Neuroinflammation is a common event in degenerative diseases of the central and peripheral nervous system, triggered by alterations in the immune system or inflammatory cascade. The pathophysiology of these disorders is multifactorial, whereby the therapy available has low clinical efficacy. This review propounds the relationship between the deregulation of T helper cells and hypoxia, mainly Th17 and HIF-1α molecular pathways, events that are involved in the occurrence of the neuroinflammation. The clinical expression of neuroinflammation is included in prevalent pathologies such as multiple sclerosis, Guillain–Barré syndrome, and Alzheimer’s disease, among others. In addition, therapeutic targets are analyzed in relation to the pathways that induced neuroinflammation. Neuroinflammation is a complex immune response of neural tissue, to restrain infection and eliminate pathogens, cell debris, and mis-folded proteins in a generic manner; it involves all the cells present within the central nervous system (CNS), including the neurons, macroglia and microglia . EmerginThe adaptive immune system has a wide array of antigen receptors that can recognize a variety of pathogens ,6,7. In The diverse subpopulations of Th cells perform different functions: Th1 cells are characterized by the secretion of interferon γ (IFN-γ) and tumor necrosis factor (TNF); they are the most common subset of memory effector T cells and offer protection against infection by bacteria, viruses, and intracellular parasites ,11. Th2 Th17 cells are a subpopulation of CD4+ lymphocytes that have been linked to the Th1 cytokine profile and are characterized by their production of IL-17 and IL-22 ,19.Previous studies have found that IL-17 is a cytokine driving autoimmune and inflammatory diseases ,21. IL17Th17 cells are not a “fixed” subset, since they are capable of converting into other lineage subsets influenced by the microenvironment . Th17 ceTregs is another lymphocyte subpopulation, characterized by the expression of high levels of CD25+, cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and the expression of transcription factor Foxp3. They have an essential role in anti-inflammatory, neurotrophic and neuroprotection functions , with a Treg cells are capable of maintaining immune homeostasis and limiting inflammatory responses, for example, by inhibiting the Th17 response. This is how these two cell types play opposite roles during inflammatory and immune responses ,29. The The molecular pathways between Treg and Th17 are reciprocally interconnected . Both thThe mechanism is explained as follows: IL-6, 21 and 23 activate the signal transducer and transcription activator 3 (Stat3), an element upstream of RORγT that induces its expression and promotes Th17 as a destination ,15,40. ICell homeostasis, which can include oxygen homeostasis, is central to physiological processes such as growth, development, regulation of metabolic energy, angiogenesis and erythropoiesis, and for pathological processes such as anemia, ischemia, inflammation and cancer ,43,44,45Under hypoxic conditions, cells respond via the increase in hypoxia inducible factor (HIF) levels, which activate genes that modulate oxygen supply and consumption ,49, and HIF-1α and HIF-2α contain two transactivation domains, an oxygen-regulated C-terminal transactivation domain (CAD) and a more centrally located transactivation domain designated N-terminal transactivation domain (NAD). Both the NAD and CAD employ recruitment of the coactivators CBP/p300, SRC proto-oncogene, non-receptor tyrosine kinase (SRC-1), and transcription intermediary factor 2 (TIF-2) . HIF-1α 2, simulating physiological conditions), the differentiation of Th17 is increased, a phenomenon that requires HIF-1α and is induced by the activation of the mammalian target of rapamycin complex 1 (mTORC1) [Several studies have shown that HIF-1α is also involved in Th17/Treg regulation, being able to promote immunity mediated by Th17cells ,26,62. H(mTORC1) . This di(mTORC1) ,63, and (mTORC1) . The mTO(mTORC1) , and reg(mTORC1) . It has (mTORC1) . Moreove(mTORC1) ,55,62.HIF-1 stimulates Th17 differentiation through RORγT induction and Treg differentiation inhibition, via an active process that promotes Foxp3 protein degradation, directly by means of ubiquitination ,46. ConvFinally, it is suggested that another factor that could determine the memory Th17 cell fate would be the activation of the HIF-1α/Notch/Bcl-2 signaling pathway, which could control the survival and apoptosis of Th17 cells ,68.All cells and tissues have the ability to respond to changes in oxygen levels ,46. The Brain tissue oxygen consumption is nearly 20% of the total oxygen consumed by the entire body. This event determined that this organ is found among the most sensitive tissues to hypoxia ,70,71,72Some of the effects observed in hippocampal cells exposed to hypoxia are as follows: apoptotic nuclei formation, decrease in neuronal survival, increase in HIF-1α expression, loss of mitochondrial membrane potential, activation of caspase 3, and others . In addiIt has been described that there are other adaptive mechanisms to hypoxia, which are part of the Warburg shunt. Some can promote neuroprotection through a reduction in cytochrome C oxidase function and by an increase in the production of mitochondrial ROS, inducing a shift in energy gain towards astrocyte-mediated glycolysis . Other mIn addition, disruptions in energy supply, because of anoxia/ischemia, cause ionic homeostasis loss and the depolarization and rapid conduction failure of central fibers ,71. In tAt the cellular level, hypoxia also generates the recruitment of microglia . MicroglMicroglia activation causes the overproduction of inflammatory cytokines, which perpetuates hypoxic brain damage resulting in the death of neurons and oligodendrocytes, leukocyte infiltration, axonal degeneration and disruption of the BBB ,70,85.Although there may be a metabolic adaptation to acute and short-term hypoxia in some cells, sustained or severe hypoxia leads to increased production of ROS in the mitochondria, which contributes to cell death . OxidatiThe pathogenic role of hypoxia, in triggering BBB anatomical and physiological disruption in neurological dysfunction, demonstrates an important protective role for vascular and glial integrity in the hypoxic brain .The BBB is a dynamic barrier that represents the microvasculature of the CNS; it is composed of endothelial cells and mural cells (smooth muscle cells and pericytes), and has a basement membrane that separates into an inner and an outer sheet . BBB alsBBB’s integrity is essential for the normal physiology of the central nervous and glial system. Its disruption is associated with neurological pathologies that determine neurodegeneration events. This continuous non-fenestrated barrier allows endothelial cells to regulate CNS homeostasis and protect it against injury; meanwhile BBB dysfunction can trigger the entry of immune cells and molecules, leading to neuronal dysfunction and degeneration , and conNeuroinflammation plays a relevant role in the development of different neurological diseases, through a response of the immune system to different noxious stimuli of the nervous system . During In the peripheral nervous system (PNS), neuroinflammation also continues, with the rupture of the blood-nerve barrier (BNB), accumulation of self-reactive T cells and macrophages due to chemotaxis and a progressive demyelination . It has Hypoxia and inflammation are two events that coexist, share multiple connections and enhance each other. In addition, both are deregulated in a large number of diseases . Hypoxia2 supply, bioenergetics and cellular survival to preserve tissue integrity. Preclinical and clinical evidence clearly demonstrates that repeated moderate hypoxic bouts, i.e., chronic intermittent hypoxia, can preserve or enhance brain functions [The adaptation to a hypoxic environment is also dependent on different inflammatory cellular and compounds induced through molecular mechanisms. Two recent mediators are described in cellular models of neurodegeneration, such as AD and PD, (i) metabolic adaptations and autophagy induction. Regarding the hypoxic reprogramming of metabolism, this is associated with the adaptation to excessive ROS production, and accompanies the mitochondrial changes in hypoxia . In thisunctions .2 availability to promote cell adaptation and survival [2) in various cell lines and has pro-survival functions by mediating hypoxia-induced autophagy. Closely-related BNIP3L is also induced by hypoxia indicating both proteins are necessary for autophagy under these stressful circumstances. HIF-1α-dependent expression of BNIP3 has also been described as essential in mitophagy, as previously mentioned [Under physiological conditions, autophagy is maintained at a low basal rate as part of quality control pathways to remove damaged proteins and organelles. However, it potently responds to external cellular microenvironments and can be influenced by nutrient and Osurvival . BNIP3  it can contribute to vascular wall atherosclerosis, causing vascular dementia and infarctions; (2) can compromise the BBB and its function, allowing leukocytes and antibodies to enter the brain; (3) brain produced antibodies can induce immune attacks, like those that occur in MS; (4) the inflammatory induced oedema; (5) some types of inflammation suppress neurogenesis; and (6) pathogens and protein can aggregate or damaged neurons can activate microglia and later kill neurons ,109,110.Autoimmune diseases are characterized by immune systems’ loss of self-tolerance, which can be caused by environmental, genetic factors, or a combination of both ,114. InfTo elucidate the role of microglia and other immune cells in neuronal damage, different genomic and transcriptomic assays have been performed in in vitro and in vivo models of AD . For exaHypoxia influences our immune system through regulation of T cell differentiation, which is important not only in alloimmune regulation after transplantation, where the organ is exposed to severe hypoxic insults, but also in the modulation of autoimmune inflammation basis of different autoimmune diseases ,49,122. In this sense, IH is strongly associated with dementia via different mechanisms, including insulin resistance, inflammation, and ischemia. However, the direct effect of IH on the development or exacerbation of cognitive impairment remains unclear . Recent In addition, hypoxia facilitates differentiation of the proinflammatory Th17 phenotype, while simultaneously inhibiting the differentiation of tolerogenic Treg cells . By contTh17 cells are important for the of the host’s mucosal defense and for mediating immunity against extracellular bacteria and fungi. However, deregulation of the Th17 response, characterized by overproduction of IL-17A and IL-17F, is particularly important as a cause or component in autoimmune diseases, and diseases associated with inflammation and destruction; these include rheumatoid arthritis, psoriasis, Parkinson, MS and Crohn’s disease ,131,132.Identifying factors driven by autoimmunity in neurodegenerative diseases remains challenging ,136. Th1In the case of neuroinflammatory diseases, several studies have analyzed HIF-1 and Th17 related contribution . Some stWithin the wide variety of diseases, we will analyze those that cause inflammatory lesions in the CNS and PNS through