPLs, differing from the case of OsSPLs.Second,  and 10d . A prelireported ; howeverications ,71,72. Iications ,74,75. TPL pairs . Unlike PL pairs ,77. IndeTaSPL genes adopted distinct expression patterns, compared with those of OsSPLs. For example, TaSPL5, 7, 10, 14, and 17 exhibited tissue-specific expression profiles, while other SPLs  were preferentially expressed in some tissues and stages, but barely expressed in some other tissues, such as leaf sheaths, roots, stems, and seeds  rice has a similar plant architecture to wheat; (2) the orthologous relationships between TaSPLs and OsSPLs are studied and reported herein; (3) rice has extensive gene expression data sets and several well-established expression databases [OsSPL members  have impacts on plant height, flowering time, panicle structure, and grain development [OsSPL genes exhibit overlapped functions with sub-functionalizations in some traits, which are likely contributed to by the differentiated expression between pairs of SPL genes [Unlike the monocot model specie rice, the genetic transformation of wheat has been established for decades ,82,83,84n system . Neverth reports ,37,44,69d stages , severald stages . For exaelopment ,36,37,50PL genes .OsSPLs, the expression patterns between OsSPLs and TaSPLs have been compared in the present study  phenotypes with less tillers, bigger panicles and bigger grains [TaSPLs could provide a novel genetic resource to modify the growth, development, and yield in cereal crops.Owing to the important relatedness between the expression patterns and gene functions as demonstrated in nt study . In suchnt study , while wnt study  [78,79,8xpressed , utilizir organs A. As a pain size . Similard TaSPL3 . Other l ligules A. The knribution . By contng seeds . Matchinn length . In riceild rice ,36,91,92OsSPLs and TaSPLs lies in the miRNAs that regulates SPL genes. In rice, miR156 and miR529 are known to target some OsSPL [TaSPL genes and their regulatory miRNAs (miR156 and miR529) are expected to be indispensable and useful in gaining a thorough understanding of the pleiotropic functions of TaSPLs in the future [Another limitation for the comparison between me OsSPL ,36,93, wme OsSPL ,94,95. Ie future .SPL genes with HMMER , and the SBP domain was obtained from SMART and InterPro databases [AetSPLs, ten TuSPLs, sixteen BdSPLs, and nineteen OsSPLs from Aegilops tauschii (v4.0 genome), Triticum urartu (v1), Brachypodium distachyon (v3.0), and Oryza sativa (IRGSP v1.0), respectively, which have been reported elsewhere, were used in the present study for phylogenetic analysis [http://plants.ensembl.org/) (accessed on 6 December 2021). Information about the TaSPLs is provided in The annotated protein-coding genes from the reference genome of the wheat cultivar Chinese Spring (CS) were used to search for atabases ,98. Eighanalysis ,43,44. TSPL gene family was performed using the maximum-likelihood method with 1000 bootstraps, using MEGA X for TaSPLs, AetSPLs, TuSPLs, BdSPLs, OsSPLs, and AtSPLs [Phylogenetic analysis of the d AtSPLs . The fulTaSPL genes were retrieved from the wheat reference genome, and the exon–intron structures of TaSPLs were analyzed using TBtools [The protein sequences of OsSPLs and the 56 TaSPL identified in the present study were aligned using ClustalW 2.0, in order to determine the SBP domain . The gen TBtools . Protein TBtools .TaSPL genes were visualized using TBtools  cultivar “Nipponbare” plants grown in greenhouse were used for rice protoplast preparation and transformation. To study the phenotypic effects of TaSPL3, transgenic lines of rice expressing TaSPL3-6A were generated  for TaSPL genes [TaSPL across different tissues and stages during development, three RNA-seq data sets were used: one from wheat cultivar Azhurnaya [cv. Chinese Spring [http://www.bioconductor.org/packages/devel/bioc/html/COMPASS.html) (accessed on 22 November 2021). When identifying sub-genome expression biases, the largest data set in wheat  was used, in order to avoid potential batch effects between the RNA-seq data sets, with the statistical differences of expression between sub-genomes calculated by two-tailed Student’s t-test  [TaSPL expression. The expression analysis of rice microarray data sets has been described elsewhere [OsSPL expression data from the RED were used to validate the OsSPL preferentially expressed tissues  and OsSPL expression data (microarray-based) first by organs and then by tissues and stages, and generally compared preferentially expressed organs and the relative expression abundance at the organ level (Two rice expression databases—RiceXpro and the rice expression database (RED)—were used to retrieve the gene expression profiles of PL genes ,80. As s period) , RXP_001lsewhere . To faci tissues . As manyan level .TaSPL genes were analyzed using quantitative PCR (qPCR) in different tissues across the developmental stages and under several abiotic stresses or phytohormone treatments.The expression profiles of TaSPL genes to various stresses, previously reported RNA-seq expression results were used  which were differentially expressed in the RNA-seq analysis (w/w), NaCl (200 mM), or cold (4 °C)—or with phytohormone addition—such as abscisic acid , indole-3-acetic acid , gibberellic acid , GR24 , methyl jasmonic acid , or brassinosteroid —for 1, 3, 6, 12, or 24 h to examine the stress tolerance-related or phytohormone-mediated regulation of TaSPL genes. Seedlings treated with distilled water were used as a control.Quantitative PCR experiments were performed to validate the expression patterns of selected analysis . To do tTaActin (TraesCS1B02G283900) was used as the internal reference gene for qPCR. The qPCR program included pre-denaturation at 95 °C for 10 min and 40 cycles of denaturation at 95 °C for 10 s, annealing at 60 °C for 30 s, and extension at 72 °C for 1 min. The primers used for qPCR are provided in Total RNA was extracted by using an RNA Extraction Kit  and reversely transcribed into cDNA for qRT-PCR as described previously . qRT-PCRTaSPL3-6A was amplified from the cDNA of wheat young spikes with the following PCR program: pre-denaturation at 98 °C for 30 s and 35 cycles of denaturation at 98 °C for 10 s, annealing at 55–65 °C for 15 s, and extension at 72 °C for 1 min .To carry out the functional study, TaSPL3-6A was fused with GFP ORF through XbaI/BamHI restriction sites to obtain the construct  for the sub-cellular localization experiment, in which the expression of TaSPL3-GFP was driven by the CaMV35S promoter. An empty vector, pSGN-GFP, was used as the negative control. Protoplasts of rice seedling leaves were prepared. Under an induction of 40% PEG-4000, empty or recombinant plasmids (pSGN-GFP or pSGN-TaSPL3-6A-GFP) were co-transformed into rice protoplasts with the marker plasmid (CFP). The transformed protoplasts were cultured at 28 °C for 8–10 h under dark conditions. A laser confocal microscope  was used to detect the sub-cellular localization of GFP proteins or TaSPL3-6A-GFP fusion proteins.The coding region of TaSPL3-6A coding region was truncated into three parts: The N-terminal (N), SBP domain (SBP), and C-terminal (C). Each of the three parts or the full-length of TaSPL3-6A was cloned into pGBKT7 plasmids through BamHI/NcoI restriction sites, in order to obtain the recombinant constructs pGBKT7-TaSPL3-N, pGBKT7-TaSPL3-SBP, pGBKT7-TaSPL3-C, and pGBKT7-TaSPL3-6A-FL, respectively. According to the manufacturer’s protocol , the abovementioned recombinant constructs, as well as pGBKT7 and the positive control, were transformed into yeast strain AH109. The transformed yeast strains were diluted at different concentrations, then dotted onto SD/-Trp or SD/-Trp/-His/-Ade medium. After culturing for four days, the trans-activation activities of full-length TaSPL3-6A or its fragments were evaluated by the columns of transformed yeasts.The TaSPL3 in rice, the open reading frame (ORF) of TaSPL3-6A was fused with the 3-myc tag and then inserted into the Agrobacterium transformation vector pCAMBIA1304, with its expression driven by the CaMV 35S promoter. Rice transformation was performed using the Agrobacterium immersion method with strain EHA105 and calluses induced from cv. Nipponbare immature embryos [0 plants, and specific PCR primers were designed to amplify the 383-bp fragment within the selection gene (hygromycin B phosphotransferase gene) or the 2161-bp fragment of the TaSPL3-6A gene. Quantitative PCR analysis was performed to evaluate TaSPL3 expression levels in the leaves of transgenic rice plants in the T0 generation  were also generated to serve as a negative control.To ectopically express  embryos . To dete2 lines of TaSPL3-OE and control lines (including both non-transformed cv. Nipponbare and VC) of rice were planted