Serum Oncomarkers in Patients with MPO-ANCA-Positive Vasculitis: Diagnostic and Prognostic Predictive Values for Interstitial Lung Disease.

PURPOSE: To explore in myeloperoxidase-antineutrophil cytoplasmic autoantibody-associated vasculitis (MPO-AAV) the value of circulating oncomarkers in identifying interstitial lung disease (ILD) and predicting prognosis. METHODS: Newly diagnosed MPO-AAV patients were evaluated retrospectively at a single center. The serum levels of carbohydrate antigen (CA) 19-9, CA125, cytokeratin fraction 21-1 (CYFRA21-1), carcinoembryonic antigen, squamous cell carcinoma antigen, and neuron-specific enolase were compared between patients with and without ILD. The strength of the oncomarkers in identifying ILD was assessed through logistic regression and receiver operating characteristic (ROC) curves. Correlation analysis was applied to detect the associations between oncomarkers and ILD severity. The significance of serum oncomarkers as prognosis predictors for MPO-AAV associated ILD was evaluated by survival analysis. RESULTS: 169 MPO-AAV patients were included and ILD was found in 101 patients. Serum CA125, CA19-9, and CYFRA21-1 were significantly higher in patients with ILD than those without ILD. The area under the ROC curve of CA19-9, CA125, and CYFRA21-1 for identifying ILD was 0.701, 0.660, and 0.711, respectively. A specificity of 98.5% for diagnosing ILD was found for CA19-9 at the recommended normal level. CA19-9 was positively correlated with HRCT fibrosis score (r = 0.498, p < 0.001) and CYFRA21-1 was correlated with ground-glass score (r = 0.316, p = 0.002). Both CA19-9 and CYFRA21-1 were independent risk factors for all-cause mortality in patients with ILD. CONCLUSION: Serum CA19-9 and CYFRA21-1 might be useful markers in the diagnosis, disease severity evaluation, and prognosis prediction of MPO-AAV-associated ILD.

Clinical features of anti-synthetase syndrome associated interstitial lung disease: a retrospective cohort in China.

BACKGROUND: Anti-synthetase syndrome (ASSD) is a chronic autoimmune condition characterized by antibodies directed against an aminoacycl transfer RNA synthetase (ARS) along with a group of clinical features including the classical clinical triad: inflammatory myopathy, arthritis, and interstitial lung disease (ILD). ASSD is highly heterogenous due to different organ involvement, and ILD is the main cause of mortality and function loss, which presents as different patterns when diagnosed. We designed this retrospective cohort to describe the clinical features and disease behaviour of ASSD associated ILD. METHODS: Data of 108 cases of ASSD associated ILD were retrospectively collected in Beijing Chaoyang Hospital from December 2017 to March 2019. Data were obtained from the Electronic Medical Record system. Patients were divided into 5 groups according to distinct aminoacyl tRNA synthetase (ARS) antibodies. RESULTS: Overall, 108 consecutive patients were recruited. 33 were JO-1 positive, 30 were PL-7 positive, 23 were EJ positive, 13 were PL-12 positive and 9 were OJ positive. The JO-1 (+) group had a significant higher rate of mechanic's hand (57.6%) than other 4 groups. Polymyositis/dermatomyositis (PM/DM) was diagnosed in 25 (23.1%) patients and no difference was observed among the 5 groups. The PL-7 (+) group had a higher frequency of UIP pattern (13.3%) than the other 4 groups but the difference was not significant, and the EJ (+) group had the most frequent OP pattern (78.2%), which was significantly higher than the PL-7 (+) (P < 0.001) and PL-12 (+) groups (P = 0.025). The median follow-up time was 10.7 months, during which no patients died. All received prednisone treatment, with or without immunosuppressants. At the 6-month follow-up, 96.3% of all patients (104/108) had a positive response to therapy, the JO-1 (+) and EJ (+) groups had a significantly higher improvement of forced vital capacity than the other 3 groups (P < 0.05), and the PL-7 group had the lowest FVC improvement (P < 0.05). The JO-1 (+) group and EJ (+) group had significantly higher anti-Ro-52 positive occurrence than the other 3 groups (P < 0.05). CONCLUSION: Anti PL-7 antibody had the same frequency as anti-JO-1 in ASSD-ILD, in which the ILD pattern was different with distinct anti-ARS antibodies. Most ASSD-ILD had a positive response to steroid therapies, with or without immunosuppressants. The PL-7 (+) group had the highest occurrence of UIP pattern, and a significantly lower response to therapy.

