Autism spectrum disorder (ASD) is a prevalent neurodevelopmental disorder characterized by impairments in social communication and social interaction, and the presence of repetitive behaviors and/or restricted interests. In the past few years, large-scale whole-exome sequencing and genome-wide association studies have made enormous progress in our understanding of the genetic risk architecture of ASD. While showing a complex and heterogeneous landscape, these studies have led to the identification of genetic loci associated with ASD risk. The intersection of genetic and transcriptomic analyses have also begun to shed light on functional convergences between risk genes, with the mid-fetal development of the cerebral cortex emerging as a critical nexus for ASD. In this review, we provide a concise summary of the latest genetic discoveries on ASD. We then discuss the studies in postmortem tissues, stem cell models, and rodent models that implicate recently identified ASD risk genes in cortical development. Autism spectrum disorder (ASD) is a neurodevelopmental disorder estimated to affect up to ~1% of the population. The condition is clinically diagnosed by persistent deficits in social communication and interaction, and the presence of repetitive behaviors and/or restricted interests. ASD severity varies greatly and can evolve along different trajectories over time ,3. AlongIn this review, we link recent genetic findings in large-scale ASD genomic studies to functional and mechanistic observations in stem cell models and rodent models. We begin by reviewing the current knowledge of the risk architecture, with a specific focus on the contribution of sequencing studies. We then discuss the most recent findings in human neuroanatomical studies and transcriptomic analyses from human postmortem samples, supporting the importance of cortical dysregulation in ASD pathophysiology. We then focus on the most recent discoveries of the mechanisms and pathways surfacing from manipulations of highly penetrant ASD risk genes in rodent models. The specific genes discussed in each section do not create an exhaustive list of all the ASD risk genes found to be involved in cortical development. We specifically selected autosomal dominant risk genes emerging from the latest exome sequencing studies on ASD ,7,8 and Enormous progress has been made in our understanding of ASD risk architecture. It is now clear that at least half of the difference in susceptibility amongst individuals depends on genetic variants that are common in the population  . Each ofIn parallel, early studies using microarrays ,22,23,24KDM5B), likely due to power limitations [Analyses are now probing the interaction between rare and common variation in shaping risk architecture ,50. The itations .Non-genetic risk factors, and in some cases their interactions with genetic factors, are also emerging ,52. One KDM5B, the gene with both GWAS [Gene discovery has brought major advances in our understanding of the biological processes and pathways associated with risk. ASD risk genes implicated by de novo variation cluster to two main biological processes: synaptic function and gene expression, most prominently chromatin remodeling ,8. KDM5Both GWAS  and sequoth GWAS  variatioCHD8) and T-Box brain transcription factor 1 (TBR1) [Chd8-depleted human and mouse progenitors [Transcriptomic analyses on postmortem brains sampled from unaffected individuals ,63,64 or1 (TBR1) ,75,76,771 (TBR1) ,72 and tgenitors ,74,75. Bgenitors ,66,68,69genitors , and RBFgenitors ,79. Impogenitors . These iGFAP and TCF7L2) [Within the cortex, single-cell RNA sequencing (scRNAseq) studies provide more granular resolution on cell types involved. Analyses on scRNAseq data from four fetal samples at mid-fetal development (post-conception weeks 17–18) show that ASD risk genes are predominantly expressed in developing glutamatergic neurons, both upper and lower layers, with interesting exceptions of genes highly expressed in oligodendrocyte precursors and pericytes . Interes TCF7L2) .TBR1 overlaps with that of visuomotor integrators in the visual and motor cortices, showing the relevance of gene expression co-regulation at a circuit level [In parallel to sequencing studies, neuroimaging and neuroanatomical studies have provided support for structural changes in cortical development in individuals diagnosed with ASD. For example, a study on postmortem brains from 11 affected children and 11 unaffected controls in the same age range revealed focal patches of altered laminar cytoarchitecture in the prefrontal and temporal cortices of the ASD group . Additioit level .During corticogenesis, a sequence of cellular events occurs to shape the developing cortex ,85 Figu. The firIn the next sections, we will discuss the evidence in rodent models implicating high-risk ASD genes ,7,8,38 iCTNBB1, CHD8, FOXP1, POGZ) and mRNA translation   A. While CTNNB1, the gene encoding for β-catenin, are associated with ASD [Ctnnb1, even via manipulation of upstream regulators, affect cortical neurogenesis [Apc, a gene necessary for β-catenin degradation [Beyond a well-established role in early brain patterning , the Wntwith ASD , ID [96,with ASD , and othwith ASD   [Chd8 with short hairpin RNAs (shRNAs) at E13 results in a decrease in progenitors from VZ/SVZ and an increase in neurons at E16, indicating a premature depletion of the NPC pool [Chd8 rescued this phenotype [Chd8 mutant lines have consistently shown megalencephaly [Chd8 mutant mice [Chd8 knockdown reduces Wnt-mediated transcriptional activity in mouse NPC in a luciferase assay, and co-expression of a resistant β-catenin construct normalized Wnt activity, the alterations in the number of NPC, and the gene expression dysregulation [CHD8 are enriched in ASD risk genes [CHD8 ,27,28,37CHD8 . Both huin mice) , and decin mice) ,106. In in mice) . For exaNPC pool . Co-exprhenotype . Howeverncephaly ,108,109,ncephaly  but see ncephaly ). While ncephaly . One of ncephaly ,72,73. Rant mice ,109 and ant mice ,72 have ant mice ,109 and/ant mice ,74,109. sk genes .DDX3X (DEAD-box helicase 3 X-linked) is another ASD risk gene [DDX3X are associated with DDX3X syndrome (OMIM 300958), an X-linked condition that manifests with ID, brain anomalies, movement disorders, and behavioral problems, including ASD [Ddx3x is highly expressed early during neurogenesis (E12.5–E14.5) in RGCs, and by E16.5 in both RGCs and neurons in the cortical plate (CP) [Ddx3x is expressed in all cortical layers and in neurons, astrocytes, oligodendrocytes, microglia, and ependymal cells [Ddx3x at E14.5 led to an increase of Pax6+ RGCs and Tbr2+ IPs, and a decrease in differentiated neurons. This imbalance is not caused by increased RGC apoptosis or reduced migration of the neurons to the CP, suggesting that Ddx3x is necessary for the differentiation of progenitors into neurons. While Ddx3x-depleted cortices have alterations in Wnt targets [RAC1  [isk gene ,39,112 tisk gene , possiblding ASD ,114. Ddxate (CP) . In postal cells . In vivo targets , a causaTPase 1) . Second,TPase 1) .PTEN (phosphatase and tensin homolog) is a tumor suppressor gene part of this cascade [PTEN are found in individuals with ASD [Pten (Pten−/−) have provided compelling evidence for a critical role of PTEN in progenitor proliferation and brain overgrowth [Pten haploinsufficient mice (Pten+/−) are calling the attention on carefully considering gene dosage when modeling ASD mutations in rodents. Pten+/− mice display brain overgrowth that appears perinatally and is maintained throughout adulthood. This phenotype is accompanied, in early phases, by increased progenitor proliferation and excess of neurons at birth, followed by postnatal apoptosis of excess neurons and excess glia in adulthood [Pten+/− mice [Pten−/− mice [Pten+/− mice, again supporting the role of β-catenin in proliferation, as opposed to both increase β-catenin and mTOR activity in Pten−/− mice [Ctnnb1 haploinsufficiency, but not Mtor in Pten+/− mice [Pten−/− mice.Another signaling pathway crucial for cell growth, proliferation, and survival is the PI3K/AKT/mTOR pathway.  cascade ,116. Mutwith ASD  and are ergrowth . Recent dulthood . Interes+/− mice  and by b−/− mice ,118,119.