Climate change is a defining challenge of the 21st century, and this decade is a critical time for action to mitigate the worst effects on human populations and ecosystems. Plant science can play an important role in developing crops with enhanced resilience to harsh conditions  and engineering efficient carbon-capturing and carbon-sequestering plants. Here, we present examples of research being conducted in these areas and discuss challenges and open questions as a call to action for the plant science community. We discuss research aimed at improving carbon sequestering capacity and climate resilience in plants to illustrate how plant science can help to mitigate climate change and enhance food security. The IPCC Sixth assessment report  of CO2 each year. In some locations, warming may benefit certain crops, and, over time, the optimal growing regions may shift farther away from the equator. However, the effects of climate change are not limited to increasing temperatures and heatwaves in many parts of the world but include changes in rainfall, more severe and frequent storms, increased drought, and increased threat of wildfires. All of these effects are anticipated to adversely affect crop yields and food security worldwide within the next 20 years (Climate change is caused by an accumulation of greenhouse gases (GHGs)  and seagrasses, followed by a section on improving photosynthesis. A third section addresses engineering climate resilience in crops (resistance or tolerance to abiotic and biotic stresses). The final section describes the vision of a sustainable global bioeconomy rooted in plant biology. We acknowledge that there are other areas, not covered here, in which plant science can play a role in mitigating adverse climate change effects, including bioenergy, forestry, and ecosystem conservation. Solutions in all of these areas are needed in the very near future, and in the longer term. We do not provide an in-depth review of these topics. Rather, the examples provided here illustrate a few of the many avenues of research being conducted by plant scientists around the world. A companion review by In this article, we explore several ways in which plant scientists are working on solutions related to carbon sequestration to help achieve net zero CO2 removal strategies, also called Negative Emissions Technologies (NETs), are needed to attain net CO2 reductions and avoid the most damaging climate change outcomes –soy (Glycine max) rotation was designed and the science to date on how management might lead to predictable increases in soil carbon. I then focus on genetic changes that are needed to create carbon-negative crops, including optimal combinations of traits that can be addressed in breeding programs.Among NET for CO decades . Current decades  and are mosphere . Here, I2O, a GHG with an effect size ∼300× that of CO2  in the form of synthetic fertilizer , which lt of CO2 . An obvit of CO2 . For thet of CO2 , a majort of CO2  and can t of CO2 . Gettingt of CO2 . Althoug2 in a few years. Getting soil carbon inputs deeper into the soil is needed to achieve greater and more durable carbon sequestration in agricultural systems , breeding during the Green Revolution led to the fixation of mutations that reduce NUE  was never bred for high N inputs, can be grown at population densities of 500,000 plants per hectare, and has greater root biomass below 50 cm than other major crops . Little is known regarding the degree to which root exudates are controlled by genetics versus the environment. Even less is known about the genetic control of the abundance and composition of root exudates, even in model species. This is due in part to the difficulty of measuring the relevant phenotypes in agriculturally relevant environments. On the soil modeling side, recent progress has been made in separating biomass and exudate inputs to soil carbon, where exudates are predicted to lead to increases in mineral-associated organic matter, which in turn is predicted to have a longer residence time than other soil carbon fractions . Finally2 from the atmosphere via photosynthesis and convert it to biomass. Earth’s soils contain a large amount of carbon, estimated at approximately 2,300 Gt carbon to 3-m depth, which constitutes about three times the current atmospheric pool of CO2 . While we are interested in trait changes that will work in the field and maintain crop productivity, it is not feasible to measure all these root traits in the field at high throughput. We therefore rely on initial screening approaches in the laboratory or the greenhouse to measure and engineer root traits, subsequently moving to in-soil or field-testing with a subset of lines that display distinctive traits. We focus on root mass in relation to depth, as the engineering or breeding goal is to direct as much root mass as possible to a deeper depth, and on enhancing the accumulation of suberin. Suberin is a highly effective apoplastic diffusion barrier and producing it everywhere in the root would be detrimental to plant health. Therefore, we focus on specific root tissues that already produce suberin such as the periderm or the exodermis, which are outer layers in mature root systems. We are targeting such tissues as suberin sinks by using tissue-specific promoters to drive suberin production, as well as utilizing genes involved in the formation of these tissues to produce additional tissue layers.To quantitatively link these root traits to carbon characteristics in the soil, we are working with soil scientists to better estimate the soil carbon impact of crop varieties that have different root mass, depth, and suberin content. We aim to test the effects of genetic alterations via gene editing or gene engineering approaches in crops over the next few years. Recent advances in high-throughput phenotyping, sequencing, and functional single-cell genomics now provide a way to leverage genes, gene constructs, and genetic variants within and between species. We aim to have the proof of concepts for enhanced crop traits within the next 3 years to then partner with both NGOs and agriculture companies to enhance varieties that are of interest to farmers.2O or methane emissions to creating carbon sequestration-friendly microbiota or mycorrhizal associations. As a community, we should think of and work toward promising plant biology-based solutions.There are numerous other opportunities for plant biologists to contribute to climate change mitigation efforts, ranging from work on traits that will reduce agricultural NMost agricultural soils have lost 50%–70% of the SOC that they had previously accumulated under native plant communities; therefore, raising the carbon levels in historically tilled agricultural soils offers the potential to partially mitigate climate change by capturing 30–60 Gt of organic carbon . The resAlthough planting long-lived perennial plants on degraded agricultural soils would be one of the most effective ways to rapidly restore soil carbon levels, this approach is limited because the herbaceous perennials currently available for use in agriculture (mainly forage crops) produce biomass that is unsuitable for direct human consumption . TherefoOryza sativa ssp. indica) and the rhizomatous perennial relative Oryza longistaminata were hybridized, and a breeding program has produced lines for flooded paddies that persist through eight harvests with yields and quality traits on par with modern rice cultivars  with perennial species  species are being evaluated for direct domestication as perennial oilseeds  in the same region  is an N-fixing semi-perennial shrub that is grown in Asia and southern Africa. Although types that can be grown for several seasons without replanting have been used in erosion control and are still grown by some farmers, annual pigeonpea is now the dominant form. A recent study in Malawi indicated that farmers are less likely to adopt erratically performing perennial pigeonpea due to social pressures and lack of trust in the technology  and NUE.Photosynthesis heavily influences crop productivity, resource use efficiency, and sensitivity to stresses. Therefore, strategic engineering of photosynthetic metabolism and the morphological features of leaves that control carbon and water fluxes can: (1) increase the food, fuel, fiber, and feed produced by crops; while (2) reducing demand for water and improving agricultural GHG balance; and (3) making crops more resilient to future climatic and atmospheric conditions. Detailed models of photosynthetic metabolism  and crop2 enhancing photosynthesis in C3 plants, global warming is expected to have profoundly negative consequences for crop photosynthesis and productivity by the middle to end of this century  was developed to recycle 2P-glycolate in the chloroplast via overexpression of plant malate synthase and Chlamydomonas (C. reinhardtii) glycolate dehydrogenase and simultaneous RNAi to downregulate a glycolate–glycerate transporter  generally enhances yields of C3 crops in major temperate growing regions , an oxalate oxidase (OsOXO3), and a catalase (OsCATC) overexpressed in rice chloroplasts , a catalase (CAT) from Escherichia coli, and a peroxisomal glycolate oxidase (GO) in Arabidopsis chloroplasts , glyoxylate carboligase (GCL), and tartronic semialdehyde reductase (TSR). Unlike the first approach, this synthetic pathway preserves 75% of carbon from two glyoxylate to produce one glycerate, which is returned to the CBB cycle (2 produced via GCL. Expressing the above genes in Arabidopsis chloroplasts increased the growth rate and biomass yield. This synthetic pathway was shown to benefit crop plants such as Camelina sativa and potato (Solanum tuberosum) in greenhouse and growth chamber conditions (The synthetic pathway originated from BB cycle . The remnditions .2 and PEP to OAA (C4), followed by splitting OAA to acetyl-CoA and glyoxylate  cycle also uses GCL, and TSR to convert two glyoxylates to glycerate, which is then converted to phosphoenolphyruvate (PEP). The oxygen-insensitive PEP carboxylase (PPC) then carboxylates COyoxylate . The glyacterium . CompareAn elegant tartronyl-CoA (TaCo) pathway was demonstrated recently, in which glycolate is activated to glycoly-CoA, which is then caboxylated to tartronyl-CoA and then to glycerate. This approach requires a new-to-nature enzyme, glycolyl-CoA carboxylase, which was developed by rational design and high-throughput screening .2-fixation pathways in microorganisms have been identified in nature –MCG –MCG , have be in vivo . The CET in vivo . An oxygThe rPS–MCG cycle consists of two parts . The fir4 plant species are overrepresented in agriculture systems and have substantially higher productivity compared to C3 crops mainly due to higher photosynthetic efficiency , oats (Avena sativa), and many vegetable and tree crops. Therefore, efforts to increase the photosynthetic efficiency and productivity of C3 crops are underway to help meet the increasing global food demand (2 saturation point for photosynthesis of C3 plants (intercellular CO2 levels ∼600 µmol mol−1) makes them more responsive to elevated CO2 than C4 plants, which are saturated for CO2 under current atmospheric CO2 levels  and a reduced rate of starch accumulation