The Biodistribution of Replication-Defective Simian Adenovirus 1 Vector in a Mouse Model.

The administration route affects the biodistribution of a gene transfer vector and the expression of a transgene. A simian adenovirus 1 vector carrying firefly luciferase and GFP reporter genes (SAdV1-GFluc) were constructed, and its biodistribution was investigated in a mouse model by bioluminescence imaging and virus DNA tracking with real-time PCR. Luciferase activity and virus DNA were mainly found in the liver and spleen after the intravenous administration of SAdV1-GFluc. The results of flow cytometry illustrated that macrophages in the liver and spleen as well as hepatocytes were the target cells. Repeated inoculation was noneffective because of the stimulated serum neutralizing antibodies (NAbs) against SAdV-1. A transient, local expression of low-level luciferase was detected after intragastric administration, and the administration could be repeated without compromising the expression of the reporter gene. Intranasal administration led to a moderate, constant expression of a transgene in the whole respiratory tract and could be repeated one more time without a significant increase in the NAb titer. An immunohistochemistry assay showed that respiratory epithelial cells and macrophages in the lungs were transduced. High luciferase activity was restricted at the injection site and sustained for a week after intramuscular administration. A compromised transgene expression was observed after a repeated injection. When these mice were intramuscularly injected for a third time with the human adenovirus 5 (HAdV-5) vector carrying a luciferase gene, the luciferase activity recovered and reached the initial level, suggesting that the sequential use of SAdV-1 and HAdV-5 vectors was practicable. In short, the intranasal inoculation or intramuscular injection may be the preferred administration routes for the novel SAdV-1 vector in vaccine development.

Redirect Tropism of Fowl Adenovirus 4 Vector by Modifying Fiber2 with Variable Domain of Heavy-Chain Antibody.

The variable domain of a heavy-chain antibody (VHH) has the potential to be used to redirect the cell tropism of adenoviral vectors. Here, we attempted to establish platforms to simplify the screening of VHHs for their specific targeting function when being incorporated into the fiber of adenovirus. Both fowl adenovirus 4 (FAdV-4) and simian adenovirus 1 (SAdV-1) have two types of fiber, one of which is dispensable for virus propagation and is a proper site for VHH display. An intermediate plasmid, pMD-FAV4Fs, was constructed as the start plasmid for FAdV-4 fiber2 modification. Foldon from phage T4 fibritin, a trigger for trimerization, was employed to bridge the tail/shaft domain of fiber2 and VHHs against human CD16A, a key membrane marker of natural killer (NK) cells. Through one step of restriction-assembly, the modified fiber2 was transferred to the adenoviral plasmid, which was linearized and transfected to packaging cells. Five FAdV-4 viruses carrying the GFP gene were finally rescued and amplified, with three VHHs being displayed. One recombinant virus, FAdV4FC21-EG, could hardly transduce human 293 or Jurkat cells. In contrast, when it was used at a multiplicity of infection of 1000 viral particles per cell, the transduction efficiency reached 51% or 34% for 293 or Jurkat cells expressing exogenous CD16A. Such a strategy of fiber modification was transplanted to the SAdV-1 vector to construct SAdV1FC28H-EG, which moderately transduced primary human NK cells while the parental virus transduced none. Collectively, we reformed the strategy of integrating VHH to fiber and established novel platforms for screening VHHs to construct adenoviral vectors with a specific tropism.

Chronic Sacral Nerve Stimulation Inhibits Visceral Hypersensitivity in Diarrhea-Predominant Irritable Bowel Syndrome Rats Model.

OBJECTIVE: Sacral nerve stimulation (SNS) is emerging as a novel treatment for irritable bowel syndrome (IBS). However, its effects are limited, and the underlying mechanisms remain largely unknown. MATERIALS AND METHODS: In this study, rats were divided into three groups (n = 12 rats per group): 1) the SNS group; 2) the sham SNS group (the sham group for short); and 3) the control group. The SNS and sham groups were exposed to chronic and acute stress to establish an IBS model. Electrode implantation surgery was performed in rats with the IBS model. The SNS group received electrical stimulation for 30 minutes every day for seven days. Abdominal withdrawal reflex (AWR) was used to evaluate the effect of SNS on visceral sensitivity in diarrhea-predominant IBS (IBS-D) rats. The frequency domain of heart rate variability (HRV) was analyzed to assess the effect of SNS on regulating the autonomic function. The expression of transient receptor potential vanilloid 1 (TRPV1) in the colon, spinal cord, and hippocampus was detected by immunohistochemistry to explore the mechanism of SNS in IBS-D rats. RESULTS: Compared with the sham group, AWR scores were significantly decreased under different gas volumes of stimulation of 0.4, 0.6, and 0.8 ml for rectal distention in the SNS group (all p < 0.05). However, there was no significant difference <1.0 ml between the two groups (p > 0.05). Compared with the sham group, the frequency domain indexes of HRV were significantly altered. Normalized low-frequency power and low frequency-to-high frequency ratio were significantly decreased, and normalized high-frequency power was significantly increased in the SNS group (all p < 0.05). Moreover, the expression of TRPV1 in the spinal cord and colon in the SNS group was significantly decreased compared with the sham group (both p < 0.05). These results suggested that chronic SNS not only improved the visceral sensitivity and autonomic dysfunction but also decreased the expression of TRPV1 in the spinal cord-gut tissue in IBS-D rats. CONCLUSION: Chronic SNS was found to have an inhibitory effect on visceral hypersensitivity in IBS-D rats, providing experimental evidence for its potential clinical application in IBS.

The association of chest computed tomography-defined visual emphysema and prognosis in patients with nonsmall cell lung cancer.

Background: Although computed tomography (CT)-defined emphysema is considered a predictor of lung cancer risk, it is not fully clear whether CT-defined emphysema is associated with the prognosis of lung cancer. We aimed to assess the clinical impact of CT-defined emphysema on the survival of lung cancer. Methods: In the prospective cohort study of nonsmall cell lung cancer (NSCLC), the correlation between CT-defined emphysema and clinical variables was analysed. A multivariable Cox regression model was built to assess the association between CT-defined emphysema and overall survival (OS) for up to 8.8 years. The differences in survival analyses were derived by Kaplan-Meier analysis and log-rank testing. Low attenuation area (LAA%) was defined as the per cent of voxels below -950 HU. Results: 854 patients were included and CT-defined emphysema was present in 300 (35.1%) at diagnosis. Epidermal growth factor receptor (EGFR) wild-type (OR 1.998; p<0.001) and anaplastic lymphoma kinase (ALK) wild-type (OR 2.277; p=0.004) were associated with CT-defined emphysema. CT-defined emphysema remained a significant predictor of prognosis adjusting for age, sex, smoking history, tumour histology and Eastern Cooperative Oncology Group Performance Status (ECOG PS), whether in I-IIIA stage (adjusted hazard ratio (HR) 1.745; p=0.017) or in IIIB-IV stage (adjusted HR 1.291; p=0.022). Stratified analyses showed that OS rate among the driver oncogene groups with different CT-defined emphysema status differed significantly (log-rank p<0.001). Furthermore, patients with centrilobular emphysema (CLE) with LAA% >17% displayed poorer survival than those with LAA% ≤17% (median 432 versus 670 days; HR 1.564; p=0.020). Conclusions: CT-defined emphysema, especially CLE with LAA%>17%, is an independent predictor of NSCLC prognosis. Moreover, prospective studies are needed to further explore this association.

Effects of pre-operative biopsy on recurrence and survival in stage I lung adenocarcinoma patients in China.

Background: Whether pre-operative biopsy affects post-operative recurrence and metastasis of lung cancer patients is still controversial. Methods: In order to clarify these disputes, we collected relevant literature to conduct a meta-analysis. To validate the results of the meta-analysis, we retrospectively analysed 575 patients with stage I lung adenocarcinoma who underwent surgical resection at our centre from 2010 to 2018 using propensity score matching and competing risk models. Results: 5509 lung cancer patients from 11 articles were included in the meta-analysis. Summary analysis showed that the total recurrence rate of the biopsy group was higher than that of the nonbiopsy group (risk ratio 1.690, 95% CI 1.220-2.330; p=0.001). After propensity score matching, we found that there was no significant correlation between biopsy and total recurrence (risk ratio 1.070, 95% CI 0.540-2.120; p=0.850). In our cohort, of 575 stage I lung adenocarcinomas, 113 (19.7%) patients underwent pre-operative biopsy. During a median (interquartile range) follow-up of 71 (57-93) months, multivariable analyses showed pre-operative biopsy in the overall observation cohort (subdistribution hazard ratio (SHR) 1.522, 95% CI 0.997-2.320; p=0.051) and in the propensity score-matched cohort (SHR 1.134, 95% CI 0.709-1.810; p=0.600) was not significantly correlated with the risk of recurrence and metastasis. Moreover, the pre-operative biopsy did not affect disease-free survival (SHR 0.853, 95% CI 0.572-1.273; p=0.438) or overall survival (SHR 0.647, 95% CI 0.352-1.189; p=0.161). Conclusion: Pre-operative biopsy might not increase the risk of recurrence and metastasis, suggesting that these procedures might be safe for patients with stage I lung adenocarcinoma whose diagnosis is difficult to determine before surgery.

The interaction and physicochemical properties of the starch-polyphenol complex: Polymeric proanthocyanidins and maize starch with different amylose/amylopectin ratios.

This study investigated the impact of polymeric proanthocyanidins (PPC) on the physicochemical characteristics of maize starch with varying amylose content, and their potential interaction mechanism. PPC with a lower content (1 %) reduced the viscoelasticity of the high amylose maize starch (HAM) system, inhibited amylose rearrangement, and enhanced its fluidity. However, excessive PPC restrained the interaction between PPC and amylose. In contrast to HAM, PPC improved the gelation ability of waxy maize starch (WAM) as PPC concentration was raised. PPC suppressed the recrystallization of starch during storage, and PPC had a superior inhibition influence on the retrogradation of WAM in comparison to HAM. This indicated that amylopectin was more likely to interact with PPC than amylose. Hydrogen bonds were the main driving force between PPC and starch chains, which was clarified by Fourier transform-infrared, nuclear magnetic resonance, X-ray diffraction, iodine bonding reaction, and dynamic light scattering data. Additionally, the mechanism of interaction between PPC and the two starch components may be similar, and variance in physicochemical attributes can be primarily credited to the percentage of amylose to amylopectin in starch.

Echocardiographic Characteristics of Pulmonary Artery Intimal Sarcoma: Comparison With CTPA.

OBJECTIVES: This study examined the echocardiographic characteristics of patients with pulmonary artery intimal sarcoma (PAIS) and compared the results with those from computed tomographic pulmonary angiography (CTPA). METHOD: Twenty-six (26) patients were diagnosed with PAIS at the current institution during the study period, and 23 were eligible for analysis. Echocardiography and CTPA examinations were performed in all enrolled patients. RESULTS: The echocardiography results showed that most lesions had expansive growth in the left pulmonary artery (PA); the right PA; or a combination of the left PA, right PA, and main PA, with extension to the pulmonary valve and/or right ventricular outflow tract. These lesions also had distinctive sieve-like echogenic signals. Echocardiography also showed that some lesions had lobulated shapes, were nearly round and echolucent or with calcifications, and moved during imaging. The lesion distribution was similar in CTPA and echocardiography (p=0.361), but CTPA was more sensitive in detection of the complete shape (p=0.023). CONCLUSIONS: The unique echocardiographic characteristics of PAIS, especially the "sieve sign", could help in the diagnosis of this cancer. Transthoracic echocardiography is a non-invasive technique that appears effective in detecting PAIS.

Left ventricular strain in patients with Takayasu arteritis with preserved ejection fraction: an analysis using cardiac magnetic resonance imaging feature tracking.

Background: The alteration of myocardial strain in patients with Takayasu arteritis (TAK) remains unclear. This study aimed to evaluate left ventricular (LV) stain in patients with TAK and preserved left ventricular ejection fraction (pLVEF) using cardiac magnetic resonance imaging feature tracking (CMR-FT) to analyze risk factors for impaired LV strain and to compare the baseline difference of LV strain between patients with reduced and nonreduced LVEF at 6-month follow-up. Methods: In all, 51 patients with TAK and 30 healthy controls were prospectively enrolled. All participants underwent multiple short- and long-axis cine scans with true fast imaging with steady-state precession sequence. In this observational study, LV global and regional longitudinal, circumferential, and radial strain and their strain rates were analyzed with FT on cine images. The relationship between LV strain and clinical data was explored. The baseline LV strain between patients with TAK and reduced and nonreduced LVEF was compared using transthoracic echocardiography (TTE) at the 6-month follow-up. Results: Patients with TAK with pLVEF showed a decline in baseline global longitudinal peak strain (GLS) [TAK (-13.35%±3.11%) vs. controls (-14.77%±1.74%), P=0.021] and circumferential peak strain (GCS) [TAK (-21.46%±2.66%) vs. controls (-22.75%±2.57%), P=0.027] in comparison with normal controls. The longitudinal peak strain (LPS) in the apical (P=0.003) and midventricular regions (P=0.027) and the circumferential peak strain (CPS) in the basal (P=0.021) and midventricular regions (P=0.008) also decreased in patients with TAK. Patients with pulmonary hypertension (PH) or myocardial late gadolinium enhancement (LGE) showed a greater reduction in strain compared with those without PH or LGE. GLS showed a negative association with erythrocyte sedimentation rate (ESR), while GCS showed a positive association with disease duration. In the 30 patients who were followed up, the baseline global and apical circumferential diastolic peak strain rates (DPSR) in patients with reduced LVEF were higher than those in patients without reduced LVEF. Conclusions: In patients with TAK and pLVEF, CMR-FT indicated that both global and segmental myocardial strain decreased. PH, male gender, long disease duration, elevated ESR, and myocardial LGE were associated with declined LV strain. Baseline increased circumferential DPSR may be associated with the decline in LVEF during follow-up.

Delayed contrast-enhanced magnetic resonance imaging enables detection of pulmonary artery lesions in Takayasu's arteritis.

Background: Delayed contrast-enhanced magnetic resonance imaging (DE-MRI) is a useful technique to identify arterial wall inflammation. The aim of this study was to explore the value of DE-MRI in the evaluation of pulmonary artery (PA) lesions in Takayasu's arteritis (TAK) compared with 18F-fuorodeoxyglucose positron emission tomography/computed tomography (18F-FDG PET/CT). Methods: Patients with TAK were recruited for this prospective, observational study. Imaging and clinical assessments were performed concurrently. Only thoracic arteries were evaluated, and they were divided into 18 segments per person. All arterial lesions were evaluated using both PET/CT and DE-MRI. Correlations between both methods were assessed in the PA and thoracic aorta. A receiver operating characteristic (ROC) curve was used to analyze the value of imaging features in detecting disease activity based on National Institutes of Health (NIH) criteria. Results: A total of 24 patients contributed 432 arterial segments. Using PET/CT, correlations between arterial wall DE, thickening, and edema in the PA were 84.52%, 67.92%, and 58.33%, respectively, with Cohen's kappa =0.69, 0.30, and 0.13, respectively; for the thoracic aorta, the values were 86.38%, 80.00%, and 75.92%, respectively, with Cohen's kappa =0.71, 0.52, and 0.372, respectively. There was a significant difference in the incidence of wall DE between the PA and thoracic aorta in patients with clinically active TAK (χ2=6.85, P=0.009). DE-MRI presented a higher area under the curve [area under the curve (AUC); 0.729, P=0.047] than wall thickening and edema in the detection of TAK activity. The wall DE combined with erythrocyte sedimentation rate (ESR) showed improved efficiency (AUC: 0.858, P=0.003). Conclusions: DE-MRI displays appreciable correlations with PET/CT findings and allows for the detection of PA inflammation in patients with TAK; it shows higher values in the thoracic aorta than in the PA. The combination of wall DE and ESR can improve the efficiency of assessing disease status.

Validation of the novel International Association for the Study of Lung Cancer grading system and prognostic value of filigree micropapillary and discohesive growth pattern in invasive pulmonary adenocarcinoma.

INTRODUCTION: The Pathology Committee of the International Association for the Study of Lung Cancer (IASLC) proposed a new histological grading system based on the combination of predominant and high-grade patterns in 2020. MATERIALS AND METHODS: Pathological sections from 631 patients with stage I-III invasive lung adenocarcinoma were reviewed. We then determined the histological grade according to the new grading system and confirmed the pathological features that included the filigree micropapillary and discohesive growth pattern. Applying of the novel IASLC grading system in prognosis stratification was verified and the clinical significance of the pathological characteristics was explored. RESULTS: Cox multivariable analysis revealed that in the stage I-III invasive lung adenocarcinoma, the IASLC grading system was significantly associated with disease-free survival (DFS) [hazard ratio (HR) = 1.419; 95 % confidence interval (CI): 1.040-1.937; P = 0.027] and overall survival (OS) (HR = 1.899; 95 % CI: 1.168-3.087; P = 0.010). In patients with IASLC Grades 1 and 2, the simultaneous presence of filigree micropapillary and discohesive growth pattern was significantly correlated with DFS (HR = 1.899; 95 % CI:1.168-3.087; P = 0.010). However, the filigree micropapillary and discohesive growth pattern did not affect the OS (HR = 2.786; P = 0.317). The competitive risk model revealed that in the stage I cohort, the simultaneous presence of filigree micropapillary and discohesive growth pattern was a risk factor for recurrence and metastasis [sub- distribution HR (SHR) = 1.987; 95 %CI: 1.122-3.518; P = 0.019]. CONCLUSION: Our study verified that the new prognostic stratification system was an effective stratification tool. Filigree micropapillary and discohesive growth pattern may also be risk factors for DFS, postoperative recurrence and metastasis.

CELO Fiber1 Knob Is a Promising Candidate to Modify the Tropism of Adenoviral Vectors.

Fowl adenovirus 4 (FAdV-4) has the potential to be constructed as a gene transfer vector for human gene therapy or vaccine development to avoid the pre-existing immunity to human adenoviruses. To enhance the transduction of FAdV-4 to human cells, CELO fiber1 knob (CF1K) was chosen to replace the fiber2 knob in FAdV-4 to generate recombinant virus F2CF1K-CG. The original FAdV4-CG virus transduced 4% human 293 or 1% HEp-2 cells at the multiplicity of infection of 1000 viral particles per cell. In contrast, F2CF1K-CG could transduce 98% 293 or 60% HEp-2 cells under the same conditions. Prokaryotically expressed CF1K protein blocked 50% transduction of F2CF1K-CG to 293 cells at a concentration of 1.3 µg/mL while it only slightly inhibited the infection of human adenovirus 5 (HAdV-5), suggesting CF1K could bind to human cells in a manner different from HAdV-5 fiber. The incorporation of CF1K had no negative effect on the growth of FAdV-4 in the packaging cells. In addition, CF1K-pseudotyped HAdV-41 could transduce HEp-2 and A549 cells more efficiently. These data indicated that CF1K had the priority to be considered when there is a need to modify adenovirus tropism.

Construction of Adenoviral Vectors using DNA Assembly Technology.

Adenoviral vectors have been used as a gene transfer tool in gene therapy for more than three decades. Here, we introduce a protocol to construct an adenoviral vector by manipulating the genomic DNA of wild-type HAdV-7 by using a DNA assembly method. First, an infectious clone of HAdV-7, pKan-Ad7, was generated by fusing the viral genomic DNA with a PCR product from plasmid backbone, comprising of the kanamycin-resistant gene and the origin of replication (Kan-Ori), through DNA assembly. This was done by designing a pair of PCR primers, that contained ~25 nucleotides of the terminal sequence of HAdV-7 inverted terminal repeat (ITR) at the 5' end, a non-cutter restriction enzyme site for HAdV-7 genome in the middle, and a template-specific sequence for PCR priming at the 3' end. Second, an intermediate plasmid-based strategy was employed to replace the E3 region with transgene-expressing elements in the infectious clone to generate an adenoviral vector. Briefly, pKan-Ad7 was digested with dual-cutter restriction enzyme Hpa I, and the fragment containing the E3 region was ligated to another PCR product of plasmid backbone by Gibson assembly to construct an intermediate plasmid pKan-Ad7HpaI. For convenience, restriction-assembly was used to designate the plasmid cloning method of combined restriction digestion and assembly. Using restriction-assembly, the E3 genes in pKan-Ad7HpaI was replaced with a green fluorescent protein (GFP) expression cassette, and the modified E3 region was released from the intermediate plasmid and restored to the infectious clone to generate an adenoviral plasmid pKAd7-E3GFP. Finally, pKAd7-E3GFP was linearized by Pme I digestion and used to transfect HEK293 packaging cells to rescue recombinant HAdV-7 virus. To conclude, a DNA assembly-based strategy was introduced here for constructing adenoviral vectors in general laboratories of molecular biology without the need of specialized materials and instruments.

Identification of key genes and pathways related to cancer-associated fibroblasts in chemoresistance of ovarian cancer cells based on GEO and TCGA databases.

BACKGROUND: Studies have revealed the implications of cancer-associated fibroblasts (CAFs) in tumor progression, metastasis, and treatment resistance. Here, in silico analyses were performed to reveal the key genes and pathways by which CAFs affected chemoresistance in ovarian cancer. METHODS: Candidate genes were obtained from the intersected differentially expressed genes in ovarian cancer, ovarian cancer chemoresistance, and ovarian CAF-related microarrays and chemoresistance-related genes from GeneCards databases. Kyoto Encyclopedia of Genes and Genomes enrichment analysis and Gene Set Enrichment Analysis were employed to identify the pathways engaged in ovarian cancer chemoresistance and ovarian CAF-related pathways. The top genes with high Degree in the protein-protein interaction network were intersected with the top genes enriched in the key pathways, followed by correlation analyses between key genes and chemotherapeutic response. The expression profiles of key genes were obtained from Human Protein Atlas database and TCGA-ovarian cancer data. RESULTS: p53, cell cycle, PI3K-Akt, and MAPK pathways were the key pathways related to the implication of CAFs in ovarian cancer chemoresistance. 276 candidate genes differentially expressed in CAFs were associated with ovarian cancer chemoresistance. MYC, IGF1, HRAS, CCND1, AKT1, RAC1, KDR, FGF2, FAS, and EGFR were enriched in the key chemoresistance-related ways. Furthermore, MYC, EGFR, CCND1 exhibited close association with chemotherapeutic response to platinum and showed a high expression in ovarian cancer tissues and platinum-resistant ovarian cancer cells. CONCLUSION: The study suggests the key genes (MYC, EGFR, and CCND1) and pathways (p53, cell cycle, PI3K-Akt, and MAPK) responsible for the effect of CAFs on ovarian cancer chemoresistance.

No Genus-Specific Gene Is Essential for the Replication of Fowl Adenovirus 4 in Chicken LMH Cells.

Essential genus-specific genes have not been discovered for fowl adenovirus (FAdV), which hampers the development of FAdV-based vectors and attenuated FAdV vaccines. Reverse genetics approaches were employed to construct FAdV-4 mutants carrying deletions or frameshift mutations covering the whole left and right ends of the viral genome. The results of virus rescue and plaque forming experiments illustrated that all the 22 designated ORFs (open reading frames) were dispensable for the replication of FAdV-4 in chicken hepatoma Leghorn male hepatoma (LMH) cells and primary embryo hepatocytes. RNA-seq data demonstrated that ORF28 and ORF29 were not protein-encoding genes, and suggested a promoter (RP1) and an intron in these regions, respectively. The promoter activity of RP1 was further confirmed by reporter gene expression experiments. GAM-1-deleted FAdV-4 formed small plaques, while deletion of GAM-1 together with ORF22 resulted in even smaller ones in LMH cells. Simultaneous deletion of ORF28, ORF29, and GAM-1 led to growth defect of FAdV-4. These facts implied that genus-specific genes contributed to and synergistically affected viral replication, although no single one was essential. Notably, replication of FAdV-4 mutants could be different in vitro and in vivo. XGAM1-CX19A, a GAM-1-deleted FAdV-4 that replicated efficiently in LMH cells, did not kill chicken embryos because virus propagation took place at a very low level in vivo. This work laid a solid foundation for FAdV-4 vector construction as well as vaccine development, and would benefit viral gene function study. IMPORTANCE Identification of viral essential genes is important for adenoviral vector construction. Deletion of nonessential genes enlarges cloning capacity, deletion of essential genes makes a replication-defective vector, and expression of essential genes in trans generates a virus packaging cell line. However, the genus-specific essential genes in FAdV have not been identified. We constructed adenoviral plasmid carrying deletions covering all 22 genus-specific ORFs of FAdV-4, and found that all virus mutants could be rescued and amplified in chicken LMH cells except those that had defects in key promoter activity. These genus-specific genes affected virus growth, but no single one was indispensable. Dysfunction of several genus-specific genes at the same time could make FAdV-4 vectors replication-defective. In addition, the growth of FAdV-4 mutants could be different in LMH cells and in chicken embryos, suggesting the possibility of constructing attenuated FAdV-4 vaccines.

Restriction-Assembly: A Solution to Construct Novel Adenovirus Vector.

Gene therapy and vaccine development need more novel adenovirus vectors. Here, we attempt to provide strategies to construct adenovirus vectors based on restriction-assembly for researchers with little experience in this field. Restriction-assembly is a combined method of restriction digestion and Gibson assembly, by which the major part of the obtained plasmid comes from digested DNA fragments instead of PCR products. We demonstrated the capability of restriction-assembly in manipulating the genome of simian adenovirus 1 (SAdV-1) in this study. A PCR product of the plasmid backbone was combined with SAdV-1 genomic DNA to construct an infectious clone, plasmid pKSAV1, by Gibson assembly. Restriction-assembly was performed repeatedly in the steps of intermediate plasmid isolation, modification, and restoration. The generated adenoviral plasmid was linearized by restriction enzyme digestion and transfected into packaging 293 cells to rescue E3-deleted replication-competent SAdV1XE3-CGA virus. Interestingly, SAdV1XE3-CGA could propagate in human chronic myelogenous leukemia K562 cells. The E1 region was similarly modified to generate E1/E3-deleted replication-defective virus SAdV1-EG. SAdV1-EG had a moderate gene transfer ability to adherent mammalian cells, and it could efficiently transduce suspension cells when compared with the human adenovirus 5 control vector. Restriction-assembly is easy to use and can be performed without special experimental materials and instruments. It is highly effective with verifiable outcomes at each step. More importantly, restriction-assembly makes the established vector system modifiable, upgradable and under sustainable development, and it can serve as the instructive method or strategy for the synthetic biology of adenoviruses.

Clot burden of acute pulmonary thromboembolism: comparison of two deep learning algorithms, Qanadli score, and Mastora score.

BACKGROUND: The deep learning convolution neural network (DL-CNN) benefits evaluating clot burden of acute pulmonary thromboembolism (APE). Our objective was to compare the performance of the deep learning convolution neural network trained by the fine-tuning [DL-CNN (ft)] and the deep learning convolution neural network trained from the scratch [DL-CNN (fs)] in the quantitative assessment of APE. METHODS: We included the data of 680 cases for training DL-CNN by DL-CNN (ft) and DL-CNN (fs), then retrospectively included 410 patients (137 patients with APE, 203 males, mean age 60.3±11.4 years) for testing the models. The distribution and volume of clots were respectively assessed by DL-CNN(ft) and DL-CNN(fs), and sensitivity, specificity, and area under the curve (AUC) were used to evaluate their performances in detecting clots on a per-patient and clot level. Radiologists evaluated the distribution of clots, Qanadli score, and Mastora score and right ventricular metrics, and the correlation of clot volumes with right ventricular metrics were analyzed with Spearman correlation analysis. RESULTS: On a per-patient level, the two DL-CNN models had high sensitivities and moderate specificities [DL-CNN (ft): 100% and 77.29%; DL-CNN (fs): 100% and 75.82%], and their AUCs were comparable (Z=0.30, P=0.38). On a clot level, DL-CNN (ft) and DL-CNN (fs) sensitivities and specificities in detecting central clots were 99.06% and 72.61%, and 100% and 70.63%, respectively. DL-CNN (ft) sensitivities and specificities in detecting peripheral clots were mostly higher than those of DL-CNN (fs), and their AUCs were comparable. Clot volumes measured with the two models were similar (U=85094.500, P=0.741), and significantly correlated with Qanadli scores [DL-CNN(ft) r=0.825, P<0.001, DL-CNN(fs) r=0.827, P<0.001] and Mastora scores [DL-CNN(ft) r=0.859, P<0.001, DL-CNN(fs) r=0.864, P<0.001]. Clot volumes were also correlated with right ventricular metrics. Clot burdens were increased in the low-risk, moderate-risk, and high-risk patients. Binary logistic regression revealed that only the ratio of right ventricular area/left ventricular area (RVa/LVa) was an independent predictor of in-hospital death (odds ratio 6.73; 95% CI, 2.7-18.12, P<0.001). CONCLUSIONS: Both DL-CNN (ft) and DL-CNN (fs) have high sensitivities and moderate specificities in detecting clots associated with APE, and their performances are comparable. While clot burdens quantitatively calculated by the two DL-CNN models are correlated with right ventricular function and risk stratification, RVa/LVa is an independent prognostic factor of in-hospital death in patients with APE.

Fibroblast activation protein imaging in reperfused ST-elevation myocardial infarction: comparison with cardiac magnetic resonance imaging.

PURPOSE: The aim of this study was to explore the correlation of 18F-labeled fibroblast activation protein inhibitor (FAPI) and cardiovascular magnetic resonance (CMR) parameters in ST-elevation myocardial infarction (STEMI) patients with successful primary percutaneous coronary intervention (PPCI) and to investigate the value of FAPI imaging in predicting cardiac functional recovery, as well as the correlation between FAPI activity and circulating fibroblast activation protein (FAP) and inflammatory biomarkers. METHODS: Fourteen first-time STEMI patients (11 men, mean age: 62 ± 11 years) after PPCI and 14 gender-matched healthy volunteers (10 men, mean age: 50 ± 14 years) who had completed FAPI imaging and blood sample collection were prospectively recruited. All patients underwent baseline FAPI imaging (6 ± 2 days post-MI) and CMR (8 ± 2 days post-MI). Ten patients had follow-up CMR (84 ± 4 days post-MI). Myocardial FAPI activity was analyzed for extent (the percentage of FAPI uptake volume over the left ventricular volume, FAPI%), intensity (target-to-background uptake ratio, TBRmax), and amount (FAPI% × TBRmax). Late gadolinium enhancement (LGE), T2-weighted imaging (T2WI), extracellular volume (ECV), microvascular obstruction (MVO), and cardiac function from CMR imaging were analyzed. Blood samples obtained on the day of FAPI imaging were used to assess circulating FAP, TGF-ß1, TNF-α, IL-6, and hsCRP in STEMI patients and controls. RESULTS: Localized but inhomogeneous FAPI uptake was observed in STEMI patients, which was larger than the edematous and infarcted myocardium, whereas no uptake was detected in controls. The MVO area showed lower FAPI uptake compared with the surrounding myocardium. FAPI activity was associated with the myocardial injury biomarkers T2WI, LGE, and ECV at both per-patient and per-segment levels (all p < 0.05), but was not associated with circulating FAP, TGF-ß1, TNF-α, IL-6, or hsCRP. Among the CMR parameters, T2WI had the greatest correlation coefficient with both FAPI% and FAPI% × TBRmax. Baseline TBRmax was inversely correlated with the follow-up left ventricular ejection fraction (LVEF) (r = - 0.73, p = 0.02). CONCLUSION: FAPI imaging detects more involved myocardium than CMR in reperfused STEMI, and is associated with myocardial damage and follow-up LVEF.

Clinical Features and Outcomes of Pulmonary Artery Sarcoma.

OBJECTIVES: A retrospective cohort study was designed to describe the clinical features and outcomes of pulmonary artery sarcoma (PAS). METHODS: Twenty-two (22) consecutive patients diagnosed with PAS by pathological examination were enrolled and followed up until they died or until January 2020. The medical records were retrospectively reviewed to evaluate the clinical characteristics, image findings, and outcomes. RESULTS: 1) Twenty-one (21, 95.5%) patients were firstly misdiagnosed. Dyspnoea was the most common presenting symptom (19 of 22, 86.4%). 2) Filling defects in the right pulmonary artery were seen in 17 patients (77.3%) with computed tomography pulmonary angiography or magnetic resonance pulmonary angiography. Among those patients, 14 underwent positron emission tomography-computed tomography detection and 13 (92.9%) were found to have increased uptake value in the pulmonary artery. 3) The median survival (from diagnosis to death or January 2020) of the total series was 11.6 months (range, 0.7-68.5 months). The estimated cumulative survival rates at 1, 2, and 3 years were 52.6%, 32.8%, and 19.7%, respectively. Patients who received surgery and/or chemo-radiotherapy treatment had a better survival rate compared with patients without treatment (the estimated cumulative survival rates at 1, 2, and 3 years were 60.3%, 39.1%, and 29.3%, respectively, vs 33.3%, 16.6%, and 0, accordingly) and better survival time (median survival 17.02 vs 3.16 months, respectively) (p=0.025). CONCLUSIONS: Pulmonary artery sarcoma is easily misdiagnosed, as the symptoms and routine image detection are nonspecific. Positron emission tomography-computed tomography may be helpful in diagnosis. Surgery and/or chemo-radiotherapy offer a chance for better outcomes.

Examining the Development of Chronic Thromboembolic Pulmonary Hypertension at the Single-Cell Level.

BACKGROUND: The mechanism of chronic thromboembolic pulmonary hypertension (CTEPH) is known to be multifactorial but remains incompletely understood. METHODS: In this study, single-cell RNA sequencing, which facilitates the identification of molecular profiles of samples on an individual cell level, was applied to investigate individual cell types in pulmonary endarterectomized tissues from 5 patients with CTEPH. The order of single-cell types was then traced along the developmental trajectory of CTEPH by trajectory inference analysis, and intercellular communication was characterized by analysis of ligand-receptor pairs between cell types. Finally, comprehensive bioinformatics tools were used to analyze possible functions of branch-specific cell types and the underlying mechanisms. RESULTS: Eleven cell types were identified, with immune-related cell types (T cells, natural killer cells, macrophages, and mast cells) distributed in the left (early) branch of the pseudotime tree, cancer stem cells, and CRISPLD2+ cells as intermediate cell types, and classic disease-related cell types (fibroblasts, smooth muscle cells, myofibroblasts, and endothelial cells) in the right (later) branch. Ligand-receptor interactions revealed close communication between macrophages and disease-related cell types as well as between smooth muscle cells and fibroblasts or endothelial cells. Moreover, the ligands and receptors were significantly enriched in key pathways such as the PI3K/Akt signaling pathway. Furthermore, highly expressed genes specific to the undefined cell type were significantly enriched in important functions associated with regulation of endoplasmic reticulum stress. CONCLUSIONS: This single-cell RNA sequencing analysis revealed the order of single cells along a developmental trajectory in CTEPH as well as close communication between different cell types in CTEPH pathogenesis.

Clinical and CT findings of adenovirus pneumonia in immunocompetent adults.

INTRODUCTION: Adenovirus pneumonia is not uncommon in children and immunocompromised patients. However, the study of the clinical and computed tomography (CT) characteristics of Adenovirus pneumonia in immunocompetent adults is still limited. OBJECTIVE: The objective of this study was to retrospectively observe the clinical and CT characteristics as well as their dynamic change of Adenovirus pneumonia in immunocompetent adults. METHODS: Twenty patients (18 males, median age, 36 years old) with Adenovirus pneumonia were retrospectively included from January 2018 to December 2019. Clinical information and chest CT at admission of all patients were reviewed. Twelve patients underwent serial CT scans, and the temporal changes of CT findings were summarized. Pneumonia severity index (PSI) was calculated according to clinical information. RESULTS: The median time interval from illness onset to admission was 6 days (interquartile range [IQR], 5-7.5 days). The clinical characteristics included the high fever (39.2 ± 0.8°C) with the normal white blood cell count, the decreased lymphocyte, and elevated C-reactive protein. Ten cases complicated with mycoplasma infection at admission. Thirteen patients were mechanically ventilated, and two patients died during hospitalization. Consolidation was a predominant pattern found during the first 2 weeks and then resolved to minimal consolidation after the fourth week. There was no significant correlation between CT score and PSI score (r = 0.15, p = 0.41). CONCLUSIONS: Predominant radiological finding of Adenovirus pneumonia was consolidation. Multilobular involvement, higher CT scores, and pleural effusion were found in more severe patients. The abnormal opacity peaked in 2 weeks of illness onset and gradually resolved after the third week. The temporal changes of radiological score are consistent with clinical findings.