Furthermore, this review discusses the association of CXCL1 with clinical aspects of gastrointestinal cancers, including its correlation with tumor size, cancer grade, tumor–node–metastasis (TNM) stage, and patient prognosis. This paper concludes by exploring CXCL1’s potential as a therapeutic target in anticancer therapy.One area of cancer research is the interaction between cancer cells and immune cells, in which chemokines play a vital role. Despite this, a comprehensive summary of the involvement of C-X-C motif ligand 1 (CXCL1) chemokine , melanoma growth-stimulatory activity (MGSA)) in cancer processes is lacking. To address this gap, this review provides a detailed analysis of CXCL1’s role in gastrointestinal cancers, including head and neck cancer, esophageal cancer, gastric cancer, liver cancer (hepatocellular carcinoma (HCC)), cholangiocarcinoma, pancreatic cancer , and colorectal cancer . This paper presents the impact of CXCL1 on various molecular cancer processes, such as cancer cell proliferation, migration, and invasion, lymph node metastasis, angiogenesis, recruitment to the tumor microenvironment, and its effect on immune system cells, such as tumor-associated neutrophils (TAN), regulatory T (T Gastrointestinal tumors are a diverse group of cancers that affect organs responsible for digestion. These tumors are categorized based on their specific organ location, including head and neck cancer, esophageal cancer, gastric cancer, liver cancer, cholangiocarcinoma, pancreatic cancer, colon cancer, and rectal cancer. In 2021, there were an estimated 5.95 million new cases of these tumors, representing 30.9% of all cancer diagnoses [One promising area of research focuses on intercellular signaling within tumor tissue, specifically the interaction between cancer cells and the immune system ,4,5,6,7.CXCL1 is a chemokine consisting of 73 amino acids and has a molecular weight of 8 kDa . Its expAnother receptor for CXCL1 is ACKR1 , though i), are directly activated by CXCR2 [Activation of the CXCR2 receptor by CXCL1 triggers signal transduction. Heterotrimeric G proteins, particularly the inhibitory guanine nucleotide regulatory protein , it is difficult to obtain a clear picture of the current knowledge in this area. To address this gap, we have reviewed the significance of CXCL1 in tumors, with a focus on gastrointestinal tumors due to the sheer volume of available information.Considerably less attention has been paid to other CXCR2 ligands in cancer research. As a result, knowledge of the significance of other CXCR2 ligands in cancer processes is incomplete. Moreover, there is a lack of available review articles that describe the significance of certain CXCR2 ligands in cancer processes, including CXCL1. With over 1300 papers available in the PubMed database on this chemokine in the context of cancer 7 and MMP9 by OSCC cells , which pCXCL1 is involved in angiogenesis in OSCC tumors ,41. FollThe high expression of CXCL1 in OSCC  and LSCCHigh levels of CXCL1 in the blood of patients with head and neck cancer are associated with failure of radiation therapy . Higher More than 604,000 new cases of esophageal cancer are diagnosed annually, which accounts for 3.1% of all diagnosed cancer cases . ESCC acCXCL1 as one of the hub genes in ESCC [CXCL1 expression is elevated in ESCC tumors relative to healthy tissue ,55, alon in ESCC . A high  in ESCC , which m in ESCC .In ESCC tumors, CXCL1 is secreted by ESCC cells as a result of high NF-κB activation, mediated by several factors, for example, laminin subunit gamma 1 (LAMC1)  and earlTumor-associated macrophages (TAM) are another source of CXCL1 in ESCC tumors . CXCL1 eCXCL1 gene as well as hypomethylation of its promoter [The increase in CXCL1 expression in Barrett’s esophagus and esophageal adenocarcinoma is also due to the frequent amplification of the promoter . Barrettpromoter . Due to CXCL1 is involved in tumorigenic processes in ESCC. CXCL1 increases the proliferation of ESCC cells . ThroughCXCL1 is also responsible for radio-resistance in ESCC cells . This prCXCL1 acts on CAF. CXCL1 causes a change in the phenotype of CAF into inflammatory CAF through activation of the CXCR2-signal transducer and activator of transcription 3 (STAT3) pathway . These aCXCL1 expression in ESCC tumors is correlated with tumor size. A larger tumor shows higher CXCL1 expression . ImmunohHelicobacter pylori infection [H. pylori causes chronic inflammation, which leads to gastritis, which after some time develops into intestinal metaplasia, atypical hyperplasia, and, finally, gastric cancer [H. pylori is cytotoxin-associated gene A (CagA) [H. pylori. Other virulence factors relevant to the pathogenesis of gastric cancer caused by H. pylori also include outer membrane inflammatory protein A (OipA), duodenal ulcer promoting gene A protein (DupA), and vacuolating cytotoxin A (VacA) [It is estimated that nearly 1.06 million new cases of gastric cancer were diagnosed in 2020, which accounted for 5.6% of all cancer diagnoses [nfection ,67. It infection . The larc cancer . An esseA (CagA) , a proteA (CagA) . CagA caA (VacA) .H. pylori infection and the effects of this bacterium cause chronic inflammation in the gastric wall, which leads to increased levels of pro-inflammatory cytokines such as TNF-α, C-C motif ligand 5 ), C-C motif ligand 3 ), C-X-C motif ligand 9 ), C-C motif ligand 20 ) and CXCR2 ligands such as CXCL8, C-X-C motif ligand 2 ) and the described CXCL1 [H. pylori increases the expression of CXCL1, as well as other CXCR2 ligands, in gastric epithelial cells [H. pylori [ed CXCL1 ,73,74. Hal cells ,73,74,75al cells , which s. pylori .H. pylori causes the translocation of CagA and fragments of peptidoglycan via TIVSS into gastric epithelial cells [H. pylori. This factor causes increases in the expression of chemokines such as C-C motif ligand 2 ), C-C motif ligand 7 ), CCL20, C-X-C motif ligand 10 ), and CXCR2 ligands [H. pylori may also indirectly increase the expression of CXCL1 and CXCL8. H. pylori activates NF-κB in a CagA-dependent manner which results in the increased expression of interleukin-32 (IL-32) [H. pylori gastritis. Neutrophils are involved in inflammatory responses in gastritis; they secrete ROS and reactive nitrogen species (RNS), which damage tissue and cause gene instability [The mechanism for the increase in CXCL1 expression involves multiple pathways. al cells , which pal cells . The bacal cells . EGFR, Eal cells ,78. At tal cells . Another ligands , which a (IL-32)  which in (IL-32) ,83. Thes (IL-32) . This meH. pylori infection to gastric cancer is associated with the CXCL1/CXCL8-CXCR2 and p53 axis. H. pylori infection increases the expression of CXCR2, the receptor for CXCL1 [Chronic inflammation leads to gastritis, which after some time develops into gastric intestinal metaplasia, then atypical hyperplasia, and, finally, gastric cancer . The proor CXCL1 , which iH. pylori infection also increases the expression of CXCL1 as well as other ligands for CXCR2 [H. pylori interfere with p53 function and increase p53 expression in gastric mucosa [H. pylori causing DNA damage [TP53 gene [H. pylori also disrupts the proper function of p53 by reducing the expression and function of upstream stimulatory factor 1 (USF1) and USF2, proteins that form a complex with p53 and are important in p53 function [H. pylori decreases p53 protein expression by increasing degradation of the protein [TP53 gene leads to escape from senescence [or CXCR2 ,74,76,82c mucosa ,86,87,88A damage , which lP53 gene . This isP53 gene , which pfunction ,93. H. p protein ,95—a resGastric cancer tumors show a higher expression of CXCL1 and CXCL8 than healthy gastric tissue ,97,98,99H. pylori infection [The elevated expression of CXCL1 in gastric cancer tumors is associated with an increase in the expression of this chemokine in an autocrine manner in gastric cancer cells ,105. Anonfection . There infection ; a micronfection . CXCL1 infection  which inThe positive correlation of CXCL1 expression with worse overall survival of gastric cancer patients is related to the fact that CXCL1 participates in tumorigenic processes. CXCL1 increases gastric cancer cell proliferation, as shown by experiments on HGC27 cells , AGS, HsGastric cancer cells secrete factors that increase CXCL1 expression in LEC ,111 as aCXCL1 induces an increase in the expression of MMP2 and MMP9 in gastric cancer ,109 and CXCL1 acts on tumor-associated cells. CXCL1 causes the recruitment of G-MDSCs , bone maGastric cancer cells can increase CXCL1 expression in tumor-associated cells. Gastric cancer cells secrete TNF-α, which increases CXCL1 expression in TAM . FurtherCXCL1 is closely associated with tumorigenesis in gastric cancer, as CXCL1 expression is positively correlated with advanced TNM stage, i.e., T invasion stage, lymph node metastasis, as well as tumor size ,112,115.Higher CXCL1 expression in gastric cancer tumors is associated with worse overall survival  97,98,9,112,115,Chronic hepatitis B virus (HBV) infection;Chronic hepatitis C virus (HCV) infection;Consuming large amounts of alcohol, which leads to alcoholic liver disease (ALD), then to liver cirrhosis, and eventually HCC;Obesity, which leads to non-alcoholic fatty liver disease (NAFLD), then liver cirrhosis, and, eventually, HCC.The most common liver cancer is HCC . An estiDue to the significance of chronic inflammation in the liver, CXCL1 plays an important role in the onset and development of HCC.CXCL1 rs4074 A allele [CXCL1 gene [CXCL1 is important in the emergence of liver cancer, as demonstrated by studies on genetic polymorphisms in patients with liver cirrhosis and HCC. Specifically, more patients with liver cirrhosis and HCC have the A allele ,119, impCL1 gene .During exposure of the liver to agents leading to liver cirrhosis and HCC, NF-κB is activated and CXCL1 expression increases. In liver cells, there is p50:p50 NF-κB , which dTGFB1 gene, which increases the expression of TGF-β1 in hepatocytes [Liver steatosis and HCC arise from chronic inflammation of the liver. This condition can be caused by either chronic HBV or HCV infection. HBV causes an increase in CXCL1 production in the liver. The hepatitis B X (HBx) antigen binds to the atocytes , a cytokatocytes . This reatocytes .Further, chronic HCV infection is associated with increased CXCL1 expression in the liver. During HCV infection, hepatocytes produce CXCL1 . HepaticAnother cause of liver cirrhosis and HCC is frequent alcohol consumption. Alcohol causes an increase in CXCL1 expression in hepatocytes . FurtherCXCL1 can also cause liver fibrosis through another pathway. CXCL1 causes infiltration of the liver by neutrophils, which activate hepatic stellate cells via ROS. Then hepatic stellate cells start producing collagen, which leads to hepatic fibrosis , which iLiver fibrosis is associated with liver cirrhosis . Liver cCXCL1 expression is higher in HCC tumors than in healthy liver tissue , and is A significant source of CXCL1 in HCC is myofibroblast-like cells , which aIn HCC tumors, many factors increase CXCL1 expression in cancer cells. In particular, CXCL1 itself is responsible for increases in NF-κB activation and thus its own expression in an autocrine manner . Other fStudies in mice have indicated that the elevated expression of CXCR2 ligands in the liver facilitates HCC formation in this organ . In miceCXCL1 increases the proliferation of HCC cells ,138,145.Further, mouse CXCR2 ligands increase the number of HCC cancer stem cells—cells that are resistant to chemotherapeutics, such as doxorubicin . TherefoCXCL1 is also important in HCC tumor growth . CXCL1 ireg, respectively [reg participate in cancer immunoevasion [CXCL1 also acts on tumor-associated cells. This chemokine induces the recruitment of neutrophils  and G-MDectively . Monocytectively , while Toevasion .The great importance of CXCL1 in tumorigenesis is confirmed by the correlation of CXCL1 expression with various clinical parameters of the cancer in question. The level of CXCL1 expression increases with HCC tumor growth. There is a positive correlation of CXCL1 expression in the tumor with TNM classification stages, macrovascular invasion, microvascular invasion, and distant metastasis ,143. HigOpisthorchis viverrini infection, HBV or HCV infection, and heavy alcohol consumption [Cholangiocarcinoma is a tumor located in the biliary tree . The incsumption ,154. StuCXCL1 inhibits the proliferation of OCUG-1 and HuCCT1 cholangiocarcinoma cells  and doesCXCL1 may also have pro-cancer properties in cholangiocarcinoma. Liver CXCR2 ligands regulate cancer immune evasion in cholangiocarcinoma, which originates in the gut. Disruption in gut barrier function leads to the passage of commensal gut bacteria into the liver, which activates toll-like receptor 4 (TLR4) on cells in that organ. This leads to an increase in the expression of CXCR2 ligands in the liver, as shown by experiments in mice . ChronicCXCL1 expression does not increase with tumor stage . The levCDKN2A, TP53, SMAD4, and BRCA2 genes [In 2020 alone, more than 495,000 new cases of pancreatic cancer were diagnosed, accounting for 2.6% of all cancers . This caA2 genes ,162. ThiIn pancreatic cancer tumors , particuKRAS gene, common in pancreatic cancer [CXCL1 gene which increases the expression of CXCL1. In a cancer cell, CXCL1 expression may also be due to the interaction of this cell with other non-cancerous cells in the tumor niche. CXCL1 expression in cancer cells can be increased by IL-17 [CXCL1 expression is found in pancreatic cancer cells ,169. Mutc cancer ,161,162,c cancer ,171. Othc cancer . p63 binby IL-17  and alsoby IL-17 . ApoE acby IL-17 .CXCL1 expression in cancer cells may also depend on pancreatic stellate cells found in a healthy pancreas that are recruited to the cancer niche during pancreatic cancer tumor development. These cells are an essential component of the cancer stroma in pancreatic cancer and play an important role in its tumorigenesis . PancreaAnother source of CXCL1 in pancreatic tumors is inflammatory CAF ,179; in CXCL1 expression in the tumor may also be increased by recruited monocytes . In partKRAS gene occur at the beginning of tumorigenesis [TP53 gene, a senescence bypass occurs [TP53 [CXCL1 is significant in the early stages of pancreatic cancer transformation. Very often mutations in the igenesis , followeigenesis  which inigenesis ,186. Thiigenesis ,182. Incrs [TP53 . Then, ars [TP53 .In advanced pancreatic ductal adenocarcinoma tumors, CXCL1 also has pro-tumorigenic properties. Other CXCR2 ligands may also either be responsible or co-responsible for the properties of CXCL1. In particular, this refers to C-X-C motif ligand 5 ) and CXCL8, which expression in pancreatic ductal adenocarcinoma tumors are the highest among CXCR2 ligands . The lowThis effect of CXCL1 on pancreatic cancer cells can be negated if they have a high expression of atypical chemokine receptor 1 ) , an atypCXCR2 gene knockout in mice leads to increased liver metastasis arising from pancreatic ductal adenocarcinoma [However, CXCL1 may be important in other cancer processes. CXCL1, together with other CXCR2 ligands, causes angiogenesis ,173,190,arcinoma , CXCL1 mCXCL1 induces the recruitment of neutrophils into the tumor niche of pancreatic ductal adenocarcinoma ,169,185 Further, CXCL1 can act on CAF where it enhances inflammatory CAF, decreases myofibroblastic-CAF phenotype , and lowCXCL1 is also involved in chemotherapy resistance in pancreatic cancer patients. Some anticancer drugs, such as gemcitabine, increase the expression of CXCL1 and CXCL2 in pancreatic ductal adenocarcinoma tumor cells ,192. ThiCXCL1 expression in tumors increases with the tumor stage . CXCL1 eColorectal cancer is one of the three most commonly diagnosed cancers. An estimated 1.88 million new cases of this cancer were diagnosed in 2020 alone, which accounted for 9.8% of all cancers diagnosed . GeneticThere is elevated CXCL1 expression in colorectal cancer tumors compared to healthy tissue ,209,210.In colorectal cancer, CXCL1 is produced by a variety of cells ,216,217.+ and CD8+ polyfunctional T cells [2 (PGE2) [UEV1A, an enzyme that catalyzes poly-ubiquitination, which results in the activation of NF-κB. This transcription factor directly attaches to the CXCL1 promoter and then increases the expression of this chemokine.Other factors increasing CXCL1 expression include IL-22, secreted by CD4 T cells , and cel T cells . CXCL1 e2 (PGE2) ,216 in a2 (PGE2) ,218. Ele2 (PGE2)  associatIncreased CXCL1 expression in colorectal cancer cells may also be associated with adiponectin ,226. CXCCXCL1 is considered a hub gene in colorectal cancer [CXCL1 in colorectal cancer tumors participates in tumorigenic processes. Due to links in tumor mechanisms with other proteins, l cancer ,230. CXCl cancer ,231,232 l cancer . In addil cancer —the reduCXCL1 also causes the migration and invasion of colorectal cancer cells ,231,232,CXCL1 may also facilitate the formation of metastasis by inducing an increase in parathyroid hormone-like hormone (PTHLH) expression in colorectal cancer cells —a factor+ T cells. In addition, CXCL1 in colorectal cancer tumors causes angiogenesis [CXCL1 also affects cells in the tumor niche in colorectal cancer. CXCL1 causes the recruitment of neutrophils ,222,223 ogenesis  and stroogenesis  which alThe level of colorectal tumor development cannot be definitely linked to the level of CXCL1 expression in the tumor, as some studies show a positive correlation and some a negative correlation between CXCL1 levels and colorectal tumor grade. Some studies show that CXCL1 expression in colorectal tumors is positively correlated with lymph node metastasis, tumor size, and tumor stage ,238,239.As described above, CXCL1 plays a significant role in the molecular processes of gastrointestinal tumors. In theory, it is possible to develop anticancer therapy targeting CXCL1. However, the most important receptor for CXCL1 is CXCR2 , which iThe most well-known and commonly tested CXCR2 antagonist as an antitumor agent is SB225002 (N-(2-hydroxy-4-nitrophenyl)-N’-(2-bromophenyl)urea) . This coAdditionally being tested are other CXCR2 antagonists that also inhibit CXCR1 activity. CXCL1 only activates CXCR2 at low concentrations, while CXCL8 activates both CXCR1 and CXCR2 at low concentrations . Therefohttps://clinicaltrials.gov/ct2/home, accessed on 5 May 2023), a search using the receptor name “CXCR2” revealed 29 distinct clinical trials involving CXCR2 antagonists. Additional search results appear when entering the names of specific drugs.CXCR2 antagonists are currently under investigation in clinical trials as potential therapeutic agents . On the Typically, a particular CXCR2 antagonist is examined for its efficacy in treating a specific group of diseases. For instance, SX-682 is being studied as a potential anticancer agent, while Ladarixin is being explored as a treatment for type 1 diabetes. Danirixin (GSK1325756), on the other hand, is being tested for lung diseases such as COPD and influenza. AZD5069 is under investigation as both an anticancer drug and a treatment for lung diseases such as COPD and asthma. Meanwhile, SB656933 is being examined as a potential therapeutic option for COPD and cystic fibrosis.https://pubmed.ncbi.nlm.nih.gov) contained over 6-fold more scientific publications discussing CXCL8 in the context of cancer compared to CXCL1. This testifies to significant knowledge gaps remain regarding the role of CXCL1 in the oncogenic mechanisms of gastrointestinal neoplasms. Future research should also explore the interrelationships among individual CXCR2 ligands in tumorigenic processes. In humans, there are seven distinct CXCR2 ligands, which exhibit similar functions; however, differences among them warrant further investigation.Among CXCR2 ligands, research predominantly focuses not on CXCL1 but on another CXCR2 ligand, CXCL8. As of 5 May 2023, the PubMed database (The role of CXCL1 in gastrointestinal malignancies has been extensively investigated and numerous studies have assessed the clinical implications of this chemokine in these tumors. Additionally, research has demonstrated the involvement of CXCL1 in a multitude of molecular mechanisms associated with these neoplasms. Presently, clinical trials exploring anticancer therapies incorporating CXCR2 antagonists are in preliminary phases. In the forthcoming years, these therapeutic agents are expected to become included in widely available cancer treatment.


Drosophila. We further show that Arf102F, a GTP-binding protein previously implicated in protein trafficking, serves downstream of Droj2 to govern neurite pruning of C4da sensory neurons. Intriguingly, our data consistently demonstrate that both Droj2 and Arf102F promote the downregulation of the conserved L1-type cell-adhesion molecule Neuroglian anterior to dendrite pruning. Mechanistically, Droj2 genetically interacts with Arf102F and promotes Neuroglian downregulation to initiate dendrite severing. Taken together, this systematic study sheds light on an unprecedented function of Droj2 and Arf102F in neuronal development.Developmental remodeling of neurite is crucial for the accurate wiring of neural circuits in the developing nervous system in both vertebrates and invertebrates, and may also contribute to the pathogenesis of neuropsychiatric disorders, for instance, autism, Alzheimer’s disease (AD), and schizophrenia. However, the molecular underpinnings underlying developmental remodeling are still not fully understood. Here, we have identified DnaJ-like-2 (Droj2), orthologous to human DNAJA1 and DNAJA4 that is predicted to be involved in protein refolding, as a developmental signal promoting dendrite sculpting of the class IV dendritic arborization (C4da) sensory neuron in  Drosophila, neurons of the larval nervous system undergo stereotyped remodeling of axons and dendrites in the central and peripheral nervous systems during early metamorphosis [Drosophila, selectively remove their larval dendrites to refine the nervous system during metamorphosis [Drosophila metamorphosis share superficial similarities with Wallerian neuronal degeneration and neurodegenerative diseases [During animal development, a vital step in the establishment and maintain functional neural circuits in the brain is selectively eliminating superfluous neuronal connections without loss of the parental neurons, referred to as pruning . Neuronaorphosis ,7. In thorphosis ,9,10. Inorphosis . Of notediseases ,13,14. Idiseases ,16. ThusDrosophila, including ubiquitin-proteasomal degradation [Dendrite pruning is governed in a cell-intrinsic manner and initiates in response to a late larval pulse of the steroid hormone ecdysone. A heterodimer complex, consisting of ecdysone receptor B1 (EcR-B1) and Ultraspiracle, works together with two epigenetic factors, Brahma and CREB-binding protein, to induce the downstream transcriptional cascade after binding to ecdysone ,11,17,18radation ,11,20, cradation ,22, compradation ,24, micrradation ,26,27,28radation ,29,30, Jradation , energy radation , and mecradation .Drosophila sensory neurons [Drosophila [It is worth noting that the dendrite severing of somatosensory neurons is triggered through both global and local endocytosis by way of Rab5/ESCRT-dependent endolysosomal degradation. It has been well documented that the conserved L1-type cell-adhesion molecule Neuroglian is internalized from the plasma membrane into early endosomes and undergoes endolysosomal degradation to trigger the onset of dendrite severing . Previou neurons . Yif1 inosophila . The memosophila . All of osophila , after wosophila . DespiteTo systematically understand the mechanisms of neurite remodeling, we took advantage of an unbiased RNA interference (RNAi) screen to identify new candidates for governing dendrite pruning. Herein, we report that DnaJ-like-2 (Droj2), orthologous to human DNAJA1 and DNAJA4, is required for dendrite pruning of the C4da neuron. A previous study implicated the crucial role of Droj2 in NF-κB signaling . MoreoveDrosophila. It has been well-documented that there are types of C4da neurons, including ddaC, v’ada, and vdaB, ranging from dorsal to ventral in each hemi-segment of Drosophila larvae. Analogical dendrite pruning manner appeared in all C4da neurons, whereas the pruning time of v’ada and vdaB neurons is marginally delayed during metamorphosis. Moreover, vdaB neurons are usually lost at the adult stage [Previous studies have unambiguously implied that both progressive and regressive events are indispensable for the establishment of mature nervous systems during an animal’s development. A variety of neurons remodel by eliminating excessive or incorrect larval connections subsequent to the regrowth of adult-specific processes during metamorphosis in lt stage . Thus, dlt stage ,37. Invalt stage ,43. Ultilt stage   and V23637 (#4), also resulted in similar pruning defects at 16 h APF (droj2 RNAi transgene (#1) with two copies of ppk-Gal4 drivers caused more severe deficiencies with a residual with an average dendritic length of 2650 μm at 16 h APF , and UAS-Droj2 overexpression, to some extent, enhances the gene expression of droj2 in Droj2 RNAi , to attenuate gene function in the C4da neuron. From this screen, we isolated two independent RNAi lines, TH02998.N (#1) and BL57382 (#2), that were delineated by a prominent deficiency in dendrite pruning. Both the RNAi lines target the gene of 16 h APF C,D,G,H. 16 h APF B,G,H. Li16 h APF . Moreove16 h APF E,G,H. In16 h APF F–H. Addioj2 RNAi . In summDrosophila C4da sensory neurons. In order to identify the downstream target of Droj2 in neurite remodeling, we performed another round of RNAi screening with our collection of RNAi lines based on the genes that might be correlated with Droj2 according to the FlyBase database. Eventually, we isolated another two independent RNAi lines, BL27268 (#1) and V12931 (#2), which displayed dendrite pruning deficiencies. Both RNAi lines correspond to the same gene, arf102f, which encodes a GTP-binding protein involved in protein trafficking. The known functions of Arf102F include the intracellular regulation of vesicle budding and uncoating within the Golgi apparatus and sustaining the integrity of the Golgi structure [arf102f via ppk-Gal4 was found to lead to deficits in dendrite pruning at 16 h APF. By contrast, all larval dendrites were completely removed in the control RNAi neurons at this time point . The secretory pathway has been well-documented to facilitate the endolysosomal degradation of Neuroglian [In an attempt to assay the molecular underpinnings for the neurite pruning deficiency resulting from the functionally compromised Droj2 and Arf102F, we first wondered whether Droj2 and Arf102F knockdown influences ecdysone-associated gene expression. However, the ecdysone target genes uroglian ,36,37. Auroglian ,45.arf102f RNAi neurons at 6 h APF  and BL37496 (#2), in Arf102F RNAi-expressing C4da sensory neurons. Notably, the expression of two independent neuroglian RNAi transgenes, which has been shown to efficiently knock down Neuroglian [arf102f RNAi C4da sensory neurons , downstream of Droj2, might also be involved in the regulation of somatosensory neurite remodeling. Therefore, further investigation is required to elucidate the unknown downstream factors that could contribute to the demonstration of a detailed regulatory mechanism of neurite sculpting by Droj2. Additionally, we also found a decreased Arf102F signal in Droj2 downregulated neurons. We propose that Droj2 is involved in the regulation of the protein processing and maturation of Arf102. The downregulation of Droj2 might cause erroneous refolding and modifications to Arf102 that could facilitate protein degradation. Future investigations are warranted to elucidate a detailed regulatory mechanism regarding the effect of Droj2 on Arf102F.Previous works have implied that the GTP-binding protein Arf102F is a regulator of vesicle budding and uncoating within the Golgi apparatus involved in protein trafficking ,49. In aDrosophila? In order to study the underlying regulatory mechanism, we next performed experiments to monitor the subcellular localization of Arf102F. It is notable that the discrete punctate signals of Arf102F-GFP were co-localized, to some extent, with the ER apparatus, and conspicuously co-localized with a Golgi marker. We next examined the distribution of the Golgi marker in Droj2 and Arf102F mutant neurons, and our data showed that the loss of Droj2 and Arf102F function resulted in a prominent decrease in Golgi marker intensity, suggesting that Arf102F might function in Golgi as an auxiliary regulator to modulate protein secretion. Previously, it has been well-documented that Neuroglian, an evolutionarily conservative L1-type cell adhesive molecule, is internalized from the plasma membrane to early endosomes via Rab5/ESCRT-dependent endocytosis and profoundly degraded by the endolysosomal pathway to activate neurite severing [Drosophila C4da neurons [Drosophila. Thus, in future studies, it would be interesting to search for the ligand or secreted protein that is regulated by Droj2 and Arf102F to trigger Neuroglian endocytosis and degradation. Of note, the human ortholog of Droj2, DnaJA1 has been shown to bind physically with Abeta, facilitating the aggregation of Abeta peptides, and downregulation of this chaperone protects against Abeta-mediated toxicity in AD models [How does Droj2/Arf102F facilitate the neurite pruning of C4da neurons during the metamorphosis of severing . The sec neurons ,36,37. ID models ,51. AddiD models . IntriguD models . Therefodroj2 RNAi #2 (BL57382), arf102f RNAi #1 (BL27268), arf102fc339 (BL16341), UAS-Arf102f-GFP (BL65866), UAS-Arf102f-HA (BL93855), neuroglian RNAi #1 (BL38215), neuroglian RNAi #2 (BL37496), UAS-GalT-RFP (BL30902), UAS-KDEL-RFP (BL30909). The following stocks were obtained from the Vienna Drosophila RNAi Centre (VDRC): droj2 RNAi #3 (V104880), droj2 RNAi #4 (V23637), arf102f RNAi #2 (V12931). The following stocks were obtained from the Tsinghua RNAi Stock Center: droj2 RNAi #1 (TH02998.N), arf102f RNAi #3 (THU2594). All the flies were raised at 25 °C on standard medium .The following stocks were obtained from Bloomington Stock Centre : mCherry RNAi (BL35785), For the rescue experiment, a UAS-Droj2-HA construct was made by cloning the full-length cDNA into the transformation vector pUAST-attB. The transgenic fly of UAS-Droj2-HA was generated by standard germline transformation. cDNA of Droj2 was obtained from reverse transcription of genomic mRNA. Fly head mRNA was extracted using TRIzol , and reverse transcribed using cDNA Reverse Transcriptase Kit .Drosophila prepupae at the WP stage were first washed in PBS buffer briefly for three times and subsequently immersed with 90% glycerol. For imaging C4da neurons, pupal cases at 16 h APF were carefully removed antecedent to mount with 90% glycerol. Images of C4da neurites were captured with Zeiss LSM 900 confocal microscope.For immunostaining, larvae and pupae were dissected in pre-cooling PBS and fixed with 4% formaldehyde for 30 min. The experimental and control samples were incubated synergistically in the same tubes. Mounting was conducted in the mounting medium of VectaShield, and the samples were directly visualized by a Zeiss LSM 900 confocal microscope. The following primary antibodies were used for immunohistochemistry at the indicated dilution: mouse anti-Neuroglian , rabbit anti-GM130 , rabbit anti-HA , mouse anti-Rab5 , mouse anti-Rab7 . The following secondary antibodies were used for immunohistochemistry at the indicated dilution: Alexa Fluor 488- and Alexa Fluor 555- conjugated secondary antibodies .The structure of the C4da neuron dendrite at the WP and 16 h APF stages were captured via live confocal imaging. The length of unpruned dendrites and percentage of severing defects are utilized to evaluate the neurite remodeling phenotype. The length of unpruned dendrites was measured in a 275 µm × 275 µm region derived from the dorsal dendritic field of C4da neurons, ranging from the abdominal segments 2–4. The percentage of dendritic severing defects was defined by the percentage of neurons with larval dendrites attached to the soma at 16 h APF.http://flybase.org/reports/FBgn0038145#interactions (accessed on 2 September 2021) (Click “Interactions” at the right sidebar of the page), from which it shows the genes that physically interact with Droj2. Subsequently, we ordered RNAi lines of these candidate genes to perform the second-round screening. Among these, we found that Arf102F is also required for neurite remodeling, and our data further confirmed that Arf102F works downstream of Droj2 in neurite remodeling.The unbiased RNAi screen is based on the stocks collected in our lab for identifying synaptic function regulators, which contain about 200 lines. Among these, we isolated Droj2, which plays a vital function in neurite remodeling. To further identify the downstream targets of Droj2 in neurite remodeling, we next searched the database at t-test was used to determine statistical significance. For multiple-group comparison, one-way ANOVA with Bonferroni’s test was used to determine significance. The error bars in all graphs represent s.e.m. Statistical significance was defined as **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05, and ns, not significant. The presented data are from three independent experiments and the number of neurons (n) in each group is shown on the bars.For pairwise comparison, a two-tailed Student’s


KRT8) genetic variants were associated with IPF. –/–Krt8 mice were protected from fibrosis and accumulation of the transitional state. Keratin 8 (K8) regulated the expression of macrophage chemokines and macrophage recruitment. Profibrotic macrophages and myofibroblasts promoted the accumulation of transitional AECs, establishing a K8-dependent positive feedback loop driving fibrogenesis. Finally, rare murine transitional AECs were highly senescent and basaloid and may not differentiate into AEC1s, recapitulating the aberrant basaloid state in human IPF. We conclude that transitional AECs induced and were maintained by fibrosis in a K8-dependent manner; in mice, most transitional cells and fibrosis resolved, whereas in human IPF, transitional AECs evolved into an aberrant basaloid state that persisted with progressive fibrosis.Idiopathic pulmonary fibrosis (IPF) is a progressive scarring disease arising from impaired regeneration of the alveolar epithelium after injury. During regeneration, type 2 alveolar epithelial cells (AEC2s) assume a transitional state that upregulates multiple keratins and ultimately differentiate into AEC1s. In IPF, transitional AECs accumulate with ineffectual AEC1 differentiation. However, whether and how transitional cells cause fibrosis, whether keratins regulate transitional cell accumulation and fibrosis, and why transitional AECs and fibrosis resolve in mouse models but accumulate in IPF are unclear. Here, we show that human keratin 8 (  Idiopathic pulmonary fibrosis (IPF) is a disease of aging in which progressive scarring of the lungs leads to death from respiratory failure . Highly Independent, robust lines of investigation have uncovered several pathologic processes activated in epithelial cells that drive fibrosis: senescence, expression of macrophage chemokines, integrin β6–dependent TGF-β activation, impaired proteostasis (endoplasmic reticulum stress), DNA damage, and cell death –25. SeneCldn4, Sfn, and TGF-β pathway genes including integrin β6  cover 98% of the alveolar surface  and mediegrin β6 . We and e causes , 50–56. e causes , 53, rese causes , in conthi transitional cells with ineffectual AEC1 differentiation may be the specific regenerative defect underlying the pathogenesis of human IPF , and organoid mouse models of regeneration and in human IPF (https://doi.org/10.1172/JCI165612DS1)  , 71–73, 5612DS1) , A and BFibrosis is characterized by the accumulation of transitional AECs with ineffectual AEC1 differentiation, suggesting that the critical regenerative defect underlying the pathogenesis of IPF may be persistence of the transitional state –58. HoweAn extensive body of work has established that epithelial cell senescence, impaired proteostasis, cell death, DNA damage, integrin β6–mediated TGF-β activation, and macrophage chemokine expression promote fibrosis –22. HoweKRT7, KRT8, KRT17, KRT18, and KRT19. Only KRT8 was associated with IPF (KRT8 SNPs had high Combined Annotation-Dependent Depletion (CADD) and Regulatory Mendelian Mutation (REMM) scores, which predict disease causality of genetic variants. These results suggest that KRT8 genetic variants may be pathogenic. Therefore, we hypothesized that keratin 8 (K8) promotes the accumulation of transitional cells and fibrosis.To further explore whether transitional cells promote fibrosis and whether keratins play a functional role in the accumulation of transitional cells and fibrogenesis, we interrogated a meta-analysis of 5 human IPF GWAS studies  for variwith IPF . The assKrt8-KO mice as long as Krt8 deficiency did not alter lung development or homeostasis. Adult –/–Krt8 mice appeared to have normal lung structure without inflammation or altered expression of other keratins  AEC1 state , but did attenuate the expression of multiple chemokines , impaired proteostasis, cell death, DNA damage, and integrin β6–mediated TGF-β activation that characterize the transitional state in vivo . To dete process . To asse+/+ AECs . No diff similar . Since p similar –63, we fanalysis , loss ofR stress . Finallyemokines . These cemokines –25, 38 apression  and the pression  during fCcr2 KO  and found that epithelial, endothelial, and immune cells expressed much lower levels of Col1a1 than did fibroblasts  resolved in vivo , with cells grown on a stiff substrate (plastic) coated with collagen. The collagen-coated stiff substrate, but not the soft laminin-based substrate, promoted the transitional state . To dete in vivo , we reex laminin . As ment laminin , 75–79.  