Many concepts have been described during the last years regarding how biosynthesis, generation products, antioxidant systems, and signal transduction involve ROS signaling, as well as its possible link with developmental processes and response to biotic and abiotic stresses. In this review, we first addressed classic key concepts in ROS and Ca2+ signaling in plants, both at the subcellular, cellular, and organ level. In the plant science field, during the last decades, new techniques have facilitated the in vivo monitoring of ROS signaling cascades. We will describe these powerful techniques in plants and compare them to those existing in animals. Development of new analytical techniques will facilitate the understanding of ROS signaling and their signal transduction pathways in plants and mammals. Many among those signaling pathways already have been studied in animals; therefore, a specific effort should be made to integrate this knowledge into plant biology. We here discuss examples of how changes in the ROS and Ca2+ signaling pathways can affect differentiation processes in plants, focusing specifically on reproductive processes where the ROS and Ca2+ signaling pathways influence the gametophyte functioning, sexual reproduction, and embryo formation in plants and animals. The study field regarding the role of ROS and Ca2+ in signal transduction is evolving continuously, which is why we reviewed the recent literature and propose here the potential targets affecting ROS in reproductive processes. We discuss the opportunities to integrate comparative developmental studies and experimental approaches into studies on the role of ROS/ Ca2+ in both plant and animal developmental biology studies, to further elucidate these crucial signaling pathways.This review explores the role of reactive oxygen species (ROS)/Ca We also provide a trans-kingdom comparative view, discussing recent advances in ROS studies in animal cells, and a comparative perspective on sexual reproduction and embryo development in both lineages. In plant and animal science, during the last decades, specific sensors have been developed that allow the in vivo detection of ROS and Ca2+ dynamics. We describe how these powerful techniques can provide novel perspectives in developmental studies. We discuss how the evolution of these techniques and also omics data can facilitate understanding ROS signaling and signal transduction. Comparative studies in plant and animal reproductive processes will give us a fruitful, comparative, and mechanistic view, taking into the account the similarities present between them. We discuss recent advances in both lineages, and propose novel key players affecting ROS and Ca2+ signal transduction, which are to be explored in the future.In response to environmental stimuli, plants are able to produce reactive oxygen species (ROS) to control many and different physiological processes, such as responses to biotic stresses, activation of programmed cell death (PCD), germination, regulation of stomatal opening, sexual reproduction, or flowering time Several ROS exist in biological systems, with a different reactivity and in different concentrations. They are reactive molecules and are therefore potentially toxic. In this section, we first consider the main ones that are produced in plant and animal cells, the antioxidant systems dedicated to their detoxification, and their role as signals.2−), hydroxyl radical (HO.), singlet oxygen (1O2), hydrogen peroxide (H2O2), and the related deprotonated anionic forms (HO2− and O22−) cyt) work as a key component in different signal transduction pathways. Depending on the stimulus, cytosolic Ca2+ rises can display the form of a single transient or repetitive Ca2+ oscillations and are commonly designated as “Ca2+ signatures”  variations to be made by recording the ratio shifts along a time-course [2+ concentration, the higher the cpVenus/CFP ratio. Traditional wide-field and confocal fluorescence microscopes able to specifically excite CFP and collect the emissions from the CFP and cpVenus can be efficiently used to measure the CFP/cpVenus FRET, as well as a fluorescent plate reader [Arabidopsis thaliana lines expressing the Cameleon YC3.6 targeted to cytosol as well as to different subcellular compartments are available and can be used in entire organs [Based on our direct experience, the Yellow Cameleon YC3.6  represen tissues ,126,127.solution . YC3.6 itide M13 . Bindinge-course . The hige reader . Arabidoe organs  . 2+ in plants essentially adopts techniques developed for analyses in animal cells; therefore, the use of Cameleon has been largely exploited to monitor the Ca2+ levels and dynamics in the cytosol, nucleus, ER, Golgi apparatus, or endosomes of single living cells  but also by the reductants themselves, such as GSH and thioredoxin. This makes the use of this probe not completely straightforward for the definition of the H2O2 levels [Arabidopsis plants, allowing to monitor in the cytosol and mitochondria the intracellular H2O2 dynamics in response to exposure to elicitors such as flg22 and chitosan [Some peroxidases have an intrinsic and powerful capacity to act as Hproteins . Hence, P2–-Orp1 . The wayces Orp1 . Because2 levels . Howeverchitosan . 2O2 ratiometric sensor is the genetically encoded fluorescent protein HyPer7 [Neisseria meningitis, which is sensitive to very low H2O2 concentrations. This allowed the detection of subtle changes in H2O2 concentrations in the cytosol and mitochondria of animal cells [A new promising Hn HyPer7 . HyPer7 al cells , but it ROS are end products of multiple and fundamental cellular processes, such as aerobic respiration and photosynthesis, for plant life. As previously described, ROS play key roles in the physiological reprogramming required for plant development . ROS, fo2 [In response to biotic and abiotic stresses, after mechanical wounding and during developmental processes, ROS are generated by reduction of O2 . At the 2 ,148,156.2 ,159,160.2 ,162,163.A. thaliana plants overexpressing thylakoidal ascorbate peroxidase, tAPX, which scavenges H2O2 (with ascorbate as electron donor), show increased resistance to paraquat-induced photooxidative stress and to nitric oxide-induced cell death; seeds of the transgenic A. thaliana line with the highest tAPX overexpression germinated later than the control seeds and also later than other tAPX transgenic lines with moderate overexpression [tAPX plants might also affect developing seeds, and hence, in turn, also their dormancy, which was prolonged in the tAPX overexpressing line. This shift in the “ROS oxidative window” would indeed explain the enhanced longevity observed in 26–30 months after-ripened tAPX seeds, when compared to their wild type line [2O2 levels in barley seed embryos after imbibition showed higher values in non-dormant seeds than in dormant seeds [2O2 during germination is multifaceted and it involves endosperm loosening and oxidation of the reserve proteins as well as of the mRNAs, together with the signaling crosstalk with ABA and GA, the two hormones acting in an antagonistic way in the regulation of the germination process, and nitric oxide (NO) [The ability of seeds to survive in harsh environments preserves their longevity. Seeds eventually germinate when the environmental conditions favor the survival and the thriving of the emerging seedlings. These are certainly among the most fascinating and challenging attributes of seed plants . The gerpression . Howeverype line . More rent seeds . These ant seeds . The actide (NO) ,204,205.ide (NO) ; indeed,ide (NO) . 2O2 works as a key developmental activator that allows the transition from stem cell maintenance into stem cell differentiation by antagonistically regulating the expression of the transcription factor WUSCHEL [2O2 is accumulated specifically in the differentiating peripheral region in order to promote stem cell differentiation and antagonistically regulate superoxide accumulation in stem cells by inhibiting key enzymes in superoxide metabolism. The differentiation program involves the repression of SOD and the concomitant activation of peroxidases, establishing the high superoxide/low H2O2 ratio in plant stem cells. During the last years, multiple studies have demonstrated the fundamental role of ROS in root differentiation [/H2O2 ratio varies along the roots: while the concentration of superoxide decreases gradually from the meristem to the transition zone, the concentration of H2O2 decreases gradually from the differentiation zone to the elongation zone in the root [ROS have multiple roles in plant stem cell regulation, as several works have shown dynamic changes in the expression pattern of ROS biosynthetic enzymes during stem cell differentiation . As eleg WUSCHEL . H2O2 isntiation ,168,208.the root ,210. Thithe root . Multiplthe root . Modulatthe root .The process of pollination, which is fundamental for sexual reproduction in almost all plants, has provided excellent examples on how ROS regulation controls developmental processes ,212. Dif2+ signal transduction in sexual reproduction in flowering plants and animals (see later). Although they constitute different biological systems, embryogenesis in plants and animals has common aspects, such as the male–female gamete interaction and subsequent events of cell proliferation, differentiation, morphogenesis, and embryo development in a maternal-containing environment . In this mutant, high levels of peroxide and mitochondrial superoxide accumulate in the central cell as well as the micropylar cells. Interestingly, a range of female gametophyte phenotypes accompanied these alterations; for instance, the mitotic arrest during megagametogenesis or the partial central cell identity determination [athemn1 mutant defective in tetrapyrrole biosynthesis, which accumulates more ROS in developing embryo sacs and has defects in female gametophyte development, with embryo sacs displaying unfused polar nuclei [athemn1 mutant. Upon fertilization, its seeds present defective endosperm development and embryo developmental arrest [Arabidopsis mutants and excellent reviews are available [In most angiosperms, the female gametophyte develops within the ovule and is formed by two antipodal cells, one central cell, two synergid cells, and one egg cell. The cells present in the embryo sac are highly polarized. For instance, while the egg cell nucleus is located toward the chalazal end of the embryo sac, the synergid and central cells have the opposite localization. During gametophyte formation, ROS levels are finely controlled, and they vary depending on the gametophyte cell type. For instance, it has been reported that superoxide and peroxide levels are present in central cells but not in antipodal cells, which later undergo cell death in mature embryo sacs . The cen via SOD . Most plmination . Anotherr nuclei . Interesl arrest . The rolvailable . Interesvailable ,216. FurCatalase1). In the central cell, some genes  were expressed at a high level and many at an intermediate level . An intermediate situation is taking place for the egg cell, where there are fewer genes expressed compared to the central cell, but usually at a moderate level. Some genes represent interesting candidates involving the cross talk among the cell types and the tissues derived from them (embryo/endosperm). For instance, Glutathione Peroxidase 2 seems to be expressed solely in the central cell, while isoform 6 is apparently expressed in all three cell types, although at different levels. For example, looking at the genes of the canonical VitC biosynthetic pathway, AT3G02870 (VTC4) is apparently expressed in both the egg cell and central cell, while AT5G55120 (VTC5) and AT3G47930  are expressed only—or almost exclusively—in the central cell. It might be worth looking at the developmental defects in preliminary developmental steps around fertilization or at early embryo development in the respective mutants. The hypothetical altered enzymatic activities of genes that act as scavengers (or that are involved in the production of scavengers) of ROS are likely to locally affect the ROS levels in specific cell types in which they are expressed, and might impact primarily the functionality of these cells. Future advances will provide knowledge on the role of those specific ROS-related regulators in plant reproduction.However, the role of many players affecting ROS in each cell has yet to be discovered. Single-cell transcriptome data are now available for specific plant structures, such as the female gametophyte . One can2O2 was detected in a specific temporal window specifically in synergid cells; after pollen arrival onto the stigma but before pollen tubes reach the ovule [fer mutants also display a pollen tube overgrowth phenotype and do not accumulate ROS in the micropyles of ovules, providing evidence that micropylar ROS levels is an important determinant controlling pollen tube reception [The role of ROS in mature female gametophyte function has also been investigated in depth during the last years and there is plenty of literature. Upon pollination, pollen grains land on the stigma of a flower. After crossing the stigmatic barrier, the pollen tube begins to grow through the style and the transmitting tract and it navigates through the funiculus, the structure that attaches the ovule to the placenta. At this stage the pollen tube exits out of the intercellular space and grows along the funiculus, reaching for the micropylar aperture of the ovule. The pollen tube enters the ovule through the micropylar region and bursts to release two sperm cells into the female gametophyte. The double fertilization in angiosperms thus requires two sexual fusions; one sperm fuses with the egg cell and leads to the diploid embryo formation, the other one fuses with the central cell, forming the triploid endosperm. The role of ROS in pollen tube growth and pollen-pistil interaction has been excellently reviewed in detail . One of he ovule . Supporthe ovule . Chemicahe ovule . Consisthe ovule . It was eception . 2+ signaling in synergid cells during pollen tube reception was also investigated [Arabidopsis synergid-specific gene NORTIA (NTA) have shown how Ca2+ oscillation in synergids is fundamental for proper pollen tube reception. nta mutants display a pollen tube overgrowth phenotype [2+ signal is required for proper pollen tube sensitivity to ovule signals in Arabidopsis thaliana; MLO5 and MLO9 can recruit a Ca2+-specific channel at the plasma membrane via SNARE proteins in order to fine-tune and modulate the Ca2+ gradients in the pollen tube [The role of Castigated ,187,223.henotype . A recenlen tube ,187. 2+ sensors during pollen tube arrival to the synergid cells in specific mutants will be essential in order to clarify the role of ROS and Ca2+-waves during reproduction in plants. The use of specific sensors targeted in different cell types appears as a promising strategy for better understanding the role of ROS in the process (as discussed previously).The last step of pollen tube reception involves pollen tube bursting in order to release the sperm cells. Pollen tube discharge triggers synergid degeneration in a coordinated way, involving a pollen tube–synergid interaction . The rolArabidopsis it was shown that the NADPH-oxidase homologues, RbohH and RbohJ, are the source for most ROS produced at the pollen tube apex [Arabidopsis KINβγ, a plant-specific subunit of the SNF1-related protein kinase 1 complex, the ROS levels of the pollen grains were reduced and concomitantly pollen adhesion to the stigmatic surface was impaired [+ channel SPIK in pollen [Brassica oleracea var. acephala), a self-incompatible species. The decreased ROS levels in kale stigma exposed to exogenous flavonoid treatment had a negative impact on the attachment and successive germination of the compatible pollen. In recent years, multiple works have investigated ROS signaling in pollen–stigma communication—we apologize for not discussing them because of space limitations [Several studies focused on the involvement of ROS in pollen functioning. Upon landing on the stigma, pollen grains undergo adhesion and hydration and trigger a germination program in order to generate the pollen tube . For insube apex . The relube apex . Those sube apex ,185. It impaired . Referrin pollen . Pollen–n pollen ,228. ROSn pollen . It has n pollen . More exitations ,229,231.In mammals, the female gamete, the oocyte, is the largest cell of the body and has the intrinsic ability to initiate embryonic development once it has undergone a process known as activation, while the paternal gamete, the spermatozoon, has developed the ability for motion and penetration of the oocyte’s investments. A schematic representation of fertilization and early embryonic development is shown in Oocyte and sperm development are complex processes that initiate very early during development of a new individual . Once tIn the female, oogenesis starts in the fetal ovary and terminates only after puberty is reached and fertilization eventually occurs. In the fetal ovary, oogonia differentiate into cells called primary oocytes, which duplicate their DNA and then enter the prophase of meiosis I, when they become arrested at the diplotene stage. Primary oocytes are covered by ovarian somatic cells , thus forming the primordial follicles, which constitute a pool from which the female will recruit follicles for growth and ovulation for the rest of the reproductive life. Follicular growth occurs when follicles are recruited from the primordial pool (follicle activation) and develop into primary, secondary, and tertiary follicles. Concomitant to follicle activation, the enclosed primary oocytes reactivate RNA synthesis and are engaged in a phase of growth during which a large amount of new molecules are produced and stored in the oocyte cytoplasm. Important morphological and functional changes, such as the synthesis of a protective glycoprotein shell—the zona pellucida—and the development of specialized cytoplasmic organelles, occur during the growth phase. At the end of the growth phase, in the tertiary follicle, transcriptional activity is again globally silenced, and the oocytes, now referred to as “fully grown” oocytes, are ready to resume meiosis. Meiotic resumption, which is cyclically triggered only after puberty by hormonal stimulation, is followed by progression to the metaphase, anaphase, and telophase of meiosis I. Completion of meiosis I is followed by an asymmetric cell division in which half of the homologous chromosomes are extruded in a small cell, called the polar body. Then, the large oocyte enters meiosis II, the chromosomes are arranged in the metaphase II plate, and meiotic progression arrests again until fertilization eventually occurs. Progression from the prophase I to the metaphase II stage is referred to as “oocyte maturation”. In most species, oocyte maturation occurs within the ovarian follicle and the metaphase II stage oocyte is released from the ovarian follicle into the oviduct, the anatomical site of fertilization, by a process called ovulation.In the male, spermatogenesis begins after puberty and includes three main steps: spermacytogenesis, meiosis, and spermiogenesis. Spermacytogenesis leads to the generation of primary spermatocytes by several mitotic divisions. Primary spermatocytes then undergo and complete meiosis, which produces haploid spermatids. Spermiogenesis follows the meiotic division and includes a series of morphological and functional changes that ultimately re-structure the round-shaped spermatids into differentiated spermatozoa. Final functional maturation of the spermatozoa, including acquisition of motility, capacitation, and acrosomal reaction, occur in the male and female genital tract.2+ waves that lead to the prevention of polyspermy, oocyte activation, and meiotic resumption. Specifically, the oocyte resumes and completes meiosis II: half of the sister chromatids are segregated into the small second polar body, while the remaining chromatids that are retained in the large ovum decondense and form the female pronucleus. The chromosomes contained in the sperm head decondense and form the male pronucleus.When the fertilizing sperm penetrates the oocyte, the maternal and paternal genomes are united in a single cell: the fertilized ovum, forming the zygote. It is well documented that fertilization triggers CaAfter DNA replication, the maternal and paternal genomes reconstitute the diploid status in a process called syngamy, chromosomes condense, and the first mitotic division of the new individual commences. Firstly, the zygote develops into the 2-cell embryo. Thereafter, the embryonic cells (the blastomeres) form a small cluster of cells referred to as the morula, which soon undergoes a process called “compaction”, forming a more uniform surface of the embryo. The outer cells develop into the trophectoderm. Subsequently, during the process of blastulation, a fluid-filled cavity, the blastocyst cavity, develops inside the trophectoderm, and the inner cells, forming the inner cell mass (ICM), gather at one pole of the embryo, which is now known as a blastocyst. The trophectoderm will participate in placenta formation while the ICM gives rise to the embryo proper. The blastocyst expands, hatches from the zona pellucida, and later, towards the end of blastulation, the ICM forms an internal and external cell layer—the hypoblast and epiblast, respectively—to establish the embryonic disc. Implantation and placental development in the uterus occur in a species-specific manner.2+ and ROS signaling has emerged in the last decades: Ca2+ signaling is essential for ROS production and, on the other hand, ROS can affect cellular Ca2+ signaling. Likewise, it is widely accepted that physiological  levels of ROS act as signaling molecules by oxidizing proteins, lipids, and nucleotides [2+ and ROS, as well as their interplay in physiological and pathological conditions, have been extensively reviewed 2+ ions can allosterically regulate the activity of many proteins and enzymes. Therefore, as already discussed for plants, Ca2+ ions are implicated in a high variety of functions in animal cells. Ca2+ signaling pathways are key in many crucial biological processes, such as cell survival, proliferation and death, contraction, secretion, metabolism, and regulation of gene expression. Animal cells have evolved complex mechanisms to control the fluctuations of cytosolic Ca2+ ions and, in turn, Ca2+-elicited cellular functions. The concentration of cytosolic Ca2+ can be altered by the controlled mobilization from two Ca2+ sources, the extracellular microenvironment, and the intracellular stores, namely, the endoplasmic reticulum (ER)—which is the main Ca2+ store—the Golgi apparatus, and the mitochondria. Animal cells exert this function through the coordinated activity of pumps and channels as well as Ca2+-binding signaling molecules, enzymes, and buffering proteins. One of the most common pathways for mammalian cells to trigger Ca2+ signaling is initiated by ligand-dependent G protein-coupled receptors, causing the synthesis of 1,4,5-inositol trisphosphate (IP3) and IP3-dependent opening of Ca2+ channels of the IP3 receptor (IP3R) family at the ER membrane , while a rise (up to 2–3 µM) in its concentration stimulates specific cellular function  are basically conserved between plants and animals, while the genes specific for muscle and nerve Ca2+ signal transduction systems  are very different [Caiew, see ,242,243)iewed in ,243). Onifferent . 2+, including those active in mitochondria and those that operate extra-mitochondrially, such as NADPH oxidases, uncoupled nitric oxide synthase, cytochrome P450s, cyclooxygenases, and many others [Many ROS-generating systems in a cell can be modulated by Cay others . 