in a randomized block field experiment with three replicates at the experimental fields . In each plot, three rows of plants were grown with 25-cm row spaces. Within each row, about 15 individual plants were grown with 20-cm of plant spacing. Regular field managements, including irrigation, fertilization, and insect, disease, and weed control, were applied.The TThe growth periods for each line of rice, including seedling date, tiller date, stem elongation date, heading date, flowering time, and maturity time were observed. In each plot, 10 plants were chosen to measure various agronomic traits, including plant height, flag leaf sizes , tiller numbers, panicle length, numbers of primary and secondary branches per panicle, grain weight per panicle, grain numbers per panicle, seed-setting percentage per panicle, length, width, and thickness of grains, thousand-grain weight, and yield per plant. The length, width, and thickness of grains were measured using seed testing instrument . The significance of differences among means of agronomic traits was determined using Tukey’s honest significant difference test.TaSPL family, identified 56 TaSPL genes, and established the orthologous relationship between TaSPLs and OsSPLs. A detailed qRT-PCR analysis pinpointed several TaSPLs, TaSPL2/6/8/10, involved in the tolerance of different abiotic stresses. Our results highlighted the conservation and divergence between TaSPLs and OsSPLs. As a proof of the functional prediction from the dry lab data, we demonstrated that TaSPL3 shares a conserved function with OsSPL3 in regulating plant height, flowering time and panicle-related traits by using transgenic lines of rice. Importantly, our work leads to a clear take-home message that the combination of evolutionary and expression analyses can serve as an efficient approach to transfer the functional knowledge from the monocot model species rice to wheat, helping to gain better understanding of the functions of TaSPLs. The approach exemplified here may also be effective in functional characterization of agronomically important gene families in wheat, such as other transcription factors. In the present study, we performed a comprehensive analysis of the


Partial epithelial-mesenchymal transition (p-EMT) is a distinct clinicopathological feature prevalent in oral cavity tumors of The Cancer Genome Atlas. Located at the invasion front, p-EMT cells require additional support from the tumor stroma for collective cell migration, including track clearing, extracellular matrix remodeling and immune evasion. The pathological roles of otherwise nonmalignant cancer-associated fibroblasts (CAFs) in cancer progression are emerging.Gene set enrichment analysis was used to reveal differentially enriched genes and molecular pathways in OC3 and TW2.6 xenograft tissues, representing mesenchymal and p-EMT tumors, respectively. R packages of genomic data science were executed for statistical evaluations and data visualization. Immunohistochemistry and Alcian blue staining were conducted to validate the bioinformatic results. Univariate and multivariate Cox proportional hazards models were performed to identify covariates significantly associated with overall survival in clinical datasets. Kaplan–Meier curves of estimated overall survival were compared for statistical difference using the log-rank test.TGFBI and HYAL1 as independent poor and protective biomarkers, respectively, for 40 Taiwanese oral cancer tissues that were all derived from betel quid users. In The Cancer Genome Atlas, TGFBI was a poor marker not only for head and neck cancer but also for additional six cancer types and HYAL1 was a good indicator for four tumor cohorts, suggesting common stromal effects existing in different cancer types.Compared to mesenchymal OC3 cells, tumor stroma derived from p-EMT TW2.6 cells was significantly enriched in microvessel density, tumor-excluded macrophages, inflammatory CAFs, and extracellular hyaluronan deposition. By translating these results to clinical transcriptomic datasets of oral cancer specimens, including the Puram single-cell RNA-seq cohort comprising ~6000 cells, we identified the expression of stromal TGFBI and HYAL1, primarily expressed by stromal CAFs and endothelial cells, respectively, could serve as robust prognostic biomarkers for oral cancer control.As the tumor stroma coevolves with cancer progression, the cellular origins of molecular markers identified from conventional whole tissue mRNA-based analyses should be cautiously interpreted. By incorporating disease-matched xenograft tissue and single-cell RNA-seq results, we suggested that  However, the 5-year survival rate (~ 56%), death ranking in all cancers , and death of middle age  have remained serious concerns in recent years , a.k.a. hybrid E/M status, EMT continuum or EMT spectrum, is referred to as varied intermediate stages where epithelial cells dedifferentiate to their mesenchymal counterparts . In contPreviously, Puram et al. revealed the ecosystems of ~6,000 cells from 18 treatment-naïve oral cancer specimens, including 5 matched lymph node metastases, by using high-resolution single-cell RNA sequencing (scRNA-seq). Their results indicated that greater than 70% of oral cavity tumors in the cancer genome atlas (TCGA) are malignant-basal type, which displays either EMT or p-EMT as hallmarks .in vitro, p-EMT TW2.6 repeatedly grew faster  in immunodeficient mice (NOG) despite their tumor-bearing rates were similar  disrupted cell cohesiveness in a 2D culture, reduced spheroid invasion in a collagen gel matrix, and suppressed angiolymphatic invasion in xenograft tissues . It is w similar .The tumor stroma is regarded as the nonmalignant part of a tumor that is interconnected by extracellular matrices (ECMs) with infiltrated immune cells, vascular or lymphatic vessels, and cancer-associated fibroblasts (CAFs). In head and neck cancer, tumor-stroma interactions, including tumor budding and tumor-stroma ratio, have emerged as powerful clinicopathological predictors for tumor aggression and patient survival –14. CAFsIn conventional bulk transcriptomic analysis, tumor- and stroma-derived transcripts are admixed, which greatly limits precise molecular stratification and cell type-driven therapies , 24, 25.FN1, TGFB2, TGFBR2, and TGFBI, dubbed the CAF index, is a poor indicator of overall survival for oral cancer (n=40) and the PANCAN  cohorts , cleaned up by RNeasy column , and subjected to an Agilent Bioanalyzer  for RNA Integrity Number (RIN) assessment. Only samples that had an RIN > 7 were selected for mRNA amplification and sequencing .Please refer to prior study for details . BrieflyAnti-Pecam1 (ab28364) was purchased from Abcam ; anti-Adgre1 (#70076S) was from Cell Signaling .Sections were dewaxed, rehydrated, and incubated with Trilogy™   at 121°C for 10 min to unmask antigens. At room temperature (RT), the slides were immersed in 3% hydrogen peroxide for 15 min to quench endogenous peroxidase activity followed by 1% bovine serum albumin for 60 min to block nonspecific antigenic sites. Slides were incubated with indicated primary antibodies at 4°C overnight . After washing with 1X TBS containing 0.05% Tween 20, slides were incubated with horseradish peroxidase-conjugated secondary antibodies and developed by chromogen diaminobenzidine using the DakoReal™ EnVision™ kit . All slides were counterstained with Mayer’s hematoxylin and scanned by a Pannoramic MIDI scanner .Alcian blue staining kit  was used to detect hyaluronan deposition in the xenograft tissues according to the manufacturer’s instruction. Briefly, dewaxed and rehydrated FFPE tissue sections were incubated with Alcian Blue Solution for 30 min at RT, slides were counterstained with Nuclear Fast Red Solution for 5 min at RT and scanned by a Pannoramic MIDI scanner (3DHISTECH).Immunohistochemistry Image Analysis Toolbox (v1.40p) . Quantitation results were visualized by R package ggplot2 (v3.3.3). Statistical differences between OC3 and TW2.6 groups were evaluated by two-sample t-test of means (compare_means), as denoted in each plot.To quantitate the immunostaining of Pecam1, Adgre1, and Alcian blue staining in the xenograft tissues, scanned images at 100× magnification were digitized and quantitated by using ImageJ plugged-in with the Cufflinks (v2.1.1). R pheatmap (v1.0.12) was used to visualize the relative expression levels of indicated genes in each cell line. Xenograft tissue dataset (GSE149496):as described previously (XenofilteR (v1.8) processed human (hg38) and mouse (mm10) aligned reads for individual genes were used to perform gene quantification in TPM values. The resulting gene numbers for human and mouse are 17,759 and 16,374, respectively. In the exploratory analysis, R package limma (v3.40.6) was used to compute differentially expressed genes followed by Volcano plots for visualization. To identify biological pathways enriched in the OC3- and TW2.6 tumor stroma, the mouse expression matrix was subjected to gene set enrichment analysis (GSEA) (ggcorrplot (v0.1.3) was used to calculate and visualize the correlation matrix comprising genes of interest. The Pearson correlation coefficient was computed; p-value < 0.05 was considered significant. R ggplot2 (v3.3.3) was used to visualize the relative expression levels of indicated genes in the OC3- and TW2.6 stroma. Unpaired two-sample t-test of means was applied to evaluate statistical differences; p-value < 0.05 was considered significant. R pheatmap (v1.0.12) was used to visualize the relative expression levels of indicated genes in each tissue. In silico enumeration of cell fractions was conducted by using CIBERSORTx according to its online documentation of which the ‘single cell RNA-seq HNSCC’ and ‘LM 22’ were used as signature matrices, respectively (Seurat (v4.0.3) : this normalized microarray dataset comprises 18,047 genes for further analysis. R ggcorrplot was used to calculate and visualize the correlation matrix composed of 20 selected stromal genes. The Pearson correlation coefficient was computed; p-value < 0.05 was considered significant. R survival (v3.2-7) was used to assess the univariate and multivariate Cox proportional hazards of indicated clinical features and genes. The Cancer Genome Atlas (TCGA) datasets: for each indicated cancer type, the expression matrix of 20 stromal genes and associated clinical information were acquired from the UCSC Xena platform .Cell line dataset (GSE150469): human reference genome (hg19) aligned reads for individual genes  were used to compute expression values in transcripts per kilobase million (TPM) by using eviously , R Xenofs (GSEA) . Enrichmectively . Puram  human hg and mousin vitro and in vivo  research reiterated the importance of associating cellular characteristics, rather than the expression of a single or a small set of molecular markers, with EMT phenotypes . To comp in vivo . In addiNext, exploratory analysis of differentially expressed genes (DEGs) in the OC3 and TW2.6 xenograft tissues revealed that (1) compared to the OC3 cells expressing various innate immunity responsive genes, TW2.6 cells expressed MYC and E2F targets involved in cell proliferation and cell adhesion . At first, this seemed perplexing since prior results showed that stronger innate immunity was present in OC3 tumor cells  and Ccl5 (Tγδ) were likely to be the leading genes contributing to the statistical enrichments  immune cell type residing in the host stroma by CIBERSORTx analysis, immunohistochemistry using an antibody against the panmacrophage marker Adgre1 (F4/80) was performed to visualize the infiltration of macrophages in each tumor section. Unexpectedly, the spatial locations of macrophages were significantly different between the OC3 and TW2.6 groups. In the majority of OC3 tissues, the tumor cells are interdigitated with irregular Adgre1-positive macrophages and are accompanied by high staining backgrounds. By contrast, in the TW2.6 group, Adgre1-positive prototypical macrophages were frequently detected in the tumor margins, a phenomenon reminiscent of ‘immune privileged’ sites detected in clinical head and neck cancer tissues . Note thvs. 50%) and faster (7 vs. 14 days) tumor-promoting incidence of otherwise nontumorigenic oral dysplastic cells in the immunodeficient mice (NSG) and significantly deeper invasion of malignant cells in the 3D biometrices constructed in vitro. The authors proposed that a switch from an earlier secretory CAF-N to a later TGFβhigh CAF-D occurred during oral cancer progression.Recent scRNA-seq studies have shown that the inflammatory subtype of CAFs (iCAFs) secretes a variety of inflammatory mediators , whereas the myofibroblastic counterpart (myCAFs) primarily expresses ECM molecules  –23. Of sTo test the hypothesis that inflammatory cytokines detected in the TW2.6 stroma might come from the iCAF cell population, in GSEA we applied customized gene matrix transposed (gmt) file of each newly identified stromal cell population as summarized in IL6, which was enriched in the iCAF population of xenograft tissues but myofibroblasts in the clinical samples, hallmark genes for myCAFs, iCAFs, and myofibroblasts were fairly consistent between the xenograft and clinical oral cancer tissues, suggesting a certain extent of conservation in non-immune stromal cell populations preserved in the current study (We were aware that two caveats are inherent to the current study: (1) immunodeficient NOG xenografts were used, which retained only limited innate immunity, and (2) human orthologous genes might not be entirely functional in mice. With these concerns, we performed cell type-gene expression analysis using the scRNA-seq dataset of oral cancer tissues in which the presence of CAF subtypes, including myCAFs and iCAFs, has been previously noted , 21, 23.HAS1 and HAS2  and hyaluronidases  was detected in the tumor cells but not in the stromal portion of TW2.6 tumors   or p-EMT TW2.6) tumor cells, which is very different from a real clinical specimen that comprises cells at variable EMT states, e.g., only tumor cells at the invasion front display a p-EMT phenotype  . SpecifiR2, FN1) Table 2,IL6, ACTA2, MYL9, TAGLN), myCAFs , iCAFs , endothelial cells , mast cells (HYAL3), T cells (HYAL4) and tumor cells (HAS3)  The expression of HYAL1 is inversely related to myCAF genes but positively related to tumor cell HAS3.First, genes identified in the xenograft experiments were validated for their primary origins of expression by cell typing of the scRNA-seq database, including myofibroblasts (s (HAS3) 5A. NextTGFBI , TGFB2, COL12A1, IL6, HAS2, and HYAL1   are derived from myCAFs. As cancer recurrence is a known prognostic factor for overall survival, we included it in multivariate Cox analysis. The results showed that TGFBI and HYAL1, respectively, remained statistically significant in multivariate Cox models .Third, a univariate Cox proportional hazards model was performed to compute the hazard ratios (HRs) of overall survival associated with each stroma-originating gene in the NCKU-OrCA-40TN cohort. Accordingly, 6 out of 20 selected genes revealed statistical significance, including =0.0039) Table 2.TGFBI, FAP and IL6 were statistically prognostic for poorer survival; the expression of HYAL1 did not reach statistical significance for better survival  and uveal melanoma  concurrently displayed TGFBI and HYAL1 as poor and good biomarkers, respectively , breast invasive carcinoma , cervical squamous cell carcinoma and endocervical adenocarcinoma , and glioblastoma multiforme ; and the expression of HYAL1 was a statistical protective marker for kidney renal papillary cell carcinoma  and pheochromocytoma (n=148) . The results indicated that while high expression levels of TGFBI and HYAL1 remained statistically significant for poor and good prognosis, respectively  were temporally linked to disease progression  and are statistically associated with nodal metastasis and perineural invasion , 9. In bgression .CXCL12/SDF-1, VEGFA, CCL2, FGFs, PDGFs, and hyaluronic acid synthase   , 23, 44.1, HAS2) ].TGFBI came from myCAFs, myofibroblasts and tumor cells  can be found below: GSE149496, GSE150469.The studies involving human participants were reviewed and approved by the Research Ethics Committee of National Health Research Institutes (Protocol No: EC1040407-E) for the use of clinical samples and patient data. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements. The animal study was reviewed and approved by the Institutional Animal Care and Use Committee of National Health Research Institutes, Taiwan (Protocol No: NHRI-IACUC-106057-M1-A).YHL, YFC, PRC, JWL, and SFL conceptualized this study. YCK, TYL, and YLC prepared xenograft tissues. YHL and YLC performed immunohistochemical assays and histological staining. YHL, TYL, YCK, and SFL performed bioinformatic analyses. JRH provides patient data. JRH, YFC, PRC, and JYC interpreted clinical data and survival analyses. SGS, JWL, JLY, and SFL participated in formal analysis, result validation, and are major contributors in writing the manuscript. All authors contributed to the article and approved the submitted version.This study was supported by the Taiwan National Health Research Institutes (NHRI CA-110-PP-05), Ministry of Health and Welfare (MOHW 110-TDU-B-212-144013 and -144026) and the Ministry of Science and Technology (MOST 108-2320-B-400-019). The NHRI institutional library paid for the open access publication fees for this manuscript.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.