−/− mice . Compati+/− mice , or by m+/− mice  or inhib+/− mice  in Pten−PTEN loss resulted in increased protein kinase B (AKT) signaling, which delayed cell cycle exit resulting in prolonged proliferation of neural progenitor, including the oRG population [PTEN, also manipulating upstream regulators converge on defects in proliferation. For example, Lei and colleagues [+ RGCs in VZ and reduced NPCs exiting the cell cycle in a conditional knockout mouse for Kdm6a , a lysine demethylase implicated in ID  affecting Pten transcription. These mutant mice have increased methylation of the Pten promoter and, consequently, decreased PTEN expression. Re-expression of PTEN rescued the abnormalities in NPC proliferation, reinforcing the importance of this phosphatase in the regulation of NPCs proliferation and differentiation [Brain organoids derived from human and mouse embryonic stem cells engineered with homozygous deletions in PTEN also support a key role for this gene in progenitor proliferation. In the human organoids, pulation . This lepulation . Rescue lleagues  describentiation .FOXP1 (forkhead box P1) is part of a subfamily of transcription factors that temporally and spatially control the expression of a wide range of genes by binding to their promoter and enhancer sequences via a forkhead DNA binding motif [FOXP1 is a high-risk gene for ASD [Foxp1 expression peaks in the VZ of the mouse cortex between E10 and E12.5, and then decreases between E13.5 and E16.5 [Foxp1 knockdown by in utero electroporation (IUE) results in reduced post-mitotic cortical neurons and increased IP in the intermediate zone (IZ) [FOXP1 affects NSC differentiation into neurons and astrocytes by repressing Jagged1—a Notch ligand expressed in NSCs that is required for their maintenance in SVZ [Foxp1 is needed early during neurogenesis for the maintenance of the apical RG (aRG) pool [Foxp1 had increased aRG self-renewal and their differentiation into early-born neurons at the detriment of late-born neurons and glia, while mice knocked out for Foxp1 disrupted early-born populations, with no effects on late-born neurons [Foxp1 expression can extend the temporal window of early neurogenesis [Finally, ASD risk genes are also associated with the Notch signaling pathway, which modulates fate decisions in embryonic and adult NSCs ,125. FOXng motif . FOXP1 i for ASD  and muta for ASD  and refend E16.5 . Foxp1 kone (IZ) . In linee in SVZ . In vitre in SVZ . More reRG) pool . Mice ov neurons . These oogenesis .POGZ .Newly generated glutamatergic neurons migrate radially from the SVZ to the preplate, situated at the pial surface. Initially, neurons acquire a unipolar morphology by extending a basal process towards the pial surface and then they translocate their nucleus and cytoplasm towards it . Later, neurons adopt a multipolar morphology while in the IZ that allows them to search for cell polarity cues in the environment. Then, they acquire a bipolar morphology and use the radial fiber of RGCs to migrate towards the pial surface (locomotion). At E50–75 in humans and E14 in mice, as neurogenesis progresses and more projection neurons reach the preplate, this region splits into the marginal zone and the cortical plate (CP). Postmitotic neurons continue to occupy the CP in an “inside-out” fashion, eventually forming six cortical layers (L1–L6). Early-born neurons will constitute deep layers (first L6 and later L5); late-born neurons will populate upper layers  ,85 Figu.CHD8 regulates both proliferation  [Tcf4 haploinsufficient mouse model [Pogz delayed migration to the CP, but that defect tracked down to impaired neuronal differentiation (see above). Importantly, the study also showed that mice engineered to carry a de novo missense mutation found in a patient with ASD had mislocalization of upper-layer neurons into lower layers, a condition that was maintained until adulthood. Interestingly, this mutation did not affect GABA+ interneurons [Defects in neuronal migration can cause cortical malformations  that are frequent in individuals with ASD ,135,136.e above, ) and mige above, . Interesl period . Similarse model . Matsumuse model  describerneurons .DDX3X [ADNP [Foxp1, loss-of-function and gain-of-function mutations appear to impact cortical migration [Foxp1 present reduced migration of late-born neurons from the SVZ/VZ to CP. The defect could be rescued by wild-type FOXP1, but not a mutant preventing sumoylation [Foxp1 mutant mouse homolog to the human recurrent nonsense mutation p.Arg525*  or wher3X [ADNP ). In theigration ,142. Micoylation . Interesoylation . FurtherArg525* , are necessary for the switch of newborn neurons from multipolar to bipolar in the IZ. Failing in this process also underlies the delayed migration of late-born neurons to the upper layers in Bcl11a-deficient mice [Fmr1 knockout mice [Sema3c expression resulting from Bcl11a loss [Chd2 levels in Fmr1 knockout mice [Other ASD risk genes, including the BAF chromatin remodeling complex subunit  617101, ) and FMRent mice  and Fmr1out mice . Impaire11a loss  or decreout mice . In bothLaminar specification is an essential process for shaping the cortex into six layers, completed at around 7 months of gestation in humans and at postnatal day 7 in mice. With cell fate specification, the developing glutamatergic projection neurons acquire subtype specificity. Two main subtypes of excitatory neurons are generated: corticofugal and intra-telencephalic projections neurons. Corticofugal projection neurons extend their axons away from the cortex into the thalamus  or subcerebral nuclei . On the contrary, intra-telencephalic projection neurons (mostly located in upper L2 and L3) extend their axons within the cortex, to the ipsilateral (associative projection neurons) or contralateral  hemisphere. Most of the latter projections cross through the corpus callosum  .BCL11A and TBR1  [Cell type specification is orchestrated by a set of transcription factors ,158,159  606053, ,161). In 606053, , Bcl11a fication ,163,164.al tract . Furthere below) . Recentle below) ,165,166,During early mid-fetal development, after neurogenesis has peaked and migration has begun, dendritogenesis ,168, axoANK2), CHD8, and the dual-specificity tyrosine phosphorylation regulated kinase 1A  [Ank2 carrying an ASD-pathogenic mutation show increased axon branching, without a concomitant change in axon length or dendritic arborization [Ank2 functions in controlling axon branching by suppressing microtubule invasion into newly formed filopodia [Chd8 shRNA [Chd8-dependent regulation of genes implicated in axonal development, as detected in human neural progenitor cells [Dyrk1a or overexpressing it as wild-type or missense mutants homologous to those found in ASD individuals reduce the axonal length in mouse cortical neurons, revealing that the dosage of Dyrk1a is critical for proper neurite and axon development [There is a large body of evidence implicating altered neuronal morphogenesis and synaptogenesis in ASD risk ,172 FigD. ASD ri 614104) ,27,28,37rization . The proilopodia . Howeverilopodia , but resd8 shRNA . One potelopment .Chd8 knockdown in L2/3 pyramidal neurons results in reduced dendritic density and complexity [ARID1B . Dendritogenesis of L5 neurons requires, among other factors, Foxp1, as the embryonic expression of an ASD-relevant Foxp1 mutant causes shorter apical dendrite in mature L5 pyramidal neurons [Foxp1 in rat cortical neurons results in dendritic malformation and shorter dendrites, with no change in axonal length [Dendritogenesis requires an intact expression of ASD risk genes. In addition to defective axonogenesis, mplexity ,106, whimplexity . ARID1B ndrites, . Arid1b-endrites . Howevernt mouse , emphasion in L6 ,163,164.operties , further neurons . FOXP1 s neurons ,177. ForSYNGAP1) [SHANK3 [GRIN2B) [v1.2 (encoded by SCN2A) [FMR1) [Pogz show an increased density of dendritic spines in L2/3 pyramidal neurons [Tbr1 in L5 or L6 have, on the contrary, reduced density of mature excitatory and inhibitory synapses accompanied by an exuberance of immature filopodia, [As mentioned, the synaptic function is a nexus of liability for ASD ,170,171.SYNGAP1) ,179,180  [SHANK3 ,182, glu[GRIN2B) , voltagey SCN2A) , synapti) [FMR1) ,186 all  neurons . Mice halopodia, , indicatCortical inhibitory interneurons develop predominantly from areas in the subpallium: the medial and caudal ganglionic eminences  of the ventral telencephalon and the preoptic region . After dCHD2  PTEN levels were significantly decreased in interneurons following the activation of pyramidal cells at postnatal day 8; and, (2) there was a higher density of PV and SST interneurons after postnatal day 10 in a mouse with ablation of Pten in postmitotic MGE interneurons or with pharmacological inhibition of PTEN [The mouse homolog of the risk gene  615369, ,188) con 615369, . In the  the MGE . Nkx2-1 ogenesis ,191 and ogenesis . The lam mutants ,77. A morneurons . The mecrneurons ,77—are s5 and 10 . Interne5 and 10 . This re of PTEN .ARID1B, which seems to affect most predominantly the birth and migration of interneurons. Haploinsufficiency of Arid1b in mice has been shown to suppress the generation of inhibitory interneurons both in vivo and in vitro, due to the combination of decreased proliferation and increased apoptosis of ventral progenitor cells in the MGE [Pvalb gene  [Arid1b-deficient pyramidal neurons; [Arid1b pathophysiology depends on the interneuron populations is a recent study using mice with Arid1b haploinsufficiency selectively in PV or SST interneurons [While all of these ASD risk genes affect both pyramidal and interneurons populations in the developing cortex, there is one gene,  the MGE . One proalbumin) . These aneurons; ). In suprneurons . While brneurons .In this review, we argue that findings in rodent models, along with human genetic, imaging, and neuroanatomical evidence, pinpoint neocortical development as a critical spatiotemporal window for ASD liability. Most ASD risk genes are involved in multiple steps of corticogenesis, emphasizing again the heterogeneity of the risk architecture .Many risk genes are functionally interrelated: perturbations in one risk gene can have repercussions on the expression of other downstream risk genes. Tackling the function of such master regulators of other risk genes is a promising future avenue to understand convergences and divergences between risk genes. Further, observations in human stem cell-derived models  will be key to identify species-specific alterations that define the unique features of social behavior in humans.