2+ homeostasis is particularly relevant [2+ [2+ concentration remains low in resting animal cells, but it rapidly increases upon cell stimulation, and that this is possibly due to the close proximity of mitochondria with Ca2+ channels in the ER that elicits the rise in cytosolic Ca2+ [2+ uniporter (MCU) and the voltage dependent anion channels (VDACs) are primarily responsible for the function that this organelle exerts in Ca2+ homeostasis . The miiewed in ).2+ uptake is in turn involved in controlling the rate of ATP production by stimulating enzymes of the Krebs cycle and oxidative phosphorylation. Nevertheless, Ca2+ control of ROS production is very complex, and the interactions depend on the cell and tissue types. Since mitochondrial ROS are produced at different sites of the electron transport chain (mostly complex I and III), it is logical to think that high metabolic rates are associated with high ROS production [2+, the metabolic state  of the mitochondria itself seems to determine the effects of Ca2+ uptake on the mitochondrial ROS level. However, in other circumstances, when mitochondria are overloaded with Ca2+, ROS production is no longer dependent upon the mitochondrial metabolic state. Furthermore, in the presence of other factors , excessive accumulation of mitochondrial Ca2+ has negative consequences: ATP production is impaired, and the production and sustained opening of the high mitochondrial permeability transition pore (mPTP) is induced [In mitochondria, Caoduction . Neverth induced ; this, i induced ,257 (rev induced ).2+ and ROS, which is quite complex in animal cells. This is also reflected in the relevance of Ca2+/ROS crosstalk in the physiopathology of the heart, skeletal muscle, neurons, and, eventually, in aging and cancer  and their elimination by antioxidative enzymes  and the iewed in ,258).2+ exerts a pivotal role in animal gametogenesis, fertilization, and embryonic development . Initia species ,261,262.iewed in ). 2+ waves, which persist for a few hours and cease when the nuclear envelope breaks down after meiotic resumption [2+ and ROS, together with a transient decrease in cyclic AMP content, destabilize MPF, thus promoting meiotic resumption of the prophase I-arrested oocyte [2+-dependent series of events that occur at fertilization (see below) is acquired during maturation [2+ store in the oocyte—the increase in intracellular Ca2+ stores, the changes in IP3R 1 expression pattern, as well as redistribution of the Ca2+-binding proteins [As anticipated, in mammals, the oocyte acquires the capability to be fertilized during oocyte growth and subsequent maturation, when the immature prophase I-arrested oocyte resumes meiosis, completes the first meiotic division by extruding the first polar body, and arrests again at the metaphase stage of the second meiotic division (MII), when fertilization will eventually occur , completion of meiosis II, formation of the pronuclei, and exocytosis of the cortical granules to prevent polyspermy. As in other mammalian cells, the Ca2+ concentration returns to its baseline by extrusion through the plasma membrane and/or re-loading in the ER, through the activity of the Ca2+ ATPases (PMCAs) and/or Na+/Ca2+ exchanger (NCX) at the plasma membrane, and the sarco-ER Ca2+ATPases (SERCAs) in the ER, respectively (see 2+ uniporter (MCU) and the secretory-pathway Ca2+-transport ATPases (SPCAs), which take up Ca2+ into these organelles. Nevertheless, a role as a Ca2+ store does not seem to be the main function of mitochondria in fertilized eggs, but rather mitochondrial Ca2+ uptake seems more related to energy production [At fertilization, when the sperm and the MII stage-arrested oocyte (egg) fuse, the sperm delivers the diffusible phospholipase C ζ (PLC ζ) into the egg cytosol. PLC ζ then hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2) into IP3, which binds its receptor, IP3R1, in the ER, which, in turn, triggers Ca cytosol ,273. Therm entry . Activatoduction ,275. Theoduction ,265). 2+ waves in fertilized oocytes has wide implications in mammalian fertility. Very recently, CRISPR/Cas9 technology was used to generate PLCζ KO mice. These studies provided the definitive evidence that PLCζ is the physiological trigger of the Ca2+ oscillations in mammals [2+ oscillations in fertilized eggs [Clearly, the control of Ca mammals ,277. In zed eggs ,279,280.For many years, ROS have been considered almost exclusively as detrimental compounds in the field of mammalian reproductive biology. Intense research has been conducted on this subject, and nowadays almost 10,000 publications are found on PubMed when the database is mined for the keywords “oxidative stress and mammalian reproduction”. Still today, even though physiological roles of ROS are widely recognized , their dNevertheless, the physiological milieu in which the female gametes are generated and then fertilized is, per se, rich in ROS. As a matter of fact, the ovary is an endocrine organ that generates ROS during steroidogenesis. Ovulation, the process by which the mature egg is released in the oviduct for fertilization, is recognized as an inflammatory-like reaction and many proinflammatory agents, such as interleukins and tumor necrosis factor, have physiological roles in the ovarian follicle . Thus, aIn males, spermatogenesis is also prone to the damaging effects of ROS. A sophisticated array of antioxidant systems that include both enzymes and free radical scavengers is set in place in testis to limit the detrimental effects of ROS ,286,287.2O2 and antioxidants, such as glutathione peroxidase 4 and 5  and peroxiredoxin 6 (PRDX6), contribute to the formation of sulfhydryl bridges between the cysteine residues of the protamines, resulting in chromatin condensation in the sperm head . For exiewed in ).2O2 in metazoan development has been revisited in the frame of the morphogenetic process during embryogenesis, regeneration, and stem cell differentiation [SOD KO mice suggest that, under oxidative stress conditions, ROS act by regulating the expression of genes of the cell cycle machinery [2+ waves [2+ oscillation at fertilization in mammals [Recently, the knowledge on the role of Hntiation . What emachinery . Neverthachinery ,290,291.2+ waves ,290,292,Very little is known about the Ca/ROS interplay during gametogenesis and early development, especially mechanistically. However, two very recent studies clearly indicate that the available technologies might be finally able to answer many unresolved questions, and likely accelerate the discoveries of new key processes in development.2+ release reduces Ca2+ store content and stimulates mitochondrial metabolism in prophase I immature oocytes [2+ waves, which persist for a few hours and cease when the nuclear envelope breaks down after meiotic resumption [2+ oscillation using the Fura-2 probe, Wakai and Fissore found that Ca2+ stored in the ER of immature oocytes constitutively “leaks” through the IP3R1 [2+ “leak” ceases around the time when the oocyte resumes meiosis and the nuclear envelope breaks down, which is when the accumulation of Ca2+ in the cellular stores, in preparation for fertilization, is firstly seen. Strikingly, during Ca2+ oscillation, Ca2+ is transferred to the mitochondria, where it stimulates metabolisms and increases the levels of ATP [2+/ROS interplay in female gametogenesis.The first study, conducted in a mouse model, demonstrated that constitutive IP3R1-mediated Ca oocytes . As alresumption ,264,275.he IP3R1 . The Ca2s of ATP . ClearlyXenopus laevis that ubiquitously expressed the H2O2 sensor HyPer, including the eggs of transgenic females [2+ waves induced by the sperm–egg fusion triggers a rapid increase in ROS levels, which in turn oscillate with each cell division. Furthermore, using inhibitors of complexes II, III, or IV of the electron transport chain, they also demonstrate that these enzymes are responsible for ROS generation. Importantly, their data also suggest that Ca2+-induced ROS signaling might control the onset of cell division, as inhibition of mitochondrial ROS production in early embryos results in cell cycle arrest, in part, via ROS-dependent regulation of Cdc25C activity. This is important in that cdc25 activates the cyclin B/cyclin-dependent kinase complex that plays a key role in cell cycle regulation to induce entry into mitosis [2+/ROS interplay has been suggested also in mammalian fertilization (see paragraph 4.3.2) and early embryos, this study sets the stage for a new area of research in mammalian early development.The second study took advantage of a transgenic  females . Using t mitosis . Since t2+ in sperm production and maturation as well as for female–male gamete interaction has been well documented both in animal and plant systems [2+, H2O2 and sensors for testing the redox status of GSH [2+ signaling. Further advances and comparative studies on the role of ROS/Ca2+ should provide new clues into the communication mechanisms between zygotic and maternal tissues in plants and animals, which are relatively poorly described in both lineages.In this review, we have revisited the literature concerning ROS signal transduction in sexual reproduction in plants and animals. Although these two systems are, apparently, biologically far different, common strategies concerning sexual reproductive patterns are shared among them. Probably this is due to the fact that sexual reproduction evolved in eukaryotes well before the divergence of plants and animals . As a ma systems ,309,310.s of GSH ,119,120.s of GSH ,314,315.2+ could support the identification of strategies to improve plant resilience to both biotic and abiotic stresses. Climate changes and global warming are posing new unprecedented challenges to crop production; efforts to understand plant resilience to environmental stresses are currently being made by different research groups [2 on plant defenses through redox signaling pathways have been recently uncovered [In agriculture, the elucidation of the complex signaling pathways triggered by plant exposure to various environmental stresses and involving ROS and Cah groups ,316. As ncovered . Advancencovered ,319, als


We collected 751 miR-93-5p targeted and AML-related genes by integrating the results of multiple databases and then used the expression profile of TCGA-LAML to construct a coexpression function network of AML WGCNA. Based on the clinical phenotype and module trait relationship, we identified two modules (brown and yellow) as interesting dysfunction modules, which have a significant association with cytogenetics risk and FAB classification systems. GO enrichment and KEGG analysis showed that these modules are mainly involved with cancer-associated pathways, including MAPK signal pathway, p53 signal pathway, JAK-STAT signal pathway, TGF-beta signaling pathway, mTOR signaling pathway, VEGF signaling pathway, both associated with the occurrence of AML. Besides, using the STRING database, we discovered the top 10 hub genes in each module, including MAPK1, ACTB, RAC1, GRB2, MDM2, ACTR2, IGF1R, CDKN1A, YWHAZ, and YWHAB in the brown module and VEGFA, FGF2, CCND1, FOXO3, IGFBP3, GSF1, IGF2, SLC2A4, PDGFBM, and PIK3R2 in the yellow module. The prognosis analysis result showed that six key pathogens have significantly affected the overall survival and prognosis in AML. Interestingly, VEGF with the most significant regulatory relationship in the yellow modules significantly positively correlated with the clinical phenotype of AML. We used qPCR and ELISA to verify miR-93-5p and VEGF expression in our clinical samples. The results exhibited that miR-93-5p and VEGF were both highly expressed in AML.Acute myeloid leukemia (AML) is a type of hematological malignancy with diverse genetic pathogenesis. Identification of the  Acute myeloid leukemia (AML) is one of the most common hematological malignancies in adults and is characterized by clonal expansion of abnormally differentiated blasts of a myeloid lineage . UsuallyFor nearly two decades, studies have found that the noncoding RNAs are closely related to tumor occurrence and development . It playMiR-93 belongs to the miR-106b-25 cluster, which can play an essential role in aging, hyperglycemia, and osteoblast calcification [miR-93-5p could participate in the occurrence, development, and drug resistance of numerous types of tumors [miR-93-5p can act as an oncogene by promoting angiogenesis. It can suppress integrin-β8 