in developing grains in rice  with CO2 responsiveness under realistic CO2 conditions using a field-based free air CO2 enrichment facility. Further, the high CO2-responsive (HCR) genotypes showed significantly higher rates of photosynthesis (A) and lower rates of NR resulting in a lower N/AR ratio as compared to the least CO2-responsive (LCR) genotypes. Interestingly, elevated CO2 was identified as the major driver influencing carbon-balance dynamics and the phenotypic response of HCR genotypes resulting in higher biomass and yield under elevated CO2 + HNT conditions, whereas the LCR genotype was severely affected by HNT despite exposure to elevated CO2.Despite the well-documented photosynthetic enhancement of Cated CO2 , active ttention . The comironment . Subsequ2, but this response was mainly confined to the HCR genotypes , which is oxygen insensitive. This carboxylation reaction occurs in the outer mesophyll cells of the leaf, with the four-carbon reaction product subsequently transported to inner bundle sheath cells for decarboxylation and re-fixation by Rubisco in the Calvin cycle . In general, C4 plants also use less water , the status quo would result in atmospheric CO2 levels of ∼500 ppm by 2050. C4 plants could thus outperform C3 plants where temperatures exceeded ∼33oC, which given climate warming predictions could be much of the global agricultural landscape for at least part of the year. Leaving predictions aside, long-term field experiments at elevated (+180 ppm) CO2 demonstrated that the biomass of C3 but not C4 grasses was enhanced over the first 12 years of the project but then C4 outperformed C3 in the following 8 years  was initiated, genes encoding all of the enzymes of the C4 pathway had been identified in maize and other C4 species, as had some of the genes encoding metabolite transporters (reviewed in 4 leaf anatomy had not been identified. The strategy to introduce C4 traits into rice was thus three-pronged: (1) introduce compartmentalized C4 metabolism into existing bundle sheath cells and the mesophyll cells immediately adjacent to them by expressing maize genes in specific cell-types of rice; (2) activate chloroplast development and photosynthesis in existing bundle sheath cells by expressing a known regulator of chloroplast development in maize  and a model cultivar of japonica rice (Kitaake), and in both cases the pathway is partly functional. Specifically, primary carboxylation by PEPCase is seen in mesophyll cells, but subsequent decarboxylation in bundle sheath cells has yet to be detected  for other mandate crops. In rice breeding, abiotic stress tolerance was not a selection target during the Green Revolution, and some evidence suggests that stress tolerance was even selected against due to tight linkage between stress tolerance loci and loci conferring unfavorable agronomic traits . Therefore, the growth stages, genetic backgrounds, and environmental conditions relevant to breeding programs should be reflected in the study systems used in upstream research. One example is in the validation of candidate genes for stress tolerance: this is frequently done in the background of On the other side of the gap, downstream science must make released varieties and advanced breeding lines more accessible to upstream scientists. Familiarity with this material is important because in some cases, key genes have been identified and advocated as promising breeding targets without the recognition that they are already present in the breeding pool. Furthermore, the availability of improved material to upstream researchers will help to ensure that target loci are effective in those genetic backgrounds. Downstream science also must stay up to date and gain access to the most recently identified traits/genes/QTLs to incorporate into the breeding program, while at the same time effectively connecting with local breeders who can conduct widespread testing and who understand the needs of farmers in stress-prone regions. This “bridge building” among scientific disciplines is critical to the development of more efficient pipelines that bring novel improvements to crops for climate change-affected farmers.To strengthen linkages between upstream and downstream development efforts, a framework has been developed that organizes and codifies trait development efforts. This “Trait Development Pipeline” developed at the International Rice Research Institute  applies The current Trait Development Pipeline is organized into six stages that link a variety of research disciplines, and the pipeline provides a structure that gives a framework for teamwork between these areas. The pipeline starts with an assessment of the trait of interest in the context of priority traits needed by farmers and consumers that are not already present in the elite breeding pool  are wild species that are closely related to domesticated crops and can be used for crop improvement. Breeders have traditionally used CWRs as sources of superior traits, including key traits for enhancing adaptation to climate change . The mai2 assimilation, deeper root systems, more efficient regulatory networks, leaf curling, and stomatal closure, as well as showing an abundance of allelic diversity within candidate genes. For example, higher WUE, higher carbon assimilation, and greater carboxylation efficiency were reported in wild lettuce  that would be a great resource for improving cultivated chickpea (Cicer arietinum). Drought-related transcription factors of the Asr  family have a high level of diversity in CWRs  under salt stress  were identified from wild rice species O. nivara and O. rufipogon, together with a likely presence of other submergence mechanisms in other wild rice accessions (qAGP1 and qAGP3) from O. nivara introgression lines  in potato  resistance in rice (Spodoptera frugiperda) resistance from wild relatives of maize  exhibited either enhanced or reduced susceptibility to infection, depending on the pathogen studied , and other crops , the causal agent of bacterial blight disease, under high temperature conditions  and salt  tolerance genes were stacked with eight pathogen and pest resistance genes in an elite rice line , which colonize host roots and are widely distributed in plants, and ectomycorrhizae, mainly associated with trees and shrubs . The soiSeveral challenges restrict the application of AMF in agriculture as a biofertilizer. Plants vary widely in response to individual mycorrhizal fungi , and resMycorrhizal fungi strongly influence host plant phosphorus acquisition. Interestingly, the phosphate starvation response was found to be a core regulator in both a direct phosphate uptake pathway via root hairs and epidermis and an indirect phosphate uptake pathway via mycorrhizal symbiosis , suggestBiological N fixation is the process by which nitrogenase (an enzyme found only in certain prokaryotes known as diazotrophs) converts dinitrogen gas from the atmosphere into ammonia and is the main path for the formation of combined N in nature. Three forms of N fixation are found in nature: free-living, associative, and symbiotic . In assoThree mechanisms have been proposed to develop N self-fertilizing cereal crops to enhance climate change resilience :Diazotrophs are present in the carbon-enriched mucilage in maize aerial roots and were found to contribute 29%–82% of the N nutrition of Sierra Mixe maize in a 5-year field experiment . EngineeMedicago truncatula and barley (Hordeum vulgare) of rhizopine, a small molecule compound synthesized by a few rhizobia, could be sensed by engineered bacteria Azorhizobium caulinodans ORS571 with a 103-fold increase in perception sensitivity (It has been shown that the engineered expression in sitivity . This prM. truncatula Nod factor receptors NOD FACTOR PERCEPTION (MtNFP) and RECEPTOR-LIKE KINASE3 (MtLYK3), respectively, triggers calcium spiking in response to a low concentration Nod factor treatment in rice (M. truncatula SHORT ROOT (MtSHR), SCARECROW (MtSCR), and LLOB-DOMAIN PROTEIN16 (MtLBD16) specify cortical cell fate with the ability to de-differentiate to form nodule primordia in response to symbiotic signals  Th in rice . (iii) T signals . This conifB, nifH, nifD, nifK, nifE, nifN, nifX, hesA, and nifV in Paenibacillus WLY78 (nif gene cassette (nifBHDKENXhesAnifV) showed moderate nitrogenase activity and resulted in higher biomass and chlorophyll compared to control plants grown in low-N or N-free medium  is a staple root crop grown on more than 28 million hectares and crucial to the food security of almost half a billion people. Cassava is uniquely positioned as one of the most climate change resilient crops due to its ability to tolerate prolonged droughts, often exceeding 5 months. The cultivation of cassava has continued to increase in tropical regions, where climate change impacts will be particularly adverse  Breeding Project initiated a multi-disciplinary effort to accelerate genetic improvement, notably using genomic selection at breeding programs in Africa and Latin America. NGC partners in Africa include the International Institute of Tropical Agriculture and the National Root Crops Research Institute in Nigeria; the West Africa Center for Crop Improvement in Ghana; the National Crops Resources Research Institute, Uganda, and Makerere University in Uganda; and the Tanzania Agricultural Research Institute in Tanzania. In South America, collaborators include EMBRAPA in Brazil and the International Center for Tropical Agriculture in Colombia. In the USA, collaborators are Cornell University, the Boyce Thompson Institute at Cornell, the University of Hawaii, and the USDA-ARS in Ithaca, NY. The details of partners and funding found at ).Instead of requiring phenotyping breeding lines over many years before selecting new parents, genomic selection enables breeders to predict performance based on genome-wide genetic markers, even at the seedling stage . GenotypFigure 10BSeveral additional innovations came to cassava under NGC, including: (1) GWAS enabling the cataloging and validation of trait-linked single-nucleotide polymorphisms used for marker-assisted selection ; (2) Cas−1) and yield observed under experimental water deficit (23.6 t.ha−1) with improved genotypes  gene resulting in greater drought tolerance through enhanced stomatal response  experiments with plants grown under standard field management practices, with various crops including soybean, sorghum (Sorghum bicolor), potatoes, wheat, barley, and rice (3 grains and legumes at the elevated CO2 concentrations predicted for 2050 (546–586 ppm). A large meta-analysis representing numerous FACE and non-FACE experiments also identified similar reductions in Zn, and in other dietary mineral nutrients such as calcium (Ca) and magnesium  and zinc (Zn), is a major problem for a significant portion of the world’s human population . Experimand rice . Free-aiand rice , showed and rice , which csium Mg; . This casium Mg; ; produci2 on mineral nutrient concentrations in crops requires concomitant consideration of elevated temperature, as they go hand in hand. Combined FACE and temperature (T-FACE) experiments have begun to address the possible impact of elevated temperatures on the carbon nutrient penalty. In soybean, elevated