Sepsis is a continuing problem in modern healthcare, with a relatively high prevalence, and a significant mortality rate worldwide. Currently, no specific anti-sepsis treatment exists despite decades of research on developing potential therapies. Annexins are molecules that show efficacy in preclinical models of sepsis but have not been investigated as a potential therapy in patients with sepsis. Human annexins play important roles in cell membrane dynamics, as well as mediation of systemic effects. Most notably, annexins are highly involved in anti-inflammatory processes, adaptive immunity, modulation of coagulation and fibrinolysis, as well as protective shielding of cells from phagocytosis. These discoveries led to the development of analogous peptides which mimic their physiological function, and investigation into the potential of using the annexins and their analogous peptides as therapeutic agents in conditions where inflammation and coagulation play a large role in the pathophysiology. In numerous studies, treatment with recombinant human annexins and annexin analogue peptides have consistently found positive outcomes in animal models of sepsis, myocardial infarction, and ischemia reperfusion injury. Annexins A1 and A5 improve organ function and reduce mortality in animal sepsis models, inhibit inflammatory processes, reduce inflammatory mediator release, and protect against ischemic injury. The mechanisms of action and demonstrated efficacy of annexins in animal models support development of annexins and their analogues for the treatment of sepsis. The effects of annexin A5 on inflammation and platelet activation may be particularly beneficial in disease caused by SARS-CoV-2 infection. Safety and efficacy of recombinant human annexin A5 are currently being studied in clinical trials in sepsis and severe COVID-19 patients. The GloDespite much research, no specific anti-sepsis treatment exists, and management relies on early administration of broad-spectrum antibiotics, resuscitative measures, and corticosteroids to reduce patient mortality . A numbeThe annexin superfamily contains over 1,000 proteins found in over 65 different species of plants and animals . All annTwelve annexin genes have been found in humans, dispersed among several chromosomes . Though Annexin A1, also known as lipocortin, is found in a number of tissues, including lung, kidney, bone marrow, intestine, spleen, thymus, brain, and seminal fluid . Its exttrans-endothelial migration , blood vessels throughout the body, and membranes in the heart, lung, and liver. Annexin A5 is an accessory protein that helps direct cytosolic phospholipase A2 to the membrane of activated platelets where it controls its activity  and is hAnnexin A5 binds the apical surface of syncytiotrophoblasts, and maintains normal blood flow through the placenta by preventing coagulation . Its shiβ, -γ, -δ, and -ε isoforms have important roles in atherosclerosis, myocardial ischemia/reperfusion injury, cardiac hypertrophy, fibrosis and heart failure (Protein kinase C (PKC) is a family of protein kinase enzymes involved in a variety of signal transduction pathways such as apoptosis, cell proliferation, differentiation, and T cell activation through phosphorylation of serine and threonine amino acid residues on the target proteins. PKCs are activated by increases in intracellular diacylglycerol (DAG). There are 15 PKC isozymes in humans. PKC-α, - failure . Annexin failure . In addi failure .E. coli, annexin A5 treatment decreases circulating EVs and dose-dependently delays the development of sepsis  reduces inflammation and attenuates myocardial dysfunction in a polymicrobial sepsis model  signaling, facilitating its internalization in the cell and signaling for release of anti-inflammatory cytokines . HoweverBecause of its high affinity binding to PS exposed on the membrane of apoptotic cells, various annexin A5 imaging probes have been used and found to be safe in human imaging studies , includiα (TNF-α) concentrations similar to control animals , demonstrating its potency  in 94 healthy subjects (NCT04217629). The trial was recently completed. Doses of intravenous injection of SY-005 from 0.75 to 20 mg per person were well tolerated without any major adverse events, suggesting that recombinant human annexin A5 is safe in healthy subjects. Another phase 1 study using annexin A5 as a therapeutic drug is recruiting healthy volunteers (NCT04850339).via angiotensin converting enzyme 2 (ACE2) (+ and CD8+ T cells) and decreased interferon-α (IFNα) expression in CD4+ T cells. SARS-CoV-2 infiltration of cells is also capable of activating inflammasomes, such as NOD-, LRR- and pyrin domain-containing protein (NLRP3), which eventually leads to pore formation on the cell surface, IL-1β and IL-18 secretion and eventual pyroptosis, which further promotes inflammation (The emergence of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) on a global scale has driven a large need for treatments of COVID-19 induced sepsis and acute respiratory distress syndrome (ARDS). In COVID-19, SARS-CoV-2 enters the host cells 2 (ACE2)  and seve2 (ACE2) . Once inammation . Thromboammation , and thoammation . The imbammation .Annexins may have therapeutic potential in viral sepsis. While the mechanism of sepsis is different between bacterial and viral insults, they share commonalities in the activated receptors and released cytokines . To thisvia inhibition of thrombin generation and platelet aggregation, and modulate the response to apoptosis, are promising therapeutic agents to reduce the severity of inflammatory processes in COVID-19. However, the exact mechanism of COVID-19 coagulopathy is not fully understood. Some studies of COVID-19 describe a pattern similar to DIC  . Heparinr to DIC , which ir to DIC . Additior to DIC . Furtherr to DIC . It is pExtracellular HMGB-1 is another potential therapeutic target. Extracellular HMGB-1 is released from dying cells and forms complexes with extracellular DNA, RNA, and other molecules after lytic cell death, which are endocytosed. It has been proposed that SARS-CoV-2 RNA may enter cells via HMGB1-assisted transfer, promoting viral infection . Plasma We have started a randomized, double blind and placebo-controlled phase 2 clinical trial to evaluate safety, tolerance and pharmacokinetics of recombinant human annexin A5 in COVID-19 patients with sepsis (NCT04748757). The intervention is SY-005, 50 or 100 μg/kg intravenously every 12 h for 7 days, with a placebo comparator. The primary outcomes are feasibility metrics of enrollment, treatment delivered per protocol, data collection per protocol, and adverse events. At the time of this submission, 12 patients were recruited with no serious adverse events.Annexins are endogenously produced proteins with anti-inflammatory, anti-apoptotic, and anticoagulant activities. Annexins and their peptide analogues, when administered in various conditions in different animal models of disease, can alleviate and improve inflammation, coagulation, and ischemia-reperfusion injury. Evidence to date suggests that annexins could be used as a treatment, prophylactic therapy, or adjuvant therapy in various contexts, most notably sepsis, coagulopathy, and ischemic-reperfusion injury. Confirmation of these benefits is required in human trials. Annexin therapy could thus be an effective therapy in various disease conditions, especially sepsis, and novel diseases such as SARS-CoV-2 infection.


Early childhood and the pre-school stage of development constitute a dynamic period for acquisition of social-emotional competencies. Yoga and mindfulness practices (YMP) have become increasingly used in schools for social emotional learning, but less is known about their utility in early childhood settings. A systematic review using PRISMA guidelines was undertaken to explore the effect of YMP on social emotional function among preschool-aged children (3–5 years). The review resulted in identification of 1115 records, of which 80 full text articles were screened, with final inclusion of 16 studies. Included studies evaluated the effect of YMP on social-emotional functioning, and identified the potential for YMP to improve regulatory skills such as behavioral self-regulation and executive function. Among studies reviewed, 13 reported improvements in these domains, but quality appraisal indicated significant variability in risk of bias across studies, and heterogeneity of outcome measurements hindered comparison. Programs appeared to produce better results when implemented for at least 6 weeks and among children who had lower baseline social-emotional functioning. YMP constitute a promising strategy for social emotional development in early childhood settings, but additional rigorously designed studies are needed to expand understanding of how and why these programs are effective. Participation in yoga and mindfulness meditation has increased over the past decade among both adults and youth in the United States ,2. AccorThe second most commonly used complementary health approach was meditation; the use of meditation increased more than threefold from 4% in 2012 to 14% in 2017. The most popular form of meditation for health in the United States is mindfulness meditation, which was introduced in 1982 at the University of Massachusetts Medical Center in the form of Mindfulness Based Stress Reduction (MBSR). Mindfulness meditation was initially defined in MBSR as intentional self-regulation of attention from moment to moment, and other definitions have since emerged centering on focal awareness of experience in the present moment . As adulSome evidence on these practices has also become available through peer-reviewed journal articles from research studies of youth-focused interventions. In 2019 alone, 164 journal articles were published on the topic of mindfulness with youth, compared with 11 journal articles on the topic having been published in 2009, just 10 years earlier  accessed on 21 August 2020, with the registration number CRD42020200206.The systematic review was conducted in accordance with PRISMA guidelines and a PRIMSA flowchart is included. The study protocol for this review was registered in the PROSPERO database   OR “yoga”[Title/Abstract] OR “Mindfulness”[MeSH Terms] OR “mindful*”[Title/Abstract] OR “self-compassion”[Title/Abstract] OR “Meditation”[MeSH Terms] OR “meditat*”[Title/Abstract] OR “contemplative”[Title/Abstract] OR “contemplation*”[Title/Abstract]) AND  OR (“early child*”[Title/Abstract])) OR (“kindergar*”[Title/Abstract])) OR (“Child Care”[MeSH Terms] OR “Child Care”[Title/Abstract] OR “children care”[Title/Abstract] OR “child day care”[Title/Abstract] OR “children day care”[Title/Abstract] OR “child daycare”[Title/Abstract])) OR ). The specific search strategy sample can be found in The review included yoga and mindfulness studies conducted in early childhood school settings, aimed at improving children’s social emotional development. Only English language studies were included. Where yoga or mindfulness was one component of a complex intervention or a complementary component, half or more of the content was required to be related to mindfulness or yoga. The intervention must have been delivered to children rather than to parents and or caregivers alone. For outcome, studies needed to report at least one child-level social-emotional skill, behavior, or symptom. The definition of “social-emotional” was intentionally broad to encompass a wide variety of skills and behaviors. According to the Collaborative for Academic, Social, and Emotional Learning (CASEL), there are five broad areas of social-emotional competence: self-awareness, self-management, social awareness, relationship skills, and responsible decision-making . StudiesIntervention studies were eligible if they were randomized controlled trials (RCTs), quasi experimental design trials (QEDs), pre-post-test designs, or otherwise used widely accepted and validated measurement and evaluation methods with statistically appropriate techniques to assess the effectiveness of intervention. The comparison groups in RCTs and QEDs included wait-list control, treatment-as-usual, or other alternative interventions. Pre-post study designs were included if the pre-post-comparison was completed. Participants in included studies were children between 3–5 years old. If a study enrolled children with an overlapping age range , the study was included if the mean age of child participants was less than six years old. Studies that enrolled children with developmental disorders, including intellectual disabilities and autism spectrum disorder, were included if the intervention was provided in a general education setting. Studies were excluded if they were conducted in a special education facility or self-contained classroom, or if they failed to provide information on participant ages.Two authors independently screened studies using prespecified inclusion and exclusion criteria. Titles and abstracts were reviewed and those deemed ineligible were excluded. Articles that met eligibility criteria upon title and abstract