Biochemical characterization of a recombinant Japanese encephalitis virus RNA-dependent RNA polymerase

<p>Abstract</p> <p>Background</p> <p>Japanese encephalitis virus (JEV) NS5 is a viral nonstructural protein that carries both methyltransferase and RNA-dependent RNA polymerase (RdRp) domains. It is a key component of the viral RNA replicase complex that presumably includes other viral nonstructural and cellular proteins. The biochemical properties of JEV NS5 have not been characterized due to the lack of a robust <it>in vitro </it>RdRp assay system, and the molecular mechanisms for the initiation of RNA synthesis by JEV NS5 remain to be elucidated.</p> <p>Results</p> <p>To characterize the biochemical properties of JEV RdRp, we expressed in <it>Escherichia coli </it>and purified an enzymatically active full-length recombinant JEV NS5 protein with a hexahistidine tag at the N-terminus. The purified NS5 protein, but not the mutant NS5 protein with an Ala substitution at the first Asp of the RdRp-conserved GDD motif, exhibited template- and primer-dependent RNA synthesis activity using a poly(A) RNA template. The NS5 protein was able to use both plus- and minus-strand 3'-untranslated regions of the JEV genome as templates in the absence of a primer, with the latter RNA being a better template. Analysis of the RNA synthesis initiation site using the 3'-end 83 nucleotides of the JEV genome as a minimal RNA template revealed that the NS5 protein specifically initiates RNA synthesis from an internal site, U₈₁, at the two nucleotides upstream of the 3'-end of the template.</p> <p>Conclusion</p> <p>As a first step toward the understanding of the molecular mechanisms for JEV RNA replication and ultimately for the <it>in vitro </it>reconstitution of viral RNA replicase complex, we for the first time established an <it>in vitro </it>JEV RdRp assay system with a functional full-length recombinant JEV NS5 protein and characterized the mechanisms of RNA synthesis from nonviral and viral RNA templates. The full-length recombinant JEV NS5 will be useful for the elucidation of the structure-function relationship of this enzyme and for the development of anti-JEV agents.</p

Seroprevalence and risk factors of Besnoitia besnoiti infection in Korean cattle – short communication

Besnoitia besnoiti is an obligate intracellular parasite that is transmitted by direct contact or via mechanical transmission by flies as vectors. Besnoitiosis causes economic losses in the cattle industry and is regarded as a re-emerging disease in Europe. This study evaluated the seroprevalence of B. besnoiti in Korean cattle using a commercial ELISA kit. Among 558 serum samples, 19 (3.4%) tested seropositive for B. besnoiti. The statistically significant risk factors included age (≥ 2 years), sex (castrated males), and region (lower latitudes) (P < 0.05). The overall seroprevalence suggested a wide distribution of B. besnoiti infection in cattle reared in Korea. Thus, the practice of intensive cattle husbandry and the regionally different seroprevalence of B. besnoiti infection in cattle in Korea warrant routine monitoring and vector control to reduce economical losses due to bovine besnoitiosis in the country

Endotracheal intubation in rabbits using a video laryngoscope with a modified blade

Rabbits are being increasingly used as companion animals, and in research; thus, the need for proper veterinary care for rabbits has increased. Surgical access is more challenging in rabbits under inhalation anesthesia compared to other animals, such as dogs and cats. Rabbits have a very narrow and deep oral cavity, large incisors, and a large tongue. Moreover, their temporomandibular joint has limited mobility, making it more difficult to approach the larynx. Various methods have been proposed to overcome this difficulty. The video laryngoscope was introduced in 1999 and is useful when airway intubation is unsuccessful using a conventional laryngoscope. We postulated that a video laryngoscope with a modified size 1 Macintosh blade (McGrath MAC Video Laryngoscope, Medtronic, USA) would facilitate the intubation of New Zealand White rabbits. Sixteen specific-pathogen-free male New Zealand White rabbits weighing 3.45–4.70 kg were studied. All rabbits were intubated using the video laryngoscope. Typically, a 3.0 mm endotracheal tube was used for rabbits weighing  4 kg. During surgery, anesthesia was well maintained, and there were no major abnormalities in the animals’ conditions. No rabbit developed breathing difficulties or anorexia after recovering from anesthesia. We established an intubation method using a video laryngoscope with a modified blade and stylet in the supine (ventrodorsal) position and successfully applied it in 16 rabbits. It is useful for training novices and for treating rabbits in veterinary hospitals with few staff members and animal research facilities where there are insufficient human resources

Expression of GA733-Fc Fusion Protein as a Vaccine Candidate for Colorectal Cancer in Transgenic Plants

The tumor-associated antigen GA733 is a cell-surface glycoprotein highly expressed in colorectal carcinomas. In this study, 3 recombinant genes were constructed as follows: GA733 tagged to the ER retention sequence KDEL (GA733K), GA733 fused to the immunoglobulin Fc fragment (GA733-Fc), and GA733-Fc fused to the ER retention sequence (GA733-FcK). Agrobacterium-mediated transformation was used to generate transgenic plants expressing recombinant genes. The presence of transgenes was confirmed by genomic PCR. Western blot, confocal immunofluorescence, and sandwich ELISA showed the expression of recombinant proteins. The stability, flexibility, and bioactivity of recombinant proteins were analyzed and demonstrated through N-glycosylation analysis, animal trials, and sera ELISA. Our results suggest that the KDEL retained proteins in ER with oligomannose glycan structure and enhanced protein accumulation level. The sera of mice immunized with GA733-FcK purified from plants contained immunoglobulins which were at least as efficient as the mammalian-derived GA733-Fc at recognizing human colorectal cancer cell lines. Thus, a plant system can be used to express the KDEL fusion protein with oligomannose glycosylation, and this protein induces an immune response which is comparable to non-KDEL-tagged, mammalian-derived proteins

Development of a Spirometry \u3cem\u3eT\u3c/em\u3e-score in the General Population

Background and objective: Spirometry values may be expressed as T-scores in standard deviation units relative to a reference in a young, normal population as an analogy to the T-score for bone mineral density. This study was performed to develop the spirometry T-score. Methods: T-scores were calculated from lambda-mu-sigma-derived Z-scores using a young, normal age reference. Three outcomes of all-cause death, respiratory death, and COPD death were evaluated in 9,101 US subjects followed for 10 years; an outcome of COPD-related health care utilization (COPD utilization) was evaluated in 1,894 Korean subjects followed for 4 years. Results: The probability of all-cause death appeared to remain nearly zero until -1 of forced expiratory volume in 1 second (FEV1) T-score but increased steeply where FEV1 T-score reached below -2.5. Survival curves for all-cause death, respiratory death, COPD death, and COPD utilization differed significantly among the groups when stratified by FEV1 T-score (P \u3c 0.001). The adjusted hazard ratios of the FEV1 T-score for the four outcomes were 0.54 (95% confidence interval, 0.48–0.60), 0.43 (95% CI: 0.37–0.50), 0.30 (95% CI: 0.24–0.37), and 0.69 (95% CI: 0.59–0.81), respectively, adjusting for covariates (P \u3c 0.001). Conclusion: The spirometry T-score could predict all-cause death, respiratory death, COPD death, and COPD utilization

Early detection of cardiac involvement in Miyoshi myopathy: 2D strain echocardiography and late gadolinium enhancement cardiovascular magnetic resonance

<p>Abstract</p> <p>Background</p> <p>Miyoshi myopathy (MM) is an autosomal recessive distal myopathy characterized by early adult onset. Cardiomyopathy is a major clinical manifestation in other muscular dystrophies and an important prognostic factor. Although dysferlin is highly expressed in cardiac muscle, the effect of dysferlin deficiency in cardiac muscle has not been studied. We hypothesized that early myocardial dysfunction could be detected by 2D strain echocardiography and late gadolinium enhancement (LGE) cardiovascular magnetic resonance (CMR).</p> <p>Method</p> <p>Five consecutive MM patients (3 male) in whom we detected the DYSF gene mutation and age-matched healthy control subjects were included. None of the patients had history of cardiac disease or signs and symptoms of overt heart failure. Patients were studied using 2D strain echocardiography and CMR, with 2D strain being obtained using the Automated Function Imaging technique.</p> <p>Results</p> <p>All patients had preserved left ventricular systolic function. However, segmental Peak Systolic Longitudinal Strain (PSLS) was decreased in 3 patients. Global PSLS was significantly lower in patients with MM than in control subjects (p = 0.005). Basal anterior septum, basal inferior septum, mid anterior, and mid inferior septum PSLS were significantly lower in patients with MM than in control subjects (P < 0.0001, < 0.0001, 0.038 and 0.003, respectively). Four patients showed fibrosis by LGE. The reduced PSLS lesion detected by 2D strain tended to be in the same area as that which showed fibrosis by LGE.</p> <p>Conclusions</p> <p>Patients with MM showed subclinical involvement of the heart. 2D strain and LGE are sensitive methods for detecting myocardial dysfunction prior to the development of cardiovascular symptoms. The prognostic significance of these findings warrants further longitudinal follow-up.</p

Neural Correlates of Transient Mal de Debarquement Syndrome: Activation of Prefrontal and Deactivation of Cerebellar Networks Correlate With Neuropsychological Assessment

Background: Mal de debarquement syndrome (MdDS) is characterized by a subjective perception of self-motion after exposure to passive motion, mostly after sea travel. A transient form of MdDS (t-MdDS) is common in healthy individuals without pathophysiological certainty. In the present cross-sectional study, the possible neuropsychiatric and functional neuroimaging changes in local fishermen with t-MdDS were evaluated. Methods: The present study included 28 fishermen from Buan County in South Korea; 15 (15/28, 53.6%) participants experienced t-MdDS for 1–6 h, and 13 were asymptomatic (13/28, 46.4%). Vestibular function tests were performed using video-oculography, the video head impulse test, and ocular and cervical vestibular-evoked myogenic potentials. Visuospatial function was also assessed by the Corsi block test. Brain imaging comprised structural MRI, resting-state functional MRI, and [18F]FDG PET scans. Results: The results of vestibular function tests did not differ between the fishermen with and those without t-MdDS. However, participants with t-MdDS showed better performance in visuospatial memory function than those without t-MdDS (6.40 vs. 5.31, p-value = 0.016) as determined by the Corsi block test. Structural brain MRIs were normal in both groups. [18F]FDG PET showed a relative hypermetabolism in the bilateral occipital and prefrontal cortices and hypometabolism in the vestibulocerebellum (nodulus and uvula) in participants with t-MdDS compared to those without t-MdDS. Resting-state functional connectivities were significantly decreased between the vestibular regions of the flocculus, superior temporal gyrus, and parietal operculum and the visual association areas of the middle occipital gyrus, fusiform gyrus, and cuneus in participants with t-MdDS. Analysis of functional connectivity of the significant regions in the PET scans revealed decreased connectivity between the prefrontal cortex and visual processing areas in the t-MdDS group. Conclusion: Increased visuospatial memory, altered metabolism in the prefrontal cortex, visual cognition cortices, and the vestibulocerebellum, and decreased functional connectivity between these two functional areas might indicate reductions in the integration of vestibular input and enhancement of visuospatial attention in subjects with t-MdDS. Current functional neuroimaging similarities from transient MdDS via chronic MdDS to functional dizziness and anxiety disorders suggest a shared mechanism of enhanced self-awareness as a kind of continuum or as overlap disorders

Substitution of Heavy Complementarity Determining Region 3 (CDR-H3) Residues Can Synergistically Enhance Functional Activity of Antibody and Its Binding Affinity to HER2 Antigen

To generate a biobetter that has improved therapeutic activity, we constructed scFv libraries via random mutagenesis of several residues of CDR-H3 and -L3 of hu4D5. The scFv clones were isolated from the phage display libraries by stringent panning, and their anti-proliferative activity against HER2-positive cancer cells was evaluated as a primary selection criterion. Consequently, we selected AH06 as a biobetter antibody that had a 7.2-fold increase in anti-proliferative activity (IC50: 0.81 nM) against the gastric cancer cell line NCI-N87 and a 7.4-fold increase in binding affinity (K-D : 60 pM) to HER2 compared to hu4D5. The binding energy calculation and molecular modeling suggest that the substitution of residues of CDR-H3 to W98, F100c, A101 and L102 could stabilize binding of the antibody to HER2 and there could be direct hydrophobic interactions between the aromatic ring of W98 and the aliphatic group of I613 within HER2 domain IV as well as the heavy and light chain hydrophobic interactions by residues F100c, A101 and L102 of CDR-H3. Therefore, we speculate that two such interactions were exerted by the residues W98 and F100c. A101 and L102 may have a synergistic effect on the increase in the binding affinity to HER2. AH06 specifically binds to domain IV of HER2, and it decreased the phosphorylation level of HER2 and AKT. Above all, it highly increased the overall level of p27 compared to hu4D5 in the gastric cancer cell line NCI-N82, suggesting that AH06 could potentially be a more efficient therapeutic agent than hu4D5.OAIID:RECH_ACHV_DSTSH_NO:T201620640RECH_ACHV_FG:RR00200001ADJUST_YN:EMP_ID:A002901CITE_RATE:2.67DEPT_NM:화학생물공학부EMAIL:[email protected]_YN:YCONFIRM: