Genetic manipulation of Patescibacteria provides mechanistic insights into microbial dark matter and the epibiotic lifestyle.

Patescibacteria, also known as the candidate phyla radiation (CPR), are a diverse group of bacteria that constitute a disproportionately large fraction of microbial dark matter. Its few cultivated members, belonging mostly to Saccharibacteria, grow as epibionts on host Actinobacteria. Due to a lack of suitable tools, the genetic basis of this lifestyle and other unique features of Patescibacteira remain unexplored. Here, we show that Saccharibacteria exhibit natural competence, and we exploit this property for their genetic manipulation. Imaging of fluorescent protein-labeled Saccharibacteria provides high spatiotemporal resolution of phenomena accompanying epibiotic growth, and a transposon-insertion sequencing (Tn-seq) genome-wide screen reveals the contribution of enigmatic Saccharibacterial genes to growth on their hosts. Finally, we leverage metagenomic data to provide cutting-edge protein structure-based bioinformatic resources that support the strain Southlakia epibionticum and its corresponding host, Actinomyces israelii, as a model system for unlocking the molecular underpinnings of the epibiotic lifestyle.

Streptomyces umbrella toxin particles block hyphal growth of competing species.

Streptomyces are a genus of ubiquitous soil bacteria from which the majority of clinically utilized antibiotics derive1. The production of these antibacterial molecules reflects the relentless competition Streptomyces engage in with other bacteria, including other Streptomyces species1,2. Here we show that in addition to small-molecule antibiotics, Streptomyces produce and secrete antibacterial protein complexes that feature a large, degenerate repeat-containing polymorphic toxin protein. A cryo-electron microscopy structure of these particles reveals an extended stalk topped by a ringed crown comprising the toxin repeats scaffolding five lectin-tipped spokes, which led us to name them umbrella particles. Streptomyces coelicolor encodes three umbrella particles with distinct toxin and lectin composition. Notably, supernatant containing these toxins specifically and potently inhibits the growth of select Streptomyces species from among a diverse collection of bacteria screened. For one target, Streptomyces griseus, inhibition relies on a single toxin and that intoxication manifests as rapid cessation of vegetative hyphal growth. Our data show that Streptomyces umbrella particles mediate competition among vegetative mycelia of related species, a function distinct from small-molecule antibiotics, which are produced at the onset of reproductive growth and act broadly3,4. Sequence analyses suggest that this role of umbrella particles extends beyond Streptomyces, as we identified umbrella loci in nearly 1,000 species across Actinobacteria.

Development and anatomy of petals with specialized nectar holder and pollen container in Fumarioideae (Papaveraceae).

MAIN CONCLUSION: Petal developmental characteristics in Fumarioideae were similar at early stages, and the specialized nectar holder/pollen container formed by the outer/inner petals. The micro-morphology of these two structures, however, shows diversity in seven species. Elaborate petals have been modified to form different types, including petal lobes, ridges, protuberances, and spurs, each with specialized functions. Nectar holder and pollen container presumably have a function in plant-pollinator interactions. In Fumarioideae, four elaborate petals of the disymmetric/zygomorphic flower present architecture forming the "nectar holder" and "pollen container" structure at the bottom and top separately. In the present study, the petals of seven species in Fumarioideae were investigated by scanning electron microscopy, light microscope, and transmission electron microscopes. The results show that petal development could divided into six stages: initiation, enlargement, adaxial/abaxial differentiation, elaborate specializations (sacs, spurs, and lobes formed), extension, and maturation, while the specialized "nectar holder" and "pollen container" structures mainly formed in stage 4. "Nectar holder" is developed from the shallow sac/spur differentiated at the base of the outer petal, eventually forming a multi-organized complex structure, together with staminal nectaries (1-2) with individual sizes. A semi-closed ellipsoidal "pollen container" is developed from the apical part of the 3-lobed inner petals fused by middle lobes and attain different sizes. The adaxial epidermis cells are specialized, with more distinct punctate/dense columnar protrusions or wavy cuticles presented on obviously thickening cell walls. In addition, a large and well-developed cavity appears between the inner and outer epidermis of the petals. As an exception, Hypecoum erectum middle lobes present stamen mimicry. Elaborate petal structure is crucial for comprehending the petal diversity in Fumarioideae and provides more evidence for further exploration of the reproductive study in Papaveraceae.

