Long-Term Survival and Recurrence Patterns in Locally Advanced Esophageal Squamous Cell Carcinoma Patients with Pathologic Complete Response After Neoadjuvant Chemotherapy Followed by Surgery.

BACKGROUND: The higher pathologic complete response (pCR) after neoadjuvant chemoradiotherapy compared with neoadjuvant chemotherapy for locally advanced esophageal squamous cell carcinoma (ESCC) has not translated into significant gains in overall survival. Data on the long-term survival of patients who obtained a pCR after neoadjuvant chemotherapy are scarce. Therefore, this study aimed to evaluate the long-term prognosis and recurrence patterns in these patients. METHODS: The study enrolled patients with locally advanced ESCC after neoadjuvant chemotherapy followed by surgery in the authors' hospital between January 2007 and December 2020. The factors predictive of pCR were analyzed. Furthermore, propensity score-matching was performed for those who did and those who did not have a pCR using 1:5 ratio for a long-term survival analysis. Finally, the survival and recurrence patterns of patients obtaining pCR after neoadjuvant chemotherapy were analyzed. RESULTS: A pCR was achieved for 61 (8.70%) of the 701 patients in the study. Univariate analysis showed that the patients without alcohol drinking had a higher possibility of obtaining a pCR, although multivariate analysis failed to confirm the difference as significant. After propensity score-matching, the 5-year overall survival was 84.50% for the patients who had a pCR and 52.90% for those who did not (p < 0.001). Among the 61 patients with a pCR, 9 patients (14.80%) experienced recurrence, including 6 patients with locoregional recurrence and 3 patients with distant metastasis. CONCLUSION: Advanced ESCC patients with pCR after neoadjuvant chemotherapy had a favorable prognosis, yet some still experienced recurrence, particularly locoregional recurrence. Therefore, for this group of patients, regular follow-up evaluation also is needed.

Mechanistic insight into the impact of interaction between goethite and humic acid on the photooxidation and photoreduction of bifenthrin.

Numerous application of pyrethroid insecticides has led to their accumulation in the environment, threatening ecological environment and human health. Its fate in the presence of iron-bearing minerals and natural organic matter under light irradiation is still unknown. We found that goethite (Gt) and humic acid (HA) could improve the photodegradation of bifenthrin (BF) in proper concentration under light irradiation. The interaction between Gt and HA may further enhance BF degradation. On one hand, the adsorption of HA on Gt may decrease the photocatalytic activity of HA through decreasing HA content in solution and sequestering the functional groups related with the production of reactive species. On the other hand, HA could improve the photocatalytic activity of Gt through extending light absorption, lowing of bandgap energy, hindering the recombination of photo-generated charges, and promoting the oxidation and reduction reaction on Gt surface. The increased oxygen vacancies on Gt surface along with the reduction of trivalent iron and the nucleophilic attack of hole to surface hydroxyl group contributed to the increasing photocatalytic activity of Gt. Electron paramagnetic resonance and quenching studies demonstrated that both oxidation species, such as hydroxyl radical (•OH) and singlet oxygen (1O2), and reducing species, such as hydrogen atoms (H•) and superoxide anion radical (O2•-), contributed to BF degradation in UV-Gt-HA system. Mass spectrometry, ion chromatography, and toxicity assessment indicated that less toxic C23H22ClF3O3 (OH-BF), C9H10ClF3O (TFP), C14H14O2 (OH-MBP), C14H12O2 (MBP acid), C14H12O3 (OH-MBP acid), and chloride ions were the main degradation products. The production of OH-BF, MPB, and TFP acid through oxidation and the production of MPB and TFP via reduction were the two primary pathways of BF degradation.

SARS-CoV-2 hijacks macropinocytosis to facilitate its entry and promote viral spike-mediated cell-to-cell fusion.

Revealing the mechanisms of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) entry and cell-to-cell spread might provide insights for understanding the underlying mechanisms of viral pathogenesis, tropism, and virulence. The signaling pathways involved in SARS-CoV-2 entry and viral spike-mediated cell-to-cell fusion remain elusive. In the current study, we found that macropinocytosis inhibitors significantly suppressed SARS-CoV-2 infection at both the entry and viral spike-mediated cell-to-cell fusion steps. We demonstrated that SARS-CoV-2 entry required the small GTPase Rac1 and its effector kinase p21-activated kinase 1 by dominant-negative and RNAi assays in human embryonic kidney 293T-angiotensin-converting enzyme 2 cells and that the serine protease transmembrane serine protease 2 reversed the decrease in SARS-CoV-2 entry caused by the macropinocytosis inhibitors. Moreover, in the cell-to-cell fusion assay, we confirmed that macropinocytosis inhibitors significantly decreased viral spike-mediated cell-to-cell fusion. Overall, we provided evidence that SARS-CoV-2 utilizes a macropinocytosis pathway to enter target cells and to efficiently promote viral spike-mediated cell-to-cell fusion.

Risk Factors for Cerebrovascular Events in Moyamoya Angiopathy Using 4D Flow MRI: A Pilot Study.

BACKGROUND: Little is known about internal carotid artery (ICA) hemodynamics in patients with moyamoya angiopathy (MMA) and its role in cerebrovascular events. PURPOSE: To characterize ICA hemodynamics in MMA patients by 4D flow MRI and investigate its relationship with cerebrovascular events. STUDY TYPE: Prospective. SUBJECTS: Seventy MMA patients (50 years old ± 9, 30 males). FIELD STRENGTH/SEQUENCE: Time-resolved three-directional velocity encoded fast field echo sequence (4D flow) MRI, T1-weighted fast field echo sequence, T2 weighted turbo spin echo sequence, diffusion weighted echo planar imaging; T2-weighted fluid-attenuated inversion recovery turbo spin echo sequence, susceptibility weighted fast field echo sequence, and time-of-flight MR angiography fast field echo sequence at 3.0T. ASSESSMENT: ICA hemodynamics (maximum and average velocity [Vmax , Vavg ], average blood flow [Flowavg ], and wall shear stress) were analyzed based on 4D flow data. Cerebral infarction, defined as the occurrence of events, in 124 brain hemispheres was determined according to clinical symptoms and conventional brain MR imaging. STATISTICAL TESTS: The independent-samples T test was used to evaluate differences in ICA hemodynamics between infarcted and non-infarcted hemispheres. Binary logistic regression was performed to investigate the relationship between ICA hemodynamics and events. A P value < 0.05 was considered statistically significant. RESULTS: Sixty-one infarcted hemispheres (eight hemispheres with acute ischemic damage, 30 with chronic ischemic damage, and 23 with chronic hemorrhagic damage) had cerebrovascular events and 63 non-infarcted hemispheres did not. The hemodynamic parameters in the infarcted hemispheres (Vmax : P < 0.001; Vavg : P = 0.003; and Flowavg : P = 0.004) were significantly lower than those in the non-infarcted hemispheres. However, Vmax (P = 0.052), Vavg (P = 0.107), and Flowavg (P = 0.074) were not significantly different among hemispheres with acute ischemic lesions, chronic ischemic lesions and chronic hemorrhagic lesions. Vmax (odds ratio 3.033, 95% CI: 1.075-8.562) was independently associated with cerebrovascular events. DATA CONCLUSION: Vmax maybe a higher risk factor of cerebrovascular events in MMA patients. EVIDENCE LEVEL: 2 TECHNICAL EFFICACY STAGE: 3.

Pre-operative oil ingestion reduces the probability of thoracic duct trunk ligation during esophagectomy.

Chylothorax is an important complication after esophagectomy. Ligation of the injured thoracic duct is the main method to prevent chylothorax after esophagectomy, but may be associated with adverse effects. Whether ligation of the injured tributary alone, keeping the main trunk intact, may suffice to prevent post-operative chylothorax is not well known. Since March 2017, 40 mL of olive oil was administered to patients posted for esophagectomy. We compared patients admitted between March 2017 and December 2019 with patients admitted between July 2014 and February 2017, who had not received pre-operative oil. The outcome measures were the need for thoracic duct main trunk or tributary ligation, development of chylothorax and missed ligation. There were 371 patients in the oil ingestion group and 308 patients in the standard control group. Chylothorax in the oil ingestion group was significantly lower than that in the standard control group (1.3% vs. 4.5%, P = 0.012). Chyle leak from thoracic duct tributaries was diagnosed in a significantly higher percentage (5.7% vs. 0.0%, P < 0.001) and missed ligation of the injured thoracic duct was significantly lower (0.3% vs. 3.9%, P = 0.002) in the oil ingestion group compared with the standard control group. The incidence of post-operative chylothorax was not statistically different (6.3% vs. 10.0%, P = 1.000) between the tributary and the trunk ligation group. Pre-operative oil ingestion can help visualize the thoracic duct trunk and its tributaries during esophagectomy. Thus, non-selected thoracic duct trunk ligation and missed ligation during esophagectomy can be reduced. Precise ligation of the injured tributary while the main trunk is intact can also prevent post-operative chylothorax.

Helicoverpa armigera GATAe transcriptional factor regulates the expression of Bacillus thuringiensis Cry1Ac receptor gene ABCC2 by its interplay with additional transcription factors.

Helicoverpa armigera is a worldwide pest that has been efficiently controlled by transgenic plants expressing Bt Cry toxins. To exert toxicity, Cry toxins bind to different receptors located in larval midgut cells. Previously, we reported that GATA transcription factor GATAe activates the expression of multiple H. armigera Cry1Ac receptors in different insect cell lines. Here, the mechanism involved in GATAe regulation of HaABCC2 gene expression, a key receptor of Cry1Ac, was analyzed. HaGATAe gene silencing by RNAi in H. armigera larvae confirmed the activation role of HaGATAe on the expression of HaABCC2 in the midgut. The contribution of all potential GATAe-binding sites was analyzed by site-directed mutagenesis using Hi5 cells expressing a reporter gene under regulation of different modified HaABCC2 promoters. DNA pull-down assays revealed that GATAe bound to different predicted GATA-binding sites and mutations of the different GATAe-binding sites identified two binding sites responsible for the promoter activity. The binding site B9, which is located near the transcription initiator site, has a major contribution on HaABCC2 expression. Also, DNA pull-down assays revealed that all other members of GATA TF family in H. armigera, besides GATAe, HaGATAa, HaGATAb, HaGATAc and HaGATAd also bound to the HaABCC2 promoter and decreased the GATAe dependent promoter activity. Finally, the potential participation in the regulation of HaABCC2 promoter of several TFs other than GATA TFs expressed in the midgut cells was analyzed. HaHR3 inhibited the GATAe dependent activity of the HaABCC2 promoter, while two other midgut-related TFs, HaCDX and HaSox21, also bound to the HaABCC2 promoter region and increased the GATAe dependent promoter activity. All these data showed that GATAe induces HaABCC2 expression by binding to HaGATAe binding sites in the promoter region and that additional TFs participate in modulating the HaGATAe-driven expression of HaABCC2.

Aminopeptidase N Is an Entry Co-factor Triggering Porcine Deltacoronavirus Entry via an Endocytotic Pathway.

Porcine deltacoronavirus (PDCoV) is a recently discovered coronavirus that poses a potential threat to the global swine industry. Although we know that aminopeptidase N (APN) is important for PDCoV replication, it is unclear whether it is the primary functional receptor, and the mechanism by which it promotes viral replication is not fully understood. Here, we systematically investigated the roles of porcine APN (pAPN) during PDCoV infection of nonsusceptible cells, including in viral attachment and internalization. Using a viral entry assay, we found that PDCoV can enter nonsusceptible cells but then fails to initiate efficient replication. pAPN and PDCoV virions clearly colocalized with the endocytotic markers RAB5, RAB7, and LAMP1, suggesting that pAPN mediates PDCoV entry by an endocytotic pathway. Most importantly, our study shows that regardless of which receptor PDCoV engages, only entry by an endocytotic route ultimately leads to efficient viral replication. This knowledge should contribute to the development of efficient antiviral treatments, which are especially useful in preventing cross-species transmission. IMPORTANCE PDCoV is a pathogen with the potential for transmission across diverse species, although the mechanism of such host-switching events (from swine to other species) is poorly understood. Here, we show that PDCoV enters nonsusceptible cells but without efficient replication. We also investigated the key role played by aminopeptidase N in mediating PDCoV entry via an endocytotic pathway. Our results demonstrate that viral entry via endocytosis is a major determinant of efficient PDCoV replication. This knowledge provides a basis for future studies of the cross-species transmissibility of PDCoV and the development of appropriate antiviral drugs.

Bacillus thuringiensis Cry1Ac Protoxin and Activated Toxin Exert Differential Toxicity Due to a Synergistic Interplay of Cadherin with ABCC Transporters in the Cotton Bollworm.

Bacillus thuringiensis Cry proteins are used worldwide for insect control. It was proposed that Cry-protoxins must be converted into activated toxin by proteases to bind midgut cell proteins to kill insects. However, Cry-protoxins also bind to midgut proteins and kill insects that have evolved resistance to activated toxins suggesting an independent toxicity pathway. Cadherin (CAD) and ABCC transporters are recognized as important receptors for Cry proteins. Here we constructed different Helicoverpa armigera mutations in these receptors by CRISPR/Cas9. HaCAD-KO mutant showed much higher resistance to Cry1Ac activated toxin than to Cry1Ac protoxin. In contrast, the HaABCC2-M and HaABCC3-M mutants showed higher resistance to Cry1Ac-protoxin than to activated toxin. However, in the double HaABCC2/3-KO mutant, very high levels of resistance were observed to both Cry1Ac protoxin and activated toxin, supporting that both ABC transporters have redundant functions for these two proteins. In addition, Hi5 cells transfected with HaCAD were susceptible only to the activated toxin but not to protoxin. In contrast, both forms of Cry1Ac were similarly toxic to Hi5 cells expressing HaABCC2 or HaABCC3. Co-expression of HaCAD with HaABCC2 or HaABCC3 revealed a more important synergistic effect for activated toxin compared to protoxin. Overall, our results show that toxicity of Cry1Ac activated toxin involves synergistic interplay of HaCAD with ABCC transporters, while the Cry1Ac protoxin toxicity is mainly mediated by ABCC transporters with little participation of HaCAD. These data help to understand the mode of action of Cry proteins that will be relevant to enhance efficacy and durability of Bt-crops. IMPORTANCE Better understanding of the mode of action of Bacillus thuringiensis toxins is beneficial for the sustainable application of Bt crops. It is generally accepted that Cry-protoxins need to be activated by proteases to bind with midgut cell proteins and exert toxicity against insects. Here, we provide new insights into the toxic pathway of Cry proteins in the cotton bollworm. First, our results demonstrate that Cry1Ac protoxin is able to exert cytotoxicity against the insect cells expressing ABCC transporters. Second, we reveal that CAD plays a critical role in the different toxicity of protoxin and toxin by facilitating a synergistic interplay with ABCC transporters. Our results provide in vivo and in vitro experimental evidence supporting that Cry1Ac protoxin exerts toxicity against H. armigera via different steps from that of toxin. These new findings on the mode of action of Cry proteins could be beneficial for efficacy enhancement and durability of Bt-crops.

Quantitative analysis of enhanced CT in differentiating well-differentiated pancreatic neuroendocrine tumors and poorly differentiated neuroendocrine carcinomas.

OBJECTIVE: To identify quantitative CT features for distinguishing well-differentiated pancreatic neuroendocrine tumors (PNETs) from poorly differentiated pancreatic neuroendocrine carcinomas (PNECs). MATERIALS AND METHODS: Seventeen patients with PNECs and 131 patients with PNETs confirmed by biopsy or surgery were retrospectively included. General demographic (sex, age) and CT quantitative parameters (arterial/portal absolute enhancement, arterial/portal relative enhancement ratio, arterial/portal enhancement ratio) were collected. Univariate and multivariate logistic regression analyses were performed to confirm independent variables for differentiating PNECs from PNETs. Receiver operating characteristic (ROC) curves for each quantitative parameter were generated to determine their diagnostic ability. RESULTS: PNECs had a much lower mean arterial/portal absolute enhancement value (19.5 ± 11.0 vs. 78.8 ± 47.2; 28.1 ± 15.8 vs. 77.0 ± 39.4), arterial/portal relative enhancement ratio (0.57 ± 0.36 vs. 2.03 ± 1.31; 0.80 ± 0.52 vs. 1.99 ± 1.13), and arterial/portal enhancement ratio (0.62 ± 0.27 vs. 1.22 ± 0.49; 0.74 ± 0.19 vs. 1.21 ± 0.36) than PNETs (all p < 0.001). After multivariable analysis, arterial absolute enhancement (odds ratio [OR]: 0.96, 95% confidence interval [CI]: 0.93, 0.99) and portal absolute enhancement (OR: 0.96, 95% CI: 0.92, 0.99) were independent factors for differentiating PNECs from PNETs. For each quantitative parameter, arterial lesion enhancement yielded the highest diagnostic performance, with an area under the curve (AUC) of 0.922 (95% CI: 0.867-0.960), followed by portal absolute enhancement. CONCLUSIONS: Arterial/portal absolute enhancements were independent predictors with good diagnostic accuracy for differentiating between PNETs and PNECs. Quantitative parameters of enhanced CT can distinguish PNECs from PNETs. KEY POINTS: • PNECs were hypovascular and had a much lower enhanced CT attenuation in both arterial and portal phases than well-differentiated PNETs. • Quantitative parameters derived from enhanced CT can be used to distinguish PNECs from PNETs. • Arterial absolute enhancement and portal absolute enhancement were independent predictive factors for differentiating between PNETs and PNECs.

Rapid visual detection of porcine reproductive and respiratory syndrome virus via recombinase polymerase amplification combined with a lateral flow dipstick.

Porcine reproductive and respiratory syndrome (PRRS) is one of the most economically devastating infectious diseases in the global swine industry. A rapid and sensitive on-site detection method for PRRS virus (PRRSV) is critically important for diagnosing PRRS. In this study, we established a method that combines reverse transcription recombinase polymerase amplification (RT-RPA) with a lateral flow dipstick (LFD) for detecting North American PRRSV (PRRSV-2). The primers and probe were designed based on the conserved region of all complete PRRSV-2 genomic sequences available in China (n = 512) from 1996 to 2020. The detection limit of the assay was 5.6 × 10-1 median tissue culture infection dose (TCID50) per reaction within 30 min at 42 °C, which was more sensitive than that of reverse transcription polymerase chain reaction (RT-PCR) (5.6 TCID50 per reaction). The assay was highly specific for the epidemic lineages of PRRSV-2 in China and did not cross-react with pseudorabies virus, porcine circovirus 2, classical swine fever virus, or porcine epidemic diarrhea virus. The assay performance was evaluated by testing 179 samples and comparing the results with those of quantitative RT-PCR (RT-qPCR). The results showed that the detection coincidence rate of RT-RPA and RT-qPCR was 100% when the cycle threshold values of RT-qPCR were < 32. The assay provides a new alternative for simple and reliable detection of PRRSV-2 and has great potential for application in the field.

Phylogenetics, Genomic Recombination, and NSP2 Polymorphic Patterns of Porcine Reproductive and Respiratory Syndrome Virus in China and the United States in 2014-2018.

Porcine reproductive and respiratory syndrome virus (PRRSV), an important pathogen that affects the pig industry, is a highly genetically diverse RNA virus. However, the phylogenetic and genomic recombination properties of this virus have not been completely elucidated. In this study, comparative analyses of all available genomic sequences of North American (NA)-type PRRSVs (n = 355, including 138 PRRSV genomes sequenced in this study) in China and the United States during 2014-2018 revealed a high frequency of interlineage recombination hot spots in nonstructural protein 9 (NSP9) and the GP2 to GP3 regions. Lineage 1 (L1) PRRSV was found to be susceptible to recombination among PRRSVs both in China and the United States. The recombinant major parent between the 1991-2013 data and the 2014-2018 data showed a trend from complex to simple. The major recombination pattern changed from an L8 to L1 backbone during 2014-2018 for Chinese PRRSVs, whereas L1 was always the major backbone for US PRRSVs. Intralineage recombination hot spots were not as concentrated as interlineage recombination hot spots. In the two main clades with differential diversity in L1, NADC30-like PRRSVs are undergoing a decrease in population genetic diversity, NADC34-like PRRSVs have been relatively stable in population genetic diversity for years. Systematic analyses of insertion and deletion (indel) polymorphisms of NSP2 divided PRRSVs into 25 patterns, which could generate novel references for the classification of PRRSVs. The results of this study contribute to a deeper understanding of the recombination of PRRSVs and indicate the need for coordinated epidemiological investigations among countries.IMPORTANCE Porcine reproductive and respiratory syndrome (PRRS) is one of the most significant swine diseases. However, the phylogenetic and genomic recombination properties of the PRRS virus (PRRSV) have not been completely elucidated. In this study, we systematically compared differences in the lineage distribution, recombination, NSP2 polymorphisms, and evolutionary dynamics between North American (NA)-type PRRSVs in China and in the United States. Strikingly, we found high frequency of interlineage recombination hot spots in nonstructural protein 9 (NSP9) and in the GP2 to GP3 region. Also, intralineage recombination hot spots were scattered across the genome between Chinese and US strains. Furthermore, we proposed novel methods based on NSP2 indel patterns for the classification of PRRSVs. Evolutionary dynamics analysis revealed that NADC30-like PRRSVs are undergoing a decrease in population genetic diversity, suggesting that a dominant population may occur and cause an outbreak. Our findings offer important insights into the recombination of PRRSVs and suggest the need for coordinated international epidemiological investigations.

Identification of critical pathways and potential therapeutic targets in poorly differentiated duodenal papilla adenocarcinoma.

BACKGROUND: Duodenal papilla carcinoma (DPC) is a rare malignancy of the gastrointestinal tract with high recurrence rate, and the pathogenesis of this highly malignant neoplasm is yet to be fully elucidated. This study aims to identify key genes to further understand the biology and pathogenesis underlying the molecular alterations driving DPC, which could be potential diagnostic or therapeutic targets. METHODS: Tumor samples of three DPC patients were collected and integrating RNA-seq analysis of tumor tissues and matched normal tissues were performed to discover differentially expressed genes (DEGs). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis were carried out to understand the potential bio-functions of the DPC differentially expressed genes (DEGs). Protein-protein interaction (PPI) network was constructed for functional modules analysis and identification of hub genes. qRT-PCR of clinical samples was conducted to validate the expression level of the hub genes. RESULTS: A total of 110 DEGs were identified from our RNA-seq data, GO and KEGG analyses showed that the DEGs were mainly enriched in multiple cancer-related functions and pathways, such as cell proliferation, IL-17signaling pathway, Jak-STAT signaling pathway, PPAR signaling pathway. The PPI network screened out five hub genes including IL-6, LCN2, FABP4, LEP and MMP1, which were identified as core genes in the network and the expression value were validated by qRT-PCR. The hub genes identified in this work were suggested to be potential therapeutic targets of DPC. DISCUSSION: The current study may provide new insight into the exploration of DPC pathogenesis and the screened hub genes may serve as potential diagnostic indicator and novel therapeutic target.

Vessel-selective 4D MRA based on ASL might potentially show better performance than 3D TOF MRA for treatment evaluation in patients with intra-extracranial bypass surgery: a prospective study.

OBJECTIVES: To compare bypass patency and intracranial collaterals from the external carotid artery (ECA) by four-dimensional MR angiography (MRA) based on super-selective pseudo-continuous arterial spin labeling (pCASL) combined with the keyhole and view-sharing techniques (4D-sPACK) versus three-dimensional time-of-flight (3D TOF) MRA in patients with extra-intracranial revascularization. METHODS: The MR data of 45 patients administered bypass surgery were collected. The image quality of 4D-sPACK was evaluated using a 4-point grading system according to whether the diagnosis of intracranial collaterals was affected. Anastomosis patency and intracranial collateral visualization from ECA were assessed by two radiologists on 4D-sPACK and 3D TOF MRA, with digital subtraction angiography (DSA) findings as reference. Intracranial collateral assessment employed another 4-point grading system according to the number of vessels shown. Interobserver agreement was assessed with the weighted kappa statistic. RESULTS: Fifty hemispheres in 43 patients were included. The image quality of 4D-sPACK was good in 47 (47/50, 94.0%) hemispheres. 4D-sPACK had a higher sensitivity than 3D TOF MRA (97.73% vs 79.55%) for visualizing anastomoses. There were significant differences between 4D-sPACK (scores, 3.22 ± 1.15) and 3D TOF MRA (scores, 1.80 ± 0.67) in the visualization of intracranial collaterals from ECA (p < 0.001). The interobserver agreement was substantial for intracranial collateral assessment (κ4D-sPACK = 0.788; κ3D TOF MRA = 0.800) and almost perfect for bypass patency (κ4D-sPACK = 0.912; κ3D TOF MRA = 0.816; κDSA = 0.811). CONCLUSION: This pilot study shows that, 4D-sPACK has a better performance than 3D TOF MRA in treatment evaluation of patients after bypass surgery, and has high consistency with DSA. KEY POINTS: • 4D-sPACK is a non-contrast-enhanced dynamic MRA method for the visualization of intracranial vessels. • 4D-sPACK has higher specificity for the diagnosis of anastomosis occlusion. • 4D-sPACK is better than 3D TOF MRA in the visualization of intracranial collaterals in patients after bypass surgery.

Recurrent hemorrhage risk associated with medial target medullary artery anastomosis from the periventricular collateral vessel in adult patients with moyamoya disease.

BACKGROUND: Although the association between periventricular target collateral anastomosis and recurrent ipsilateral hemorrhage has been evaluated in adult patients with moyamoya disease (MMD), no studies have investigated the relationship between target anastomotic territory and recurrent ipsilateral hemorrhage. The goal of this study was to assess this association. METHODS: Consecutive adult MMD patients who had experienced initial intracranial hemorrhage and undergone conservative treatment were included. Two readers assessed angiographic results to identify the target anastomotic territory (medial medullary artery, lateral medullary artery, multiple medullary arteries, or nonmedullary artery) responsible for the hemorrhage. Cox proportional hazard regression models were used to estimate the risk of recurrent hemorrhage. RESULTS: In the 36 hemispheres with initial hemorrhage, the target anastomotic territory was in the anastomotic territory of the medial medullary artery in 10 (27.8%), lateral medullary artery in 15 (41.7%), multiple medullary arteries in 2 (5.6%), and a nonmedullary artery in 9 (25.0%) hemispheres. During 45.1 ± 40.0 months of follow-up, recurrent ipsilateral hemorrhage occurred in 44.4% (16/36) of hemispheres. The target anastomotic territories responsible for the recurrent event were in the anastomotic territory of the medial medullary artery in 9 (56.3%) hemispheres, lateral medullary artery in 6 (37.5%) hemispheres, and multiple medullary arteries in 1 (6.3%) hemisphere. The anastomotic territory of the medial medullary artery was associated with recurrent hemorrhage before (HR = 2.94; 95% CI, 1.07-8.08; p = 0.037) and after (HR = 6.65; 95% CI, 1.32-33.60; p = 0.022) adjustments were made for confounding factors. CONCLUSIONS: The incidence of recurrent ipsilateral hemorrhage varies with the target anastomotic territory in adult patients with MMD. Medial target medullary artery anastomosis is a significant risk factor for recurrent ipsilateral hemorrhage.

Systematic review and network meta-analysis assess the comparative efficacy and safety of transsphenoidal surgery for pituitary tumor.

To quantitatively synthesize the comparative efficacy and safety of the most common surgical approaches including endonasal transsphenoidal endoscopic surgery (ETES), sublabial transsphenoidal microsurgery (STMS) and endonasal transsphenoidal microsurgery (ETMS) for all kinds of pituitary tumors. This systematic review and network meta-analysis was performed on randomized controlled trials (RCTs) and comparison studies from databases of Pubmed, EMBASE, and the Cochrane library. We selected the rate of gross complete resection as our primary outcome of efficacy. And the incidence of all complications, cerebrospinal fluid (CSF) leak, diabetes insipidus, nasal septal perforation, death, and bleeding were designed as our primary outcomes of safety. Twenty-seven studies with 2618 patients were included in this network meta-analysis. On efficacy, there was no statistical difference among the three methods including ETES, STMS, and ETMS. As for safety, results indicated that the incidence of total complications of STMS (OR = 4.74; 95% CI 1.03, 40.14) is significantly superior to ETES. And the incidence of diabetes insipidus of ETMS (OR = 2.21; 95% CI 1.31, 3.81) was significantly superior to that of ETES. Besides, there was no statistical difference in the other complications including CSF leak, nasal septal perforation, death, and bleeding. We clarified the overpraise of the efficacy of endoscopy especially the endonasal transsphenoidal approach, and verified that all the approaches owned similar efficacy. Moreover, we recommended the endoscopy to be the first choice for pituitary tumors, because it demonstrated the best safety.

Bacillus thuringiensis cry toxin triggers autophagy activity that may enhance cell death.

Although it is well known that Bacillus thuringiensis Cry toxins kill insect pest by disrupting midgut cells of susceptible larvae through their pore formation activity, it is not clear what intracellular events are triggered after pore formation on the cell membrane of the target cells. Here we analyzed the role of Cry toxins on autophagy activation using several cell lines as models as well as in Helicoverpa armigera larvae. The selected insect cell lines (Hi5, Sl-HP and Sf9) were susceptible to activated Cry1Ca toxin, but only Sl-HP cells were also susceptible to activated Cry1Ac toxin. In contrast, the mammalian cell line 293 T was not susceptible to Cry1Ac or to Cry1Ca. Results show that Cry toxins induced autophagy only in the susceptible cell lines as shown by the analysis of the changes in the ratio of Atg8-PE to Atg8 and by formation of autophagosome dots containing Atg8-PE. The Cry1Ac enhanced autophagy in the midgut tissue of H. armigera larvae. Silencing expression of specific genes by RNAi assays confirmed that the autophagy induced by activated Cry toxins was dependent on AMPK and JNK pathways. Finally, inhibition of autophagy in the cell lines by specific inhibitors or RNAi assays resulted in delayed cell death triggered by Cry toxins, suggesting that the increased autophagy activity observed after toxin intoxication may contribute to cell death.

Single Virus Tracking with Quantum Dots Packaged into Enveloped Viruses Using CRISPR.

Labeling viruses with high-photoluminescence quantum dots (QDs) for single virus tracking provides a visual tool to aid our understanding of viral infection mechanisms. However, efficiently labeling internal viral components without modifying the viral envelope and capsid remains a challenge, and existing strategies are not applicable to most viruses. Here, we have devised a strategy using the clustered regularly interspaced short palindromic repeats (CRISPR) imaging system to label the nucleic acids of Pseudorabies virus (PRV) with QDs. In this strategy, QDs were conjugated to viral nucleic acids with the help of nuclease-deactivated Cas9/gRNA complexes in the nuclei of living cells and then packaged into PRV during virion assembly. The processes of PRV-QD adsorption, cytoplasmic transport along microtubules, and nuclear entry were monitored in real time in both Vero and HeLa cells, demonstrating the utility and efficiency of the strategy in the study of viral infection.

Streptococcus suis Serotype 2 Infection Causes Host Immunomodulation through Induction of Thymic Atrophy.

Streptococcus suis serotype 2 is an important bacterial pathogen of swine and is also an emerging zoonotic agent that may be harmful to human health. Although the virulence genes of S. suis have been extensively studied, the mechanisms by which they damage the central immune organs have rarely been studied. In the current work, we wanted to uncover more details about the impact and mechanisms of S. suis on specific populations of thymic and immune cells in infected mice. Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL) assays revealed that S. suis infection induced apoptosis in CD3+, CD14+, and epithelial cells from the thymus. S. suis infection resulted in a rapid depletion of mitochondrial permeability and release of cytochrome c (CytC) and apoptosis-inducing factor (AIF) through upregulation of Bax expression and downregulation of Bcl-xl and Bcl2 expression in thymocytes. Moreover, S. suis infection increased cleavage of caspase-3, caspase-8, and caspase-9. Thus, S. suis induced thymocyte apoptosis through a p53- and caspase-dependent pathway, which led to a decrease of CD3+ cells in the thymus, subsequently decreasing the numbers of CD4+ and CD8+ cells in the peripheral blood. Finally, expression dysregulation of proinflammatory cytokines in the serum, including interleukin 2 (IL-2), IL-6, IL-12 (p70), tumor necrosis factor (TNF), and IL-10, was observed in mice after S. suis type 2 infection. Taken together, these results suggest that S. suis infection can cause atrophy of the thymus and induce apoptosis of thymocytes in mice, thus likely suppressing host immunity.

Host BAG3 Is Degraded by Pseudorabies Virus pUL56 C-Terminal 181L-185L and Plays a Negative Regulation Role during Viral Lytic Infection.

Bcl2-associated athanogene (BAG) 3, which is a chaperone-mediated selective autophagy protein, plays a pivotal role in modulating the life cycle of a wide variety of viruses. Both positive and negative modulations of viruses by BAG3 were reported. However, the effects of BAG3 on pseudorabies virus (PRV) remain unknown. To investigate whether BAG3 could modulate the PRV life cycle during a lytic infection, we first identified PRV protein UL56 (pUL56) as a novel BAG3 interactor by co-immunoprecipitation and co-localization analyses. The overexpression of pUL56 induced a significant degradation of BAG3 at protein level via the lysosome pathway. The C-terminal mutations of 181L/A, 185L/A, or 181L/A-185L/A in pUL56 resulted in a deficiency in pUL56-induced BAG3 degradation. In addition, the pUL56 C-terminal mutants that lost Golgi retention abrogated pUL56-induced BAG3 degradation, which indicates a Golgi retention-dependent manner. Strikingly, BAG3 was not observed to be degraded in either wild-type or UL56-deleted PRV infected cells as compared to mock infected ones, whereas the additional two adjacent BAG3 cleaved products were found in the infected cells in a species-specific manner. Overexpression of BAG3 significantly suppressed PRV proliferation, while knockdown of BAG3 resulted in increased viral yields in HEK293T cells. Thus, these data indicated a negative regulation role of BAG3 during PRV lytic infection. Collectively, our findings revealed a novel molecular mechanism on host protein degradation induced by PRV pUL56. Moreover, we identified BAG3 as a host restricted protein during PRV lytic infection in cells.

Two Residues in NSP9 Contribute to the Enhanced Replication and Pathogenicity of Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus.

Highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) possesses greater replicative capacity and pathogenicity than classical PRRSV. However, the factors that lead to enhanced replication and pathogenicity remain unclear. In our study, an alignment of all available full-length sequences of North American-type PRRSVs (n = 204) revealed two consistent amino acid mutations that differed between HP-PRRSV and classical PRRSV and were located at positions 519 and 544 in nonstructural protein 9. Next, a series of mutant viruses with either single or double amino acid replacements were generated from HP-PRRSV HuN4 and classical PRRSV CH-1a infectious cDNA clones. Deletion of either of the amino acids led to a complete loss of virus viability. In both Marc-145 and porcine alveolar macrophages, the replicative efficiencies of mutant viruses based on HuN4 were reduced compared to the parent, whereas the replication level of CH-1a-derived mutant viruses was increased. Plaque growth assays showed clear differences between mutant and parental viruses. In infected piglets, the pathogenicity of HuN4-derived mutant viruses, assessed through clinical symptoms, viral load in sera, histopathology examination, and thymus atrophy, was reduced. Our results indicate that the amino acids at positions 519 and 544 in NSP9 are involved in the replication efficiency of HP-PRRSV and contribute to enhanced pathogenicity. This study is the first to identify specific amino acids involved in PRRSV replication or pathogenicity. These findings will contribute to understanding the molecular mechanisms of PRRSV replication and pathogenicity, leading to better therapeutic and prognostic options to combat the virus.IMPORTANCE Porcine reproductive and respiratory syndrome (PRRS), caused by porcine reproductive and respiratory syndrome virus (PRRSV), is a significant threat to the global pig industry. Highly pathogenic PRRSV (HP-PRRSV) first emerged in China in 2006 and has subsequently spread across Asia, causing considerable damage to local economies. HP-PRRSV strains possess a greater replication capacity and higher pathogenicity than classical PRRSV strains, although the mechanisms that underlie these characteristics are unclear. In the present study, we identified two mutations in HP-PRRSV strains that distinguish them from classical PRRSV strains. Further experiments that swapped the two mutations in an HP-PRRSV strain and a classical PRRSV strain demonstrated that they are involved in the replication efficiency of the virus and its virulence. Our findings have important implications for understanding the molecular mechanisms of PRRSV replication and pathogenicity and also provide new avenues of research for the study of other viruses.