RNA m6A modification regulates L1 retrotransposons in human spermatogonial stem cell differentiation in vitro and in vivo.

The maintenance of genome integrity in the germline is crucial for mammalian development. Long interspersed element type 1 (LINE-1, L1) is a mobile genetic element that makes up about 17% of the human genome and poses a threat to genome integrity. N6-methyl-adenosine (m6A) plays an essential role in regulating various biological processes. However, the function of m6A modification in L1 retrotransposons and human germline development remains largely unknown. Here we knocked out the m6A methyltransferase METTL3 or the m6A reader YTHDF2 in human embryonic stem cells (hESCs) and discovered that METTL3 and YTHDF2 are crucial for inducing human spermatogonial stem cells (hSSCs) from hESCs in vitro. The removal of METTL3 or YTHDF2 resulted in increased L1 retrotransposition and reduced the efficiency of SSC differentiation in vitro. Further analysis showed that YTHDF2 recognizes the METTL3-catalyzed m6A modification of L1 retrotransposons and degrades L1 mRNA through autophagy, thereby blocking L1 retrotransposition. Moreover, the study confirmed that m6A modification in human fetal germ cells promotes the degradation of L1 retrotransposon RNA, preventing the insertion of new L1 retrotransposons into the genome. Interestingly, L1 retrotransposon RNA was highly expressed while METTL3 was significantly downregulated in the seminal plasma of azoospermic patients with meiotic arrest compared to males with normal fertility. Additionally, we identified some potentially pathogenic variants in m6A-related genes in azoospermic men with meiotic arrest. In summary, our study suggests that m6A modification serves as a guardian of genome stability during human germline development and provides novel insights into the function and regulatory mechanisms of m6A modification in restricting L1 retrotransposition.

Palmitoylation of vacuole membrane protein 1 promotes small extracellular vesicle secretion via interaction with ALIX and influences intercellular communication.

BACKGROUND: Small extracellular vesicles (EVs), exemplified by exosomes, mediate intercellular communication by transporting proteins, mRNAs, and miRNAs. Post-translational modifications are involved in controlling small EV secretion process. However, whether palmitoylation regulates small EV secretion, remains largely unexplored. METHODS: Vacuole Membrane Protein 1 (VMP1) was testified to be S-palmitoylated by Palmitoylation assays. VMP1 mutant plasmids were constructed to screen out the exact palmitoylation sites. Small EVs were isolated, identified and compared between wild-type VMP1 or mutant VMP1 transfected cells. Electron microscope and immunofluorescence were used to detect multivesicular body (MVB) number and morphology change when VMP1 was mutated. Immunoprecipitation and Mass spectrum were adopted to identify the protein that interacted with palmitoylated VMP1, while knock down experiment was used to explore the function of targeted protein ALIX. Taking human Sertoli cells (SCs) and human spermatogonial stem cell like cells (SSCLCs) as a model of intercellular communication, SSCLC maintenance was detected by flow cytometry and qPCR at 12 days of differentiation. In vivo, mouse model was established by intraperitoneal injection with palmitoylation inhibitor, 2-bromopalmitate (2BP) for 3 months. RESULTS: VMP1 was identified to be palmitoylated at cysteine 263,278 by ZDHHC3. Specifically, palmitoylation of VMP1 regulated its subcellular location and enhanced the amount of small EV secretion. Mutation of VMP1 palmitoylation sites interfered with the morphology and biogenesis of MVBs through suppressing intraluminal vesicle formation. Furthermore, inhibition of VMP1 palmitoylation impeded small EV secretion by affecting the interaction of VMP1 with ALIX, an accessory protein of the ESCRT machinery. Taking SCs and SSCLCs as a model of intercellular communication, we discovered VMP1 palmitoylation in SCs was vital to the growth status of SSCLCs in a co-culture system. Inhibition of VMP1 palmitoylation caused low self-maintenance, increased apoptosis, and decreased proliferation rate of SSCLCs. In vivo, intraperitoneal injection of 2BP inhibited VMP1 palmitoylation and exosomal marker expression in mouse testes, which were closely associated with the level of spermatogenic cell apoptosis and proliferation. CONCLUSIONS: Our study revealed a novel mechanism for small EV secretion regulated by VMP1 palmitoylation in Sertoli cells, and demonstrated its pivotal role in intercellular communication and SSC niche.

H9N2 Avian Influenza Virus Downregulates FcRY Expression in Chicken Macrophage Cell Line HD11 by Activating the JNK MAPK Pathway.

