CADH5 and KLF2, and lncRNA G8399 with LARP6 and EOMES were found to be important for follicular development in GCs. Thus, the results would provide a rich resource for elucidating the reproductive biology of geese and accelerate the improvement of the egg-laying performance of geese.Granulosa cells (GCs) are decisive players in follicular development. In this study, the follicle tissues and GCs were isolated from the goose during the peak-laying period to perform hematoxylin-eosin staining and RNA-seq, respectively. Moreover, the dynamic mRNA and lncRNA expression profiles and mRNA-lncRNA network analysis were integrated to identify the important genes and lncRNAs. The morphological analysis showed that the size of the GCs did not significantly change, but the thickness of the granulosa layer cells differed significantly across the developmental stages. Subsequently, 14,286 mRNAs, 3,956 lncRNAs, and 1,329 TUCPs  were detected in the GCs. We identified 37 common DEGs in the pre-hierarchical and hierarchical follicle stages, respectively, which might be critical for follicle development. Moreover, 3,089 significant time-course DEGs  and 13 core genes in 4 clusters were screened during goose GCs development. Finally, the network lncRNA G8399 with  AMH (Anti-Mullerian Hormone) (FSHR (Follicle Stimulating Hormone Receptor) (CYP17 (Cytochrome P450 Family 17) (Smad9 (SMAD Family Member 9) (BMP4 (Bone Morphogenetic Protein 4) (Granulosa cells (GCs) not only regulate follicular development in poultry through proliferation, differentiation, apoptosis, and steroidogenesis but also provide an essential microenvironment for follicular growth by synthesizing and secreting various hormones . In poulHormone) , FSHR (Feceptor) , CYP17 (mily 17) , Smad9 (ember 9) , and BMPotein 4) .Unlike mammals, the follicles in poultry possess unique characteristics. The follicles are classified according to the size, as pre-hierarchical or hierarchical follicles. The pre-hierarchical follicles differ from the hierarchical follicles in undergoing a process of raising and selection; under normal circumstances, with no follicular selection, the number of hierarchical follicles remains unchanged . Moreovecis-and trans-target genes were predicted and the mRNA-lncRNA network was constructed to analyze the important genes and lncRNAs. Our findings thus aim to broaden our understanding of follicle development, providing a new robust resource for gene discovery and validation in goose.This study investigated the phenotypic differences in the size of the GCs, and the thickness of the granulosa layer cells at these developmental stages by HE staining. Then, the temporal expression profiles of the differentially expressed genes (DEGs) and differentially expressed long noncoding RNAs (DElncRNAs) were determined across the GCs, and the genes or lncRNA (Long non-coding RNA) in GCs crucial during the follicular development were screened. Furthermore, the All the animal experiments were carried out strictly according to the guidelines of the Animal Care and Welfare Committee of Chongqing Academy of Animal Science (CAAS), China. The Sichuan White goose population was reared individually in the cages (600 × 800 × 900 mm) and fed rice grains ad libitum  under naThe follicular tissues were fixed at 4°C with paraformaldehyde (4%). After 24 h of fixation, the tissues were buried in paraffin and sliced by a fully automatic dehydrator along the axes for histological staining using hematoxylin-eosin (HE). The follicle samples were analyzed using an Olympus BX51 microscope  equipped with dry lenses and a microscope digital camera, Olympus DP70 . After staining each slice with hematoxylin and eosin (H&E staining), the structure of the follicular tissues at 100× and 400× magnifications, including the thickness of the granular layer and the size of GCs in each stage were analyzed using the Image-Pro Plus software (version 6.0). More than five follicles at each stage were randomly selected, with more than five different regions being randomly selected from each follicle for slicing, while more than five different regions were also randomly selected from the follicles at each of F5–F1 for slicing.http://www.novogene.com).The GCs were collected from the follicular tissues in the phosphate-buffered saline (PBS) and immediately frozen in liquid nitrogen, followed by storage at −80°C for RNA-seq and real-time PCR. The total RNA was extracted using the Trizol reagent  and further purified using an RNeasy column , according to the manufacturer’s instructions. The NanoDrop ND-1000  and 1% agarose gel electrophoresis were employed to analyze the concentration and integrity of RNA, respectively. Finally, a total of 45 total RNA samples (4 μg each) were isolated from the nine stages of the five individuals. The mRNA isolated from the total RNA by binding oligo (dT) magnetic beads was cleaved into fragments (approximately 155 bp). The 45 cDNA libraries were constructed using these fragments, as described previously , and the-g -u -b --library-type fr-first strand using the Cufflinks (2.2.1) (https://code.google.com/archive/p/assemblyline/) was used to filter the transfrags produced by the background noise (http://cpc2.cbi.pku.edu.cn/) , the remaining transcripts were identified to be long non-coding RNAs. We classified the lncRNAs into five types  as stated previously  . Then, tnd noise . Next, and noise  to removedu.cn/)  was run eviously . To ideneviously . Also, teviously .p-value. A 0.05 FDR and 1.5-fold change were set as the threshold to define DEGs. Moreover, the next-maSigPro method of the maSigPro program R package was used to identify the genes with differential temporal expression profiles from SWF to F1 GCs  was obtained by adjusting the o F1 GCs . The difcis-target genes of lncRNA were defined as the mRNA located within 100 kb, whereas the trans-target genes were those having a Pearson correlation coefficient more than 0.95 or less than −0.95 with p-value < 0.05. The cis-target genes and trans-target genes of lncRNA, and the four gene expression patterns were integrated. Then, the co-expression mRNA-lncRNA network was generated by analyzing using the weighted gene co-expression network analysis (WGCNA) (The  (WGCNA) . The gen (WGCNA) .GAPDH) gene was selected as an internal control gene for normalization. The primers of the six genes were designed for qRT-PCR analyses  were calculated using the 2−ΔΔCt method   during the peak-laying period (45 weeks) were selected as experimental animals . Our res < 0.01) . There w < 0.01) . The HE  < 0.01)  showed n < 0.01) .A total of 315.98 Gb raw reads were obtained from these 45 libraries ranging from 4.74 to 9.74 Gb. After quality control, we acquired 311.84 Gb clean data (∼6.93 Gb per each) of all samples ranging between 4.68 and 9.63 Gb, and the qualities and alignment ratios were shown in Then, the expression level of PCG, lncRNA, and TUCP were normalized to TPM (Transcripts Per Kilobase of exon model per Million mapped reads) with the software Kallisto , and PCGIn this study, 2,269, 396, 744, 3,095, 104, 716, 515, and 1,323 DEGs  were detWASL (Wiskott-Aldrich syndrome protein), GPR63 (G-protein coupled receptor 63), FST (Follistatin precursor), AtTTP (At1g68200), NNT , and P2RY6 (Pyrimidinergic Receptor P2Y6) genes  . We foun6) genes . The resTo investigate the genome-wide mRNA and lncRNA expression pattern profiles during the development of the follicular GCs, the nine-stage  of goose GCs were considered with temporal changes to determine the progression patterns, similar to that of the GCs development. The maSigPro method was employed to identify 3,089 significant time-course DEGs  and wereOverall, the 995 gene expression in cluster 1 reached the top point in LYF, and then consistently decreased to the lowest point at the SWF stage . The expThe gene functional enrichment analysis was performed by the Metascape website to investigate the biological characteristics of the genes in the 4 clusters during the GC development. 1) The gene function analysis showed that the genes in cluster 1 were significantly enriched in mRNA processing (GO:0006397), ncRNA metabolic process (GO:0034660), covalent chromatin modification (GO:0016569). 2) The result of functional enrichment showed that the genes in cluster 2 significantly enriched in the steroid biosynthetic process (GO:0006694), steroid metabolic process (GO:0008202), organic hydroxy compound metabolic process (GO:1901615). 3) The functional analysis showed that the genes in cluster 3 were significantly enriched in the regulation of protein kinase activity (GO:0045859), regulation of cell adhesion (GO:0030155), regulation of cell adhesion (GO:0043009). 