Taurine is a naturally occurring sulfur-containing amino acid that is found abundantly in excitatory tissues, such as the heart, brain, retina and skeletal muscles. Taurine was first isolated in the 1800s, but not much was known about this molecule until the 1990s. In 1985, taurine was first approved as the treatment among heart failure patients in Japan. Accumulating studies have shown that taurine supplementation also protects against pathologies associated with mitochondrial defects, such as aging, mitochondrial diseases, metabolic syndrome, cancer, cardiovascular diseases and neurological disorders. In this review, we will provide a general overview on the mitochondria biology and the consequence of mitochondrial defects in pathologies. Then, we will discuss the antioxidant action of taurine, particularly in relation to the maintenance of mitochondria function. We will also describe several reported studies on the current use of taurine supplementation in several mitochondria-associated pathologies in humans. Indearginine ,30. In aarginine , which iarginine , the mit2, produced via the tricarboxylic acid (TCA) cycle, deliver electrons along the electron transport chain to reduce molecular oxygen to water. The influx of electrons along the electron transport chain creates a transmembrane proton gradient that drives ATP synthesis via the ATP synthase (FoF1 complex synthase) [2−·) [2−· is dismutated to hydrogen peroxide (H2O2) and molecular oxygen. H2O2 can be further partially reduced to a hydroxyl radical (OH−), which is a highly reactive species [Mitochondria are cellular organelles that regulate various essential cellular processes ,35,36,37ynthase) . A consee) [2−·) . When ca species .2− into water and H2O2 [2O2 into water and oxygen [The mitochondria are the main source of superoxide production, primarily via Complex I and Complex III of the electron transport chain ,44. Undeand H2O2 . Both cad oxygen ,70. Whend oxygen ,72. Ofted oxygen ,74.Impairment of mitochondrial function has been commonly reported in pathologies such as aging, cardiovascular diseases, mitochondrial diseases, metabolic syndrome, cancer and neurological disorders ,13,14,75Bos taurus [5) is synthesized in the liver from methionine (1) or cysteine (2) to produce hypotaurine (4) by cysteine dioxygenase and cysteine sulfonic acid decarboxylase (CSAD). Cysteine dioxygenase converts methionine or cysteine (1–2) to cysteinesulfinate (3), while CSAD converts cysteine sulfinate (3) to hypotaurine (4). Hypotaurine (4) is then readily oxidized to taurine (5), which may be excreted directly or as a conjugate with bile salts such as taurocholate (6) [Taurine or 2-aminoethane-sulfonic acid is a unique amino acid as it has a sulfonyl group on the C-terminus and an amino group residing on the β-carbon a rather s taurus . As desclate (6) .In most mammals including humans, rodents and some primates, taurine is considered a conditionally essential amino acid, as cysteine dioxygenase and CSAD are present abundantly. Mammals primarily depend on taurine biosynthesis in vivo  and partTauT). This TauT has a higher affinity for β-amino acids, such as taurine, but a lower affinity for α-amino acids [TauT gene [Taurine is ubiquitously expressed in most tissues, particularly in the excitable tissues such as the heart, retina, brain and muscles . The intno acids . The impauT gene ,127, as auT gene ,129 or gauT gene ,131.TauT (TauTKO) were generated by Ito’s group [TauT, it is convincing that taurine indeed has multiple physiological roles that include maintaining mitochondrial function.Two mouse models lacking the ’s group  and Wars’s group . Both Ta’s group ,127. As ’s group ,133,134.’s group . Further’s group . In addi’s group . The Tau’s group ,137. Def’s group . InteresSimilarly, pharmacological inhibition of taurine transport using β-alanine or GES in both in vitro and in vivo studies resulted in significant pathological conditions, which include atrophic cardiac remodeling ,139,140,Leu(UUR). Shetewy et al. [Various in vitro and in vivo studies have reported that taurine supplementation protects against mitochondrial dysfunction. Homma et al.  recentlyy et al.  showed ty et al.  also shoy et al. . The afo5u) and 5-taurinomethyl-2-thiouridine (τm5s2u). These conjugates are linked to the role of taurine as an antioxidant. Taurine conjugates with uridine at the anticodon wobble position of mitochondrial tRNALeu(UUR) or mitochondrial tRNALys to form τm5u and τm5s2u, respectively [Taurine is primarily a free amino acid, although it does conjugate with bile acids to form taurocholate . In 2002ectively ,146. Theectively , are reqectively .Leu(UUR). The importance of post-transcriptional modifications in tRNAs lies not only in ensuring proper codon recognition for translation accuracy, they are also essential in improving the efficiency of tRNA and facilitating the codon recognition by elongation factors or aminoacyl-tRNA synthetases. Consequently, a mutation in the post-transcriptional modification of mitochondrial tRNALeu(UUR) can affect protein synthesis as it can decrease RNase P processing, tRNA stability and aminoacylation, resulting in an abnormal tRNA conformation [Based on the wobble hypothesis, the nucleoside at the first position of the anticodon forms hydrogen bonds with the third nucleoside of the codon, forming a wobble base pair. Normally, uridine at the anticodon wobble position base pairs with either adenine (A) or guanine (G) at the codon position of mRNA, which translates for leucine codons (UUA and UUG). An unmodified uridine at the anticodon wobble position can base pair with all four bases, A, G, cytosine (C) and uracil (U) at the third position of the codon . These pormation .Leu(UUR), which prevents the formation of 5-taurinomethyl-uridine, has been implicated in mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS), as well as myoclonus epilepsy associated with ragged-red fibers (MERRF) [Leu(UUR) weakens the binding affinity for the UUG codon, which could result in inefficient synthesis of mitochondrial proteins. Among the 13 polypeptides that encode mitochondrial proteins, ND5, ND6 and cytochrome b contain two, eight and two UUG codons, respectively. A deficiency in taurine conjugation of mitochondrial tRNALeu(UUR), therefore, may affect the synthesis of mitochondrial proteins. Indeed, Jong et al. [Leu(UUR), as well as an improvement in the mitochondrial function in the iPSC generated from a MELAS patient. While many other studies [Leu(UUR). However, this matter will require further validation.A defect in taurine conjugation of mitochondrial tRNA (MERRF) ,148,149.g et al.  have shog et al. ,129. Indg et al.  showed tg et al.  observed studies ,117,145 2O2) in the presence of chloride ions, producing N-chlorotaurine [There have been many studies showing taurine as an antioxidant with a role in protecting against oxidative stress in the mitochondria ,136,145.otaurine ,152. Theotaurine ,154,155.While recent studies have suggested that the prevention of superoxide in the mitochondria is linked to the taurine conjugation of the mitochondrial tRNA ,84,147, Several studies from El-Idrissi’s group have shown that taurine regulates intracellular calcium homeostasis and protects against glutamate-induced mitochondrial damage and cell death ,167,168.It has been thought that taurine acts at the level of mitochondria to inhibit apoptosis. Indeed, Jong et al.  showed tSeveral clinical studies investigating the therapeutic potential of taurine have been reported, particularly in relation to its role as an antioxidant in improving mitochondrial function. Below, we report several known clinical studies on taurine supplementation in various pathologies in humans.10, no significant improvement in the systolic left ventricular function was observed [10 is an antioxidant that mediates the transfer of electrons from complex I and complex II to complex III [10 were reduced and supplementation with coenzyme Q10 was shown to improve the symptoms among heart failure patients [10, in improving myocardial energy in heart failure suggests the significant effect of taurine as an antioxidant, potentially by improving the myocardial energy production. In heart failure, a disturbance in ATP production affects myocardial contraction [Taurine was first used to treat heart failure patients by Azuma’s group in Japan ,30,181. plex III . In hearpatients ,183,184.traction ,186, thetraction . Again, Taurine has also been supplemented among patients with hypertension. Taurine supplementation of 1.6 g per day for at least 12 weeks in prehypertensive patients or 6 g per day for 7 days in hypertensive patients lowered blood pressure and improved vascular function ,188. In Taurine supplementation among patients with type II diabetes mellitus also showed reduced oxidative stress and inflammation, as well as reduced diabetic complications, such as nephropathy, retinopathy and neuropathy ,199,200.Despite the effectiveness of taurine in reducing diabetic complications in several clinical trials, there are a few studies contradicting the clinical effectiveness of taurine in diabetic patients. Firstly, Franconi et al.  reportedTaurine has also been administered in overweight or obese individuals, which has shown promising therapeutic effects on lipid profiles. Rosa et al.  reported5u) and 5-taurinomethyl-2-thiouridine (τm5s2u) [Leu(UUR), as well as improvement of the mitochondrial function in the iPSC generated from a MELAS patient. Indeed, oral taurine supplementation of either 9 g or 12 g per day for 52 weeks in MELAS patients showed prevention of stroke-like episodes, as well as increased modification of taurine-conjugated uridine of mitochondrial tRNALeu(UUR) [Recent studies on mitochondrial diseases such as MELAS and MERRF revealed a reduction in the formation of taurine-modified uridine of mitochondrial tRNAs, specifically 5-taurinomethyluridine (τm(τm5s2u) ,148,149.(τm5s2u)  observedLeu(UUR) ,28. In aLeu(UUR) , plasma Leu(UUR) .Currently, there are no reported clinical studies on taurine supplementation among patients with neurodegenerative diseases. However, there have been some in vivo studies assessing the therapeutic effects of taurine in several mouse models of neurodegenerative diseases, which include Parkinson’s disease ,226 and The clinical effectiveness of taurine in neurodevelopmental disorders has been reported in several studies by Erickson et al. ,242,243.Leu(UUR) or tRNALys(UUU) for proper codon–anticodon interaction to facilitate synthesis of mitochondrial-encoded proteins. Inefficiency of taurine modification of mitochondrial tRNA promotes inefficient translation of mitochondrial proteins, which are components of the electron transport chain. As a result, the assembly and stability of the respiratory chain complexes are impaired, which causes a sluggish response in the electron flux, yielding superoxide production. Taurine also regulates intracellular calcium homeostasis and intracellular antioxidant activity and inhibits apoptosis. As evidenced in many in vitro, in vivo and clinical studies on taurine supplementation, the occurrence of mitochondrial dysfunction, reduced energy production, oxidative stress and apoptosis are mostly inhibited. Taurine therapy, therefore, could potentially improve mitochondrial health, particularly in mitochondria-targeted pathologies, such as cardiovascular diseases, metabolic diseases, mitochondrial diseases and neurological disorders. Whether the protective mechanism on mitochondria primarily relies on the taurine modification of mitochondrial tRNA requires further investigation.Taurine is a simple but unique sulfur-containing amino acid that has multiple physiological functions, including the maintenance of mitochondria health. While taurine is widely known as an antioxidant, its underlying mechanism remains unclear as taurine is not a radical scavenger. Studies conducted by Suzuki and colleagues have shown that taurine conjugates with mitochondrial tRNA


Novel secondary metabolites from marine macroorganisms and marine-derived microorganisms have been intensively investigated in the last few decades. Several classes of compounds, especially indole alkaloids, have been a target for evaluating biological and pharmacological activities. As one of the most promising classes of compounds, indole alkaloids possess not only intriguing structural features but also a wide range of biological/pharmacological activities including antimicrobial, anti-inflammatory, anticancer, antidiabetic, and antiparasitic activities. This review reports the indole alkaloids isolated during the period of 2016–2021 and their relevant biological/pharmacological activities. The marine-derived indole alkaloids reported from 2016 to 2021 were collected from various scientific databases. A total of 186 indole alkaloids from various marine organisms including fungi, bacteria, sponges, bryozoans, mangroves, and algae, are described. Despite the described bioactivities, further evaluation including their mechanisms of action and biological targets is needed to determine which of these indole alkaloids are worth studying to obtain lead compounds for the development of new drugs.  Marine natural products (MNPs) have drawn much attention from both natural product chemists and pharmacologists due to their potential bioactivities. These chemical compounds have made a major contribution for the treatment of a myriad of diseases including cancer and infectious diseases as well as other therapeutic areas such as multidrug resistances (MDRs), cardiovascular diseases, and multiple sclerosis ,2. Compaviz. simple indole alkaloids, prenylated indoles, bis-/tris-indoles, and annelated indoles . . FusariuDichotomomyces cejpii F31-1, obtained from the inner tissue of a soft coral Lobophytum crassum that was collected from the Hainan Sanya National Coral Reef Reserve, China, yielded an indole-containing amide, dichotomocej D (3) (4)   8]. Pse. PseDich (3) (4) , a metyle, China . Engyodontium album (IVB1b), isolated from a marine sponge Ircinia variabilis that was collected at İlyosta–Ayvalık in Balıkesir, Turkey, produced 1-(4-hydroxybenzoyl)indole-3-carbaldehyde (5) (A marine fungus hyde (5)  10]. . EngyodoScedosporium apiospermum F41-1, also obtained from the inner tissue of the soft coral L. crassum that was collected from the Hainan Sanya National Coral Reef Reserve, China, yielded scequinadoline J (6)  spectra , wh, whEurotn C (12)  were obtt sample . 13), variecolorin M (14), and (+)-variecolorin G (15)    in 18 was determined by comparison of the calculated and experimental ECD spectra while the absolute configurations of C-3, C-11, and C-11 in 19 were established by X-ray diffraction analysis using CuKα radiation ..A. sulphAspergillus versicolor from the mud, collected in the South China Sea, also produced rare linearly fused dimethypyranoindoles containing an amine fused-imine pyrrole ring system, asperversamides A–E (34–38)  at 223 nm (+25.9) and negative CE at 242 nm (−28.6) in the experimental ECD spectrum and confirmed by a single-crystal X-ray diffraction analysis. The absolute configurations of the stereogenic carbons in 37 and 38 were established based on a similarity of their ECD spectra to that of the previously reported compound, (+)-6-epi-stephacidin A, and in the case of 37, its absolute structure was confirmed by X-ray analysis naphthalene ring system. The relative configurations of 48 and 49 were established by NOESY correlations. The absolute configurations of the estereogenic carbons of 48 were established by X-ray analysis, while those of 49 were established by a comparison of the calculated and experimental ECD spectra . Co. Co54–5859–62) . In particular, 59 and 60 contain a spiro 2-oxindole, similar to that found in 35, 36, 39, and 40   from the, and 40 . The strat of 59 .63–66)  , paxilinerations .67–70)   are indoat of 67 .71a and 71b) - and (+)-asperginulin A (and 71b) , was obtskeleton .Aspergillus austroafricanus Y32-2, isolated from a seawater sample, which was collected from the Indian Ocean at the depth of 30 m, yielded two diketopiperazine-containing prenylated indole dimers, di-6-hydroxydeoxybrevianamide E (72) and dinotoamide J (73)  . The strBis-and tris-indoles can be derived either from two (or three) direct linkage between the same indole monomers to give homodimers (or homotrimers) or different indole monomers to give heterodimers (or heterotrimers). Moreover, these indole monomers can also link together through different side chains.74), a bis-indole derivative comprising two indole moieties linked through C-2 and C-2′, and fusariumindole A (75)   were als5  were a76a and 76b) -hydroxymethyl-2-oxindole. The racemic mixture was separated by the HPLC chiral column into 76a and 76b. The stereostructure of 76a was established by X-ray analysis as (3S)-(3′-methyleneindole)-hydroxymethyl-2-oxindole, whereas 76b was identified as (3R)-(3′-methyleneindole)-hydroxymethyl-2-oxindole through comparison of its CD spectrum with that of 76a.Shaker et al.  describeand 76b)  from the77) and B (78) propan-2-ol, whereas 78 was identified as 3,3′bis(1H-indole). However, the absolute configuration of the stereogenic carbon in both compounds was not determined  assay showed that 141 was almost equally cytotoxic to the NIH 3T3 mouse fibroblast cell line (IC50 = 7.8 µM), indicating no selectivity for the malaria parasite [Dragmacidin G (141)  was assaparasite .162) and 6-bromotrisi3ndoline (163)  and 13.47 (72 h), and 12.35 (48 h), and 10.27 (72 h) µM, respectively [5-Bromotrisindoline (ne (163)  were evaectively ,24–30  , which is the most active inhibitor of C. marina alkaline phosphatase (CmAP) described to date. On the other hand, 129 showed weak inhibitory activity (IC50 = 100 μM) whereas 126, 127, and 130 did not inhibit this enzyme [Guitarrins A–E (126–130)  were asss enzyme .73) (73 exhibited a significant effect in a dose-dependent manner. Ginsenoside Rg1 (120 µg/mL) was used as a positive control [Dinotoamide J (73)  was test control .In order to facilitate the localization of the bioactive indole alkaloids from different sources and their biological and pharmacological activities, the source organisms, biological/pharmacological activities, compound numbers, and the references cited are summarized in Marine organisms have been proven to be valuable sources of structurally diverse and unique indole compounds. Their structures vary from simple to complex and incorporate different types of compounds ranging from simple prenyl groups to diterpene moieties or other molecules or scaffolds such as pyrazine, diketopiperazine, guanidine, and quinazoline. The indole ring system can be present as an intact indole or undergoes oxidation or/and rearrangements to form different scaffolds or even incorporate a nitrogen atom to form azaindole derivatives or halogen and sulfur atoms to form bromoindoles, sulfonyl, and sulfinyl side chains. Moreover, these marine-derived indoles can have a single indole unit or contain two or three indole units as found in bis-indoles, tris-indoles, homo-, and heterodimers of indole compounds. This structural diversity of marine-derived indoles is reflected by a variety of biological and pharmacological activities that they manifest such as antibacterial, antiviral, anticancer, antiparasitic activities, and enzyme inhibition. Thus, marine-derived indole compounds are excellent sources to be explored for pharmacophores to serve for the development of drug leads or even therapeutic agents for the treatments of infectious diseases, cancer, or even some metabolic diseases.


Takayasu arteritis (TAK) is a chronic granulomatous large vessel vasculitis with multiple immune cells involved. Chemokines play critical roles in recruitment and activation of immune cells. This study aimed to investigate chemokine profile in the peripheral blood and vascular tissue of patients with TAK.A total of 58 patients with TAK and 53 healthy controls were enrolled. Chemokine array assay was performed in five patients with TAK and three controls. Chemokines with higher levels were preliminarily validated in 20 patients and controls. The validated chemokines were further confirmed in another group of samples with 25 patients and 25 controls. Their expression and distribution were also examined in vascular tissue from 8 patients and 5 controls. Correlations between these chemokines and peripheral immune cells, cytokines, and disease activity parameters were analyzed. Their serum changes were also investigated in these 45 patients after glucocorticoids and immunosuppressive treatment.+CD4+ T cell numbers. Close localization of CCL22, CXCL11, or IL-16 with inflammatory cells was also observed in TAK vascular tissue. No correlations were found between these chemokines and cytokines  or inflammatory parameters . No differences were observed regarding with these chemokines between active and inactive patients. After treatment, increase of CCL22 and decrease of RANTES and CXCL16 were found, while no changes were showed in levels of CXCL11 and IL-16.Patients and controls were age and sex-matched. Twelve higher chemokines and 4 lower chemokines were found based on the chemokine array. After validation, increase of 5 chemokines were confirmed in patients with TAK, including CCL22, RANTES, CXCL16, CXCL11, and IL-16. Their expressions were also increased in vascular tissue of patients with TAK. In addition, levels of RANTES and IL-16 were positively correlated with peripheral CD3CCL22, RANTES, CXCL16, CXCL11, and IL-16 were identified as the major chemokines involved in the recruitment of immune cells in the vascular tissue of patients with TAK. Additionally, the persistently high levels of CCL22, CXCL11, and IL-16 observed after treatment indicate their role in vascular chronic inflammation or fibrosis and demonstrate the need for developing more efficacious treatment options.The online version contains supplementary material available at 10.1186/s13075-022-02740-x. Takayasu arteritis (TAK) is a type of large vessel vasculitis that predominantly occurs in female patients of reproductive age (<40 years) and leads to vascular thickness, stenosis, and even occlusion of the aorta and its branches . TAK is +T cells, CD14+ monocytes, and neutrophils cells have been found in the peripheral blood of patients with TAK  and serum C-reactive protein [CRP]) and immune cell numbers or proportions in the peripheral blood. These immune cells were detected by routine flow cytometry, including monocytes . According to the instructions of the manufacturer, 31 chemokines were detected . These chemokines were CCL22, RANTES, CXCL16, CXCL11, IL-16, CCL1, CCL17, CCL19, CCL20, CXCL1, CX3CL1, and XCL1. After preliminary verification, five chemokines  with higher levels in TAK cases were further detected in the remaining 25 patients with TAK and 25 controls for further confirmation.To clarify the effects of glucocorticoids and immunosuppressants on the expression of the chemokines, the serum levels of these five chemokines were also detected in the 45 patients with TAK after treatment. To investigate immune responses in the patients with TAK, the expression of relevant cytokines, including IL-6, IL-17, and IFN-γ was also detected in the 45 patients (baseline and post-treatment) and 45 controls. Commercial ELISA kits purchased from Boster Bio  were used to detect these chemokines and cytokines.To evaluate vascular infiltration upon vascular tissue, immunofluorescence for CD3 , CD19 , and CD68  staining was performed. During this process, the deparaffination, rehydration, antigen retrieval, and blocking process were conducted as previously described . During For the immunohistochemistry analysis, primary antibodies against CCL22 , RANTES , CXCL16 , CXCL11 , and IL-16 , and their corresponding secondary antibodies were applied.2) upon 400X magnification within the greatest inflammatory infiltrates were selected from each slide. The average number of CD3 or CD19 or CD68-positive cells were calculated using ImageJ and graded as +++ (the average number > 100/0.15mm2), ++ (the average number 50–100/0.15mm2), + (the average number 10–50/0.15mm2), or - (the average number <10/0.15mm2). Chemokine expressions were semi-quantified by IHC profiler in ImageJ. The results were presented as negative (-), weak positive (+), moderate positive (++), and strong positive (+++).All the slides were digitally scanned using a 3DHISTECH scanning microscope, and images were viewed and selected using CaseViewer 2.4.0 . In the quantification process, 10 views  numbers in the peripheral blood were analyzed to investigate their potential role in immune cell recruitment. In addition, correlations between these chemokines and cytokines  were analyzed to explore their potential role in TAK immune response. Moreover, the levels of these five chemokines were compared between patients with active and inactive disease, and their correlations with inflammatory parameters (ESR and CRP) were examined to clarify their role in disease activity. Finally, changes in these chemokines were analyzed after glucocorticoids and immunosuppressive treatment.Distribution of the identified five chemokines  within inflammatory cells in vascular tissue and correlations between chemokine levels and immune cell . Graphs were generated using GraphPad Prism 5 . P values <0.05 were considered to indicate statistical significance.Measurement data, such as chemokine levels, ESR, and CRP, were expressed as mean ± standard deviation, while enumeration data, such as patients with active/inactive disease, were presented as frequencies and percentages. Student’s p = 0.27) and female to male ratio . The clinical characteristics of patients with TAK included in the serum examination are listed in Table p > 0.05 for all parameters, Table n = 5) selected for chemokine screening and those selected for chemokine validation (n = 45).The patients with TAK and healthy controls selected for serum examination were matched for age  and controls (n = 5) selected for vascular tissue examination are listed in Supplementary Table n = 7) and renal artery occlusion (n = 1). Thus, ascending aortic specimens (n = 7) and a renal artery specimen (n = 1) were obtained. In addition, apparently normal specimens (control) of the ascending aorta (n = 3) and abdominal aorta (n = 2) were obtained from donors for heart transplantation (n = 3) and liver transplantation (n = 2).The clinical characteristics of patients     in vascular adventitia  exhibited a different pattern. Surprisingly, no correlations were found between CCL22 and immune cell numbers in peripheral blood  and IL-17 (p = 0.004) in patients with TAK in contrast to the healthy controls , cyclophosphamide , tocilizumab , or methotrexate . The median treatment duration was 6 (interquartile range: 5–6) months. After treatment, 14 (31.11%) patients had active disease according to their NIH score.p = 0.03, Fig. p = 0.001) and CXCL16   vs. 74.01 , p = 0.48, IFN-γ: 22.56  vs. 20.88 , p = 0.39, Fig. After treatment, a further increase was observed in CCL22 levels 85.66 52.39, 114.64) vs. 111.20 , 9, 114.649, 114.64We found that changes in the examined chemokines after treatment differed across individual patients. Therefore, immune cell numbers, ESR, CRP, and cytokines changes in the peripheral blood after treatment were compared between patients with increased and decreased levels of each chemokine. However, no differences were observed in these indicators between the two groups (data not shown). Correlations between the changes in these chemokines were also analyzed, and the results indicated similar trends in the changes in CCL22, CXCL16, and IL-16 levels after treatment (Supplementary Fig. A summary of the characteristics of these five chemokines in patients with TAK is shown in Table The present study demonstrates that the chemokines CCL22, RANTES, CXCL16, CXCL11, and IL-16 are elevated in the peripheral blood as well as vascular tissue of patients with TAK, and expression of CCL22, CXCL11, and IL-16 are distributed in vascular inflammatory lesions. These phenomena indicated that they may play a role in the recruitment of immune cells in vascular lesions of TAK. Further, after routine glucocorticoids and immunosuppressant treatment, RANTES and CXCL16 are decreased, while CCL22, CXCL11, and IL-16 present a persistent high levels. Thus, CCL22, CXCL11, and IL-16 may participate in the vascular chronic inflammation or fibrotic process of TAK.The key functions of the five TAK-related chemokines, namely, CCL22, RANTES, CXCL16, CXCL11, and IL-16, identified in this study are reviewed in Table RANTES is an inflammatory chemokine that exhibits chemotaxis towards multiple inflammatory cells, especially T cells via CCR5 , 20. RANCXCL16 participates in various pathological processes such as immune cell recruitment, cell interactions, angiogenesis etc. , 21, leaCXCL11 acts as a chemokine for T cells and macrophages via binding to the receptor CXCR3 . The CXC+ T cells [+ T lymphocyte infiltration in multiple disorders [+ T cell number and the close distribution of IL-16 to lymphocyte infiltrates also indicate an important role for IL-16 in CD4+ T cell recruitment. Further, a persistent high levels of IL-16 were observed in TAK after glucocorticoids and immunosuppressive treatment. Whether high levels of IL-16 contribute to chronic fibrosis in vascular lesions needs further exploration.IL-16 has chemotactic function in immune cells, especially CD4 T cells , 23. Itsisorders –48. In aisorders  and was isorders . In the In the present study, although no correlations were observed between these chemokine levels and the disease activity markers ESR or CRP, high levels of IL-6 and IL-17 were detected along with the high levels of chemokines. This finding, combined with the close distribution of chemokines with vascular immune cells, implies that these chemokines may also be indicators of active immune responses in TAK. Additionally, the persistently high levels of CXCL11 and IL-16 after treatment probably indicates uncontrolled immune activation or their role in chronic vascular fibrosis. The correlations observed between CCL22, CXCL16, and IL-16 indicate that these chemokines may function in a synergistic way.This study comprehensively investigated the chemokine profile of TAK and preliminarily explored the potential role of five identified chemokines in vascular immune cell recruitment. They may play different roles in vascular lesions by attracting different cell types. These findings provide important evidence for the pathogenesis of TAK, but there are also some limitations to this study. Firstly, in this study, four chemokines with lower levels screened from array had not been further validated. Lower levels of chemokines may reflect the usage of them by immune cells. Thus, it was worthy to have more research on them in future TAK studies. In addition, only five chemokines validated in the serum were detected in vascular tissue. Thus, certain critical chemokines in vascular tissue could have been overlooked due to inconsistencies between the peripheral blood and vascular tissue findings. Moreover, the treatment period was relatively short, so the effects of glucocorticoids and immunosuppressive agents on these chemokines need further verification Finally, the origins and roles of these chemokines remained to be explored further.CCL22, RANTES, CXCL16, CXCL11, and IL-16 are identified as the major chemokines that may involve in the recruitment of immune cells in the vascular tissue of patients with TAK. Additionally, the persistently high levels of CCL22, CXCL11, and IL-16 observed after treatment might suggest their participation in vascular chronic inflammation or fibrosis and demonstrate the need for developing more efficacious treatment options.Additional file 1: Supplementary Figure 1. Flow chart depicting the study design. Supplementary Figure 2. Gating strategies in flow cytometry for peripheral immune subsets analysis. Supplementary Figure 3. Results of chemokine array in the present study. Supplementary Figure 4. Vascular infiltration in patients with TAK. Supplementary Figure 5. Peripheral cytokine levels and their correlation with chemokine levels in patients with TAK. Supplementary Figure 6. Correlations between chemokines and disease activity markers in patients with TAK. Supplementary Figure 7. Correlations among different chemokines and changes in their levels after treatment. Supplementary Table 1. Clinical characteristics of the patients included for vascular tissue examination. Supplementary Table 2. Key functions of the five major chemokines.


Tbr2-GFP mice, cell sorting, and microarrays. These data were used in combination with in situ hybridization to ascertain gene sets specific for IPs, RGPs, PNs, interneurons, and other neural and non-neural cell types. RGP-selective transcripts (n = 419) included molecules for Notch receptor signaling, proliferation, neural stem cell identity, apical junctions, necroptosis, hippo pathway, and NF-κB pathway. RGPs also expressed specific genes for critical interactions with meningeal and vascular cells. In contrast, IP-selective genes (n = 136) encoded molecules for activated Delta ligand presentation, epithelial-mesenchymal transition, core planar cell polarity (PCP), axon genesis, and intrinsic excitability. Interestingly, IPs expressed several “dependence receptors”  that induce apoptosis in the absence of ligand, suggesting a competitive mechanism for IPs and new PNs to detect key environmental cues or die. Overall, our results imply a novel role for IPs in the patterning of neuronal polarization, axon differentiation, and intrinsic excitability prior to mitosis. Significantly, IPs highly express Wnt-PCP, netrin, and semaphorin pathway molecules known to regulate axon polarization in other systems. In sum, IPs not only amplify neurogenesis quantitatively, but also molecularly “prime” new PNs for axogenesis, guidance, and excitability.Cerebral cortex projection neurons (PNs) are generated from intermediate progenitors (IPs), which are in turn derived from radial glial progenitors (RGPs). To investigate developmental processes in IPs, we profiled IP transcriptomes in embryonic mouse neocortex, using transgenic  Intermediate progenitors (IPs) are a type of cortical progenitors “intermediate” in the lineage from radial glial progenitors (RGPs), which produce IPs, and projection neurons (PNs), which are generated from IPs . RGPs haCxcl12 (SDF-1), which binds to Cxcr4 and Ackr3 receptors on INs to guide their tangential migration  to identify genes that are selectively expressed in IPs; (2) to analyze the pathways of IP-specific genes, using context-specific annotations from previous studies of neocortex; and (3) to identify developmental processes that are selectively activated in IPs, and compare them to those in RGPs and PNs. As part of this analysis, we ascertained gene sets for other cell types and features of E14.5 mouse neocortex, including neocortex-specific properties such as rostrocaudal patterning and PN laminar fate.Tbr2 (MGI: Eomes), but also exhibited some transcriptome differences. That transcriptome study accorded with histological results showing that Tbr2+ IPs occupy distinct bands in the VZ and SVZ (Tbr2-GFP mouse neocortex to compare IP gene expression across embryonic ages (Tbr2) were not ascertained. More recently, single-cell analyses of temporally defined RGP-IP-PN lineages reported transcriptional waves associated with PN differentiation (in situ hybridization (ISH), and we have found that many putative markers of RGPs, IPs, and PNs from that study do not show expected patterns on ISH. For example, some proposed IP markers  show strong expression in the CP on ISH, consistent with postmitotic PNs. In the present study, we hypothesized that a new approach and analysis of IP transcriptomes could improve our knowledge of IP-selective genes and developmental mechanisms.Previous studies of mouse IP transcriptomes, using different approaches, have produced distinct perspectives. An early single-cell transcriptome study using microarrays and unbiased cluster analysis distinguished RGPs, two types of IPs, and new PNs as cell types in the embryonic mouse VZ and SVZ . That st and SVZ . Subsequnic ages ; howeverntiation , 2019. HTbr2-GFP mouse neocortex, then compared their transcriptomes using microarrays. Partial analyses of these data have been published previously  14.5 eviously , but thieviously , we deteNon-neural cell types in developing neocortex are known to include microglia, leptomeninges, vascular, and blood cells. The vascular and blood elements are each further divided into multiple types: endothelium and pericytes for vascular; erythrocytes, monocytes, T lymphocytes, and B lymphocytes for blood. The gene sets for these cell types were useful to evaluate interactions between neural and non-neural cells during cortical development, such as those between RGPs and meningeal cells , and betOur results demonstrate: (1) that each cell type in E14.5 mouse neocortex is characterized by the expression of specific gene sets; (2) that different cell types activate distinct signaling pathways; (3) that IPs likely play previously unsuspected roles in defining neuronal polarity and axogenesis; and (4) that extensive interactions occur between diverse cell types to coordinately regulate the growth, organization, and homeostasis of this complex brain region.Tbr2-GFP neocortex (E14.5) was dissociated, and cells were sorted into GFP+ and GFP− bins, after first enriching for progenitor cells on the basis of DNA content  in 4,685 genes, and GFP− cells were significantly enriched in 3,262 genes. Gene enrichment was expressed quantitatively as log2 of the fold change (log2FC), which was positive for genes enriched in GFP+ cells, and negative for genes enriched in GFP− cells. The raw microarray results are presented in RNA was amplified from GFP+ and GFP− cells for hybridization on Affymetrix Mouse Gene 430 2.0 microarrays . In thisTbr2-GFP neocortex labels not only IPs, but also postmitotic PNs (due to passive GFP inheritance), GFP+ cells encompassed the entire IP-PN lineage, including Cajal-Retzius and subplate neurons. Conversely, GFP– cells included RGPs and other non-Tbr2-expressing cell types, including all non-neural lineages. We evaluated the top 300 enriched genes in GFP+ cells and GFP− cells