laminin . In thisce of K8 , i.e., tSince fibrosis maintained the transitional state , we nexthttps://github.com/kriemo/lung-regeneration-meta). We performed unbiased clustering. Clusters were annotated on the basis of expression of canonical markers   , a highl Cdkn2a) , 27, 82. Cdkn2a) . Cluster Cdkn2a) . Of note Cdkn2a) , 53, 62  Cdkn2a) . Moreove Cdkn2a) . These r Cdkn2a) , as prev Cdkn2a)  50)..Cdkn2b, Fblim1, Palld, and Pdlim7. Interestingly, by day 7 of 2D culturing, AECs had upregulated cluster 7 genes and were highly senescent   , 58, we +) , suggest+) . Human Ias KRT17 , 58. Moras KRT17 . This dial cells . Moreovell genes . To conf markers . Of the  markers  and in v markers . Althougluster 7 . Howeverstaining . The basstaining  and in tstaining  can aris+KRT5–  and ABI2 , or “tra+KRT5– , respectll genes . Likewisll genes  and assuulturing . Thus, tid state . Taken tKRT17+KRT5– state, which was associated with loss of an AEC1 fate and nonresolving fibrosis, we next asked whether a murine model of nonresolving fibrosis would be characterized by prevalent aberrant basaloid cells with a paucity of AEC1s. We treated mice with multiple doses of bleomycin and stained lung sections for the cluster 7 markers PDLIM7 and K17. Aberrant basaloid cells were more abundant and may have existed in a histologic pattern of “bronchiolization” (hi) but not aberrant basaloid  cells  cells . We spechi transitional cells uniquely activated multiple profibrotic processes, thus unifying diverse, independent lines of investigation. Genetic variants in KRT8 were associated with IPF  were conserved in the human aberrant basaloid state  (hi), but not permanently senescent (p16+), basaloid (K17+) state  . Similar/CDKN2A) , consist/CDKN2A) –33, 82. +) state . Presuma+) state , retain +) state .Ultimately, the impact of this work will be realized by future mechanistic and clinical studies. Whether mechanisms other than chemokine expression mediate the role of K8 in fibrosis and whether keratins other than K8 regulate alveolar regeneration and fibrogenesis should be studied. Conditional-KO mice will be needed to confirm that K8 expressed by transitional cells arising from AEC2s and/or club-like cells , 54 reguIn summary, we demonstrate here that a keratin, K8, promoted fibrosis and accumulation of transitional cells at the expense of AEC1 differentiation. K8 regulated chemokine expression and the recruitment of macrophages, which, together with transitional epithelial cells, activated fibroblasts. Macrophages and activated fibroblasts in turn promoted the accumulation of transitional AECs. This positive feedback loop ultimately resolved in mice, with AEC1 differentiation and the resolution of fibrosis. However, in human IPF, transitional AECs evolved into a bonafide senescent, aberrant basaloid state that existed in a self-amplifying feedback loop of epithelium-macrophage-fibroblast crosstalk, in which fibrosis begat further fibrogenesis, driving the progressive and ultimately fatal clinical disease. Modulation of K8 or progression from the transitional to the aberrant basaloid state may be effective therapeutic strategies for pulmonary fibrosis.Details on the methods are provided in the RNA-Seq data were deposited in the NCBI’s Gene Expression Omnibus (GEO) database (GEO GSE223302). Values for all data points in graphs can be found in the Supplemental FW and RLZ designed research studies. FW, CT, MD, KF, NP, NK, JN, BMV, JAW, EC, EFR, and XZ conducted experiments. FW, CT, MD, KAR, AL, EC, DWHR, EFR, DMT, XZ, BBM, PAC, MBO, and RLZ analyzed data. DMT and MBO provided reagents. RLZ wrote the manuscript. CT, KAR, JN, BMV, EC, DWHR, XZ, DMT, BBM, PAC, and MBO revised the manuscript.


TNNI1), myosin light chain 9(MYL9), and serpin family C member 1(SERPINC1)) and high-IMF pigs (CD4 molecule (CD4), CD2 molecule (CD2), and amine oxidase copper-containing 2(AOC2)) were identified, and then verified by quantitative real-time PCR (qRT-PCR) in semimembranosus muscles. Additionally, the gene set enrichment analysis and single-sample gene set enrichment analysis of hallmark gene sets were collectively performed on the identified biomarkers. Finally, the transcription factor-biomarker and lncRNA-miRNA-mRNA (biomarker) networks were predicted. The identified potential sex-specific biomarkers may provide new insights into the molecular mechanisms of IMF deposition and the beneficial foundation for improving meat quality in pig breeding.Intramuscular fat (IMF) content is a key determinant of pork quality. Controlling the genetic and physiological factors of IMF and the expression patterns of various genes is important for regulating the IMF content and improving meat quality in pig breeding. Growing evidence has suggested the role of genetic factors and breeds in IMF deposition; however, research on the sex factors of IMF deposition is still lacking. The present study aimed to identify potential sex-specific biomarkers strongly associated with IMF deposition in low- and high-IMF pig populations. The GSE144780 expression dataset of IMF deposition-related genes were obtained from the Gene Expression Omnibus. Initially, differentially expressed genes (DEGs) were detected in male and female low-IMF  and high-IMF pigs . Moreover, hub genes were screened via PPI network construction. Furthermore, hub genes were screened for potential sex-specific biomarkers using the least absolute shrinkage and selection operator machine learning algorithm, and sex-specific biomarkers in low-IMF (troponin I ( Intramuscular fat (IMF) content is an important trait in the pig industry, which is positively correlated with meat quality, and affects the tenderness, flavor, and juiciness of the meat ,2. The pACAA1) is involved in fatty acid oxidation and lipid metabolism, and is strongly linked to PPAR signaling and fatty acid metabolism pathways  and generated using semimembranosus muscles (SM) from Italian Large White pigs   × 100. Considering the weight and IMF content as factors, three male pigs and three female pigs exhibiting exceptionally high IMF content and three male pigs and three female pigs displaying remarkably low IMF content were selected for the subsequent validation analysis.The IMF content of SM was measured by the Soxhlet extraction method . Firstly® Premix Ex Taq™ II (Tli RNaseH Plus)  on a qPCR system . The reaction system was prepared for qPCR reaction. It was performed using a two-step amplification, Stage1 (predenaturation): 95 °C, 30 s; Stage2: repeat 40, 95 °C, 30 s, 60 °C, 30 s; Stage3: Dissociation. The gene-specific qPCR primers are listed in −ΔΔCt method; GAPDH was used as the internal control for normalization. The relative mRNA expression levels of male and female pigs were compared by unpaired t-test using SAS software (version 9.2), p < 0.05 was considered significant, and p < 0.01 was considered extremely significant.The total RNA was extracted from SM samples using an RNA sample total Extraction Kit . Briefly, the SM sample was frozen in liquid nitrogen and ground to a fine powder. Then, chloroform was added, the aqueous phase was collected and translated to RNase-Free columns, and the RNA was precipitated by the addition of absolute ethanol. Subsequently, buffer RD and RW were stepwise added to an RNA pellet to obtain RNA. Finally, RNA was eluted from the column by 50 µL of RNase-free water. Reverse transcription was performed using PrimeScript™ RT reagent Kit with gDNA Eraser  according to the manufacturer’s instructions. The first step considers: the reaction conditions of system1: 42 °C, 2 min; 4 °C, 18 min; second, the reaction conditions: 37 °C, 15 min; 85 °C, 5 s; 4 °C, 18 min. qPCR assay was performed using TB Greenp < 0.05), 64 of which were significantly up-regulated, and 98 were significantly down-regulated in males  and inter-α-trypsin inhibitor heavy chain 1 (ITIH1) were up-regulated in high-IMF male pigs, but down-regulated in male pigs with low IMF.The up- and down-regulated DEGs between the low- and high-IMF groups were further compared, as shown in p < 0.05)   . The top < 0.05) . The topIn the high-IMF group, GO analysis showed that a total of 5 BPs, 5 CCs, and 2 MFs were significantly enriched . KEGG enOnly one of all enriched GO terms (GO:0010951: negative regulation of endopeptidase activity) was found to overlap in a functional enrichment analysis. The results also suggested that the molecular regulatory mechanisms of male and female pigs differ substantially between the low- and high-IMF groups.From the low-IMF group, 162 DEGs were submitted to the STRING database, which identified a PPI network with 152 nodes and 165 edges A. A highFrom the high-IMF group, 202 DEGs were submitted to the STRING database, which identified a PPI network with 184 nodes and 150 edges L. One moTNNI1 (score = 1092), myosin Light Chain 2 (MLC2V) (score = 1032), myosin heavy chain 6(MYH6) (score = 1008), myosin light chain 3(MYL3) (score = 888), troponin C1(TNNC1) (score = 774), myosin binding protein C3(MYBPC3) (score = 744), ryanodine receptor 2(RYR2) (score = 722), MYL9 (score = 318), tropomyosin 3(TPM3) (score = 264), and SERPINC1 (score = 230) in the whole PPI network , protein tyrosine phosphatase receptor type C (PTPRC) (score = 305), selectin l (SELL) (score = 289), CD69 molecule(CD69) (score = 288), CD3E molecule(CD3E) (score = 240), CD2 (score = 121), AOC2 (score = 120), C-X-C motif chemokine receptor 4(CXCR4) (score = 38), serpin family B member 2(SERPINB2) (score = 26), and MYC proto-oncogene (MYC) (score = 15) in the whole PPI network , respectively, all of which showed a higher effectiveness. In particular, TNNI1 may be the sex-specific biomarker with the highest potential in low-IMF pigs ; MYL9 was positively correlated with complement, apoptosis, and androgen response (p < 0.05); and SERPINC1 was not observed with significant correlation with hallmark gene sets (p > 0.05).In the low-IMF group, HALLMARK coagulation in males was lower than that in females, and HALLMARK_glycolysis was higher A. To theAOC2 was negatively correlated with wnt_β_catenin_signaling (p < 0.05) and notch_signaling (p < 0.01) and positively associated with myc_targets_v2 (p < 0.05), myc_targets_v1 (p < 0.05), and mtotic1_signaling (p < 0.001). CD2 was negatively correlated with wnt_β_catenin_signaling (p < 0.05) and positively associated with oxygen species pathway (p < 0.05), myc_targets_v1 (p < 0.05), and adipogenesis (p < 0.05). However, no significant correlations were observed between CD4 and hallmark gene sets (p > 0.05)In the high-IMF group, there were no significant differences between males and females in hallmark gene sets C. As shoTNNI1, 10 TFs predicted by MYL9, and 20 TFs predicted by SERPINC1. Subsequently, a potential sex-specific transcriptional regulatory network was constructed  were predicted. Finally, the ceRNA network was constructed using Cytoscape J. In theThis set of samples consisted of 12 individuals, chosen from the population of thirty tested Saba pigs. For their extreme and divergent contents of SM IMF, the animals were divided into two groups, low- and high-IMF groups, each with six pigs , as shown in TNNI1, MYL9, and SERPINC1 in low-IMF pigs and CD4, CD2, and AOC2 in high-IMF pigs in the SM tissues were consistent with the results of the transcriptome analysis. In addition, significant differences in mRNA expression levels of TNNI1 (p = 0.0266) and MYL9 (p = 0.0286) between the females and males in the low-IMF group were observed (CD2 (p = 0.0009) and AOC2 (p < 0.0001) in the high-IMF group were observed  is considered crucial for maintaining the uterine surface glycocalyx during placental attachment in pigs, according to a prior study [GADD45GIP1 and ITIH1 on pork IMF contents of different sexes have not been reported. Our study may provide a novel insight into the role of GADD45GIP1 and ITIH1 in IMF deposition between the sexes, which requires further exploration and confirmation.In the present study, 162 DEGs, including 64 up- and 98 down-regulated genes, were detected between males and females in the low-IMF group; 202 DEGs, including 147 up- and 55 down-regulated genes, were screened in the high-IMF group. Eleven overlapping DEGs were identified in low- vs. high-IMF pigs. Among the overlapping genes, function . ITIH1 ior study . Up to nFunctional analysis indicated that the DEGs in the low-IMF group were primarily associated with BPs, such as ventricular cardiac muscle tissue morphogenesis, biomineral tissue development, cardiac muscle contraction, and muscle contraction. These GO terms correspond with previous studies identifying DEGs related to modulating adipogenesis in intramuscular adipocytes , the impTNNI1, MYL9, and SERPINC1 genes were identified as potential sex-specific biomarkers in low-IMF pigs using the LASSO algorithm. Among them, TNNI1 might be the sex-specific biomarker in low-IMF pigs with the highest potential. The functional analysis of TNNI1 also indicated that it might be involved in the muscle regulatory response. TNNI1 is a slow-twitch skeletal muscle isoform expressed solely in slow-twitch skeletal fibers and is related to the muscle fiber type. A previous study has shown that TNNI1 gene polymorphisms were related to IMF content and meat color in the biceps femoris of pigs [TNNI1 expression was detected in L-arginine-supplemented pigs [TNNI1 might play a positive regulatory role in IMF content and meat quality improvement. Abundant TNNI1 expression was also found in cardiac muscles [TNNI1 could act as a marker of pork quality [TNNI1 was expressed at a higher level in males, and it had a significant positive correlation with TPM3, TNNC1, and TPM3 expression levels, which were related to muscle formation and development [MYL9, which encodes myosin light chain, may regulate muscle contraction by modulating the ATPase activity of myosin heads. Previous studies have shown that MYL9 was correlated with ATP kinase activity regulation, muscle cell control, and signal transduction [MYL9 displayed higher transcriptome and proteome expression levels in myostatin-knockout Meishan pigs, and it was hypothesized that MYL9 might play an important role in skeletal muscle growth and development [SERPINC1, a member of the serpin superfamily, contributes to the regulation of the blood coagulation cascade. A previous study showed higher SERPINC1 expression in female vs. male livers due to the different growth hormone secretion patterns between the sexes [SERPINC1 played a critical role in immune responses [SERPINC1 expression is inherently associated with immune cell infiltration by B cells, T cells, CD4+ T cells, macrophages, neutrophils, and dendritic cells [SERPINC1 gene was essentially related to fat metabolism and development during beef cattle growth [TNNI1, MYL9, SERPINC1 and muscle formation, and IMF deposition in male and female low-IMF pigs, we speculated that these biomarkers might be involved in advancing IMF deposition through their involvement in muscle development based on previous studies. Our results also further indicate that TNNI1, MYL9, and SERPINC1 are closely associated with muscle development and IMF content.Subsequently, 10 hub genes were selected from the PPI network via the topology analysis method—MCC, and were mainly involved in muscle fiber and tissue development-associated processes, especially in the development of muscle organs, such as cardiac chamber morphogenesis, ventricular cardiac muscle tissue development, and cardiac muscle tissue morphogenesis; this was consistent with the results of our previous functional analysis of DEGs. Furthermore,  of pigs . Additioted pigs , suggest muscles . One stu quality . In our elopment ,48,49. Msduction . Severalelopment . SERPINChe sexes . A recenesponses . SERPINCic cells . Reportse growth . AlthougTNNI1, MYL9, and SERPINC1 included muscle development-related and immunity-related pathways, such as cardiac muscle contraction, calcium signaling pathway, complement and coagulation cascades, and bile secretion, which are consistent with the DEG functional analysis results described above. We also identified energy metabolism-related signaling pathways, such as the cholesterol metabolism, cAMP signaling pathway, TCA cycle, and p53 signaling pathway. CAMP, synthesized from ATP by the adenylate cyclase family, played a crucial role in both adipogenesis and lipid allocation in adipose tissue [The GSEA functions of e tissue . CAMP ple tissue . Previoue tissue . The TCAe tissue . The TCAe tissue . P53 wase tissue .TNNI1 and MYL9 differed significantly between male and female in low-IMF pigs, which further indicated that TNNI1 and MYL9 might be reliable biomarkers. Especially in the TNNI1, the expression was significantly up-regulated in the male pigs in qRT-PCR and RNA-seq results.In addition, the qRT-PCR results showed that the expression of PPAR signaling pathway in lipid metabolism [In the high-IMF group, BPs of DEGs were mainly enriched in inflammation/ immunization-related signaling pathways, such as the regulation of interleukin-8 (IL-8) production and leukocyte migration. IL-8 facilitates macrophage infiltration in adipose tissue, which induces topical and systemic inflammation . Obese itabolism ,69. The tabolism ,71.CD4, CD2, and AOC2 genes were selected as potential sex-specific biomarkers in high-IMF pigs and further investigated. Among them, AOC2 may be the most promising sex-specific biomarker in high-IMF pigs. CD4, which encodes the CD4 membrane glycoprotein of T lymphocytes, is primarily expressed in T lymphocytes. Some investigations have shown the association between CD4+ T cells and the progression of obesity and obesity-related diseases, suggesting the role of CD4 in controlling immune and adipose tissue [+ T cells in adipose tissue play a vital role in maintaining the pro-inflammatory state in obesity [+ T cells might be activated through the expression of the main histocompatibility complex class II and co-stimulatory membrane receptors of adipocytes to satisfy the adaptive immune response that adjusted adipose tissue inflammation [+ T cells is regulated by adipocyte-derived factors, such as adipokines and lipids [CD2, a glycoprotein expressed on the surface of most human T cells and natural killer cells, is crucial for mediating cell attachment in both T-lymphocytes and in signal transmission [CD2 is crucial during the initial steps of the immune response. Several studies have documented the role of anti-CD2 monoclonal antibodies in interfering with afferent immunity, inducing CD2-antigenicity and downregulating CD3, CD4, and CD8 cell surface expression [AOC2 is involved in oxidation by cytochrome P450 and meta-pathway biotransformation phases I and II, which are associated with median neuropathy and amyotrophic lateral sclerosis. The up-regulated AOC2 expression during adipocyte differentiation in vitro showed that this gene might play a vital role in adipogenesis [AOC2 was similar to that of the adiponectin and PPARc biomarkers [CD4, CD2, and AOC2 in male and female high-IMF pigs at present. The GSEA functions of CD4, CD2, and AOC2 included IMF deposition and immunity-related pathways, such as the JAK-STAT and T cell receptor signaling pathways; this was consistent with the results of the above DEG functional analysis.In addition, ten hub genes were selected from the PPI network. The functional analysis results of the above DEGs were supported by the hub genes’ considerable associations with immunity