4) The results of the functional enrichment showed that the genes in cluster 4 were significantly enriched in cell division (GO:0051301), microtubule cytoskeleton organization (GO:0000226), mitotic cell cycle phase transition (GO:0044772). The DNA repair (GO:0006281), regulation of cell cycle process (GO:0010564), the centrosome (GO:0005813), cell projection assembly (GO:0030031), plasma membrane-bounded cell projection assembly (GO:0120031) were common in clusters 1 and 2 .GON4L (GON-4-like protein), NUMA1 (Nuclear mitotic apparatus protein 1), SAFB1 (scaffold attachment factor B1) (RASF8 (Ras association domain-containing protein 8) (APJ (Apelin receptor), CATK (Cathepsin K), CD34 (Hematopoietic progenitor cell antigen CD34), EOMES (Eomesodermin), KLF2 (Krueppel-like factor 2), VGFR1  (PLPL7(Patatin-like phospholipase domain-containing protein 7), RFA2 (Replication Protein A2), and TMOD1 (Tropomodulin-1) (Thirteen hub genes were screened from the four clusters . There wctor B1) ; In clusotein 8) . There weptor 1) . There wdulin-1) .cis-lncRNAs or trans-lncRNAs using the co-expression analysis or the WGCNA method in the 4 clusters. In cluster 2, the G3427 co-expressed with RL4 , RS25 , RL32 , RS27A , RS4 , and RL22  (CADH5 (Cadherin 5), KLF2 (Kruppel Like Factor 2), CD34 (CD34 Molecule), and the G4610 were associated with APCD1 (APC Down-Regulated 1). The G8399 co-expressed with ESRP2 , ADAD1 (Adenosine Deaminase Domain Containing 1), EOMES (Eomesodermin), DHB2 (Estradiol 17-beta-dehydrogenase 2), LRMP (Lymphoid-restricted membrane protein), and LARP6  and B910  genes  . In clusn) genes .The pre-hierarchal and preovulatory follicles clusters in goose are subjected to stages like initiation, development, and selection. The number of follicles in the goose was found to decrease from SWF to LYF, while the diameters and the weights increased simultaneously , which iFST gene, have multiple roles in follicular development, steroid hormone synthesis, and granulose cell proliferation (WASL gene, which modulates the Wnt signaling and is necessary for the hair follicle cycling in mice (The PCA analysis showed that the nine GCs groups were clustered into two groups: pre-hierarchical and hierarchical follicles . These rferation ; these g in mice .The growth and proliferation of the GCs from the pre-hierarchical follicles are the key to initiation , and theGon4l, NUMA1, and SAFB1 (Gon4l was conserved within the animal species (Gon4l has been reported to participate in cell proliferation, cell survival, and mesoderm-derived tissue specification including those of the blood and somites in the zebrafish, (Gon4l in zebrafish results in cellular apoptosis and failure of the cell to differentiate, blocking the mitotic cell division in the developing embryos (NUMA1, is involved in the maintenance and formation of the spindle poles and mitotic spindle organization during meiotic cell division.(NUMA1 resists cell death in the PTC-318 cell (SAFB1 regulates RNA processing and neuronal function rendering the chromatin permissive for DNA damage signaling and myogenic differentiation (SAFB1 absence in mice to reduce the mice growth by affecting the IGF-1 level, affecting the reproductive ability of the female mice by reducing the levels of estradiol and progesterone. On the contrary, SAFB1 overexpression tends to shorten the S phase of the cell cycle, thereby accelerating cell apoptosis (The hub genes identified in cluster 1 were nd SAFB1 . Gon4l w species , with cr species . Moreovebrafish, . Deficie embryos . One of division.. The abs318 cell , and the318 cell . SAFB1 rntiation . Previoupoptosis . In concRASSF8 is provital for embryonic development in Drosophila, where it is involved in pupal wing cell hexagonal packing promoting wing elongation and epithelial ordering (In cluster 2, ordering . RASSF8 ordering . Moreoveordering .VEGFR1, in cluster 3, is vital for angiogenesis, blood vessel patterning on the retinal astrocytes, neuronal precursors during fetal and retinal development (CatK is to regulate bone resorption in mammals (CatK to be associated with diseases, including skeletal diseases, renal disease, cardiovascular system, central nervous system, respiratory system, autoimmune diseases. (CatK is also associated with thyroid development, brain development, lipid homeostasis, and metabolism (elopment . One of  mammals , Previouiseases. . Moreovetabolism .TMOD1 critically mediates the actin dynamics, participating in the cell migration, motility, proliferation, cycle, morphology, neurite outgrowth, and spine formation (TMOD1 deficiency in mice is known to lead to embryonic lethality with cardiac defects (TMOD1 still play an important role during goose GCs development.In cluster 4, ormation . TMOD1 d defects . In concCADH5, CD34 (CADH5 has a multi-functional biological function in the endothelial cells, such as regulating cell proliferation, apoptosis, cell survival, cell adhesion, cell shape, cell motility (KLF2, is essential for the development and differentiation of the lungs (KLF2 null mice (CADH5 and KLF2 by participating in the granulosa cells in embryonic development, cell proliferation, and apoptosis. However, further investigations are required for an in-depth understanding of the molecular mechanisms for the lncRNA G15904in the future.In cluster 3, the lncRNA G15904 co-expressed with H5, CD34 . Interesmotility . The CDHmotility . Moreovehe lungs , inhibitull mice . Taken tLARP6, one of the co-expressed genes associated with the lncRNA G8399 encodes a protein that prevents cell death until it is triggered by dephosphorylation and degraded (EOMES, one of the target genes for the lncRNA G8399, is involved in the trophoblast differentiation as well as gastrulation, regulating both the mesodermal delamination, endodermal specification, which are essential for developing the tissues extraembryonic to the uterus during the mammalian embryogenesis (degraded . We alsoogenesis . Thus, tIn conclusion, this study measured the expression of the genes in the nine stages of development of goose GCs. Furthermore, the genome-wide mRNA and lncRNA expression pattern profiles were investigated during the development of the follicular GCs and the function of the genes of four clusters was explored during the nine developmental stages. Furthermore, two lncRNA-gene regulatory networks were also identified in goose GCs that could be explored in the future.


Angelica gigas Nakai as a main constituent, has been reported to have various biological activities, including anti-inflammatory, anticancer, and antioxidant effects. This study aimed to predict and confirm the pharmacological relevance of Decursin on chemotherapy-induced alopecia (CIA) with the underlying molecular mechanisms. Decursin-targeted genes were compared with the gene set of alopecia and investigated through functional enrichment analysis. CIA was induced in C57BL/6J mice by injection of cyclophosphamide, and 1, 10, and 100 μM of Decursin were topically treated to depilated dorsal skin. KGF+ expression was detected in the dorsal skin tissues. Based on the predicted results, caspase, PIK3/AKT, and MAPKs protein expressions by Decursin were analyzed in the TNF-α-induced keratinocytes. The Decursin network had 60.20% overlapped genes with the network of alopecia. Biological processes, such as cellular response to chemical stimulus, apoptosis, PI3K-AKT signaling pathway, and MAPK signaling pathway, were derived from the Decursin network. In the Decursin-treated skin, there was morphological hair growth and histological restoration of hair follicles in the CIA mice. The KGF+ fluorescence and protein expressions were significantly increased by Decursin treatment. In addition, caspase-3, -7, and -8 expressions, induced by TNF-α, were dose-dependently decreased along with the inhibition of PI3K, AKT, ERK, and p38 expressions in Decursin-treated keratinocytes. These findings indicated that Decursin would be a potent therapeutic option for hair loss, in response to chemotherapy.Decursin, a pyranocoumarin compound from the root of  A hair follicle is the essential organ for the generation of hair, along with physical protection, thermal insulation, camouflage, sebaceous dispersion, sensory perception, and social interactions . The haiMultiple treatments have been developed and used for CIA. Scalp cooling during chemotherapy is a primary method approved by U.S. Food and Drug Administration to reduce blood flow to hair follicles . UnfortuAngelica gigas Nakai as a main constituent. In our previous study, the root of Angelica sinensis inhibited apoptosis-derived catagen regression, inducing hair regrowth in depilated mice [A. gigas root extract promoted hair growth by regulating pro- and/or anti-inflammatory cytokines in mice [Decursin has been extensively investigated to exert various biological activities including anti-inflammatory, anticancer, and antioxidant effects . It is ated mice . Additio in mice . RecentlDecursin had 100 genes co-occurrences, gathered from PubChem database in literatures. Two genes were sorted out due to low confidence and relevance, resulting in construction of the Decursin network, consisting of 98 target genes. A total of 3840 genes composes the gene set of alopecia, derived from GeneCards database with the relevance score. As a result, to compare between the Decursin Network and the gene set of alopecia, based on its related genes respectively, 59 genes were overlapped between Decursin and the alopecia network. The Decursin network included 59 intersecting genes of the 98 whole genes between alopecia, leading to about 