review were retrieved and reviewed in depth. Full-text articles were then screened for inclusion and exclusion. The study selection process and reasons for full-text exclusion are shown in A standard data abstraction form was adopted from the Cochrane Collaboration  to colleThe following information was obtained from each study: citation information , methods  participants , interventions , outcomes , data analysis , and general information .n = 6) and randomized trials (n = 10), respectively. For both tools, “Low risk” correspond to the risk of bias in a high quality study. Disagreements in risk of bias were resolved by discussion and consensus. Both tools include domain items relating to deviations from the intended interventions, missing outcome data, measurement of the outcome, and selection of the reported result. The ROBINS-I also includes items relating to confounding, selection of participants, and classification of intervention. The RoB 2 includes items relating to the randomization process. Risk of bias was assessed for each domain and pooled for an overall risk of bias rating for each study. All papers provided enough information to produce a final rating.Three review authors independently assessed risk of bias for each study using risk of bias tools from Cochrane. The ROBINS-I assessment tool  and the Data was synthesized by a tabulated and narrative summary of included studies. Demographic and descriptive information including participants, methodology, interventions, outcome measures, follow up, statistical significance, effect sizes, conclusions, and recommendations of intervention studies were synthesized.Where sufficient detail was available from the selected studies, information on sub-topics of interest has been presented in addition to an overall synthesis. For example, information is presented on the following: participant characteristics , intervention content , intervention duration, evaluation method, domains of social-emotional development , and study design (randomized/non-randomized).The flow diagram of search results is shown in n = 15) [n = 14) [n = 4) [n = 4) [n = 2) [n = 1) [n = 1) [n = 1) [n = 1) [Study characteristics are listed in n = 15) ,44,45,46[n = 14) ,44,45,46 [n = 4) ,38,43,46 [n = 4) ,39,40,44 [n = 1) , Journal [n = 1) , Journal [n = 1) , and Ear [n = 1) . Two inc [n = 1) ,41.n = 5) were parallel RCTs [n = 5) were cluster RCTs [n = 2) [n = 2) [n = 1) [n = 1) [The included studies utilized a variety of study designs, with the most common being a randomized controlled trial (RCT). A total of 10 trials used an RCT design ,44,45,46lel RCTs ,37,45,46ter RCTs ,36,40,44ter RCTs ,40,43,44ter RCTs ,46. The  [n = 2) ,45, impl [n = 2) ,35, or e [n = 1)  or unspe [n = 1) .n = 11) of the trials were conducted in the United States [This review features studies from multiple countries and early childhood settings. More than half  [n = 3) [n = 2) [Twelve studies ,43,45,46n = 2) ,43, Hisp [n = 3) ,42,45, o [n = 2) ,46. One  [n = 2)  recruiteAs described earlier, studies that enrolled children with developmental disorders, including intellectual disabilities and autism spectrum disorder, were included only if they were conducted in an integrated or general education setting. Only three studies ,33,41 ren = 4) [n = 1) [n = 1) [The included studies included mindfulness, yoga, or both, and some interventions had additional social-emotional learning components. Five studies ,43,44,46n = 4) ,35,36,37 [n = 1) , or emot [n = 1) .n = 12) [n = 2) [n = 1) [n = 1) [Data on program implementation varied widely between the included papers. Six interventions were delivered by a certified yoga teacher or mindfulness instructor ,39,40,41n = 12) ,43,44, s [n = 2) ,45, indi [n = 1) , or in an = 15) [Included studies n =  [15,32,3n = 15) ,44,45,46n = 15) ,42,43,45n = 15) ,40,44,45n = 15)  specificn = 15) ,39,40,46n = 15) ,41,44 exSome of the included studies also examined other domains of social-emotional functioning beyond executive function and self-regulation. Seven studies ,43,44,45The overall ratings indicated significant variability in risk of bias across the sample. For the ten RCT studies ,44,45,46n = 4) [n = 3) [n = 2) [n = 1) [n = 3) [n = 2) [The source of bias differed between RCT and non-RCT studies. Specifically, for the RCT studies, bias in outcome measurement was the most frequent source of high risk of bias (n = 4) ,35,36,44 [n = 3) ,37,46, b [n = 1) . The bia [n = 3) ,41,42 fo [n = 2) ,41.n = 13) studies [ studies ,43,44,45 studies ,41,46 fa studies ,41 were Self-regulation refers to processes that allow children to manage their thoughts, behavior, and emotions . Seven sOther measures used to measure behavioral self-regulation included the Delay of Gratification Task , the ToyUnder the umbrella of self-regulation, emotion regulation describes how children manage affective states . Two stun = 2) [n = 2) [n = 2) [n = 1) [n = 1) [n = 1) [n = 1) [Six studies ,42,43,45n = 2) ,42, Peg/ [n = 2) ,45, Flan [n = 2) ,43, Minn [n = 1) , Dimensi [n = 1) , and theOverall, the effect of yoga/mindfulness participation on children’s executive function was mixed. For example, using the BREIF-P, a teacher-rated questionnaire of children’s executive function, Thierry et al., 2016  found poSimilar inconsistencies emerged in the remaining studies that used other EF measures. For the Flanker task, only one  found a Six studies ,39,40,46Four studies ,33,41,44Seven studies ,43,44,45n = 3) [Five studies ,38,39,45n = 3) ,39,45 reStudy results were consistent across different demographic groups. Interventions were effective across a broad range of participant groups representing children of diverse racial/ethnic backgrounds and SES levels. However, only two studies ,45 explin = 3) [n = 13) [Duration  appeared to potentially influence program effectiveness. When comparing the studies that had null results (n = 3) ,41,46 to[n = 13) ,43,44,45n = 6) [n = 5) [n = 2) [n = 1) [n = 1) [Frequency  differed widely across studies, making it difficult to draw conclusions about frequency as a potential moderator. Programs were implemented on a daily basis (n = 6) ,42,43,45 [n = 5) ,34,40,41 [n = 2) ,44, four [n = 1) , or six  [n = 1) . One stu [n = 1)  evaluateStudies among children with lower baseline social-emotional functioning described the largest increase in social emotional function from yoga/mindfulness interventions. A 2015 study  found thThis systematic review is the first to assess the effects of YMP on social emotional outcomes in preschool-aged children. Our results align with those of previous systematic reviews and meta-analyses ,62,63 inBehavioral self-regulation was one of the most frequently studied outcomes, assessed in 7 of 16 (44%) studies. A majority of the studies (71% or 5 of 7 studies) evaluating behavioral self-regulation found an improvement following the intervention. This improvement varied depending on the outcome measurement used. Whereas improvement was found in all studies utilizing the HTKS measure , and oneExecutive function, the cognitive processes underlying behavioral self-regulation, was the second most highly studied outcome (38% or 6 of 16 studies). However, considerable heterogeneity in measurement tools and results make it difficult to draw conclusions on the effect of mindfulness and yoga intervention. BRIEF-P , FlankerChildren between 3–5 years old experience rapid growth and development of regulatory abilities. Interventions delivered in the preschool period may occur during a sensitive period in development where these skills are first coming “online” . Self-reMany of the interventions explicitly taught kindness and/or social skills, or hypothesized that participation in YMP would have a downstream effect on peer interactions. Therefore, outcomes related to prosocial behavior were assessed in 7 of 16 (44%) studies. However, measurement tools and effects for prosocial behavior varied across studies. A majority of studies (4 of 7 or 57%) reported a positive impact of yoga/mindfulness interventions. The remaining 3 studies (43%) did not report significant effects. The mixed results here are not surprising given the variety of programs represented in the review. Direct instruction of social skills using role plays and activities may be the best way to teach prosocial behaviors in early childhood classrooms .A number of studies examined attentional capacities (38% or 6 of 16 studies), or deficits in attention by looking at ADHD symptoms (25% or 4 of 16 studies). Most studies (83%) found significant improvements in attentional capacities following intervention. As unique measurement tools were used in each study, this finding did not correspond to measurement type. In regard to ADHD symptoms, all four studies reported at least one significant finding linking yoga/mindfulness participation with reductions in ADHD symptoms. These findings align with prior research suggesting that yoga is a promising intervention for children with attention problems . YMP mayIt is important to examine what interventions work for different populations and under what conditions. The second research question examined whether results were consistent across populations or dimensions of implementation . There were no differences based on population or frequency of intervention delivery. Variation was noted, however, according to duration, or the total length of time that children received the YMP intervention. For interventions lasting from 15 min to 4 weeks, null results were reported. In contrast, interventions lasting at least six weeks reported at least one favorable SEL-related result. Out of all possible dimensions of implementation, duration seemed to matter the most. Children who participate in YMP programs for a longer period of time receive a higher “dosage” of the intervention, and dosage may an important predictor of child outcomes in educational settings .In regard to other moderators of program effect, children with poorer baseline skills showed the most improvement following yoga/mindfulness interventions. This was true in the case of children with lower levels of social competence and executive functioning. , for chiFuture research should continue to investigate how yoga/mindfulness interventions may improve SEL outcomes for other at-risk populations of young children. In the adult literature , mindfulThe current study has several limitations. Firstly, the success of an intervention hinges on the quality of implementation, but data on implementation was limited. Future studies should provide more in-depth implementation data on acceptability, feasibility, and student engagement. Secondly, quantitative analysis was not conducted in the present study, limited by the diversity of measurement tools in each outcome. This makes it impossible to draw conclusions based on pooled effect size estimates. The review was also unable to determine with confidence if yoga and mindfulness interventions might work best for certain populations of children. This would require future research to conduct moderation analyses of program effects.Lastly, while review distinguished between different SEL domains, there is substantial overlap between self-regulation and executive function, for example. Constructs were classified based on the language used to describe the measures along with precedent. The results should be interpreted with the understanding that social-emotional functioning often involves multiple, coordinated skills that are difficult to parse apart.The results of this study can be used to inform YMP programming in early childhood settings. In general, teachers in elementary  and earln = 16) included in the review and risk of bias, more research is needed. Future research should continue to investigate the efficacy and effectiveness of YMP interventions in diverse contexts. It is important to determine under what conditions and for whom these interventions are best suited. Our review of the literature indicated that children with the lowest baseline social-emotional skills may benefit the most from YMP interventions. Early childhood centers may use universal screening using an instrument to identify children with lower baseline scores. The quality of implementation matters in health and education prevention programs [The results of this systematic review may also inform educational policy and practice in early childhood settings, as well as contribute to additional rigor and planning of future research on yoga and mindfulness with young children. Given the relatively small number of studies . The review identified YMP having favorable effects on several regulatory domains, as well as on attentional capacities, peer and prosocial behavior, and general well-being, but due to heterogeneity of measurement of social-emotional outcomes and risk of bias, the level of evidence remains moderate. Additional methodologically rigorous studies are required to assess pooled data and to increase confidence in the level of evidence. As YMP continue to be used in schools, a clearer understanding of how and why it may be beneficial for young children will emerge.