The cardioprotective effects of dietary nitrate from beetroot in healthy and hypertensive individuals are undeniable and irrefutable. Nitrate and nitrate-derived nitrite are precursors for nitric oxide synthesis exhibiting an effect on cardiomyocytes and myocardial ischemia/reperfusion, improving endothelial function, reducing arterial stiffness and stimulating smooth muscle relaxation, decreasing systolic and diastolic blood pressures. Beetroot phytochemicals like betanin, saponins, polyphenols, and organic acids can resist simulated gastrointestinal digestion, raising the hypothesis that the cardioprotective effects of beetroots result from the combination of nitrate/nitrite and bioactive compounds that limit the generation of reactive oxygen species and modulate gene expression. Nitrate and phytochemical concentrations can be adjusted in beet formulations to fulfill requirements for acute or long-term supplementations, enhancing patient adherence to beet intervention. Based on in vitro, in vivo, and clinical trials, beet nitrate and its bioactive phytochemicals are promising as a novel supportive therapy to ameliorate cardiovascular diseases. These compounds, termed functional nutrients, provide benefits for the promotion and maintenance of human health ,2. Epidepressure ,5,6.Furthermore, secondary metabolites found in vegetables are involved in protective responses to different abiotic plant stresses . In the Beta vulgaris L. species) is a source of bioactive compounds, including dietary NO3−, betanin, antioxidant substances, and phenolic compounds (PCs), as well as a source of dietary fiber, minerals  and vitamins  [3− supplementation from beetroot requires smart formulations, to provide convenient serving portions while also containing effective concentrations of NO3− and bioactive compounds as a feasible alternative to the consumption of whole in natura vegetables.Red beetroot (complex) . However3− concentrations, making it difficult to convince individuals to adhere to certain proposed nutritional interventions [Traditional beetroot formulations, such as the cooked vegetable or fresh juice, must be offered in large amounts to reach pharmacological NOventions ,11,12.Advances in beetroot formulations and regimen administration are discussed herein, addressing the features of designed formulations regarding nutritional composition, functional phytochemicals, antioxidant capability, and the potential to improve NO production, enhance cytoprotective and ensure positive effects on hemodynamic parameters in healthy individuals and in patients presenting risk factors for developing cardiovascular diseases.Beta vulgaris belongs to the Betoideae subfamily, within the Amaranthaceae/Chenopodiaceae alliance. Although originated in Europe and North Africa, red beets are now naturalized in several countries worldwide. This species develops better in deep, looser, acid soils rich in organic matter and in mild (20 °C) to cold temperatures (10 °C). In cold weather, the beetroot plant goes through the reproductive stage of its cycle and the vegetable attains its best color, taste, and quality [ quality . The app quality . The roo quality ,15.3− accumulation in vegetables, although some agricultural management aspects, such as farming systems, soil fertilization, nutrient availability, and herbicide use must be considered to achieve this crop’s growth and development [Abiotic conditions, such as atmospheric humidity, extreme temperatures, low and high, exposure time to sunlight, and brightness can affect NOelopment .3− concentration capable of producing the cardioprotective effects. A combination of concentrated fresh beetroot juice and beetroot chips in different amounts can adjust bioactive compound concentrations and result in attractive and convenient NO3−-rich beetroot products. Formulations must be maintained at appropriate serving portions [Despite these factors, the consumption of a regular serving portion of beetroot in nature or minimally processed cannot reach the effective NOportions , avoidin3− sources to stimulate NO production and promote beneficial health effects [Several beetroot formulations have been designed and tested according to the group population to be supplemented, to produce convenient and attractive dietary NO effects ,12,18,19All distinct beetroot formulations, presenting particular physicochemical characteristics and nutritional composition, have been rated in clinical trials, to evaluate their health effects on distinct populations. Beetroot formulated as fresh concentrated juice or fermented juice, bread, powder, chips or crunchy slices, gel, and cereal-bar have all been used to supplement healthy and/or unhealthy volunteers ,3,20,21.3− supplementation [3− beetroot juice was obtained by the removal of NO3− by anion-exchange chromatography using PuroliteA-520E resin. Depleted-NO3− juice displays similar sensory characteristics and is indistinguishable in color, taste, appearance, and texture from NO3−-rich beetroot juice.Beetroot juice is the original formulation for dietary NOentation  and has Fermented beetroot juice can be formulated by spontaneous lactic acid fermentation or by use of starter cultures, enriching betalain content in a probiotic product used to supplement healthy men ,22,23,243− during sports competitions. The beetroot gel was prepared to mix the fresh and concentrated beetroot juice and powder obtained by crashing beetroot chips. The chips were prepared from frozen and freeze-dried beet slices crushed in a portable blender to prepare beet powder. The gel was then prepared with a mixture of beetroot juice, beetroot powder, and carboxymethyl cellulose at a 90:17:3 ratio. A depleted-NO3− beetroot gel was formulated by mixing depleted NO3− beetroot juice and Fuji apple  puree, in substitution to beetroot chips [A beetroot gel was formulated to supplement athletes with dietary NOot chips .3− administration to individuals who present risk factors for developing cardiovascular diseases (1). Beetroot-cereal bars were produced from the compaction of concentrated fresh beetroot juice and cereals, such as oats, wheat, soybeans, corn, and rice. This beetroot-cereal bar formulation is rich in nutrients and packed into 60 g pieces measuring 10 cm × 3 cm × 1.5 cm [Crunchy beetroot slices can be produced by means of microwave rotating vacuum drying in industrial facilities, resulting in a beet formulation retaining the taste, odor, and nutritious characteristics of their fresh equivalents . Recentldiseases . Beetroo× 1.5 cm ,29. Beet3− and saponin when compared to beetroot gel, chips, and juice, considering 100 g of each formulation (2−) (<0.5 mmol/100 g) and lipids were present at <1 mmol/100 g. Beetroot chips presented the highest carbohydrate content (and energy) when compared to other beetroot formulations.Beetroot-cereal bars presented the highest contents of protein, lipids, total dietary fibers, total sugars, fructose, glucose, sucrose, NOmulation . Maltose3− supplementation included the binding phase ingredients or carboxymethyl cellulose, respectively, increasing carbohydrate content and energy but maintaining low lipid concentrations in both formulations. The beetroot cereal-bar and gel are considered low-fat foods, according to Brazilian legislation for solid foods [The beetroot-cereal bar and beetroot-gel designed for NOid foods , but loww/w) fibers, in accordance with the Brazilian legislation [Beetroot-cereal bar, gel, and chips can be classified as dietary fiber sources, since they contain over 3% (islation . Beetroow) may promote undesirable modifications, such as non-enzymatic browning and crispness reduction, sensory attributes inherent to cereal bars and chips. In addition, moisture-rich food matrices can favor the growth of spoilage microorganisms, consequently decreasing product shelf-life [The physicochemical characteristics of beetroot food interventions were considered to design the new formulations, since high water activity  when compared to beetroot gel (6.30 ± 0.01 mmol), chips (6.90 ± 0.02 mmol), and juice (4.10 ± 0.01 mmol). NO2− contents ranged in low concentrations, from 0.10 ± 0.02 mmol to 0.20 ± 0.01, with no physiological significance  than beetroot juice and gel, previously used to treat individuals at risk of developing CVD. Considered a snack, beetroot-cereal bars can be administered between meals, facilitating adhesion to NO3− nutritional interventions. Due to the mixture of fresh juice and powder, NO3− amounts can be adjusted and the beetroot-cereal bar can be used for both acute or chronic NO3− supplementations, presenting beneficial cardiovascular system effects for both healthy and patient populations.Dietary NOificance . Most beentation ,33. On tFew studies report the saponin content of beetroot products, but it is known that saponin content and types may vary according to the plant cultivar and food matrix processing . SaponinBeta vulgaris L. roots have been shown to promote hypoglycemic effects in rats. [Several beneficial bioactivities are attributed to isoprenoid or terpenoid compounds, where an aglycone is attached by a covalent bond to one or two sugar chains, forming a mono- or di-desmoside. Furthermore, oleanoic acids, betavulgarosides II, III, and IV, found in in rats. .Beetroots are rich in OAs, similar to most plants, where these acids are used to cope with nutrient deficiencies, metal detoxification, and tolerance, and pathogens, as well as endophytic and symbiotic-microbe interactions operating at the root-soil interface . Humans Beetroot-cereal bars present the highest total OA content (9.19 ± 0.71 mg/g) compared to chips (5.34 ± 0.35 mg/g), gel (4.17 ± 0.35 mg/g), and juice (2.84 ± 0.7 mg/g) . Six dis+) salts, such as those formed by citrate, malate, and, less efficiently, by oxalate, and their contents can be influenced by soil characteristics, temperature and precipitation regimes, conventional or organic farming systems and post-harvest processing  [In roots, OAs are present as partially neutralized potassium (Kr chips) .Phosphoric acid and citric acid are predominant in beetroot juice, followed by oxalic acid and malic acid. Subsequently, shikimic acid, the precursor for the synthesis of aromatic amino acids such as phenylalanine, tyrosine and tryptophan, and betalains, are detected in high concentrations in organic and conventional farming beets, while citric acid, malic acid, and fumaric acid are also observed, but at lower concentrations . Malic aSome OAs are involved in the beneficial effect promoted by certain foods against oxidative stress, aiding in chronic and degenerative conditions, including cardiovascular diseases .2+) renal stone disease, due to its potential ability to complex with Ca2+ in urine, preventing the formation of Ca2+ oxalate (CaOx), the main kidney stone component. Malic alkalizing effects increase citrate excretion, improving hypocitraturia [Malic acid is a putative adjuvant in the conservative treatment of calcium -butenedioic acid, present in beetroot formulations cereal bars at 0.81 ± 0.1 mg/g, in chips at 0.63 ± 0.1 mg/g, gel at 0.41 ± 0.2 mg/g, and juice at 0.18 ± 0.1 mg/g , is a di3−.Succinic acid (butanedioic acid) was detected at 0.51 ± 0.01 mg/g in beetroot cereal bars . This acA large body of epidemiological evidence and meta-analyses has demonstrated that dietary PCs  can aid p-coumaric, and syringic phenolic acids, are not yet widely available. When tested in cell cultures and animals, some of these compounds have shown antibacterial, anti-inflammatory, antioxidant, anti-tumoral, and protective effects against reperfusion ischemia injury [To the best of our knowledge, human intervention trials assessing the direct effect of each beetroot compound, such as betagarin, betavulgarin, flavonoids, vanillic, a injury ,86,87,88p-coumaric, chlorogenic, p-hydroxybenzoic, syringic and vanillic acids, quercetin, and myricetin [Beetroot is a dietary source of PCs, although their concentrations vary according to the plant part, high in plant skin, and less concentrated in the crown and flesh ,90. PCs yricetin ,91.p-coumaric acid, rosmarinic acid, syringic acid, and vanillic acid , gel (25.45 ± 1.42 mg/100 g), and juice (18.00 ± 0.155 mg/100 mL) . PCs idelic acid .Gallic acid is described as the most abundant beetroot polyphenol ,91,92. BBeetroot products present caffeic acid concentrations of over 