Clinically Applicable Pan-Origin Cancer Detection for Lymph Nodes via Artificial Intelligence-Based Pathology.

INTRODUCTION: Lymph node metastasis is one of the most common ways of tumour metastasis. The presence or absence of lymph node involvement influences the cancer's stage, therapy, and prognosis. The integration of artificial intelligence systems in the histopathological diagnosis of lymph nodes after surgery is urgent. METHODS: Here, we propose a pan-origin lymph node cancer metastasis detection system. The system is trained by over 700 whole-slide images (WSIs) and is composed of two deep learning models to locate the lymph nodes and detect cancers. RESULTS: It achieved an area under the receiver operating characteristic curve (AUC) of 0.958, with a 95.2% sensitivity and 72.2% specificity, on 1,402 WSIs from 49 organs at the National Cancer Center, China. Moreover, we demonstrated that the system could perform robustly with 1,051 WSIs from 52 organs from another medical centre, with an AUC of 0.925. CONCLUSION: Our research represents a step forward in a pan-origin lymph node metastasis detection system, providing accurate pathological guidance by reducing the probability of missed diagnosis in routine clinical practice.

Response characteristic and potential molecular mechanism of tail resorption in Bufo gargarizans after exposure to lead and copper, alone or combined.

Tail resorption during amphibian metamorphosis is one of the most dramatic processes that is obligatorily dependent on thyroid hormone (TH). Heavy metals could result in thyroid gland damages and disturb TH homeostasis. Lead (Pb) and copper (Cu) often co-exist in natural aquatic ecosystems. However, there is still little information on how tail resorption responds to alone or combined exposure to Pb and Cu. Our study investigated the effects of Pb and Cu alone or combined exposure on the morphological parameters of the tail, histological changes of thyroid gland and tail, and gene expression programs involved in cell death of the tail in Bufo gargarizans tadpoles at the climax of metamorphosis. Results demonstrated that Pb, Cu and Pb-Cu mixture exposure resulted in a significantly longer tail compared with control. Damages to notochord, muscle, skin and spinal cord of the tail were found in Pb and Cu exposure groups. The colloid area, the height of follicular cells and number of phagocytic vesicles of thyroid gland in Pb-Cu mixture exposure groups were significantly reduced. In addition, the expression levels of TH, apoptosis, autophagy, degradation of cellular components and oxidative stress-related genes in the tail were significantly altered following Pb and Cu exposure. The present work revealed the relationship between environmental pollutants and tail resorption, providing scientific basis for amphibian protection.

Splicing complexity as a pivotal feature of alternative exons in mammalian species.

BACKGROUND: As a significant process of post-transcriptional gene expression regulation in eukaryotic cells, alternative splicing (AS) of exons greatly contributes to the complexity of the transcriptome and indirectly enriches the protein repertoires. A large number of studies have focused on the splicing inclusion of alternative exons and have revealed the roles of AS in organ development and maturation. Notably, AS takes place through a change in the relative abundance of the transcript isoforms produced by a single gene, meaning that exons can have complex splicing patterns. However, the commonly used percent spliced-in (Ψ) values only define the usage rate of exons, but lose information about the complexity of exons' linkage pattern. To date, the extent and functional consequence of splicing complexity of alternative exons in development and evolution is poorly understood. RESULTS: By comparing splicing complexity of exons in six tissues (brain, cerebellum, heart, liver, kidney, and testis) from six mammalian species (human, chimpanzee, gorilla, macaque, mouse, opossum) and an outgroup species (chicken), we revealed that exons with high splicing complexity are prevalent in mammals and are closely related to features of genes. Using traditional machine learning and deep learning methods, we found that the splicing complexity of exons can be moderately predicted with features derived from exons, among which length of flanking exons and splicing strength of downstream/upstream splice sites are top predictors. Comparative analysis among human, chimpanzee, gorilla, macaque, and mouse revealed that, alternative exons tend to evolve to an increased level of splicing complexity and higher tissue specificity in splicing complexity. During organ development, not only developmentally regulated exons, but also 10-15% of non-developmentally regulated exons show dynamic splicing complexity. CONCLUSIONS: Our analysis revealed that splicing complexity is an important metric to characterize the splicing dynamics of alternative exons during the development and evolution of mammals.

Head-to-head comparison of perfluorobutane contrast-enhanced US and multiparametric MRI for breast cancer: a prospective, multicenter study.

BACKGROUND: Multiparametric magnetic resonance imaging (MP-MRI) has high sensitivity for diagnosing breast cancers but cannot always be used as a routine diagnostic tool. The present study aimed to evaluate whether the diagnostic performance of perfluorobutane (PFB) contrast-enhanced ultrasound (CEUS) is similar to that of MP-MRI in breast cancer and whether combining the two methods would enhance diagnostic efficiency. PATIENTS AND METHODS: This was a head-to-head, prospective, multicenter study. Patients with breast lesions diagnosed by US as Breast Imaging Reporting and Data System (BI-RADS) categories 3, 4, and 5 underwent both PFB-CEUS and MP-MRI scans. On-site operators and three reviewers categorized the BI-RADS of all lesions on two images. Logistic-bootstrap 1000-sample analysis and cross-validation were used to construct PFB-CEUS, MP-MRI, and hybrid (PFB-CEUS + MP-MRI) models to distinguish breast lesions. RESULTS: In total, 179 women with 186 breast lesions were evaluated from 17 centers in China. The area under the receiver operating characteristic curve (AUC) for the PFB-CEUS model to diagnose breast cancer (0.89; 95% confidence interval [CI] 0.74, 0.97) was similar to that of the MP-MRI model (0.89; 95% CI 0.73, 0.97) (P = 0.85). The AUC of the hybrid model (0.92, 95% CI 0.77, 0.98) did not show a statistical advantage over the PFB-CEUS and MP-MRI models (P = 0.29 and 0.40, respectively). However, 90.3% false-positive and 66.7% false-negative results of PFB-CEUS radiologists and 90.5% false-positive and 42.8% false-negative results of MP-MRI radiologists could be corrected by the hybrid model. Three dynamic nomograms of PFB-CEUS, MP-MRI and hybrid models to diagnose breast cancer are freely available online. CONCLUSIONS: PFB-CEUS can be used in the differential diagnosis of breast cancer with comparable performance to MP-MRI and with less time consumption. Using PFB-CEUS and MP-MRI as joint diagnostics could further strengthen the diagnostic ability. Trial registration Clinicaltrials.gov; NCT04657328. Registered 26 September 2020. IRB number 2020-300 was approved in Chinese PLA General Hospital. Every patient signed a written informed consent form in each center.

PhieABEs: a PAM-less/free high-efficiency adenine base editor toolbox with wide target scope in plants.