The H9N2 avian influenza virus causes reduced production performance and immunosuppression in chickens. The chicken yolk sac immunoglobulins (IgY) receptor (FcRY) transports from the yolk into the embryo, providing offspring with passive immunity to infection against common poultry pathogens. FcRY is expressed in many tissues/organs of the chicken; however, there are no reports investigating FcRY expression in chicken macrophage cells, and how H9N2-infected HD11 cells (a chicken macrophage-like cell line) regulate FcRY expression remains uninvestigated. This study used the H9N2 virus as a model pathogen to explore the regulation of FcRY expression in avian macrophages. FcRY was highly expressed in HD11 cells, as shown by reverse transcription polymerase chain reactions, and indirect immunofluorescence indicated that FcRY was widely expressed in HD11 cells. HD11 cells infected with live H9N2 virus exhibited downregulated FcRY expression. Transfection of eukaryotic expression plasmids encoding each viral protein of H9N2 into HD11 cells revealed that nonstructural protein (NS1) and matrix protein (M1) downregulated FcRY expression. In addition, the use of a c-jun N-terminal kinase (JNK) activator inhibited the expression of FcRY, while a JNK inhibitor antagonized the downregulation of FcRY expression by live H9N2 virus, NS1 and M1 proteins. Finally, a dual luciferase reporter system showed that both the M1 protein and the transcription factor c-jun inhibited FcRY expression at the transcriptional level. Taken together, the transcription factor c-jun was a negative regulator of FcRY, while the live H9N2 virus, NS1, and M1 proteins downregulated the FcRY expression through activating the JNK signaling pathway. This provides an experimental basis for a novel mechanism of immunosuppression in the H9N2 avian influenza virus.

The change of state-trait anxiety among patients undergoing orthognathic surgery: A longitudinal study.

INTRODUCTION: This study aimed to assess state-trait anxiety level changes in Chinese patients with dentofacial discrepancies before and after orthognathic surgery and to explore the feasibility of developing a reference index for the preoperative screening of postoperative patients with high anxiety. METHODS: A total of 96 Chinese patients with dentofacial discrepancies who underwent orthognathic surgery were included in this study. Data were collected before orthognathic surgery and at 2 weeks (T2), 3 months, and 6 months (T4) after surgery using the State-Trait Anxiety Inventory. Receiver operating characteristic and linear regression analyses were performed to screen for preoperative indicators of postoperative high-state anxiety. RESULTS: State-trait anxiety levels in patients with dentofacial discrepancies decreased after surgery (F = 18.95, P <0.01; F = 6.90, P <0.01). Trait Anxiety Inventory can be used to screen patients with high-state anxiety from T2 to T4 (area under cover 95% confidence interval: T2, 0.74 [0.62-0.86]; 3 months, 0.79 [0.69-0.90]; T4, 0.77 [0.66-0.87], P <0.01), corresponding to cutoff values of 48.5, 46.5, and 45.5, respectively. CONCLUSIONS: All participants' state-trait anxiety levels improved after surgery compared with their preoperative levels. Preoperative trait anxiety levels can be used as a reference indicator to screen patients who may have high-state anxiety levels after orthognathic surgery. The creation of a screening scale will assist health care professionals to more pertinently help patients with high anxiety.

[Mandibular condyle localization in orthognathic surgery based on mandibular movement trajectory and its preliminary accuracy verification].

OBJECTIVE: To establish and assess the precision of pre-surgical condyle position planning using mandibular movement trajectory data for orthognathic surgery. METHODS: Skull data from large-field cone beam computed tomography (CBCT) and dental oral scan data were imported into IVSPlan 1.0.25 software for 3D reconstruction and fusion, creating 3D models of the maxilla and mandible. Trajectory data of mandibular movement were collected using a mandibular motion recorder, and the data were integrated with the jaw models within the software. Subsequently, three-dimensional trajectories of the condyle were obtained through matrix transformations, rendering them visually accessible. A senior oral and maxillofacial surgeon with experience in both diagnosis and treatment of temporomandibular joint disease and orthognathic surgery selected the appropriate condyle position using the condyle movement trajectory interface. During surgical design, the mobile mandibular proximal segment was positioned accordingly. Routine orthognathic surgical planning was completed by determining the location of the mandibular distal segment, which was based on occlusal relationships with maxilla and facial aesthetics. A virtual mandible model was created by integrating data from the proximal and distal segment bone. Subsequently, a solid model was generated through rapid prototyping. The titanium plate was pre-shaped on the mandibular model, and the screw hole positions were determined to design a condylar positioning guide device. In accordance with the surgical plan, orthognathic surgery was performed, involving mandibular bilateral sagittal split ramus osteotomy (SSRO). The distal segment of the mandible was correctly aligned intermaxillary, while the proximal bone segment was positioned using the condylar positioning guide device and the pre-shaped titanium plate. The accuracy of this procedure was assessed in a study involving 10 patients with skeletal class Ⅱ malocclusion. Preoperative condyle location planning and intraoperative positioning were executed using the aforementioned techniques. CBCT data were collected both before the surgery and 2 weeks after the procedure, and the root mean square (RMS) distance between the preope-rative design position and the actual postoperative condyle position was analyzed. RESULTS: The RMS of the condyle surface distance measured was (1.59±0.36) mm (95%CI: 1.35-1.70 mm). This value was found to be significantly less than 2 mm threshold recommended by the expert consensus (P < 0.05). CONCLUSION: The mandibular trajectory may play a guiding role in determining the position of the mandibular proximal segment including the condyle in the orthognathic surgery. Through the use of a condylar positioning guide device and pre-shaped titanium plates, the condyle positioning can be personalized and customized with clinically acceptable accuracy.