Plants remodel their root architecture in response to a salinity stress stimulus. This process is regulated by an array of factors including phytohormones, particularly auxin. In the present study, in order to better understand the mechanisms involved in salinity stress adaptation in rice, we compared two contrasting rice cultivars—Luna Suvarna, a salt tolerant, and IR64, a salt sensitive cultivar. Phenotypic investigations suggested that Luna Suvarna in comparison with IR64 presented stress adaptive root traits which correlated with a higher accumulation of auxin in its roots. The expression level investigation of auxin signaling pathway genes revealed an increase in several auxin homeostasis genes transcript levels in Luna Suvarna compared with IR64 under salinity stress. Furthermore, protein profiling showed 18 proteins that were differentially regulated between the roots of two cultivars, and some of them were salinity stress responsive proteins found exclusively in the proteome of Luna Suvarna roots, revealing the critical role of these proteins in imparting salinity stress tolerance. This included proteins related to the salt overly sensitive pathway, root growth, the reactive oxygen species scavenging system, and abscisic acid activation. Taken together, our results highlight that Luna Suvarna involves a combination of morphological and molecular traits of the root system that could prime the plant to better tolerate salinity stress. The plant root is the vital organ that serves a wide range of functions and regulates crop productivity. As roots are in direct interface with the soil, they act as the primary site for perceiving environmental stress-related signals for plants ,2. AmongThe potential of several phytohormones to ameliorate the damaging effects of salinity stress has attracted the attention of researchers in the recent past ,12. AmonAlthough roots are the critical site for the perception of salinity stress signals and are responsible for triggering stress-related mechanisms in plants, very little attention has been paid to analyzing this underground part of the plant in the context of understanding salinity tolerance. Physiological, biochemical and genetic studies have provided ample evidence in support of the key role of auxin in triggering abiotic stress-mediated differential modifications in the root system architecture of plants . The keyPorteresia coarctata with two rice cultivars variable in salt sensitivity—IR64  and Pokkali —suggested that, in the leaves of Porteresia coarctata, several proteins exhibited up-regulation that could provide it a physiological advantage under salinity stress [For finding significant clues on the adaptive behavior of plants to salinity stresses, studies at the protein level might be a better option compared to at the transcript level since many post-transcriptional and post-translational changes often take place in plants and hence, the rate of transcription and the translation will not necessarily always correlate. Hence, proteome-based approaches involving two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS) are often utilized for unraveling proteins associated with induced changes in plants as they are very reliable, sensitive and powerful technologies ,23. For y stress . HoweverThe differences in the morphological parameters of the two cultivars were clearly observable when cultivated in normal conditions A,B. An aOsYUCCA5, OsYUCCA7, and OsYUCCA8 exhibited significant up-regulation of 2.79, 3.53, and 2.58 fold, respectively, in the roots of the salt-tolerant cultivar LS as compared to the salt-sensitive cultivar IR64 of rice under normal conditions   A. On the control A. The trOsPIN genes, OsPIN2, OsPIN5a, and OsPIN5b exhibited higher transcript level accumulation of 1.95, 2.36, and 2.46 fold, respectively, in the roots of untreated LS as compared to the IR64 , β-glucosidases 12 (β-gluc), phytosulfokines 3 (PSK), and B3 domain-containing protein (B3D) were observed to be 1.09, 1.02, 3.64, and 1.78 fold higher, respectively, in LS roots compared to IR64 under normal conditions , delta-pyrroline-5-carboxylate synthase (P5CS), cyanate hydratase (CHS), DEAD-box ATP-dependent RNA helicases 53 (DEAD), plant intracellular Ras group related LRR protein 2 (RAS-LRR), and minichromosome maintenance 6 (MCM6) was 0.69, 0.38, 0.30, 0.21, 0.64, and 0.22 fold lower, respectively, in the roots of tolerant cultivar LS   was perfnditions . Howevertivar LS  with res control . On the rol IR64 . However.65 fold  lower trPlant roots perceive the salinity stress signals and promptly pass them to the shoot to activate various stress-responsive pathways ,2,9,10. iaam-OX transgenic lines (with higher endogenous IAA level) and wild-type plants of Arabidopsis pretreated with IAA exhibited resistance towards drought stress [yuc1yuc2yuc6, which were deficient in endogenous IAA content, showed decreased resistance towards drought stress [iar4 mutants, reduced root meristem activity and root growth were reported due to diminished auxin distribution in root tips, indicating the key role of auxin in root growth and development [It is believed that phytohormones are critical signaling molecules that function downstream of environmental stimuli and regulate various stress adaptive pathways . In prevt stress . Howevert stress . Moreovet stress . It was t stress ,36. In Aelopment . The exoelopment ,33,37,38YUCCA genes—YUCCA3, YUCCA5, YUCCA7, YUCCA8, and YUCCA9—express highly in Arabidopsis roots, playing an essential role in the root development [YUCCA genes and salinity stress adaptation has never been evaluated in rice. Interestingly, in the present study, it was observed that the transcript level accumulation of different OsYUCCA genes was higher in the roots of LS. It might be the primary reason for more auxin biosynthesis and its accumulation in LS roots. Further, in response to salinity stress, the transcript level accumulation of OsYUCCA3, OsYUCCA4, OsYUCCA5, OsYUCCA6, OsYUCCA7, and OsYUCCA9 was enhanced in LS roots. In Arabidopsis, YUCCA8 and YUCCA9 have been linked with the development of lateral roots, while their mutants develop shorter primary roots suggesting their key role in the development of root system architecture [OsYUCCA genes mediated enhanced auxin accumulation and subsequently better developed root system architecture for the acquisition of salinity stress tolerance in rice.It is well realized that the process of auxin-mediated root development is regulated by a complex interplay between auxin metabolism, its signaling and transport leading to the spatio-temporal distribution of auxin ,39,40. Telopment . Howeveritecture . Hence, OsPINs in rice [OsPIN1a, OsPIN2, OsPIN3a, OsPIN5a, and OsPIN5b, were found to be higher in the LS root. Moreover, under salinity stress, the expression of auxin transport genes, particularly OsPIN1a, OsPIN1b, OsPIN2, OsPIN3a, and OsPIN5b, was up-regulated in the roots of LS. OsPIN1b and OsPIN9 have been suggested to participate in root development in rice, by regulating auxin-cytokinin interaction [OsPIN2 expresses highly in roots and enhances shoot to root auxin transport [Once IAA is biosynthesized, it is transported to the area of its requirement with the help of cell-to-cell auxin transport mediated by  in rice . Earlier in rice . Moreove in rice ,44. Inteeraction . Furtherransport . Thus, tOsGH3 genes [OsGH3.13 was analyzed in the roots of salt-sensitive (IR64) and salt-tolerant cultivar (LS) of rice. It was observed that the expression of OsGH3.13 was significantly higher in the roots of LS, which is contradictory to the observed higher IAA content in LS. Thus, it can be inferred that the role of the OsYUCCA genes is probably more critical in regulating auxin content in rice as compared to OsGH3. Further, the analysis of OsGH3.8 at transcript level suggests no difference in expression level between IR64 and LS roots. However, under salinity stress, the transcript level accumulation of OsGH3.13 was down-regulated in the roots of LS compared to IR64. On the contrary, OsGH3.8 exhibited up-regulation in the root but not to a significant level. It indicates that the lower expression of OsGH3.13 under NaCl application in LS root might be responsible for providing tolerance against salinity stress, probably by enhancing IAA levels.The optimum concentration of IAA is maintained in a cell through their conjugation and degradation by H3 genes . Hence, OsAFB2 and OsTIR1 are the auxin signaling receptors affected by salinity stress [OsTIR1, OsAFB2, and OsABP1 were found to be elevated in the roots of LS with respect to IR64 under normal conditions. This identification of the enhanced expression of auxin receptors can be linked to higher auxin content in the roots of LS compared to IR64. Under salinity stress, the expression of OsTIR1 showed down-regulation in LS roots with respect to control IR64. On the contrary, the transcript levels of OsAFB2 and OsABP1 exhibited up-regulation in the roots of LS compared to IR64 under the salt application. The expression of various auxin signaling genes, OsARF1, OsARF2, OsARF16, OsAUX/IAA1, and OsAUX/IAA4, was also found to be higher in the roots of LS compared to IR64 under both control as well as salinity stress conditions, demonstrating elevated endogenous IAA level in LS root compared to IR64. Previous studies have also linked elevated auxin concentration with increased auxin transport and downstream signaling genes [OsAUX/IAAs) and 25 auxin activator (OsARFs) genes that participate in auxin signaling were observed to be suppressed by cold, heat and drought stress [OsAUX/IAA, such as OsAUX/IAA 6,9,18,19,20 and 28 and OsARF 4,11,13,14,16,18 and 19, were induced by at least one among