60.20% matching A. ScreenThe Decursin network was constructed from 98 genes. The network was composed of 98 nodes, which are target genes of Decursin, and 7595 edges, which show the connections between nodes A. Based Using a digital dermatoscope, we observed that cyclophosphamide (CYP) injection led to the dystrophic changes of hair growth in depilated skin. Topical treatment of Decursin induced newly growing hair fiber through epidermis. At all concentrations of Decursin, as well as dexamethasone (DEX) treatment, hair growth was shown in dorsum compared to mice in the CYP group A. Histol+ expression in the skin tissues, compared to skin tissues of the CTR group. Concentrations of 1, 10, and 100 μM of Decursin had an increasing effect on KGF+ expression of skin tissues in CIA mice (CYP injection led to reduction of keratinocyte (KGF)CIA mice A. In addCIA mice B. In theCIA mice C.The caspase expressions in the HaCaT keratinocytes were significantly higher than that in the non-treated cells. Increased rates of caspase-3, -7, and -8 in the TNF-α-sensitized cells were 5.07, 2.52, and 13.70, respectively, compared to the non-treated cells. Treatment with 10 nM of Decursin dose-dependently decreased the increment of protein levels of caspase-3, -7, and -8, induced by TNF-α in HaCaT cells . The perAbout 1.56 and 2.70 times of phosphoinositide 3-kinase (PI3K) and protein kinase B (AKT) were phosphorylated in the HaCaT cells by TNF-α sensitization, compared to non-treated cells. The protein levels of phosphorylated PI3K and AKT were significantly decreased by 10 nM of Decursin treatment, by 28.79% and 56.34%, respectively A. The phThere was a significant correlation between Decursin and alopecia, showing 60.20% intersecting common rate of Decursin-targeted genes with alopecia-related genes. Through functional enrichment analysis of the Decursin network composing 98 nodes and 7595 edges, biological terms, including cellular response to chemical stimulus, response to chemical, regulation of cell death, negative regulation of apoptotic process, and regulation of apoptotic process, were derived from GO Process. From those results, the potential anti-CIA effects of Decursin were predicted.+ immunofluorescence expression and protein level when compared with the skin tissue of the CYP group. Additionally, the protein expression of KGF was significantly increased in the TNF-α-sensitized and Decursin-treated HaCaT keratinocytes. These results suggest that Decursin could effectively restore the damaged hair cycle and induce hair regrowth in CIA, and support the prediction that Decursin has a relative connection to alopecia, particularly the chemical stimulus response.Chemotherapy is reported to induce damaged hair follicles with matrix deterioration; as a result, dystrophic catagen is formed and hair shedding is increased . EspeciaTo detect coherent pathways of Decursin on CIA, functional enrichment analysis was performed on KEGG Pathways. As a result, PI3K-AKT signaling pathway, apoptosis, and MAPK signaling pathway were predicted as pathways of the Decursin network with the high FDR-value. Taken together with the GO Process and KEGG Pathways, the pathways imply that Decursin might affect apoptosis and cell death process. At the stage of catagenic hair follicle, apoptosis-driven involution is activated by caspase production . Cell deIn the current study, KEGG enrichment analysis of the Decursin network revealed that the biological terms; PI3K-AKT signaling pathway, apoptosis, and MAPK signaling pathway, showed high matching rates with Decursin-targeted genes. The PI3K/AKT signaling pathway is regarded as one of the important factors to initiate and promote the primordial follicle development . Chemothhttps://pubchem.ncbi.nlm.nih.gov/; accessed on 11 May 2021), which is an open database of accessible chemical information, including physical properties, biological activities, literature citations, and more. One hundred genes, which had co-occurrences in the literature with Decursin, were gathered from PubChem. The confidence score was set > 0.7 and max add 20 relevance through STRING database  based on the previous literatures [http://www.genecards.org/; accessed on 15 May 2021), genes related to alopecia were collected to create an alopecia gene set -13-046). Male C57BL/6J mice aged 5 weeks were purchased from RAONBIO Inc. . All mice were maintained in plastic cages at 22 ± 1 °C temperature and 50 ± 5% humidity with 12 h light/dark cycle. After 1 week of adaptation, mice were randomly assigned to 6 groups; (1) CTR, normal control group; (2) CYP, CYP-induced alopecia group; (3) DEX, DEX-treated in CYP-induced alopecia mice as a positive control group; (4) D1, 1 μM of Decursin-treated in CYP-induced alopecia mice; (5) D10, 10 μM of Decursin-treated in CYP-induced alopecia mice; and (6) D100, 100 μM of Decursin-treated in CYP-induced alopecia mice. CYP, DEX, and Decursin were purchased from Sigma-Aldrich . To synchronize the phase of hair growth cycle, black fur of dorsal skin in all mice, except the CTR group, was shaved using shaving cream on Day 0. After 9 days, 150 mg/kg of CYP was dissolved in saline and administered intraperitoneally once to all mice, except the CTR group. Injection of CYP induces the phase of hair follicles into dystrophic catagen. From Day 9 to Day 16, 100 μL of 1, 10, and 100 μM of Decursin in saline containing 0.1% dimethyl sulfoxide, were topically treated to shaved dorsal skin, once a day. An amount of 0.1% DEX, as a positive control, was topically administered to the depilated region on Days 1, 3, 5, 7, and 9 to 16 . CTR andThe digital dermatoscope used in this study was Smart Microscope Pro , with 10× magnification. Dermoscopic images were acquired in the same area; the central region of the dermal skin treated samples, in all mice.After sacrifice, dorsal skin tissues of all mice were collected and incubated in 10% neutralized formalin for 24 h. It was dehydrated in ethanol and xylene, and then embedded in paraffin. The skin specimens were sliced into 7 μm thick sections and stained with hematoxylin and eosin (H&E) solution.Paraffin tissue sections were hydrated with xylene and ethanol, and then blocked in 1% bovine serum albumin. The slides were incubated with primary anti-rabbit KGF antibody  overnight at 4 °C. Secondary horseradish peroxidase (HRP)-conjugated fluorescence antibody  was treated in the tissue slides at room temperature for 1 h. The stained skin tissues were observed under a fluorescence microscope .v/v fetal bovine serum (Gibco), 2 mM glutamine, 100 IU/mL penicillin, and 100 μg/mL streptomycin (Gibco), at 37 °C in an atmosphere containing 5% CO2 with 95% humidity. The 1 × 106 cells/well HaCaT cells were seeded in 6 well plates. After stabilization, cells were co-treated with 1 μM of DEX, and 0.1, 1, and 10 nM of Decursin, in the presence of 100 ng/mL of TNF-α , for 24 h.Human keratinocyte HaCaT cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) , supplemented with 10% Proteins were extracted from the collected skin tissues and HaCaT cells, using radioimmunoprecipitation assay lysis buffer  containing protease inhibitor cocktail . The lysates were quantified by Bradford method and denatured with sodium dodecylsulfate buffer at 98 °C for 5 min. The protein was electrotransferred onto a polyvinylidene fluoride membrane. The membranes were consecutively incubated with primary anti-KGF, caspase-3, -7, -8, PI3K, AKT, ERK, JNK, and p38 antibodies , and then secondary HRP-conjugated anti-rabbit and anti-mouse antibodies . Relative band densities were observed under ImageQuant TL (IQTL) software  and quantified using a computerized densitometry system . Heatmap was designed to visualize mean values from experiments, based on clustering methods, in range from 1 to 13.57, which were the lowest and highest value.p < 0.05 was taken to indicate statistical significance.Significance was determined by one-way analysis of variance (ANOVA) and Tukey’s multiple comparison tests. In all analyses, The findings of the present study focused on the prediction of efficacy and the underlying mechanism of Decursin on CIA through the network pharmacology, and the subsequent confirmation via in vivo and in vitro. The Decursin-targeted genes exerted high matched rate with alopecia, indicating the relevance of Decursin and alopecia disease. Decursin showed ameliorative effects on hair loss in CYP-treated mice. Through the enrichment analysis, cellular response to chemical stimulus, apoptosis, PI3K/AKT signaling pathway, and MAPKs signaling pathway were expected to have a correlation with the underlying mechanism of Decursin. Correspondingly, Decursin significantly inhibited the apoptotic factors, such as caspase-3, -7, and -8, and the phosphorylation of PI3K, AKT, ERK, and p38 protein expressions. In conclusion, Decursin might be one of leading candidates for CIA by inhibiting apoptosis, PI3K/AKT, and MAPKs signaling. Based on our investigation, the cross-work of prediction derived from network pharmacology analysis and confirmation in cell and rodent models, would provide effective therapeutic strategies to develop new drugs.


