Comparison of CT-derived Plaque Characteristic Index With CMR Perfusion for Ischemia Diagnosis in Stable CAD.

BACKGROUND: Coronary computed tomography angiography (CCTA) and cardiac magnetic resonance (CMR) have been used to diagnose lesion-specific ischemia in patients with coronary artery disease. The aim of this study was to investigate the diagnostic performance of CCTA-derived plaque characteristic index compared with myocardial blood flow (MBF) and myocardial perfusion reserve (MPR) derived from CMR perfusion in the assessment of lesion-specific ischemia. METHODS: Between October 2020 and March 2022, consecutive patients with suspected or known coronary artery disease, who were clinically referred for invasive coronary angiography were prospectively enrolled. All participants sequentially underwent CCTA and CMR and invasive fractional flow reserve within 2 weeks. The diagnostic performance of CCTA-derived plaque characteristics, CMR perfusion-derived stress MBF, and MPR were compared. Lesions with fractional flow reserve ≤0.80 were considered to be hemodynamically significant stenosis. RESULTS: Nighty-two patients with 141 vessels were included in this study. Plaque length, minimum luminal area, plaque area, percent area stenosis, total atheroma volume, vessel volume, lipid-rich volume, spotty calcium, napkin-ring signs, stress MBF, and MPR in flow-limiting stenosis group were significantly different from nonflow-limiting group. The overall accuracy, sensitivity, specificity, positive predictive value, and negative predictive value of lesion-specific ischemia diagnosis were 61.0%, 55.3%, 63.1%, 35.6%, and 79.3% for stress MBF, and 89.4%, 89.5%, 89.3%, 75.6%, 95.8% for MPR; meanwhile, 82.3%, 79.0%, 84.5%, 65.2%, and 91.6% for CCTA-derived plaque characteristic index. CONCLUSIONS: In our prospective study, CCTA-derived plaque characteristics and MPR derived from CMR performed well in diagnosing lesion-specific myocardial ischemia and were significantly better than stress MBF in stable coronary artery disease.

A chimeric recombinant infectious hematopoietic necrosis virus induces protective immune responses against infectious hematopoietic necrosis and infectious pancreatic necrosis in rainbow trout.

Infectious pancreatic necrosis virus (IPNV) and infectious hematopoietic necrosis virus (IHNV) are two common viral pathogens that cause severe economic losses in all salmonid species in culture, but especially in rainbow trout. Although vaccines against both diseases have been commercialized in some countries, no such vaccines are available for them in China. In this study, a recombinant virus was constructed using the IHNV U genogroup Blk94 virus as a backbone vector to express the antigenic gene, VP2, from IPNV via the reverse genetics system. The resulting recombinant virus (rBlk94-VP2) showed stable biological characteristics as confirmed by virus growth kinetic analyses, pathogenicity analyses, indirect immunofluorescence assays and western blotting. Rainbow trout were immunized with rBlk94-VP2 and then challenged with the IPNV ChRtm213 strain and the IHNV Sn1203 strain on day 45 post-vaccination. A significantly higher survival rate against IHNV was obtained in the rBlk94-VP2 group on day 45 post-vaccination (86%) compared with the PBS mock immunized group (2%). Additionally, IPNV loads decreased significantly in the rBlk94-VP2 immunized group in the liver (28.6-fold to 36.5-fold), anterior kidney (21.7-fold to 44.2-fold), and spleen (14.9-fold to 22.7-fold), as compared with the PBS mock control group. The mRNA transcripts for several innate and adaptive immune-related proteins (IFN-γ, IFN-1, Mx-1, CD4, CD8, IgM, and IgT) were also significantly upregulated after rBlk94-VP2 vaccination, and neutralizing antibodies against both IHNV and IPNV were induced on day 45 post-vaccination. Collectively, our results suggest that this recombinant virus could be developed as a vaccine vector to protect rainbow trout against two or more diseases, and our approach lays the foundations for developing live vaccines for rainbow trout.

Identification of the optimal insertion site for expression of a foreign gene in an infectious hematopoietic necrosis virus vector.

Infectious hematopoietic necrosis virus (IHNV) was developed as a vector to aid the construction of vaccines against viral diseases such as viral hemorrhagic septicemia virus, spring viremia of carp virus, and influenza virus H1N1. However, the optimal site for foreign gene expression in the IHNV vector has not been determined. In the present study, five recombinant viruses with the green fluorescence protein (GFP) gene inserted into different genomic junction regions of the IHNV genomic sequence were generated using reverse genetics technology. Viral growth was severely delayed when the GFP gene was inserted into the intergenic region between the N and P genes. Real-time fluorescence quantitative PCR assays showed that the closer the GFP gene was inserted towards the 3' end, the higher the GFP mRNA levels. Measurement of the GFP fluorescence intensity, which is the most direct method to determine the GFP protein expression level, showed that the highest GFP protein level was obtained when the gene was inserted into the intergenic region between the P and M genes. The results of this study suggest that the P and M gene junction region is the optimal site within the IHNV vector to express foreign genes, providing valuable information for the future development of live vector vaccines.

Recovery of recombinant infectious hematopoietic necrosis virus strain Sn1203 using the mammalian cell line BHK-21.

Reverse genetics systems are powerful tools for understanding the virulence mechanisms and gene functions of negative-sense RNA viruses. The reverse genetics systems commonly used for recombinant infectious hematopoietic necrosis virus (IHNV) are based on vaccinia virus infection. To avoid the potential biological safety risks associated with vaccinia virus, a recombinant IHNV virus strain Sn1203 (rIHNV-Sn1203) was rescued in this study using a mammalian cell line, BHK-21. The genome sequence authenticity of rIHNV-Sn1203 was confirmed using two silent genetic tags introduced by site-directed mutagenesis. Indirect immunofluorescence assays and transmission electron microscopy revealed that rIHNV-Sn1203 and wild-type IHNV-Sn1203 (wtIHNV-Sn1203) had identical immunogenicity and virion morphology. The virulence and pathogenicity of rIHNV-Sn1203 were assessed in vitro and in vivo. Although rIHNV-Sn1203 displayed trends toward delayed intracellular viral replication and lower virion yields compared with wtIHNV-Sn1203, statistical analyses revealed no significant differences between these two viruses. Moreover, rainbow trout challenged with rIHNV-Sn1203 and wtIHNV-Sn1203 showed indistinguishable mortality. Together, these results show that IHNV was successfully rescued using BHK-21 cells. This method is very convenient and may also be suitable for use in the recovery of other Novirhabdoviruses.

De novo assembly and characterization of the Hucho taimen transcriptome.

Taimen (Hucho taimen) is an important ecological and economic species that is classified as vulnerable by the IUCN Red List of Threatened Species; however, limited genomic information is available on this species. RNA-Seq is a useful tool for obtaining genetic information and developing genetic markers for nonmodel species in addition to its application in gene expression profiling. In this study, we performed a comprehensive RNA-Seq analysis of taimen. We obtained 157 M clean reads (14.7 Gb) and used them to de novo assemble a high-quality transcriptome with a N50 size of 1,060 bp. In the assembly, 82% of the transcripts were annotated using several databases, and 14,666 of the transcripts contained a full open reading frame. The assembly covered 75% of the transcripts of Atlantic salmon and 57.3% of the protein-coding genes of rainbow trout. To learn about the genome evolution, we performed a systematic comparative analysis across 11 teleosts including eight salmonids and found 313 unique gene families in taimen. Using Atlantic salmon and rainbow trout transcriptomes as the background, we identified 250 positive selection transcripts. The pathway enrichment analysis revealed a unique characteristic of taimen: It possesses more immune-related genes than Atlantic salmon and rainbow trout; moreover, some genes have undergone strong positive selection. We also developed a pipeline for identifying microsatellite marker genotypes in samples and successfully identified 24 polymorphic microsatellite markers for taimen. These data and tools are useful for studying conservation genetics, phylogenetics, evolution among salmonids, and selective breeding for threatened taimen.

Thrombin induced platelet-fibrin clot strength in relation to platelet volume indices and inflammatory markers in patients with coronary artery disease.

Platelet aggregation and inflammation are both implicated in coronary artery disease (CAD). Thrombin induced platelet-fibrin clot strength (MAThrombin) measured by thrombelastography (TEG) has been proved to be a novel marker of platelet aggregation. The aim of this study was to investigate the correlation of MAThrombin to platelet volume indices (PVIs) or to inflammatory markers in different types of CAD. 206 patients with different types of CAD were enrolled. MAThrombin, PVIs, including mean platelet volume (MPV), platelet distribution width (PDW), and platelet-large cell ratio (P-LCR) as well as inflammatory markers, including high-sensitivity C-reactive protein (hs-CRP) and fibrinogen (Fbg) were measured. Multiple linear regression models were used to analyze the association between MAThrombin, PVIs, and inflammatory markers. MAThrombin and inflammatory markers both varied with CAD types (P<0.001). MAThrombin was correlated to PVIs in NSTEMI individuals (MPV, r=0.393, P=0.007; PDW, r=0.334, P=0.023; P-LCR, r=0.382, P=0.008), but had inner-link with inflammatory markers in STEMI cases (hs-CRP, r=0.499, P<0.001; Fbg, r=0.500, P<0.001). These findings may suggest different mechanisms of platelet aggregation in different types of CAD. Moreover, MAThrombin may be used as a potential parameter to evaluate platelet aggregation and inflammation together.

Autophagy induced by infectious hematopoietic necrosis virus inhibits intracellular viral replication and extracellular viral yields in epithelioma papulosum cyprini cell line.

Infectious hematopoietic necrosis virus (IHNV) is a common pathogen that causes severe disease in the salmonid aquaculture industry. Recent work demonstrated that autophagy plays an important role in pathogen invasion by activating innate and adaptive immunity. This study investigated the relationship between IHNV and autophagy in epithelioma papulosum cyprini cells. The electron microscopy results show that IHNV infection can induce typical autophagosomes which are representative structures of autophagy activation. The punctate accumulation of green fluorescence-tagged microtubule-associate protein 1 light chain 3 (LC3) and the protein conversion from LC3-I to LC3-II were respectively confirmed by confocal fluorescence microscopy and western blotting. Furthermore, the effects of autophagy on IHNV replication were also clarified by altering the autophagy pathway. The results showed that rapamycin induced autophagy can inhibit both intracellular viral replication and extracellular viral yields, while autophagy inhibitor produced the opposite results. These findings demonstrated that autophagy plays an antiviral role during IHNV infection.

Preliminary study of an oral vaccine against infectious hematopoietic necrosis virus using improved yeast surface display technology.

Infectious hematopoietic necrosis virus (IHNV) is a common pathogen that causes severe disease in the salmonid aquaculture industry. Because oral vaccines induce more efficient mucosal immunity than parenteral immunization, an oral vaccine was developed with an improved yeast cell surface display technology to induce an immune response to IHNV. The oral yeast vaccine, designated EBY100/pYD1-bi-G, was delivered orally to rainbow trout (Oncorhynchus mykiss) on days 1 and 32, and the nonspecific and specific immune responses were measured 50days after the first vaccination. In the hindgut, spleen, and head kidney, the expression of IFN-1 and Mx-1 was significantly upregulated after oral vaccination with EBY100/pYD1-bi-G, and the highest expression of IFN-1 and Mx-1 was observed in the spleen (7.5-fold higher than the control group) and head kidney (3.9-fold higher than the control group), respectively. Several markers of the adaptive immune response (IgM, IgT, CD4, and CD8) were also significantly upregulated, and the highest expression of these markers was observed in the hindgut, suggesting that the mucosal immune response was successfully induced by oral vaccination with EBY100/pYD1-bi-G. Sera from the orally vaccinated rainbow trout showed higher anti-IHNV neutralizing antibody titers (antibody titer 81±4) than the control sera (antibody titer 7±3), and the relative percentage survival after IHNV challenge was 45.8% compared with 2% in the control group. Although the protection afforded by this orally delivered vaccine was lower than that of a DNA vaccine (83%-98%), it is a promising candidate vaccine with which to protect larval fish against IHNV, which are most susceptible to the virus and difficult to inject with a DNA vaccine.

An efficient and simple method to increase the level of displayed protein on the yeast cell surface.

BACKGROUND: The development of oral vaccines using yeast surface display technology is an area of intensive study in vaccine development, but the protein level displayed on yeast surfaces is not currently high enough to obtain a robust immune response. METHODS: To address this issue, we established an efficient and simple method of increasing the level of displayed protein on the yeast cell surface. We used the single chain variable fragment (scFv) of an antibody against the infectious hematopoietic necrosis virus isolate Sn1203 as a target display protein. The yeast-derived scFv was first displayed on the yeast surface by galactose induction, and then Escherichia coli-derived scFv was also displayed on the same yeast via an artificial anchoring condition to increase the total scFv level on the yeast surface. RESULTS: The levels of yeast- and E. coli-derived scFv displayed on the yeast cell surface were analyzed by flow cytometry, western blotting, and fluorescent microscopy. The flow cytometry results indicated that when the cells were suspended in phosphate-buffered saline with 1mmol/L glutathione, 0.2mmol/L oxidized glutathione, and 5% dimethyl sulfoxide at 4°C for 6h, the E. coli-derived scFv protein was stably anchored to the yeast cell surface. The mean fluorescence intensity in these experiments, which is an indirect quantitative representation of the surface scFv expression, was three times higher in the treated cells than that in control cells. The western blotting results show two specific protein bands, the smaller of which was identified as the E. coli-derived scFv that was displayed on the yeast cell surface. Cell immunofluorescence is a more direct way to detect differentially produced proteins that are displayed on the yeast cell surface. The fluorescence microscopy results show that both fluorescence corresponding to the yeast-derived scFv and fluorescence corresponding to the E. coli-derived scFv can exist on the cell surface of same yeast cell. This confirms that the E. coli-derived scFv protein was successfully displayed on the yeast cell surface. CONCLUSIONS: This method provides a rapid, simple, and high-efficiency strategy to increase the level of displayed protein on the yeast cell surface. Application of this technique may allow the yeast surface display system to be used to generate potential oral vaccines.

Recombinant scFv antibodies against infectious pancreatic necrosis virus isolated by flow cytometry.

Infectious pancreatic necrosis is a significant disease of farmed salmonids in China. In this study, a single chain variable fragment (scFv) antibody library derived from rainbow trout (Oncorhynchus mykiss) and viral protein VP2 of a Chinese infectious pancreatic necrosis virus (IPNV) isolate ChRtm213 were co-expressed by a bacterial display technology. The library was subjected to three rounds of screening by flow cytometry (FCM) to select IPNV specific antibodies. Six antibody clones with different mean fluorescence intensities (MFI) were obtained by picking colonies at random. The antibody clones were expressed and purified. The purified IPNV-specific scFv antibodies were used successfully in Western blotting, enzyme linked immunosorbent assay (ELISA) and an immunofluorescence antibody test (IFAT). This method provides a high throughput means to screen an antibody library by flow cytometry, and isolate a panel of antibody that can be used as potential reagents for the detection and study of IPNV that are prevalent in China.

Nine-month angiographic and two-year clinical follow-up of novel biodegradable-polymer arsenic trioxide-eluting stent versus durable-polymer sirolimus-eluting stent for coronary artery disease.

BACKGROUND: Despite great reduction of in-stent restenosis, first-generation drug-eluting stents (DESs) have increased the risk of late stent thrombosis due to delayed endothelialization. Arsenic trioxide, a natural substance that could inhibit cell proliferation and induce cell apoptosis, seems to be a promising surrogate of sirolimus to improve DES performance. This randomized controlled trial was to evaluate the efficacy and safety of a novel arsenic trioxide-eluting stent (AES), compared with traditional sirolimus-eluting stent (SES). METHODS: Patients with symptoms of angina pectoris were enrolled and randomized to AES or SES group. The primary endpoint was target vessel failure (TVF), and the second endpoint includes rates of all-cause death, cardiac death or myocardial infarction, target lesion revascularization (TLR) by telephone visit and late luminal loss (LLL) at 9-month by angiographic follow-up. RESULTS: From July 2007 to 2009, 212 patients were enrolled and randomized 1:1 to receive either AES or SES. At 2 years of follow-up, TVF rate was similar between AES and SES group (6.67% vs. 5.83%, P = 0.980). Frequency of all-cause death was significantly lower in AES group (0 vs. 4.85%, P = 0.028). There was no significant difference between AES and SES in frequency of TLR and in-stent restenosis, but greater in-stent LLL was observed for AES group (0.29 ± 0.52 mm vs. 0.10 ± 0.25 mm, P = 0.008). CONCLUSIONS: After 2 years of follow-up, AES demonstrated comparable efficacy and safety to SES for the treatment of de novo coronary artery lesions.

Short-term safety and efficiency of cryoablation for renal sympathetic denervation in a swine model.

BACKGROUND: Renal sympathetic nerves are involved in the reflective activation of the sympathetic nervous system in circulatory control. Catheter-based renal denervation (RDN) ameliorated treatment-resistant hypertension safely, but 10%-20% of treated patients are nonresponders to radiofrequency denervation. The purpose of this study was to investigate the safety and efficiency of cryoablation for sympathetic denervation in a swine model and to explore a new way of RDN. METHODS: Seven swines randomly assigned to two groups: Renal cryoablation (CR) group and control group. The control group underwent renal angiogram only. The CR group underwent renal angiogram plus bilateral renal cryoablation. Renal angiograms via femoral were performed before denervation, after denervation and prior to the sacrifice to access the diameter of renal arterial and the pressure of aorta abdominalis. Euthanasia of the swine was performed on 28-day to access norepinephrine (NE) changes of the renal cortex and the changes of renal nerves. RESULTS: Cryoablation did not induce severe complications at any time point. There was no significant change in diameter of renal artery. CR reduced systolic blood pressure (BP) from 145.50 ± 9.95 mmHg at baseline to 119.00 ± 14.09 mmHg. There was a slight but insignificant decrease in diastolic BP. The main nerve changes at 28-day consisted of necrosis with perineurial fibrosis at the site of CR exposure in conjunction with the nerve vacuolation. Compared with the control group, renal tissue NE of CR group decreased by 89.85%. CONCLUSIONS: Percutaneous catheter-based cryoablation of the renal artery is safe. CR could effectively reduce NE storing in the renal cortex, and the efficiency could be maintained 28-day at least.

The complete mitochondrial genome sequence of white-spotted char (Salvelinus leucomaenis).

The complete sequence of the mitochondrial genome of the white-spotted char (Salvelinus leucomaenis) was determined to be 16,658 bp in length, which contains the control region (CR), the origin of light-strand replication (OL), 22 transfer RNA genes, 2 ribosomal genes and 13 protein-coding genes. Overall, base composition of the complete mitochondrial DNA was 28.20% A, 26.51% T, 28.39% C, 16.90% G, with 54.71% AT.

Epitope mapping of the infectious hematopoietic necrosis virus glycoprotein by flow cytometry.

The glycoprotein of infectious hematopoietic necrosis virus was truncated to ten overlapping fragments. All fragments were displayed on the inner membrane of the Escherichia coli periplasm. After disruption of the outer membrane, spheroplasts that had anchored with the glycoprotein fragment were incubated with an anti-glycoprotein polyclonal antibody. Prey pairs were detected and quantitated by flow cytometry with all fragments but one, G2, reacting with the polyclonal antibody. The antigenicity of all ten fragments was analyzed using conventional methods, and epitopes were localized in all fragments, except for G2 and were consistent with FCM analysis. Antigenicity of purified glycoprotein fusion proteins was confirmed by western blotting and ELISA. This method provides a rapid, quantitative and simple strategy for identifying linear B cell epitopes of a given protein.

The Complete mitochondrial genome of Spinibarbus denticulatus (Oshima).

Spinibarbus denticulatus (Oshima) is a rare and commercial fish. The complete mitochondrial genome sequence of S. denticulatus will help us to study the genetic of conversation, such as the genetic diversity and genetic structure, and provides the basis for the study in evolution. In this paper, the complete mitochondrial genome of S. denticulatus was determined to be 16,549 bp in length by Sanger sequencing technology. Thirteen protein-coding genes, 22 tRNA genes and 2 ribosomal genes were characterized. We also analyzed the structure of control region, 6 CSBs (CSB-1, CSB-2, CSB-3, CSB-D, CSB-E and CSB-F) and 1 TAS were identified, the control region also included an AT unit tandem repeat with 17 repeat times.

Mitochondrial DNA sequence of Mongolian redfin (Chanodichthys mongolicus).

Monglian redfin is a kind of freshwater aquaculture species which has an important economic value in China. In this study, we report the complete sequence of mitochondrial genome of Monglian redfin. The complete mitochondrial genome sequence is determined to be 16,621 base pairs (bp) in length and contain 13 protein-coding gene (PCGs), 22 transfer RNA genes, large (rrnL) and small (rrnS) ribosomal RNA, and the non-coding control region. Its total A+T content is 55.98%. We also analyzed the structure of control region, six conserved sequence blocks (CSBs) (CSB-1, CSB-2, CSB-3, CSB-D, CSB-E and CSB-F) and one potential termination-associated sequence were detected and the control region also included a 2-bp tandem repeat with eight repeat times.

[Effects of delayed first feeding on growth and survival of Hucho taimen larvae].

This paper studied the effects of delaying first breeding Hucho taimen larvae for different days on the larvae growth, survival, and body size. Five treatments were installed, i. e. , feeding begins on the first eating day (control, S0) and on the 9th, 12th, 15th, and 18th days after the first eating day (S1 -S4) at 10.4-14.9 degree C, respectively. By the end of the experiment (36-day), the growth rate and initial feeding rate in S1 was higher than that in S0, and the overall mortality rate in S1 was lower, but the body size and mass in S1and S0 had no significant difference. Compared with S0, S2 had higher growth rate, initial feeding rate, total mortality, and self-mutilation mortality, the body mass was significantly lower, but the body size had less difference. S3 had higher first feeding rate, body size, total mortality, and self-mutilation mortality, but significantly lower body mass than S0, whereas the growth rate had less difference. In S4, the growth rate and body mass were lower, and the total mortality and self-mutilation mortality were higher than those in S0. It was suggested that under the same conditions, delaying first feeding for 9 days would induce H. taimen larvae presenting "completely compensatory growth", and this feeding way could be applied for the culture of H. taimen larvae in their initial feeding period.

[Optimal feeding strategy for juvenile Hucho taimen].

Three experiments including starvation and re-feeding, starvation and re-feeding recovery, and feeding frequency per day were conducted to approach the optimal feeding strategy for the growth and survival of juvenile Hucho taimen. In the experiment of starvation and re-feeding, all groups of restricted feeding showed non-compensatory growth. However, in the experiment of starvation and re-feeding recovery, different degrees of compensatory growth appeared in different starving groups, among which, the half a day starvation and half a day feeding group (S1/2) had a weight increment approximately the same as the control, and showed completely compensatory growth, indicating that the S1/2 could be a useful feeding strategy for the juvenile H. taimen at its early growth stage with the body mass from 0 to 2 g and at the water temperature from 9 to 15.3 degrees C. In feeding frequency experiment, the group T3 (three meals per day) had the highest body length, body mass, specific growth rate, and relatively high food conversion ratio, indicating that three meals a day could be more effective for improving the growth performance of juvenile H. taimen at its late stage with the body mass from 2 to 21 g and at the water temperature from 8.8 to 15.5 degrees C.

[Prokaryotic expression and immunogenicity analysis of glycoprotein from infectious hematopoietic necrosis virus].

In order to detect Infectious hematopoietic necrosis virus with immunological methods, the surface glycoprotein of a recent IHNV-Sn isolated from farmed rainbow trout ( Oncorhynchus mykiss ) in China was amplified and cloned into pET27b(+) vector (designated as pET27b-G ). The expression of recombinant plasmid pET27b-G in E. coli BL21(DE3) was induced and determined by SDS-PAGE analysis. The predicted molecular weight of glycoprotein protein was approximately 55 kD and was confirmed in this study. The inclusion body of glycoprotein was treated with urea at different urea concentrations, and dialyzed into PBS buffer. Purified glycoprotein with high concentration was obtained after dialyzed in the PBS buffer. Antisera against glycoprotein were produced from immunized rabbits. The prepared antisera could react specifically with both the recombinant glycoprotein and natural glycoprotein of the IHNV-Sn isolated in the test of indirect ELISA, and the titer against the recombinant glycoprotein was 1:20,000. IFA showed that the antisera can recognize the glycoprotein located on the surface of IHNV-Sn and IHNV reference strain. These results indicated that the expressed glycoprotein was immunogenical and antigenical and could be functional as the natural IHNV glycoprotein. These results established a foundation for further study on vaccine and rapid diagnosis of IHNV.