and inflammation regulation-related processes, such as the regulation of IL-8 production, T cell receptor signaling pathway, and primary immunodeficiency. Subsequently, e tissue . Activat obesity . Previouammation . Additiod lipids . CD2, a smission . CD2 is pression . AOC2 isogenesis ,79. Studomarkers . HoweverAOC2 was significantly up-regulated in male pigs of the high-IMF group, which further indicated that AOC2 might be a reliable sex-specific biomarker.In addition, both RNA-seq and qRT-PCR results revealed that Although sex-specific biomarkers have been identified, and some genes have been validated in previous studies, some limitations of the current study must be noted. First, the sex-specific biomarkers were obtained through a bioinformatics method; biomarker expression levels should be validated via the assessment of larger sample sizes and more pig breeds via more accurate methods. Second, the specific functions of biomarkers need to be verified using overexpression or knockdown methods in a cell or animal.TNNI1, MYL9, and SERPINC1) and high-IMF groups . In the low-IMF group, TNNI1 was evaluated as the most valuable biomarker. AOC2 was evaluated as the most valuable biomarker in the high-IMF group. The different expression patterns of DEGs, hub genes, and potential sex-specific biomarkers were shown in the male and female pigs with different IMF level, with more genes enriched in low-IMF pigs for muscle fiber and organ-formation-associated processes, such as cardiac chamber morphogenesis, ventricular cardiac muscle tissue development, and muscle contraction, but with more genes enriched in high-IMF pigs for metabolic processes, inflammation, and the immune system, such as the MAPK signaling pathway, cytokine-cytokine receptor interaction, JAK-STAT signaling pathway, the regulation of IL-8 production, and the T cell receptor signaling pathway. The results of the present study showed that IMF deposition was closely related to sex. In addition, there were differences in the molecular and regulatory mechanisms of IMF deposition between male and female pigs with varying IMF levels. These findings provided new insights into the molecular mechanisms of pig IMF deposition and meat quality improvement.In summary, we screened potential sex-specific biomarkers in male and female pigs in the low-IMF (


We assigned clinical significance for all variants and highlighted the prevalence and characteristics of PGVs. Our primary analysis of the tumor-normal sequencing cohort revealed that 152 patients (4.1%) harbored PGVs in 15 CSGs. Among 783 germline variants, 113 were annotated as PGVs, 70 as benign variants, and 600 as variants of uncertain significance. The number of PGVs identified was highest in BRCA1/2, with 56, followed by TP53, with 18. PGVs were the most prevalent in ovarian and peritoneal cancers, including among cancer types common in Asia. In the tumor-only sequencing cohort, of the 5,184 pathogenic somatic variants across 26 CSGs, 784 (15.1%) were extracted according to the European Society for Medical Oncology recommendations for germline-focused tumor analysis. The prevalence of PGVs was similar to that previously reported in Europe and the United States. This is the largest analysis based on real-world tumor-normal sequencing tests in Asia. The more widespread use of the tumor-normal sequencing CGP test could be reasonable for evaluating PGVs.Cancer genome profiling (CGP) occasionally identifies pathogenic germline variants (PGV) in cancer susceptibility genes (CSG) as secondary findings. Here, we analyzed the prevalence and clinical characteristics of PGVs based on nationwide real-world data from CGP tests in Japan. We analyzed the genomic information and clinical characteristics of 23,928 patients with solid cancers who underwent either tumor-only (BRCA1/2 and TP53, in CSGs. It highlights the prevalence of PGVs in Asian populations and supports the broader adoption of tumor-normal sequencing CGP tests for PGV evaluation.We analyzed real-world data from over 23,000 patients in Japan, revealing 4.1% harbored PGVs, particularly in  In clinical settings, massively parallel next-generation sequencing (NGS)-based cancer genome profiling (CGP) has become a routine practice for patients with advanced solid cancers worldwide for identifying clinically actionable somatic alterations for systemic therapy selection . Most coIn Japan, three types of NGS-based CGP tests, OncoGuide NCC Oncopanel System (NOP), FoundationOne CDx (F1CDx), and FoundationOne Liquid CDx (F1L), are reimbursed by the national health insurance system and implemented in routine oncological practice. F1CDx is a tumor-only genome profiling test commonly used in the United States, whereas NOP is a paired tumor-normal test for both tumor and normal (blood) DNA, uniquely implemented in Japan. Genomic information and clinical characteristics, including multiple cancers and family history, of patients who underwent CGP tests are collected at the Center for Cancer Genomics and Advanced Therapeutics (C-CAT), established in the National Cancer Center . The C-Cn = 3,739), or F1CDx  and had their genomic analysis and clinical data registered with the C-CAT from June 2019 to December 2021. All patients had advanced or recurrent cancers that were refractory to standard chemotherapy. Patients with NOP tests were our main cohort for germline variant analysis. Written informed consent, including permission for possible data use for research and registration for the C-CAT, was obtained from all patients before NGS-based CGP tests. This study was conducted in accordance with the Declaration of Helsinki, and this study was approved by the ethics committee of the National Cancer Center Hospital (no. 2020-067).This study included 23,928 patients who received tissue-based CGP tests, NOP (NGS-based CGP tests (NOP or F1CDx) covered by the Japanese National Health Insurance System were selected at the discretion of the physician or patient. F1 L was not included in this study.APC, BRCA1, BRCA2, MLH1, MSH2, PTEN, RB1, RET, SMAD4, STK11, TP53, TSC1, and VHL included from the beginning; NF1, PALB2, and SMARCB1 included from October 2020) from June 2019 to January 2021 (2 area and 50 µm thickness) with tumor concentration ratios ≥ 20% and peripheral blood (2 mL) cells using a QIAamp DNA FFPE Tissue kit (Qiagen) and a Maxwell RSC Blood DNA kit (Promega), respectively. The extracted DNA was quantified using a Qubit dsDNA BR Assay Kit (Thermo Fisher Scientific) and Qubit 3.0 Fluorometer (Thermo Fisher Scientific). Sequencing libraries were prepared from 50–800 ng DNA using the SureSelect XT reagent (Agilent Technologies) and KAPA Hyper Prep kit (KAPA Biosystems) and then analyzed on the Illumina MiSeq or NextSeq platform (Illumina) with 150 bp paired-end reads.NOP, a paired tumor-normal test, analyzed tumor and germline DNAs of 114 cancer-associated genes, 12 fusion genes, and tumor mutation burden (TMB) and provided germline variants for 16 selected genes . F1CDx cannot directly identify germline variants. As the data of Asian SNPs are not incorporated into the F1CDx database, F1CDx cannot exclude SNPs unique to Asians.F1CDx analyzes only tumor DNAs for genomic alterations in 324 cancer-related genes , MSI, and TMB . F1CDx rAPC, BRCA1, BRCA2, MLH1, MSH2, NF1, PALB2, PTEN, RB1, RET, SMAD4, STK11, TP53, TSC1, and VHL) were included in NOP before revision (NOP Ver.1), and an additional 11 CSGs  were included after revision  recommendations for secondary findings, ESMO guidelines, National Comprehensive Cancer Network clinical practice guidelines, and the Japan Agency for Medical Research and Development . Among tSequencing data of the CGP tests were submitted to the C-CAT along with clinical data from each patient, including diagnostics, treatment, and outcome information. The C-CAT has constructed a cancer knowledge database (CKDB) optimized for Japan to assist decision-making . AlthougBAP1, FH, FLCN, NF1, POLE, and TP53), early (<30 years) onset , and a high conversion ratio of somatic to germline variants . Filters with VAF ≥ 30% can cause misleading and should be used with caution, but we evaluated CSG variants based on the ESMO recommendations, which were established with the intent of avoiding excessive diversion of effort and resources towards “germline follow-up testing” of vast numbers of variants in tumor-only CGP tests.F1CDx, a tumor-only CGP test, cannot directly detect germline variants. We evaluated CSGs with high actionability based on the ESMO recommendations . Germlinχ2 test or Fisher exact test. The median values of the variables were compared using Kruskal–Wallis test. Statistical significance was set at P < 0.05. Statistical analysis was performed using EZR software version 1.38 . Institutional review at your institution and C-CAT data utilization review are required, but these data are publicly available. Derived data supporting the findings of this study are available from the corresponding author upon request.Raw data for this study were generated from C-CAT Research-Use Portal site  had PGVs. Eight patients had PGVs in the 11 CSGs added to the germline analysis target in NOP Ver.2. PGVs were observed in several tumor types, and the number of identified PGVs was highest in