Adenine base editors (ABEs), which are generally engineered adenosine deaminases and Cas variants, introduce site-specific A-to-G mutations for agronomic trait improvement. However, notably varying editing efficiencies, restrictive requirements for protospacer-adjacent motifs (PAMs) and a narrow editing window greatly limit their application. Here, we developed a robust high-efficiency ABE (PhieABE) toolbox for plants by fusing an evolved, highly active form of the adenosine deaminase TadA8e and a single-stranded DNA-binding domain (DBD), based on PAM-less/free Streptococcus pyogenes Cas9 (SpCas9) nickase variants that recognize the PAM NGN (for SpCas9n-NG and SpGn) or NNN (for SpRYn). By targeting 29 representative targets in rice and assessing the results, we demonstrate that PhieABEs have significantly improved base-editing activity, expanded target range and broader editing windows compared to the ABE7.10 and general ABE8e systems. Among these PhieABEs, hyper ABE8e-DBD-SpRYn (hyABE8e-SpRY) showed nearly 100% editing efficiency at some tested sites, with a high proportion of homozygous base substitutions in the editing windows and no single guide RNA (sgRNA)-dependent off-target changes. The original sgRNA was more compatible with PhieABEs than the evolved sgRNA. In conclusion, the DBD fusion effectively promotes base-editing efficiency, and this novel PhieABE toolbox should have wide applications in plant functional genomics and crop improvement.

Coordinative Combination of Nitroamine and Gem-Dinitromethyl with Fused Ring for Enhanced Oxygen Balances and Detonation Properties.

Oxygen balance and heat of formation are closely related to the nitrogen and oxygen content in a molecule and have a significant effect on the detonation performance of energetic materials. Here a new family of 1,2,4-triazolo [4,3-b][1,2,4,5]-tetrazine containing gem-dinitromethyl and nitroamine with high nitrogen-oxygen content was synthesized and characterized. Moreover, the structure of the guanidine salt (3) and TATOT salt (4) were confirmed by single-crystal X-ray diffraction. The nitrogen and oxygen content of ammonium salt 2 reached 82.5%, with a high density (1.805 g cm-3) and high detonation properties (D = 8900 m s-1; P = 32.4 GPa), which were similar to those of RDX.

An Efficient Marker Gene Excision Strategy Based on CRISPR/Cas9-Mediated Homology-Directed Repair in Rice.

In order to separate transformed cells from non-transformed cells, antibiotic selectable marker genes are usually utilized in genetic transformation. After obtaining transgenic plants, it is often necessary to remove the marker gene from the plant genome in order to avoid regulatory issues. However, many marker-free systems are time-consuming and labor-intensive. Homology-directed repair (HDR) is a process of homologous recombination using homologous arms for efficient and precise repair of DNA double-strand breaks (DSBs). The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 (Cas9) system is a powerful genome editing tool that can efficiently cause DSBs. Here, we isolated a rice promoter (Pssi) of a gene that highly expressed in stem, shoot tip and inflorescence, and established a high-efficiency sequence-excision strategy by using this Pssi to drive CRISPR/Cas9-mediated HDR for marker free (PssiCHMF). In our study, PssiCHMF-induced marker gene deletion was detected in 73.3% of T0 plants and 83.2% of T1 plants. A high proportion (55.6%) of homozygous marker-excised plants were obtained in T1 progeny. The recombinant GUS reporter-aided analysis and its sequencing of the recombinant products showed precise deletion and repair mediated by the PssiCHMF method. In conclusion, our CRISPR/Cas9-mediated HDR auto-excision method provides a time-saving and efficient strategy for removing the marker genes from transgenic plants.

Factors associated with information literacy of nursing undergraduates in China.

OBJECTIVE: Information literacy is an important foundation for evidence-based nursing practice. Quantitative studies using validated questionnaires on information literacy of nursing undergraduates in China are rare. The students' baseline information literacy must be evaluated before exploring ways to improve their level of information literacy. This study aimed to investigate the factors potentially involved in the information literacy of nursing undergraduates. METHODS: In this cross-sectional study, male and female nursing undergraduates (n = 710) from Inner Mongolia, China were included in the final sample. The Information Literacy Competency Scale of the Applied Undergraduate Student (ILCSAUS) was used for evaluation. Multivariate stepwise linear regression analysis was performed to assess the association between various factors associated with information literacy. RESULTS: The students' information literacy score was 105.00 (94.00-119.00). The highest score in the four dimensions was information awareness dimension, and the average score was 4.00 (3.80-4.40). Multivariable stepwise linear regression analysis showed that received training in medical statistics, received training in literature retrieval and utilization, and birth place were independently associated with information literacy scores (p < 0.05). CONCLUSIONS: Results indicate that learning about medical statistics, literature retrieval and utilization, and paying attention to students born in countries and towns can help improve information literacy in the nursing undergraduates.

