Expression characteristics and in vitro antibacterial properties of C-type lysozyme in crucian carp infected with Aeromonas salmonicida.

Aeromonas salmonicida is an ancient fish pathogen. Lysozymes are important molecules in the innate immune system that fight bacterial infections. The expression characteristics of C-type lysozyme in crucian carp infected with A. salmonicida and its antibacterial effect against A. salmonicida had not been investigated. Thus, we used bioinformatics to analyze the gene and protein characteristics of C-type lysozymes in crucian carp. Changes in C-type lysozyme expression before and after crucian carp infection with A. salmonicida were detected, and the in vitro antibacterial effect of recombinant carp C-type lysozyme on A. salmonicida was validated. The results showed that the coding DNA sequence region of the lysozyme gene sequence was 438 bp long, encoding 145 amino acids and containing two conserved catalytic sites: Glu53 and Asp69. Phylogenetic analysis revealed that crucian carp C-type lysozymes clustered with Cyprinus carpio lysozyme C. After crucian carp were infected with A. salmonicida, the gene and protein expression of C-type lysozymes in the liver, spleen, kidney, and hindgut were significantly upregulated, with the liver showing the highest upregulation that was 15 times higher than that in the uninfected group. In addition, recombinant C-type lysozyme exhibited significant antibacterial activity against A. salmonicida, with an average inhibition zone radius of 0.92 cm when using 40 µg recombinant lysozyme. In conclusion, this study reveals the important role of C-type lysozymes in the innate immune response of crucian carp and provides a theoretical basis for preventing crucian carp infection with A. salmonicida.

Comparative transcriptomics and histopathological analysis of crucian carp infection by atypical Aeromonas salmonicida.

Aeromonas salmonicida is a ubiquitous fish pathogen known to cause furunculosis. With the emergence of new subtypes and the expansion of the host range, it has threatened the health of a variety of marine and freshwater fish, particularly the non-salmonids, manifesting differently from the classical furunculosis. Although there have been reports of infection by atypical strains on the crucian carp, the pathogenesis and tissue pathology remain unclear. In this study, transcriptomics and histopathology were used to analyze the immune response and lesions of crucian carp infected with A. salmonicida. Comparative analysis showed 6579 differentially expressed genes (DEGs) (3428 down-regulated and 3151 up-regulated) were identified on day 5 post-infection (5 dpi). Further annotation and analysis revealed that the DEGs were enriched in enzyme regulator activity, response to oxidative stress, iron ion homeostasis and other functions, and mitogen-activated protein kinase (MAPK), nuclear factor-κB (NF-κB), toll-like receptor (TLR), and nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) etc., and immune-related signaling pathways. Meanwhile, the four C-type lysozyme genes found in all DEGs were significantly up-regulated after infection. In addition, there was severe bleeding on the body of the infected fish. Also, the intestine, liver, spleen, and kidney showed varying degrees of inflammatory damage, especially the goblet cell hyperplasia of intestinal mucosa epithelium and degeneration and necrosis of renal tubular epithelium cells. Additionally, with the increase in pathogen concentration, the cumulative mortality increased, the severity of lesions in the hindgut and head-kidney tissues increased. The relative expression levels of four immune-related genes (TNF-α, IL-1ß, IL-11, C-lysozyme) were also significantly upregulated, compared with the control (P < 0.05). In conclusion, this study provides a scientific basis for further study on the immune response, pathological diagnosis, and prevention of crucian carp infection caused by atypical A. salmonicida.

MicroRNA-421 promotes proliferation and invasion of non-small cell lung cancer cells through targeting PDCD4.

Recent evidence highlights that microRNAs serve as crucial regulators of tumorigenesis, including non-small cell lung cancer (NSCLC). The present study was designed to investigate the expression profile, clinical significance and biological role of miR-421 in NSCLC. The results showed that miR-421 expression was markedly increased in NSCLC tissues and cell lines. Further experimental data indicated that knockdown of miR-421 significantly inhibited NSCLC cell proliferation and induced cell cycle arrest in vitro. The migratory and invasive abilities of NSCLC cells were also attenuated following miR-421 knockdown. Furthermore, PDCD4 was identified as a direct target of miR-421, and its expression was inversely correlated with miR-421 expression in NSCLC tissues. PDCD4 also abrogated the oncogenic role of miR-421 in NSCLC cells. Collectively, our study revealed that miR-421 is significantly upregulated in NSCLC and might represent a potential therapeutic target for NSCLC patients.

A recombinant adenovirus targeting typical Aeromonas salmonicida induces an antibody-mediated adaptive immune response after immunization of rainbow trout.

Aeromonas salmonicida, the oldest known fish pathogen and currently endemic throughout most of the world in both fresh and marine waters, causes severe economic losses to the salmon farming industry. Although there have been many studies on the prevention of furunculosis over the past few decades, it is still prevalent in many fisheries. In this study, a recombinant adenovirus vaccine candidate harboring the highly immunogenic Vapa gene (pAd-easy-cmv-Vapa) was successfully constructed and tested. The immune protection rate and specific antibody levels in the peripheral blood were then determined after immunizing rainbow trout. In addition, relative levels of IgM and IgT in the head kidney and hindgut before and after immunization were measured by quantitative reverse transcription PCR. Western blotting results indicated that the recombinant adenovirus could infect HEK-293 cells and express the A layer protein (encoded by Vapa). Further, survival analysis of fish 28 days after challenge showed that immunization significantly lowered the mortality rate (40%) compared to that in the control group (76.6%) and empty vector group (73.6%). This also led to an increase in specific antibodies in peripheral serum. In addition, levels of IgM and IgT in the head kidney and hindgut were increased to varying degrees. In conclusion, our research provides a candidate vaccine for the prevention of Aeromonas salmonicida A450 infection in rainbow trout and lays the foundation for future research on adaptive immune mechanisms associated with rainbow trout antibodies.

Effects of Bombyx mori nuclear polyhedrosis virus on serpin and antibacterial peptide expression in B. mori.

The silkworm (Bombyx mori) is a typical and economically important lepidopteran species, and research has resulted in the development and accumulation of breeding lines. Studies of immune-related silkworm genes not only promote our understanding of silkworm immune response mechanisms, but they also inform insect immune molecular diversity research. Here, silkworm proteins were screened using proteomics after Bombyx mori nuclear polyhedrosis virus (BmNPV) infection, and 2368 silkworm proteins were identified, including six antimicrobial peptides and 12 serpins. The mRNA expression levels of these 18 proteins were examined at different times. The results indicated that attacin had the highest expression level, while serpin-5 and cecropin-D exhibited a negative regulatory correlation. These results provide a significant step toward a deeper understanding of B. mori immunoregulation.

Baculoviral infection reduces the expression of four allergen proteins of silkworm pupa.

Silkworm (Bombyx mori) larvae are widely used to express exogenous proteins. Moreover, some silkworm pupal proteins can be used as drug-loading materials for selfexpressed oral tolerance drugs. However, several proteins expressed in silkworm pupae cause severe allergic reactions in humans and animals. Interestingly, some baculovirus vectors have been shown to alter the host gene and its expression in insect cells, but this has not been confirmed in silkworm. Here, we analyzed the effects of infection with an empty B. mori baculovirus (BmNPV) vector on silkworm pupal protein expression. Using a proteomics approach, the allergens thiol peroxiredoxin (Jafrac1), 27-kDa glycoprotein (p27k), arginine kinase, and paramyosin as well as 32 additional differentially expressed proteins were identified. Downregulation of the messenger RNA expression of the four known allergens was observed after BmNPV infection; subsequent changes in protein expression were confirmed by the western blot analysis using polyclonal antibodies prepared with recombinant proteins of the four allergens. Collectively, these data indicate that the four known allergens of silkworm pupae can be reduced by infection ith an empty BmNPV vector to increase the safety of silkworm pupa-based exogenous protein expression and drug delivery of oral pharmaceuticals. In addition, the four recombinant allergen proteins may contribute to the diagnosis of allergic diseases of silkworm pupa.

Cementless Total Hip Arthroplasty With a High Hip Center for Hartofilakidis Type B Developmental Dysplasia of the Hip: Results of Midterm Follow-Up.

BACKGROUND: Acetabular reconstruction in adults with Hartofilakidis type B developmental dysplasia of the hip is a major technical challenge. The purpose of this retrospective study was to evaluate hip function and radiographic outcomes regarding high hip center at midterm follow-up. METHODS: From January 1, 2007 to December 31, 2009, 37 patients who had Hartofilakidis type B developmental dysplasia of the hip underwent a primary total hip arthroplasty using a high hip center technique. Functional, radiographic, and survivorship outcomes were evaluated. RESULTS: Of the 37 patients, 31 patients (83.8%) were available for the mean follow-up of 6.1 years (range, 1.5-7.6 years). Thirty-one cementless cups were located at an average vertical distance of 38.1 ± 3.3 mm and at a mean horizontal distance of 35.5 ± 3.4 mm. The mean ratio of the height of the hip center was 2.4% (range, 2.0%-2.9%). The Harris Hip Scores were improved from 50.3 points (range, 38-63 points) preoperatively to 92.3 points (range, 85-100 points) at the final follow-up (P < .001). Four patients continued to present with Trendelenburg gait pattern at the last follow-up. With use of revision for any reason and aseptic loosening as the end point, the 5-year survival rates were 90.3% (95% CI, 79.9%-100%) and 93.3% (95% CI, 84.3%-100%), respectively. CONCLUSIONS: The high hip center technique in conjunction with a cementless acetabular component seems to be a valuable alternative to achieve satisfactory midterm outcomes for Hartofilakidis type B developmental dysplasia of the hip.

Biomechanical effects of pedicle screw adjustments on the thoracolumbar burst fractures.

BACKGROUND: Posterior pedicle screw device is widely used in treatment of thoracolumbar burst fractures. As the clinical operation is not based upon quantitative data of adjustments, the results are not optimal. At present, no study has assessed the associations between the device adjustments and the restoration of stiffness. We investigated the biomechanical effects that adjustments of a pedicle screw device had on the burst fracture, and explored an optimal adjustment. METHODS: Burst fractures were produced at L1 vertebra in 24 fresh calf spines (T12-L3). The specimens were divided into four groups at random. Pedicle screw devices were attached to T13 and L2. Four device adjustments, consisting of distraction and extension, were applied. Adjustment 1 was pure 6° extension, adjustment 2 was pure 5 mm distraction, adjustment 3 was 6° extension followed by 5 mm distraction, and adjustment 4 was 5 mm distraction followed by 6° extension. The effect of each adjustment on the stiffness restoration, anatomical reduction, and neural decompression for the burst fractures was analyzed and evaluated. RESULTS: Pure extension (Group 1) produced the closest segment height and the least restoration of the canal to the intact. Pure distraction (Group 2) restored stiffness most, but with only 60% stiffness of the intact value, and lost the segmental angle most to the intact. The combination of extension-distraction (Group 3 and Group 4) produced the maximum reduction of the anatomy and restoration of the canal in the burst fracture, and the least stiffness restoration. The sequence of extension and distraction did not affect stiffness restoration, anatomical reduction, and neural decompression. CONCLUSIONS: The device adjustments affected stiffness restoration, anatomical reduction, and neural decompression. The combined extension-distraction adjustment may be the most suitable considering the anatomical reduction and neural decompression, but the stiffness decreased the most; it should be considered to reconstruct L1 vertebra.