by ISH, using online databases and previous studies as described . Genes expressed mainly in aIPs were designated IP-a (n = 12), and genes mainly in bIP cells were designated IP-b (n = 69). Thus, genes expressed in aIPs included IP-a and IP-ab genes, while genes expressed in bIPs included IP-b and IP-ab genes. The standard for determining localization in VZ and SVZ was Tbr2 , some were expressed predominantly in the pallium  on ISH, with little expression in subpallial forebrain. These pallial-selective genes, which may be important in cortex-specific differentiation, were designated PN-cp, PN-iz, PN-svz, or PN-vz, according to zonal expression patterns . Genes were confirmed as PN laminar fate or axon target markers if they were enriched in GFP+ cells, and exhibited zonal expression consistent with the proposed identity . For exaTranscriptional markers of proliferative activity have been established in previous studies . These wn = 15; n = 9; Nes  and progenitor (NSPC) identity  were scr= 9; Nes  and Msi1es , Notch1, Glul (glutamine synthetase), Sox9, and Vim (vimentin) , two Spalt , and two Tcf/Lef (Tcf7l1 and Tcf7l2) family TFs, all linked to NSC maintenance and/or repression of neuronal differentiation. Pax6, previously characterized as a possible marker of RGPs  regulates mRNA translation spatially and in response to signaling activity   column Iimentin) , 6 RNA-b of RGPs , did not of RGPs . Other T markers . Interesactivity . Presumaransport . Among mn = 159) or DNA damage response (n = 10), the latter also active during the mitotic cycle (n = 29) or IP/neuronal differentiation (n = 18) were also numerous among RGP-selective genes. Other RGP-selective transcripts indicated functions such as membrane trafficking (n = 8), phagocytosis (n = 3), and GPCR trafficking .Pathways were inferred from gene expression, with the caveat that protein signaling activity may not reflect mRNA expression. With this caveat in mind, pathway data  showed tic cycle . Genes tRela), p50 (Nfkb1), and TBL1 (Tbl1x) were all RGP-selective, as were several upstream positive and negative regulators. These findings indicate that the p65/p50-dependent NF-κB pathway is largely RGP-selective in developing neocortex. The function of NF-κB signaling in RGPs is primarily to maintain NSC identity and block IP genesis (Tnfrsf1a) activation or DNA damage (Ripk1)  .Gas1, a Shh co-receptor that binds Shh and potentiates its activity; Gli3, a downstream effector of Shh that promotes proliferation and represses IP genesis; and Tulp3, an intracellular repressor of Shh signaling. The coordinated RGP-selective expression of both activators and inhibitors of signaling pathways was a theme observed with not only NF-κB and Shh, but also apoptosis, canonical Wnt, and hippo pathways (Efnb1) signaling pathway, which represses neuronal differentiation , were all RGP-selective. Moreover, we observed that Ephb6, a ligand for ephrin-B1, is highly expressed by IPs and PNs. Potentially, Ephb6 from IPs and PNs might provide feedback to repress neuronal differentiation of RGPs. Since EphB6 lacks an intracellular kinase domain, its feedback to Efnb1 could be an example of pure reverse signaling.Additional pathways with RGP-selective components included Shh, Wnt, Ras-MAPK, hippo, IGF, FGF, and purinergic signaling . The Shhpathways . In addintiation . Efnb1, Tjp1)  surface, RGPs have robust adherens junctions (AJs) that form a belt-like zonula adherens . But unlTjp1) . IPs andTjp1) . In RGPsTjp1) , but no Ctnna1; αE-catenin) and “neural”  AJ molecules , they express several TJRPs for enhanced adhesion and paracellular calcium diffusion. In addition, RGPs selectively expressed Fat1, which associates with AJs and promotes F-actin; Rhoa, a key regulator of AJ integrity; Efhd2, which stabilizes F-actin; Lima1 (eplin), which promotes formation of the zonula adherens  and Cdc42, are essential to maintain AJs and apical surface integrity of the neocortex , and Cdc42 . Some coeocortex . Perhapsntiation . No MalsLrp2  was RGP-selective, but Prom1 was not . Among many dozens of known primary cilium molecules, only one (Ift74) was RGP-selective.The plasma membrane at the ventricular surface of RGPs, known as the apical plasma membrane, gives rise to the RGP primary cilium, and contains specific proteins for functions such as endocytic uptake and membrane remodeling. Among these, Lama2, Lamb1, and Lamb2) and collagens (such as Col3a1). RGPs selectively produced only a few ECM molecules, including Vit, Ccdc80, and Bcan. To attach to the basement membrane, RGPs require three basal attachment complexes built around dystroglycan (Dag1), which binds Lama2; GPR56 (Adgrg1), which binds Col3a1; and integrin-α6β1 , which binds laminins and promotes focal adhesions. Of these, only Dag1 showed RGP-selective expression, while Adgrg1 and the integrin genes were also expressed in other cell types (Dag1) or their signaling pathways cause cobblestone-like cortical malformations  and Bmpr2, although not RGP-specifically , mostly from aIPs , Mfng (a glycosyltransferase that modifies Dll1 and Notch), as well as Hes6 and Hey1 TFs, were IP-selective. Dll3 was bIP-selective, and acts cell autonomously to dampen Notch signaling. The Delta-Notch signaling interaction between IPs and RGPs is illustrated in Activation of Notch receptors is essential for RGP self-renewal, and is driven by presentation of Delta-like 1 .Several mitochondrial molecules, including egulated . Also, mvβ8 (Itgav and Itgb8) by RGPs is essential for cortical vasculature development. Also, TGF-β and VEGF signals from RGPs promote vascular development. Many molecules that mediate RGP-endothelial interactions were selectively expressed by RGPs or endothelial cells, illustrating the extensive, highly specific interactions between these cell types , an IP-specific transcriptional coregulator that is first induced by Neurog2, then binds and inactivates Neurog2  in RGPs, to Ctnna2 (αN-catenin) in IPs and differentiating PNs. Conversely, aIPs and bIPs selectively expressed Ptprk, a homophilic adhesion molecule. These data revealed a rapid, profound change in both the strength and quality of AJs occurs concomitantly with RGP-IP differentiation. Interestingly, while necroptosis pathways were reduced in IPs, apoptosis of IPs may occur by other pathways, mediated by “dependence receptors” that promote apoptosis if ligand is not bound (see below). Consistent with this idea, IPs and neurons express Ppp2r2b, which drives apoptosis in response to growth factor deficiency.Compared to RGPs, IPs markedly down-regulated proliferation, AJ components, and RGP-specific pathways such as NF-κB, hippo, and necroptosis. Not only were AJ components overall downregulated in IPs, but the isoform of α-catenin also changed from Boc was selectively expressed in aIPs  functions to repress Notch signaling and, together with Bcor (an abIP-selective gene), represses Hes5 (Hey1 (abIP-selective) represses Hes1 and Gas1, while promoting self-renewal of NSPCs  in the Shh pathway, may support IP division despite overall reduced proliferative activity of IPs compared to RGPs.Notch pathway molecules were differentially and selectively expressed between not only RGPs and IPs, but also between aIPs and bIPs. Expression of elective , while DIP cells . At the ses Hes5 . Hey1 , although best known for mediating RGP interactions with the meninges , both of which stabilize TGF-β, and thus potentially enhance TGF-β signaling from RGPs to blood vessels  in bIPs , which is expressed in the IP-PN lineage beginning in the SVZ, may contribute to EMT  . Consist in bIPs . By ISH,P to bIP . In addie to EMT . Partiale to EMT . In addie to EMT .Chn2 , a Rac-GEF. Both Chn2 and Prex1 have been linked to regulation of multipolar migration, indicating the importance of Rac1 (which is widely expressed) in this process. Interestingly, Chn2 links EphA receptor signaling with Rac1 inactivation to suppress migration  are expressed in the SVZ and IZ, our data suggest that EphA activation restricts migration. Lateral dispersion of PN clones away from source RGPs is also regulated by EphA signaling , reported to mediate repulsive responses to CCK . Potentially, CCK signaling may restrict IPs from entering the CP.Other known regulators of bIP and new PN migration include Rnd3 , which w new PNs . Anothers to CCK , was selChrna3 selectively in abIPs, and Chrnb2 in IPs and new neurons beginning in the SVZ. Cholinergic receptors can be activated in vivo to provoke inward Ca(2+) currents , some of which have multiple gene isoforms. We found that a suite of three core PCP molecules were expressed selectively in abIPs: Celsr1, Vangl2, and Ankrd6. Together with broadly expressed isoforms of other core PCP components — including Fzd3, Prickle2, and Dvl2 — IPs thus uniquely acquire a full complement of core PCP components  in the cortical hem and hippocampus; and by Sfrp2 (a Wnt antagonist) from the rostrolateral antihem , an activity-induced cell surface protein that promotes axon growth; Sstr2, which stimulates axon outgrowth upon activation by somatostatin , which is linked to axogenesis; and Dusp14, a phosphatase that inhibits MAPK signaling and negatively regulates axon growth  is expressed by Tbr2+ IPs in developing human cortex , which causes membrane depolarization upon glutamate binding; Kcnd2 (KV4.2), which mediates repolarization; and Fgf11, a member of the FGF-homologous factor