cold, heat, and drought stress [AUX/IAA in LS root compared to IR64 might be extrapolated to the salt tolerance and a higher number of roots detected in the tolerant cultivar.Various findings have suggested that y stress ,48. Howeng genes , thus prt stress . On the t stress . Hence, t stress . Howevert stress ,51. Henc2+ sensing proteins of the CIPK gene family, have been previously linked with the enhanced tolerance against salinity stress conditions in Arabidopsis [salt overly sensitive 3 (SOS3) encodes for CBL, which functions in sensing the cytosolic Ca2+ concentration by directly binding to it [2+ bound CBL proteins directly activate their interacting partners, such as CIPK6, which are involved in auxin transport, regulation of root length and lateral root development [NAC, PIN2, and P5CS genes, which promote salinity stress resistance in plants [Pisum sativum PsMCM6 in tobacco confers salinity stress tolerance without affecting yield [RD29A, RD29B, RAB18, ABI1, ERD15, KIN, ERD10, and COR15a) [CIPK, CCoA, P5CS, PSK, CHS, and MCM6 genes exhibited down-regulations with respect to the control IR64, which might lead to salinity stress susceptibility in the sensitive cultivar. The present finding also indicates that, in IR64, the protein turnover rate might be high, probably leading to targeting of the salinity stress responsive proteins towards degradation, leading to salinity stress sensitivity. On the contrary, in LS upon salinity stress application, higher transcript accumulation of CIPK, CCoA, PSK, CHS, DEAD and MCM6 was observed, which could be linked to its acquisition of the salinity stress resistance property.The comparative proteomics study revealed that some proteins were specifically present in the roots of LS compared to IR64. Among these, CIPK21 and CIPK29, the Cabidopsis ,53. It wng to it ,54. The elopment ,54. CIPKn plants . P5CS prn plants ,56. Thusn plants . Thus, tn plants ,58,59. Fn plants ,61. PSK,n plants . PSK alsn plants ,63,64. Hn plants ,66. It an plants . Hence, n plants ,68. Thern plants . In one n plants . It was ng yield ,69. Furtng yield . Further COR15a) . The pro COR15a) ,73. Thes COR15a) . In IR64Oryza sativa L.) cultivars were procured from Punjab Agricultural University, Ludhiana, India, and the Central Rice Research Institute (CRRI), Cuttack, India, respectively. LS can tolerate the salt stress up to 8 dSm−1. The seeds were surface sterilized with 70% ethanol (v/v) for 1 min and treated with 0.4% sodium hypochlorite solution containing a drop of tween-20 for 30 min. The seeds were washed thrice with autoclaved distilled water and were then dried on autoclaved Whatman paper (3 mm) for 5 min. After surface sterilization, the seeds were inoculated in the plastic tray containing autoclaved sand moistened with sterile distilled water and were incubated in the culture room at 25 °C (day/night), 70–80% relative humidity (day/night), and 14 h photoperiod for 14 days. After 14 days, IR64 and LS seedlings were treated with 100 mM NaCl for 8 h for imposing salinity stress. The roots were later separated for protein and RNA extraction to conduct 2-dimensional gel electrophoresis and gene expression studies, respectively.The certified and disease-free seeds of salinity stress-sensitive IR64 and salinity stress-tolerant LS rice  for IAA estimation. Further, the elution was carried out with 100 % methanol (HPLC grade): Water (Formic acid 0.05% v/v), 35:65, at a flow rate of 1 mL·min−1. The column elutes were passed through a UV detector at 254 nm, and IAA was estimated with reference to an authentic standard of IAA (1 mM) . The readings were taken in the replicates of three and the average of peaks was obtained.To estimate the content of IAA, 5 g fresh roots of IR64 and LS were crushed finely in liquid nitrogen and extracted in chilled 80% ethanol (15 mL/g) containing butylated hydroxytoluene (BHT) (100 mg/L) . The homhttp://www.invitrogen.com, last accessed on 20 July 2021), as per the manufacturer’s instructions. RNase-free DNase  was used to remove the genomic DNA and 2 µg of RNA was used to synthesize cDNA in a total volume of 10 µL reaction using the iScript cDNA synthesis kit  as per the manufacturer’s recommendations.A total of 150 mg of root sample of IR64 and LS was homogenized in liquid nitrogen using pestle and mortar. Total RNA was isolated using Trizol reagent  (T (∆∆CT) and normalized against OsUBQ5 (LOC_Os1g328400) used as an endogenous control.qRT-PCR was performed to study the differential expression of auxin homeostasis genes in the roots of IR64 and LS. The nucleotide sequences of different genes involved in auxin homeostasis were retrieved from the rice annotation project database (RAP-DB) and the gene-specific primers were designed using Integrated DNA Technologies, USA (ly 2021) . The qRTA phenol-based method was used for extracting proteins from 1 g roots of IR64 and LS as reported previously . The samThe protein pellets were suspended thoroughly in rehydration buffer . Protein concentration was quantified with a Bradford protein estimation assay  using bo® 3/10 ampholyte, 0.001% bromophenol blue  and passively rehydrated over IPG strips  overnight at 20 °C. After rehydration, the strips were focused at 250V for 40 min, 4000 V for 2 h with linear voltage amplification, and finally to 10,000 V h with rapid amplification. After IEF, the strips were incubated with equilibration buffer I, containing 6 M urea, 375 mM Tris-HCL pH 8.8, 2% SDS and 2% DTT for 15 min for reduction . For alkylation of the proteins, the strip was further incubated with 2.5% iodoacetamide dissolved in equilibration buffer II containing 6 M urea, 375 mM Tris-HCL pH 8.8 and 2% SDS (ReadyPrep 2-D Starter Kit Equilibration Buffer I #1632108) for 15 min. The second-dimensional electrophoresis was performed using 12% polyacrylamide gel. After mounting the strip on the gel, it was sealed with 0.5% agarose containing 0.1% bromophenol blue, and the protein molecular marker was also loaded. Electrophoresis was performed at a constant voltage of 100 V for 2 h in tris-glycine-SDS containing running buffer.For isoelectric focusing (IEF), 150 µg of protein was dissolved in a total of 130 µL of rehydration buffer containing 8 M urea, 2% CHAPS, 50 mM DTT, 0.2% Bio-LyteAfter 2-DE, gels were stained with Coomassie brilliant blue and were stored in 5% acetic acid until further analysis. Gel imaging was conducted using the Molecular Imager Gel Doc XR system  and the images were analyzed using PDQuest 8.0.2 software.4HCO3/50% ACN solution and washed twice with 200 µL of Milli-Q water for 5 min each and were dehydrated using 100 µL of acetonitrile. The samples were subjected to trypsinolysis in 25 µL of trypsin solution  with a concentration of 20 µg/mL in 25 mmol/L NH4HCO3, and were incubated overnight at 37 °C. Each digested peptide was further extracted from the gels using 50% trifluoroacetic acid/ 50% acetonitrile, twice at room temperature. The extracted peptides were mixed with 0.5 µL of α-cyano-4-hydroxy-cinnamic acid (Bruker) of a concentration of 20 mg/mL prepared in 0.1% trifluoroacetic acid, 30% (v/v) acetonitrile and dried at room temperature. The trypsin digested protein samples were subjected to mass spectrometric analysis using an UltrafleXtreme™ mass spectrometer . The instrument was calibrated and fine-tuned with a mass standard starter kit (Bruker) and standard tryptic digested BSA . TOF spectra were recorded in positive ion reflector mode between mass ranges of 700–3500 Da. For protein characterization, the obtained MS spectra were searched against a non-redundant database (SwissProt database) using a MASCOT search engine with these parameters: taxonomy: Oryza sativa (rice); parent ion mass tolerance was set at ± 1.2 Da and MS/MS tolerance at 100ppm; variable modifications, oxidation of methionine (M) and carbadomethylation of cysteine (C) and trypsin enzyme.Proteins spots showing variations in their intensities, presence and absence were manually excised from Coomassie brilliant blue-stained gels and were subjected to mass spectrometric analysis . The excp < 0.05 were considered significantly different.All the data obtained from different experiments were evaluated using statistical analysis. An unpaired t-test and a one-way analysis of variance (ANOVA)  test were conducted to compare the mean differences using Sigma Stat version 3.5. Comparisons with The present study shows that salt tolerant rice cultivars present salinity stress adaptive root traits, likely due to an elevated endogenous auxin content and augmented levels of key salinity stress providing proteins in its roots. Salt tolerant rice LS cultivars exhibited higher transcript-level expression of different genes involved in auxin homeostasis both under control and salinity stress conditions. Thus, our study suggests that an elevated level of auxin and a higher buffering capacity of the auxin homeostasis process may be critical for the acquisition of salinity stress adaptation in rice. Upon 2-DE and MS analysis, several salinity stress tolerance providing proteins were detected that exhibited higher constitutive expression in the roots of LS with respect to IR64. In LS roots, the transcript level of some identified stress marker proteins exhibited lower expression; on the contrary, their protein accumulation was higher in the tolerant cultivar, LS. It indicates that their protein turnover rate might be low. Taken together; these results highlight morphological and molecular features that are critical for rice adaptation towards salinity stress and reveal that this process is multifactorial. Moreover, our results pinpoint several candidate genes that could be artificially overexpressed to increase salinity stress tolerance in rice.