Differential interactions of missing in metastasis and insulin receptor tyrosine kinase substrate with RAB proteins in the endocytosis of CXCR4.

Missing in metastasis (MIM), an inverse Bin-Amphiphysin-Rvs (I-BAR) domain protein, promotes endocytosis of C-X-C chemokine receptor 4 (CXCR4) in mammalian cells. In response to the CXCR4 ligand stromal cell-derived factor 1 (SDF-1 or CXCL12), MIM associates with RAS-related GTP-binding protein 7 (RAB7) 30 min after stimulation. However, RAB7's role in MIM function remains undefined. Here we show that RNAi-mediated suppression of RAB7 expression in human HeLa cells has little effect on the binding of MIM to RAB5 and on the recruitment of CXCR4 to early endosomes but effectively abolishes MIM-mediated CXCR4 degradation, chemotactic response, and sorting into late endosomes and lysosomes. To determine whether I-BAR domain proteins interact with RAB7, we examined cells expressing insulin receptor tyrosine kinase substrate (IRTKS), an I-BAR domain protein bearing an Src homology 3 (SH3) domain. We observed that both MIM and IRTKS interact with RAB5 at an early response to SDF-1 and that IRTKS binds poorly to RAB7 but strongly to RAB11 at a later time point. Moreover, IRTKS overexpression reduced CXCR4 internalization and enhanced the chemotactic response to SDF-1. Interestingly, deletion of the SH3 domain in IRTKS abolished the IRTKS-RAB11 interaction and promoted CXCR4 degradation. Furthermore, the SH3 domain was required for selective targeting of MIM-IRTKS fusion proteins by both RAB7 and RAB11. Hence, to the best of our knowledge, our results provide first evidence that the SH3 domain is critical in the regulation of specific endocytic pathways by I-BAR domain proteins.

Missing-in-metastasis protein downregulates CXCR4 by promoting ubiquitylation and interaction with small Rab GTPases.

Surface expression of chemokine receptor CXCR4 is downregulated by missing-in-metastasis protein (MIM; also known as MTSS1), a member of the inverse BAR (I-BAR)-domain protein family that recognizes and generates membranes with negative curvature. Yet, the mechanism for the regulation is unknown. Here, we show that MIM forms a complex with CXCR4 by binding to E3 ubiquitin ligase AIP4 (also known as ITCH) in response to stromal cell-derived factor 1 (SDF-1; also known as CXCL12). Overexpression of MIM promoted CXCR4 ubiquitylation, inhibited cellular response to SDF-1, caused accumulation and aggregation of multivesicular bodies (MVBs) in the cytoplasm, and promoted CXCR4 sorting into MVBs in a manner depending on binding to AIP4. In response to SDF-1, MIM also bound transiently to the small GTPase Rab5 at 5 min and to Rab7 at 30 min. Binding to Rab7 requires an N-terminal coiled-coil motif, deletion of which abolished MIM-mediated MVB formation and CXCR4 internalization. Our results unveil a previously unknown property of MIM that establishes the linkage of protein ubiquitylation with Rab-guided trafficking of CXCR4 in endocytic vesicles.

Mxd1 mediates hypoxia-induced cisplatin resistance in osteosarcoma cells by repression of the PTEN tumor suppressor gene.