expression, which is ultimately associated with the tumor, and overexpression of miR-93 is associated with cell spreading, growth, migration, and tube formation of cancer cells [miR-93-5p has been found in other types of tumors such as neuroblastoma, non-small-cell lung cancer, and lacrimal adenoid cystic carcinoma [miR-93-5p is associated with the poor prognosis of gastric cancer and squamous cell carcinoma of the head and neck [miR-93-5p. It can promote tumor growth, blood vessel formation, and cell proliferation in AML. Also, the expression levels of miR-93 are closely associated with VEGF formation in AML. These studies proved that miR-93 is associated with several cancers, including AML [miR-93-5p participates in AML in pos-transcriptional regulation still needs further study. Therefore, the current study aimed to determine the expression of miR-93-5p and its targeted genes in AML based on integrative bioinformatics analysis and clinical validation.fication . Recentlf tumors –18. Meaner cells . The higarcinoma –22. Furtand neck , 24. StimiR-93-5p, we used the miRNet [https://www.mirnet.ca/miRNet/home.xhtml) to obtain miR-93-5p target genes from miRTarBase v8.0 [miR-93-5p. Moreover, we obtained a gene set that contains 985 genes that closely interact with the pathogenesis of AML from the National Center for Biotechnology Information (NCBI-Gene) database [n = 151) [Firstly, to collect the target genes of e miRNet   , we haveFurthermore, we constructed a coexpression gene network in AML-related genes profile based on weighted gene coexpression network analysis (WGCNA) . We analβ = 5 as the soft threshold to ensure the connections between genes in the network and obey the scale-free network distribution. The minimal number of genes in an individual module is set as 20.WGCNA is a newly developed method that aims to identify gene modules cooperatively expressed by characteristic genes, explore the association between gene networks and phenotypes of interest, and identify critical genes in a specific phenotype . This neFinally, the hierarchical clustering tree was constructed through the correlation coefficients between genes, and the similarity coexpression module was shown as the topological overlap matrix (TOM). Besides, to determine the correlation between clinical phenotype and modules, we used the person method to calculate the relationship between module eigengenes and the phenotypes of AML.To explore the genes' biological functions and signal pathways in dysfunctional modules, we used the GSEA  to perfoThe biomarker is helpful for the diagnosis, staging, or classification of diseases. Based on WGCNA, we have established a coexpression network of AML-related genes, and we use the Cytoscape (version 3.8.0) for screening the module hub genes . Then, thttps://portal.gdc.cancer.gov/) in January 2020. The difference between the two groups was compared using KM survival analysis and log-rank.To analyze the prognosis value of screened hub genes in AML patients, the clinical information was obtained from the profile of TCGA-LAML  with 95% confidence interval (CI) and the gression . The anaP value, HR, and 95% CI between each variable. The multivariate Cox proportional hazard analysis results were used to develop a nomogram for predicting the 3-year overall recurrence. The nomogram provided a graphical contribution of the factors, which can be used to estimate the risk of recurrence for an individual patient by the points associated with each risk factor through the “rms” R package.The univariate and multivariate Cox regression analysis was used to identify the significant variables to establish the nomogram . The “fomiR-93-5p and VEGF. Bone marrow samples from 18 patients who were newly diagnosed with AML were collected at our institution between January 2019 and September 2019. Diagnose AML according to the classification criteria of the World Health Organization MICM  [To verify the expression of two molecules from the most significant modules that are still not clearly defined, we have validated the expression of biology) . The incmiRNA-93 were quantified using SYBR Green Master Mix , and the U6 gene was used as an internal control. The following primers were used: miR-93-5p, 5′-CAAAGTGCTGTT CGTGCAGGTAG -3′ and U6 5′-GGATGACACGCAAATTCGTGAAGC-3′. qRT-PCR was performed on ViiATM 7 System software . The results were normalized to the expression of the U6 gene and are presented as the fold change (2CT−ΔΔ). VEGF protein expression by bone marrow mononuclear cells was quantified for media samples collected from different experiments using VEGF ELISA kit per manufacturer protocol .cDNA was generated using the Reverse Transcription Kit . The expression levels of miR-93-5p regulatory target genes in the pathogenesis of AML, we collected a large number of results from multiple online databases. First, we obtained 2624 target genes of miR-93-5p from the miRNet, and these genes were predicted by three commonly used databases . Second, to further screen these target genes related to AML, we sought to obtain 3900 AML-related genes from NCBI-Gene and OMIM databases. Then, we screened 751 interact genes between miR-93-5p target genes and AML-related genes to determine the essential regulatory functions of miR-93-5p and its target genes in AML. The Venn diagram is shown in To systematically study the critical role of miR-93-5p targeting AML-related genes, we downloaded the RNA-seq profile of 151 AML samples from the TCGA database. The clinical information is represented in β = 5) to ensure the stability of the scale-free network, and we established eight coexpressed gene modules. The clustering of module gene expression behaviors in AML is shown in In the current research, to match and construct an expression matrix of R2 = 0.61 and R2 = 0.51) (R2 = 0.28), and brown modules were negatively correlated with cytogenetic risks (R2 = -0.32) (We found that two modules are significantly correlated with the FAB classified subtypes and cytogenetic risks . Accordi = 0.51) . Further= -0.32) . What isAs the two modules (yellow and brown) have been observed with the highest correlated modules with AML subtype and cytogenetic risk, we performed the GO enrichment on characteristic genes in these two modules, including biological process (BP), cellular component (CC), and molecular function (MF). The mainly enriched terms in the brown module were regulation of protein metabolic and modification process, regulation of intracellular signal transduction, regulation of phosphorus metabolic process, and positive regulation of molecular function. The significant GO terms of the brown module are visualized in MAPK1, ACTB, RAC1, GRB2, MDM2, ACTR2, IGF1R, CDKN1A, YWHAZ, and YWHAB in the brown module  network in brown (83 nodes and 243 edges) and yellow modules (41 nodes and 103 edges) based on the STRING database. Then, we screened the top 10 hub genes in these two modules separately, including n module , and VEGw module . These glog-rank test (P < 0.05). Highly expressed ACTR2 and YWHAB are associated with poor prognosis. Low expression of IGF2, IGFBP3, SLC2A4, and IGF1R is related to poor prognosis contrarily  is a highly aggressive malignant tumor caused by abnormal cloning of hematopoietic stem cells . CytogenmiR-106b-25 cluster, located in intron 13 of the MCM7 gene [miR-93-5p involves many kinds of human tumors [miR-93-5p is associated with the development of multidrug resistance in various types of cancer [miR-93-5p was abnormally expressed in AML, and the abnormal expression of miR-93-5p may affect the prognosis of AML [miR-93-5p in AML is still unclear. Therefore, to fully explore the specific mechanism of miR-93-5p regulating target genes to mediate in the pathogenesis of AML, we adopted comprehensive bioinformatics research. In this study, we obtained 2,624 miR-93-5p target genes through the prediction of target genes from multiple online databases. At the same time, to further determine these miR-93-5p target genes are closely associated with the pathogenesis of AML, we collected a gene list related to AML through NCBI-GENE and OMIM. Then, we obtained 751 interacted genes between miR-93-5p target genes and AML associated genes. Besides, based on the RNA-seq profile of TCGA-LAML, we constructed an expression matrix for WGCNA analysis. Our new ideas about collecting disease-related genes and gene cluster modularization may provide a feasible research method for other diseases like AML that lack strict controls. As the result of WGCNA, we conducted eight dysfunction modules to characterize the pathogenesis of AML. Interestingly, we found that two modules were significantly correlated with the phenotype of AML. Furthermore, GO analysis shows that these two modules are involved in protein metabolism and modification, intracellular signal transduction, and cell differentiation and proliferation regulation. KEGG pathway analysis also indicates that these two modules were mainly involved in the cancer pathways, such as MAPK signaling pathway, insulin signaling pathway, p53 signaling pathway, TGF-beta signaling pathway, mTOR signaling pathway, VEGF signaling pathway, and other different pathways that are closely connected with the pathogenesis of AML [MicroRNA-93 is a member of the CM7 gene . It is an tumors , 48. Thef cancer , 49. Recs of AML . Howevers of AML .VEGF has been a core regulatory position in the yellow module, and MAPK1 also acts as a core regulatory role in the brown module. Vascular endothelial growth factor (VEGF) is an essential factor in promoting angiogenesis in vivo, promoting the proliferation, differentiation, and infiltration of tumor cells [VEGF could lead to an increase of leukemic cells, and patients with higher expression levels of VEGF are more likely to have poor prognoses [VEGF are more likely to relapse [VEGF/VEGFR complex's critical regulatory part in angiogenesis may be the key to studying the pathogenesis of AML [VEGF may affect the pathogenesis and treatment of AML in three forms: higher levels of VEGF can be detected in the BMSCs of leukemia patients, which resists the apoptosis of leukemia cells by chemotherapeutics through autocrine pathways and promotes leukemia Cell resistance; VEGF can stimulate endothelial cells to secrete growth factors and reversely bind to receptors on leukemia cell membranes, thereby promoting vascular endothelial proliferation and secreting cytokines that encourage cell leukemia proliferation and indirectly promote the pathogenesis of leukemia through the paracrine pathway; VEGF can also promote the expansion of leukemia stem/progenitor cells [VEGF signaling pathway's downstream regulatory mechanism will guide the development of new treatments for AML.As we have found in our research, the hub genes with core regulatory roles in brown and yellow modules were further identified. Using connectivity to calculate the relationship between module genes, we screened ten hub genes in each module. or cells . It has rognoses . Studies relapse , 55. As s of AML . Furtheror cells . TherefoVEGF and miR-93-5p in our clinical samples. The result shows that these two regulators are both highly expressed in AML compared to those in the healthy group. miRNA-93 is a member of the miR-106b-25 cluster, which plays an essential regulatory function in promoting oncogenes' expression and inhibiting the expression of apoptotic proteins [miRNA-93 can promote osteosarcoma proliferation and angiogenesis [miRNA-93 is closely related to the increase of VEGF. The proliferation of VEGF expression level is also clinically associated with AML patients' prognosis [miR-93-5p and VEGF. However, after predicting target genes through the TargetScan Human database (http://www.targetscan.org), we discovered that miRNA-93 might be targeting hypoxia-inducible factor 1α (HIF-1α) affecting the expression of VEGF [miRNA-93 consensus sequence also exists in the 3′UTR region of VEGF mRNA, which inspires our next research: miRNA-93 may mediate the occurrence development of AML by regulating the expression of HIF-1α/VEGF [miR-93 regulating the expression of HIF-1α/VEGF in AML and whether its expression characteristics are related to the treatment and prognosis of AML are scientific issues worthy of further study.In addition, we verified the expression of proteins . Studiesogenesis . Besidesrognosis . These rrognosis . Unfortu of VEGF . At the -1α/VEGF . HoweverMiR-93-5p may be associated with the translational signaling of AML and a broader spectrum of hematological malignancies. The mechanistic study revealed that miR-93 was found to inhibit the phosphorylation of AKT (pAKT) [miR-93 promoted the proliferation, invasion, progression, and metastasis of cancer cells through activation of PI3K/AKT signaling [miR-93 has associated with drug resistance in cancer cells through the miR-93/PTEN/AKT signaling pathway [VEGF from myeloid cells and the elevated levels were detected in the AML microenvironment [miR-93 may be associated with regulating the miR-93/AKT/mTOR/VEGF pathway in AML, and clinical investigation will be warranted. However, our findings still need more clinical samples and broader prospective studies to validate further.T (pAKT) . In contignaling , 63, 64. pathway . Besides pathway . Similar pathway , 68. Int pathway , 70. Theironment , which imiR-93-5p targeted and AML-related genes and verifying the expression of clinical samples, we initially explored the molecular mechanism of miR-93-5p targeting and regulating VEGF-mediated pathogenesis of AML. Further, we laid a theoretical foundation for the research on targeting miR-93-5p to treat AML.In summary, our work provides comprehensive and novel insights into the pathogenesis of AML. By constructing a coexpression module of