3.17 ± 0.45 mg/g .p-coumaric acid and ferulic acid in inhibiting copper-mediated oxidative modifications of human LDL, and consequently, in reducing the pathogenesis of atherosclerosis [Caffeic acid displays antioxidant and anticancer activities ,125,126,clerosis ,138,139.Chlorogenic acid is a class of compounds formed by hydroxyl cinnamic esters with quinine acid ,141. BeeA growing body of evidence supports the therapeutic effects of chlorogenic acid, including antioxidant activities, hepato, and cardioprotective properties, anti-inflammatory, anti-obesogenic and anti-hypertensive abilities, its ability to influence glucose homeostasis, and a neuroprotective role ,143,144.Caryophyllales order. Betalains are synthetized from tyrosine via the shikimate pathway [Betalains are aromatic indole derivatives, comprising nitrogen-containing and water-soluble pigments that confer beetroot color. These pigments are widely distributed in plant tissues and organs of members belonging to the  pathway ,155,156. pathway ,159,160 Red beetroot is an excellent source of betanin (75–95%) but it also contains lower concentrations of isobetanin, betanidin, and betaxanthin ,163. BetConsidering the betanin content found in beetroots and prospecting the amount in different beetroot formulations, beet chips would show the highest content (1274 mg/g) followed by juice, gel, and cereal bars  Table 2Table 2.In the food industry, betanin obtained from beetroot is used in sorbets, dairy derivatives like yogurts and ice creams, as well as meats , since betanin display good stability in a wide pH range (pH 3–7). The use of betanin as a natural red-violet dye for food is regulated by the Food and Drug Administration (FDA) and European Food Safety Authorities, under E-number E162 ,167. BetThe exact mechanisms of betanin absorption, metabolic breakdown, and route excretion in humans have not yet been completely elucidated, and identification of chemical intermediates, such as glucuronides, sulfates, or conjugates of methylated betalain, in plasma and urine is still scarce. It is known that the bioavailability of betanin can be influenced by the source matrix  and by human interindividual variability such as genetics, sex, age, and health conditions, which alter its absorption and excretion profile ,170,171.3+-TPTZ) to the ferrous ion (Fe2+-TPTZ) was demonstrated in the ferric reducing ability of plasma (FRAP), as well as in the reduction of the 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS+) radical in the trolox equivalent antioxidant capacity (TEAC) and oxygen radical antioxidant capacity (ORAC) assays [Betanin stability and antioxidant ability have been evaluated in assays mimicking in vitro human digestion and ex vivo colonic fermentation . Over ha) assays . In agre) assays .Human betanin bioavailability is low as 2.7% of total oral intake is excreted in urine and feces ,173,174.In vascular tissue, betanin antiradical activity maintains endothelial function and reduces the atherogenesis process . In addiSince excessive ROS are removed by betanin, NF-κB activation, and cytokine expression down-regulation are noted . BetaninTherefore, the effects of betanin on inflammation, oxidative stress, and diabetes in rodent models are well documented, these findings have not yet been confirmed in humans.3− from beetroot and green leafy vegetables may provide a physiological substrate for the generation of NO and other bioactive nitrogen oxides, leading to vasodilation and consequent improvement in cardiovascular function [Vegetables are important health-promoting foods in a balanced diet, due to the bioactivities of their phytochemicals ,190. It function .3− is well absorbed in the upper gastrointestinal tract. About 25% of dietary NO3− is captured by the salivary glands, where it is reduced to NO2− by commensal bacteria that express and secret NO3−-reductase enzyme in saliva [Granulicatella, Actinomyces, Veillonella, Prevotella, Neisseria, Haemophilus, and Rothia genera that live on the tongue can directly influence the NO3− to NO metabolism. Individuals with a higher abundance of NO3−-reducing bacteria were able to generate more salivary NO2− and, consequently, NO at a faster rate following the ingestion of dietary NO3− [3− to NO2− may be disrupted by antibiotic use or mouth rinsing with an anti-bacterial mouthwash. Oral nitrate-reducing microbiota are beneficial to the host and participate in the control of cardiovascular NO homeostasis [Dietary NOn saliva ,191. Theary NO3− . In conteostasis ,192,193.3− to NO2− in the oral cavity, the NO2− in the saliva is swallowed and reaches the stomach, where NO2− is non-enzymatically decomposed into NO and other bioactive nitrogen oxides in this acidic environment, by vitamin C or polyphenols. In addition to dietary sources, NO3− and NO2− can be endogenously originated from NO synthetized by the three isoforms of the nitric oxide synthase (NOS), family from the amino acid L-arginine and O2, namely the neuronal (nNOS or NOS-I) and endothelial (eNOS or NOS-III) isoforms, both constitutive and dependent on Ca2+-calmodulin, and the inducible isoform (iNOS or NOS-II). In addition, L-arginine is metabolized by arginase to L-ornithine and urea to eliminate excess nitrogenous compounds [After the conversion of dietary NOompounds .2− and NO3−. NO displays an affinity for lipophilic environments and accumulates in the lipid milieu, such as cell membranes and lipoproteins [NO is a low molecular weight compound (30.01 g/mol) with a short-life from 5 to 10 s) produced in gas form, containing 11 electrons in its valence shell with an unpaired electron. This radical character confers high reactivity to this compound, since it rapidly oxidizes to NOproteins . In huma to 10 s proteins ,194,195.Multiple pathways are used by NO to promote these actions, which depend on the cell tissue and the amount of produced NO . As ment•−), generating peroxynitrite (ONOO−), which, in turn, causes lethal damage to pathogens or tumoral cells by attacking copper and iron-metalloproteins [In immune cells, NO is produced as part of the inflammatory response by macrophages and other immune system cells, which express the inducible isoform type II NO synthase. The formed NO reacts with the superoxide anion  from guanosine-5′-triphosphate (GTP) catalyzed by the soluble guanylate cyclase (sGC), cGMP then activates protein kinases triggering phosphorylation of target enzymes, activating or inhibiting them [2−Tyr via ONOO− formation), and PKG-dependent phosphorylation of Ser residues of the target proteins [NO formed by the neuronal NO synthase (nNOS) acts as a neurotransmitter in the central and peripheral nervous systems, mediating synapse plasticity in nerve impulse transmission and favoring the secretion of neurotransmitters or hormones in neuronal junctions. The nervous impulse transmission occurs when glutamate, the main excitatory neurotransmitter, diffuses from the presynaptic terminal to bind to the N-methyl-D-aspartate type (NMDA) receptors at the postsynaptic terminal. NMDA receptors are coupled to Caing them . Howeverproteins .2+ through increasing Ca2+ extrusion rates and sarcoplasmic reticulum Ca2+-ATPase and decreased Ca2+ input from the extracellular medium. NO promotes phosphorylation of thromboxane-2 receptor and down-regulates P-selectin expression, preventing platelet activation and adhesion [− pathway, thereby inhibiting thromboxane-2 synthesis [Both endothelium- and platelet-derived NO prevent platelet aggregation and fibrin formation, inhibiting the spread of thrombi generation . NO exeradhesion . In addiynthesis .2+ pump, decreasing intracellular Ca2+ and promoting vasodilation as a result of diminished vascular tone [NO regulates vascular tone by diffusing across endothelial cells, reaching vascular smooth muscle cells and, through sGC, activates the sarcoplasmic Calar tone .2 levels and pH, any member of the NO2− reductase class enzymes, including xanthine, aldehyde oxidases, aldehyde dehydrogenase type 2, carbonic anhydrase, or deoxyhemoglobin, can reduce NO2− to NO [2− from the dietary-NO3− conversion improves oxidative phosphorylation efficiency, evidenced by an increased P/O ratio, indicating no uncoupling mechanisms, such as proton leaks towards ATP synthesis and turnover, improving ATP supply to skeletal muscle [Under low O2− to NO . The NO l muscle .3− in the stimulation of NO production and biochemical, hemodynamic, and vascular parameters following the intake of doses ranging from 6.3 to 22.0 mmol. Different beetroot product interventions have been formulated with distinct nutritional compositions and tested to achieve their claimed health effects  and 24 h ambulatory systolic (SBP) and diastolic blood pressure (DBP) [3− in 100 g of beetroot gel to 25 healthy and physically active runners for 1 week promoted increases in urinary NO3−, creatinine, and NO2− after 90 min of beetroot ingestion and after exercise. However, urinary levels of nitrous compounds were not related to changes in oxygen volume (VO2peak), time to fatigue during treadmill running, respiratory quotient, SBP, and DBP [However, to obtain the maximum cardioprotective effect of NOre (DBP) . A suppl and DBP .3− in 200 mL of beetroot juice, by 14 non-hypertensive obese males increased NO3− + NO2− (NOx) plasma concentrations (from 9.9 ± 8.4 μM to 47.0 ± 16.9 μM), which remained elevated until 1h post-intervention (54.7 ± 10.1 μM), while no changes in 24 h ambulatory SBP and DBP were detected [3− on brachial SBP, not sustained over 24 h, and carotid to femoral pulse wave velocity (cfPWV) [3− and a proved improvement in NO synthesis, these aforementioned results indicate a critical role of vascular impairment caused by some chronic non-communicable diseases such as hypertension, dyslipidemia, obesity, and aging, impairing NO effects in target tissues [3− supplementation benefits on physical performance have been suggested as more meaningful in healthy, but non physically active, individuals, rather than active ones. Physiological adaptations of endurance training may stimulate the expression and activity of the NOS enzyme through the endogenous pathway (via L-arginine/NO), increasing NO bioavailability. Due to the activation of the NO endogenous biosynthesis, the dependency of NO bioavailability derived from dietary NO3− supplementation seems to be reduced [Acute ingestion, of higher doses, of ≈13.0 mmol of NOdetected . Minimal (cfPWV) . Regardl tissues . Further reduced .3− if long-term supplementation is performed [3− in 70 mL beetroot juice to 24 older and overweight volunteers promoted an increase in NO synthesis, estimated by urinary and salivary NO3− and NO2−, resulting in SBP decreases of up to 7.3 mm Hg [3− by 34 drug-naive hypertensive patients for 4 weeks increased NO synthesis and cGMP levels, accompanied by decreases in arterial stiffness and a ≈20% improvement in endothelial function proven by decreases in 24 h ambulatory and home BPs [3− for 3 weeks by five patients presenting at least three risk factors for the development of CVD promoted increases in the NO synthesis and improvements in cutaneous microvascular conductance peak decreases in arterial stiffness  and decreases in SBP and DBP [However, in individuals presenting one or more risk factors for the development of cardiovascular diseases, the reversal of endothelial dysfunction evaluated by decreased large-artery stiffness and BP is achieved following the intake of up to 6.0 mmol of NOerformed ,204,218..3 mm Hg . The inthome BPs . The int and DBP .3− and beetroot supplementation can improve endothelial function. Beetroot juice intake provoked decreases in SBP  and DBP  [3− effects on endothelial function were associated with dose, age, body mass index (BMI), and previous SBP.A systematic review and meta-analysis study of randomized controlled trials demonstrated that inorganic NO8 mm Hg)  associat3− dose , age  and BMI . A critical review of experimental data confirmed that NO3− is a positive vascular endothelium effector, promoting vasodilatation and reducing blood pressure in both normal and hypertensive individuals. Beneficial effects were shown to be dependent on both NO3− dosage and continued intervention. Furthermore, beetroot product interventions designed to fulfill healthy effects are well tolerated even during long-term administration of super-concentrated beet