CO2 caused a decrease in seed Fe and Zn concentrations (as previously observed), while elevated temperature had the opposite effect; but the combined effect of elevated temperature and CO2 restored seed Fe and Zn concentrations   . Routes irgatum) . When puirgatum) .Maximizing the recovery of biomass carbon into fuels and co-products requires flexible design capabilities to produce cell wall architectures that can be easily and completely deconstructed for current and future conversion processes . As robuMaterial use is tightly coupled to energy use, GHG emissions, land and water use, and waste flows. About one-third of global GHG emissions comes from industrial manufacturing. To decarbonize this economic sector will require changing the means of manufacture as well as the nature of material inputs . We mighGossypium hirsutum), tobacco, hemp, hops (Humulus lupulus), and biofuel feedstocks, to name just a few, can be of considerable value to growers and mitigate financial risk. The diversification of food and nonfood plant products within a single cropping system, or a single crop, coupled with principles of sustainability and climate change resilience, could thus be an advantage toward achieving both food and energy security. The decarbonization of agriculture could include the use of bioenergy crops to displace fossil fuels as a source of hydrogen for ammonia production (Food security is a paramount concern and there are land use issues to consider in raising crops for nonfood versus food production. Nonfood cash crops, such as—traditionally—cotton (oduction  as well In the USA, an annual sustainable resource of over 1.6 billion tons of lignocellulosic biomass could be considered a strategic carbon reserve . This quWe presented examples of some plant biology-based solutions that we believe show promise toward enhancing terrestrial carbon sequestration and engineering climate resilient crops. Although we addressed several disparate topics, a few overarching conclusions emerge.Some of the ideas described here may seem far-fetched to today’s readers, but we believe that for our planet to remain inhabitable and sustainable, many of the ideas proposed here—or others like them—will need to be realized, and we will need plant scientists to help achieve them.By definition, efforts to mitigate global climate change must be large scale. Plant scientists contributing meaningfully in this arena most likely will be those who seek out effective collaboration—not only with other plant scientists but also with those in other disciplines, including for example agronomy, bioinformatics, data science, engineering, forestry, and soil science. Improved communication and collaboration across disciplines and between academia and industry can also be viewed as a low-tech effort that can have a strong impact. Identifying and seeking out potential collaborators who can link the research to impactful pathways should be a primary goal early in the planning stages of research projects for maximum benefit. Socio-economic and political perspectives will also be crucial in determining which approaches will be adopted and how quickly they will be implemented. Networking, discussion, and collaboration in the socio-political arena and with industry, governmental, and nongovernmental organizations may also be crucial.2 emissions, including reducing food and agricultural waste, shifting to plant-based diets, reducing deforestation coupled with afforestation/reforestation, and restoring coastal wetlands. Some technological solutions also have potential in the shorter term, including direct air capture, biochar, enhanced rock weathering, and bioenergy combined with carbon capture and storage. To date, none of these options has been implemented globally due to cost, timeline, inefficiency, lack of scalability, or an uncertain and evolving carbon price and market. Estimates for the potential of available technologies vary widely and will depend on the ability of nations to realize effective measures (Many current practices have substantial potential for mitigating COmeasures . We have4 photosynthesis into rice, symbiotic N fixation into cereals, and crops that produce a variety of synthetic products. Although time is pressing, this does not make them unworthy of attention. First, aspects of these longer-term goals may provide significant benefits in the short term, and second, the need for carbon capture and enhancing crop resilience and food security will continue in the future. The need is urgent for every plant biologist to consider today how their research can contribute to addressing climate change, ensuring food security, and achieving a sustainable biomass-based bioeconomy.Some of the goals of examples discussed may require years to realize fully, such as engineering C


Patients with recurrent pregnancy loss (RPL) account for approximately 1%-5% of women aiming to achieve childbirth. Although studies have shown that RPL is associated with failure of endometrial decidualization, placental dysfunction, and immune microenvironment disorder at the maternal-fetal interface, the exact pathogenesis remains unknown. With the development of high-throughput technology, more studies have focused on the genomics, transcriptomics, proteomics and metabolomics of RPL, and new gene mutations and new biomarkers of RPL have been discovered, providing an opportunity to explore the pathogenesis of RPL from different biological processes. Bioinformatics analyses of these differentially expressed genes, proteins and metabolites also reflect the biological pathways involved in RPL, laying a foundation for further research. In this review, we summarize the findings of omics studies investigating decidual tissue, villous tissue and blood from patients with RPL and identify some possible limitations of current studies. Recurrent pregnancy loss (RPL), defined as two or more consecutive clinically recognized spontaneous pregnancy losses before 20 weeks of gestation and includes embryonic or fetal loss, is a frequently occurring human infertility-related disease that affects 1-5% of parturients . AlthougOmics studies generate a large amount of information concerning the biomarkers, molecular mechanisms and biological pathways involved in complex diseases . More revia nuclear magnetic resonance, gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry. By exploring the relative relationship between metabolites and physiopathological changes, it is possible to find biomarkers for the diagnosis of disease. By studying and integrating data obtained using different omics approaches, knowledge of the underlying molecular interactions and associated longitudinal effects may be discovered and understood more deeply , insulin-like growth factor, Krüppel-like factor 17, atrial natriuretic peptide may play important roles in the formation of the maternal-fetal vasculature   . Anotheran women . A recenan women . UterineHMGB2 [associated with replicative senescence of human fibroblasts  was significantly increased  were closely related to embryonic development  and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses -binding protein family (CREB5 has been shown to promote the invasion of tumor cells (GREB5 promoter regions upregulated the mRNA and protein expression levels of CREB5 in RPL (CREB5 increased migration and apoptosis in the HTR8-S/Vneo (human chorionic trophoblast cell line in vitro) and JEG-3 (human choriocarcinoma cell) cell lines and prolonged the cell cycle  levels and decreasing the interleukin-10 levels in plasma  is a transcription inhibitor that plays a role in embryonic development  mRNA expression was reduced in the trophoblasts of RPL , and then exacerbated the inflammatory response of trophoblastic cells by enhancing the release of TNF-α and IL-1β  and Prothrombin (F2), were associated with early embryonic development . Among tResearch concerning the metabolomics of villous tissue in RPL is limited. A recent study showed that low succinate accumulation in villi participated in the occurrence of RPL by reducing the invasion and migration of trophoblastic cells . Omics oCAPS) might be the potential pathogenesis of RPL  gene was specifically hypermethylated in RPL , Guangdong Basic and Applied Basic Research Foundation (2019A1515011315), Shenzhen Fundamental Research Program , and National Natural Science Foundation of China .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Even though the functional role of mRNA molecules is primarily decided by the nucleotide sequence, several properties are determined by secondary structure conformations. Examples of secondary structures include long range interactions, hairpins, R-loops and G-quadruplexes and they are formed through interactions of non-adjacent nucleotides. Here, we discuss advances in our understanding of how secondary structures can impact RNA synthesis, splicing, translation and mRNA half-life. During RNA synthesis, secondary structures determine RNA polymerase II (RNAPII) speed, thereby influencing splicing. Splicing is also determined by RNA binding proteins and their binding rates are modulated by secondary structures. For the initiation of translation, secondary structures can control the choice of translation start site. Here, we highlight the mechanisms by which secondary structures modulate these processes, discuss advances in technologies to detect and study them systematically, and consider the roles of RNA secondary structures in disease. We discuss the consequences of secondary structure formation for splicing and translation, with particular focus in G-quadruplexes, hairpins and long range interactions. We also discuss the contribution of secondary structures in the regulation of mRNA splicing and in translation initiation and discuss the mechanisms involved.mRNAs are essential molecules in the cell as they are key to extracting information stored in the DNA. Although the function of mRNA molecules is primarily determined by the nucleotide sequence, some properties are determined by secondary structures. Secondary structures are defined as distinct features, including hairpins, long range interactions, G-quadruplexes, R-loops and pseudoknots and they are formed as a consequence of the interactions of non-adjacent nucleotides. Their presence can impact various processes involving the mRNA, including synthesis, splicing and translation. Secondary structures are dynamic and can be modulated by multiple proteins, in particular RNA binding proteins (RBPs), and as they cannot be predicted solely from the primary sequence they are challenging to study. Nevertheless, several assays are available for both 2In RNA, intra and intermolecular long-range interactions, including hairpins, pseudoknots, and G-quadruplexes, are commonly observed. Hairpins are composed of a hybridized stem and a single stranded loop a and b ain vitro, while more recent methods can deduce structures in vivoin vitro, and RNA G-quadruplexes have also been visualized in cellulo using a specific antibody A number of methods that probe RNA structures have been developed. Methods such as selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE)-seq 3S. cerevisiae and S. pombe, folding energy and GC content in the transcription