activation of the immune system, such as MS and inflammatory demyelinating disease, respectively, and inflammatory lesions neurodegenerative in nature, as with Alzheimer’s and Parkinson’s Disease ,45,139.Guillain–Barré syndrome (GBS) is an acute inflammatory disorder mediated by the immune system in the PNS, and characterized by inflammatory infiltration and damage to the myelin and axon ,99. ExpePathologically, EAN is characterized by the BNB rupture, accumulation of self-reactive T cells and macrophages in the PNS by chemotaxis and demyelination . In the Multiple sclerosis (MS) is a disease characterized by neuroinflammation in the CNS, and one of its animal models is EAE ,125,142.Pathologically, in MS there is disruption of the BBB, inflammation ,133 and The neurological deficits and expression of HIF-1α have correlated quantitatively, temporally and spatially with the hypoxia generated in the gray and white matter in EAE, relating functional deficits to the neuroinflammation caused by tissue hypoxia . PatientThe etiological cause of AD involves accumulation of the beta amyloid protein, chronic inflammation reactions, oxidative stress, proteasome inhibition, and high cholesterol levels, where all these mechanisms are associated by a common factor: neuroinflammation ,146. In However, it has been mentioned that previous to amyloid accumulation, other alterations arise that could cause the onset of AD. This includes cerebral hypometabolism, triggered by events related to hypoxia or oxidative stress , or vascDue to the existing relation between Th17, HIF-1α and inflammation in neurodegenerative diseases, new therapeutic targets have been developed that could be used in the treatment of diseases with neuroinflammation.The pathological niche created by hypoxia contributes to the development of inflammatory and autonomic diseases in a tissue- and context-dependent manner . The eneHIF-1 inhibitors, which increase the hydroxylation activity of PHD that may increase its degradation, could prevent the rupture of the BBB, associated with insult due to hypoxic damage and brain ischemia ,96. InhiThe increase in the Th17 response in the brain and peripheral tissues has a synergic effect on neuroinflammation and neurodegeneration in EAE, since the increase in IL17 and IL22 in the peripheral blood promotes the rupture of the BBB so that more Th17 cells migrate to the cerebral parenchyma, exacerbating the damage ,148. TheBecause pro- and anti-inflammatory cytokines play an important role in the T cell immunomodulation, the reduction in proinflammatory cytokines and the increase in cytoprotective factors (such as antioxidants with a neuroprotective capacity) may be a potential therapeutic target ,153. TheIL17-producing cells play a key role in promoting disease progression through amplification of the local immune response . Some thThe unregulated activation of the JAK/STAT signaling pathway has been implicated in several autoimmune and neurodegenerative diseases . STAT3 sCurrently, neuroinflammation defines pathophysiological events, including several molecular pathways that regulate differentiation and maturation of the immune system. In fact, it is thought that the alteration of the metabolic environment in cells by hypoxia is what guides the activation of HIF, creating a feedback loop that connects the metabolism of immune cells with inflammation . In thisThe pharmacological approach, which attenuates the response related to these molecular pathways, with mechanisms, for example, that sense hypoxia and its signaling pathway, could be used as a potential therapeutic target in the treatment of the disorders associated with neuroinflammation .The different subpopulations of T lymphocytes and their plasticity under specific conditions, play a key role in neuroinflammatory diseases. For this reason, it is important to continue studying the contribution of each one, how they are able to change in the face of different stimuli, and how they trigger the onset or progression of the disease.Finally, it is essential to determine the correct experimental model that would allow the study of these dynamic processes efficiently.


NF-κB, IκB, p38, IL-1β, TNF-α, Beclin, ATG7, ATG5, LC3α, BaK, Pum, Caspase-3, RIP1, RIPK3, PERK, IRE1, elF2α, GRP78, CHOP2, ZO-1, ZO-2, Occludin, E-cadherin, CaM, MLC, MLCK, Rho, and RhoA in the LSe group were significantly increased. The mRNA levels of IL-10, p62 BcL-2 and BcL-w were significantly decreased in the LSe group compared with the CSe group. These results suggest that changes in the abundance of Lactobacillus, bifidobacterium, ileum, and Romboutsia may be associated with cellular inflammation, autophagy, endoplasmic reticulum stress, apoptosis, tight junction, and abnormal smooth muscle contraction. Intestinal flora may play an important role in chronic diarrhea caused by selenium deficiency.Selenium (Se) is a micronutrient that plays a predominant role in various physiological processes in humans and animals. Long-term lack of Se will lead to many metabolic diseases. Studies have found that chronic Se deficiency can cause chronic diarrhea. The gut flora is closely related to the health of the body. Changes in environmental factors can cause changes in the intestinal flora. Our study found that Se deficiency can disrupt intestinal flora. Through 16s high-throughput sequencing analysis of small intestinal contents of mice, we found that compared with CSe group, the abundance of Lactobacillus, Bifidobacterium, and Ileibacterium in the low selenium group was significantly increased, while Romboutsia abundance was significantly decreased. Histological analysis showed that compared with CSe group, the small intestine tissues of the LSe group had obvious pathological changes. We examined mRNA expression levels in the small intestine associated with inflammation, autophagy, endoplasmic reticulum stress, apoptosis, tight junctions, and smooth muscle contraction. The mRNA levels of  Selenium (Se) is a micronutrient that plays a vital role in various physiological processes in humans and animals. Lots of previous research have confirmed the biological effects of Se. Se has antioxidant properties, anti-cancer, enhances immunity, and other efficacy –3. HumanThe various bacteria that live in the gut are called the gut microbiome, and there are about 10 trillion of them. It contains 100 to 1000 bacterial species and 50 phyla. The gut microbiota is called the second gene . With thDiarrhea is associated with intestinal inflammation, disruption of the intestinal mucosal barrier, and abnormal contraction of the intestinal smooth muscle –37. PrevZO-1, ZO-2, and Occludin are classic tight junction proteins and can be used as common indicators to observe tight junction damage  to form a Ca2+-CaM complex (MLCK) in the cytoplasm. The non-calcium-dependent pathway is associated with RhoA/Rho kinase (ROCK) signaling pathway , and myosin light chain (MLC)  . Many stin (MLC) . The imbIn this study, we sequenced the intestinal microflora of the mouse model. This study investigated the effect of selenium deficiency on intestinal epithelial cell tight junction, intestinal inflammation, and intestinal smooth muscle contraction due to changes in the intestinal flora. It provides theoretical guidance for the follow-up research.Female c57 mice aged 4-5 weeks  were selected. These mice were divided randomly into two groups and 20 mice in each group. The environmental parameters such as temperature, relative humidity, wind speed, and the illumination of each group were controlled and kept consistent. Low selenium group (LG): 20 mice were fed with Se-deficient mouse diet (Se content: 0.01mg Se/kg). Normal Se group (NG): 20 mice were fed with normal Se content mouse diet (Se content: 0.15mg Se/kg). Rat food is customized for selenium deficiency rat food and normal rat food from Trophic Animal Feed High-tech Company, China. The mice were free to eat and drink. The mice died of cervical dislocation after being fed at room temperature for 50 days and were quickly sampled and stored in a -80°C refrigerator. The animal study was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Jilin University.Small intestinal tissue was fixed in 4% paraformaldehyde and paraffin-embedded overnight. The small intestine sections were cut into two μm thick and soaked overnight in ZGSJ (Masson A). The small intestine sections were stained with Weigert’s hematoxylin  for 1 minute, differentiated by 1% acid ethanol, Then it was stained in scarlet magenta solution (Masson D) for 6 min, differentiated in phospho-molybdenum-phosphotungstate solution (Masson E) for 1 min, and transferred directly (without rinsing) to aniline blue solution (Masson F) for 2-30s. Then, rinse briefly in distilled water for 2-5 minutes. Finally, dehydration through anhydrous ethanol, xylene transparent, and neutral sealant. The sections were scanned by Pannoramic 250 slide scanner (3D HISTECH). Micrographs were analyzed by the blind method. Collagen volume fraction (CVF) was observed by image analysis system software . Technical support was provided by Servicebio, Inc. .Chromosomal DNA double-strand or single-strand breaks produce sticky 3’-OH ends. Under the catalysis of deoxyribonucleotide terminal transferase (DTT), dUTP with fluorescein molecule is labeled to the 3’end of DNA. Observe the stained apoptotic cells through a fluorescence microscope. Dewax the paraffin sections to water, break the membranes, incubate the Tunel reaction solution, microwave repair, incubate the primary antibody overnight at 4°C, add the secondary antibody, DAPI staining the nucleus, anti-fluorescence quenching and mounting, microscopic examination and taking pictures, and observe the results.Total RNA was extracted from frozen intestinal tissue. The concentration and purity of RNA solution were determined by UV spectrophotometry at 260nm and 280nm. Targeting specific primers were synthesized by reverse transcription design of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) based on known sequences. The ABI PRISM7700 processing system was used for real-time quantitative PCR. For each gene to be measured, the cDNA template and sample cDNA of the expressed gene is selected for PCR reaction. There are 40 cycles, such as 94°C for 30 seconds, 94°C for 5 seconds, and 60°C for 30 seconds. Each experiment was repeated three times, and each sample was repeated three times. GAPDH was used as an endogenous internal standard control. The primers were purchased from Sangon Biotech, Shanghai. Primer sequences are shown in ® High Fidelity PCR Master Mix with GC Buffer and High Fidelity enzyme for PCR amplification efficiency and accuracy. PCR products were detected by electrophoresis with 2% agarose gel. The qualified PCR products were purified by magnetic beads, quantified by enzyme standard, and mixed in equal quantities according to the concentration of PCR products. After full mixing, PCR products were detected by 2% agarose gel electrophoresis, and the target bands were recovered using gel recovery kits provided by Qiagen. TruSeq® DNA PCR-free Sample Preparation Kit was used for library construction. The constructed library was quantified by Qubit and Q-PCR. After the library was qualified, NovaSeq6000 was used for machine sequencing. Each sample data was separated from the off-machine data according to the Barcode sequence and PCR primer sequence. Qiime software (Version 1.9.1) was used