family, whose members function as intracellular modulators of voltage-gated sodium channels, and may localize with them to the AIS (Kcnq2 (KV7.2) was also expressed in IPs, albeit not selectively. Molecular markers of stabilized axon initial segments, such as Ank2, Ank3, and Cacna2d1, were not expressed until PNs reach the IZ and CP , another molecule linked to axon initial segment stabilization, was expressed in bIPs; but this function of Nfasc is thought to be Ank3-dependent.A recent study unexpectedly found that voltage-gated sodium channel n cortex . In the  in bIPs . These i the AIS . In addiZ and CP . Thus, dZ and CP . We spec patches , with prnd KV7.2 . Also, NSlc17a6 (VGLUT2), a vesicular glutamate transporter that packages glutamate for release as a neurotransmitter , a synaptic vesicle molecule enriched in the IP-PN lineage beginning in bIPs , a tyrosine phosphatase that functions to promote release of Nptx1. Interestingly, GluA1 (Gria1), as well as GluA4 (Gria4), are selectively expressed by migrating INs  and Plxna2 . These findings suggested that Ntn4 may be important in cortical development. Indeed, Ntn4 deficient rats show reduced thalamocortical innervation , and was expressed in the SVZ, IZ, and CP, consistent with bIPs and PNs . PNs in the CP did, however, express high levels of Dcc, Neo1 , Dscam, and Draxin . The receptor for Sema5a consists of Nrp2 with Plxna2, both expressed selectively by bIPs  defies simple predictions about effects on axon guidance, it seems clear at least that the high rostrocaudal gradients of secreted semaphorins  may have additive or redundant effects, especially on cortical axons destined to cross the midline  expressed in bIPs in the SVZ, is the receptor for Ntf3, and is part of a neurogenic feedback mechanism from PNs to progenitor cells that promotes a switch to upper layer fates   . In the adherin) . Also, w in aIPs , consistCelsr1, Ankrd6, and Vangl2, completing, along with more broadly expressed molecules, a core PCP program in IPs (Fzd3 (the Fzd most often linked to PCP) along with Celsr2 or Celsr3 instead of Celsr1  and Lyn mediate axon interactions; however, Cntn2 null mice have no significant axonal defects, and in our assays, Lyn was detected only in non-neural cells of E14.5 neocortex.The functions of core PCP in developing neocortex have previously been linked mainly to directional growth of axons  caused severe cortical thinning  is, like several other Wnts    and may  center) . Moreove center) .Wnt5a and other Wnts could potentially orient PCP in IPs by Wnt-PCP signaling. But could this system orient axons? Previous studies have demonstrated that Wnt-PCP signaling can specify axon orientation and steer axon growth in diverse species , a netrin receptor transducing repulsive responses. One proposed function of Unc5d is to regulate cell migration through interactions with Flrt2; however, Unc5d null mice exhibit no histological defects in cortical layer formation  between sorted cell populations (GFP+ and GFP−), and were expressed above a minimal detection threshold (2.279), determined empirically to accord with ISH detection. For genes with multiple probes, the gene was excluded if different probes conflicted by indicating significant enrichment in both GFP+ and GFP− cells. If multiple probes were differentially expressed and in agreement, the probe with highest absolute value of log2FC was used to represent the gene for integrated analysis  were confirmed as C-R markers. Additional genes were also screened from candidate C-R neuron markers (Cacna2d2 and Rcan2) were included.C-R neuron-selective genes  were sig markers , of whicTbr2-GFP− cells on microarray, and predominant expression in choroid plexus epithelium (not fibrovascular core) on ISH. Ttr, a known marker of choroid plexus, met these criteria and was included.Genes  were selTbr2-GFP− cells on microarray, and showed expression predominantly in the marginal zone and SVZ. Although most IN subtypes do not differentiate until postnatal ages, we also assessed peptide markers of IN subtypes . All except Pvalb (which was below the detection threshold) were significantly expressed and enriched in Tbr2-GFP− cells. Putative IN genes from previous studies were also screened (Interneurons  were enrscreened .IP genes  were assThese genes  were selGenes for this set  were selThese  were selAif1 (Iba-1 gene), Cx3cr1, and Cd68 met these criteria and were among the included genes.Genes  were selMeningeal genes  were enrThese genes , represeThese genes , represeThese genes  were higThese markers of general neuronal differentiation  were enrOlig1 and Pdgfra) were significantly enriched in Tbr2-GFP− cells on microarray, and were expressed by scattered cells in progenitor and differentiation compartments , suggesting that a few immature oligodendrocyte precursors are present in E14.5 neocortex.Oligodendrocytes do not differentiate until postnatal ages. the possibilities that oligogenic lineages might exist in E14.5 cortex, or might differentiate prematurely in mutant mice, prompted us to select markers of oligodendroglia in postnatal cortex as candidate oligodendrocyte identity genes . Two genThese genes , represeThese genes , represeThese genes , represeThese genes , represeTbr2-GFP+ cells on microarray, and predominant expression in the subplate on ISH. Proposed markers of the embryonic subplate  and/or IZ, possibly extending into the upper CP. Notable exclusions were Cux1 and Cux2, due to non-enrichment in Tbr2-GFP + cells (possibly reflecting expression in INs). Tac2 (Genes previously implicated in genesis of upper layers in publications were selected  if they s). Tac2  was beloLower layers are comprised of mainly corticothalamic and subcerebral projection neurons. Genes were selected  if they Genes  were selThese gene sets  have beeBy ISH, these genes  were expBy ISH, these genes  were expBy ISH, these genes  were expBy ISH, these genes  were expCaudal identity in each zone  was asseGenes  were firThese  were aggThese were designated according to These were designated according to GSE22371 (GPL1261) at www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GPL1261.Publicly available datasets were generated for this study. This data can be found here: NCBI GEO accession number The animal study was reviewed and approved by The University of Washington Institutional Animal Care and Use Committee .FB and RH: study conception and design, acquisition of data, analysis and interpretation of data, critical revision, and final approval of the version to be published. RH: drafting of manuscript. Both authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


MicroRNAs (miRNAs) are endogenous, non-coding small functional RNAs that govern the post-transcriptional regulatory system of gene expression and control the growth and development of plants. Ginger is an herb that is well-known for its flavor and medicinal properties. The genes involved in ginger rhizome development and secondary metabolism have been discovered, but the genome-wide identification of miRNAs and their overall expression profiles and targets during ginger rhizome development are largely unknown. In this study, we used BGISEQ-500 technology to perform genome-wide identification of miRNAs from the leaf, stem, root, flower, and rhizome of ginger during three development stages.SPL, MYB, GRF, SCL, and NAC genes, respectively.In total, 104 novel miRNAs and 160 conserved miRNAs in 28 miRNA families were identified. A total of 181 putative target genes for novel miRNAs and 2772 putative target genes for conserved miRNAs were predicted. Transcriptional factors were the most abundant target genes of miRNAs, and 17, 9, 8, 4, 13, 8, 3 conserved miRNAs and 5, 7, 4, 5, 5, 15, 9 novel miRNAs showed significant tissue-specific expression patterns in leaf, stem, root, flower, and rhizome. Additionally, 53 miRNAs were regarded as rhizome development-associated miRNAs, which mostly participate in metabolism, signal transduction, transport, and catabolism, suggesting that these miRNAs and their target genes play important roles in the rhizome development of ginger. Twelve candidate miRNA target genes were selected, and then, their credibility was confirmed using qRT-PCR. As the result of qRT-PCR analysis, the expression of 12 candidate target genes showed an opposite pattern after comparison with their miRNAs. The rhizome development system of ginger was observed to be governed by miR156, miR319, miR171a_2, miR164, and miR529, which modulated the expression of the This is a deep genome-wide investigation of miRNA and identification of miRNAs involved in rhizome development in ginger. We identified 52 rhizome-related miRNAs and 392 target genes, and this provides an important basis for understanding the molecular mechanisms of the miRNA target genes that mediate rhizome development in ginger.The online version contains supplementary material available at 10.1186/s12864-021-08273-y. MicroRNAs (miRNAs) are endogenous noncoding small RNA molecules with a length of 20–24 nucleotides (nt). In the past few decades, it has been shown that many miRNAs are evolutionarily conserved across diverse plant species and play essential roles in the post-transcriptional regulation of growth and development, biotic/abiotic responses, and other important biological processes of plants , 2.SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) genes in diverse flowering