Hypoxia-induced chemoresistance remains a major obstacle to treating osteosarcoma effectively. Mxd1, a member of the Myc/Max/Mxd family, was shown to be involved in the development of drug resistance under hypoxia. However, the effect of Mxd1 on hypoxia-induced cisplatin (CDDP) resistance and its mechanism in osteosarcoma have not been fully elucidated. In this study, we demonstrated that HIF-1α-induced Mxd1 contributed to CDDP resistance in hypoxic U-2OS and MG-63 cells. The knockdown of Mxd1 expression elevated PTEN expression at both protein and RNA levels in these hypoxic cells. Using Luciferase reporter and ChIP assays, we confirmed that Mxd1 directly bound to the E-box sites within the PTEN promoter region. We further demonstrated that PTEN knockdown decreased CDDP sensitivity in Mxd1 siRNA-transfected U-2OS and MG-63 cells under hypoxia. Our results also showed that Mxd1 deficiency in hypoxic U-2OS and MG-63 cells lead to inactivation of PI3K/AKT signaling, which is the downstream of PTEN. Furthermore, blockade of PI3K/AKT signal re-sensitized hypoxic U-2OS and MG-63 cells to CDDP. Taken together, these findings suggest that HIF-1α-induced Mxd1 up-regulation suppresses the expression of PTEN under hypoxia, which leads to the activation of PI3K/AKT antiapoptotic and survival pathway. As a result CDDP resistance in osteosarcoma cells is induced.

The SH3 domain distinguishes the role of I-BAR proteins IRTKS and MIM in chemotactic response to serum.

The family of inverse BAR (I-BAR) domain proteins participates in a range of cellular processes associated with membrane dynamics and consists of five distinct members. Three of the I-BAR proteins, including insulin receptor tyrosine kinase substrate (IRTKS), contain an SH3 domain near their C-termini. Yet, the function of the SH3 domain of IRTKS remains uncharacterized. Here we report that in contrast to MIM, which is a prototype of I-BAR proteins and does not contain an SH3 domain, IRTKS promoted serum-induced cell migration along with enhanced phosphorylation of mitogen activated kinases Erk1/2 and p38, and activation of small GTPases Rac1 and Cdc42. In addition, cells overexpressing IRTKS exhibited an increased polarity characterized by elongated cytoplasm and extensive lamellipodia at leading edges. However, a mutant with deletion of the SH3 domain attenuated both cellular motility and p38 phosphorylation but had little effect on Erk1/2 phosphorylation. Also, a chimeric mutant in which the N-terminal portion of MIM is fused with the C-terminal IRTKS, including the SH3 domain, was able to promote chemotactic response to serum and cellular polarity. In contrast, a chimeric mutant in which the N-terminal IRTKS is fused with the C-terminal MIM failed to do so. Furthermore, treatment of cells with SB203580, a selective inhibitor of p38, also neutralized the effect of IRTKS on cell migration. These data indicate that the SH3 domain distinguishes the function of IRTKS in promoting cell migration and inducing signal transduction from those of SH3-less I-BAR proteins.

Cryo-EM Visualization of Nanobubbles in Aqueous Solutions.

The detection of nanobubbles on surfaces is well established (e.g., AFM and optical microscopy methods), but currently no methods exist for the direct detection of bulk nanobubbles. Here, cryo-electron microscopy (cryo-EM) has been employed to observe bubbles in aqueous solutions for the first time. Nitrogen bubbles generated by a chemical reaction were observed in amorphous ice trapped between two carbon films. The cryo-EM images of bubbles showed the same features as predicted by theory. The fact that no bubbles were observed near an air-water interface suggests that bubbles may diffuse to the nearby air-water interface and escape. The estimate of the bubble diffusion coefficient is about 30-250 µm2/s.

Proteomic analysis of solid pseudopapillary tumor of the pancreas reveals dysfunction of the endoplasmic reticulum protein processing pathway.