to calculate and analyze the data. This experiment was commissioned by Novogene Tianjin Company.In this study, genomic DNA was extracted from intestinal contents by CTAB or SDS. After that, agarose gel electrophoresis was used to detect the purity and concentration of DNA. An appropriate amount of DNA was taken into the centrifuge tube and diluted to 1ng/μ L with sterile water. Using diluted genomic DNA as a template, specific primers with barcodes were used according to the 16S V3-V4 region of the sequencing area. New England Biolabs PhusionSPSS 22.0 statistical software was used for statistical analysis. All data were expressed as ± standard deviation (SD) measurements. The data were compared by the T-test method. The significance of the difference criterion is P<0.05.The species accumulation curve is used to determine whether the available sample size is sufficient for a high-throughput community analysis of the species. If the upward trend of this curve is always steep, it indicates that the sample size is too small to support the following analysis. As the number of samples increases, the upward trend of the curve is relatively gentle, indicating that the original sample size is large. Therefore, it is necessary to measure the cumulative curve of species before all kinds of analyses of fecal flora. If the cumulative curve always shows an upward trend, it indicates that the sample size is insufficient to support the analysis of species composition structure. On the contrary, if the curve rises gently with the increase of samples, it indicates that the sample size is sufficient to reflect the species composition of the community. It can be seen in The representative sequences of the top100 genera were obtained by multiple sequences alignment to further study the phylogenetic relationships of genus-level species. The resulting display was shown in The rarefaction Curve and Rank Abundance curve are usually describing the diversity of samples within groups. In Beta Diversity is a comparative analysis of microbial community composition of different samples. As shown in PCA  is a method to extract the most important elements and structures in data based on Euclidean Distances. PCA analysis can extract two coordinate axes that reflect the difference between samples to the greatest extent. The differences in multi-dimensional data are reflected in the two-dimensional coordinate graph, and the simple rules under the complex data background are revealed. The more similar the community composition of the samples is, the closer they are to the PCA diagram. As shown in To characterize the overall differences in the gut microbiome between groups, we performed a principal coordinate analysis (PCoA) of unweighted UniFrac distances. There were significant differences between the LSe group and CG  is a ranking method suitable for ecological studies. NMDS is a nonlinear model based on bray-Curtis distance, which is reflected in a two-dimensional plane in the form of points according to the species information contained in the sample. It can better reflect the nonlinear structure of ecological data. NMDS analysis can reflect the differences between and within groups of samples. NMDS analysis results based on OTU level are shown in LEfSe is used to detect OTUs or bacterial system types (phylum to order level) in different feeding groups. In Tax4Fun function prediction is used by the nearest neighbor method based on the minimum 16SrRNA sequence similarity. The specific method was to extract the 16S rRNA gene sequence of the whole genome of prokaryotes from the KEGG database and compare it with the SILVA SSU Ref NR database (BLAST bitscore >1500) using the BLASTN algorithm to establish a correlation matrix. The whole-genome functional information of prokaryotes annotated by UProC and PAUDA in the KEGG database was corresponding to the SILVA database to realize the functional annotation of the SILVA database. SILVA database sequence was used as a reference sequence to cluster OTU and obtain functional annotation information. Changes of Intestinal microflora induced by Selenium Deficiency and their relationship with Human Diseases, Environmental Information Processing, Metabolism, Organismal Systems, Cellular Processes Genetic information processing. The result of the annotation is shown in Histological analysis was shown in NF-κB, IκBα, p38, IL-1β, and TNF-α in the LSe group were significantly increased, while the mRNA levels of IL-10 were significantly decreased. It is evidence of inflammation in the small intestine. The mRNA levels of Beclin, ATG7, ATG5, and LC3α were significantly increased in the LSe group, while the mRNA levels of p62 were slightly decreased compared with the CSe group. It indicates that the cells of the small intestine are autophagy. Compared with CSe, mRNA levels of PERK, IRE1, elF2α, GRP78, and CHOP2 were significantly increased. It indicates endoplasmic reticulum stress in the cells of the small intestine. The mRNA levels of BaK, Pum, Caspase-3, RIP1, and RIPK3 were significantly increased in the LSe group, while the mRNA levels of BcL-2 and BcL-w were significantly decreased in the LSe group compared with the CSe group. It indicates that the cells of the small intestine have undergone apoptosis. The mRNA levels of tight junction proteins such as ZO-1, ZO-2, Occludin, and E-cadherin were significantly increased. It suggests that the tight junctions of the small intestine were broken in the LSe group. The expression of LSe smooth muscle contraction-related genes such as CaM, MLC, MLCK, Rho, and RhoA increased. It indicates abnormal contraction of the small intestine smooth muscle.We examined gene levels in the small intestine related to inflammation, autophagy, endoplasmic reticulum stress, apoptosis, tight junctions and smooth muscle contraction. The results were shown in Se is a micronutrient. It has biological functions such as anti-oxidation, anti-cancer, and enhancing immunity , 3. Se dIntestinal flora has become a new hot research field. The interaction between intestinal flora and Se in food mainly focuses on the composition of symbiotic flora, the regulation of the metabolic process, and the function of the intestinal mucosal barrier. To further evaluate the effects of Se deficiency on intestinal microbiota in mice, we extracted the contents of the small intestine for 16S high-throughput sequencing analysis. The results showed that the intestinal microbiota of the LSe group changed significantly compared with that of CG. The strains with the highest abundance in the CG were Dubosiella, Romboutsia, Lactobacillus, and Faecalibaculum. Dubosiella, Bifidobacterium, Lactobacillus, and Ileibacterium had the highest abundance in the LSe group. The composition of microflora changed obviously. Compared with CG, the abundance of Lactobacillus, Bifidobacterium, and Ileibacterium with LSe significantly increased, while the abundance of Romboutsia significantly decreased or almost disappeared. The abundance changes of Ileibacterium and Romboutsia are the most obvious. The abundance changes of these two bacteria likely cause the abundance changes of Dubosiella and Lactobacillus. But this still needs to be further studied. The result suggests that selenium deficiency can lead to changes in the composition of intestinal flora.IL-1β, TNF-α, and IL-6 were significantly elevated. The LSe group also significantly increased the expression of NF-κB and the IκBα gene in the classic inflammatory signaling pathway. This confirmed that selenium deficiency can cause inflammation of the small intestine through intestinal flora.Our study showed significant pathological changes in small intestinal tissues of mice in the LSe group compared with CSe. The studies on gene levels showed that cytokines Beclin is an autophagy-related gene and participates in the formation of autophagosomes  of Jilin University.FW, NS, and WZ conceived and designed the research; FW, NS, HZ, and YG performed the research and acquired the data, and FW analyzed and interpreted the data and wrote sections of the manuscript. All authors contributed to manuscript revision, read, 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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Each country has their own national breeds that are well adapted to the local climate and other conditions. Our study examines two national sheep breeds from Slovakia, the Original and the Improved Valachian sheep. The main goal of the study was to identify parts of their genome which contain genes enabling such adaptations. These are the so called selection signatures. Within the breed we look for genomic regions that were remarkably similar among a large proportion of genotyped individuals. The results point towards adaptation to high altitude pastures and resistance to parasites. The signatures of selection between the Original and Improved Valachian sheep focus on regions with striking difference in the same region of the genome between the two breeds. These results highlighted genes that improve the milk, meat and wool production, and their quality characteristics.This study explored the genomic diversity and selection signatures in two Slovakian national breeds, the Original Valachian and the Improved Valachian sheep. As they are an important animal genetic resource within the country, but with decreasing population size, our aim is to identify potentially valuable genomic regions. A total of 97 sheep  from the Original Valachian, and 69 sheep  from the Improved Valachian populations were genotyped using the GeneSeek GGP Ovine 50 K chip. The inbreeding levels were assessed with runs of homozygosity (ROH). The selection signatures within breeds were identified based on the top 1% of most homozygous regions within the breed, the so-called ROH islands. The selection signatures between breeds were assessed based on variance in linkage disequilibrium. Overall, we have identified selection signatures with quantitative trait loci (QTL) and genes pointing towards all three production purposes of the Valachian sheep, milk, meat, and wool, including their quality characteristics. Another group with apparent large importance was the various traits related to health and resistance to parasites, which is well in line with the sturdy nature of this breed. The domestication of various livestock species is one of the greatest achievements of humankind. This process inherently resulted in permanent changes in the domesticated species, related to their morphological characteristics, behavior, and production levels. Parallel to the changes at the phenotype level, there were changes also occurring on the genomes. These detectable changes are called selection signatures, and they are of great interest to evolutionary biologists and livestock researchers alike. Sheep was one of the prime targets of domestication efforts, due to the wide variety of useful products, ability to adapt to different climatic conditions, and manageable size . SubsequThe Original Valachian sheep a national, autochthonous breed from Slovakia. The small population of ca. 2900 animals is located in the northern mountainous regions, as it is well adapted to the harsh conditions of high-altitude pastures there. It was traditionally kept for combined triple-use: milk, meat and wool. The production is low, with ca. 80 kg milk per lactation, body weight of 35–55 kg, and average daily gain of 200 g until