FLT3 internal tandem duplication (FLT3‐ITD) mutation and co‐mutations with NPM1 and DNMT3A are overrepresented in female AML. Here, we have investigated the relationship between sex and FLT3‐ITD mutation status by comparing clinical data, mutational profiles, gene expression and ex vivo drug sensitivity in four cohorts: Beat AML, LAML‐TCGA and two independent HOVON/SAKK cohorts, comprising 1755 AML patients in total. We found prevalent sex‐associated molecular differences. Co‐occurrence of FLT3‐ITD, NPM1 and DNMT3A mutations was overrepresented in females, while males with FLT3‐ITDs were characterized by additional mutations in RNA splicing and epigenetic modifier genes. We observed diverging expression of multiple leukaemia‐associated genes as well as discrepant ex vivo drug responses, suggestive of discrete functional properties. Importantly, significant prognostication was observed only in female FLT3‐ITD‐mutated AML. Thus, we suggest optimization of FLT3‐ITD mutation status as a clinical tool in a sex‐adjusted manner and hypothesize that prognostication, prediction and development of therapeutic strategies in AML could be improved by including sex‐specific considerations.Incidence, molecular presentation and outcome of acute myeloid leukaemia (AML) are influenced by sex, but little attention has been directed at untangling sex‐related molecular and phenotypic differences between female and male patients. While increased incidence and poor risk are generally associated with a male phenotype, the poor prognostic  FLT3) gene are linked to poor prognosis in acute myeloid leukaemia (AML). Here, we investigated sex‐related differences in mutation, gene expression and drug sensitivity profiles in FLT3‐ITD‐mutated AML. We demonstrated significant sex‐associated molecular and functional differences, and surprisingly identified significant prognostication related to FLT3‐ITD only in female AML patients.Internal tandem duplications (ITDs) in the FMS‐like tyrosine kinase 3 ( AMLacute myeloid leukaemiaARallelic ratioAUCarea under the curveBMbone marrowCEcapillary electrophoresisDGEdifferential gene expressionFLT3Fms‐like tyrosine kinase 3ITDinternal tandem duplicationMDSmyelodysplastic syndromeNGSnext‐generation sequencingVAFvariant allele frequency1Sex influences regulatory mechanisms of the haematopoietic system as well as innate and adaptive immune responses , 2. StroFLT3‐ITD, NPM1 and DNMT3A mutations are overrepresented in females [RUNX1, ASXL1, SRSF2, STAG2, BCOR, U2AF1 and EZH2 are more prevalent in males [DNMT3A, NPM1 and FLT3‐ITD mutations has also been reported [Androgens have been used to treat various bone marrow (BM) failure syndromes since the 1960s . Yet, li females , 12, whireported .FLT3). FLT3‐ITD is present in approximately 25% of the AML cases and is a negative prognostic marker that is integrated in standard risk stratification guidelines in AML as well as guiding FLT3‐targeted therapy [FLT3‐ITD mutations has not been explored in depth. Here, we present results from genomic, functional and time‐to‐event‐analyses of four well‐annotated cohorts, including the Beat AML cohort [FLT3 mutation status are described in Table Despite the demographic differences in survival and somatic mutation profiles, sex‐specific considerations are currently not made in therapeutic assessment or clinical risk stratification. Among the mutations with reported sex‐dependent discrepancies are internal tandem duplications (ITDs) of the FMS‐like tyrosine kinase 3  analyses and exploration of Beat AML  and LAMLBeat AML  are publhttp://www.vizome.org/aml/geneset/ on the 01.11.2018. This included 608 samples from 519 individuals. At inclusion, 15 individuals had two samples acquired from different tissues. The sample with the lowest number of recurrent mutations was discarded. We filtered the remaining samples based on the variable ‘dxAtSpecimenAcquisition’ and retained samples annotated as ‘Acute myeloid leukaemia (AML) and related precursor neoplasms’, resulting in a total of 571 samples. For descriptive analysis of somatic variants, we included only the first sample when serial samples were present, resulting in 498 samples from 498 individuals. For DGE and drug response, we analysed 390 samples from 360 individuals and 359 samples from 322 individuals, respectively, including only samples with sample IDs overlapping with the exome sequencing data set  newly diagnosed AML patients aged 15–80 years treated on various study protocols of the Dutch‐Belgian Hemato‐Oncology Cooperative Group (HOVON) and the Leukaemia Group of the Swiss Group for Clinical Cancer Research (SAKK) during the period 1987 to 2013. The sample selection analysed here was restricted to treatment naïve non‐APL AML patients with known 4, HO04a , HO29 [14, HO04a , 19, HO44, HO04a , HO43 [24, HO04a  and HO104, HO04a . The stu4, HO04a . Patientde novo AML patients, including 92 females and 108 males ranging from 18 to 88 years. Due to its selective and unrepresentative composition, we have mainly used this cohort for integrated survival analyses. Single cohort comparative analyses of sex differences related to clinical parameters were not performed. The TCGA‐AML data analysis is based exclusively on variables as presented in the file ‘SuppTable01.xlsx’, downloaded from https://gdc.cancer.gov/node/876. For survival analysis, the variable ‘OS months 3.31.12’ is used.LAML‐TCGA is a strongly selected sample cohort, composed to cover the major cytomorphologic and cytogenetic groups recognized in AML prior to 2013 . The coh2.2All clinical trials were approved by local ethics committees and performed in accordance with the Declaration of Helsinki.2.3bioconductor r package (limma version 3.38.3) [P‐values were adjusted by the Benjamini–Hochberg method, and threshold was set at P ≤ 0.05. All statistical analyses were performed in r‐studio (version 1.1.453) and r (version 3.5.0) . Graphs were made with ggplot2  [For comparison of continuous variables, we applied the nonparametric Wilcoxon rank sum test/Mann–Whitney test. The Fisher exact test was applied for comparison of categorical data. For DGE analysis, we log‐transformed the CPM matrix provided in ‘Table  3.38.3) . For exprse.org)  and figu33.1FLT3‐ITD‐mutated subgroup. We identified 28 highly recurrently mutant genes, of which 23 genes are autosomal and five are X‐linked, including BCOR, BCORL1, PHF6, STAG2 and ZRSR2. The frequency of FLT3  and DNMT3A  mutations was higher in females, although not statistically significant. However, seven other genes were significantly different; NPM1  was overrepresented in females, while RUNX1 , ZRSR2 , SRSF2 , U2AF1 , ASXL1  and EZH2  were overrepresented in males  exclusively in females, while BRCC3  and SMC3  were both significantly overrepresented in males  and CCND3  only in male FLT3‐ITD AML  were significantly more frequent in females, RUNX1 , SRSF2 , U2AF1 , ZRSR2  and EZH2  were significantly characteristic of males  and U2AF1  remained significant. Additionally, MXRA5  and NF1  were identified as more frequently mutated in females, while PHF6  was significantly overrepresented in males . Similarly, 10% female vs 23% male individuals had at least one mutation in one of the four epigenetic modifier genes: ASXL1, BCOR, EZH2 and BCORL1 . The relationships remained significant in the nontransformed group for both RNA splicing genes  and epigenetic modifier genes . The association was also significant within the FLT3‐ITD‐negative group, where both epigenetic modifier genes  and RNA splicing genes  remained significantly associated with males. In the FLT3‐ITD‐positive group, the same trend was apparent , although not significant by adjusted P‐value . The combination of FLT3‐ITD and NPM1 mutation without DNMT3A mutation  and the combination of FLT3‐ITD, NPM1 and DNMT3A mutation  were both significantly associated with females  of the genes mutated at least 10 times in the Beat AML cohort and found that VAF was significantly higher in male compared to female individuals in five genes. As expected, this included the X‐linked genes les Fig. .FLT3 was not among the genes identified with significantly different VAF across the sexes. It has previously been shown that the allelic ratio (AR) of FLT3‐ITD mutations has prognostic implications [FLT3‐ITD AR among these patients, we calculated the AR from the VAF  [FLT3 mutational burden in AML patients [ications , 29. To sis (CE) , 31. Herpatients . As was 3.2FLT3‐ITD and co‐mutations, we questioned whether there was sex disparity in gene expression in the FLT3‐ITD‐mutated group. We analysed 380 specimens in the Beat AML cohort, comprising 163 female and 217 male samples, respectively, of which 51 female and 47 male were FLT3‐ITD‐positive samples. We identified a total of 39 differentially expressed genes: 16 upregulated and 23 downregulated in the female FLT3‐ITD‐positive subgroup, including 14 Y‐linked, 2 X‐linked, 7 nonannotated and 16 autosomal genes  and FLT3‐ITD‐negative specimens (n = 282) irrespective of sex, we found that GLI2, CCL1, JPH1, MDGA1, RASGRF1, AE000661.37, HOXB‐AS3, IRX5 and RP11‐713C5.1 were all significantly upregulated in FLT3‐ITD‐positive AML, while GPR126 was significantly downregulated.Based on the sex‐related pattern of nes Fig. . Subtracles Fig.  and S9. NETO1, TMSB4Y, TTTY10, HMGA2 and FAT1. In multivariate analysis including these five transcripts, only NETO1 remained significant  and higher sensitivity in females. When comparing les Fig. . CompariFLT3‐ITD status: Females were more sensitive to AT7519, palbociclib and quizartinib (AC220), while males were more sensitive to cediranib (AZD2171) and pazopanib (GW786034). Further, we identified three compounds that were significantly more potent in female FLT3‐ITD‐negative samples, including AT7519, palbociclib and trametinib (GSK1120212). Importantly, we identified 14 inhibitors that demonstrated significantly lower potency in female FLT3‐ITD‐positive samples, with an overrepresentation of inhibitors targeting tyrosine kinase receptors and downstream targets known to be influenced by FLT3‐ITD signalling, including idelalisib, BEZ235, MGCD‐265, masitinib (AB 1010), NVP‐ADW742, tivozanib (AV‐951), S31‐201, afatinib (BIBW‐2992), PRT062607, PI‐103, cediranib (AZD2171), lapatinib, STO609 and axitinib (AG‐013736)  Fig. .FLT3‐ITD‐positive samples. We found that sensitivity to NVP‐ADW742 correlated with increasing gene expression of 7/13 RNA transcripts identified