MPEG1 mutations were observed in SN patients, while SE patients were associated with upregulation of TGF-β signaling pathway and downregulation of T-cell receptor signaling pathway. SE patients also presented immunosuppressive status with lower activity of killing of cancer cells and recruiting dendritic cells. Extranodal involvement had no influence on progression-free survival (PFS) or overall survival (OS) in localized DLBCL. Serum lactate dehydrogenase >3 upper limit of normal was an independent adverse prognostic factor for OS, and ATM mutations were related to inferior PFS. Although the overall prognosis is satisfactory, specific clinical, genetic, and microenvironmental factors should be considered for future personalized treatment in localized DLBCL.The clinical and molecular characteristics of localized diffuse large B-cell lymphoma (DLBCL) with single nodal (SN) or single extranodal (SE) involvement remain largely elusive in the rituximab era. The clinical data of 181 patients from a retrospective cohort and 108 patients from a phase 3 randomized trial NHL-001 (NCT01852435) were reviewed. Meanwhile, genetic aberrations, gene expression pattern, and tumor immunophenotype profile were revealed by DNA and RNA sequencing of 116 and 53 patients, respectively. SE patients showed similar clinicopathological features as SN patients, except for an increased percentage of low-intermediate risk in the National Comprehensive Cancer Network–International Prognostic Index. According to the molecular features, increased  Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin's lymphoma and represents a heterogeneous entity with various clinical, immunophenotypic, and molecular features , 2. AntiMYD88 and CD79B were frequently observed in primary CNS, breast, female genital tract, and testicular DLBCL  and single extranodal (SE) group. Other than lymph node, Waldeyer's ring and spleen are considered as nodal tissue , while Gar DLBCL , 11 but ar DLBCL , 13. In ar DLBCL . TherefoIn the present study, we analyzed the clinical characteristics and prognostic features of localized DLBCL both in retrospective and prospective cohorts, and evaluated the molecular heterogeneity between SN and SE including genetic aberrations, gene expression pattern, and tumor microenvironment profile, which may be helpful for future personalized treatment in localized DLBCL.2), R-CEOP70 (epirubicin 70 mg/m2), or R-CEOP90 (epirubicin 90 mg/m2) regimen as previously described  performance status, serum LDH, IPI, NCCN-IPI, and bulky tumors (>7.5 cm). A total of 14 common sites with lymphoma involvement were analyzed, including lymph node, Waldeyer's ring, spleen, GI tract, breast, skin, bone, thyroid, ovary, nasal, lung, salivary glands, liver, and adrenal, as previously described . Immunohn = 51), whole-exome sequencing (WES) (n = 51), or whole-genome sequencing (WGS) (n = 14) was performed on 116 patients (including 52 SN and 64 SE patients) with frozen or FFPE tumor tissue samples. Among 65 patients with WES or WGS, the DNA sequencing data of 64 patients were from our previous report on extranodal DLBCL (n = 17) and WES (n = 25) were performed on 42 matched peripheral blood samples to exclude germ-line polymorphisms. The detailed procedures for DNA sequencing and variant calling were carried out as previously described . For formalin-fixed paraffin-embedded (FFPE) samples, genomic DNA was extracted using a GeneRead DNA FFPE Tissue Kit (Qiagen). Targeted sequencing  using frozen tumor tissue samples. RNA sequencing was performed on 53 patients (including 32 SN and 21 SE patients). Among them, the RNA sequencing data of 47 patients were from our previous report on extranodal DLBCL , and the2-test or Fisher's exact-test for qualitative data and independent-sample t-test or Mann–Whitney U-test for quantitative data. Progression-free survival (PFS) was calculated from the date of diagnosis to the date when the disease progression was recognized or the date of last follow-up . Overall survival (OS) was measured from the date of diagnosis to the date of death or the date of last follow-up. Survival analyses were estimated using the Kaplan–Meier method and compared by log-rank test. Univariate hazard estimates were generated with unadjusted Cox proportional hazards models. Clinical and pathological covariates demonstrating significance with P-value < 0.100 on univariate analysis were included in the multivariate model. Statistical significance was defined as P-value < 0.050. All statistical analyses were carried out using R software  and Statistical Package for the Social Sciences (SPSS) 22.0 software .The baseline and molecular characteristics of patients were analyzed using Pearson's χP < 0.001) and in the prospective cohort .As listed in PIM1 , TET2 , KMT2D , BTG2 , BTG1 , MYD88 , ARID1A , HIST1H1E , MPEG1 , TNFAIP3 , TP53 , CREBBP , FAS , GNA13 , and TMSB4X . No significant differences in total mutation frequency between SN and SE groups were observed  were observed in SN than SE patients  and MYD88  mutations than those without GI tract involvement. Patients with lymphoma involvement in breast had higher MYD88  mutations than those without breast involvement.Targeted sequencing, WES, and WGS were performed on 51, 51, and 14 patients, respectively, including 52 of 126 SN patients and 64 of 163 SE patients with available tumor tissue samples. A total of 55 genes related to the tumorigenesis of DLBCL according to literature were analyzed . At leasobserved . As for observed , signifipatients . Among cassessed . PatientTGFB2, BMP2, and BMP4 was significantly increased in SE than SN patients  were observed in SE, as compared to SN patients, while the other six steps showed no obvious differences between SN and SE groups (P = 0.010) than SN patients  was significantly decreased in SE as compared to SN patients  months. For a total of 289 stage I DLBCL patients, the 4-year PFS and OS rates were 90.3 and 94.1%, respectively . Among aATM mutations were prognostic for inferior PFS. In multivariate analysis, serum LDH >3 ULN was an independent adverse prognostic factor for OS (P < 0.001).In univariate analysis, serum LDH >3 upper limit of normal (ULN) was significantly prognostic for inferior PFS and OS . Other cr for OS . The 4-yAmong localized DLBCL patients, 56.4% were extranodal in origin, consistent with the previous report . The GI ATM mutations were related to inferior PFS. As an important cell cycle checkpoint kinase, ATM mutations also predicted inferior prognosis in GCB–DLBCL patients  can be found below: The studies involving human participants were reviewed and approved by Ruijin Hospital Ethics Committee. The patients/participants provided their written informed consent to participate in this study.WZ, PX, LW, and SC designed the study. WQ, HY, LD, QSo, XJ, and ZL acquired data. WQ, QSh, WZ, PX, DF, HH, and JH analyzed the data and made the figures. WZ, PX, WQ, and DF drafted the manuscript. All authors contributed to the article and approved the submitted version.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. 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