weaning. The Improved Valachian sheep was created to address these low production characteristics, while keeping the adaptation and climate resistance traits. A targeted and planned crossbreeding scheme was implemented between 1950s and 1980s, using Texel, Hampshire Down, Lincoln Longwool, and other breeds. The Improved Valachian sheep has higher milk production of ca. 100 kg per lactation, much higher body weight of 50 to 75 kg and average daily gain to weaning about 220 g.Such diversity of breeds could evolve due to the wide variety of natural and artificial selection pressures on the populations. The selection itself leaves detectable changes on the genome caused by the so-called genetic hitchhiking effect . Here, tOne of the most widespread methods to detect the reduced variance in genomic regions of selection signatures is based on so-called runs of homozygosity (ROH) islands. The ROH are fully homozygous genomic segments that occur due to the relatedness of the two parents. Because of this assumption, they are often used to determine the inbreeding coefficient of animals . HoweverAnother possibility to explore the variation, and therefore the impact of selection on the genome is via patterns of LD. The LD is the population-wide correlation of nearby variants and characterizes the strength of connection between pairs of loci. The so-called LD blocks build up as a result of selection, via the genetic hitchhiking effect. In short, the allele at which the selection is acting is not inherited independently, but together with the loci that are nearby. If the selection pressure is strong enough, the allele is widespread in the population, together with its neighboring neutral alleles. This process results in a strong correlation, thus strong LD within this region. The differences in LD blocks between breeds than indicate differences in selection. This method was used to detect selection signatures in cattle ,12,13, pFor our current study, we intend to fill the gap in the knowledge in genomic characterization of small indigenous breeds. We focus in particular on the exploration of diversity and selection signatures of the two Slovakian national sheep breeds, the Original Valachian and the Improved Valachian. They are an important animal genetic resource with diminishing population size; thus, identifying their characteristic and potentially valuable genomic regions is of great importance. Therefore, our aim in this paper is to explore the selection signatures within and between the Original and Improved Valachian sheep populations.p < 10−7 were excluded from the data set for the LD based selection signature analyses. The minor allele frequency threshold was not considered for run of homozygosity analyses. The SNPs from unplaced or sex chromosomes were removed for all analyses. After quality control, 96 animals and 38,236 SNPs remained for the Original Valachian sheep, and 68 animals and 39,240 SNPs were left for the Improved Valachian sheep population.A total of 97 sheep  from the Original Valachian, and 69 sheep  from the Improved Valachian populations were genotyped using the GeneSeek GGP Ovine 50 K chip, with DNA obtained from hair follicles. The quality control was done using PLINK 1.9 , with thk, and L is the length of the autosomal genome covered by SNPs.The inbreeding levels were computed using the software cgaTOH , based oFurther, the overlap between the ROH regions with minimum length of 1 Mb was examined for each breed. Such overlaps were called ROH islands, and were considered as selection signatures. The exact cut-off value for the ROH islands to be analyzed in the follow up was defined as the top 1% of most homozygous regions. The results were visualized using R, and the regions were compared to the sheep QTLdb  to determine their potential functions.The linkage disequilibrium was computed as squared correlations using PLINK’s—r2 option, between each SNP within 1000 kb windows. The SNPs were ordered based on the inter marker distance, and the LD decay was visualized as the average within non-overlapping sliding windows of 5 SNPs.In addition, the varLD software  was usedThe inbreeding levels for the two populations are summarized in The overlap of ROH segments with minimum length of 1 Mb in both populations was assessed and visualized in In both breeds, there were a number of QTLs related to the main production of the breed, meat, milk and wool. These were represented by QTLs for both the amount of production alone, but also quality parameters, such as fat and protein content of milk, pH and color traits of meat, and similar. Interestingly, in the Original Valachian sheep, there we fewer, but larger regions, also containing a range of adaptation and disease resistance traits, such as response to parasitic infections. In the Improved Valachian sheep the most homozygous regions were more scattered, and not as widespread within the population . Such results could be expected due to the history of crossbreeding, with admixture breaking down longer, almost fixed genomic regions into shorter segments. The QTL content of these smaller segments throughout the genome was clearly related to production, mostly milk and meat, also including their quality parameters, but also QTLs related to parasite resistance and health traits. On chromosome 10, around the highest signal, there were a number of QTLs related to horn traits. This might be of significance and could merit a further study, due to the peculiar horn shape of the Valachian sheep. Another interesting region in the Improved Valachian sheep was a small region on chromosome 25 (12.4–12.6 Mb), with a large number of QTL related to wool production. Although wool is currently not of great importance, the previous breeding process placed some emphasis on the improvement of this traits as well, which might have left some detectable signatures behind.The much lower inbreeding  in the ISimilarly to ROH results, the consequences of the applied breeding strategy were also visible for the comparison of LD decay in After a breed-specific analysis of QTL functions in the identified regions, the two breeds were compared to identify regions where they differ from each other. As for the top 0.1% highest signal, there was only one region on chromosome 20, between 29.9 and 32.7 Mb. There seems to be a gap in the signal between 30.9 and 31.9 Mb. This relatively wide signal does not have a pronounced peak, indicating that the whole genomic region could be of interest, rather than any specific point. The SNP indicating the highest signal within this region was located at 30.7 Mb. The top signal for the entire genome was found on chromosome 22. The top 0.1% region was found between 10.3 and 11.6 Mb. The region exceeding the 0.01% threshold was found between 10.8 and 11.0 Mb on chromosome 22.In general, the more relaxed the thresholds were, the wider the section signatures became. This could be a possible reason why other studies, such as  and 14]14] evaluLIPA, IFIT2, IFIT3, IFIT5, SLC16A12 and three pseudogenes. The LIPA gene was found to be involved in the lipid mechanism and composition of lipoproteins [IFIT genes was shown to have antiviral functions [SLC16A12 gene was also shown to have various immune related functions, related to bacterial infections [Our top signal directly overlaps with the genes proteins , responsproteins , but alsproteins . The famunctions . Similarunctions . The SLCfections ,26.A large number of pseudogenes and protein coding genes were found in the other regions identified by the VarLD approach. Here, they are listed according to the chromosome number for the regions indicated above, and those with possible larger relevance are further described.USP24, PHF11, DHCR24, TTC22 and TTC4. The gene PHF11 appears to inhibit replication of certain retroviruses [DHCR24 appears to have an important function related to the biosynthesis of cholesterol, but also involved in neurological disorders, such as the Alzheimer disease [For chromosome 1, these protein coding genes were oviruses  and playoviruses . The DHC disease .DAPK1, FBP1, FBP2, CTSL, ISCA1, GOLM1, NTRK2, SLC28A3, RMI1, HNRNPK, KIF27, GKAP1, UBQLN1, FRMD3, IL11RA, ARID3C, DCTN3, DNAI1, C9orf24, NUDT2, KIF24, UBAP1, DCAF12, UBAP2, UBE2R2, NOL6, AQP3, AQP7, NFX1, BAG1, B4GALT1, DNAJA1, APTX, EPHX2, CHRNA2, PTK2B, DPYSL2, BNIP3L, PPP2R2A, EBF2, CDCA2, DOCK5, NEFL, NEFM, ADAM7, ADAM28, STC1, LOXL2, ENTPD4, RHOBTB2, PEBP4, EGR3, BIN3, SORBS3, DLIM2, PPP3CC, SLC39A14, PIWIL2. Even though it is a very wide region with a high number of genes, there is a remarkable diversity in gene functions, which is relevant for productive, reproductive and health aspect of animals’ life. These associations were highlighted in a wide range of studies. Unfortunately, there were only a few studies done directly in sheep, but relevant studies were found in humans, but also in livestock, mostly cattle.On chromosome 2, there was a very wide signal harboring a large number of protein coding genes, such as DAPK1 gene had an identified relationship to somatic cell score in cattle [FBP1 gene plays an important role in the glucosynthesis [FBP2 gene was shown to be related to milk productions traits [CTSL (cathepsin L) seems to be interesting from multiple angles, as it had been shown to influence carcass traits, production of milk [ISCA1 was found to be associated with multiple mitochondrial dysfunctions [NTRK2 was shown to be a novel candidate gene for litter size in sheep [RMI1 was shown to be associated with growth in sheep [KIF27 gene was involved in fertility [UBQLN1 is involved male fertility, more precisely in ubiquitination during spermatogenesis [IL11RA gene was also shown to be reproduction related, with its involvement in the placentation in multiple species [ARID3C was involved in immunoglobulin gene transcription [DNAI1 gene was identified to be relevant for spermatogenesis in cattle [UBAP2 gene was found to be implicated in hypoxia and heat stress in sheep and goats [AQP3 and APQ7 gene [B4GALT1 gene was found to be associated with milk production in cattle [DNAJA1 gene was highly expressed in both mammary gland and milk transcriptome, suggesting involvement in milk synthesis and production [APTX gene was found in a study for marbling score in cattle [PTK2B gene was recently found to be involved in regulation of bovine mastitis [DPYSL2 gene, also known as CRMP2, is a gene of major interest, as it could be involved in an extensive collection of neurodegenerative, sensory and motor neuron, and central disorders [BNIP3L, PPP2R2A, CDCA2 and NEFL genes were related to reproductive traits, namely, to ovarian follicle development and with lambings out of season [EBF2 was shown to be involved in regulation of body fat [ADAM7 and ADAM28 genes were previously found in genomic regions related to male fertility. In particular, ADAM28 showed significant non-additive effects for sire conception rate in cattle [STC1 gene [LOXL2 gene was found to be related to meat production in cattle [ENTPD4 gene seems to be influencing both milk production [PEBP4 which was found to simultaneously affect growth and fertility in cattle [SORBS3, PPP3CC and PIWIL2 were located in a region associated with growth and wool traits in [PPP3CC gene was linked to heat tolerance in cattle [PIWIL2 was associated with reproduction traits [SLC39A14 gene was found to be associated with feed efficiency in cattle [The n cattle . The FBPynthesis , while ts traits , activats traits . The gen of milk  and repr of milk . The ISCunctions . Accordiin sheep . RMI1 wain sheep . The KIFertility . The genogenesis . The IL1 species . ARID3C cription . The DNAn cattle . The UBAnd goats . In addind goats . The colPQ7 gene , also inn cattle . The DNAoduction . At the oduction . Similarn cattle . The PTKmastitis . The DPYisorders , which cf season . The EBFbody fat . The ADAn cattle . The femTC1 gene . The LOXn cattle . The ENToduction  and fertoduction . Anothern cattle . The genraits in . In addin cattle , and then traits . The SLCn cattle .TMBIM1 and PNKD, connected to meat color in cattle [USP37, involved in regulation of reproduction in mice [VIL1, related to meat tenderness [CTDSP1, related to milk fatty acid synthesis in goats [SLC11A1, with significant disease resistance related effects, in particular paratuberculosis, tuberculosis and brucellosis in a number of livestock species [The second signal on chromosome was only a single significant SNP at 220.4 Mb. The genes around this SNP were: n cattle ; USP37,  in mice ; VIL1, rnderness ; CTDSP1,in goats ; SLC11A1 species ,69.LYN, PLAG1, RPS20 and MOS were located. From these the most important was the PLAG1, with a widely recognized effect on stature and growth traits in multiple species, including sheep [LYN, MOS) but also fertility and dystocia (RPS20) [SYBU gene, which was related to milk fat synthesis, transport, and metabolism [The next signal was on chromosome 9, around 36Mb, where the genes such as ng sheep ,71. The  (RPS20) . The sectabolism ,74.PDE7A, MTFR1, DNAJC5B, TRIM55, ADHFE1, CSPP1, COPS5, ARFGEF1, and CPA6. From these genes, the most important were the PDE7A related to growth traits, through its significantly upregulation in lambs with muscle hypertrophy, callipyge [TRIM55 gene was upregulated for beef tenderness, juiciness, and flavor [COPS5 gene seems to be involved in multiple pathways, as it regulates the endometrial function in goats [ARFGEF1, and CPA6 were involved in weight and carcass traits in cattle [The wider region on chromosome 9 between 42.7 and 44.9 Mb contained protein coding genes such as allipyge . The TRId flavor . The COPin goats , was shoin goats , and carin goats . The ARFn cattle .SPAG4, NFS1, and ROMO1. The SPAG4 gene was shown to be connected to reproduction traits and spermatogenesis [On chromosome 13 at 64.8 Mb were the following genes: ogenesis ,82.KIF2A gene was located, which plays an important role during the meiosis, and its downregulation might interfere with the spermatogenesis [IPO11, which was shown to be upregulated in mastitis [On the signal on chromosome 16, the ogenesis . One othmastitis , but alsmastitis .FAT4 gene, associated with milk fat composition [The signal on chromosome 17 was remarkably sparse, with almost no protein coding genes along the 3 Mb genomic region. The only gene in the region was the position .PRKD1, associated with BMI and height in humans [The signal on chromosome 18 at 37.8 Mb contains the gene n humans ,88, but n humans .ANK3 and CCDC6. The CCDC6 gene was of particular interest, as it was previously associated with body weight [Chromosome 25 contained a narrow signal indicated by two nearby SNPs at around 15 Mb. This region contained only two protein coding genes, y weight , but alsy weight .In the study, we carried out analysis of selection signatures within the Original and Improved Valachian sheep populations based on homozygosity and LD patterns on their genome. The results could be used in practice to improve the genomic characterization of the two breeds. The unique selection signatures for the respective breeds could serve as to clearly distinguish between the breeds on a genomic level, and aiding the decisions related to the conservation of genetic diversity. Overall, we have identified selection signatures with QTLs and genes pointing towards all three production purposes of the Valachian sheep, milk, meat and wool, including their quality characteristics. Another group with apparent large importance was the various traits related to health traits and resistance to parasites, which is well in line with the sturdy nature of this breed, kept mostly on high altitude pastures. More importantly, the genomic regions related to health and disease resistance, along with others pointing to various production traits, were also apparent in the Improved Valachian sheep. This indicates that the improvement process was a success that brought in increased productive capacity while retaining the adaptation characteristics of this Slovak traditional breed.


Research on the development of its spikelets is not only directly related to the yield and economic value of foxtail millet but also can provide a reference for studying the fertility of other C4 crops. In this study, a hybrid population containing 200 offspring was constructed from the Xinong8852 and An15 parental lines, and two extreme trait populations were constructed from the F2 generation for analysis of the spikelet sterility. The F2 population conformed to a 3:1 Mendelian segregation ratio, and it was thus concluded that this trait is likely controlled by a single recessive gene. Bulk segregant analysis sequencing (BSA-Seq) was used to determine the candidate regions and candidate genes related to the development of foxtail millet spikelets. Additionally, the functional analysis of differentially expressed genes in populations with different traits was conducted by bulk segregant RNA sequencing (BSR-Seq). Finally, conjunctive analysis of BSA-Seq and BSR-Seq results, combined with biological information analysis, revealed six genes on chromosome VII that were ultimately identified as candidate genes controlling foxtail millet spikelet development. This study provides a new reference for research on foxtail millet sterility and lays a solid foundation for the examination of fertility in other gramineous crops.Foxtail millet has gradually become a model gramineous C Thus, BSA-Seq enables the rapid identification of genes associated with qualitative traits controlled by a single gene or major genes associated with quantitative traits. Bulked segregant RNA sequencing (BSR-Seq) is the combination of BSA-Seq and RNA-Seq, and it has been utilized in the localization of the maize yellowing mutation gene , the locIn this study, we used the Xinong8852 and An15 parental lines of foxtail millet to construct a single F2 population that contained 200 individual plants. After field investigation, the traits related to the fertility of the F2 population conformed to a 3:1 Mendelian segregation ratio, and it was thus concluded that this trait is likely controlled by a single recessive gene. Then, BSA-Seq was used to sequence the two populations with extreme traits, which revealed the AF of SNPs from the two populations. This was followed by a correlation analysis based on the differentially expressed genes (DEGs) obtained by BSR-Seq analysis. Thus, this study aimed to localize infertility-related genes and loci and analyze the molecular mechanism of floral organ sterility in foxtail millet.In our study, Xinong8852 (XAP1) and An15 (AYP1), as well as the F2 population constructed by their cross, were used as experimental materials and planted in the Crop Teaching Sample Area of Northwest A&F University . The parents and F2 progeny were planted separately by dibble seeding. At the flowering stage of foxtail millet, the traits were observed, and one parent plant was marked. In the F2 generation, 30 plants with normal spikelet development (KY) and 30 with abnormal spikelet development (BY) were marked. After labeling, fresh leaves were selected from the parents and the extreme population at the same time and stored at –80°C for subsequent DNA extraction for BSA-Seq. Before reaching full bloom, samples were taken from the spikes, respectively, and stored at –80°C for subsequent RNA extraction for BSR-Seq.The modified cetyltrimethyl ammonium bromide (CTAB) method  was usedAfter filtering the raw reads obtained by sequencing to remove the adapter sequences, BWA software  was usedSingle-nucleotide polymorphism index values were used to represent the sequence difference between the offspring population and their parents, and the SNP index value of each variant locus after quality control in the KY and BY bulk samples was calculated. The loci with SNP index values less than 0.3 and SNP depths less than 7 in the KY and BY bulk samples were filtered, and the homozygous and non-missing SNP loci were then retained. At the same time, the differences between the SNP index values of the two progeny pools were calculated to obtain the delta (SNP index) values. After 1,000 permutation tests, 95% CI were applied as the screening threshold to determine candidate sites. Additionally, functional annotation and analysis of genes corresponding to candidate sites were performed.The RNA of the two parents and two bulk samples of offspring with extreme phenotypes were extracted using the Trizol method. The concentration and purity of the extracted RNA were detected using the NanoDrop instrument , and the integrity of RNA fragments was detected using the Agilent 2100 platform . Thus, four bulk samples were obtained for BSA-Seq. Then, after quality control, Beijing Ovison Gene Technology Co. Ltd. completed the sample database construction and sequencing using an Illumina high-throughput sequencing platform . FastQC (version 0.11.9) was used to evaluate the quality of the sequence data. The original data was filtered using Fastp software (version 0.20.0) to remove adapters, uncalled bases, and low-quality sequences to obtain high-quality data .p-values were calculated based on the negative binomial  distribution. Finally, multiple hypothesis testing correction (via the Benjamini–Hochberg procedure) was performed to control the false discovery rate. DESeq2  is an internationally standardized gene function classification system that can be used to classify DEGs according to their molecular function, biological process, and cell composition (cellular component) ontologies, respectively. We used GOseq software (2) not only comprises all characterized metabolic pathways but also comprehensively annotates the enzymes that catalyze each step of such reactions, including amino acid sequences and PDB library links   via the Applied Biosystems ABI 7500 Fast real-time PCR machine . The cycling conditions were 95°C for 1 min, 40 cycles at 95°C for 15 s, and 60°C for 15 s. Each target gene had three technical replicates, and the relative expression level of the target gene was calculated by the 2–ΔΔCt method.In order to further verify the accuracy of the above experimental results and the expression of candidate genes in foxtail millet corresponding with differences in fertility, quantitative reverse transcription PCR (RT-qPCR) experiments were conducted using RNA extracted from the panicles of millet in the two progeny pools before flowering. Each sample  was used for the extraction of total RNA. RNA samples were treated with DNase I  to remove genomic DNA contamination, followed by cleanup using the GeneJET RNA Cleanup and Concentration Micro Kit . A NanoDrop2000 spectrophotometer (Thermo Fisher) and 1% agarose gel electrophoresis were used to evaluate the total RNA concentration and quality of each sample. The validated RNA was uniformly mixed to form the BY sample (from the BY pool) and KY sample (from the KY