products.Chronic beetroot juice ingestion improved vascular performance, evaluated by flow-mediated dilation (FMD) and endothelium functional effects according to the administered NO3− reported by several independent clinical trials performed worldwide are incontestable, increasing the importance of considering the individual susceptibilities and health status of each organism. Successful NO3− supplementation depends on the dosage and supplementation regimen, as well as the dietary source of NO3−. Impaired NO status can result from unsatisfactory production and/or reduced bioavailability, explaining why high NO3− supplementation dosages are necessary to fully overcome deficient endogenous NO synthesis or force the absorption rates. Therefore, beetroot consumption has emerged as an alternative, convenient and attractive way to obtain the cardioprotective NO3− effects in healthy individuals presenting risk factors for CVD risk, due to the higher concentration of NO3− per vegetable weight. However, the large serving portion of beetroot formulations necessary to achieve the effective dose of dietary NO3− can cause gastrointestinal adverse effects, limiting adherence to long term treatments. Furthermore, the aforementioned studies suggest that frequent daily doses of dietary NO3− for long periods would be necessary to result in beneficial effects on blood pressure and endothelial function and should be recommended to populations with compromised vascular responsiveness.The data compiled herein shows that the cardioprotective effects of beetroot NO3− intervention may be due to up- and down-gene expression in endothelial function regulation and platelet and macrophage recruitment and vasodilation, while also reducing imbalances in the redox state of the cardiovascular system, associated with mRNA inhibition of endogenous ROS generators, as well as NADPH oxidases. Meanwhile, activations of GPx, CAT, and SOD gene expressions are also noted, increasing the availability of scavenging enzymatic effectors [The cellular and systemic improvements observed after dietary NOffectors .3− supplementation highlight changes in the expression of genes encoding the calcium-signaling pathway, as well as in detoxification and antioxidant defenses. As a long-term effector, NO3− promoted up-regulation of genes encoding Ca2+—signaling proteins, including those able to increase Ca2+ in the cytosol, such as the sarcoplasmic Ca2+ channel, the ryanodine receptor 2 (Ryr2), the inositol triphosphate receptor ; and L-type calcium channel (Cacna1d and Ppapdc2), and also the broad spectrum protein regulators, like Ca2+/calmodulin-dependent protein kinase II  which, together, can cause smooth muscle cell relaxation [Transcriptional patterns in aged mice whole thoracic aortas after chronic NaNOlaxation ,222,223.3− intake indicates the up-regulation of genes enrolled in the lipid and carbohydrate metabolisms and the intracellular transport of molecules, as well as genes related to protein synthesis, turnover, and repair, including those encoding glucokinase, pyruvate dehydrogenase kinase, acetyl coenzyme A acetyltransferase 2, acyl CoA synthetase short-chain,17-dehydrocholesterol reductase, retinol dehydrogenase 11, farnesyl diphosphate synthase, nucleoside transporter, sodium/bile acid co-transporter family member, carbonic anhydrase 3, G2 cyclin, Rho GTPase, activating protein 9, glutamyl aminopeptidase and beta-lactamase 2 [A transcriptome analysis of ischemic stress responses following NOtamase 2 .Betanin promotes healthy benefits to the cardiovascular system due to its anti-radical scavenger effect, reducing the reactivity of these molecules, protecting from endothelial tissue from damage. Simultaneously, betanin down-regulates the mRNA of pro-inflammatory mediators while reinforcing endogenous antioxidant defenses. Furthermore, several lines of evidence implicate betanin in the transcriptional regulation of metabolic and antioxidant/detoxification genes . In humaBetanin may, therefore, be a supportive therapeutic alternative to attenuate the main mechanisms involved in CVD without any harmful effects. Although the exact mechanisms by which betanin exerts its cardioprotective role have not been yet fully elucidated, its ability to act directly on ROS/RNS species alongside the induction of the antioxidant and cytoprotective Nrf2-ARE pathway and suppression of the inflammatory NFk-B pathway in CVD can account for all betanin health-promoting benefits ,226. Fur3− from beetroot are reported as affecting cardiovascular and metabolic functions by regulating the gene expression patterns or modulating the activity of proteins and enzymes involved in these cellular processes. The cytoprotective effects of NO-derived from NO3−-NO2−/NO pathway may be collectively reinforced by certain bioactive compounds naturally found in beetroot.Interventions with dietary NOPCs and OAs identified at high concentrations in beetroot should also be considered antioxidant defense adjuvants in health promotion and chronic disease prevention. However, the most remarkable compound found in beetroot seems to be betanin. Thus, betanin could be a putative candidate to attenuate the oxidative stress status in humans.If previously described betanin effects in rodent models are confirmed in humans, it can be expected that short-term betanin intake will be able to attenuate the redox state of human cells by cytoprotective effects, regulating glucose and lipid metabolisms, controlling insulin resistance and lipid peroxidation, and, thus, protecting the cardiovascular system, liver, and kidneys from damage.


Coronavirus disease 2019 (COVID-19) is an acute viral pneumonia that had recently been found in humans. The first case was discovered in Wuhan, Hubei province, China, in December 2019. In this article, we aimed to demonstrate the clinical and radiological characteristics of COVID-19 patients in Somalia from 20 March 2020 to 20 April 2020.Twenty-seven patients that had a positive RT-PCR test between 20 March 2020 and 20 April 2020 were retrospectively observed. This study included 19 (70.4%) males and 8 (29.6%) females, and the mean age and range were 43 years (SD ± 14.0) and 27–70 years, respectively. The majority (59.3%) of COVID-19-infected patients had no obvious history of exposure to infected patients. The participants of our study mostly presented with dry cough 24 (88.9%) patients, fever 19 (70.4%), myalgia 18 (66.6%), and sore throat 16 (59.3%). Twenty-five of 27 patients had abnormal chest CT, while 2 (7.4%) patients had normal chest CT. The most common patterns of abnormality seen on chest CT in patients with COVID-19 were ground-glass opacity (GGO) 74.1%, crazy paving pattern 18.5%, consolidation 14.8%, and mixed GCO 11.1%. Also, the most common predominant lesion distributions were bilateral lung involvement (88.9%), peripheral distribution (77.8%), and lower lung predominance (63%). Particularly, lung cavitation, discrete pulmonary nodules, pleural effusion, and underlying pulmonary fibrosis or emphysema had not been observed.Dry cough, fever, myalgia, and sore throat were the most clinical presentations. GGO, crazy paving pattern, patchy consolidation, and mixed GCO were the typical chest CT manifestations. Coronavirus disease 2019 (COVID-19) is an acute viral pneumonia that was recently found in humans. The first case was discovered in Wuhan, Hubei province, China, in December 2019. COVID-19 is described by the World Health Organization (WHO) as an international public health emergency of public concern and also declared COVID-19 as a pandemic .Chen et al. demonstrated that history of exposure, clinical features, results of lab tests, and chest computed tomography (CT) findings were the basic diagnostic parameters of COVID-19 . Fever, The medical ethics committee approval letter of this retrospective study was received from the institutional review board of the hospital, and written patient informed consent was waived.From March 20 to April 20, 2020, a clinical and chest CT imaging data of 27 patients  with laboratory-confirmed reverse transcriptase polymerase chain reaction (RT-PCR) assay with throat swab samples diagnosed as SARS-CoV-2 pneumonia were included in this study.The throat swab samples were collected for RT-PCR in accordance with the WHO guidelines . The dia.All demographic characteristics, clinical signs and symptoms, laboratory results, and chest CT imaging data were retrospectively reviewed from the patient’s electronic medical records in Mogadishu Somalia Turkey Training and Research Hospital and De Martino Hospital information system (HIS)The clinical data analyzed were as follows: age, sex, comorbid conditions, exposure history, signs and symptoms , and laboratory results .All the patients in this study underwent non-enhanced chest CT examinations for detecting SARS-CoV-2 pneumonia in the supine position during end-inspiration.CT data was obtained using a 16-slice configuration CT scanner . The protocols were as follows: tube voltage, 120 kVp; automatic tube current modulation, 30–70 mA; and rotation time, 0.75 s. All images were then reconstructed with a slide thickness of 0.75–1.50 mmTwo radiologists (with 12 and 10 years of experience in thoracic CT) evaluated all imaging features blindly and independently in consensus.The epidemiological history, clinical signs and symptoms , and laboratory tests were available for both radiologists.We described three distributions: lung region, craniocaudal, and transverse. The main CT features included ground-glass opacities (GGO), mixed GGO, consolidation, crazy paving, air bronchogram, reticular pattern, subpleural linear opacity, bronchial dilatation, cavitation, intrathoracic lymph node enlargement, and pleural effusions. All terms were defined in accordance with the Fleischer Society guidelines .Statistical software SPSS  was used for all statistical analyses. Categorical variables were displayed as counts and percentages, and continuous variables were reported as mean ± standard deviation and range.Clinical, laboratory, and radiological data of 27 patients performed oropharyngeal swab tests and confirmed as 2019-nCoV were included in this study. Epidemiologically, there were two main groups of patients: 11 (40.7%) were healthcare workers who had close contact with patients at the hospital who had confirmed COVID-19 pneumonia and the remaining 16 (59.3%) did not have any obvious history of exposure.There were 19 (70.4%) males and 8 (29.6%) females studied; the mean age of patients was 43 ± 14.0 ranging from 27 to 70 years  patients were in the normal range, while in 4 (14.8%) patients below the normal range and in 2 (7.4%) patients above the normal range. Lymphocytes were below the normal range in most of the patients 21 (77.8%), while 19 (70.4%) patients had neutrophils above the normal range. Patients with increased CRP were more than those within the normal range , which appeared predominantly in the peripheral zones and most often involved lower lung lobes and segments which are consistent with the results of previous studies of COVID-19 and other viruses including SARS and MERS –15. FurtIn our study, we had several limitations; first, it was a retrospective study, and we had a comparatively small number of 27 patients. On the one hand, we only analyzed the clinical and chest CT manifestations at the initial presentation. Further studies are needed including clinical and radiological follow-ups.d-dimer, lactate dehydrogenase, and serum ferritin.Second, not all laboratory tests were done in all patients, including procalcitonin, AST, ALT, creatinine, urea, albumin, procalcitonin, Finally, the article only included non-pregnant adults. The clinical characteristics and radiological findings of pregnant women and children infected with COVID-19 are not clear.This study represents an early investigation of clinical characteristics and chest CT findings in patients with 2019 novel coronavirus (2019-nCoV). Dry cough, fever, myalgia, and sore throat were the most clinical presentations.GGO, crazy paving pattern, patchy consolidation, and mixed GCO were typical chest CT manifestations. The most common predominant lesion distributions were bilateral, peripheral and lower lung zone predominance. A small number of patients with COVID-19 pneumonia had had no obvious symptoms, and there chest CT scans were normal.