bubble have been correlated with RNA polymerase distribution, and RNA structures within nascent transcripts promote forward translocation of the polymerase and limit back-tracking A variety of features are associated with RNAPII speed. For instance, the presence of introns and the length of the first intron are both positively correlated with RNAPII speed 4Pre-mRNA splicing is a key biological process that enables the removal of introns and the joining of intervening exons, eventually resulting in a mature mRNA molecule. Alternative splicing affects approximately 90–95% of mRNA transcripts in humans Splicing is mediated through the spliceosome complex which recognizes splice signals, the key members being the 5′ splice site (5′ss), the 3′ splice site (3′ss), and the branch point. The recognition of these consensus sequences is commanded by U1 and U2 small nuclear ribonucleoproteins (snRNPs) and other auxiliary protein factors that are involved in early spliceosomal assembly. Since higher-eukaryotic genes are often interrupted by long introns, early spliceosomal complex assembly over exons recognizes both splice sites during a process commonly known as exon definition cis-regulatory elements can be modulated by the presence of RNA structures in nascent transcripts and in mature mRNAs ATE1 gene, where splicing of two mutually exclusive exons is regulated by competing long-range hairpin structures that span up to 30 kB Even though the RNA structural code has been less explored 5During the mRNA lifecycle, RBPs regulate to a significant extent diverse transcriptional and post-transcriptional stages including splicing, transportation, translation, stability and degradation. They bind to pre-mRNA molecules in the nucleus and regulate its maturation and transportation to the cytoplasm where they regulate translation and degradation. The number of proteins that can bind to RNA in humans is estimated to be more than 1,500, adding complexity to all the aforementioned programs in vivo as demonstrated by high-throughput experiments that identify the sites where RBPs bind to endogenous RNAs such as cross-linking immunoprecipitation followed by high-throughput sequencing (CLIP-seq). One possible explanation is that RNA structures provide additional contextual features beyond the primary motif sequences  or CRISPR-based technologies have enabled systematic interrogation of the roles of diverse proteins, such as RBPs, helicases, and topoisomerases 7G-quadruplex sequences are enriched at promoters and they have been extensively studied in this context CD44 gene in vitro RBP binding to mRNA molecules One of the first exemplary cases of RNA G-quadruplex mediated regulation of alternative splicing was found in the hTERT gene, which encodes for the catalytic subunit of the telomerase enzyme, and one of its exon skipping events is promoted by the stabilization of intronic G-quadruplexes The genome-wide effect of RNA G-quadruplex formation over splicing factor binding remains unclear. High-throughput screening of chemical compounds via dual-color splicing reporters has identified two small molecules, emetine and cephaeline, that disrupt RNA G-quadruplex formation 8D. melanogaster for the DSCAM gene, where RNA-RNA interactions, mediated through multiple structures, regulate the selection of exons within arrays of mutually exclusive exons Long range interactions are important for splicing modulation Hairpins may also directly affect exon skipping events by a mechanism known as “looping-out”, whereby inter-intronic base-pairing RNA interactions can loop out exons to promote their skipping FGFR2, the formation of a hairpin structure is required for efficient splicing from two mutually exclusive exons and its splicing effect is not dependent on its primary nucleotide composition as shown using minigene assays The formation of RNA structures can also enhance RBP regulatory range by bringing distal regulatory elements in close proximity with their exon targets The fibronectin EDA exon is controlled by seven hairpins and a key exonic splicing enhancer is found in the loop of one of the hairpins, which is in turn bound by splicing regulatory proteins such as SRSF1 9The half-life and decay rates of mRNA transcripts in human cells influence protein expression levels. A number of features determine transcript stability including GC content, transcript length, polyA tail length, RBP sites, microRNA binding sites, and mRNA secondary structures Massively parallel reporter assays are high-throughput technologies that enable rapid measurements of thousands of sequences for their regulatory activity and have received widespread adoption in recent years 10Translation can be divided into four phases, initiation, elongation, termination and ribosome recycling Although the vast majority of eukaryotic translation start sites have an AUG codon, often the first AUG codon is bypassed, resulting in usage of more distal AUG codons and alternative protein isoforms. This process is referred to as leaky scanning, with a proportion of ribosomes initiating translation from downstream start codons. Leaky scanning and translational efficiency are influenced by the presence of secondary structures Secondary structures can conceal or expose binding sites for translation regulators, and it has been shown that certain RBPs bind preferentially at structured RNA while others have a preference for linear forms E. coli the lysine riboswitch when lysine is present it restricts translation initiation and also exposes RNase E cleavage sites Riboswitches are components of mRNA molecules that can bind a small molecule and directly control gene expression through RNA conformational changes, without proteins being involved. They are found in both prokaryotes and eukaryotes, with most discovered riboswitches being present in bacteria and archaea BRCA1, a tumor suppressor gene, a longer 5′UTR isoform is expressed only in breast cancer cells, resulting in a 10-fold decrease of translational efficiency due to the formation of a stable complex secondary structure RNA structures can directly interact with the translational machinery and influence the recognition of the translation start 11Early studies indicated that hairpin formation can influence translation efficiency Hairpins at the 5′UTR of ferritin-H and ferritin-L mRNAs act as an iron-responsive element controlling iron levels and are highly dynamic response elements to environmental changes 12RNA G-quadruplexes are enriched at 5′UTRs  where they show a higher frequency at the template strand, suggesting a relative depletion of G-quadruplexes at the RNA level It has also been shown that G-quadruplexes at 5′UTRs of eukaryotic genes can promote translation by favoring recognition of the IRES A study that used massively parallel reporter assays to investigate mRNA translation found that G-quadruplexes in the 5′UTR act as translational inhibitors, and that knockdown of G-quadruplex resolving helicases aggravated these phenotypes 13Regions that are predisposed to secondary structure formation, such as G-quadruplexes have an excess of germline and somatic mutations U2AF1 or SRSF2 mutants, mutations that are commonly observed in myelodysplastic syndrome, results in the accumulation of R-loops MAPT gene, also known as tau, in the interface between exon 10 and intron 10, there is a hairpin structure which can mask the splice site SMN2 and interference with the structure formation is a therapeutic target against the spinal muscular atrophy molecular phenotype Mutations of alternative splicing factors can lead to R-loop accumulation, which may compromise genomic stability and be relevant in the context of cancer pathogenesis TP53 and TERT, where they can modulate splicing and protein isoforms RNA G-quadruplex structures have been identified in several cancer genes, including C9orf72 gene which results in frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). These repeats form different secondary structures including G-quadruplexes, R-loops and hairpins The expansion of microsatellite repeats at 5′UTRs has been associated with aberrant translation and has been implicated in multiple disorders 14RNA secondary structures are pervasive, interact with RNA binding proteins and are linked to a large number of important functions, including transcription, splicing and translation. Even though the functional importance of secondary structures has been repeatedly demonstrated, the contribution of RNA structures in these processes remains incompletely understood due to the difficulties in identifying dynamic RNA structures and their mechanisms of action. High-throughput technologies enable the systematic investigation of RNA secondary structures and the design of experiments to quantify their contribution in transcription, splicing and translation enables directly testing their mechanisms of action. New methods to dynamically identify RNA secondary structures are gradually revealing their widespread and diverse contributions in gene regulation. However, it remains difficult to capture their dynamic changes across cellular conditions and their interplay with proteins. The degree to which RNA secondary structure formation is influenced by the tissue and cell type remains largely unstudied. The availability of large scale single cell assays will enable the investigation of associations between secondary structures, the presence of various sequence motifs, and expression levels of RBPs across different cell types. Even more interesting could be the combination of single cell technologies with different small molecules that stabilize specific structures.