plants [NAC DOMAIN CONTAINING PROTEIN 1 (NAC1), participate in the formation of lateral roots in Arabidopsis and maize [As an evolutionarily conserved microRNA, miR156 regulates the vegetative phase transition by modulating the expression of a subset of g plants . miR164 nd maize .GpECH2 module participated in the stem-to-rhizome transition and probably promoted cell expansion by indole-3-butyric acid (IBA)-to-indole-3-acetic acid (IAA) conversion, and the GpmiR166e-GpSGT-like module promoted rhizome formation in Gynostemma pentaphyllum. In rice, overexpressing OsmiR397a and OsmiR397b transgenic lines show strongly nodding panicles in comparison with wild-type plants. Further molecular and genetic results determined that miR397 leads to an increase in grain yield by the downregulation of its target gene LAC, a regulator involved in the sensitivity of plants to brassinosteroids [It has been shown that a crucial regulatory module, miR396-GRF/GIF, controls the growth of multiple tissues and organs in Arabidopsis –12, tomasteroids .Caenorhabditis elegans [https://www.mirbase.org/; Release 22.1, Oct 2018), a total of 38,589 mature miRNA sequences from 271 different species (ranging from viruses to humans) have been currently identified. However, few studies have been conducted on the identification of miRNAs in ginger.In 1993, the first miRNA, lin-4, was identified from  elegans . In plan elegans –23. The  elegans . Therefo elegans . In 2005 elegans . Since t elegans –29. BaseZingiber officinale Rosc.), belonging to the genus Zingiber in the family Zingiberaceae, is recognized not only as a spice but also as a traditional Eastern medicine for treating chronic inflammation, such as in asthma, rheumatoid arthritis, and other ailments [Ginger , 2nd I_d (second internode), 3rd I_d (third internode) of ginger and then used to construct 7 small RNA libraries. Then, the 7 small RNA libraries were sequenced using the BGI-tech Standard Small RNA sequencing platform and underwent bioinformatics analysis. A total of 28,804,171 raw tag counts from the leaf, 29,640,807 from the root, 29,630,116 from the 1st I_d of the rhizome, 29,084,284 from the 2nd I_d of the rhizome, 28,757,890 from the 3rd I_d of the rhizome, 29,115,222 from the stem, and 28,991,447 from the flower were obtained. After removal of adaptors, low quality reads, and contaminants, 26,648,833 clean tag counts from the leaf, 24,815,451 from the root, 26,324,400 from the 1st I_d of rhizome, 26,825,995 from the 2nd I_d of rhizome, 26,253,384 from the 3rd I_d of rhizome, 26,823,604 from the stem, and 26,988,071 from the flower were kept  ranged from 10,000 to 50,000 at least in one tissue. However, the other miRNA families  had fewer than 5000 reads in all 7 tissues  for further comparative analysis. Based on a criteria of |log2 (fold change)| ≥ 1 and qvalue< 0.005, differential expression patterns of 90 of the most abundant conserved miRNAs and 62 novel miRNAs in 7 libraries were analyzed.We found that 17, 9, 8, 4, 13, 8, and 3 conserved miRNAs exhibited significant tissue-specific expression patterns in leaf, stem, root, flower, 1st I_d, 2nd I_d, and 3rd I_d of the rhizome, respectively , approximately 22 conserved miRNAs were downregulated, and 8 miRNAs were conserved in the 2nd I_d in comparison with the 1st I_d. Additionally, 8 conserved miRNAs were downregulated, and 15 conserved miRNAs were upregulated in the 3rd I_d in comparison with the 2nd I_d , LOB domain-containing protein, meristem (NAM) protein, DCL1, NAC domain transcription factor, AGO1, APETALA2, ATP-dependent RNA helicase, squamosa promoter-binding proteins (SPL), and growth-regulating factors (GRF), which are involved in various aspects of secondary metabolic substance biosynthesis and the tissue growth and development of plants. However, some predicted target genes of several conserved miRNAs in ginger were different from those in other plants, including pirin-like protein, (MIR156) homeobox-leucine zipper protein (MIR166), and TIFY (MIR167)  and GRF4-like (Maker00009062), were significantly downregulated, respectively. In contrast, miR529-5p and miR172_2 were significantly down-regulated during the development of ginger rhizome, and their targets were significantly up-regulated. Like miR156a, miR529-5p also targeted SPL13 transcription factor genes  with different target sites.miRNAs are conserved across plant species, which offers the possibility of miRNA identification through homologue identification in a new plant species , 35. In SPL, MYB, GRF, SCL, and NAC genes, respectively , Maker00052423 (AthSPL13 homolog), and Maker00060741 (AthSPL3 homolog) were identified as under negative regulation by miRNA156a. Interestingly, the expression level of miR156_2 was obviously increased during rhizome growth and development, and its targets Maker00010413 (AthCAT5 homolog), Maker00022469 (AthSPL9 homolog), Maker00023004 (AthSPL2 homolog), Maker00025444 (AthSPL2 homolog), Maker00027061 (AthSPL13 homolog), Maker00031959 (AthSPL9 homolog), Maker00044264 (AthSPL13 homolog), Maker00064385 (AthSPL13 homolog), and Maker00064398 (AthSPL2 homolog) also belong to the SPL gene family. This indicates that members of SPL family of ginger could have essential roles in rhizome development and were under negative regulation of miRNA156a and positively regulation of miR156_2. Furthermore, we found Maker00064385 (SPL13 homologue) was also targeted by miR529, but the target sites were different from those of miR156a.miR156 family members are well studied and function across various plant developmental traits . In Arabansition . Three hMaker00030506 (MYB33 homolog), exhibited the opposite expression profile. In Arabidopsis, MYB33 targeted by miR159 act as modifiers of the juvenile-to-adult transition by regulateing the expression of miR156 [Maker00030506 (MYB33 homolog) was more abundantly expressed in the juvenile stage. Along with the development of rhizomes, miR319 expression was increased, and the expression of Maker00030506 and miR156a were decreased during the same stage. These indicate that the miR319c/Maker0030506 regulator may be involved in rhizome development. The other target of miR319 was Maker00009476, which is the TCP4 homolog of Arabidopsis. Schommer et al. [TCP4 activity has an immediate impact on leaf cell number by significantly reducing cell proliferation. In this study, we proved that the TCP4-like gene (Maker00009476) was found in greater abundance in Rz_1 and was downregulated in Rz_2 and Rz_3. Thus, decreased expression of Maker00009476 may promote the rhizome expansion of ginger. Maker00055205 (TCP24 homolog) modulates secondary cell wall thickening, which is negatively regulated by miR319. Along with rhizome development, the biosynthesis of the secondary cell wall was decreased. miR319/Maker00055205 may be involved in rhizome fiber biosynthesis.The expression of miR319c was more abundant in the 1st I_d, and then it gradually decreased in the 2nd and 3rd rhizome development stages. One of its targets, f miR156 . In the r et al.  reportedMaker00010557 (NAC1 homolog) was negatively regulated by miR164. In Arabidopsis, NAC1 was negatively regulated by miR164, which regulates lateral root formation [Maker00009062 and Maker00009306 are homologs of GRF4 in Arabidopsis, which coordinates cellulose biomass production and utilization. In the current study, we found that Maker00009062 and Maker00009306 were targeted by miR396h and miR172a. These two miRNAs were decreased in rhizome development. Wang et al. [SCL6 was involved in regulating shoot branching. Negative expression of miR171 was found during rhizome development. It was found that nine SCL6 homologous genes were negatively regulated by miR171 and decreased during rhizome development, indicating that miR171 and its targets could be involved in rhizome branching. Although, we found some tissue specific novel miRNAs were differently expressed during the rhizome development, their targets mainly were genes that coding uncharacterized proteins or that not involved in any GO categories , which belongs to the genus Zingiber, is known as a perennial herbaceous plant that grows from a rhizome. It is among the most important edible and medicinal crops in east and southeast Asia. To identify miRNAs in ginger and understand their potential functions, we constructed and sequenced 7 small RNA libraries from the flower, leaf, root, stem, and rhizome of Zingiber officinale Roscoe cultivar “LAIWU 2 J”. We subsequently identified 160 conserved miRNAs and 104 novel miRNAs from seven small RNA libraries through high-throughput sequencing, and predicted their putative targets using Target Finder software. This study presents a first report on the investigation of miRNAs and their targets involved in the rhizome development of ginger. Our findings may also be helpful in developing strategies aimed at enhancing abnormal stem production based on miRNAs. Indeed, further experiments are required to characterize the role of miRNAs in the regulation of rhizome development of this important edible and medicinal plant.Ginger  according to the manufacturer’s protocol. Finally, an Agilent 2100 Bioanalyzer  and 1% agarose gel electrophoresis were used to confirm the integrity of total RNA. The analysis of each library was repeated three times.The plants of the Small RNA libraries of root, leaves, stem, flowers, and rhizome were constructed following the methods