as upregulated in female FLT3‐ITD‐positive AML. Conversely, increasing GLI2 expression correlated with reduced sensitivity to 4/14 drugs that were less potent in female FLT3‐ITD‐positive AML  had a median follow‐up of 113.7 months, while HOVON 2 (n = 625) had a median follow‐up of 42.3 months. The Beat AML cohort had a short median follow‐up of 15.2 months. For survival analyses, only newly diagnosed cases were included (n = 303). LAML‐TCGA (n = 200) had a median follow‐up 47.2 months.Finally, we investigated the relationships between sex and the prognostic strength of ly Table a–e. HOVOFLT3‐ITD status being a recognized negative prognostic marker in AML, we found a significant association to poor outcome only in the HOVON 1 cohort , we found as expected that FLT3‐ITD mutation status was associated with significantly lower survival   nor older (≥ 60 years) patients  Fig. . Howevernts Fig. .4ex vivo drug response patterns in FLT3‐ITD‐mutated AML cases across four well‐characterized AML cohorts. FLT3‐ITD mutation status is integrated in standard risk stratification guidelines in AML. Yet, in the datasets we explored, FLT3‐ITD mutation status separated the disease outcome only in female AML. This may indicate that the prognostic utility of FLT3‐ITD mutation status is different across sexes. Of note, the prognostic value of FLT3‐ITD mutations is reportedly influenced by the mutational burden. Several studies have shown that a high FLT3‐ITD AR is linked to poor prognosis [FLT3‐ITD AR lacking NPM1 mutations have a worse prognosis [FLT3‐ITD VAF nor AR as significantly different across sexes, suggesting that a difference in the mutational burden of FLT3 is likely not the cause of the sex discrepancies observed in outcome.In this work, we demonstrated sex disparity in somatic variant composition, gene expression and rognosis , 29. Furrognosis . In the FLT3‐ITD‐positive subgroup. While males more frequently present with somatic variants in epigenetic modifier genes and/or RNA splicing genes, co‐mutations of FLT3‐ITD, NPM1 and DNMT3A were overrepresented in females. The mutation pattern characterizing female patients in this subgroup is previously shown to be associated with adverse prognosis in FLT3‐ITD‐mutated AML [FLT3‐ITD‐negative subgroup, characterized by an abundance of WT1 mutations in female specimens contrasted by overrepresentation of mutations in RUNX1, SRSF2, U2AF1, ZRSR2 and EZH2 in the male subgroup. These differences are most pronounced in the Beat AML cohort, where the mutated genes dominating the male FLT3‐ITD‐negative subpopulation are associated with poor outcome and with myelodysplastic syndrome (MDS) and secondary AML [SRSF2 and U2AF1, are reportedly overrepresented in male MDS [Another plausible explanation is related to discrepant distribution of age, disease presentation pattern and poor risk molecular features. We identified significant differences in the distribution of co‐mutations within the ated AML . Importadary AML . Whetherdary AML . Similardary AML . Additiomale MDS .FLT3‐wt‐ and FLT3‐ITD‐mutated subgroups. Specifically, age differences could potentially result in sex disparities of treatment , which might be an important confounder for this study. In the HOVON cohorts, however, we found no significant differences between males and females in the proportion of transplanted patients  AML patients, but not within the older population (60–74 years) [FLT3‐ITD mutations.Sex disparity in AML demography is well known, with a progressive male excess with increasing age . As the ts Table . Due to 4 years) . We corrThe sex‐specific age distribution characterizing AML demography suggests that cohort composition is an important confounder. The age composition of the Beat AML cohort resembles the reported demographic distribution of AML far better than the strongly selected LAML‐TCGA cohort, in part explaining the lack of sex‐biased mutations in this cohort. One could argue that age‐matching and/or use of a population‐based sample selection is the optimal condition for comparison. What remains, however, is to identify the cell‐intrinsic and cell‐extrinsic mechanisms underlying the sex disparity in AML incidence and molecular presentation. We hypothesize a significant contribution of sex‐specific leukaemia‐host interactions related to disease development. Mutations are stochastic events, and there are thus no apparent reasons to suggest that male and female haematopoietic stem cells acquire discrete mutations. What is plausible, however, is that sex is an important contextual contributor in determining the translational effect in comparative fitness of novel gene variants. Interestingly, one of the very first papers characterizing the FLT3 receptor suggested FLT3‐mediated regulatory function not only in the haematopoietic compartment but also in the gonads, placenta and brain ; tissuesFLT3‐ITD‐positive AML, the hedgehog signalling mediator GLI2 has been associated with FLT3‐ITD‐mutated leukaemia [HOXB‐AS3 is reportedly upregulated in NPM1‐mutated AML, and has been implied to contribute in regulation of AML cell‐cycle progression [FLT3‐ITD‐positive AML, FAT1 was previously shown to be somatically mutated in FLT3‐ITD‐positive AML and significantly in combination with NPM1 and DNMT3A [FAT1 exerted tumour suppressor activity specifically in FLT3‐ITD‐positive AML, perhaps providing a partial explanation for the lack of prognostic impact of FLT3‐ITD in this subpopulation.The DGE analysis showed distinct expression of leukaemogenesis‐associated genes, suggesting functionally relevant cell‐intrinsic sex‐dependent differences. Of the genes more highly expressed in female eukaemia , while Hgression . NETO1, gression , and a rd DNMT3A . This stex vivo drug response patterns and clinical outcome analysis suggests that response to therapeutic intervention in FLT3‐ITD‐mutated AML may be influenced by sex. Interestingly, this is in line with observations from the phase III clinical trials RATIFY and QuANTUM‐R, treating FLT3‐ITD‐positive de novo AML with FLT3‐targeting inhibitors , both reporting significant survival benefit in the male subpopulation only [ex vivo drug sensitivity analyses did not, however, show a similar sex difference in the sensitivity to FLT3 inhibitors. It has previously been shown that FLT3‐ITD dependency in ex vivo assays is contingent on the availability of growth factors, and thus, the response to FLT3‐targeting drugs is permissive of the culture conditions [ex vivo drug screens, and conclusions regarding the translational value of such data should be drawn with caution. Notably, sex‐related ex vivo drug response patterns have also been reported in the study of the Cancer Cell Line Encyclopedia [The sum of the ion only , 46. Unfnditions . This coclopedia  and in cclopedia . Thus, tThe impact of sex on disease presentation and outcome is likely a composite picture involving multiple factors, including societal influence and socialization . Most importantly, however, sex is associated with major physiological differences, the impact of which has been insufficiently explored in haematology and in medicine in general. The field of cardiovascular disease is a noteworthy exception, where sex disparity is now widely recognized and where sex considerations are becoming integrated into research, diagnostics and clinical disease management , 51. Sim5FLT3‐ITD mutation status is well established as a biomarker in AML. However, important questions remain regarding its optimal application and utility. Our observations suggest that FLT3‐ITD mutation status could be optimized as a clinical tool in a sex‐adjusted manner. Furthermore, we suggest that sex‐specific considerations should be considered in preclinical and clinical experimental design and biomarker analyses in AML. Sex should represent an independent stratification factor when randomizing to clinical trials, and be systematically included when analysing and reporting on clinical data in AML. We hypothesize that addressing sex‐related regulation of molecularly defined subgroups of AML could advance pathophysiological understanding, perhaps ultimately revealing new therapeutic possibilities.Assessment of mutation status is becoming an integrated part of the diagnostic characterization of adult AML patients. This information is subsequently incorporated into risk stratification models and clinical decision‐making. The authors declare no conflict of interest.The study was designed by CE and BTG. CE analysed and interpreted data, prepared figures and wrote the paper. MH interpreted data, prepared figures and wrote the paper. TG, BL and PJMV collected data from the HOVON cohorts and contributed with data interpretation. All authors contributed in manuscript preparation.https://publons.com/publon/10.1002/1878‐0261.13035.The peer review history for this article is available at Fig. S1. Overview of the Beat AML sample selection analysed.Fig. S2. Distribution of somatic variants in the Beat AML sample selection.Fig. S3. Sex‐specific distribution of somatic mutations in the Beat AML cohort.Fig. S4. Age‐ and sex distribution of samples with somatic mutations, presented by gene class.Fig. S5. Sex‐specific distribution of comutations of FLT3‐ITD, NPM1 and DNMT3A in the HOVON1, HOVON2, Beat AML and LAML‐TCGA cohorts.Fig. S6. Sex‐specific distribution of VAF of mutations detected in a minimum of 10 samples in the Beat AML sample selection.Fig. S7. Sex‐specific distribution of FLT3‐ITD allelic ratio.Fig. S8. Expression level of genes identified as differentially expressed between male and female FLT3‐ITD‐positive samples.Fig. S9. Pairwise comparison of gene expression in FLT3‐ITD and non‐FLT3‐ITD samples in genes identified as differentially expressed between male and female FLT3‐ITD‐positive samples.Fig. S10. Cox Proportional‐Hazards model including the five genes where expression was identified as significantly correlated with outcome by univariate analysis.Fig. S11. Kaplan–Meier curves comparing patients with high and low expression of NETO1, split by FLT3‐ITD mutation status and sex.Fig. S12. Overview of drugs and drug classes.Fig. S13. Comparison of drug sensitivity scores between FLT3‐ITD‐mutated male and female samples, presented by drug class.Fig. S14. Comparison of drug sensitivity scores of drugs identified with significantly different potency in male and female FLT3‐ITD‐mutated samples.Fig. S15. Kaplan–Meier curves comparing the outcome of male and female FLT3‐ITD and FLT3‐wt patients in the HOVON1, HOVON2, Beat AML and LAML‐TCGA cohorts.Fig. S16. Kaplan‐Meier curve comparing the outcome of female and male patients across the four cohorts separated by FLT3‐ITD mutation status.Table S1. (A) Cohort Composition – Beat AML sample cohort . (B) Cohort Composition – Beat AML sample cohort (no FLT3‐ITD). (C) Cohort Composition – Beat AML sample cohort (FLT3‐ITD). (D) Cohort Composition – Beat AML sample cohort  split by FLT3‐ITD.Table S2. Cohort Composition HOVON1.Table S3. Cohort Composition HOVON2.Table S4. (A) Somatic mutations in the Beat AML sample cohort . (B) Somatic mutations in the Beat AML sample cohort (FLT3‐ITD). (C) Somatic mutations in the Beat AML sample cohort (no FLT3‐ITD). (D) Somatic mutations in the Beat AML sample cohort (not transformed).Table S5. Somatic mutations in the LAML‐TCGA cohort .Table S6. Mutations in Beat AML cohort categorized by gene product function.Table S7.FLT3‐ITD, DNMT3A and NPM1 comutations in the Beat AML, LAML‐TCGA HOVON1 and HOVON2 cohorts.Table S8. Beat AML cohort – Differential gene expression analysis.Table S9. Univariate Cox regression analysis of differentially expressed genes in the Beat AML cohort.Table S10. Number of FLT3‐ITD and FLT3‐ITD wt male and female samples screened for each drug in the in drug screen.Table S11. P‐values for comparison of drug sensitivity.Table S12. (a) Survival analysis of all cohorts combined. (b) Survival analysis of the Beat AML cohort. (c) Survival analysis of the LAML‐TCGA cohort. (d) Survival analysis of the HOVON1 cohort. (e) Survival analysis of the HOVON2 cohort.Click here for additional data file.