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Immunomodulatory biomaterials have the potential to stimulate an immune response able to promote constructive and functional tissue remodeling responses as opposed to persistent inflammation and scar tissue formation. As such, the controlled activation of macrophages and modulation of their phenotype through implant surface modification has emerged as a key therapeutic strategy. Methods: Online databases were searched for in vitro studies between January 1991 and June 2020 which examined the effect of titanium implant surface topography on the adherent macrophage phenotype at either the gene or protein level. Results: Thirty-nine studies were subsequently included for review. Although there was significant heterogeneity between studies, treatment of titanium surfaces increased the surface roughness or hydrophilicity, and hence increased macrophage attachment but decreased cell spreading. Physical coating of the titanium surface also tended to promote the formation of cell clusters. Titanium and titanium-zirconium alloy with a micro- or nano-scale rough topography combined with a hydrophilic surface chemistry were the most effective surfaces for inducing an anti-inflammatory phenotype in adherent macrophages, as indicated by significant changes in cytokine gene expression and or cytokine secretion profiles. Conclusions: The published data support the hypothesis that incorporation of specific topographical and physiochemical surface modifications to titanium can modulate the phenotypic response of adherent macrophages. Implants manufactured from titanium are a well-established treatment modality for the anchorage of prosthetic devices into bone in a process known as osseointegration. Titanium has been the material of choice for many medical devices such as hip, knee and dental implants due to its excellent mechanical properties and high degree of biocompatibility . BiomateImmune system biology has shown that within injured tissue including bone following implant placement, immune response mediators such as macrophages polarize into different phenotypes depending upon the signals received during their activation. These signals arise from their interaction with the titanium surface and thus differential macrophage responses may be critical in the overall osseointegration process given macrophages play dual roles as a major modulator of the initial healing response, as well as in the formation of osteoclasts involved in the later remodeling phase of bone homeostasis . BiomateThe phenotype of macrophages present at the implant site can be broadly defined by their functional properties and are generally referred to as having either a M1 or an M2 phenotype mimicking the Th1/Th2 nomenclature described for T-helper cells . The M1 For an implant to become osseointegrated, it therefore needs to trigger an overall osteo-formative response around the titanium surface at both cortical and cancellous bone levels. The interplay between M1 and M2 dominated microenvironments and the temporal modulation of the transition from M1 to M2 driven by interaction with the implant surface, may therefore be critical in determining the implant tissue response and ultimately the fate of the implant through the release of anti-inflammatory and proinflammatory cytokines, respectively.Despite its superior mechanical properties compared to other biomaterials, titanium does not possess any osteoconductive or osteoinductive properties by itself. Hence, to try and improve the rate and or degree of bone formation around implants, significant research on the modification of titanium’s surface properties such as its topography  and physiochemistry  has been performed in this regard. For example, studies using topographically modified implants that aimed to reproduce the morphology of native bone, clearly showed that microtopography and or hydrophilic implants performed better clinically at influencing contact osteogenesis ,11,12.Following these attempts to further improve the rate and or degree of osseointegration, and thereby subsequently enhance the ultimate success rate of implants, there has been a paradigm shift in the development of titanium implants away from being classically inert, to being immunomodulatory, i.e., able to stimulate a host immune response that provides an osteogenesis-enhanced environment for bone producing cells. However, in vitro and in vivo studies using the same biomaterials have often been shown to yield varying results . While sThe review search question was formulated using the ‘PICO’ framework , with deElectronic searches of the PubMed, Ebsco, Embase, Scopus, Wiley Online and Ebsco Dent databases were performed. The search strategy used in PubMed, comprising free keywords and Medical Subject Headings is presented in Articles were included for review if the studies considered the effect of titanium implant surfaces on macrophage phenotype at either the gene or protein level. Studies carried out entirely in vitro, or those in vivo studies which included some in vitro analysis, were included. Articles were excluded if implants other than dental implants were investigated, i.e., studies reporting on the effect of macrophages related to artificial joint replacement, plates for maxillofacial rehabilitation and other implants outside the oral cavity for purposes other than dental restoration. Studies on microorganisms which influence the regulation of the immune system and reactive lesions in patients that used dental implants except as a control group were also excluded. Review articles, letters, personal opinions, book chapters, conference abstracts and articles published in a language other than English were also excluded.All results retrieved from the searches were exported into reference management software and the duplicates removed. Those articles found to be appropriate through title and abstract screening were considered for further full-text review. Summary data from the selected full-text articles were extracted and split into six tables under the following subject headings: Reference, Author, Cell Type, Surface Modification, Surface Coating, Morphological Changes, Proliferation Changes, Osteogenic Activity, Gene and Cytokine Expression Changes and Study Conclusion(s). These subject heading were selected as the most consistent across the selected articles that broadly described (1) the methodology utilised ; and (2) the macrophage phenotypic changes observed . A glossary of abbreviations used is provided as To avoid any risk of bias in manuscript selection process, the ‘ToxRTOOL Ver. 1.0’ assessment tool was used to assess the data provided . This toA flow chart demonstrating the records retrieved from the database searches, included and excluded based on eligibility criteria is presented in The most studied biomaterial was commercially available titanium alloy (Ti-6Al-4V) used in either a disc or screw shape of various diameters and dimensions with a range of surface modifications . The surMacrophages studied included both primary human (7 studies) and rodent cells (5 studies) and those from human  and rodent monocytic cell lines, i.e., J774.A1 (3 studies) and RAW 264.7 (22 studies) .Cell shape changes have often been associated with different functional states of cells. McWhorter 2013) demonstrated that macrophages polarized toward different phenotypes in vitro also exhibited dramatic changes in cell shape, with M2 cells exhibiting an elongated shape compared with M1 cells . As such demonstrOverall, of the 39 studies selected in this review, 25 studies (64%) also reported morphological changes in cells that were attached to the test (modified) surface when compared to the control surface . To helpp-value < 0.001).Subsequent statistical analysis clearly supports the proposal that a rough titanium implant topography promotes the activation of an M2 macrophage phenotype in adherent cells , similarly downregulated pro-inflammatory cytokines IL-1β and IL-6, producing an anti-inflammatory microenvironment by inducing macrophage activation similar to the anti-inflammatory M2-like state and increased levels of the cytokines IL-4 and IL-10 [As for native titanium, hydrophilic modification of a zirconia-titanium alloy  indicating that data from these studies is reliable without any restrictions . None ofThe findings of the studies  includedMore specifically, titanium surface roughness is well known to increase the surface area of implants and ultimately enhance osseointegration when compared with smooth surfaced implants, however further modification of this rough surface to increase surface energy thus promoting super-hydrophilicity, not only down-regulates the initial pro-inflammatory response by macrophages, but up-regulates an anti-inflammatory phenotype able to further promote wound healing. Topography-directed macrophage polarization is therefore a biologically feasible mechanism to assist in the design of implant surfaces aimed at promoting osteogenesis and osseointegration. Unfortunately, this review of in vitro studies only does not allow any determination of whether an amelioration of inflammation or promotion of anti-inflammatory mediators may be responsible for improved osseointegration seen clinically with specific surface modified titanium implants.Of the 39 papers reviewed in this study, only one, Morra et al. (2015), provided data that suggested some caution should be used when assessing the potential impact of surface modification on the subsequent crosstalk between cells of the immune and skeletal systems . These aThe mechanism through which the presence of a rough, hydrophilic surface topography and chemistry affects the osteoimmune response of macrophages in comparison to other distinct topographies is not yet understood. Li et al. (2020) showed higher hydrophilicity and surface free energy in anisotropic nanowire-like textured titanium promoted the availability of RGD binding domains in fibronectin and fibrinogen adsorbed onto the titanium surface . These tIn a subsequent coculture model, MSCs on the nanostructured surface exerted greater effects on naïve and M1 macrophages, causing them to adopt a less inflammatory macrophage profile characterized by reduced expression of IL-6 and TNF-α and concurrent increased expression of IL-10 and Arg1.Alternatively, Pan et al. (2017) suggested the immunomodulatory properties of a plasma sprayed nanotextured surface, whereby macrophages were found to switch to M2 phenotype with decreased levels of inflammatory gene expression as well as increased expression of anti-inflammatory genes, were probably regulated by the ‘decisive’ role of cytoskeleton tension induced by specific cell shape when macrophages were cultured on this surface .2 nanotube layers could stimulate RAW 264.7 macrophages to secrete BMP-2 in contrast to smooth surfaces [Regardless of the precise mechanism(s) responsible, biomaterial surface cues from immuno-modulatory surfaces interpreted by macrophages, results in the secretion of distinct cytokine profiles that are able to modulate osteogenic gene expression in osteoblasts in a paracrine fashion. Previous studies by our group have shown this may occur as a result of upregulation of the TGF-β/BMP signalling pathway . Sun et surfaces . Increassurfaces .An unfortunate limitation of this study was that a meta-analysis of the included papers was not able to be performed due to the significant heterogeneity found in the methods and outcome data presented by the study authors. Semiquantitative analysis where possible however , did supFinally, to help establish the veracity of our PICO question without the complications of unknown or unaccounted for systemic effects, this review has focused only on data arising from in vitro studies. Given the positive outcome, further systematic assessment of appropriate in vivo studies is now required to delineate the role of the biomaterial surface on the modulation of macrophage phenotype on in vivo osteogenesis.In attempts to try and mimic the native tissue microenvironment, surface-modified titanium has been shown to modulate the function of adherent macrophages. Whilst any implanted device will result in an initial inflammatory response, modification of the device’s physiochemical properties to make it hydrophilic, treatment to add nanotube structures to the surface, or the addition of bio-functional surface coatings such as hydroxyapatite may reduce this initial inflammatory response and up-regulate a more regenerative phenotype in adherent macrophages, as suggested by the selected papers reviewed in this study. In vivo studies are now required to determine if these various modifications of potential implant surfaces will facilitate an enhanced rate or degree of osteogenesis not only in healthy individuals but also in immune-compromised patients.