and TP53 . PGVs weBRCA1/2 variants were not found in patients under 30 years of age but were common PGVs in patients over 30 years of age, whereas 40% (10 patients) of TP53 variants were detected in patients under 30 years of age. Various PGVs were observed in individuals over 70 years of age. Pathogenic germline TP53 variants were found not only in the younger generation (≤40 years), which is one of the diagnostic criteria for Li-Fraumeni syndrome (LFS) but also in the older generation . We determined whether these variants were pathogenic based on CKDB, ClinVar, 1000 Genomes, iJGVD, and ACMG/AMP guidelines, and variants were assigned VUS if not listed in these databases. eded 70% .TP53, APC, SMAD4, and PTEN. A total of 1,622 variants were annotated as PGVs. The ratio of somatic to germline pathogenic variants varied by gene  pathway, and MLH1, MSH2, and MSH6, involved in the mismatch repair (MMR) pathway, also had germline variants at a relatively high ratio of 30%–40%.Paired tumor-normal NOP tests detected 1,534 somatic variants across the 26 CSGs. Among these variants, 1,509 were annotated as PGVs, most frequently in  by gene . For exaPOLE (22%) and FH (20%). In comparison, for other CSGs having a high conversion ratio of somatic to germline variants, more than 40% of variants met the recommendations criteria. The variants of HRR-related genes  and MMR genes  met the recommendations at a particularly high percentage.The tumor-only F1CDx test detected 5,184 somatic variants annotated as PGVs across 26 CSGs listed in the ESMO recommendations for germline-focused tumor analysis. Among these somatic variants, 784 (15.1%) met the criteria for the ESMO recommendations . For CSGBRCA2 and TSC2 had significantly higher rates of pathogenic variants conforming to the ESMO recommendations in F1CDx than the rate of PGVs in NOP , and prostate cancers, whereas BRCA2 somatic variants were not found in ovarian cancer, were increased in gastrointestinal and prostate cancers, and were less common in breast and ovarian cancers than germline variants. Somatic variants were more common in males. In F1CDx, BRCA2 mutations were more common in the prostate than in breast or pancreatic cancers.In contrast, in NOP, BRCA1 and BRCA2, somatic variants were associated with a higher median patient age than germline variants. There were no significant differences in the presence or absence of family history and multiple cancers among variants in NOP and F1CDx.In MLH1, PMS2, PALB2, RB1, and TP53, had a VAF of less than 30%, and other PGVs were 30%–70%.We examined the distribution of non-tumor  VAF for PGVs detected in NOP . The medMSH6, PMS2, RAD51C and RAD51D, and NOP Ver.2 still did not include SDHB, SDHC, SDHD, and MUTYH, which were included in ACMG and ESMO recommendations  had core LFS cancer, whereas nearly all of the patients in the younger generation (<45 years) had core LFS cancers. Y220C variant was found only in patients older than 60 years, all with VAF less than 30%. Y220C has been reported to be relatively more frequent in clonal hematopoiesis Click here for additional data file.Table S2Detailed information about patients with BRCA1/2 and TP53 pathogenic germline variantsClick here for additional data file.Table S3Comparison of pathogenic germline variantsClick here for additional data file.Table S4Characteristics of patients with BRCA1/2 variantsClick here for additional data file.Figure S1The time-related changes in the OncoGuideTM NCC oncopanel systemClick here for additional data file.Figure S2Distribution of variant allele frequencyClick here for additional data file.


Escherichia coli is not favored. This suggests that eukaryotic cells with more complex expression systems are a better host choice for hLYZ productionLysozyme (LYZ) is an oval-shaped antimicrobial agent with good bacterial lytic properties and is widely distributed in human, animal, plant, and even some microbial tissues. It can destroy the walls of bacterial cells by cleaving the chemical bond linking N-acetylglucosamine and N-acetyl carbamate in the peptidoglycan layer. Due to its superior bactericidal and anti-inflammatory properties, human LYZ (hLYZ) is used as an alternative to antibiotics, food additive, feed additive, and anti-infection agentAspergillus niger is one of the most widely used eukaryotic expression systems in biotechnology. It is a highly safe and food-grade strain with a short fermentation cycle, complete protein synthesis and modification system, and excellent protein secretion ability2 to 103 timesA.niger expression system, so exploring more fusion proteins ideal for the A. niger expression system is highly innovative. In this study, we selected five types of highly expressed endogenous proteins, including endoglucanase (EglA), endo-1,4-beta-xylanase (XynB), feruloyl esterase (FaeA), tannase (Tan), and glucose oxidase (GOD), ofA.niger as carrier proteins and five different linker peptides, including (GGGGS)1‒3, (EAAAK)2, and (AP)5. The efficient expression of hLYZ inA.niger was taken as the research object to develop more fusion expression strategies suitable for theA.niger expression system to achieve the efficient expression of heterologous proteins in this system.Currently, there are few fusion proteins suitable for theSupplementary Table S1 andSupplementary Figure S1). Subsequently, AGL1-positive transformants were cocultured withA.niger TH-2 . Recombinant strains with different carrier proteins EglA, XynB, FaeA, Tan, and GOD were named ELF, XLF, FLF, TLF, and GLF, respectively. All of these recombinant strains used GGGGS as the linker peptide. The recombinant control strain without carrier protein was named LF.First, we successfully constructed recombinant expression vectors containing different carrier proteins and linker peptides using PCR amplification and pSZHG6R vectors stored in our laboratory. Second, the recombinant expression vector was transferred into AGL1 by the freeze-thaw method, and the AGL1-positive transformants were screened by PCR using primers P15 and P16  of the supernatant and theand ELF B. This ictively C. TherefhLYZ gene in ELF, XLF, FLF, TLF, and GLF were 2.68 times, 4.20 times, 2.42 times, 2.58 times, and 1.21 times as high as that of strain LF, respectively . Then, real-time quantitative PCR was performed. The results showed that the transcription levels of thectively D. This o1‒3, (EAAAK)2, and (AP)5 between XynB and hLYZ, were constructed and named XLF, XG2LF, XG3LF, XE2LF and XALF, respectively. SDS-PAGE and enzyme activity results of recombinant strains with different linker peptides showed that compared with the original strain TH-2, the protein band of hLYZ was approximately 14.7 kDa, and the fusion protein band was approximately 35 kDa. More importantly, we found that compared with strain XLF, some hLYZ in other recombinant strains could be secreted independently, and the linker peptide in the fusion protein could be broken (3LF was highest (546.58 U/mL), and the independent expression of hLYZ in strain XE2LF was the best among all recombinant strains. This may be because the secondary structure of the flexible linker peptide (GGGGS)n is a mainly random curly state, which allows the carrier protein to freely regulate the distance between the target protein and the carrier protein, thereby promoting the correct folding and secretion of hLYZn has a hydrogen bond and a tightly arranged main chain, and the secondary structure is an α-helix structure, so the structure is more rigid and stable. The rigid linker peptide (EAAAK)2 can maintain a stable distance between the carrier protein and the target protein while building fusion proteins without affecting the independent function2LF.Five recombinant strains with different linker peptides, including (GGGGS) broken A. At the3 LF>XG2LF>XLF (546.58 U/mL>346.67 U/mL>46.58 U/mL) (2LF (486.62 U/mL). The reason may be that the rigid linker peptide (XP)n contains a proline preference sequence, which can increase the rigidity of the linker peptide, makes it difficult to break, and is not conducive to protein interaction and expressionFurthermore, we compared the enzyme activity of the recombinant strains containing flexible linker peptides and found that the order from high to low was XG8 U/mL) B. This ihLYZ increased by 4.20 times, 3.37 times, 4.76 times, 3.60 times, and 3.46 times, respectively 3 has the best effect. The rigid linker peptide (EAAAK)2 was more effective in the independent expression of fusion proteins. Since the difference in enzyme activity between XG3LF and XE2LF is only 11%, both linker peptides can be selected.The transcription levels of the recombinant strains with different linker peptides showed that the transcription levels of the target genectively C, comparA.niger expressing hLYZ was successfully constructed in this study, which provided a safe and efficient production mode for hLYZ. More importantly, this study explored the possibility of more carrier proteins and linker peptides suitable for theA .niger expression system. This provided strong support for the ability of theA.niger system to express more foreign proteins.In conclusion, a recombinant strain ofActa Biochimica et Biophysica Sinica online.Supplementary data is available at23122Supplementary_Data