Solid pseudopapillary tumor of the pancreas (SPTP) is a low-grade malignant tumor with a favorable prognosis after surgery. Many previous studies have focused on clinical features or pathological biomarkers of the disease, but a better understanding of the molecular mechanisms underlying SPTP may help guide future therapeutic strategies. Here, we used isobaric tags for relative and absolute quantitation (iTRAQ) technology integrated with liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis to identify differentially expressed proteins in SPTP specimens. A total of 1171 proteins with a threshold of a 1.5-fold change and a p value ≤ 0.05 between SPTP tissue and matched normal pancreas tissue were identified for bioinformatics analysis. Mass spectrometry results were then further confirmed by assessing six representative proteins (ACADL, EPHX2, MSI2, DKK4, JUP, and DAD1) in individual specimens with immunohistochemistry. Upon mapping of the differentially expressed proteins to the Kyoto Encyclopedia of Genes and Genomes pathways database, we found several new cell-adhesion molecules that could be used as pathologic biomarkers. Furthermore, we observed that many endoplasmic reticulum-associated proteins were altered, suggesting that endoplasmic reticulum stress may play an important role in SPTP tumorigenesis. Seven proteins (ERO1LB, TRIM1, GRP94, BIP, SEC61B, P4HB, and PDIA4) in this pathway were further validated by immunohistochemistry, and six of them (except SEC61B) coincided to the LC-MS/MS results. This first comprehensive analysis of the SPTP proteome confirms proteins that have been implicated in earlier reports and reveals novel candidates and pathways that could be investigated further for clinical applications.

Controlled Endolysosomal Release of Agents by pH-responsive Polymer Blend Particles.

PURPOSE: A key step of delivering extracellular agents to its intracellular target is to escape from endosomal/lysosomal compartments, while minimizing the release of digestive enzymes that may compromise cellular functions. In this study, we examined the intracellular distribution of both fluorecent cargoes and enzymes by a particle delivery platform made from the controlled blending of poly(lactic-co-glycolic acid) (PLGA) and a random pH-sensitive copolymer. METHODS: We utilized both microscopic and biochemical methods to semi-quantitatively assess how the composition of blend particles affects the level of endosomal escape of cargos of various sizes and enzymes into the cytosolic space. RESULTS: We demonstrated that these polymeric particles enabled the controlled delivery of cargos into the cytosolic space that was more dependent on the cargo size and less on the composition of blend particles. Blend particles did not induce the rupture of endosomal/lysosomal compartments and released less than 20% of endosomal/lysosomal enzymes. CONCLUSIONS: This study provides insight into understanding the efficacy and safety of a delivery system for intracellular delivery of biologics and drugs. Blend particles offer a potential platform to target intracellular compartments while potentially minimizing cellular toxicity.

Inhibiting CB1 receptors improves lipogenesis in an in vitro non-alcoholic fatty liver disease model.

BACKGROUND: The endocannabinoids system (ECs) mediated mainly by CB1 and CB2 receptors plays an important role in non-alcoholic fatty liver disease by regulating lipid metabolism. This study is to further investigate the expression of CB1 and CB2 in the fat accumulation liver cells and to identify possible underlying mechanism by detecting the key lipogenesis factors. METHODS: Sodium oleate and sodium palmitate were added into the HepG2 cell line for forming fat accumulation liver cell. MTT assay was used to test the cell's cytotoxicity. The accumulation rate of fat in HepG2 cell was analyzed by the fluorescent staining. The mRNA and protein expression levels of CB1, CB2, SREBP-1c, ChREBP, L-PK, ACC1, FAS, LXRs and RXR were detected by RT-PCR and Western blot before and after the use of the antagonist. RESULTS: The receptors of CB1 were expressed in HepG2 cells with low levels while in HepG2 fatty liver cells with higher levels (p < 0.05). However, after the application of antagonist, the expressions were significantly decreased (p < 0.05). The expressions of SREBP-1c, ChREBP and LXRs were detectable in HepG2 cells and the expressions were increased in HepG2 fatty liver cells (p < 0.05). After using the antagonists, the expressions of SREBP-1c, ChREBP, LXRs, ACC1 and FAS were significantly decreased (p < 0.05). But L-PK and RXR changed little in two groups (p > 0.05). CONCLUSION: Results of the present study demonstrated that CB1 receptors had important pathophysiological effects on the formation of fatty liver. CB1 receptors could be regulated by SREBP-1c, ChREBP and LXRs. Therefore, targeting CB1 receptors for the treatment of NAFLD might have a potential application value.

[Protein interaction site of Toxoplasma gondii microneme protein 6 and aldolase determined by site-directed mutagenesis].

OBJECTIVE: To identify the protein interaction site of Toxoplasma gondii microneme protein 6 (MIC6) and aldolase by using site-directed mutagenesis. METHODS: Based on Toxoplasma gondii MIC6 gene sequence (GenBank Accession No. AF110270), the specific primers were designed. Tryptophan (W)-348 of MIC6 C terminus (MIC6C) was mutated to valine (V) via site-directed mutagenesis. MIC6C W/V gene was obtained from cDNA library by PCR amplification and subcloned into pGEX-4T-1. The mutant protein GST-MIC6C W/V was expressed in E. coli, induced by 0.8 mmol/L IPTG, and purified by affinity chromatography. Glutathione sepharose beads were incubated with GST-MIC6C W/V and GST-MIC6C, respectively, and then incubated with T. gondii tachyzoites lysate, and bound proteins were eluted using sample buffer. Bound products were resolved by SDS-PAGE and Western blotting. Glutathione sepharose beads were incubated with GST-MIC6C W/V and GST-MIC6C, respectively, and then incubated with aldolase-His6. After incubation, the resin was washed and subjected to SDS-PAGE. RESULTS: The MIC6C W/N gene was obtained, and the recombinant plasmid MIC6C W/V/pGEX-4T-1 was successfully constructed. The mutant protein GST-MIC6C W/V was expressed and purified in vitro. SDS-PAGE analysis indicated that GST-MIC6C was co-precipitated with aldolase from T. gondii tachyzoites lysate or aldolase-His6, whereas GST-MIC6C W/V failed to precipitate aldolase from T. gondii tachyzoites lysate or aldolase-His6. Western blotting analysis using anti-aldolase antibody indicated that GST-MIC6C could pull-down aldolase from T. gondii tachyzoites lysate. CONCLUSION: Tryptophan (W348) was the interaction site of MIC6 and aldolase in T. gondii.

Comparison of the safety profiles for pirfenidone and nintedanib: a disproportionality analysis of the US food and drug administration adverse event reporting system.

Background: Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive interstitial lung disease of unknown etiology. Pirfenidone (PFD) and nintedanib (NDN) were both conditionally recommended in the clinical practice guideline published in 2015. Safety and tolerability are related to the risk of treatment discontinuation. Therefore, this study evaluated and compared the adverse events (AEs) of PFD and NDN in a large real-world population by analyzing data from the FDA Adverse Event Reporting System (FAERS) to provide a reference for their rational and safe use. Methods: The AEs of PFD and NDN were extracted from the FAERS database. The pharmacovigilance online analysis tool OpenVigil 2.1 was used to retrieve data from the FAERS database from the first quarter of 2012 to the second quarter of 2022. The reporting odds ratio (ROR) and proportional reporting ratio were used to detect the risk signals. Results: The database included 26,728 and 11,720 reports for PFD and NDN, respectively. The most frequent AEs of PFD and NDN were gastrointestinal disorders. The RORs for these drugs were 5.874 and 5.899, respectively. "Cardiac disorders" was the most statistically significant system order class for NDN with an ROR of 9.382 (95% confidence interval = 8.308-10.594). Furthermore, the numbers of designated medical events of PFD and NDN were 552 and 656, respectively. Notably, liver injury was reported more frequently for NDN (11.096%) than for PFD (6.076%). Conclusion: This study revealed differences in the reporting of AEs between PFD and NDN. The findings provide reference for physicians in clinical practice. Attention should be paid to the risks of cardiac disorders and liver injury associated with NDN.

Structural characterization and anti-oxidative activity for a glycopeptide from Ganoderma lucidum fruiting body.

A water-soluble glycopeptide (named GL-PWQ3) with a molecular weight (Mw) of 2.40 × 104 g/mol was isolated from Ganoderma lucidum fruiting body by hot water extraction, membrane ultrafiltration, and gel column chromatography, which mainly consisted of glucose and galactose. Based on the methylation, FT-IR, 1D, and 2D NMR analysis, the polysaccharide portion of GL-PWQ3 was identified as a glucogalactan, which was comprised of unsubstituted (1,6-α-Galp, 1,6-ß-Glcp, 1,4-ß-Glcp) and monosubstituted (1,2,6-α-Galp and 1,3,6-ß-Glcp) in the backbone and possible branches that at the O-3 position of 1,3-Glcp and T-Glcp, and the O-2 position of T-Fucp, T-Manp or T-Glcp. The chain conformational study by SEC-MALLS-RI and AFM revealed that GL-PWQ3 was identified as a highly branched polysaccharide with a polydispersity index of 1.25, and might have compact sphere structures caused by stacked multiple chains. Moreover, the GL-PWQ3 shows strong anti-oxidative activity in NRK-52E cells. This study provides a theoretical basis for further elucidating the structure-functionality relationships of GL-PWQ3 and its potential application as a natural antioxidant in pharmacotherapy as well as functional food additives.

Fibronectin-mediated cell spreading requires ABBA-Rac1 signaling.

ABBA was reported to be an actin dynamics regulator. However, the molecular mechanism of action of ABBA is still totally obscure. Here, we show that ABBA is ubiquitously expressed in all the examined cultured cells. We found that expression of ABBA in NIH3T3 cells promotes cell spreading. ABBA binds to and markedly promotes cell spreading-induced Rac1 activation. Cell spreading stimulates ABBA activation probably by inducing it tyrosine phosphorylation, which endows ABBA much higher activity to activate Rac1, and attenuates the interaction between ABBA and Rac1. Loss of function suggests that deletion of ABBA in C6-R cells markedly inhibits Rac1 activation and cell spreading; this suggests that and the interaction between ABBA and activated Rac1 is required for ABBA-promoted cell spreading. Taken together, our results indicate that ABBA is activated in response to cell spreading, which markedly promotes cell spreading, and ABBA is required for Rac1 activation and cell spreading.

Genotyping of Pneumocystis jirovecii isolates from Chinese HIV-infected patients based on nucleotide sequence variations in the internal transcribed spacer regions of rRNA genes.

Genetic diversity of Pneumocystis jirovecii isolates based on internal transcribed spacer (ITS) of the nuclear rRNA locus has previously been reported. The information about ITS genotype and epidemiology of this organism in Chinese human immunodeficiency virus-infected patients has not been available. In this study, 12 bronchoalveolar lavage fluid specimens obtained from HIV-infected patients were analyzed by PCR followed by cloning, sequencing and typing. Three ITS1 genotypes (E, B and 'H') and four ITS2 genotypes (b, g, i and r) as previously reported were identified, the most common of which were E, b and i. Five ITS haplotypes (Eg, Eb, Bi, Er and 'H'r) and 19 new combination types were also identified with the most common types being Eg (four of 12 patients, 10 of 60 clones), Eb (three of 12 patients, 11 of 60 clones) and Bi (three of 12 patients, 10 of 60 clones). Nine patients were found to be co-infected with more than one ITS genotype of P. jirovecii. The prevalence of ITS genotypes in HIV patients from one Chinese hospital did not seem to be significantly different when compared to reports from other countries.

Dimerization is necessary for MIM-mediated membrane deformation and endocytosis.

MIM [missing in metastasis; also called MTSS1 (metastasis suppressor 1)] is an intracellular protein that binds to actin and cortactin and has an intrinsic capacity to sense and facilitate the formation of protruded membranous curvatures implicated in cellular polarization, mobilization and endocytosis. The N-terminal 250 amino acids of MIM undergo homodimerization and form a structural module with the characteristic of an I-BAR [inverse BAR (Bin/amphiphysin/Rvs)] domain. To discern the role of the dimeric configuration in the function of MIM, we designed several peptides able to interfere with MIM dimerization in a manner dependent upon their lengths. Overexpression of one of the peptides effectively abolished MIM-mediated membrane protrusions and transferrin uptake. However, a peptide with a high potency inhibiting MIM dimerization failed to affect its binding to actin and cortactin. Thus the results of the present study indicate that the dimeric configuration is essential for MIM-mediated membrane remodelling and serves as a proper target to develop antagonists specifically against an I-BAR-domain-containing protein.

Predicting mini-tablet dissolution performance utilizing X-ray computed tomography.

Mini-tablets (MTs) have been utilized as an alternative to monolithic tablets due to their ease of use for pediatric populations, dose flexibility and tailoring of drug release profiles. Similar to monolithic tablets, MTs can develop film coat and internal core defects during manufacturing processes that may adversely affect their dissolution performance. The use of x-ray computed microtomography (XRCT) is well documented for monolithic tablets as a means of identifying internal defects, but applications to MTs have not been well studied. In this study, we have developed a workflow that analyzes reconstructed XRCT images of enteric-coated mini-tablets using deep learning convolutional neural networks. This algorithm was utilized to extract key physical features of individual MTs, such as micro-crack volume and enteric coat thickness. By performing dissolution studies on individual MTs, correlations were established based on the physical parameters obtained by XRCT and the dissolution performance, enabling prediction of dissolution performance utilizing non-destructive imaging data. This workflow provides insight into the physical variability of MT populations that are generated during manufacturing, enabling optimization of critical tableting and coating parameters to achieve the target dissolution criteria. Through this mechanistic understanding, quality is built into the final drug product through rational development of formulation and process parameters.

Automatic Identification and Segmentation of Orbital Blowout Fractures Based on Artificial Intelligence.

Purpose: The incidence of orbital blowout fractures (OBFs) is gradually increasing due to traffic accidents, sports injuries, and ocular trauma. Orbital computed tomography (CT) is crucial for accurate clinical diagnosis. In this study, we built an artificial intelligence (AI) system based on two available deep learning networks (DenseNet-169 and UNet) for fracture identification, fracture side distinguishment, and fracture area segmentation. Methods: We established a database of orbital CT images and manually annotated the fracture areas. DenseNet-169 was trained and evaluated on the identification of CT images with OBFs. We also trained and evaluated DenseNet-169 and UNet for fracture side distinguishment and fracture area segmentation. We used cross-validation to evaluate the performance of the AI algorithm after training. Results: For fracture identification, DenseNet-169 achieved an area under the receiver operating characteristic curve (AUC) of 0.9920 ± 0.0021, with an accuracy, sensitivity, and specificity of 0.9693 ± 0.0028, 0.9717 ± 0.0143, and 0.9596 ± 0.0330, respectively. DenseNet-169 realized the distinguishment of the fracture side with accuracy, sensitivity, specificity, and AUC of 0.9859 ± 0.0059, 0.9743 ± 0.0101, 0.9980 ± 0.0041, and 0.9923 ± 0.0008, respectively. The intersection over union (IoU) and Dice coefficient of UNet for fracture area segmentation were 0.8180 ± 0.0093 and 0.8849 ± 0.0090, respectively, showing a high agreement with manual segmentation. Conclusions: The trained AI system could realize the automatic identification and segmentation of OBFs, which might be a new tool for smart diagnoses and improved efficiencies of three-dimensional (3D) printing-assisted surgical repair of OBFs. Translational Relevance: Our AI system, based on two available deep learning network models, could help in precise diagnoses and accurate surgical repairs.

Elevated neutrophil extracellular traps by HBV-mediated S100A9-TLR4/RAGE-ROS cascade facilitate the growth and metastasis of hepatocellular carcinoma.

BACKGROUND: Neutrophil extracellular traps (NETs) are considered significant contributors to cancer progression, especially metastasis. However, it is still unclear whether NETs are involved in hepatitis B virus (HBV)-related hepatocarcinogenesis and have potential clinical significance during evaluation and management for hepatocellular carcinoma (HCC). In this study, we aimed to investigate the functional mechanism of NETs in HBV-related hepatocarcinogenesis and their clinical significance. METHODS: A total of 175 HCC patients with and without HBV infection and 58 healthy controls were enrolled in this study. NETs were measured in tissue specimens, freshly isolated neutrophils and blood serum from these patients, and the correlation of circulating serum NETs levels with malignancy was evaluated. The mechanism by which HBV modulates NETs formation was explored using cell-based studies. In addition, in vitro and in vivo experiments were further performed to clarify the functional mechanism of NETs on the growth and metastasis of HCC. RESULTS: We observed an elevated level of NETs in blood serum and tissue specimens from HCC patients, especially those infected with HBV. NETs facilitated the growth and metastasis of HCC both in vitro and in vivo, which were mainly dominated by increased angiogenesis, epithelial-mesenchymal transition (EMT)-related cell migration, matrix metalloproteinases (MMPs)-induced extracellular matrix (ECM) degradation and NETs-mediated cell trapping. Inhibition of NETs generation by DNase 1 effectively abrogated the NETs-aroused HCC growth and metastasis. In addition, HBV-induced S100A9 accelerated the generation of NETs, which was mediated by activation of toll-like receptor (TLR4)/receptor for advanced glycation end products (RAGE)-reactive oxygen species (ROS) signaling. Further, circulatory NETs were found to correlate with viral load, TNM stage and metastasis status in HBV-related HCC, and the identified NETs could predict extrahepatic metastasis, with an area under the ROC curve (AUC) of 0.83 and 90.3% sensitivity and 62.8% specificity at a cutoff value of 0.32. CONCLUSIONS: Our findings indicated that activation of RAGE/TLR4-ROS signaling by HBV-induced S100A9 resulted in abundant NETs formation, which subsequently facilitated the growth and metastasis of HCC cells. More importantly, the identified circulatory NETs exhibited potential as an alternative biomarker for predicting extrahepatic metastasis in HBV-related HCC.

A cryo-EM structure of KTF1-bound polymerase V transcription elongation complex.

De novo DNA methylation in plants relies on transcription of RNA polymerase V (Pol V) along with KTF1, which produce long non-coding RNAs for recruitment and assembly of the DNA methylation machinery. Here, we report a cryo-EM structure of the Pol V transcription elongation complex bound to KTF1. The structure reveals the conformation of the structural motifs in the active site of Pol V that accounts for its inferior RNA-extension ability. The structure also reveals structural features of Pol V that prevent it from interacting with the transcription factors of Pol II and Pol IV. The KOW5 domain of KTF1 binds near the RNA exit channel of Pol V providing a scaffold for the proposed recruitment of Argonaute proteins to initiate the assembly of the DNA methylation machinery. The structure provides insight into the Pol V transcription elongation process and the role of KTF1 during Pol V transcription-coupled DNA methylation.