IntroductionIt has been shown that the expression of the epidermal differentiation marker, Cornulin, declines with the progression of squamous cell carcinomas of several tissue types.ObjectivesThis study aims to examine Cornulin expression at the cellular level in various cell lines representative of the successive progression steps of oral squamous cell carcinoma (OSCC), a major type of head and neck cancer. This can pave the way for the utilization of this novel biomarker as a diagnostic and prognostic indicator for oral cancer and help guide treatment options.Study designWestern blotting was performed to measure Cornulin expression levels using standardized cell lysates from four different cell lines representing the successive steps of OSCC progression. Specifically, primary gingival keratinocytes, dysplastic oral keratinocytes (DOK), squamous cell carcinoma 25 (SCC25) cells, and Detroit 562 cells were used to represent normal oral keratinocytes, DOKs, locally invasive OSCC cells, and metastatic OSCC cells, respectively.ResultsCornulin expression was found to be downregulated with the progression from normal to premalignant to malignant cells. Quantitative analysis revealed that Cornulin is significantly downregulated by 3.4 folds in DOK cells, by 23.7 folds in SCC25 cells, and by 5.2 folds in Detroit 562 cells compared to normal gingival keratinocytes. Interestingly, Cornulin was upregulated by 4.5 folds in the metastatic Detroit 562 cell line compared to the locally invasive SCC25 cells.ConclusionAltogether, Cornulin proved to be differentially expressed at the cellular level in cell lines representative of the successive steps of OSCC progression. Specifically, we documented a gradual decrease in Cornulin expression with the progression from normal to premalignant to malignant cells. Notably, there is a significant increase in the expression of Cornulin in the metastatic cell line Detroit 562 compared to the locally invasive cell line SCC25, suggesting a possible correlation with the biological behavior and unique characteristics of these two different phenotypes. Oral cancer consists of a family of neoplasms that could arise in any part of the oral cavity, pharyngeal region, or salivary glands . Oral sqCRNN gene, is a 495 amino acid protein belonging to the family of S100 fused-type proteins, is located on chromosome 1q21 locus, and has tumor suppressor characteristics [Cornulin, encoded by the eristics , esophageristics , cutaneoeristics , and OSCeristics . This stCell cultureA total of four different cell lines were used to model the successive steps of OSCC progression. The squamous cell carcinoma 25 (SCC25) human OSCC cell line, Detroit 562 metastatic pharyngeal squamous cell carcinoma (SCC), and primary gingival epithelial cells were acquired from American Type Culture Collection (ATCC) , and the dysplastic oral keratinocytes (DOKs) were acquired from the European Collection of Authenticated Cell Cultures (ECACC)  via Millipore Sigma . All cell lines were cultured and maintained in their respective specialized media according to vendors’ instructions.Western blotThe four cell lines were separately harvested for their total protein content and the protein concentration was determined using the BCA protein assay . The cell lysate was prepared for the SDS-PAGE electrophoresis in radioimmunoprecipitation assay buffer (RIPA buffer) containing dithiothreitol (DTT), and the samples were then boiled for 5 minutes. An XCell SureLock system and Novex precast protein gels  were used for the SDS-PAGE electrophoresis, and the gel was run at two different settings: the first at 60 V for 40 minutes and the second at 140 V for 1 hour and 35 minutes. Once complete, the wet transfer was set up using a Mini-PROTEAN Tetra system  and Immobilon-P PVDF transfer membranes . This was carried out at 100 V for 1 hour in a pre-chilled transfer buffer.The resulting membranes were then blocked using 5% milk dissolved in 1x tris-buffered saline with 0.01% Tween (TBST). Membranes were blocked for 2 hours. The membranes were then incubated with the primary antibody in blocking buffer overnight at 4°C. A 1:100 dilution was used for the anti-Cornulin mouse monoclonal antibody . Membranes were then washed and incubated with the secondary antibodies for 1 hour at room temperature. A 1:5,000 dilution was used for the mouse IgGκ light chain binding protein secondary antibody . Subsequent labeling of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was utilized as a loading control. A 1:10,000 dilution was used for GAPDH mouse monoclonal antibody . Membranes were subsequently imaged using a BioRad ChemiDoc XRS+ system (Bio-Rad) and Amersham ECL western blotting detection reagent .Quantitative analysist-test.The images produced by the BioRad ChemiDoc system (Bio-Rad) were analyzed using ImageJ software . The pixel intensity of each sample was assessed compared to the GAPDH control and plotted as a fold difference in protein expression of Cornulin compared to GAPDH. Sample sets of three repeats were compared to each other using an unpaired Based on existing evidence on the role of Cornulin as a tumor suppressor gene product, we put forward a hypothesis predicting a gradual decrease in Cornulin expression in correlation to the stepwise progression from normal oral keratinocytes to premalignant keratinocytes to invasive and metastatic malignant OSCC keratinocytes Figure .To test our hypothesis, we utilized established human cell lines including primary gingival keratinocytes to model the normal oral mucosa, DOKs to represent the premalignant lesion known as leukoplakia, SCC25 cell line to represent locally invasive SCC cells, and Detroit 562 cell line as a representative for the metastatic phenotype Table .Western blot proteomic analysis on the four cell lines confirmed that Cornulin expression declines with the progression from normal oral keratinocytes to premalignant dysplastic keratinocytes to malignant OSCC cells Figure . StatistCornulin was first referred to as C1Orf10 and is known to be expressed in esophageal, oral, anal, cervical, and skin squamous epithelia ,13. It iSignificant differences in Cornulin expression were observed in dysplastic keratinocytes (DOK), locally invasive cancerous cells (SCC25), and metastatic cancerous cells (Detroit 562) in comparison to the normal gingival keratinocytes. Interestingly, a statistically significant increase in Cornulin expression was noted between the locally invasive SCC25 cells and the metastatic Detroit 562 neoplastic cells, possibly pointing to a role for Cornulin in the process of metastasis or perhaps as a result of the natural selection of few cells with metastatic abilities and higher than average Cornulin levels from the heterogeneous cell population of the primary tumor with variable Cornulin expression levels.A limitation of this study is the reliance on established human cell lines to represent the steps in progression of oral squamous carcinoma lesions. Although in vitro models for tumor progression, such as the immortalized, dysplastic, and cancer cell lines used in this study, have the advantages of fast growth rate , unlimitIn the clinical practice, OSCC, leukoplakia, and several non-cancerous oral lesions are difficult to distinguish from each other by physical examination alone ,18. ThisThis study has demonstrated significant quantitative differences in the expression of Cornulin across the successive steps of OSCC progression. These findings allude to the potential utility of Cornulin as a predictive and prognostic indicator. Follow-up studies to monitor Cornulin levels throughout the clinical course of disease and relative survival rate data are needed to firmly establish this promising biomarker in the clinical setting to supplement current pathological assessment and prognosis for patients afflicted with oral cancer.


Porphobilinogen deaminase (PBGD) haploinsufficiency  is characterized by neurovisceral attacks associated with high production, accumulation and urinary excretion of heme precursors, δ-aminolevulinic acid (ALA) and porphobilinogen (PBG). The estimated clinical penetrance for AIP is extremely low (<1%), therefore it is likely that other factors may play an important role in the predisposition to developing attacks. Fasting is a known triggering factor. Given the increased prevalence of insulin resistance in patients and the large urinary loss of succinyl-CoA to produce ALA and PBG, we explore the impact of reduced availability of energy metabolites in the severity of AIP pathophysiology. Classic studies found clinical improvement in patients affected by AIP associated with the administration of glucose and concomitant insulin secretion, or after hyperinsulinemia associated with diabetes. Molecular studies have confirmed that glucose and insulin administration induces a repressive effect on hepatic ALA Synthase, the first and regulatory step of the heme pathway. More recently, the insulin-mimicking α-lipoic acid has been shown to improve glucose metabolism and mitochondrial dysfunction in a hepatocyte cell line transfected with interfering RNA targeting PBGD. In AIP mice, preventive treatment with an experimental fusion protein of insulin and apolipoprotein A-I improved the disease by promoting fat mobilization in adipose tissue, increasing the metabolite bioavailability for the TCA cycle and inducing mitochondrial biogenesis in the liver. In this review, we analyze the possible mechanisms underlying abnormal hepatocellular carbohydrate homeostasis in AIP. Acute intermittent porphyria AIP, MIM 176000) is an autosomal dominant disease caused by a partial deficiency of the hepatic porphobilinogen deaminase PBGD, EC 4.3.1.8), the third enzyme of the heme synthesis pathway. AIP is characterized by acute attacks of abdominal pain, nausea, vomiting and fatigue that can be triggered by endogenous or exogenous factors, such as a low carbohydrate diet or fasting, that up-regulate the expression of the first enzyme of the pathway, δ-aminolevulinate (ALA) synthase 1  . Th. Th39]. To our knowledge, no molecular mechanism has been described that could explain the pancreatic hypersecretion of insulin in some patients with AIP. The most widely accepted model of the pathogenesis of type 2 diabetes postulates that a high-fat diet leads to obesity and insulin resistance . AccordiShanik et al.  describeAlthough the appearance of insulin resistance is not fully explained, our results suggest that the increase in insulinemia could be associated with the stabilization of porphyria, from both a clinical and biochemical point of view. This fact is supported by the known repressive effect of insulin on the transcription of PGC1α, a cofactor with the ability to repress ALAS1 transcription. Thus, insulin administration might be an innovative therapeutic tool for treating acute crises of porphyriaOne study already reported in 1970 showed how some patients experienced a clinical improvement when small amounts of insulin were administered together with carbohydrates during crises . ExperimHowever, a possible drawback of using this drug could be the iatrogenic induction of hypoglycemia . This coThe Ins-ApoAI protein has a natural tropism for the liver ,51 and aMore recently, the administration of glucose and the insulin-mimicking α-lipoic  acid impHowever, we found that prophylactic administration of the recombinant Ins-ApoAI protein and glucose was not sufficient to achieve biochemical protection against a severe attack induced after recurrent phenobarbital administration . The hepHeme synthesis is fully integrated into the metabolism network because it uses glycine and succinyl-CoA from the TCA cycle. Hence, stimulation of heme biosynthesis during an acute attack requires TCA cycle replenishment, driving pyruvate and acetyl-CoA from the degradation of glucose, fatty acid β-oxidation, and ketogenic and glucogenic amino acids. All these data suggest the close integration of heme biosynthesis into carbohydrate, lipid and protein metabolism.However, several studies have addressed the higher prevalence of insulin resistance and other alterations in glucose metabolism in patients with porphyria compared to healthy individuals. This alteration can be related to various causes such as the dysfunction of glutathione-insulin transhydrogenase and the chronic carbohydrate overload to which patients with AIP are exposed . HoweverIn addition, fasting secondary to nausea and vomiting and major losses of hepatic succinyl-CoA during an acute attack, as well as impaired glucose metabolism, reduce the availability of energy metabolites and could play a role in modulating the severity of porphyria attacks.The intravenous administration of an experimental insulin-ApoAI protein or oral supplementation with a molecule that mimics insulin can improve glucose therapy by the repressive effect of insulin on the transcription of hepatic ALAS1, by increasing the energy supply to replenish the hepatocyte TCA cycle, as well as by enhancing mitochondrial respiration.