pool). The cDNA was then prepared from 1 μg of total RNA using All-in-one First-Strand cDNA Synthesis SuperMix , following the manufacturer’s instructions, for RT-qPCR analysis. Actin was used as an internal reference gene, and all primers were designed with Primer 5 . All RT-2 test of the F2 survey results confirmed that the fertility segregation rate of the F2 generation was consistent with a 3:1 ratio  content of these reads was 46.31–46.94%, and the quality of the sequencing data was high  . Next, wANNOVAR is an efficient software tool that can use the latest information to perform functional annotations of genetic variants detected from multiple genomes. We used it to annotate the functions of detected SNP. By screening the SNPs detected by the unified genotype model of gatk3.7 software, 1,945,742 SNPs were obtained. By further annotating the SNPs with ANNOVAR, it was found that 73,739 SNPs were located in the exon region, including “stop gain” mutations, which cause a full coding sequence gene to contain a stop codon; “stop-loss” mutations, which cause a gene to lose its stop codon; “synonymous” mutations, which do not affect the amino acid sequence; and “non-synonymous” mutations, which alter the amino acid sequence.To determine the mutation sites that exist in the BY bulk sample of foxtail millet, the SNP index method was used to analyze the detected SNPs. First, after filtering out the heterozygous SNPs from the filial generation, the SNP index was less than 0.3, with a depth less than 7, and the sites in each F2 pool lacked SNPs; we identified 926,529 such SNPs from 1,945,742 SNPs. Then, we found a total of 9,055 mutation sites outside the CI by calculating the delta SNP index in the KY and BY bulk samples and screening with a 95% CI , and theIn considering the polymorphisms associated with the occurrence of sterility, we filtered out the sites with synonymous mutations in 523 loci and obtained 3 regions and 211 polymorphisms sites related to sterility from chromosomes III, VI, and VII. By associating the location information of these 211 polymorphic sites with genome annotation information, we identified 90 candidate genes, 70 of which were located between 20,557,247 bp and 31,479,141 bp on chromosome VII, 19 of which were located between 33,984,600 bp and 35,805,805 bp on chromosome VI, and a single candidate gene located between 4,908,634 and 4,912,328 on chromosome III. To further analyze the subcellular localization, molecular function, and biological processes associated with these candidate genes, we performed GO annotation and enrichment analysis for 90 candidate genes. All genes were annotated with 705 GO terms, of which 54 genes were significantly enriched within 23 functional groups, including 13 biological processes, 3 cellular components, and 7 molecular functions. Among the biological processes, these genes are mainly involved in the reproduction process, the development of seeds and fruits, the development of the reproductive system, and the reproductive structure. Among the cellular components, these genes are mainly enriched in the components of the cell membrane. Lastly, among molecular functions, these genes are mainly involved in functions such as binding and catalytic activity.In order to further clarify the transcription process associated with organ sterility in foxtail millet, we used the two parents and two extreme trait groups to construct four pools. After establishing three replicates in each bulked pool, we performed RNA sequencing of 12 cDNA libraries on an Illumina (HiSeq™2500/4000) high-throughput sequencing platform. After reads with low quality and adapter sequences were removed, we obtained 187,104,828 clean reads from 193,102,338 raw reads from four samples . Then, tp-value > 0.05 and | log2(fold change)| > 1 were selected as the DEGs. Spikelet sterility only appeared in the BY sample, but both parents and the KY sample showed normal spikelet phenotypes. We then compared the samples with abnormal floral organs with other samples with normal development to obtain genes related to floral organ sterility in foxtail millet. By comparing differential gene expression between BY with KY, BY with AYP1, and BY with XAP1, we obtained 1,814, 3,132, and 2,022 upregulated DEGs, and 4,537, 3,703 and 4,278 downregulated DEGs, respectively , a protein phosphorylation gene (Setit009293mg), a gene related to sugar transport (Setit009865mg), a serine/threonine-protein phosphatase PP2A-1 catalytic subunit gene (Setit011746mg), a gene related to DNA binding factor (Setit011801mg), and a gene related to ethylene (Setit009909mg).p < 0.001). However, the expression levels of Setit009865mg, Setit009909mg, and Setit011746mg were significantly lower than those of fertile samples (p < 0.001) (p < 0.05)  , and the < 0.05) . Thus, tIn most crops, flower development directly affects its economic value. However, when crops are affected by the external environment or have various mutations, infertility is often a consequence. A thorough study of crop infertility and exploring its occurrence mechanism is not only important for improving crop yields and economic value but also for artificial interference breeding and the effective utilization of crop heterosis. Numerous studies have shown that the development of crop spikelets is a precise and complex regulated process, and any factor change in the process may lead to infertility .ch in foxtail millet, and the existence of multiple alleles causing male sterility in foxtail millet was further confirmed at the level of cytogenetics , can usually interact with transcription factors or themselves to regulate various physiological and biochemical processes in plants (Among the candidate genes, monosaccharides and sucrose transporters play a pivotal role in saccharide transport from source to sink . StudiesAPETALA3 . The VQ APETALA3 . A studyn plants . Ethylenn plants . Ethylenn plants . Addition plants .As a reaction catalyzed by protein kinases, protein phosphorylation is one of the most important post-translational protein modifications, and it is related to the regulation of many biological activities and functions in cell signaling. When plants encounter stress, trauma, or stimulation by other cytokines and hormones, serine/threonine protein kinase is rapidly phosphorylated and activated at serine and threonine residues. It further activates downstream signal molecules through cascade phosphorylation, activates specific signal transduction pathways, and finally transmits external signals to the nucleus to activate or inhibit the expression of specific genes .Based on the functional analysis of the above genes and the published literature, we speculated that the six candidate genes in Through the combination of BSA-Seq and BSR-Seq, candidate genes related to millet spikelet development were preliminarily identified, and their functions and possible related pathways were preliminarily examined and confirmed by RT-qPCR. This study provides a reference for the improvement of millet yield and the study of yield-related traits in other gramineous crops. However, there are still some limitations in the functional verification and mechanism of action of genes in the experiment. In future research, we will continue to verify and explore the detailed mechanism of foxtail millet sterility based on the identified candidate genes.4 crops.Three regions associated with abnormal floret development were identified on chromosomes III, VI, and VII by BSA-Seq, and 90 candidate genes were identified. Combined with BSR-Seq results and biological analysis, six candidate genes related to foxtail millet sterility were finally determined, and Setit009293mg was considered to be the main gene controlling the sterility phenotype. This study lays a foundation for future breeding and map-based cloning of related genes in foxtail millet and other CThe original contributions presented in the study are publicly available. This data can be found here: PRJNA792301YG: methodology, formal analysis, visualization, writing—review, and editing. LD: conceptualization and resources. QM: investigation and data curation. YY: validation, writing—review, and editing. JL: methodology and data curation. HS and BF: supervision and funding acquisition. All 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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The use of lymph node (LN) tracers can help obtain a complete dissection of the lymph nodes and increase the detection rate of LNs and metastatic LNs. Carbon nanoparticle suspension injection (CNSI) and indocyanine green (ICG) have been widely used in radical gastrectomy in recent years. Nevertheless, the comparison of their clinical effects has not been studied.The FUTURE-01 trial will be the first randomized, open-label, single-center trial to compare CNSI and ICG. The study started in 2021 and enrolled 96 patients according to a prior sample size calculation. The primary outcome is the number of LNs retrieved. The secondary outcomes are LN staining rate, LN metastasis rate, stained LN metastasis rate, perioperative recovery and survival.By comparing the safety and efficacy of CNSI and ICG tracer-guided LN dissection in patients with gastric cancer, we can determine the most appropriate LN tracer at present. With the help of LN tracers, the operation is simplified, and the prognosis of these patients is improved. Our study is a prospective exploration of the safety, efficacy, and prognosis of CNSI and ICG.https://clinicaltrials.gov/ct2/show/NCT05229874?cond=NCT05229874&draw=2&rank=1, identifier NCT05229874. DespiteFor GC, complete resection with standardized LN dissection (D2) is important . AlthougD2 LN dissection is complex and tricky, and it is performed by removing perivascular fat and the LNs containing adipose baring fat tissue. In the process of lymphadenectomy in gastrectomy, one of the common and severe intraoperative complications is vessel injury. With the aim of easing the difficulty of LN dissection, an increasing number of surgeons are trying to use LN tracers to make the affected LNs distinct. Initially, surgeons used methylene blueand tattoo ink as LN tracers . Becausein vivo after approximately 3-4 months. Multiple studies have reported that CNSI has a respectably high safety profile, and no significant adverse effects were seen while the detection rate of LNs and metastatic LNs was increased , and the other is indocyanine green (ICG). In 2004, the CNSI  made in China was launched, which reduced the price of CNSI and the cost medical expenditure and promoted the development of LN tracers. This product is a stable suspension of carbon nanoparticles 150 nm in diameter, which are smaller than the lymphatic capillary endothelial cell gap 120-500 nm) and larger than the capillary endothelial cell gap (30-50 nm) . Therefo0-500 nm ncreased , 13. AddICG, as a kind of fluorescent dye, can be applied intraoperatively and used to sort LNs in postoperative specimens. When injected into the submucosa of the gastric wall of GC patients, ICG combines with serum albumin in the circulation and can be found in the LNs. Once excited by infrared light , ICG can emit infrared fluorescence , so ICG fluorescence imaging can guide intraoperative LN dissection , 15. ICGConcerning the LN metastasis rates, there was not much difference between the CNSI, ICG, and control groups. Hence, we concluded that CNSI was more likely to be the best LN tracer at present. However, this is a retrospective single-center study that includes the well-known limitations of such a study design. It is therefore worthwhile to carry out a prospectively designed study to compare the safety and efficacy of CNSI and ICG tracer-guided LN dissection during radical gastrectomy.The present study therefore aims to compare the safety and efficacy of CNSI and ICG tracer-guided LN dissection during radical gastrectomy using a randomized clinical trial design and to provide a theoretical basis for further investigation.22.1The current study is a prospective, randomized, open label, single-center, noninferiority clinical trial. The study takes place in the Fourth Hospital of Hebei Medical University. Patient enrollment started on 20 January 2021, and the trial is expected to end on January 20 2025. 