Skin aging is a specific manifestation of the physiological aging process that occurs in virtually all organisms. In this study, we used data independent acquisition mass spectrometry to perform a comparative analysis of protein expression in volar forearm skin samples from of 20 healthy young and elderly Chinese individuals. Our quantitative proteomic analysis identified a total of 95 differentially expressed proteins (DEPs) in aged skin compared to young skin. Enrichment analyses of these DEPs (57 upregulated and 38 downregulated proteins) based on the GO, KEGG, and KOG databases revealed functional clusters associated with immunity and inflammation, oxidative stress, biosynthesis and metabolism, proteases, cell proliferation, cell differentiation, and apoptosis. We also found that GAPDH, which was downregulated in aged skin samples, was the top hub gene in a protein-protein interaction network analysis. Some of the DEPs identified herein had been previously correlated with aging of the skin and other organs, while others may represent novel age-related entities. Our non-invasive proteomics analysis of human epidermal proteins may guide future research on skin aging to help develop treatments for age-related skin conditions and rejuvenation. Aging is a normal physiological phenomenon related to progressive deficits in various physiological variables such as cellular redox status, immunity, and metabolism, which contribute to disruption of tissue homeostasis. Skin aging is a specific manifestation of organ aging in the human body and results also from the combined effects of the above factors. Specific features of skin aging include thinning of the epidermis, degeneration of elastic tissues, reduction of melanocyte numbers, and impaired barrier function, which manifest as wrinkles, decreased elasticity, dryness, and dyschromia . Many inCellular senescence as the basis of endogenous  aging is largely determined by gradual, age-dependent shortening of the telomeres, small DNA sequences present at the ends of chromosomes. Telomerase enzymes act as reverse transcriptases to extend the telomeres and slow cellular aging; however, this topic is still controversial. For instance, increased telomere length may be associated with increased risk of melanoma, while shortened telomeres may confer greater risk for cutaneous squamous cell carcinomas .ROS are natural byproducts of cellular respiration. Imbalances between ROS generation and elimination can cause DNA mutations and cell damage, hinder protein synthesis, and induce apoptosis of skin cells . ResearcEnvironmental factors can also reduce skin elasticity and increase collagen fiber damage. UV radiation is the main cause of skin photoaging. Interestingly, repeated UV radiation causes damage to the dermis and dermal extracellular matrix by promoting chronic inflammation . UV raysThrough LC-MS-based proteomics and bioinformatics analyses, the present study evaluated differences in the expression of epidermal proteins between healthy young and elderly subjects to identify differentially expressed proteins (DEPs) possibly involved in skin aging. We also addressed the potential implications of our findings on skin aging mechanisms such as oxidative stress, metabolic reprogramming, and chronic inflammation induced by either physiological aging or photoaging.Conventional data dependent acquisition (DDA) mass spectrometry was used to establish and analyze a spectral library of human volar skin proteins obtained from 20 healthy subjects, i.e. 10 young and 10 elderly Chinese individuals. As a result, 9005 peptides and 1631 proteins were identified. To evaluate the functional significance of all identified proteins, Blast2GO software was used to perform gene ontology (GO) annotation analysis. Protein information and visualization of results are provided in Next, we carried out GO enrichment analysis of the identified DEPs . Based oNext, we predicted the potential functions of the identified DEPs using EuKaryotic orthologous groups (KOG), a database for the classification of protein orthologs . The mosThen, we conducted KEGG enrichment analysis to further characterize the biological functions of the identified DEPs . ResultsWe next used WoLF PSORT software to predict the subcellular localization of the identified DEPs . ResultsThe DEPs were next imported into the STRING database (STRING 11.0) to perform network interaction analysis of protein-protein relationships in the first 100 confidence intervals . Two maiAs the largest organ of the human body exposed to the external environment, the skin controls water loss and acts as a physical, anti-microbial, and immune barrier. Over time, the skin is subjected to both natural  aging and photoaging caused by UV rays. Main features of skin aging are thinning of the epidermis, degeneration of elastic tissue, loss of melanocytes, and decreased barrier function. The molecular basis of skin aging involves cell senescence, oxidative stress, chronic inflammation, reduced DNA repair ability, telomere shortening, point mutations in nuclear mitochondrial DNA, increased frequency of chromosome abnormalities, gene mutations, etc. . In thisGo and KEGG enrichment analyses indicated that several upregulated DEPs identified herein play a role in chronic inflammation, a common contributor to skin aging. KEGG analysis showed that ‘platelet activation’ and ‘complement and coagulation cascade’ were the two most enriched pathways. UV light induces the accumulation of ROS, which are recognized by the complement system, causing chronic inflammation. At the same time, excessive UV induction can directly increase the synthesis of complement factors in the skin . MoreoveWe found that several DEPs upregulated in aged skin are related to coagulation, immunity, and inflammation. Alpha-1-acid glycoprotein1 (ORM1) is a 41-43-kDa glycoprotein involved in the liver acute phase response to inflammation. ORM1 is also one of the most important plasma binding proteins, involved in the transport of several endogenous ligands related to inflammation. In addition, ORM1 can interact with leptin receptors , and itsMany blood particles and coagulation cascade proteins were identified as DEPs in aged skin. These include hemoglobin, haptoglobin (HP), antithrombin III (SERPINC1), and fibrinogen. A normal adult hemoglobin tetramer is composed of two alpha chains (HBA) and two beta chains (HBB). Hemoglobin mediates oxygen transport and is also an indicator of skin’s physiological function . HP is cAt present, the "brick wall" model is most commonly used to describe the skin barrier. Keratinocytes and their internal structural proteins form the "brick" of the skin barrier, and skin lipids serve as the "mud." Together they maintain skin stability and rejuvenation. Interestingly, studies on lipid metabolism and aging reported that the lifespan of experimental animals can be extended by manipulating genes involved in lipid synthesis (e.g. lipase overexpression), and even by fat removal. Indeed, it was suggested that blood lipids may be used as biomarkers of human aging . ResearcPhospholipase A2 group IVB (PLA2G4B) and phospholipase A2 group IVE (PLA2G4E) are members of the calcium-dependent phospholipase A2 family, also associated with lipid metabolism. Their role in the lipid signaling network in the skin may be mediated by binding to the phospholipase A2 receptor (PLA2R1) to influence pro-inflammatory signaling, autoimmunity, apoptosis, and aging. In this regard, Griveau et al.  reportedVarious protease inhibitors were also differentially upregulated in aged skin samples. Alpha-1-antichymotrypsin (SERPINA3) is present in senile plaques and accelerates the formation of Aβ fibers associated with Alzheimer's disease (AD) . In addiAmong the metal-binding proteins found to be upregulated in aged skin was melanotransferrin (MELTF) and ceruloplasmin (CP). Melanotransferrin is a transferrin homolog involved in iron metabolism and transport, whose activity impacts angiogenesis, plasminogen activation, cell migration, and tumorigenesis . CerulopATP6AP2 participates in the assembly of the vacuolar (V)-ATPase proton pump, thereby controlling proteolysis, autophagy, and glycosylation events. Accordingly, missense mutations in ATP6AP2 lead to decreased V-ATPase activity and defects in autophagy and glycosylation , 48. CarAged skin samples showed upregulated expression of clusterin (CLU), a chaperone that inhibits both accumulation of elastin in photoaged skin  and melaGlypican 1 (GPC1) is associated with control of cell growth, proliferation, and differentiation. A study showed that GPC1 is the predominant GPC isoform in human keratinocytes and its expression decreases significantly with age . This reSeveral other functional proteins were also found to be upregulated in aged skin. GC (vitamin D-binding) protein (GC) is involved in the transport and storage of vitamin D, clearance of extracellular actin, and chemotaxis of neutrophils and macrophages during inflammation , 72. TraWe detected 38 downregulated DEPs in our aged skin samples. Among them were five keratins , which is consistent with decreased epidermal barrier function with aging. Besides keratins, actin, proline-rich small protein 1 (SPRR1), S100 proteins, and loricrin are among the best studied skin barrier proteins. Several S100 proteins were downregulated in aged skin. They contain two EF-hand calcium binding motifs and are involved in the regulation of many cellular processes, such as cell cycle progression and differentiation. S100 proteins are one of the components of the cornified envelope (CE) in the skin . Our resExtracellular matrix protein 1 (ECM1) supports the structure and function of human skin. In the epidermis, ECM1 affects the proliferation and differentiation of keratinocytes. In the dermis, ECM1 binds to perlecan, type IV collagen, and laminin 332 and acts as a “biogel” helping to assemble and combine the basement membrane and dermal interstitium . ECM1 paInterestingly, all the DEPs with vesicle trafficking functions identified in this study were downregulated with age. These included synaptosomal-associated protein 23 (SNAP-23), synaptic vesicle membrane protein VAT-1 homolog (VAT-1), vesicle transport through interaction with t-SNAREs homolog 1B (VTI1B), and AP-2 complex subunit mu (AP2M1). SNAP-23 regulates the exocytosis of various inflammatory mediators by mast cells . VAT-1 pOther skin DEPs downregulated with age participate in cell proliferation, differentiation, and apoptosis. Rap1A, a member of the Ras oncogene family, is a small G protein involved in cell differentiation. Studies have shown that Rap1A regulates osteoblast differentiation through ERK/p38 signaling and promotes the transformation of epithelium to mesenchyme through the AKT signaling pathway , 89. BRIOther downregulated DEPs included enzymes related to biosynthesis and metabolism. Among these, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was the most interconnected (hub) node in our PPI analysis. GAPDH is a key enzyme in anaerobic glycolysis and its activity influences many processes, including carbohydrate metabolism, apoptosis, autophagy, vesicle trafficking, nuclear membrane fusion, signal transduction, transcriptional regulation, and telomere stability and maintenance –96. DysrHypoxanthine phosphoribosyltransferase 1 (HPRT1) plays a key role in