Transcranial direct current stimulation (tDCS) is a non-invasive brain stimulation technique (NIBS) that has been proven to promote beneficial effects in a range of neurological and psychiatric disorders. Unfortunately, although has been widely investigated, the mechanism comprehension around tDCS effects presents still some gaps. Therefore, scientists are still trying to uncover the cellular and molecular mechanisms behind its positive effects to permit a more suitable application. Experimental models have provided converging evidence that tDCS elicits improvements in learning and memory by modulating both excitability and synaptic plasticity in neurons. Recently, among tDCS neurobiological effects, neural synchronization and dendritic structural changes have been reported in physiological and pathological conditions, suggesting possible effects at the neuronal circuit level. In this review, we bring in to focus the emerging effects of tDCS on the structural plasticity changes and neuronal rewiring, with the intent to match these two aspects with the underpinning molecular mechanisms identified so far, providing a new perspective to work on to unveil novel tDCS therapeutic use to treat brain dysfunctions. Transcranial direct current stimulation (tDCS) is a low-intensity constant electric current given through the scalp by using two electrodes. During stimulation, the current delivered flows through the brain layers and, depending upon the polarity of the applied stimulation, tDCS can have a depolarizing  or hyperpolarizing  effect . Differe2, 5 min; 2, 10 min). At the functional level, tDCS application over the motor cortex of healthy subjects enhanced motor learning and motor task performance . Later, the application of tDCS over different human brain areas showed multiple beneficial effects on both cognitive and motor function domains primarily modulating short- and long-lasting synaptic plasticity (Early tDCS studies demonstrated how tDCS was able to induce long-lasting and polarity-specific excitability changes in the human motor cortex and changes in synaptic efficacy including long-term potentiation (LTP)-like and long-term depression (LTD)-like effects . At last, in the patients with stroke, tDCS was able to ameliorate fine motor control as well as the recovery of upper limb function , and Alzheimer’s disease (AD) . It has function . DespiteIndeed, cellular and molecular studies mainly performed in animal models, provided evidence of plasticity mechanisms based on tDCS after-effects thus justifying and supporting its therapeutic potential for brain disorders based on impaired synaptic plasticity .Within this frame, in this review, we will summarize recent findings reporting plastic and metaplastic effects of tDCS and the beneath molecular mechanisms, focusing on those involved in the synaptic and dendritic spine changes, which are at the basis of neuronal network and connectivity rearrangements. In particular, we narrowed our review on preclinical and clinical works reporting positive functional outcomes specifically linked to tDCS-induced changes in plasticity and/or connectivity. Unless otherwise stated, the efficacy of treatment has been reported by the cited studies compared with the control groups receiving sham stimulation.Experimental and clinical results will be discussed and paralleled to pin down a clearer picture of the mechanisms underlying tDCS modulatory effects providing elements to perfection the therapeutic application of this non-invasive brain stimulation technique (NIBS) and to use its properties to shape the plastic elements of the brain.Many works have documented plasticity-related effects of tDCS in animal models. The ability of tDCS to modulate Hebbian plasticity might well explain its positive effects on motor learning and cognitive enhancement observed in animal and human models both in physiological and pathological conditions.2 for 20 min) and shed light on the molecular mechanisms underlying tDCS plastic effects. Basically, tDCS transiently increases intracellular Ca2+ initiating a molecular cascade that leads to an increased level of pCREBSer133 and its binding on BDNF promoter I. This event facilitates CREB binding protein (CBP) recruitment on the promoter region which, in turn, increases H3K9 promoter acetylation thus enhancing BDNF exon I expression .Hence, while the immediate effects of tDCS can be explained by membrane potential changes, long-term after-effects are the result of intracellular calcium dynamics and secondary synaptic plasticity key elements modulation . This ac2, 15 min) over the primary motor cortex (M1) followed by a repetitive transcranial magnetic stimulation (TMS) protocol elicits polarity-dependent facilitation on motor-evoked potentials (MEPs) in the healthy human subjects  preconditioning of the primary visual cortex followed by the application of TMS repetitive stimulation protocol can induce and modulate synaptic strength in healthy subjects, proving the occurrence of tDCS metaplastic mechanisms  when these were separated by a time interval resulting in an increase in MEPs as a metaplastic outcome in healthy subjects.tDCS metaplastic outcomes have been observed in several clinical studies. Indeed, unilateral anodal tDCS  induced metaplastic-like effects resulting in motor skill performance improvements similarly to what observed in humans (Ser831), a residue particularly important for the channel permeabilization and LTP expression providing the evidence of tDCS effect on LTP through a metaplastic modification.In line with this, n humans . SpecifiPhysiologically dendritic spines respond to synaptic plasticity stimulation by modifying their structural and functional features, contributing to learning and memory formation . On the Spines undergo activity-dependent changes playing a fundamental role in LTP and LTD through spine enlargement and shrinkage, respectively . In addiex novo spine formation or spine enlargement. The first event is represented by calcium entrance inside the cell where it binds to Ca2+-binding protein calmodulin (CaM) which in turn activates the holoenzyme CaMKII  on healthy rats  and on rats with altered auditory cortex structural plasticity . In this work comes up very clearly that tDCS exerts a global trophic action by increasing the number of dendritic spines in the auditory cortex, targeting apical dendrites of pyramidal neurons of layer II-III and V-VI in NH rats, while in NIHL rats, tDCS specifically targeted the most noise-affected layer II-III. In addition, the authors demonstrated that tDCS can modulate dendritic spine shape, increasing the number of both thin- and mushroom-shaped spines in NIHL, while in NH rats it increased only the number of thin spines. These results suggest that in the lesioned auditory cortex tDCS induces the formation of new spines but at the same time it stabilizes those already existing to preserve plasticity. TDCS-related structural changes were accompanied by increased synaptophysin levels and causally linked to increased level of BDNF in both NH and NIHL rats. TDCS effects on spinogenesis were, indeed, completely abolished in presence of ANA-12, the BDNF/TrkB receptor blocker.In this view, 2 for 20 min, single daily sessions for 3 consecutive days). This result was matched by enhanced forelimb strength and motor skilled performance and by increased synaptic transmission and plasticity at M1 layer II-III horizontal connections in tDCS-mice compared with the relative sham-controls. At the cellular level, tDCS increased neurotransmitter release and AMPA/NMDA ratio, events that are consistent with the increased spine density observed in tDCS mice. At the molecular level, the dendritic spine structural changes were accompanied by an increased level of BDNF and by an increased level of phosphorylation of key synaptic and structural plasticity players including CaMKII, CREB, and GluA1. Moreover, tDCS activated nitric oxide pathway leading to GluA1 S-nitrosylation and consecutively GluA1 phosphorylation at Ser831 (pGluA1Ser831) thus increasing single-channel conductance  has an effect on functional network synchronization not only within the target motor area but also coupling the former with neighbored premotor and sensorimotor areas.In line with this, Neuroplastic events and the reorganization of motor cortical connections have also been described as crucial processes in stroke recovery . In pati2, 15 min; 2, 12 min). 2, 30 min) applied over M1-modulated FC in patients with stroke. In particular, seed-based analysis of FC established that tDCS-enhanced FC within the motor and premotor regions in the lesioned hemispheres 1 week after the end of the stimulation protocol.In keeping with this, bilateral tDCS effects in stroke recovery have been tested in clinical studies showing encouraging results . Further2, 20 min, single daily sessions for 3 consecutive days) in a mouse model of ischemic stroke and showed that tDCS accelerated motor recovery by enhancing forelimb strength and ameliorating performances in both skilled and non-skilled motor tasks. The authors looked at possible effects on FC by recording local field potentials through epidurally implanted electrodes over M1 and somatosensory cortices and analyzing total coherence (TotCoh)—an index expressing global functional coupling of the LFP rhythms  on EEG and functional MRI signals and at mechanism of action on dendritic spine structure and dynamics to optimize its use to modulate, preserve, and restructure neural circuits.In the last two decades, tDCS’ positive impact on the cognitive and motor functions has been proven in the subjects with diverse psychiatric and neurological disorders. This NIBS has also been used on animal models to clarify the mechanisms behind its effects on brain plasticity and to understand, more broadly, the relationship between structural and functional changes at synapses.In literature, tDCS has been shown to modulate synaptic plasticity and, at the same time, to regulate the expression, activation, and localization of plasticity-related proteins such as BDNF, CaMKII, and GluA1. Intriguingly, these molecules are part of well-known signaling pathways at the basis of spine remodeling and actin polymerization.Recent works have indeed shown that tDCS can modify the number and structure of dendritic spine, indicating that this NIBS not only changes the efficacy of a synapse, but it also shapes it. These structural changes at synapses might favor rewiring processes improving the neuronal networks and resulting in enhanced learning and memory as well as amelioration of cognitive and motor functions clinically and experimentally observed following tDCS.In conclusion, in this review, we recapitulated the most recent evidence around tDCS’ plastic effects bringing into focus its capability to trigger dendritic spine rearrangement in neurons, thus, changing neuronal wiring patterns. Currently, treatments based on boosting rewiring are explored by the scientific community to treat acute brain damage as well as permanent and progressive brain dysfunctions. A deeper comprehension of tDCS effects on rewiring and structural plasticity at the basis of neuronal network rearrangement will allow tDCS to be exploited therapeutically for treating brain pathologies characterized by the widespread brain wiring abnormalities.SAB conceived the work and drafted the manuscript. MVP conceived the work and contributed making a critical revision of the manuscript. CG contributed by making a critical revision of the manuscript. All authors read and approved the final version of the review.


Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease that is characterized by the degeneration of both upper motor neurons and lower motor neurons, leading to motor and extra-motor symptoms. Although ALS has a low prevalence, its high mortality makes it one of the most intractable diseases. Mean survival time from the onset is 3–5 years, but some people could live for more than 10 years while slowly losing mobility. The pathogenesis of ALS remains unknown; however, recent research suggests that genetic factors may play an important role.The objective of the Research Topic “New Insights into the Molecular Mechanism of Amyotrophic Lateral Sclerosis Pathogenesis” was to gather original research articles and reviews illustrating the recent advances concerning the molecular mechanism of ALS pathogenesis. This Research Topic consists of four original articles.Zhang et al. proposed 27 candidate loci that were most possibly linked with sALS, which were identified for further analysis employing sequenom massARRAY technology and DNA sequencing in a separate case/control cohort of 239 sALS patients and 261 control subjects of Han ancestry from mainland China (HACM) ethnicity. They discovered that the polymorphism rs2619566 located within the contactin-4 (CNTN4) gene, rs10260404 in the dipeptidyl-peptidase 6 (DPP6) gene, and rs79609816 in the inositol polyphosphate-5-phosphatase B (INPP5B) gene were strongly associated with sALS in subjects of Han ancestry of China mainland. They also demonstrated the polymorphisms of rs2619566, rs10260404, and rs79609816 may affect the splicing, transcription, and translation of CNTN4, DPP6, and INPP5B genes and may play roles in the pathogenesis of ALS.Barton et al. used human pathology material from sporadic ALS patients, genetic ALS patients (carrying the C9orf72 mutation), and age- and sex-matched non-neurological controls to explore myelination at an RNA, protein, and structural level. They used electron microscopy to achieve (i) quantitative spatial profiling of the myelin basic protein (MBP) mRNA transcript, (ii) quantification of MBP protein, and (iii) the first quantitative structural assessment of myelination in ALS post-mortem tissues. Although significant dysregulation of subcellular transport of MBP mRNA was found in ALS patients compared with controls, there were no differences in MBP protein levels or myelination ultrastructure. They confirm that whilst there is a cell-autonomous mRNA transport defect affecting oligodendrocytes in ALS, it has no effect on myelin structure.Xu et al. compared the transcriptomic profile of the anterior horns of the lumbar spinal cord (AHLSC) between SOD1G93A mice and their wild-type (WT) littermates. When comparing pre-symptomatic/symptomatic ALS mice to WT mice, they discovered that bone marrow stromal cell antigen 2 (BST2) was considerably greater in the AHLSC of pre-symptomatic/symptomatic ALS mice. Immunofluorescent staining further confirmed that BST2 is mainly expressed on microglia in the AHLSC of ALS mice. These findings support the view that immune-related neuroinflammation plays a role in the early stages of ALS pathogenesis, and that BST2 could be used to treat microglia-mediated neuroinflammation later in the disease.Bigelow et al. explored the β-sitosterol β-D-glucoside (BSSG) model with a focus on motor function, and associated immunohistochemical markers. They used BSSG modeling to detect behavioral changes and immunohistochemical markers in mice. The results revealed that no changes in behavior were observed at any time point. Animals were processed for immunohistochemistry markers of substantia nigra integrity after behavioral testing. In the substantia nigra, immunohistochemistry indicated no alterations in the microglial marker Iba1 or the dopaminergic integrity marker tyrosine hydroxylase (TH) at any assessment point. The lack of any behavioral or immunohistochemical changes across groups implies an inability to duplicate earlier findings. Before the BSSG model can be used in preclinical studies, more research into the sources of variability in the model is required.In summary, the articles presented in the Research Topic “New Insights into the Molecular Mechanism of Amyotrophic Lateral Sclerosis Pathogenesis” cover four different aspects of ALS research, expanding our understanding of the disease and provide a valuable source of information concerning the molecular mechanism of ALS pathogenesis.All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.This study was supported by grants the Committee of National Natural Science Foundation of China , Education Department of Jiangxi Province (GJJ13198 and GJJ170021), Jiangxi provincial department of science and technology  and Health and Family Planning Commission of Jiangxi province (20181019) (All to RX).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Neurod1 whereas RGC development depends on Atoh7 and related bHLH genes. Lancelet  does not express Neurod or Atoh7 and possesses a few neurosensory cells with cilia that reach out of the opening of the neural tube. Sea-squirts (Ascidians) do not express Neurod and express a different bHLH gene, Atoh8, that is likely expressed in the anterior vesicle. Recent data indicate the neurosensory cells in lancelets may correspond to three distinct eye fields in ascidians, which in turn may be the basis of the vertebrate retina, pineal and parapineal. In this review we contrast the genetic control of visual structure development in these chordates with that of basal vertebrates such as lampreys and hagfish, and jawed vertebrates. We propose an evolutionary sequence linking whole-genome duplications, initially to a split between photoreceptor and projection neurons (RGC) and subsequently between pineal and lateral eye structures.Early in vertebrate evolution, a single homeobox (Hox) cluster in basal chordates was quadrupled to generate the Hox gene clusters present in extant vertebrates. Here we ask how this expanded gene pool may have influenced the evolution of the visual system. We suggest that a single neurosensory cell type split into ciliated sensory cells  and retinal ganglion cells . In vertebrates, development of photoreceptors is regulated by the basic helix-loop-helix (bHLH) transcription factor  Opsin-e>bipolar  , which ne cells . Lampreyne cells  express ne cells  with morne cells . One senne cells  yet alsone cells . In jawene cells  and, unlne cells . Furtherne cells . The difne cells . On the ne cells  shows thAtoh1 and Neurod1  which is not regulated by Atoh7  mouse  has been established following the initial discovery of proliferation of a column of retinal cells . The eartebrates . Downstrby Atoh7 . The nex(Neurod1  and inteod1 (Crc . The besy) mouse .Mitf, Pax6, Rax, Otx2, Six3 and Lhx2  signal transduction pathways to retain RPE under Mitf, BMP, RA and β-catenin control  require additional interactions under wingless / integrated  and Zic2 interact with Shh to regulate closure of the neural tube and reduced eye development is one consequence of failure of neural tube closure , Nodal and BMPs that provide the signals that will, eventually, close the neuropore  that cause the craniorachisis in mutant mice, implying these signal pathways in closure of the neuropore  from a single cell type (neurosensory cells) in lancelet and ascidians (Neurod1) and a set of neurons (under control of Atoh7) is proposed to parallel that in gnathostomes, which subsequently have developed many more basic neuron types  and four Neurod  genes that will allow to establish the bHLH genes in chordates.We now propose that the increased gene pool also allowed divergence of more complex visual structures, that is, lateral eyes and pineal that develop next to the neuropore. Neurosensory cells have dual function of light transduction and central projection, represented by cells that have a ciliary process with its own axon in the lancelet and ascidians. These cell types form three distinct clusters in the blastocyst life stage of the tunicate sea squirt scidians , we suggon types . Further6Neurod1) connecting via ribbons to the Atoh7-regulated cell families that send the visual information to the brain. The three different branches of vertebrate eyes  could have originated in the three retinal outputs present in ascidians that all develop near the neuropore.Central projection of visual information in vertebrates has its evolutionary origin in a single neurosensory, central projecting, cell type. This single cell type split into a sensory ciliated cell .BF was supported by 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.