Fast Response Organic Tandem Photodetector for Visible and Near-Infrared Digital Optical Communications.

Organic photodetectors (OPDs), which usually work as photodiodes, photoconductors, or phototransistors, have emerged as candidates for next-generation light sensing. However, low response speed caused by low carrier mobility and resistance-capacitance (RC) time constant, severely hinders the commercialization of OPDs. Herein, the authors demonstrate a state-of-the-art OPD with a record response speed of 146.8 ns by employing tandem structure to simultaneously reduce both the carrier transit time and RC time constant of the device, which is faster than that of previously reported OPDs as far as they know. Moreover, benefitting from the multi-level barrier enhancement and voltage division engendered by tandem structure, an ultralow noise current of 7.82 × 10-14 A Hz-1/2 is obtained, as well as a wide detection range in 300-1000 nm. In addition, the tandem OPDs are successfully integrated into the optical communication system as signal receivers, demonstrating the precise digital signal communication from visible to near-infrared light. It is believed that tandem OPDs have promising application potential in the wireless transmission system.

Non-coenzyme role of vitamin B1 in RANKL-induced osteoclastogenesis and ovariectomy induced osteoporosis.

Vitamins B are co-enzymes participating in energy metabolic pathways. While some vitamins B are known affecting bone homeostasis, the effects of vitamin B1 (thiamine) on bone health remains unclear. In our study, we used cell counting kit-8, tartrate-resistant acid phosphatase stain, actin cytoskeleton stain, and pit formation assay to evaluate the effect of thiamine on osteoclast differentiation, formation, and function, respectively. Then we used dichloro-dihydro-fluorescein diacetate assay to investigate reactive oxygen species (ROS) generation and removal. Osteoporosis model by ovariectomy was established for animal experiments. We found that thiamine had inhibitory effect on osteoclast differentiation. And its inhibitory role on osteoclast differentiation is in a dose-dependent way. Mechanistically, ThDP suppresses intracellular ROS accumulation and unfolded protein response signaling during osteoclastogenesis via inhibiting Rac-Nox1/2/4 and intracellular inositol-requiring protein-1α/X-box-binding protein pathways, respectively. Osteoporotic mice treated with thiamine rich dietary showed better bone strength relative to thiamine deficient dietary. Our study explored the non-coenzyme inhibitory functions of B1 vitamin in receptor activator of nuclear factor κB ligand induced osteoclastogenesis and uncovered the significance of B1 vitamin in bone health.

A primary benign cardiac tumor misdiagnosed as cardiac metastasis in the right atrium.

We present a patient with breast cancer with multiple metastases who had an unusual cardiac mass in the right atrium. The cardiac mass was initially diagnosed as malignant metastasis by transthoracic echocardiogram (TTE) but subsequently diagnosed as benign by contrast-enhanced ultrasonography, cardiac magnetic resonance imaging (MRI), and positron emission tomography/computed tomography (PET/CT). TTE is the preferred imaging method for examination of cardiac masses. However, this case demonstrates that contrast-enhanced ultrasonography, MRI, and PET/CT are useful to differentiate between diagnoses of benign and malignant tumor. The combination of multiple diagnostic imaging modalities is necessary to confirm the diagnosis of cardiac tumors.

Right atrial appendage aneurysm complicated with atrial septal defect: A rare case report.