2.2The inclusion criteria are as follows: (1) aged 18–75; (2) histologically proven gastric adenocarcinoma on biopsy ; (3) proven clinical stage of cT1-4a N0/+ M0 by ultrasound endoscopy, enhanced CT/MRI examination or diagnostic laparoscopy according to the TNM classification of the American Joint Committee on Cancer ; (4) without distant metastasis and no invasion of adjacent organs; (5) a preoperative Eastern Cooperative Oncology Group (ECOG) score of 0 or 1; (6) a preoperative American Society of Anesthesiologists (ASA) score of I-III; and (7) signed a written informed consent form.The exclusion criteria are as follows: (1) pregnant or lactating women; (2) patients with severe mental disorder; (3) a history of upper abdominal surgery (with exception of laparoscopic cholecystectomy); (4) a history of gastrectomies, endoscopic mucosal resection (EMR) or endoscopic submucosal dissection (ESD); (5) enlarged or bulky regional LNs according to preoperative imaging and larger than 3 cm at the long diameter; (6) the tumor invades the duodenum or esophagus; (7) Borrmann type IV GC; (8) history of other malignant tumors within the last 5 years; (9) received previous neoadjuvant radiotherapy or chemotherapy; (10) history of myocardial infarction or unstable angina within the last 6 months; (11) history of cerebrovascular accident within the last 6 months; (12) undergoing sustained systemic corticosteroid treatment within 1 month prior to surgery; (13) emergency surgery due to GC complications ; and (14) FEV1 <50% predicted value.2.3The patients were enrolled by a dedicated surgeon on our team. For eligible patients treated with GC at The Fourth Hospital of Hebei Medical University, thoracoabdominal CT, echocardiography, ultrasound of supraclavicular LNs, sonography of lower extremity veins, electrocardiogram and lung function examination should be performed before the operation. The patients who meet the inclusion criteria and are feasible for radical onco-surgery are then randomized to receive either endoscopic injection of CNSI (CNSI group) or endoscopic injection of ICG (ICG group) during gastroscopy at a 1:1 ratio. Randomization has been achieved using a random number table by the data manager, and the allocation is not concealed. While the blinding of the surgeons or participants is impossible, the pathologists are blinded to the types of surgical approaches.2.4In the CNSI group, the patients are given injections of CNSI (50 mg/dose) produced by Chongqing Lummy Pharmaceutical Co., Ltd. in the endoscopy division 1 day before surgery. CNSI is injected submucosally at 4 points  0.5-1 cm from the tumor edge under endoscopy. The optimized dose for each point is approximately 0.25 ml  produced by Dandong Yichuang Pharmaceutical is marked in the endoscopy division 1 day before surgery and is injected submucosally at 4 points  0.5-1 cm from the tumor edge under endoscopy. The optimized dose for each point is approximately 0.5 ml , 800 ml of normal saline is routinely used to wash the area near the carcinomatous foci of GC and is then re-collected as much as possible. It is important to collect at least 300 ml of the flushing fluid from the pelvic cavity. Cytology examination is then immediately performed on the flushing fluid collected. If the peritoneum is free of metastasis and peritoneal lavage cytology is negative, standard laparoscopic or robotic radical gastrectomy with D2 lymphadenectomy is then performed by the designated and experienced team of surgeons according to the Japanese Gastric Cancer Treatment Guidelines 2014. Distal gastrectomy for LN dissection included LN station 1, 3, 4 sb, 4d, 5, 6, 7, 8a, 9, 11p, 12a.Before starting to retrieve LNs from the GC patients, the surgeon should determine the positions of each station LN on the specimen in detail and plan the sequence of retrieving the LNs. After washing and flattening the specimen, we began to retain the soft tissues of each station of LNs by cutting off the useless tissues in the specimen Figure 32.5The primary outcome of this study is to determine the number of LNs retrieved. The secondary outcomes are to determine the LN staining (or fluorescence) rate , LN staining (or fluorescence) rate at each station, stained (or fluorescent) LN positive rate, stained (or fluorescent) LN negative rate, nonstained (or nonfluorescent) LN positive rate, nonstained (or nonfluorescent) LN negative rate, LN metastasis rate, number of metastatic LNs, complication and mortality rates within 30 days, 3-year disease-free survival (DFS), 3-year overall survival (OS), recurrence pattern within 3 years, intraoperative blood loss volume, time spent retrieving LNs, and postoperative recovery .Any complications should be recorded and reported. There are mainly two complications related to the procedure: intraoperative complications and postoperative complications. The former includes bleeding, injuring the viscera, lymphatic leakage and so on. The latter should be classified according to the Clavien−Dindo classification system.2.6Adverse events (AEs) refer to patient injuries caused by medical care . Serious2.7The sample size for this study was calculated based on previous research by our team and computed by PASS version 11. Eligible subjects were randomly assigned (1:1) into the CNSI group and the ICG group. All analyses were two-sided with α=0.05 and β=0.80. the prospective mean number of the LNs retrieved in the CNSI group was 56.93. The prospective mean number of the LNs retrieved in the ICG group was 50.52. The total sample size was 96 (48 per group) after taking into account a 10% dropout rate in each group. The planned recruitment period is 1 year, and the follow-up duration is 3 years.2.8via follow-up up until 3 years after the surgical operations. The follow-up should consist of a 3-month interval from 0–2 years and a 6-month interval in the third year. The main follow-up items included routine physical examination, tumor marker detection , abdominal CT, and annual electronic gastroscopy. The follow-up included an outpatient follow-up, a telephone follow-up and a short message platform follow-up. Further evaluation and treatment should be performed if relapse occurs.Designated and trained surgeons collect the data from the pathology reports and operative reports. The focus of the data should contain clear information about the tumor location, tumor size, histopathologic type, tumor differentiation degree, Lauren classification, vascular tumor embolus, nerve invasion, immunohistochemistry results and pathological findings of the LNs at each station. Long-term prognosis data are collected The purpose of this study is to compare the safety and efficacy of CNSI and ICG during radical gastrectomy. The primary objective is to determine the number of LNs retrieved. Comparability analysis will be performed on the baseline data to determine whether the two groups are comparable. The number of patients, mean, standard deviation, median, maximum, and minimum will be listed. Descriptive data will be presented as the mean ± SD, while categorical data will be presented as the number and percentage (%). The independent samples t test or nonparametric test will be used to analyze continuous data, and the chi-square test or Fisher’s exact test will be used to analyze categorical variables. It is valuable to analyze the influence of CNSI (ICG) staining or other factors on the number of LNs detected according to the statistical analysis. We can also research the effect of CNSI (ICG) staining on the operative time, intraoperative bleeding volume, intraoperative transfusion volume and postoperative complications. Survival analysis will be performed using the Kaplan−Meier method, and the log rank test will be used to test the difference in survival rates between the two groups. SPSS 26.0 statistical software will be used for statistical analysis. P<0.05 will be considered to indicate statistically significant differences.3Despite the serious burden of GC in China, the public awareness of risk factors or warning symptoms and screening is low, and there is a lack of screening programs . TherefoA number of interesting differences were identified through our analyses of CNSI and ICG. First, taking an appropriate amount of time to dissect LNs as an example, the LNs are stained black using CNSI, while the LNs labeled with ICG have the ability to generate strong fluorescence emission in certain cases. When utilizing CNSI as a tracer of LNs, surgeons can dissect the LNs under direct vision without the assistance of other instruments. However, if surgeons intend to use indocyanine fluorescence imaging to guide the postoperative nodal dissection, the fluorescence imaging mode must be available, and the wear and tear of the instrument and the burden on surgeons and operating room nurses will increase, which may prolong the time needed for dissecting the LNs. Second, compared with CNSI, ICG may have the potential to decrease the risk of anastomotic fistula with the assistance of examining the blood supply of the anastomosis. Third, CNSI extravasation into gastric serosa will contaminate the surgical fields and increase the difficulty of surgically dissecting the gastric tumor, but surgeons can switch between the fluorescence mode and normal mode to avoid the adverse effects caused by ICG extravasation .There are a number of previous reviews on the effectiveness of CNSI and ICG. Chen pointed out that their team analyzed 129 GC patients in the ICG group and 129 control subjects and concluded that ICG tracer-guided LN dissection enabled the retrieval of a higher number of LNs . Two retWhether the results of retrospective analysis can be confirmed by prospective randomized controlled clinical trials still needs to be further studied. Therefore, we are currently conducting a clinical trial to compare CNSI and ICG in detail to choose the optimal and suitable LN tracer for radical gastrectomy.The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.The studies involving human participants were reviewed and approved by the Ethical Review Committee of Hebei Medical University. The patients/participants provided their written informed consent to participate in this study.Conceptualization: QZ and YT. Data curation: YT, YP, PY, HG, YaL, ZeZ, PD, and TZ. Investigation and experiments: YoL, LF, ZhZ, XZ, BT, DW, and QZ. Methodology: YT. Formal analysis: YT, YP, and PY. Supervision: QZ. Writing – original draft: YT and YP. Writing review and editing: YoL, LF, ZhZ, XZ, BT, DW, and QZ. All authors contributed to the article and approved the submitted version.


This study aimed to identify factors associated with diabetes management self-efficacy in pregnant women with gestational diabetes mellitus (GDM) in Korea.A total of 173 pregnant women with GDM in Korea were recruited by posting announcements at two Korean online communities focusing on pregnancy and GDM. Participants completed a structured online survey from July to September 2018. Thirteen inappropriate responses were excluded and a total of 160 questionnaires were used in the final analysis. Descriptive statistics were calculated and multiple regression with the enter method was done to identify the associations of depressive mood, anxiety, emotional intelligence, and sleep quality with diabetes management self-efficacy.p<.001). Other factors, in descending order of influence, were sleep quality  and exercise . Taken together, the aforementioned factors explained 34.6%  of the total variance. Respondents reported a moderately depressive mood , low to moderate anxiety , above-average emotional intelligence , moderate sleep quality , and above-average diabetes management self-efficacy . The major factor associated with diabetes management self-efficacy of pregnant women with GDM was emotional intelligence (β=.51, The results of this study suggest that to improve the diabetes management self-efficacy of pregnant women with GDM, it is necessary to develop an education program that can also enhance emotional intelligence, sleep quality, and exercise. Gestational diabetes mellitus (GDM) is diagnosed when glucose intolerance develops after 20 weeks of pregnancy in a woman who previously did not have diabetes . AccordiSelf-efficacy in diabetes management reflects individual beliefs and refers to the patient’s degree of self-confidence that they can practice healthy behaviors and manage their diabetes. This confidence has been reported as the strongest influencing factor in diabetes self-management and healthy behavior practices . HoweverPatients with diabetes who are in a negative psychological state experience more difficulty in implementing self-management due to impaired emotional strength . PregnanConcerns about fetal health are the primary cause for anxiety in pregnant women and pregnant women with GDM experience greater anxiety than pregnant women without GDM . The negEmotional intelligence helps pregnant women with GDM maintain rational thinking even in a negative emotional state, helping them to empathize with others and maintain hope, to motivate themselves even in exhausting situations, and to control impulses for immediate gratification . These bThe physical effects resulting from impaired sleep and poor sleep quality are experienced by people with negative emotions . In a reTherefore, we hypothesized that diabetes management self-efficacy was crucial for pregnant women with GDM to successfully practice diabetes management, and that various psychological, emotional, and physical factors could influence that self-efficacy. However, the existing literature has been focused on the changing trends in the treatment of pregnant women with GDM ,3, the iThus, the purpose of this study was to examine diabetes management self-efficacy among pregnant women with GDM and to assess the effects of depression, anxiety, emotional intelligence, and sleep quality on self-efficacy. The ultimate goal was to provide basic data for developing interventions that enhance diabetes management self-efficacy in pregnant women with GDM.The specific objectives of this study were as follows: (1) to identify differences in diabetes management self-efficacy according to the general and clinical characteristics of pregnant women with GDM; (2) to assess the degree of depression, anxiety, emotional intelligence, sleep quality, and diabetes management self-efficacy; (3) to evaluate the relationship between diabetes management self-efficacy and depression, anxiety, emotional intelligence, and sleep quality; and (4) to investigate factors associated with diabetes management self-efficacy.Ethics statement: This study was approved by the Institutional Review Board of Chonnam National University (No. 1040198-180418-HR-023-03). A document substituting for an informed consent form was obtained from the participants online.https://www.strobe-statement.org/).This descriptive correlational study was designed to identify the factors affecting diabetes management self-efficacy in pregnant women with GDM. The description followed the STROBE  reporting guidelines  a participant could withdraw from the study at any time if she did not want to participate, even after completing the survey, (2) the anonymity of personal information would be guaranteed, (3) the data would be used only for research purposes, and (4) the data would be retained for 3 years. Anonymity was maintained throughout the survey process and, after the survey was completed, a predetermined online gift certificate was provided to the participants by e-mail or text message.The accessible population of this study was pregnant women with GDM who joined and used online communities. Convenience sampling was conducted to recruit participants from two communities on the Naver online portal. The inclusion criteria were (1) pregnant women who were diagnosed with GDM after 20 weeks of gestation and used an online GDM community and (2) women who understood the purpose of this study, voluntarily consented to participate in the study, and submitted a document substituting for a consent form online. Women who had previously been diagnosed with type 1 or type 2 diabetes or were not pregnant at the time of the study were excluded.2) of .15, and a power (1-β) of .80 for multiple regression analysis involving 20 independent variables, which resulted in 157 participants. The effect size was determined based on the median effect size of f2=0.15 for multiple regression analysis according to the Cohen criteria [Sample size calculation was done using G*Power 3.1.9.2  with the significance level (α) set at .05, an effect size -X, which was developed by Spielberger  and adapThe Emotional Intelligence Scale (EIS), which was developed by Wong and Law  and was The Verran & Snyder-Halpern Sleep Scale, which had been developed by Snyder-Halpern and Verran  and was The general characteristics and clinical characteristics of the participants were evaluated based on previous studies . GeneralDescriptive statistics were utilized to analyze the participants’ general and clinical characteristics, depression, anxiety, emotional intelligence, sleep quality, and diabetes management self-efficacy. The differences in diabetes management self-efficacy according to the participants’ general and clinical characteristics were analyzed using the independent t-test, and Pearson correlation coefficients were used to determine the relationship between anxiety, emotional intelligence, sleep quality, and diabetes management self-efficacy. For factors affecting the diabetes management self-efficacy among the pregnant women with GDM, multiple regression analysis with the enter method was performed, and the normal distribution of continuous variables was confirmed by the Kolmogorov-Smirnov test. IBM SPSS ver. 24.0  was used for the data analysis.The mean age of the 160 pregnant women with GDM who participated in this study was 33.21±3.15 years  and the mean number of weeks of gestation was 29.46±3.98 weeks . The number of participants with a college degree or higher  was greater than those who had a high school diploma or less . Eighty-eight women (55.0%) were religious while 72 (45.0%) were non-religious. More participants had no child  than those who had at least one child , and a greater number of women had a job  than those who had no job . The majority of participants  responded that their perceived financial status was below average .2 . Of the total participants, 12 (7.5%) had a history of GDM and 59 (36.9%) had a family history of diabetes or GDM. There were 103 patients (64.4%) who had participated in GDM education. The GDM management methods included regular glucose checks (90.6%), dietary management (96.3%), and exercise (69.4%), and 21 participants (13.1%) underwent insulin treatment , and the average pre-pregnancy BMI was 23.19±4.41 kg/mreatment .The average depression score of the study participants was 10.36±5.30 points (51.9% of the participants were in the high-risk group with 10 points or less), and the average score for anxiety was 41.65±11.16 points. The average score for emotional intelligence was 78.04±13.70 points. For each subdomain the scores were as follows: self-emotion appraisal, 20.94±3.91 points; emotion appraisal of others, 19.81±3.15 points; use of emotions, 18.16±4.28 points; and regulation of emotions, 18.98±4.24 points. The average score for sleep quality was 42.01±10.09 points, and that for diabetes management self-efficacy was 52.29±12.38 points .p=.010). An analysis of the differences in diabetes management self-efficacy according to the clinical characteristics of the study participants (p=.043).Analysis of the differences in diabetes management self-efficacy according to the general characteristics of the study participants  showed ticipants  showed tp=.022) and anxiety , whereas it showed positive correlations with emotional intelligence  and sleep quality .The results of analyzing the relationship between depression, anxiety, emotional intelligence, sleep quality, and diabetes management self-efficacy of the study participants are as follows : the diaThe results of stepwise multiple regression analysis performed to identify factors affecting the self-efficacy of diabetes management in pregnant women with GDM are presented in One data point that was an outlier  was removed, and then multiple regression analysis was performed on 159 people. In the examination of autocorrelation (independence) of the errors, the Durbin Watson statistic was close to 2 , indicating there was no autocorrelation between the error terms. In the multicollinearity test, the range of tolerance was >0.1  and the variance inflation factor was also ≤10 , indicating there was no problem of multicollinearity. In addition, the assumptions of linearity, normality, and equal variance of residuals were met, and the Cook distance value for detecting outliers did not exceed 1.0, showing there were no outliers.p<.001) with an explanatory power of 38.6%. Evaluation of the relative importance of the input variables showed emotional intelligence  was followed by sleep quality  and exercise .As a result of the multiple regression analysis, the diabetes management self-efficacy model for pregnant women with GDM showed a statistical significance (F=17.58, Due to the absence of previous studies that investigated diabetes management self-efficacy and emotional intelligence among pregnant women with GDM, it was difficult to compare the findings of this study directly to those of other studies. However, a previous study , which sIt is necessary to improve diabetes management self-efficacy by developing a program that considers emotional intelligence, sleep quality, and exercise so that pregnant women with GDM can recognize their emotions, express negative emotions, and find support in the diabetes management process.Conversely, results of the multiple regression analysis showed that depression and anxiety were not significant influencing factors for diabetes management self-efficacy. However, in a study by Jeong , depressEducational background and exercise levels were statistically significant factors related to the level of diabetes management self-efficacy according to the general and clinical characteristics of the participants of this study; specifically, those who had a college degree or higher and those who exercised had higher diabetes management self-efficacy scores. Although few studies have directly analyzed subjects’ educational background in relation to their diabetes management self-efficacy, one previous study  showed tIn this study, depression and anxiety were inversely correlated with diabetes management self-efficacy, and emotional intelligence and sleep quality were positively correlated with diabetes management self-efficacy. The depression score of the pregnant women with GDM was 10.36 in the current study. In a previous study  that useThe results of multiple regression analysis showed that predictive variables for diabetes management self-efficacy in pregnant women with GDM were emotional intelligence, sleep quality, and exercise. In other words, a higher level of emotional intelligence, higher sleep quality, and more exercise were associated with greater diabetes management self-efficacy.A limitation of this study is that it was based on cross-sectional data obtained only from pregnant women with GDM using online communities, which may reflect sampling bias of women who are active. Another limitation is that although postpartum depression was measured, the study did not include physical symptoms of the participants. However, while many studies used depression, anxiety, and self-efficacy as independent variables to verify the effectiveness of programs for pregnant women with GDM, few studies have demonstrated negative emotions as influencing factors on diabetes management self-efficacy. In addition, this study provides basic data on individual factors that affect diabetes management self-efficacy in pregnant women with GDM, unlike most previous studies that investigated diabetes management self-efficacy as a factor influencing diabetes management outcomes.In conclusion, to improve diabetes management self-efficacy in pregnant women with GDM, it is necessary to apply methods that can improve both physical and emotional health. Specifically, strategies should be developed to enhance emotional intelligence, sleep quality, and exercise. Furthermore, as the causal relationship between the major variables affecting diabetes management self-efficacy may be bidirectional, not unidirectional, future studies that explore a research model that verifies the direction of causality among the influencing variables would be beneficial.