Antitumor Effect of Apcin on Endometrial Carcinoma via p21-Mediated Cell Cycle Arrest and Apoptosis.

OBJECTIVE: Endometrial carcinoma (EC) is a prevalent gynecological malignancy characterized by increasing incidence and mortality rates. This underscores the critical need for novel therapeutic targets. One such potential target is cell division cycle 20 (CDC20), which has been implicated in oncogenesis. This study investigated the effect of the CDC20 inhibitor Apcin on EC and elucidated the underlying mechanism involved. METHODS: The effects of Apcin on EC cell proliferation, apoptosis, and the cell cycle were evaluated using CCK8 assays and flow cytometry. RNA sequencing (RNA-seq) was subsequently conducted to explore the underlying molecular mechanism, and Western blotting and coimmunoprecipitation were subsequently performed to validate the results. Animal studies were performed to evaluate the antitumor effects in vivo. Bioinformatics analysis was also conducted to identify CDC20 as a potential therapeutic target in EC. RESULTS: Treatment with Apcin inhibited proliferation and induced apoptosis in EC cells, resulting in cell cycle arrest. Pathways associated with apoptosis and the cell cycle were activated following treatment with Apcin. Notably, Apcin treatment led to the upregulation of the cell cycle regulator p21, which was verified to interact with CDC20 and consequently decrease the expression of downstream cyclins in EC cells. In vivo experiments confirmed that Apcin treatment significantly impeded tumor growth. Higher CDC20 expression was observed in EC tissue than in nonmalignant tissue, and increased CDC20 expression in EC patients was associated with shorter overall survival and progress free interval. CONCLUSION: CDC20 is a novel molecular target in EC, and Apcin could be developed as a candidate antitumor drug for EC treatment.

18F-FAPI PET/CT performs better in evaluating mediastinal and hilar lymph nodes in patients with lung cancer: comparison with 18F-FDG PET/CT.

BACKGROUND: The aim of this study was to evaluate the efficacy of fluorine 18 (18F) labeled fibroblast activation protein inhibitor (FAPI) in identifying mediastinal and hilar lymph node metastases and to develop a model to quantitatively and repeatedly identify lymph node status. METHODS: Twenty-seven patients with 137 lymph nodes were identified by two PET/CT images. The sensitivity, specificity, accuracy, positive predictive value (PPV), and negative predictive value (NPV) of lymph node status were analyzed, and the optimal cut-off value was identified by ROC analysis. RESULTS: The SUVmax of metastatic lymph nodes on 18F-FAPI was higher than that on 18F-FDG PET/CT (10.87 ± 7.29 vs 6.08 ± 5.37, p < 0.001). 18F-FAPI presented much greater lymph node detection sensitivity, specificity, accuracy, PPV and NPV than 18F-FDG PET/CT (84% vs. 71%; 92% vs. 67%; 90% vs. 69%, 84% vs. 52%, and 92% vs. 83%, respectively). Additionally, the diagnostic effectiveness of 18F-FAPI in small lymph nodes was greater than that of 18F-FDG PET/CT (specificity: 96% vs. 72%; accuracy: 93% vs. 73%; PPV: 77% vs. 33%, respectively). Notably, the optimal cut-off value for specificity and PPV of 18F-FAPI SUVmax was 5.3; the optimal cut-off value for sensitivity and NPV was 2.5. CONCLUSION: 18F-FAPI showed promising diagnostic efficacy in metastatic mediastinal and hilar lymph nodes from lung cancer patients, with a higher SUVmax, especially in small metastatic nodes, compared with 18F-FDG. In addition, this exploratory work recommended optimal SUVmax cutoff values to distinguish between nonmetastatic and metastatic lymph nodes, thereby advancing the development of image-guided radiation. Trial registration ClinicalTrials.gov identifier: ChiCTR2000036091.

Manipulating the processing window of directed self-assembly in contact hole shrinking with binary block copolymer/homopolymer blending.