purine nucleotide production through the purine rescue pathway . Malate Heat shock protein family E (Hsp10) Member 1 (HSPE1) has chaperone function and complexes with Hsp60 to assist protein folding in the mitochondrial matrix . ProteasOther DEPs downregulated in aged skin included copper homeostasis protein cutC homolog (CUTC), a shuttle protein that along with CP helps maintain copper homeostasis and is essential for energy generation, ROS removal, coagulation, and connective tissue cross-linking ; endoplaSeveral cytoskeleton-related proteins were also differentially downregulated in aged skin. Echinoderm microtubule-associated protein-like 4 (EMAL4) stabilizes microtubules . Myosin The functions of other DEPs found to be downregulated in aged skin are less clear. For example, V-set and immunoglobulin domain-containing 8 (VSIG8) may regulate hair follicle keratinization and hair stem differentiation , 119. PoThe results of our differential protein expression analysis in young vs aging skin may allow distinction between "young factors", i.e. proteins involved in angiogenesis, vesicle transport, skin barrier structure, cell proliferation and differentiation, and "aging factors", i.e. proteins related to chronic inflammation, oxidative stress, DNA damage, mitochondrial dysfunction, and signal transduction. Several of the DEPs identified in aged skin are associated to chronic inflammation, an important factor leading to skin aging, while others are related to the development of cancer or AD. Among the 95 DEPs identified, some had already been linked to skin rejuvenation or aging. For other DEPs, age-related expression was proved in other tissues and organs, but had not yet been demonstrated in skin tissue. In turn, and to the best of our knowledge, many other DEPs identified herein had not previously been related to aging. Therefore, this proteomics study provides reference proteins for future research on skin aging and may help develop treatments for age-related skin conditions and rejuvenation.Twenty healthy Chinese volunteers participated in the study. Mean age was 26.4 ± 3.06 years (range 20-35 years) in the youth group (control) and 59.9 ± 6.24 years (range 50-75 years) in the elderly group. Age grouping was not influenced by gender distribution, as determined by Fisher's exact probability method test (P = 0.370). The inclusion criterion was to have well preserved skin integrity at the sampling site. Donors with skin disease, other systemic diseases, or who experienced allergic reactions to the skin sampling tape were excluded from the study. No humectants or other cosmetics were used on the day of the experiment. The study followed the recommendations of the Medical Ethics Committee of Anhui Medical University, and all subjects provided informed consent.Sodium dodecyl sulfate (SDS), polyacrylamide, SDS-free L3 lysate, ethylenediamine tetraacetic acid (EDTA), dithiothreitol (DTT), Coomassie brilliant blue G-250, 40% ethanol, 10% acetic acid, acrylonitrile (ACN), and trypsin. Empore™ C18 47 mm extraction discs (Model 2215), Pierce C18 tips (10 μl bed), Thermomixer (MS-100), CentriVap vacuum concentrator, and related accessories were obtained from Thermo Fisher Scientific . Ultrafiltration membranes  were obtained from Pall Corp. . A Q Exactive HF mass spectrometer and an UltiMate 3000 HPLC system  were used for sample analyses.Stratum corneum samples from the volar forearm skin were collected using 3M medical tape. To this end, the sampling area was gently cleaned with a sterile cotton ball, the tape was applied onto the skin, and uniform pressure was applied intermittently for 1 min before tape removal. Five collections were sequentially performed at the same location. To minimize variability, in all cases the procedure was carried out by the same technician.Protein extraction was performed as follows: (1) tape-skin samples were cut into small pieces (0.5 x 0.5 cm) with a sterile blade and deposited inside a glass plate prior to transfer to a 1.5 ml centrifuge tube; (2) Add appropriate amount of lysis buffer without SDS to the sample, add 2mM EDTA, 1XCocktail, and place on ice for 5 minutes. Then, 10 mM DTT were added and samples were left to soak overnight; (3) following centrifugation at 25,000g, 4 °C for 15 min, the supernatants were recovered and treated with 10 mM DTT on a 56 °C bath for 1 h; (4) samples were then treated with 55 mM IAM, incubated at RT in the dark for 45 min, and centrifuged at 25,000g, 4 °C for 15 min to obtain the final protein solution in the resulting supernatant. Protein concentrations were next measured using the Bradford assay , and theAll samples were mixed equally (10μg/sample), and 200μg sample was diluted with 2 mL buffer A  and injected onto a Shimadzu LC-20AB liquid phase system. A 5 μm, 4.6 x 250 mm Gemini C18 column was used for liquid phase separation of samples. Gradient elution was applied at a flow rate of 1 mL/min: 5% buffer B  for 10 min, 5% to 35% buffer B for 40 min, and 35% to 95% buffer B for 1 min. Phase B lasted 3 min and 5% buffer B was equilibrated for 10 min. The elution peak was monitored at a wavelength of 214 nm and one component was collected every minute. The sample was combined with the elution peak of the chromatogram to obtain 10 components, and then freeze-dried.The dried peptide sample was re-dissolved with buffer A , centrifuged at 20,000 g for 10 min, and the supernatant was injected. Separation was performed on a Thermo Corporation UltiMate 3000 UHPLC. The sample first entered a trap column for enrichment and desalting. Then the sample was connected in series with a self-packed C18 column . Peptides were separated at a flow rate of 500 nL/min through the following effective gradient: 0-5 min, 5% buffer B ; 5-160 min, 5% to 35% buffer B; 160-170 min, 35% to 80% buffer B; 170-175 min, 80% buffer B; 176-180 min, 5% buffer B. The nanoliter liquid separation end is directly connected to the mass spectrometer. The peptides separated by liquid phase were sprayed into the nanoESI source and then entered a Q-Exactive HF tandem mass spectrometer for DDA and DIA analysis. DDA MS parameters were set to: (1) MS: 350–1,500 scan range (m/z); 60,000 resolution; 3e6 AGC target; 50 ms maximum injection time (MIT); 30 loop count; NCE 28; (2) HCD-MS/MS: 15,000 resolution; 1e5 AGC target; 100 ms MIT; charge exclusion, exclude 1, 7, 8, >8; filter dynamic exclusion duration 30 s; 2.0 m/z isolation window. The same nano-LC system and gradient are used for DIA analysis. DIA MS parameters were set to: (1) MS: 350–1,500 scan range (m/z); 20 ppm MS tolerance; 120,000 resolution; 3e6 AGC target; 50 ms MIT; 50 loop count; (2) HCD-MS/MS: 1.7 m/z isolation window; 30,000 resolution; 1e5 AGC target; automatic MIT; 50 loop count; filter dynamic exclusion duration 30s; stepped NCE: 22.5, 25, 27.5.DDA data were identified using MaxQuant (version 1.5.3.30) . PeptideSupplementary Figure 1Supplementary Table 1Supplementary Table 2Supplementary Table 3Supplementary Table 4Supplementary Table 5Supplementary Table 6Supplementary Table 7Supplementary Table 8


P2RX1, CAB39L, BLK, CSMD3, GPR65, ADRB2, CSMD1, PLPP4, ATF3, PRLL, STMN3, RORB, PIK3R1, PERP1, ACSBG1, MRTO4, CDKN1A, and EDA2R were identified to be potentially related to egg production. Furthermore, Kyoto Encyclopedia of Genes and Genomes analysis indicated neuroactive ligand-receptor interaction, cell adhesion molecules, peroxisome proliferator-activated receptor pathway, and cAMP signaling pathway might elicit an important role in formation of egg-laying traits by influencing ovarian follicle development. This study represents the first transcriptome analysis of various-sized follicles between HR and LR hens. These results provide useful molecular evidence for elucidating the genetic mechanism underlying ovarian follicle development associated with egg production in chicken.Egg production is an important economic trait in the commercial poultry industry. Ovarian follicle development plays a pivotal role in regulation of laying hen performance and reproductive physiology. However, the key genes and signaling pathways involved in the various-stages of laying hen follicular development remain poorly understood. In this study, transcriptomes of ovarian follicles at three developmental stages, the large white follicle (LWF), small yellow follicle (SYF), and large yellow follicle (LYF), were comparatively analyzed in hens with high (HR) and low (LR) egg-laying rates by RNA-sequencing. Eighteen cDNA libraries were constructed and a total of 236, 544, and 386 unigenes were significantly differentially expressed in the LWF, SYF, and LYF follicles of HR and LR hens, respectively. Among them, 47 co-transcribed differentially expressed genes (DEGs) in LWF and SYF, 68 co-expressed DEGs in SYF and LYF, and 54 co-expressed DEGs in LWF and LYF were mined. Thirteen co-expressed DEGs were found in LWF, SYF, and LYF follicles. Eighteen candidate genes, including  Egg-laying production efficiency is an important economic trait in the world-wide poultry industry. Efficiency is determined by the number of ovarian follicles destined for ovulation or atresia and the capacity of the oviduct to transform the ova into a hard-shelled egg . FolliclZP2, WNT4, AMH, IGF1, and CYP17A1, which serve as potential candidate genes for improving egg production  that were identified in the ovaries of Jinghai Yellow chickens related to egg production including oduction . It was oduction . These r MANBAL1 . Additiobreeding .FSHR, ESR1, ESR2 and PRLR, and FTH serving as representative candidate genes  in the ovaries of pre-laying and laying geese, with te genes . Luan ette genes . Ren et elopment . Wu et aelopment . These rThe Lohmann Brown (LB) layer, a commercial egg-laying breed, is characterized as possessing a high rate of egg production (HR), high laying peak, and great consistency in laying performance . In contTo reveal the potential candidate genes associated with egg production, high-throughput transcriptome technology was performed to enrich and analyze differentially expressed genes of follicles sized 3.5–5.5, 6.0–8.0, and 8.5–10.5 mm in diameter between the LB and JB breeds. The objective of this study was to identify key functional genes implicated in ovarian follicular growth and development (including follicle selection) that directly influences egg-laying productivity in hens, and to explore molecular functions, biological processes, and signaling pathways by which the candidate genes contribute to egg-laying production. The present study has laid a foundation for further work ascertaining the exact roles and regulatory mechanisms of the promising genes in ovary development. Finally, these results can be utilized for discovering potential molecular markers of egg-laying traits that can facilitate layer breeding and genetic improvement programs.All procedures executed in chickens were approved by the Institutional Animal Care and Use Committee (IACUC) of Jilin Agricultural University . All birds were sacrificed prior to the removal of organs based upon the IACUC Guidelines for the euthanasia of experimental animals [Permission No. GR (J) 19-030]. Euthanasia of the chickens is conducted fully compliant with Chinese applicable laws and regulations concerning care and use of laboratory animals, which was issued on the basis of the Regulations for the Administration of Affairs Concerning Experimental Animals of the State Council of the People’s Republic of China (1988). All of efforts have been made to minimize the suffering of the animals.Lohmann Brown and Jilin Black hens were reared in laying battery cadges according to the husbandry practices as previously reported . Under tTotal RNA was isolated from follicles of each hen using Trizol Reagent  according to the recommended manufacturer’s protocol, and concentration, quality, and integrity were determined by a Thermo NanoDrop 8000 Spectrophotometer . Three micrograms of RNA were used as input material for RNA sample preparations. The other parts of the samples were stored at –80°C for validation experiments.cDNA libraries were generated using the TruSeq RNA Sample Preparation Kit . Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. First strand cDNA was synthesized using random oligonucleotides and SuperScript II after fragmentation was performed utilizing divalent cations under elevated temperature in an Illumina proprietary fragmentation buffer. Second strand cDNA synthesis was subsequently carried out by using DNA polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities and enzymes were removed. Following adenylation of the DNA fragment 3′ends, Illumina PE adapter oligonucleotides were immediately ligated to prepare for hybridization. To screen cDNA fragments 200 bp in length, library fragments were purified by the AMPure XP system . DNA fragments with ligated adaptor molecules on both ends were selectively enriched by applying Illumina PCR Primer Cocktail in a 15 cycle PCR reaction. Finally, products were purified (AMPure XP system) and quantified utilizing the Agilent high sensitivity DNA assay on a Bioanalyzer 2100 system .Effective concentration of libraries were greater than 2 nM and libraries were qualified, sequenced, and paired-end sequencing with the 150 bp sequencing read length was performed. The cDNA libraries were sequenced on a Hiseq platform  by Shanghai Personal Biotechnology Cp. Ltd.1 were processed by filtering out low-quality reads and trimming the adaptor sequences using in-house perl scripts. Meanwhile, Q20, Q30, and N (fuzzy base) content of the raw data were evaluated. Follow-up analyses were based on clean data with high quality. All of the clean reads were aligned with the reference genome2 by using sequence alignment program HISAT 2.1.0 (To obtain clean reads (clean data), raw data (raw reads) of FASTQ formatAT 2.1.0 .3 based upon the normalized FPKM + 1 value between case group and control group. The resulting p-values were adjusted using the Benjamini and Hochberg’s approach for controlling the false discovery rate and an absolute value of the |log2FoldChange| (Log2FC) was used as the threshold for judging significance of the gene expression. Candidate genes with an adjusted p-value (padj) < 0.05 and Log2FC > 1 were identified as differentially expressed. Volcano plots to visualize up- and down-regulated genes of each sample were generated by using the R package “ggplot2” (version 3.5.0), based on the values of padj and Log2FC (4.HTSeq (ver. 0.6.1) software was used to count the reads mapped to each gene. The gene expression level was normalized based on its FPKM (Fragments per Kilobase of transcript per Million mapped reads) values using the Cufflinks package (v2.1.1). The expression pattern assessment for differentially expressed genes (DEGs) was performed by the Multi-Experiment Viewer (MeV) software (version 4.9.0)d Log2FC . To expl5), was utilized to annotate and analyze the molecular and functional characteristics of the DEGs , cellular component (CC), and molecular function (MF) of the GO database. A topGO version 3.0 package6 provided the tools for testing GO terms while accounting for the topology of the GO graph.An online biological tool, Gene Ontology , provided the comprehensive database resources for the KEGG pathway enrichment analysis of the DEGs. In this step, four databases were utilized to reveal high-level functions and biological systems of the DEGs, including Reactome8, KEGG pathway9, PANTHER10, and STRING11. Results with p < 0.05 were considered significantly enriched by DEG. Additionally, the UniProt (UniProt Knowledgebase12) database, EC (Enzyme Commission13) database, and eggNOG  database were used for functional and biological analysis of the enriched DEGs.Another online biological tool, KEGG  according to our previously described method . The prit-test for comparisons between the RNA-Sequencing and RT-qPCR determination after confirmation of normal distributions for non-parametric analysis. Values were presented as mean ± SEM and bars with superscript symbols that indicate the significant difference compared with control groups at ∗∗p < 0.01, ∗p < 0.05.Statistical analysis was fulfilled using the SPSS12.0 software package . Data weIn this study, a total of 18 cDNA libraries were constructed from HR and LR ovarian follicles ranging in size from 3.5 to 5.5 mm (LWF), 6.0 to 8.0 mm (SYF), and 8.5 to 10.5 mm (LYF) in diameter. Raw reads of each library ranged from 41.61 million up to 53.22 million reads. Among the raw reads, more than 97.35 and 93.87% had quality scores at the Q20 and Q30 levels, respectively. The highest N (base) content of the raw data was 0.0071% . By qual15 of the Ensembl database, unigenes were analyzed and annotated by using the NCBI and five other databases , cellular component (CC), and molecular function (MF) of the GO database. Based upon comparison of LWF follicles between HR and LR hens, 236 DEGs were assigned to 203 different GO terms covering 162 BP, 179 CC,159 MF, and the top 10 significant GO terms for BP, CC and MF are presented in P < 0.05, Comparison of significant GO terms of DEGs in the three follicle groups demonstrated the average top-10 term frequency in SYF follicles from HR and LR hens was much greater than undifferentiated LWF and preovulatory LYF –5. This To reveal what signaling pathways were significantly associated with egg production, and to further identify which DEGs annotated by the GO ontology were implicated in those transducing activities, enrichment analysis of the KEGG pathway was accomplished on the follicles between HR and LR hens. In LWF follicles, most relevant signaling pathways remarkably enriched consisted of cell adhesion molecules , PPAR signaling pathway (ko 03320), neuroactive ligand-receptor interaction (ko 04080), fatty acid biosynthesis (ko 00061), AMPK signaling pathway (ko 04152), cAMP signaling pathway (ko 04024), adipocytokine signaling pathway (ko 04920), ECM-receptor interaction (ko 04512), p53 signaling pathway (ko 04115), and insulin secretion (ko 04911). Collectively, there were 44 DEGs (24 up- and 20 down-regulated) to be significantly enriched in these 10 KEGG pathways .In SYF follicles, the prominently enriched KEGG pathways included phagosome (ko 04145), CAMs (ko 04514), the NF-kappa B signaling pathway (ko 04064), the calcium signaling pathway (ko 04020), the Toll-like receptor signaling pathway (ko 04620), neuroactive ligand-receptor interaction (ko 04080), and the GABAergic synapse (ko 04727). In total, 87 DEGs (80 up- and 7 down-regulated) were implicated in the seven signaling pathways .O-glycan biosynthesis (ko 00512), the PPAR signaling pathway (ko 03320), D-arginine and D-ornithine metabolism (ko 00472), carbohydrate digestion and absorption (ko 04973), the JAK-STAT signaling pathway (ko 04630), the cAMP signaling pathway (ko 04024), and cytokine-cytokine receptor interactions (ko 04060). Totally, 71 DEGs (57 up- and 14 down-regulated) were encircled in the 11 signaling pathways , CAMs (ko 04514), circadian entrainment (ko 04713), glutamatergic synapse (ko 04724), mucin type pathways .ACSBG1, CNTN1, DMB2, FABP7, GRIK1, GABRA1, GABRB2, GABRG1, GABRQ, GALR2, GRIA2, GRIA4, GRIN2A, HRH1, HRH2, HTR1A, HTR2A, ITGB2, LEPR, LRRC4C, NCAM1, NLGN1, NRCAM, PIK3R1, PRLR, PTPRC, P2RX7, RYR2, VCAM1, VCAN, VIP, and VIPR2, et al.  in LWF, SYF, and LYF follicles and two co-enriched pathways  in LWF and LYF follicles of HR and LR hen ovaries. The relevant up-regulated DEGs included , et al. –13. The , et al. –13.P2RX1, CAB39L, BLK, CSMD3, GPR65, and ADRB2  were up-regulated in LWF and SYF follicles sampled from HR and LR hens. For the candidate genes from SYF and LYF follicles, three genes  were up-regulated and two genes (PRLL and STMN3) were down-regulated at the different development stages examined, but candidate RORB was down-regulated at the SYF stage, while up-regulated at the LYF stage. For the candidate genes co-expressed in LWF and LYF follicles, three genes  were up-regulated; however, three genes  were down-regulated. For the DGEs co-expressed in LWF, SYF and LYF follicles, one gene (GABRA1) was up-regulated, two genes (NDUFAB1 and NCAM2) were down-regulated, and a novel gene (LOC424014) was down-regulated in the LWF and LYF follicles, while it was up-regulated in the SYF group. Our results show the significantly differential expression levels of the most transcripts detected by RT-qPCR analysis were consistent with the observations determined by RNA-seq  and one novel gene (LOC424014) were characterized.In this study, high-throughput transcriptome technology was performed to analyze and identify DEGs of the follicles sized 3.5–5.5, 6.0–8.0, and 8.5–10.5 mm in diameter between the LB and JB chicken breeds. To our knowledge, this is the first study to highlight potential pivotal genes associated with high and low rates of egg production in hen ovarian follicles at various developmental stages via RNA sequencing analysis. Our data showed 47 co-expressed DEGs in LWF and SYF follicles, 68 co-expressed DEGs in SYF and LYF, and 54 co-expressed DEGs in LWF and LYF. These DEGs were significantly related with various physiological functions of follicle growth and development, which have potential to be involved in hen egg-laying production. Moreover, by further enrichment and analyses of the DEGs, 13 of the co-expressed DEGs were obtained in LWF, SYF, and LYF follicles of HR and LR hens. Among them, three known genes , which is one member of the gamma-aminobutyric acid signaling pathway of the PANTHER subfamily (PTHR18945:SF514). Previously, studies demonstrated the γ-aminobutyric acid type A receptor  is a ligand-gated Cl– ion channel , NDUFAB1 subunit of mitochondrial complex I (NADH: ubiquinone oxidoreductase), which is involved in cytosolic and mitochondrial fatty acid synthesis (FAS) pathways. The active form, holo-ACP, serves as a FAS platform, using its 4′-phosphopantetheine group to present covalently attached FAS intermediates to enzymes responsible for the acyl chain elongation process , encoded by the culation . It was culation . In thisLOC424014 gene encodes a putative methyltransferase that interacts with histone modifications to catalyze DNA methylation to inhibit gene transcription and induce gene silencing  of Jilin Agricultural University . All birds were sacrificed prior to the removal of organs based upon the IACUC Guidelines for the euthanasia of experimental animals [Permission No. GR (J) 19-030].XC, XS, and RX conceived and designed the experiments. XC, XS, IC, NQ, XX, SL, and RX performed the experiments. XC, RX, XX, and JG analyzed the data. XC and RX wrote the manuscript. JG reviewed and edited the manuscript. All the 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.