Obesity, insulin resistance, type 2 diabetes mellitus (T2DM) and hypertension (HTN) are common that are associated with low-grade systemic inflammation. Diet, genetic factors, inflammation, and immunocytes and their cytokines play a role in their pathobiology. But the exact role of sodium, potassium, magnesium and other minerals, trace elements and vitamins in the pathogenesis of HTN and T2DM is not known. Recent studies showed that sodium and potassium can modulate oxidative stress, inflammation, alter the autonomic nervous system and induce dysfunction of the innate and adaptive immune responses in addition to their action on renin-angiotensin-aldosterone system. These actions of sodium, potassium and magnesium and other minerals, trace elements and vitamins are likely to be secondary to their action on pro-inflammatory cytokines IL-6, TNF-α and IL-17 and metabolism of essential fatty acids that may account for their involvement in the pathobiology of insulin resistance, T2DM, HTN and autoimmune diseases. Both type 2 DM (T2DM) and hypertension (HTN) are common diseases in almost all countries of the world. It is estimated that about 50% of the population above the age of 45 years have HTN and another 10-20% of the population have T2DM (detected or undetected). Almost 50% of the patients who have T2DM have or develop HTN eventually. Thus, both T2DM and HTN are present in many of the patients (population). In addition, obesity is also common. Patients with both T2DM and HTN are generally obese. It is noteworthy that obesity, T2DM and HTN are all associated with insulin resistance. In fact, insulin resistance is the first to develop in those who eventually develop HTN and T2DM. It is now recognised that obesity, T2DM and HTN are all low-grade systemic inflammatory conditions. Coronary heart disease (CHD) eventually occurs in some, if not all, of the patients with insulin resistance, obesity, T2DM and HTN. Thus, all these conditions have overlapping features and one condition may lead to the other(s) . InsulinOf all the dietary factors that are known to influence the development of HTN and T2DM, salt, potassium, essential fatty acids, minerals, trace elements, vitamins and calorie intake are important. Salt does not influence the development of HTN in all but in a subset of patients who have salt sensitive HTN, whereas potassium seems to suppress the pro-hypertensive action of salt , 10. In It is well documented that increased consumption of high calorie diet or high fat diet (HFD) enhance the risk of development of insulin resistance, obesity, T2DM and HTN. Similarly, high dietary content of trans-fats and saturated fats enhance the incidence of insulin resistance, obesity and T2DM. In contrast to this, adequate exercise is beneficial in reducing the risk of insulin resistance, obesity, T2DM and HTN and consequently improve cardiovascular health. In addition, deficiency or sub-optimal intake of minerals, trace elements and vitamins increase the risk of development of obesity and T2DM . SeveralIncreased salt (sodium chloride) intake enhances the induction of human TH17 cells by activating the p38/MAPK pathway and serum/ glucocorticoid-regulated kinase 1 (SGK1)  resultinHypertension is common in many countries. It is estimated that the prevalence of hypertension among adults was 29.0% and was similar among men (30.2%) and women (27.7%) in USA. Approximately ~ 50% of patients with T2DM have and/or develop HTN that enhances risk of vascular diseases  due to DM. The risk for cardiovascular disease (CVD) is ~ four-fold higher in those with both DM and HTN compared to the normotensive non-diabetics. DM enhances the risk for coronary heart disease (CAD), stroke and deaths from cardiovascular cause, including heart failure, cardiac arrhythmia, sudden death, hypertensive disease, and aortic aneurysms . The enhNADPH oxidase (nicotinamide adenine dinucleotide phosphate oxidase) is a plasma membrane-bound enzyme complex that is used by neutrophils to inactivate microorganisms by producing superoxide free radical. The NADPH oxidase complex is dormant under normal circumstances but is activated during respiratory burst. Superoxide anion kills bacteria directly by attenuating their superoxide dismutase (SOD) genes and generating hydrogen peroxide and other reactive oxygen species (ROS). These free radicals inactivate several metabolic enzymes, initiate lipid peroxidation, damage iron-sulphur clusters, and allows the generation of indiscriminate oxidants. Vascular NADPH oxidases are important players in vascular remodeling and disease. Thrombin, platelet-derived growth factor (PDGF), tumor necrosis factor-α (TNF-α), interleukin-1, oxidized LDL and arachidonic acid can activate NDPH oxidase –35. In t2). Thus, MnSOD clears reactive oxygen species (ROS) and protects cells from apoptosis. Thus, MnSOD protects various cells/tissues from oxidative stress induced by various agents and from inflammatory cytokines [Manganese-dependent superoxide dismutase (MnSOD) is an enzyme which is a member of the iron/manganese superoxide dismutase family that binds to the superoxide byproducts of oxidative phosphorylation and converts them to hydrogen peroxide and diatomic oxygen , reducing endothelial NO generation, enhancing the formation of asymmetrical dimethyl arginine (ADMA), oxidative stress secondary to excessive production of ROS, inflammation, impaired insulin-mediated vasodilatation, increased sympathetic nervous system (SNS) activation, dysfunctional immune responses, and abnormal renal handling of sodium , 59–64. Recent studies revealed that increased salt (sodium chloride) intake augments the activity of TH17 cells via the p38/MAPK pathway and serum/glucocorticoid-regulated kinase 1 (SGK1)  that resMagnesium is a co-factor needed for the normal physiological activity of desaturases , 89, 90 ++ concentrations need to be tightly controlled. Under normal physiological conditions, sodium and potassium ions have opposite actions. Increased sodium chloride concentrations enhance the induction of TH17 cells by activating the p38/MAPK pathway and serum/glucocorticoid-regulated kinase 1 (SGK1) [H17 cell generation and production of IL-17 cytokine and thus, promote autoimmune diseases. Thus, increased dietary salt, high fat diet and high sugar all enhance the risk of developing obesity, HTN and T2DM and autoimmune diseases  through the induction of pathogenic TH17 cells [+ concentration suppresses T cell function by inhibiting Akt and mTOR protein kinases [+ and K+ across the cell membrane is critical not only for T cell function but also for other cells such as endothelial cells to prevent inappropriate production of pro-inflammatory cytokines  is intracellular and only (~2%) a small portion of it is extracellular. In view of its actions on the cardiac muscle, the extracellular K1 (SGK1) , 107–11217 cells , 107–113 kinases , 115. Heytokines ). Thus, The observation that salt enhances the production of IL-17, a pro-inflammatory molecule, implies that HTN could be an immunological disorder. Increased salt intake induced elevation in plasma angiotensin-II levels  that, in+ in renal cortex that was reverted to near normal by Treg cells transfer. These results suggest that angiotensin-induced hypertension and other features are due to its pro-inflammatory action mediated by Teff cells and this can be reversed or prevented by Treg cells that have anti-inflammatory actions [Furthermore, angiotensin-II mediates induction of HTN by acting on T effector (Teff) cells. Under normal physiological conditions, T regulatory cells (Tregs) suppress Teff lymphocytes. In experimental animals, infusion of angiotensin-II (1 μg/kg/minute subcutaneous) for 14 days increased systolic blood pressure by more than 40 mm Hg, enhanced NADPH oxidase activity in aorta and heart by more than 1.5 fold, impaired acetylcholine vasodilatory action by ~70% compared to control and enhanced vasculature stiffness and mesenteric artery vascular cell adhesion molecule expression by more than 2 fold and aortic macrophage and T cell infiltration, events that could be reversed by Treg cell adaptive transfer. Angiotensin-II induced decrease in Foxp3 actions –125. Thu actions . This is actions –130. The actions . Since n actions –144  and alpha-linolenic acid ALA, 18:3 n-3) are essential for the survival of humans. EFAs are needed for integrity of skin, normal immune function, and inflammation. EFAs deficiency can result in death due to dehydration, desquamation of skin, poor wound healing, growth retardation, immunosuppression and increased infections, hepatic and splenic dysfunction and abnormal inflammation that eventually can lead to death. But EFAs deficiency is rare since they are present in almost all food that we consume. EFAs deficiency is common in those who are on total parenteral nutrition , suffer from long-standing and severe bowel disease, following massive bowel resection surgery, pancreatic surgery, and gastric bypass surgery and have significant cystic fibrosis –153. Due n-3 are  n-6 and On exposure to different stimuli, naïve T cells get activated, form three distinct effector TH subsets: TH1 cells produce IFN-γ; TH2 cells produce IL-4, IL-13 and IL-25; the third subset of TH cells known as TH17 cells produce IL-17. TH17 cells also produce IL-21 and IL-22. TGF-β, IL-6, IL-21, IL-23, and IL-1β have a role in the formation of TH17 cells. High concentrations of TGF-β inhibit IL-6-induced IL-22 expression, whereas a combination of TGF-β and IL-6 induce generation of