In this process, rearrangement of the actin cytoskeleton, such as calcium-activated actin reset (CaAR), may construct actin filaments at the cell cortex or other subcellular domains that provide a scaffold to launch Hippo pathway activators. This article will review studies demonstrating Ca2+-mediated Hippo pathway modulation and discuss its implication in understanding the role of actin cytoskeleton in regulating the Hippo pathway.The Hippo pathway is a conserved signaling network regulating organ development and tissue homeostasis. Dysfunction of this pathway may lead to various diseases, such as regeneration defect and cancer. Studies over the past decade have found various extracellular and intracellular signals that can regulate this pathway. Among them, calcium (Ca The Hippo pathway is a conserved signaling network governing organ development and tissue homeostasis. Various diseases, such as distorted tissue regeneration and cancer, have been linked to dysfunction of this pathway . The Hip2+ is a signaling messenger important for a variety of cellular functions. Recent studies found that Ca2+ can regulate YAP/TAZ in various situations and is an emerging signal for the Hippo pathway regulation. This article will review the studies supporting this notion and discuss the potential mechanism underlying regulation of the Hippo pathway by Ca2+.Growth control or homeostatic signals emitted extracellularly or intracellularly can regulate YAP/TAZ through Lats1/2-dependent or -independent mechanisms. Previous studies have found that the Hippo pathway can respond multiple upstream signals, including mechanical forces, cell polarity and adhesion, soluble factors, as well as various cellular stress. The Hippo pathway regulation by these signals have been extensively reviewed recently . Ca2+ is2+ level by enhancing store-operated Ca2+ entry (SOCE) -mediated Ca2+-induced actin remodeling drives accumulation of protein kinase C (PKC) beta II in an actin cytoskeletal compartment. Such translocation is critical for PKC beta II to activate Lats1/2 , a Ca2+ channel responsible for Ca2+ releasing from acidic organelles  can inhibit the epithelial-to-mesenchymal transition. This is through increasing cellular Ca2+ level and expression of E-cadherin, which then promotes YAP phosphorylation through activating Lats1/2  . The ele Lats1/2 . The stuganelles , in metaganelles . In thestivation . In brea Lats1/2 .2+ level caused by directly perturbating Ca2+ channels, the Hippo pathway regulation by mechanical force appears to involve Ca2+. 2+-dependent manner. In this process, transient receptor potential cation channel subfamily V member 4 (TRPV4) is responsible for the mechanical compression-induced Ca2+ influx and RhoA inhibition. Along with the reduction of RhoA activity, YAP translocates to cytoplasm from the nucleus when cells are compressed. However, the YAP translocation is suppressed when Ca2+ is eliminated from the compressed cells  and Drosophila wing epithelium development. ADPKD is caused by mutations in PKD1 and PKD2 genes. Their protein products PC1 and PC2, respectively, form a plasma membrane calcium channel complex .In human neural stem/progenitor cells, spontaneous Caverslips . The Ca2 surface , this obh events . This stmpressed . Notably2+ release through activating inositol triphosphate receptor (IP3R), resulting in activation of RhoA, which can then activate TAZ through inhibiting Lats1/2. Ca2+ signals derived from ER perturbation can also regulate the Hippo pathway in Drosophila wing development. 2+ regulator, can synergize with RasV12 to induce tumor growth through inhibiting the Hippo pathway. In this process, Emei mutation reduces Ca2+ level in ER and subsequently activates JNK. Perturbation of another ER Ca2+ regulator, SERCA, indicated that reducing ER Ca2+ can synergize with RasV12 to promote tumor growth. Because preventing cytosolic Ca2+ from importing into ER by disrupting these ER Ca2+ importers may cause ER Ca2+ imbalance, which can trigger plasma membrane Orai Ca2+ channel through Stim to import extracellular Ca2+, the Hippo pathway response in this case could be a consequence of increasing the cytosolic Ca2+ level. In this study, whether the cytosolic Ca2+ level is increased has not been directly tested. Notably, the aforementioned study reported that cells with SERCA and Orai double mutations in the wing epithelium also show an disrupted Hippo pathway ,  pathway , suggest2+ was further demonstrated by a recent study through a real-time single-cell visualization of YAP subcellular localization and its target gene transcription dynamics  are interrelated or independent needs to be resolved.It is still unclear how Cativation . The roltivation . Whetherf Merlin , an essef Merlin . The ubitivation , Merlin tivation . Current by Ca2+ . It was function . It woulThe actin cytoskeleton appears to be a central mediator of various upstream biochemical and mechanical signals to YAP/TAZ through the core kinase cascade-dependent and -independent manners. How the cytoskeleton executes this role is unclear . Because2+ can induce a characteristic actin rearrangement    . A formit (CaAR) . In INF2 by Ca2+ . It was  by Ca2+ . Therefolocation . Collect (HUVEC) . Such ph minutes , whereas minutes . As the 2+ level can regulate the Hippo pathway (2+ could be an intracellular messenger for the Hippo pathway in responding to upstream signals, such as mechanical forces and soluble factors. Intriguingly, the Hippo pathway responses to Ca2+ appear to be variable when examined in different experimental settings. The distinct responses suggested that the Hippo pathway regulation by Ca2+ is complex, and that the Hippo pathway responses may be determined by the stages after the initial Ca2+ influx as well as the subcellular domains where Ca2+ influx occurs. Therefore, it is important for the future study to examine these temporal and spatial factors when assessing the Hippo pathway responses as well as Ca2+ effectors involved in the Hippo pathway regulation. An emerging principle of Ca2+ regulation of the Hippo pathway is the involvement of actin cytoskeleton rearrangement, such as INF2-mediated CaAR and RhoA-mediated stress fiber formation. The transition among these actin filaments may modulate the effectors, such as PKC and NEDD4L, which are responsible for the Hippo pathway regulation (Multiple studies in various circumstances have demonstrated that changes of intracellular Ca pathway . These fgulation . FurtherYW and WL wrote the manuscript and approved the submitted version. Both 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.


Mycoplasma pneumoniae (MP) infection. Urticarias’ symptoms and signs were refractory to regular antihistamines dose but showed marked improvement or complete resolution following clarithromycin administration. CU response to antibiotics pointed strongly to a potential causative role of MP in the pathogenesis of chronic spontaneous and chronic inducible urticarias. It is not clear if MP was the etiopathogenic cause or just the trigger. Nevertheless, refractory to antihistamines urticarias associated with MP infection may respond to antibiotics, which should be considered as an alternative therapeutic approach.Acute childhood urticaria is a common disorder that has been associated with infections. In a few children, it may last for more than six weeks, thereafter it is characterized as chronic urticaria (CU). We report two cases, one suffering from chronic spontaneous urticaria and one chronic inducible urticarias (dermographism and cold urticaria). Both children had concomitant respiratory symptoms that were associated with  Mycoplasma pneumoniae (MP) is a common pathogen that mostly affects the respiratory tract. Infection is often subclinical, but in patients, the clinical manifestations may vary from mild upper respiratory tract infection to bronchitis and pneumonia. MP infections are more common in young children and adolescents and tend to rise in summer and peak in winter . Inclusion criteria were: (1) clear distinction between chronic urticaria versus other allergies; (2) clear distinction of chronic spontaneous or idiopathic urticaria as opposed to acute urticaria; (3) studies in humans; and (4) published articles written in the English language. Additional potentially relevant studies were scrutinized in the reference lists of the eligible publications. Interestingly, only four relevant articles were identified.Lim et al. reported that in 6.8% of adult patients, who presented at a private dermatology practice with chronic urticaria, there was evidence of acute MP infection. Nevertheless, the question of whether patients with acute urticaria and an untreated MP infection are at greater risk for developing chronic urticaria remains intriguing yet unanswered .Mycoplasma-specific IgG (26). Unfortunately, the absence of a normal control population without urticaria renders the drawing of conclusions about the pathogenetic role of MP in chronic urticaria an elusive task ) is a reasonable alternative with excellent specificity up to 97%, especially for the IgM determination with CFT .According to the conducted systematic review, Case 1 is the first MP-associated dermographism in the literature. However, this developed cold urticaria, as well. More than one inducible urticarias can coexist in the same patient and may have a common pathophysiological background that can explain the simultaneous or sequential onset.It has been suggested that most cases of childhood urticaria may be related to infections as associated triggers or even as etiopathogenic factors . It can Mycoplasma pneumoniae infection may cause or trigger urticaria, especially in susceptible individuals. In the cases with recent respiratory infection and persistent urticaria, refractory to standard doses of antihistamines, serological examination of common respiratory pathogens is suggested. Appropriate antibacterial treatment should be considered in MP seropositive cases as it may constitute an alternative therapeutic option to control or even treat urticaria.Chronic urticaria and prior upper or lower respiratory illnesses have been pathophysiologically linked.