Directed self-assembly (DSA) lithography has demonstrated significant potential in fabricating integrated circuits. However, DSA encounters limited processing windows due to the requirement for precise matching between the period of block copolymers (BCPs) and graphoepitaxy templates. We propose a binary BCP/homopolymer blending strategy to manipulate the self-assembly behavior and the processing window of graphoepitaxy DSA in contact hole shrinking. By carefully tailoring the blending rates of poly(methyl methacrylate) (PMMA) with different molecular weights in cylindrical polystyrene-b-poly(methyl methacrylate) (PS-b-PMMA), we manipulate the period and morphology of BCP/homopolymer self-assembly. Specifically, we employ BCP/homopolymer blending to fine-tune the critical dimension (CD) of contact holes with PS-affined topographical templates. Subsequent pattern transferring is achieved by selectively etching defect-free shrinkable cylinders as hard masks. Furthermore, self-consistent field theory (SCFT) simulation was employed to explore the self-assembly of BCP/homopolymer blending in confined cylindrical space and the results were in good consistency with the experimental results.

Properties of [18F]FAPI monitoring of acute radiation pneumonia versus [18F]FDG in mouse models.

OBJECTIVE: In this study, the uptake characteristics of [18F]fibroblast activation protein inhibitor (FAPI) molecular imaging probe were investigated in acute radiation pneumonia and lung cancer xenografted mice before and after radiation to assess the future applicability of [18F]FAPI positron emission tomography/computed tomography (PET/CT) imaging in early radiotherapy response. METHODS: Initially, the biodistribution of [18F]FAPI tracer in vivo were studied in healthy mice at each time-point. A comparison of [18F]FAPI and [18F]fluorodeoxyglucose (FDG) PET/CT imaging efficacy in normal ICR, LLC tumor-bearing mice was evaluated. A radiation pneumonia model was then investigated using a gamma counter, small animal PET/CT, and autoradiography. The uptake properties of [18F]FAPI in lung cancer and acute radiation pneumonia were investigated using autoradiography and PET/CT imaging in mice. RESULTS: The tumor area was visible in [18F]FAPI imaging and the tracer was swiftly eliminated from normal tissues and organs. There was a significant increase of [18F]FDG absorption in lung tissue after radiotherapy compared to before radiotherapy, but no significant difference of [18F]FAPI uptake under the same condition. Furthermore, both the LLC tumor volume and the expression of FAP-ɑ decreased after thorax irradiation. Correspondingly, there was no notable [18F]FAPI uptake after irradiation, but there was an increase of [18F]FDG uptake in malignancies and lungs. CONCLUSIONS: The background uptake of [18F]FAPI is negligible. Moreover, the uptake of [18F]FAPI may not be affected by acute radiation pneumonitis compared to [18F]FDG, which may be used to more accurately evaluate early radiotherapy response of lung cancer with acute radiation pneumonia.

Effects of pomegranate (Punica granatum L.) peel on the growth performance and intestinal microbiota of broilers challenged with Escherichia coli.

The effects of pomegranate peel on the growth performance, intestinal morphology, and the cecal microbial community were investigated in broilers challenged with avian pathogenic Escherichia coli (APEC) O78. A total of 240 one-day-old chicks (120 males and 120 females) were randomly and evenly allotted into 4 treatment groups (each with 6 biological replicates each of 10 chicks), i.e., negative control (NC), positive control (PC), and 2 experimental groups treated with 0.2% fermented pomegranate peel (FP) and 0.2% unfermented pomegranate peel (UFP), respectively, with PC, FP, and UFP groups challenged with APEC O78 (5 × 108 CFU) on day 14. Results showed that the challenge of APEC O78 decreased the body weight (BW) and average daily gain (ADG) of broilers from 1 to 28 d (P < 0.01). These broilers exhibited more pathological conditions in the heart and liver and higher mortality rates in 28 d compared to the NC group. Diet supplemented with pomegranate peel (either fermented or unfermented) significantly increased BW, ADG, and the villus height/crypt depth ratio (VCR) of small intestine in 28 d compared to the NC group (P < 0.05). Results of the taxonomic structure of the gut microbiota showed that compared to the NC group, the APEC challenge significantly decreased the relative abundance of Bacteroidetes and increased the relative abundance of Firmicutes (P < 0.01). Compared to the PC group, the relative abundance of Ruminococcus_torques_group in FP group was increased, while the relative abundance of Alistipes was decreased. In summary, our study showed that the dietary supplementation of pomegranate peel could maintain the intestinal microbiota at a state favorable to the host, effectively reduce the abnormal changes in the taxonomic structure of the intestinal microbiota, and improve the growth performance in broilers treated with APEC.