IL-17 by TH17 cells. IL-22 production by TH17 cells need the co-operation of IL-23. Thus, IL-22 is the end point effector cytokine secreted by TH17 cells –171 , and PGE2 by macrophages and other cells . TH17 isTH17 development is regulated by the transcription factor, the orphan nuclear receptor ROR-ct and TGF-β plus IL-6. Furthermore, Treg cells and TH17 cells have a reciprocal relationship between them. This is supported by the observation that IL-2, a growth factor for Treg cells, inhibits the generation of TH17 cells, whereas lack of IL-2 reduces the number of Treg cells and increases TH17 cells and leads to the development of inflammatory diseases that can be suppressed Treg cells. IL-4, IL-25, IL-27 and IFN-γ inhibit the expansion of TH17 cells –172.But surprisingly antibodies against IL-17 are not effective against inflammatory bowel disease, an autoimmune disease. In contrast, targeting IL-23 is effective . PUFAs .Several studies revealed that EFAs and their metabolites can regulate blood pressure and prevent development of HTN, at least, in part by restoring IL-17 and Treg balance, decreasing the formation of angiotensin-II and suppressing the expression of angiotensin-II receptors , 173–181+ T cells. TXA4, a pro-inflammatory molecule derived from AA, also facilitates IL-17A production from Vγ4+ γδ T cells [PGE2, derived from AA, induces the expression of IL-23R in naïve CD4 T cells –193. Lip T cells –193. But T cells . But, in T cells –193  [It is evident from the preceding discussion that increased salt intake  results in an: (i) increase in IL-17 production; (ii) decrease in the activity of desaturases leading to low levels of GLA, DGLA, AA, EPA and DHA and their anti-inflammatory, vasodilator, and platelet anti-aggregator metabolites LXA4, resolvins, protectins and maresins that have the ability to suppress IL-16, IL-6, IL-17 and TNF-α production; (iii) increase in PGE2, LTs, TXA2 production that have pro-inflammatory action , 196; (iolecule) . The redolecule) , 206. Itolecule) , 208 andolecule) , 210 expSodium-glucose cotransporter 2 inhibitors (SGLT2i) are hypoglycemic drugs that target SGLT2, the major glucose transporter in the kidney responsible for about 90 percent of glucose reabsorption from primary urine . SGLT2i In this context, it is noteworthy that regulatory T cells (Tregs) express FOXP3, which is needed for natural Tregs (nTregs) along with TGFβ and retinoic acid for induced Tregs (iTreg). Interestingly, the signature transcription factor for Th17 cells, RORγt, is induced by TGFβ, thus linking the differentiation of the Treg and TH17 lineages. FOXP3 can inhibit RORγt function and drive Treg differentiation. However, when the cell receives a signal from IL-6, FOXP3 function is inhibited and the differentiation pathway is induced. Thus, the balance between FOXP3 and RORγt function determines CD4 T cell fate and the type of immune response that will be generated . It is iBased on these results, it is suggested that in addition to reducing the intake of salt, carbohydrate and energy dense foods, it is worthwhile to supplement various EFAs, especially AA/EPA/DHA, their co-factors that are essential for the normal physiological action of desaturases such as vitamin C, folic acid, B1, B6, B12, zinc, and magnesium to prevent the development of HTN and T2DM and to inhibit their progression if they are already present.


Cydia pomonella, is a quarantine pest that causes extensive damage to many important pome fruits. To control this pest, insecticides are frequently used, leading to the development of resistance. In this study, we analyzed resequencing data of two resistant and one susceptible strains of codling moth, detecting the positively selected genes under the insecticide selective pressure. Coupled with transcriptome data, we discussed the potential role in insecticide resistance of these positively selected genes. Our results identified eight genes including CYP6b2, CYP307a1, 5-hydroxytryptaminereceptor, cuticle protein, and acetylcholinesterase, which are potentially involved in cross-resistance to azinphos-methyl and deltamethrin. Overall, our finding indicated that the insecticide resistance mechanism in C. pomonella is a complex physiological and biochemical process.The codling moth, Cydia pomonella L. , is a serious invasive pest of pome fruits. Currently, C. pomonella management mainly relies on the application of insecticides, which have driven the development of resistance in the insect. Understanding the genetic mechanisms of insecticide resistance is of great significance for developing new pest resistance management techniques and formulating effective resistance management strategies. Using existing genome resequencing data, we performed selective sweep analysis by comparing two resistant strains and one susceptible strain of the insect pest and identified seven genes, among which, two (glycine receptor and glutamate receptor) were under strong insecticide selection, suggesting their functional importance in insecticide resistance. We also found that eight genes including CYP6B2, CYP307a1, 5-hydroxytryptamine receptor, cuticle protein, and acetylcholinesterase, are potentially involved in cross-resistance to azinphos-methyl and deltamethrin. Moreover, among several P450s identified as positively selected genes, CYP6B2, CYP4C1, and CYP4d2 showed the highest expression level in larva compared to other stages tested, and CYP6B2 also showed the highest expression level in midgut, supporting the roles they may play in insecticide metabolism. Our results provide several potential genes that can be studied further to advance understanding of complexity of insecticide resistance mechanisms in C. pomonella. The codling moth,  Cydia pomonella L. , is a serious insect pest of many economically important pome fruit trees including apples, pears, and walnuts  v1). We used the fastp (v0.20.0) to filter out the low-quality reads and trim adapters with the default parameters . After b(v2.1.0) . The FPKC. pomonella reference genome. A total of 1.45 million high-quality SNPs were detected among all samples. PCA revealed a clear split between resistant and susceptible strains  and Raz  strains in comparison with the susceptible strain (5.372 × 10−3) (Genomic resequencing data of 18 samples belonging to three strains were mapped to the  strains a. The fi strains . Further × 10−3) b, suggesC. pomonella genome regions with the top 5% FST to detect signatures of positive selection, and subsequently found 784 and 809 genes from the Raz and Rde strains, respectively  approach and found 21 PSGs which were not detected in the GWAS analysis . These 2omonella b.ST and θπ (susceptible/resistant) values for further analysis, including 431 from the Raz strain and 424 from the Rde strain  and θπ ratio  compared to neighboring regions. Their roles were also confirmed by lower values of Tajima’s D in Raz and Rde strains, respectively . Interee strain , and Taje strain . In partectively . In addiectively . CYP307a1 (geneID: CPOM09450) and CYP6B2 (geneID: CPOM05212) showed high FST in both Raz and Rde strains  and life stages  (CYP6B2 (geneID: CPOM03544) was expressed at the highest level in the midgut (CYP6B2 (geneID: CPOM05212), CYP4C3 (geneID: CPOM08186), and CYP4C1 (geneID: CPOM18543), showed the highest expression in larva, the most feeding-active life stages, suggesting their possible roles in insecticide metabolism.To investigate how these positively selected P450s were expressed across d adult) . In compe midgut . Given t+ channels, persistent depolarization, and repetitive firing lead to seizure, paralysis, and death of insects [C. pomonella. These PSGs are mainly P450s, cuticle proteins, and target receptors , where prolonged opening of Na insects ,32. By c insects ,34. In teceptors , making hemicals ,37, and osphates . P450-deosphates . Our stus-methyl , and CYPecdysone ,40. Despn avenae . In addin avenae . The epin avenae , and then avenae . Insect n avenae ,45. In Dto drugs . Becausehickness , and thehickness . BecauseST, by comparison of allele frequencies among different subgroups; (ii) increased LD, such as XP-EHH, by comparison of haplotype homozygosity among different subgroups; and (iii) nucleotide polymorphism, with a lower level of polymorphism indicating stronger selection. Application of at least two of these features is an effective strategy to predict strong selective genomic signatures with reduced false positive rates. In the present study, we used the FST and θπ ratio (θπ(susceptible/resistant)) approaches to detect particularly strong signs of selective sweeps presumably associated with insecticide-resistance and identified seven resistance-related genes which were not detected previously using the GWAS approach [+ channels [Natural or artificial selection of favorable mutations leads to reduced polymorphism and increased LD (linkage disequilibrium) and allele frequency ,49,50. Aapproach . Among tapproach ,52 and happroach ,54,55. Bapproach , and pyrchannels ,57,58. TNote that though the candidate genes listed in


DOCK8 deficiency is a primary immunodeficiency characterized by recurrent infections, severe allergic disease, and autoimmunity. Here, we report a patient with DOCK8 deficiency that was initially presented as systemic lupus erythematosus (SLE) without recurrent infections and treated with hematopoietic stem cell transplantation (HSCT).A 16-month-old boy with a previous history of eczema developed high fever and hand and foot swelling. Over time, multiple purpura, oral ulcers, and oliguria developed with a persistent fever. His laboratory findings showed anemia, thrombocytopenia, and coagulopathy with a high level of C-reactive protein (CRP). No definite pathogens were identified. The complement fractions C3, C4, and CH50 were low. Autoantibodies including antinuclear antibody (ANA) and anti-ds DNA antibody were positive. He definitively satisfied the 2015 ACR/SLICC revised criteria for the diagnosis of SLE (7 points out of 16); therefore, he was treated with a steroid. Lupus nephritis was confirmed by renal biopsy later. Considering the early-onset SLE, partial exome sequencing was performed.One heterozygous missense variant, c.5536A>G (p.Lys1846Glu), which was inherited from his father, and heterozygous deletion of exon 1 to 8 inherited from his mother were found. Through the results of the genetic testing, the patient was confirmed to have DOCK8 deficiency.At the age of 28 months, he received haploidentical HSCT from his mother as a donor.Laboratory findings including complement fractions C3, C4, CH50, anti-ds DNA antibody, and the ANA became normal after HSCT. Currently, at 12 months post-HSCT, he is doing well, without any autoimmune features or infections.DOCK8 deficiency can be presented as autoimmune disease such as SLE. Encountering a child diagnosed with SLE at a very young age, pediatricians should consider immunodeficiency syndrome including DOCK8 deficiency. DOCK8 deficiency is the most common cause of autosomal recessive hyper-immunoglobulin E syndrome (HIES) and is mainly expressed as recurrent infections and severe allergic diseases affecting the skin. In addition, autoimmune features, including systemic lupus erythematosus (SLE), vasculitis, and autoimmune hemolytic anemia may be presented in DOCK8 deficiency.–5Dedicator of cytokinesis 8 protein (DOCK8) deficiency is a primary immunodeficiency disease caused by loss-of-function mutations in the  SLE is one of the autoimmune disorders and results from the loss of immunological tolerance. Therefore, defective Tregs are possible pathogenic mechanisms of SLE. DOCK8 deficiency might overlap with these autoimmune characteristics of SLE via defects in Tregs function.Autoimmune characteristics in DOCK8 deficiency result from the impairment of regulatory T cells (Tregs) that maintain self-tolerance by suppressing autoreactive B cells.Here, we report a case of a 16-month-old boy diagnosed with DOCK8 deficiency that was initially presented as SLE without recurrent infections. Informed written consent was obtained from his parents for publication of this case report.2A 16-month-old boy was transferred from an outside hospital because of a high fever and skin rash. He was born via normal vaginal delivery at 40 weeks of gestation, with a birth weight of 3670 g. He was previously healthy except for atopic dermatitis and a history of suspected viral meningitis at the age of 100 days, which was resolved without complication. Age-appropriate vaccination was done without specific adverse events following the administration of the vaccines, which included the Bacillus Calmette–Guérin (BCG), measles/mumps/rubella, and varicella vaccines. His language and motor development were within the normal ranges. On the day of admission, he had a weight of 11.5 kg (50th–75th percentile) and a height of 81.9 cm (50th–75th percentile).He was admitted to the outside hospital because of a high fever of up to 40°C for 3 days. On physical examination, abnormal findings were not shown other than hand and foot swelling. The initial complete blood count (CBC), blood urea nitrogen (BUN), creatinine (Cr), and serum albumin levels were normal. However, his C-reactive protein (CRP) level was high, at 10.37 mg/dL . He was treated with an initial empiric antibiotic of intravenous cefotaxime. On his third day of hospitalization, perioral ecchymosis and mild coagulopathy developed, with a prothrombin time (PT) international normalized ratio (INR) of 1.30, activated partial thromboplastin time (aPTT) of 67.0 seconds, and D-dimer of > 1000 ng/mL. No bacterial or viral pathogens were identified in the initial blood culture and respiratory viral polymerase chain reaction (PCR), respectively. Besides, oliguria (urination of 0.5 cc/kg/h) developed, along with persistent high fever and swollen extremities. He was transferred to our hospital on the sixth day of fever.On the first day at our hospital, his vital signs were as follows: a blood pressure of 149/91 mm Hg (reference: 86–103/40–55); a pulse of 189 bpm (reference: 70–160); and a body temperature of 38.2°C. He appeared acutely ill. Generalized edema, oral ulcers, and multiple purpura, including perioral ecchymosis, were observed on physical examination  G, M, and A levels were normal . However, the immunoglobulin E (IgE) level was high, at 1679 kU/L , and the complement fractions C3, C4, and CH50 were low, at 80.0 mg/dL , 6.7 mg/dL , and 13.3 U/mL, respectively. The antinuclear antibody (ANA) was positive, at 1:320 with homogenous fluorescence, and the anti-double stranded (anti-ds) DNA antibody was also positive, at 10.8 IU/mL. The tests for anti-SSA, anti-SSB, anti-ribonucleoprotein, anti-scleroderma 70, and anti Jo1 antibodies were all negative.Two weeks after he was hospitalized, leukopenia developed, and thrombocytopenia became worse, along with the persistent fever. The complement fractions were decreased to 37.9 mg/dL of C3, < 1.5 mg/dL of C4, and under 10.0 U/mL of CH50, respectively. The ANA titer increased to 1:1280 with homogenous fluorescence, and the anti-ds DNA antibody level increased to 84.4 IU/mL. The lupus anticoagulant and anticardiolipin antibody were all positive, while other autoantibodies were all negative. To control the fever, he was started on naproxen on the 30th day of fever, and his fever subsided from 32nd day of fever. He definitively satisfied the 2015 American College of Rheumatology (ACR)/Systemic Lupus Erythematosus International Collaborating Clinics (SLICC) revised criteria for the diagnosis of SLE (7 points out of 16); therefore, he was treated with a steroid . The anti-ds DNA antibody level and complement fractions were improved after steroid therapy, and his fever did not recur.2) for 3 days, followed by cyclophosphamide (500 mg/m2). Then, steroid (deflazacort), mycophenolate mofetil, and cyclosporin were used for his SLE, and the disease was well controlled.Approximately 1 week after starting steroid treatment, his steroid dose was reduced to 1.0 mg/kg/day, orally. Two weeks after the first dose reduction, his steroid dose was again reduced to 0.5 mg/kg/day. However, at this point, his fever returned, and his anti-ds DNA antibody titer increased. Accordingly, his steroid dose was increased to 1.0 mg/kg/day, and his fever disappeared. One week later, fever, the swelling of the hand and foot, and proteinuria (Urine Protein/Creatinine ratio of 0.484 mg/mg) appeared again. A renal biopsy confirmed the diagnosis of lupus nephritis . Transthoracic echocardiography showed vegetation in the tricuspid valve, which was thought to be indicative of Libman-Sacks endocarditis. He was treated with intravenous methylprednisolone  and SIFT . This variant has been classified as likely to be pathogenic, according to the American College of Medical Genetics and Genomics (ACMG). To rule out the possibility of a large exonic deletion of DOCK8, multiple ligation-dependent probe amplification (MLPA) analysis  was done in the peripheral leukocytes of both the patient and his parents, which revealed a heterozygous deletion of exon 1 to 8 in the patient and his mother. Through the results of the genetic testing, he was confirmed to have DOCK8 deficiency.Considering the early-onset SLE, partial exome sequencing for 4813 OMIM genes was performed in his peripheral leukocytes. Exomes were captured using the TruSight One Panel (Illumina Inc.), which enriches a 12 Mb region spanning 4813 genes. Sequencing was performed using the NextSeq platform (Illumina Inc.). Sequence reads were aligned to the reference genome, hg19, using a Burrows–Wheeler aligner (BWA) . The mean depth of coverage was 90× (> 10× = 98%). One heterozygous missense variant, c.5536A>G (p.Lys1846Glu), was found, which was inherited from his father. This variant was not reported previously. The mutation was confirmed using in silico prediction tools, such as PolyPhen-2 . From that point, he experienced skin and soft tissue infections several times, which is compatible with ecthyma gangrenosum.At the age of 22 months, when the patient's anti-ds DNA antibody titer and complement levels were normal as a result of the use of immunosuppressants, he newly experienced skin and soft tissue infections and bacteremia caused by At the age of 28 months, he received haploidentical hematopoietic stem cell transplantation (HSCT) from his mother as a donor, with a non-myeloablative conditioning regimen that included antithymocyte globulin, thiosulfan, and fludarabine. WBCs were grafted on the 10th day following HSCT, and no significant HSCT-related morbidity, including graft versus host disease (GvHD), occurred during a follow-up period of 1-year post-HSCT. At 1 month post-HSCT, the complement fractions C3, C4, and CH50 were normal, at 113.0 mg/dL, 22.7 mg/dL, and 51.2 U/mL, respectively. The ANA was positive at 1:160, with homogenous fluorescence, and the anti-ds DNA antibody level had decreased to 13.0. The IgE level was high, at 526 kU/L. However, the anti-ds DNA antibody normalized at 3 months post-HSCT, and the ANA and IgE became normal at 6 months post-HSCT. He had 20% and 33% donor T-cell chimerism at 3 months and 6 months post-HSCT, respectively. Currently, at 12 months post-HSCT, he is doing well, without any autoimmune features or recurrent infections  and WASP-interactin protein (WIP), or the activation of STAT3.,14 Recently, it has been shown that DOCK8 regulates the suppressive activity of Treg cells through interleukin (IL)-2 in STAT5 signaling. As observed in DOCK8 deficiency, defective Treg cells play a role in the pathogenesis of SLE. Treg defects in SLE are also associated with IL-2 deficiency, as well as with dysregulation of tumor necrosis factor-alpha (TNF-α), major histocompatibility class II and III, and complement components.In DOCK8-deficient patients, the number and the suppressive activity of Tregs, which are responsible for immune tolerance, are decreased. These defective Tregs increase the peripheral B cell autoreactivity and enhance the ability of B cells to produce autoantibodies to self-antigens.Since the discovery of the DOCK8 mutation in 2009 as one of the causes of the autosomal recessive form of HIES, genetic evaluation has been conducted to confirm the cause of HIES, and novel mutations in DOCK8 have been reported. Our patient's genetic analysis through gene sequencing identified c.5536A>G in exon 43. This sequence variant, c.5536A>G, predicts an amino acid change of lysine to glutamine at codon 1846 of the DOCK8 protein (p.Lys1846Glu). This sequence variant is of unknown significance, as it has not yet been shown that this mutation alters DOCK8 protein expression. However, this variant was classified as “likely pathogenic,” which provides enough evidence that physicians can use the information in clinical decision making when combined with the presence of other evidence of the disease. HSCT should be considered as early as possible to prevent life-threatening infections, nonreversible parenchymal organ damage, and complications related to autoimmune disease therapy. Reports of the successful treatment of DOCK8 deficiency through HSCT from human leukocyte antigen (HLA)-matched siblings or unrelated donors have been published.–20 More recently, HSCT from haploidentical related donors using post-transplantation cyclophosphamide has been found to be effective in patients with DOCK8 deficiency who lack a fully HLA-matched donor. Ghosh et al and Shah et al reported success in haploidentical HSCT using both Treg depletion and post-transplant immunosuppressants for preventing GvHD. Our patient underwent haploidentical HSCT successfully using a non-myeloablative conditioning regimen without post-transplantation immunosuppressants. Although HSCT cures infection susceptibility and eczematous dermatitis, it remains undetermined whether HSCT cures the autoimmune complication in DOCK8 deficiency. However, our patient has lived for 1 year after HSCT without any recurrence of his autoimmunity. It is necessary to confirm the usefulness of haploidentical HSCT in DOCK8 patients and the curability of autoimmunity via continuous follow-up evaluations after HSCT from all types of donors.Allogeneic HSCT is a fundamental treatment of autoimmune disease and primary immune deficiency, and it has been reported as curative in DOCK8 deficiency.4DOCK8 deficiency can be presented as autoimmune disease such as SLE. When a child is diagnosed with an unusual early-onset SLE, pediatricians should suspect other immunodeficiency diseases. Besides, HSCT can be considered as an attractive and promising option for the treatment of DOCK8 deficiency with autoimmune features.All authors have made substantial contributions to the conception of the work. ES, JHL, YSP, HJI, and JL are the treating physicians. BHL conducted the genetic investigation and provided the treatment recommendations. All authors were involved in drafting the work, reviewing, and revising the manuscript. All authors approved the final manuscript as submitted and agree to be accountable for all aspects of the work.Conceptualization: Euri Seo, Beom Hee Lee, Joo Hoon Lee, Young Seo Park, Ho Joon Im, Jina Lee.Writing – original draft: Euri Seo, Jina Lee.Writing – review & editing: Euri Seo, Beom Hee Lee, Joo Hoon Lee, Young Seo Park, Ho Joon Im, Jina Lee.


Triple-negative breast cancer (TNBC) can be further classified into androgen receptor (AR)-positive TNBC and AR-negative TNBC or quadruple-negative breast cancer (QNBC). Here, we investigated genomic instability in 53 clinical cases by array-CGH and miRNA expression profiling. Immunohistochemical analysis revealed that 64% of TNBC samples lacked AR expression. This group of tumors exhibited a higher level of copy number alterations (CNAs) and a higher frequency of cases affected by CNAs than TNBCs. CNAs in genes of the chromosome instability 25 (CIN25) and centrosome amplification (CA) signatures were more frequent in the QNBCs and were similar between the groups, respectively. However, expression levels of CIN25 and CA20 genes were higher in QNBCs. miRNA profiling revealed 184 differentially expressed miRNAs between the groups. Fifteen of these miRNAs were mapped at cytobands with CNAs, of which eight , and presented concordance of expression and copy number levels. Pathway enrichment analysis of these miRNAs/mRNAs pairings showed association with genomic instability, cell cycle, and DNA damage response. Furthermore, the combined expression of these eight miRNAs robustly discriminated TNBCs from QNBCs (AUC = 0.946). Altogether, our results suggest a significant loss of AR in TNBC and a profound impact in genomic instability characterized by CNAs and deregulation of miRNA expression. Triple-negative breast cancer (TNBC) is a highly metastatic breast cancer subtype that afflicts nearly a half million women in the US and accounts for 10–20% of newly diagnosed breast cancers worldwide . It follGenomic instability has been recognized as one of the drivers of tumorigenesis and a factor facilitating the hallmarks of cancer ,24. It cThe expression of several miRNAs is dysregulated in breast cancers, enhancing cancer aggressiveness by modulating signaling pathways involved in cell proliferation, differentiation, and metastasis ,38. In TA total of 53 full-face TNBC tissue sections were stained for AR A. AR wast-test, p < 0.05; DNA copy number analysis was performed to determine the impact of the loss of AR expression on genomic instability in breast tumors. To this end, 14 TNBC samples and 19 QNBC samples were subjected to array-CGH. A total of 204 CNAs were observed among the TNBC samples, with an average of 14.57 ± 3.49 alterations per sample. Among QNBC samples, a total of 484 CNAs were identified, with an average of 25.47 ± 3.73 alterations per sample. The number of CNAs was significantly higher in QNBC samples than in TNBCs . In the QNBC group, gains at 8p12-p11.11 were present in 78.9% of the samples. In QNBC samples, significant copy number gains were also observed at the 1q21.1-q44, 6p25-p12.1, and 9p24.3-p13.1 cytobands; these CNAs were present in 63.2% of the samples. In both groups, copy number losses were observed at only two cytobands: 4p16.3-p12 and Xp22.33-p11.21 . Overall, QNBC samples exhibited significantly higher numbers of CNAs (except for CNAs at the 3q11.1-q29 region) than TNBC samples  .FOXM1, CNAP1, and RAD51AP1) showed differences between QNBC and TNBC groups in the interval-based analysis. These genes presented significantly higher copy numbers in QNBC samples than in TNBC samples  between QNBC and TNBC samples . CIN25 sn = 9) and AR high (n = 81). Our results indicated that CA20 and CIN25 signature genes were expressed at significantly higher levels in patients with AR-low tumors than in those with AR-high tumors . Thus, the TNBC samples were categorized as AR-low . Among these, 112 miRNAs were downregulated, and 72 were upregulated in QNBC samples. Supervised hierarchical clustering based on the differentially expressed miRNAs distinctly clustered TNBC and QNBC samples except for five samples  are shown in Global miRNA expression profiling was performed in 32 (12 TNBC and 20 QNBC) tissue samples. A total of 184 miRNAs were found to be differentially expressed between QNBC and TNBC samples , axon guidance (hsa04360), Hippo signaling pathway (hsa04390), pathways in cancer (hsa05200), ErbB signaling pathway (hsa04012), Rap1 signaling pathway (hsa04015), N-glycan biosynthesis (hsa00510), Ras signaling pathway (hsa04014), renal cell carcinoma (hsa05211), and glioma (hsa05214) . t-test p > 0.05) between these groups. Using the Ensembl and miRbase database, we mapped the 184 differentially expressed miRNAs between QNBC and TNBC samples to cytobands exhibiting CNAs; only cytobands affected in ≥30% of QNBC samples were used. We identified eight miRNAs presenting expression levels in concordance with the observed CNAs at their respective genome locus . Six miRNAs  were mapped to cytobands with copy number gains and two (miR-23c and miR-943) to cytobands with copy number losses A. A casePathway enrichment analysis of the eight miRNAs resulted in 25 potentially affected pathways , with twWe also assessed for predicted miRNA targets potentially affected by the regulation of the eight miRNAs and that were altered by CNAs. We identified 4,050 predicted target genes (predicted in at least two independent databases), which were compared to the list of the 17,521 genes presenting CNAs ; 1239 geA comparison of a list of 221 target genes involved in selected KEGG pathways with the 1239 genes with CNAs resulted in 58 genes . Most ofACER2, ATP6V1C1, B3GNT5, B4GALT4, B4GALT5, CERS2, CHST7, CPEB2, FUT9, GALNT1, JARID2, STK4, ST8SIA1, and UST. Moreover, to assess the potential role of the selected eight miRNAs in genomic instability, we compared the list of target genes with the list of genes involved in the gene ontology (GO) biological process “cellular response to DNA damage stimulus” (677 genes) and the list of genes involved in KEGG pathway “cell cycle” (hsa04110—124 genes)  analysis. Considering PPIs with the highest confidence (interaction score > 0.9) and removing nodes without connections, we generated a network with eight miRNAs and 44 out of the 58 target genes C. The ge4 genes) .To determine the ability of each individual miRNA and the combined miRNA panel in discriminating TNBC from QNBC, we performed receiver operating characteristic (ROC) curve analysis considering the selected eight miRNAs. A moderate power was observed for the individual miRNAs in discriminating the two groups, with the area under the curve (AUC) values ranging between 0.6667 (for miR-1267) and 0.7583 (for miR-567). However, ROC analysis of the eight miRNAs combined provided an AUC value of 0.946, suggesting robust discrimination of TNBC from QNBC .t-test, p < 0.05).The relationship between the expression levels of the eight miRNAs and the clinicopathological characteristics of patients with QNBC was evaluated. The expression levels of the eight miRNAs were not associated with age at diagnosis, tumor size, or lymph node status. The expression levels of five miRNAs were associated with distant metastasis. Specifically, high expression levels of miR-548ai, miR-567, miR-1265, and miR-1267 were associated with distant metastasis, whereas high expression levels of miR-23c were associated with no metastasis , BRIP1 (miR-567 target), and CHEK2 (miR-943 target) genes, which are known to be involved in breast and ovarian cancers [BARD1 and BRIP1 are both tumor suppressors, that act in the repair of double-stranded DNA damage, with proteins, interact with BRCA1. The function of these proteins is essential for maintaining the stability of genetic information in the cells [NBN gene target, is part of the MRN complex (MRE11/RAD50/NBN), with a fundamental role in the maintenance of chromosomal stability. After DNA damage, it participates and/or coordinates several repair activities, such as DNA resection, activation of the DNA damage checkpoint, chromatin remodeling, and recruitment of the repair machinery [CHEK2 gene, on the other hand, in response to DNA damage regulates cell division by preventing cells from entering mitosis or arresting cell cycle in gap 1 phase (G1). Therefore, CHEK2 is essential for cell cycle regulation, and its abnormal expression could lead to cancer [RAD51AP , which was observed with differences in CNAs in the QNBC and TNBC samples. CNAs on this gene, however, should be validated by non-genome-wide methodologies, such as FISH using locus-specific probes.The target prediction and functional enrichment analysis of the miRNAs associated with CNAs revealed their involvement in cancer and genome instability pathways, such as the ones that control cell cycle and mismatch repair pathways. For example, the  cancers ,75. BARDhe cells . The NBNachinery . CHEK2 go cancer . MoreoveIn most of the breast cancer studies, the expression levels of the identified miRNAs were not investigated in association with AR expression. However, we suggest that they may reflect AR status, considering their high power in discriminating QNBCs from TNBCs. Altogether, the findings of the integrated analyses of CNAs and miRNA expression profiles support the profound impact of CNAs on the expression of miRNAs and on miRNA-mediated regulation of gene expression, pointing out to cancer drivers that contribute to genomic instability in QNBC. However, it is important to mention that the relationship between cancer-related regions and miRNA locations is not uniformly reported. CNAs can affect miRNA in a tissue-specific manner and not correspond to the frequent dual action of miRNAs in tumorigenesis. Furthermore, miRNA expression can be regulated by several other mechanisms, not directly involving CNAs. In conclusion, our results showed loss of AR expression in a high number of TNBCs and a significant impact of this loss in the tumors’ genomic instability, as evidenced by CNAs and deregulation of miRNA expression. Our results also suggest that in TNBCs with AR loss, CNAs affect miRNA expression levels and their corresponding involvement in signaling pathways associated with cancer aggressiveness and patient outcomes. The general workflow of the study is presented in A total of 53 formalin-fixed, paraffin-embedded (FFPE) TNBC samples were retrieved from the Histopathology and Tissue Shared Resources (HTSR) of Lombardi Comprehensive Cancer Center, Georgetown University. Clinical samples were collected during surgical excision of the primary tumor before any systemic treatment. All the aspects of the study, including study protocols, sample procurement, and study design, were approved by the Institutional Review Board (IRB). The specimens were decodified with no patient identifiers under the HTSR IRB-approved protocol (IRB#1992-048). Clinicopathological data, patient survival information, and tissue blocks were available for all patients.Immunohistochemistry (IHC) was performed as previously described . BrieflyTissue samples with at least 80% of tumor area were considered for DNA and RNA isolation Tumor areas were selected in unstained FFPE samples (5 μm), and consecutive tissue sections from the same tissue blocks were microdissected to isolate DNA and RNA while ensuring a direct correlation of DNA copy number and miRNA expression profiles. DNA was isolated from tumor tissues using a phenol–chloroform protocol optimized for FFPE samples ,44. DNA Genome-wide copy number profiling of FFPE samples was performed using the SurePrint G3 Human CGH Microarray  as previously described ,81. BrieAURKA, CCNA2, CCND1, CCNE2, CDK1, CEP63, CEP152, E2F1, E2F2, LMO4, MDM2, MYCN, NDRG1, NEK2, PIN1, PLK1, PLK4, SASS6, STIL, TUBG1) and CIN25 , C20orf24-TGIF2, MCM2, H2AFZ, TOP2A, PCNA, UBE2C, MELK, TRIP13, CNAP1, MCM7, RNASEH2A, RAD51AP1, KIF20A, CDC45L, MAD2L1, ESPL1, CCNB2, FEN1, TTK, CCT5, RFC4) signatures [t-test; p < 0.05 was considered statistically significant.The copy number status of the genes composing the CA20  Data Portal . A totalhttp://www.mirbase.org, accessed on 13 August 2020) targeting 827 human miRNAs, six positive controls, eight negative controls, three ligation positive controls, three ligation negative controls, five internal reference genes , and five spike-in controls . Raw data were processed using NanoString nCounter RCC collector  and normalized using NanoString nSolver 4.0 software  and the following settings: background subtraction, geometric mean of negative controls; technical normalization, geometric mean of positive controls; codeset content normalization, all genes geometric mean. Normalized data were log2—transformed and analyzed using the MultiExperiment Viewer software (MeV 4.9.0)  [miRNA expression analysis was performed using NanoString nCounter Human v3a miRNA Expression Assay  as previously described ,36. The st 2020) , GraphPahttp://www.mirbase.org, accessed on 13 August 2020). The second step involved the identification of common gene targets of the selected miRNAs that may be affected by both CNAs and miRNA expression alterations. For previously selected miRNAs, gene targets were identified using available miRNA target databases: Diana micro-T-CDS v. 5.0 , miRDB , and TargetScan Release 7.1 . Only miRNA target genes that were present in two out of the three miRNA databases were selected. Most differentially expressed miRNAs between TNBC and QNBC samples were integrated with array-CGH data from the same tissue sample by two distinct steps ,36. The http://snf-515788.vm.okeanos.grnet.gr/, accessed on 14 August 2020) to assess the potential role of dysregulated miRNA and their involvement in regulating biological pathways. Enrichment of Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways among miRNA target genes was analyzed, and enriched pathways with p-value < 0.05 (FDR corrected) were considered significant.Diana miRPath v.3.0 was used  and miRnet  databases were used to identify interactions between miRNAs, and target genes validated based on strong  and less strong  experimental assays. STRING v. 11  was used to verify protein-protein interactions (PPI) between the validated target genes; a minimum interaction score of 0.9 (highest confidence) was used. Cytoscape v. 3.8.2   [miRTarBase   was usedt-test and logistic regression were used for binary clinical variables . p < 0.05 was considered statistically significant. These analyses were performed using GraphPad Prism 8.3.0 . Univariable linear regression was used to determine the correlation of miRNAs (identified by the integration of array-CGH and miRNA expression data) and continuous clinical variables (age and tumor size); miRNA log2 values were used as outcomes, and clinical data were used as regressors. The student’s t-test or Wilcoxon–Mann–Whitney test.Statistical analyses were carried out using R version 3.6.0. Differences in clinicopathological characteristics between TNBC and QNBC groups were determined using the chi-squared test or Fisher’s exact test. Differences in continuous clinicopathological variables between AR-positive and AR-negative groups were analyzed using a two-tailed