Right atrial appendage aneurysms (RAAAs) are rare heart malformations, presenting as isolated anomalies or co-existing with other structural heart diseases. We describe a rare case of RAAA complicated with an atrial septal defect (ASD). The diagnosis was established using transthoracic echocardiography and confirmed using cardiac magnetic resonance imaging. To treat the ASD and reduced right atrium volume load, ASD transcatheter closure was performed. On echocardiography performed 3 months post discharge, the RAAA was observed to have reduced in size compared to that presurgery. Six years later, she was in good condition without any adverse events.

[Impact of Smoking on the Histological Subtype and Outcome of the Primary Lung Adenocarcinoma].

OBJECTIVE: To explore the effect of smoking on the histological subtype and prognosis of patients with lung adenocarcinoma (LAC) in China. METHODS: According to the new International Association for the Study of Lung Cancer/American Thoracic Society/European Respiratory Society(IASLC/ATS/ERS)classification, 266 donors with primary LAC were reclassified. The correlation between clinicopathological factors including smoking status and the histological subtype was analyzed, and survival analysis was used to analyze the prognosis of primary LAC. RESULTS: There were four main histological subtypes including acinar predominant adenocarcinoma (APA) 30.1%, papillary predominant adenocarcinoma (PPA) 26.7%, solid predominant adenocarcinoma (SPA) 25.9%, and lepidic predominant adenocarcinoma (LPA) 11.7%.Smoking was associated with the histological subtype.The proportion of smokers was significantly higher than non-smokers in the SPA group, and the proportion of non-smokers was higher in other subtypes group. Cox regression model showed that the histological subtype and TNM stage were the independent predictors of prognostic in all patients.TNM stage was the predictor of postoperative survival in both smokers and non-smokers, and histological subtypes was the predictor only in smokers (ß=0.898, RR=2.455). Compared with the non-SPA group, the prognosis of the SPA group was significantly worse. CONCLUSION: Smoking is associated with SPA subtype, which affect the prognosis of primary LAC.

Expression of TBX3 in Hepatocellular Carcinoma and Its Clinical Implication.

BACKGROUND Hepatocellular carcinoma (HCC) is the fifth most common malignancy in China, and China's annual number of new cases accounts for about 45% of the world total. This research was aimed to study the expression of TBX3 protein in HCC and exploring its clinical significance. MATERIAL AND METHODS We collected tumor tissues and adjacent non-tumoral tissues of 174 patients with HCC undergoing surgical resection. The expression of TBX3 protein in different tissues and cell lines in vitro (LO2, HHL-5, MHC97-L, MHC97-H) was detected by immunohistochemistry or Western blotting, and the relationship between TBX3 expression and clinical data of patients with HCC was analyzed. RESULTS The expression of TBX3 protein in HCC was significantly correlated with histological grade, tumor size, cancer cell metastasis, hepatitis B surface antigen, and the expression of Ki-67 in tumor tissues (P<0.05), and it was positively correlated with serum AFP level (r=0.766, P<0.05). The expression of TBX3 increased with increased histological grade in HCC (P<0.05). Cox regression analysis showed that the expression of TBX3 protein in HCC was an independent risk factor for prognosis (OR=0.524, 95% CI=0.283-0.964). The 5-year survival rate of patients with HCC that highly expressed TBX3 protein was 20.83%, which was significantly lower than the 40.20% rate in patients with low expression (P<0.05). CONCLUSIONS The expression of TBX3 in HCC patients undergoing surgical resection is high, and its expression increases with the degree of tumor differentiation. It is related to the metastasis of tumor cells and is positively correlated with the serum level of AFP and may affect the survival time of HCC patients undergoing surgical resection.

Persistent distention of colon damages interstitial cells of Cajal through Ca2+ -ERK-AP-1-miR-34c-SCF deregulation.

Gastrointestinal motility disorders (GMDs) are attributed to loss of interstitial cells of Cajal (ICC), whose survival and function are deeply dependent on the activation of KIT/SCF signalling. Based on the facts that gastrointestinal distention is common in GMD patients and SCF produced by smooth muscle cells (SMCs) is usually decreased before ICC loss, we considered a possible contribution of persistent gastrointestinal distention/stretch to SCF deficiency. In this study, chronic colonic distention mouse model, diabetic gastrointestinal paresis mouse model, cultured mouse colonic SMCs and colon specimens from Hirschsprung's disease patients were used. The results showed that SCF was clearly decreased in distent colon of mice and patients, and microRNA array and real-time PCR indicated a concomitant increase of miR-34c in distent colon. A negative regulation of miR-34c on SCF expression was confirmed by luciferase reporter assays together with knock-down and overexpression of miR-34c in cultured colonic SMCs. Using EMSA and ChIP assays, we further consolidated that in response to persistent stretch, the transcription factor AP-1/c-Jun was highly activated in colonic SMCs and significantly promoted miR-34c transcription by binding to miR-34c promoter. Knock-down or overexpression of AP-1/c-Jun in cultured colonic SMCs leads to down- or up-regulation of miR-34c, respectively. In addition, the activation of AP-1/c-Jun was through ERK1/2 signalling provoked by Ca2+ overload in colonic SMCs that were subject to persistent stretch. In conclusion, our data demonstrated that persistent distention/stretch on colonic SMCs could suppress SCF production probably through Ca2+ -ERK-AP-1-miR-34c deregulation, resulting in ICC loss or impairment and GMD progress.

Phosphorylation of ETV4 at Ser73 by ERK kinase could block ETV4 ubiquitination degradation in colorectal cancer.

It was reported that Src-mediated and RTK-dependent accumulation of key transcription factor, ETV4, which played an important role in the migration of embryonic cells and tumor cells, were regulated by their common downstream MAPK molecules. However, the detailed mechanism was not completely clear. In the present study, we revealed that ETV4 protein was significantly enhanced by ERK kinase activation in the colorectal cancer (CRC) patients and mouse models as well as in the CRC cell lines. It was further confirmed that the activation of ERK kinase led to the phosphorylation of ETV4 at Ser73 and the ETV4 phosphorylation could block its binding to COP1, thereby stabilized ETV4 via avoiding its ubiquitination degradation. In addition, this effect was not due to altering an E3 ubiquitin ligase, COP1 amount or p-COP1/COP1 ratio. Our results will help understand the mechanism of ETV4 overexpression in CRC patients and provide a clue to search new therapeutic target to treat the related tumors in clinical practice.

SCF/C-Kit/JNK/AP-1 Signaling Pathway Promotes Claudin-3 Expression in Colonic Epithelium and Colorectal Carcinoma.

Claudin-3 is a major protein of tight junctions (TJs) in the intestinal epithelium and is critical for maintaining cell-cell adhesion, barrier function, and epithelium polarity. Recent studies have shown high claudin-3 levels in several solid tumors, but the regulation mechanism of claudin-3 expression remains poorly understood. In the present study, colorectal cancer (CRC) tissues, HT-29 and DLD-1 CRC cell lines, CRC murine model (C57BL/6 mice) and c-kit loss-of-function mutant mice were used. We demonstrated that elevated claudin-3 levels were positively correlated with highly expressed c-kit in CRC tissues based upon analysis of protein expression. In vitro, claudin-3 expression was clearly increased in CRC cells by overexpressed c-kit or stimulated by exogenous recombinant human stem cell factor (rhSCF), while significantly decreased by the treatment with c-kit or c-Jun N-terminal kinase (JNK) inhibitors. Chromatin immunoprecipitation (ChIP) and luciferase reporter assay showed that SCF/c-kit signaling significantly promoted activator protein-1 (AP-1) binding with CLDN-3 promoter and enhanced its transcription activity. Furthermore, decreased expression of claudin-3 was obtained in the colonic epithelium from the c-Kit loss-of-function mutant mice. In conclusion, SCF/c-kit-JNK/AP-1 signaling pathway significantly promoted claudin-3 expression in colonic epithelium and CRC, which could contribute to epithelial barrier function maintenance and to CRC development.