described in Evers et al. (2015). Briefly, small RNA fragments 10–30 nt in length were purified using a 15% denaturing polyacrylamide gel, followed by ligating with 5′ and 3′ adapters. Adapter-ligated RNAs were reverse-transcribed by Superscript II reverse transcriptase (Invitrogen) and amplified by PCR. Then, approximately 20 μg products from each sample was sequenced using the BGISEQ-500 platform .Chip adaptor sequences, low quantity reads, and contaminations were removed. Bowtie2 is used to map clean reads to the reference genome and to other sRNA databases. Then, the clean data were used to search against Rfam using the program cmsearch. Their default alignment parameters are as follows: Bowtie2: -q -L 16 --phred64 -p 6 cmsearch: --cpu 6 --noali. In the annotation information of different RNAs, some small RNA tags may be mapped to more than one category. To make sure each unique small RNA is mapped to only one category, we follow the priority rule: MiRbase> pirnabank> snoRNA (plant) > Rfam > other sRNA. We use miRA (for plants) to predict novel miRNA by exploring the characteristic hairpin structure of miRNA precursor. In order to find more accurate targets, multiple types of software are used. Generally, we use psRobot, TAPIR or Target Finder to predict plant targets. The default parameters are as follows: psRobot: -gl 17 -p 8 -gn 1, TargetFinder: -c 4, and TAPIR: --score 5 --mfe_ratio 0.6.http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/ RNAfold.cgi). All the obtained RNA sequences were used for target prediction using the TargetFinder and psRNA Target programs.Desired sequences were small RNAs with no more than two mismatches, which were then aligned against ginger RNA sequences. Their flanking sequences were used to predict secondary structures with the assistance of MFOLD software. Conserved miRNAs were identified following the rule of the filtered small RNA containing a perfect stem-loop structure. miRDeep2 software was used to identify novel miRNAs and confirm the secondary structures of putative pre-miRNAs with modified parameters. The equation: MFEI = AMFE/(G + C)% was used to calculate the minimum free energy index (MFEI). The adjusted minimal folding free energy (AMFE), representing the MFE of 100 nucleotides, was calculated using the RNAfold webserver . Then, the total RNA was reverse-transcribed to generate cDNA using the stem-loop RT primer and the PrimeScript RT reagent kit  following the manufacturer’s protocol. All the primers listed in Table S−ΔΔCT method was used to calculate the relative expression levels [RBP genes were used as references. All primers used in this study are shown in Supplementary Table SP < 0.05.The qRT-PCR reactions were performed using the following conditions: 95 °C for 5 min, 40 cycles of denaturation consisting of 15 s at 95 °C, 30 s at 60 °C, and 30 s at 72 °C, using SYBR Premix Ex Taq II solution . For the qRT-PCR analysis of the miRNA targets, total RNA was used as a template to generate reverse transcripts using the PrimeScript RT reagent kit following the manufacturer’s instructions. For each miRNA target, specific primer pairs were designed to amplify the cDNA (Supplementary Table Sn levels . To normAdditional file 1: Supplementary Table S1. Statistics of sequencing reads from leaf, root, rhizome, stem and flower libraries of Z. officinale Roscoe.Additional file 2: Supplementary Table S2. Conserved miRNAs identified from the flower, leaf, root, stem, and rhizome libraries of Z. officinale Roscoe.Additional file 3: Supplementary Table S3. Novel miRNAs identified from the flower, leaf, root, stem, and rhizome libraries of Z. officinale Roscoe.Additional file 4: Supplementary Figure S1. The stem-loop structures of conserved Z. officinale Roscoe miRNA precursors.Additional file 5: Supplementary Figure S2. Expression profile of conserved miRNAs during rhizome development. Rz_1: Rhizome 1st I_d; Rz_2: Rhizome 2nd I_d; Rz_3: Rhizome 3rd I_d.Additional file 6: Supplementary Figure S3. Expression profiles of novel miRNAs during rhizome development. Rz_1: Rhizome 1st I_d; Rz_2: Rhizome 2nd I_d; Rz_3: Rhizome 3rd I_d.Additional file 7: Supplementary Table S4. Conserved and novel miRNA targets involved in rhizome development of Z. officinale Roscoe.Additional file 8: Supplementary Table S5. All the primers used in this study.


DMRT1 gene, which directly or indirectly activates the male factors, HEMGN, SOX9 and AMH. Recent single cell RNA-seq has defined cell lineage specification during chicken testis development, while comparative studies point to deep conservation of avian testis formation. Lastly, we identify areas of future research on the genetics of avian testis development.As in other vertebrates, avian testes are the site of spermatogenesis and androgen production. The paired testes of birds differentiate during embryogenesis, first marked by the development of pre-Sertoli cells in the gonadal primordium and their condensation into seminiferous cords. Germ cells become enclosed in these cords and enter mitotic arrest, while steroidogenic Leydig cells subsequently differentiate around the cords. This review describes our current understanding of avian testis development at the cell biology and genetic levels. Most of this knowledge has come from studies on the chicken embryo, though other species are increasingly being examined. In chicken, testis development is governed by the Z-chromosome-linked  In the  factors ,50,72. T factors ,69. Thes factors . Outside factors , the sitand SOX9 C,D . PA. PAPAX2,l system ,88,89, bogenesis . Of thes chicken ,67, and  chicken . A recenDMRT1 expression in ZZ chicken embryos operates as the genetic switch for testis formation. The DMRT1 gene is a deeply conserved among vertebrates, with a conserved role in testis development and function across all major clades . Th. ThDMRT1DHH singaling, expression of this factor has not been detected in the embryonic chicken testis . At. AtSry/S CYP17A1  [86]. Fuc stages . In the c stages . This isDMRT1 in genetic males is repression of aromatase expression, possibly via antagonism of the FOXL2 transcription factor [In birds, estrogen production by the female gonadal medulla (embryonic pre-granulosa cells) is essential for proper ovary formation ,172,173.n factor . Howevern factor . Howevern factor .in situ hybridisation and RNA-seq have shown that the embryonic chicken testis expresses all enzymes required for testosterone synthesis from cholesterol  from the early stages of gonadal sex differentiation (at least as early as E6/stage 31) [CYP19A1 (encoding aromatase) commences at E6/ stage 30. However, as noted above, there is no evidence that testosterone plays a decisive role in avian testicular morphogenesis [More recent approaches such as RNA tage 31) ,20. Basetage 31) . Howevertage 31) ,178. In ogenesis . Gonads ogenesis ,180,181.ogenesis .CYP26B1 enzyme expressed in Sertoli cells is thought to degrade retinoic acid (RA), creating a physical barrier to meiosis in the adjacent PGCs [Cyp26b1 null mouse mutants have impaired steroidogenesis and mild ovotestis development [CVH (VASA) gene, which encodes an ATP-dependent RNA helicase [STRA8) [RALDH2 and CYP26B1 [The primary role of the gonads is to facilitate gametogenesis . As the ent PGCs . Insteadelopment . In the elopment . The PGCelopment ,192. Thehelicase . While m [STRA8) , entry o [STRA8) , suggest [STRA8) . In the  CYP26B1 . It seem CYP26B1 . Germ ce CYP26B1 , suggest CYP26B1 ,195.CVH immunostaining and flow cytometry have been used to study the dynamics of chicken embryonic germ cells ,197. GerDCN, POSTN and TCF21. In E8.5 gonads, male interstitial cells can be differentiated from their female counterparts by up-regulation of ACTA2 (α-Smooth Muscle Actin), suggesting this interstitial population to be also the source of peritubular myoid cells  is sufficient to drive specification of the pre-Sertoli cell lineage. In males, PAX2, OSR1 and WNT4 expression decline as DMRT1 expression increases. DMRT1 is required for SOX9, HEMGN and AMH expression, while inhibiting the FOXL2/ Aromatase female pathway. A subset of Sertoli cells up-regulate steroidogenic markers, giving rise the fetal Leydig cells. Signals from the Sertoli cell lineage must also drive interstitial cell development, and formation of the squamous surface epithelium, and induction of germ cell mitotic arrest  gene (methylation and histone lysine methylation) and described how these marks are only partially modified when ZZ gonads are femininsed with estradiol [DMRT1, especially given the recent finding that temperature can induce histone modifications to the DMRT1 locus as a molecular mechanism underling temperature-dependent sex determination in some reptiles. In the red-eared slider turtle, Trachemys scripta, thermosensitive H3K27 methylase, KDM6B, directly promotes the transcription of DMRT1 by eliminating trimethylation of H3K27 near its promoter [Another area for research is epigenetics. At present, there is very little known about the epigenetic landscape of the embryonic chicken gonad. One study reported sex-specific epigenetic marks on the chicken stradiol . This supromoter . HistoneLastly, knowledge of the interaction between Sertoli cells and germ cells will be important for poultry science and for saving endangered bird species. Most recently, methods of harvesting, storing and manipulating avian germ cells have been perfected ,209,210