The first-order static hyperpolarizability and second-order susceptibility were estimated by in vacuo and in-crystal density functional theory calculations, and compared with the experimental SHG response of powdered samples. The parameters influencing the SHG response of these compounds were investigated by comparison with similar previously analysed MOFs, to understand how to design more efficient materials to be used as nanoprobes by exploiting crystal engineering techniques.In the last few decades optical imaging techniques based on nonlinear optical properties have been of interest for biosensing applications. This work focuses on two isostructural and isomorphous sugar-derived metal–organic frameworks (MOFs) with second-harmonic generation (SHG) properties, in order to investigate their possible application as biosensors in view of their high biocompatibility. Combining 2-deoxy- The advantage of SHG-based nanoprobes is that in principle they do not bleach or blink, and the second-harmonic signal does not saturate with increasing illumination intensity  properties, and in particular the second-harmonic generation (SHG), of materials have been of interest for biosensing applications  ]. We also analysed similar MOFs with similar composition  but different structures and stoichiometries  showed the most promising SHG response. Thus, we oriented our synthesis towards MOFs containing this saccharide, along with alkali earth halogenides, hoping that it would impart high SHG efficiency to the crystals.The aim of the present work is to analyse the role of the sugar in determining the SHG properties in this type of MOF. In the past, by screening experimentally the SHG efficiencies of about 150 powdered saccharides, Bourhill  al. 1993 observedet al.,1972et al., 1970P21, with all the molecular units adopting the β-2-deoxy-d-galactopyranose ring form , we obtained two new isomorphous MOFs of formula [Ca(C6H12O5)2]X2, named CaDGalBr and CaDGalI, respectively, for which the SHG is expected to be higher than that of fructose-based MOFs, if the contribution of the sugar is what causes the SHG efficiency. In order to understand this perspective, the two compounds were characterized by single-crystal X-ray diffraction. The first-order static hyperpolarizability and second-order susceptibility were estimated by in vacuo and in-crystal density functional theory (DFT) calculations, and compared with experimental SHG values obtained for powdered samples.By combining 2-deoxy-2.2.1.d-galactose in stoichiometric ratios of 2:1 (0.200:0.075 g), 1:1 (0.100:0.075 g) and 1:2 (0.050:0.075 g) were dissolved in ethanol (1 g). The solvents were evaporated slowly at room temperature and after a few days colourless crystalline powders were formed. The powders were washed with a few drops of ethanol and dried in an oven at 323 K.Calcium bromide and 2-deoxy-2.2.d-galactose in stoichiometric ratios of 2:1 (0.180:0.050 g), 1:1 (0.090:0.050 g) and 1:2 (0.045:0.050 g) were dissolved in ethanol (1 ml). The solutions were heated up to 353 K for 30 min, cooled to room temperature and the solvent was evaporated for two days in an oven at 323 K to give colourless crystalline powders. The powders were washed with a few drops of ethanol and dried in an oven at 323 K.Calcium iodide and 2-deoxy-2.3.Kα radiation (1.5418 Å). The CrysAlisPro : monoclinic, space group P21, Z = 2. Cell parameters are reported in Table 1Rint = 0.0374). R1 = 0.0350, wR2 = 0.0764 .Crystal data for CaDGalBr (Mw = 658.22): monoclinic, space group P21, Z = 2. Cell parameters are reported in Table 1Rint = 0.0434). R1 = 0.0316, wR2 = 0.0792 .Crystal data for CaDGalI  and CIF files (CCDC 2058083 and 2058084).All atoms except H atoms were refined with anisotropic displacement parameters. Due to the low number of reflections collected, even if the H-atom peaks were observed in the difference Fourier maps, the H atoms were calculated and refined as riding with 2.4.2.4.1.GAUSSIAN09 and GAUSSIAN16 set of programs  approach as implemented in the 2.5.The SHG efficiency of the powdered compounds was measured by the method of Kurtz & Perry 1968. SamplesI2ω), and re-collected with a Hamamatsu R 5108 photomultiplier tube. The SHG efficiency was evaluated by taking as reference the SHG signal of ground sucrose powders (I2ω/I2ωsucrose).The non-resonant 1064 nm wavelength of a Nd:YAG pulsed laser beam was directed onto capillaries containing the samples. The scattered radiation was collected by an elliptical mirror, filtered to select only the second-order contribution at 532 nm  and do not exhibit any disorder. As expected, the I-containing crystal has a slightly larger (by 6.9%) cell volume, but the large iodine ions do not imply any significant change in the packing motifs Table 1. Thus, a2+ ion, two X− anions, two sugar molecules and two water molecules coordinated to the metal atom  . In the end, the coexistence of smaller and larger rings in this structure allows an efficient occupation of space, in agreement with Kitaigorodskii’s principle of close packing 3I]I .In the present compounds, the Ca–furanose parallel chains are reciprocally connected in the crystal through strong hydrogen bonds involving the halogen anion, the water molecules and the free OH groups of the pyranose sugar molecules  we demonstrated the inverse proportionality between the size of the ground particles and the grinding energy of a planetary mill (grinding time and number of revolutions per minute).For experimental applications, the MOFs have to be reduced to particles of a few tens of nanometres, which can be obtained by vigorously grinding the crystals. In fact, in our previous work  and six water molecules, while Fragment2 in Fig. 5d-galactose molecules, six anions (Br− or I−) and six water molecules. Since sucrose is the usual reference compound for SHG measurements, the same types of calculation were carried out on a model of bulk sucrose, composed of four sucrose units. The atomic coordinates of sucrose were obtained from the Cambridge Structural Database  calculations: the natural atomic charges on Ca, Br and I and the group charges of the galactose and water molecules are reported. No significant differences were observed between the complexes or between the different fragments.tot and the second-order susceptibility χ(2) for the two compounds and the two different fragments. The ratio between the second-order susceptibility of the compounds to that of sucrose is also reported, in order to compare the computational results with the experimental second harmonic measurements.Table 4tot is defined as:The total dipole moments μ and the mean polarizabilities α in a Cartesian frame are defined as:N is the number of particles per unit volume and F is the local field factor. F depends upon the crystal symmetry. It is related to the crystal’s refractive index, and it can vary if different compounds are considered. Values between 1 and 2 are generally reported  values, we assumed F = 1.The relationship between the macroscopic second-order susceptibility, the quantity that correlates to the second-harmonic intensity, and the microscopic total hyperpolarizability is given by equation  values are similar for the two fragments of the same complex, underlying that the geometry of the fragments does not affect this result, while a difference is observed by comparing the χ(2) values of the Br− versus I− complexes. The complexes containing the larger and more polarizable I− anions show a higher value of χ(2), confirming the trend already observed in our previous work.The calculated static susceptibility χ3.2.2.supporting information) included the coupling of an external electric field with the crystal field, allowing us to extract from the Bloch-consistent periodic wavefunction information on optical axes, dielectric tensors and first- and second-order polarizabilities. This approach bears several advantages against the more classical sum-over-state (SOS) method under Unsöld’s approximations : the r.m.s.d.s on the coordinates of C, O, Ca2+ and halogen atoms within the whole unit cell do not exceed 0.12/0.28 Å in the compounds CaDGalBr/CaDGalI. As expected, the largest deviations affect H atoms, and particularly the relative orientation of Ca-coordinated water molecules . Thus, symmetry breaking is not the main trigger of the NLO response. This is consistent with our Kurtz–Perry measurements, even though the bulk DFT simulations predict that, under ideal conditions, the iodine chromophore should perform better than the bromine one  while the DGal sugar crystallizes in the same monoclinic space group (e Table 6. Any difM(sugar)X MOFs. Instead, the two DGal MOFs analysed in this work do not show the expected enhancement of SHG efficiency with respect to the analogous fructose-based ones studied in our previous work. Thus, the nature of the sugar can influence the SHG behaviour of the compounds only because it entails a change in the crystal structure.As mentioned above, we expected that substituting fructose with DGal would impart a higher SHG efficiency to the The SHG measurements and the theoretical calculations agree on the fact that the SHG efficiency of CaDGalI is greater than that of CaDGalBr. Since the two compounds are isostructural and isomorphous, this behaviour is necessarily imputable to the larger polarizability of iodine ions and was observed in all the isostructural and isomorphous compounds previously analysed. It is noteworthy that in the bulk calculations the difference in the responses of the two MOFs is greater than for the other two methods. This behaviour might be traced back to cooperativity effects, that is, to the symmetry-constrained alignment of polar molecules in the bulk crystal. It is known, for example, that an external electric field can partially align polar molecules, eliciting a temperature-dependent contribution to SHG even in an otherwise isotropic achiral liquid  CaDgalBr_new, CaDgalI_new. DOI: 10.1107/S2052520621004637/um5049CaDgalBrsup2.hklStructure factors: contains datablock(s) CaDgalBr_new. DOI: 10.1107/S2052520621004637/um5049CaDgalIsup3.hklStructure factors: contains datablock(s) CaDgalI_new. DOI: 10.1107/S2052520621004637/um5049sup4.pdfAdditional details, including refinements, calculations and hydrogen bonds. DOI: 2058083, 2058084CCDC references: