Efficient cross-protection against serotype 4/8a fowl adenoviruses (FAdVs): recombinant FAdV-4 with FAdV-8a Fiber.

IMPORTANCE: Epidemiological data reveal that FAdV-4 and FAdV-8a are the dominant serotypes of FAdVs in the poultry industry in China. Although three commercial inactivated vaccines against FAdV-4 have been licensed in China, the bivalent vaccine against both FAdV-4 and FAdV-8a is not available. Here, we used CRISPR-Cas9 and Cre-LoxP system to generate a recombinant virus FAdV4-F/8a-rF2 expressing the Fiber of FAdV-8a. Notably, FAdV4-F/8a-rF2 was highly attenuated and could provide efficient protection against both FAdV-4 and FAdV-8a in the chicken infection model, highlighting the applaudable application of FAdV4-F/8a-rF2 as a novel live-attenuated bivalent vaccine against the diseases caused by the infection of FAdV-4 and FAdV-8a.

An efficient double-fluorescence approach for generating fiber-2-edited recombinant serotype 4 fowl adenovirus expressing foreign gene.

Recently, the infection of serotype 4 fowl adenovirus (FAdV-4) in chicken flocks has become endemic in China, which greatly threatens the sustainable development of poultry industry. The development of recombinant FAdV-4 expressing foreign genes is an efficient strategy for controlling both FAdV-4 and other important poultry pathogens. Previous reverse genetic technique for generating the recombinant fowl adenovirus is generally inefficient. In this study, a recombinant FAdV-4 expressing enhanced green fluorescence protein (EGFP), FA4-EGFP, was used as a template virus and directly edited fiber-2 gene to develop an efficient double-fluorescence approach to generate recombinant FAdV-4 through CRISPR/Cas9 and Cre-Loxp system. Moreover, using this strategy, a recombinant virus FAdV4-HA(H9) stably expressing the HA gene of H9N2 influenza virus was generated. Chicken infection study revealed that the recombinant virus FAdV4-HA(H9) was attenuated, and could induce haemagglutination inhibition (HI) titer against H9N2 influenza virus at early time points and inhibit the viral replication in oropharynx. All these demonstrate that the novel strategy for constructing recombinant FAdV-4 expressing foreign genes developed here paves the way for rapidly developing attenuated FAdV-4-based recombinant vaccines for fighting the diseases caused by both FAdV-4 and other pathogens.

A novel recombinant serotype 4 fowl adenovirus expressing fiber-2 protein of duck adenovirus 3.

Recently, the highly pathogenic serotype 4 fowl adenovirus (FAdV-4) and duck adenovirus 3 (DAdV-3) were outbroken and widespread, causing substantial economic losses to the duck industry. Therefore, there is an urgent need to generate a recombinant genetic engineering vaccine candidate against both FAdV-4 and DAdV-3. In this study, a novel recombinant FAdV-4 expressing the Fiber-2 protein of DAdV-3, designated as rFAdV-4-Fiber-2/DAdV-3, was generated based on CRISPR/Cas9 and Cre-LoxP systems. Indirect immunofluorescence assay (IFA) and western blot (WB) showed that the Fiber-2 protein of DAdV-3 in rFAdV-4-Fiber-2/DAdV-3 was expressed successfully. Moreover, the growth curve revealed that rFAdV-4-Fiber-2/DAdV-3 replicated efficiently in LMH cells and even showed a stronger replication ability compared to the wild type FAdV-4. The generation of the recombinant rFAdV-4-Fiber-2/DAdV-3 provides a potential vaccine candidate against both FAdV-4 and DAdV-3.

[Seasonal Variations, Size Distributions, and Sources of Chemical Components of Submicron Particulate Matter in Nanjing].

Size-segregated particulate matter (PM) samples were collected in different seasons from 2016 to 2017 at the Xianlin Campus of Nanjing University. Mass concentrations of water-soluble inorganic ions, carbonaceous components, and elements were analyzed for PM with an aerodynamic diameter ≤ 1.1 µm (PM1.1; <0.4 µm, 0.4-0.7 µm, and 0.7-1.1 µm). The results showed that PM1.1, OC, NO3-, SO42-, and NH4+ exhibited higher ambient levels in fall-winter than those in spring-summer, which was attributed to the changes in local diffusion conditions, evaporation, and decomposition of non-refractory components. Elemental carbon (EC) reached its maximum concentration[(1.87±0.98) µg·m-3]in spring due to the increase in industrial and road dust resuspension. According to the characteristic ratio between bulk components, the anions in PM1.1 were dominated by NO3-, SO42-, and Cl- in Nanjing, and the carbonaceous components were mainly from fossil fuel combustions and associated aging processes. As the ambient temperature increased, the size distributions of thermo-unstable components including NH4+, NO3-, and OC shifted towards finer particles, whereas EC became more enriched in coarse particles, possibly due to the increase in emission intensity of motor vehicles and fugitive dust contributions. Since high relative humidity (>70%) is often accompanied by high temperature (>20℃) and improved diffusion conditions, a relative humidity of 60%-70% was more conducive to the formation of secondary inorganic ions in PM1.1. Source apportionment results based on the speciation data of PM1.1 showed that secondary formation processes[(66.6±18.3)%]and dust resuspension[(16.8±14.8)%]were the main contributors to PM1.1 in Nanjing, and further control of the emissions of gaseous precursors and dust is necessary.

Electro-enhanced sulfamethoxazole degradation efficiency via carbon embedding iron growing on nickel foam cathode activating peroxymonosulfate: Mechanism and degradation pathway.

The combination of peroxymonosulfate (PMS) activation by hetero-catalysis and electrolysis (EC) attracted incremental concerns as an efficient antibiotics degradation method. In this work, carbon embedding iron (C@Fe) catalysts growing on nickel foam (NF) composite cathode (C@Fe/NF) was prepared via in-situsolvothermal growth and carbonization method and used to activate PMS toward sulfamethoxazole (SMX) degradation. The EC-[C@Fe/NF(II)]-PMS system exhibited an excellent PMS activation, with 100% SMX removal efficiency achieving within 30 min. Reactive oxygen species (ROS) generation and their roles in SMX degradation were confirmed by quenching experiments and electron paramagnetic resonance. It was found that singlet oxygen (1O2) and surface-bound radicals were responsible for SMX degradation, and 1O2 contributed the most. Furthermore, the possible SMX degradation pathways were proposed on the base of the detected degradation intermediates and density functional theory (DFT) calculation. Toxicity changes were also assessed by the Ecological Structure Activity Relationships (ESAR). This work provides a practicable strategy for synergistically enhancing PMS activation efficiency and promoting antibiotics removal.

An Efficient and Rapid Assay for Detecting Neutralizing Antibodies Against Serotype 4 Fowl Adenovirus.

Currently, the outbreak of serotype 4 fowl adenovirus (FAdV-4) has spread worldwide and caused tremendous economic loss to the poultry industry. Although inactivated vaccines have been licensed against FAdV-4 in China, a rapid and efficient serological method for measuring the titer of neutralizing antibodies (NAbs) specific for FAdV-4 post-infection or vaccination is rarely reported. Classical virus neutralization test (VNT) is superior in sensitivity and specificity for detecting NAbs but is either time-consuming or laborious. In this study, a recombinant virus FA4-EGFP expressing EGFP-fiber-2 fusion protein, rather than wild type (WT) FAdV-4 was used to develop a novel VNT for detecting FAdV-4 NAbs. Specificity analysis showed that the approach only reacted with the sera against FAdV-4, not with the sera against other avian pathogens tested. The novel VNT was effective in the detection of NAbs against FAdV-4 in sera from both experimentally infected and clinically vaccinated chickens, and had good linear correlation with the classical VNT. Moreover, the novel VNT not only significantly simplifies the procedure for detection of NAbs, but also shortens the timeline to 24 h in comparison with the classical VNT with 3-4 d. All these data demonstrate that the FA4-EGFP based VNT developed here provides an efficient diagnostic method for monitoring the immunological state of the vaccination or diagnosing the clinical infection of FAdV-4 in a quick and funding-saving manner.

Fiber-1 of serotype 4 fowl adenovirus mediates superinfection resistance against serotype 8b fowl adenovirus.

In recent years, hepatitis-hydropericardium syndrome (HHS) and inclusion body hepatitis (IBH) caused by serotype 4 fowl adenovirus (FAdV-4) and serotype 8b fowl adenovirus (FAdV-8b), respectively, are widely prevalent in China, causing huge economic losses to the poultry industry. Numerous studies have revealed the mechanism of the infection and pathogenesis of FAdV-4. However, little is known about the mechanism of infection with FAdV-8b. Among the major structural proteins of fowl adenoviruses, fiber is characterized by the ability to recognize and bind to cellular receptors to mediate the infection of host cells. In this study, through superinfection resistance analysis and an interfering assay, we found that Fiber-1 of FAdV-4, rather than hexon, penton, and fiber of FAdV-8b, conferred efficient superinfection resistance against the infection FAdV-8b in LMH cells. Moreover, truncation analysis depicted that the shaft and knob domains of FAdV-4 Fiber-1 were responsible for the inhibition. However, knockout of the coxsackie and adenovirus receptor (CAR) in LMH cells inhibited the replication of FAdV-8b only at early time points, indicating that CAR might not be the key cell receptor for FAdV-8b. Overall, our findings give novel insights into the infection mechanism of FAdV-8b and provide a new target for the prevention and control of both FAdV-4 and FAdV-8b.

Reconstruction of soft tissue defect after maxillectomy of ameloblastic carcinoma with submental island flap: a case report and literature review.

Ameloblastic carcinoma (AC) is an extremely rare malignant odontogenic tumor. The mean age of occurrence for all 141 AC cases analyzed in our systematic review study was 43.59±19.51 years. Males were more affected than females, and the mandible was predominantly affected compared with the maxilla. The main clinical manifestation was a painful or painless swelling with ulceration and radiographic features usually displayed as mixed cystic or solid changes. Surgical resection was the first recommended method of management. Fourteen cases had cervical lymph node spread, 19 had distant metastasis (most commonly in the lung), and 33 had recurrence. We present a rare case of AC involving the maxillary region. Locally extensive surgical resection was carried out. Ablative defects after maxillectomy resulted in the communication of oral cavity and nasal cavity/maxillary antrum and would bring about difficulties in mastication, deglutition, and speech. A submental island flap was applied to close the oronasal and oroantral fistula. The flap and the wounds healed well, with excellent outcomes in terms of appearance, the function of speech, and swallowing on follow up. The submental island flap provides a relatively thin, easy-to-harvest, and well-vascularized tissue, which makes it a reliable option in soft tissue reconstruction of the oral and maxillofacial region.

A gene signature associated with prognosis and immune processes in head and neck squamous cell carcinoma.

BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) has a poor prognosis that has not significantly improved in the past several decades. A prognostic-related signature was needed. METHODS: The Cancer Genome Atlas and GSE41613 databases were downloaded as a training and validation set, respectively. We identified 12 genes that demonstrated progression and prognostic value, and then, a gene signature was constructed. RESULTS: This classification could reflect distinct characteristics, phenotypically and molecularly, among HNSCC tumors. It could stratify patients with significantly different survival rates (median survival: 2083 days vs 927 days; P = 3.85E-08) in the training cohort and validation cohort (P = 0.007) and was significantly involved in immune/inflammatory response and tumor progression processes. CONCLUSIONS: This bioinformatics-based signature suggested the presence of two distinct populations of patients with HNSCC with distinguishable phenotypic characteristics and clinical outcomes and might provide insight for new types of immune therapy.

[Combined application of non-specific activated immunocytes and cisplatin to treat hemangioendothelioma EOMA xenografts].

OBJECTIVE: To investigate the possibility of combined non-specific activated splenocytes (aSplenocytes) and cisplatin in treating murine hemangioendothelioma EOMA cell line. METHODS: The effects of cisplatin, aSplenocytes or both on EOMA cells were measured by MTT assay and trypan blue exclusive method, respectively. EOMA-bearing mice were established by subcutaneous inoculation of 1.5x10(6) EOMA cells in 6-week old male BALB/c nu/nu mice, divided into untreated control, cisplatin group, aSplenocytes group, and combination group, 5 mice per group. Tumor was treated with 1.5x10(6) aSplenocytes per 100 microL via intratumoral injection, and 24 hours later, followed by cisplatin treatment via intraperitoneal injection. Cisplatin treatment was repeated once a week at the dose of 8 mg/kg of body weight. Tumor volume was measured at indicated time. RESULTS: Both cisplatin and aSplenocytes had inhibitory effects on EOMA cells. 47.7%+/-5.9% EOMA cells were found in 100 micromol/L cisplatin-treated EOMA cells for 24 hours. 42.8%+/-2.6% and 45.3%+/-2.2% EOMA cells were in survival in 1x10(5) /mL aSplenocytes-treated EOMA cells in cell-cell direct contact co-culture system and in transwell system for 48 hours, respectively. When EOMA cells were sensitized with 1x10(5) aSplenocytes in cell-cell direct contact system or in transwell system for 24 hours, followed with 100 micromol/L cisplatin for additional 24 hours, only 25.0%+/-2.7% and 27.5%+/-3.8% EOMA cells were in survival. Compared with single-agent alone treatments, aSplenocytes plus cisplatin significantly inhibited the growth of EOMA cells (P<0.05). There was no significant difference in EOMA cell survival between the two co-culture systems (P>0.05). In vivo assay showed the tumor volume was (680.3+/-68.0) mm3, (825.2+/-71.8) mm3, (535.3+/-74.5) mm3 and (351.5+/-79.5) mm3 in the control, aSplenocytes, cisplatin and aSplenocytes plus cisplatin group, respectively, on the 18 th day after treatment. aSplenocytes plus cisplatin markedly delayed EOMA tumor growth (P<0.05). CONCLUSION: aSplenocytes could enhance the cytotoxicity of cisplatin in EOMA cells in vitro and in vivo.

[A preliminary study of murine hemangioma model by delivery of recombinant adeno-associated virus mediated human vascular endothelial growth factor-121 gene].

OBJECTIVE: To investigate the feasibility of establishing a murine hemangioma model with injection of recombinant adeo-associated virus mediated human vascular endothelial growth factor-121 (rAAV-hVEGF(121)) gene. METHODS: rAAV-hVEGF(121) was constructed, identified and then implanted to the left back ear of each mouse (1.0 x 10(11)VG in 50 microl per mouse and 10 nude mice received the injection), the rights served as controls with an injection of the same volume of phosphate buffered solution (PBS). The skin color and swelling of left back ear were observed every other day. Histological examination was carried out after mice were sacrificed 2, 4, 6, 8, 12 weeks after injection. RESULTS: The rAAV-hVEGF(121) was correctly constructed and confirmed by restriction endonuclease analysis, polymerase chain reaction and DNA sequencing analysis. The skin of left back ear became red 2 weeks after injection and gradually exhibited a red lump which was at its utmost 12 weeks after injection. Such phenomena were not observed in right back ear. Histological examinations showed aggregates of endothelial cells by 2 weeks and at 8 weeks the swollen tissue contained many cysts filled with a mass of red cells. CD-34 staining suggested most of the newly-formed cells were endothelial cells. CONCLUSIONS: A hemangioma model was established in mice with injection of recombinant rAAV-hVEGF(121) gene.

[Diagnosis and therapy on hemangiomas and vascular malformation in view of the new classification].

Vascular birthmarks are the most common disease. The morbidity is about 2.5%, most of the lesions occur in oral and maxillofacial regions which accounts for 40%-60% of the total lesions. In 1982, Mulliken and Glowacki proposed a biologic classification of vascular birthmarks on the basis of their clinical manifestations, histopathological features, and natural history. They defined hemangiomas as vascular tumors with a growth phase, marked by endothelial proliferation and hypercellularity, and an involutional phase. They recognized that many entities referred to as hemangiomas are actually structural malformations of the vasculature, derived from capillaries, veins, lymph vessels, or arteries or from a combination of these sources. The classification was confirmed and issued by International Society for the study of vascular anomaly (ISSVA) in 1988. Waner and Suen amended the above category in 1995. This paper presents the new classification of vascular birthmarks and the developments in this field in recent years, including the pathology, clinical features and the therapy. For example, the classification of venular malformation categorized by Waner in 1989; the classification of lymphous malformation by Waner and Suen in 1995; and the treatments according to above classifications.

[Therapeutic effects of Avastin on the murine hemangioendothelioma].

OBJECTIVE: Investigated the efficacy of Avastin on murine hemangioendothelioma in vitro and in vivo using a mouse hemangioendothelioma-derived cell line----EOMA. METHODS: The in vitro effect of Avastin on cell proliferation of EOMA cell line was measured by CCK-8 assay at 0, 50, 100, 200 mg/L concentration of Avastin. When tumors produced by subcutaneous injection of EOMA cells reached a volume of 100 mm(3), animals were treated by intra-tumor injection with Avastin (1 mg/kg body weight) or with vehicle alone (PBS) twice a week. Mice were weighted and tumors were measured three times weekly. At the end of the experiment, the mice were sacrificed and their tumors were excised and processed for histology. Immunohistochemical study of apoptosis was conducted using a TUNEL kit, tumor cell proliferation was assessed with anti-proliferating cell nuclear antigen (PCNA) antibody. Cardiac puncture was performed under deep anesthesia for collection of serum, serum vascular endothelial growth factor (VEGF) level was tested by VEGF-ELISA assay. RESULTS: Avastin exhibited cell inhibited rate of 24.21%, 26.26% and 34.58% at 50, 100 and 200 mg/L, respectively. When experiment was terminated, the tumor volume in the PBS-treated mice was (1 860.10+/-146.96) mm3, being significantly larger than that in the mice that were treated by Avastin [(681.45+/-63.01) mm3, P<0.01]. Avastin-treated tumors showed decreased tumor cell proliferation and increased cell apoptosis. The VEGF level in mice treated with Avastin [(594.65+/-118.79) ng/L] was significantly lower than that in PBS-treated mice [(802.24+/-238.41) ng/L, P<0.05]. CONCLUSION: The results suggest that topically applied Avastin provides an effective and safe approach to treat hemangioendotheliomas and might be used as a novel treatment of angiomatous diseases in the future.