CircRNA PVT1 promotes proliferation and chemoresistance of osteosarcoma cells via the miR-24-3p/KLF8 axis.

OBJECTIVE: To investigate the regulatory effect and mechanism of circular RNA PVT1 (circPVT1) in proliferation and chemoresistance of osteosarcoma (OS) cells. METHODS: The expression of circPVT1 in human OS and adjacent normal tissues was detected. The correlation between circPVT1 expression and clinical features of OS was analyzed. The expressions of circPVT1 and miR-24-3p in OS cells resistant to cisplatin, doxorubicin or methotrexate and parental OS cells were detected after cell transfection. CCK-8 and colony formation assay assessed the viability and proliferative ability of OS cells. qRT-PCR and Western blotting measured the expression of KLF8. Dual-luciferase reporter and RNA pull-down assays verified the targeting relationships of circPVT1/miR-24-3p and miR-24-3p/KLF8. RESULTS: CircPVT1 was over-expressed in OS tissues and cells, and correlated with clinical features of OS. Over-expressed circPVT1 reduced the survival of OS patients. CircPVT1 was up-regulated in chemoresistant OS cells compared to their parental cells. CircPVT1 inhibition suppressed the proliferation and chemoresistance of OS cells. MiR-24-3p was under-expressed in OS cells and further down-regulated in chemoresistant cells. CircPVT1 could bind and down-regulate miR-24-3p. MiR-24-3p overexpression inhibited the proliferation and chemoresistance of OS cells. KLF8 was over-expressed in OS cells and further up-regulated in chemoresistant cells. MiR-24-3p negatively regulated the expression of KLF8. CONCLUSION: CircPVT1 mediates proliferation and chemoresistance of OS cells via the miR-24-3p/KLF8 axis. The findings may provide guidance for clinical treatment of OS.

Systemic immune-inflammation index is associated with disease activity in patients with ankylosing spondylitis.

BACKGROUND: The systemic immune-inflammation index (SII) is a recently developed indicator for systemic inflammatory response. We aimed to explore the association between SII and disease activity in patients with ankylosing spondylitis (AS). METHODS: This retrospective study included 136 patients with AS and 63 healthy controls. Patients were divided into two groups according to Bath Ankylosing Spondylitis Disease Activity Index (BASDAI); active group (n = 60) and remission group (n = 76). Clinical, laboratory, and demographic characteristics were recorded. Spearman's correlation analysis was used to determine correlations of SII with C-reactive protein (CRP) level, erythrocyte sedimentation rate (ESR), and BASDAI in AS patients. Binary logistic regression analysis was used to assess risk factors for AS disease activity. Receiver operating characteristic curve analysis was used to evaluate the diagnostic value of SII and the above variables for the active group compared with the remission group. RESULTS: Systemic immune-inflammation index levels were higher in AS patients than in healthy controls (p < 0.001). SII levels were higher in the active group than in the remission group (p < 0.001). For patients with AS, SII correlated positively with CRP (rs  = 0.483, p < 0.001), ESR (rs  = 0.374, p < 0.001), and BASDAI (rs  = 0.667, p < 0.001). SII (OR = 1.009, 95% CI = 1.006-1.012, p < 0.001) was an independent risk factor affecting AS disease activity. The specificity and sensitivity of SII using a cutoff value of 513.2 were 83.33% and 86.84%, respectively, for the active group. CONCLUSION: Systemic immune-inflammation index was increased in AS. SII may be a novel indicator for monitoring AS disease activity.

The preparation of endotoxin-free genetically engineered murine B1 antisense RNA.

One of the major limitations in the production of genetically engineered RNA from Escherichia coli (E. coli) is contamination by endotoxin. Here we report the first method that is capable of removing endotoxin from genetically engineered RNA. As a proof of concept, we transformed E. coli with a plasmid containing a tandem short interspersed nuclear elements from the mouse genome (SINE B1 elements). We then evaluated several extraction methods (SDS-NaCl centrifugation, SDS-NaCl filtration, TRIzol and SDS hot-phenol) and refinements thereof, and measured the resulting RNA yield, RNA purity, RNA integrity and endotoxin content. SDS-NaCl filtration with 2 mol/L NaCl, incorporating DEPC as an RNA protective agent, effectively removed endotoxin and resulted in a good RNA yield. Triton X-114 phase separation further reduced the endotoxin content of SDS-NaCl filtration-extracted RNA. RNA extracted by SDS-NaCl filtration with Triton X-114 phase separation did not cause adverse reactions in BALB/c mice and did not induce fever in rabbits when injected into these animals. The RNA met the requirements of nucleic acid reagents for in vivo experiments on animals.

GENETIC CHARACTERISTICS OF CANINE DISTEMPER VIRUSES CIRCULATING IN WILDLIFE IN THE UNITED STATES.

Canine distemper virus (CDV) is a highly contagious disease of wild and domestic mammals. Maintenance of CDV among wildlife plays an important role in the disease epidemiology. Wild animals, including raccoons (Procyon lotor) and gray foxes (Urocyon cinereoargenteus), serve as reservoirs of CDV and hamper the control of the disease. Recently, we discovered that at least three different CDV lineages (America-3 [Edomex], America-4, and America-5] that are genetically different from the available vaccine strains are circulating in domestic dogs in the United States. Because wildlife serve as a reservoir for the virus, it is important to determine if wildlife play a role in the maintenance and spread of these lineages. To determine the genetic characteristics of circulating strains of CDV in wildlife in various geographic regions in the United States, we studied the nucleotide sequences of the hemagglutinin (H) gene of 25 CDV strains detected in nondomestic species. The species included were free-ranging wildlife: three fishers (Martes pennanti), six foxes, one skunk (Mephitis mephitis), 10 raccoons, two wolves (Canis lupus), and one mink (Neovison vison). Strains from two species in managed care, one sloth (Choloepus didactylus) and one red panda (Ailurus fulgens), were also evaluated. Phylogenetic analysis of the H genes indicated that in addition to America-3, America-4, and America-5 lineages, there are at least two other lineages circulating in US wildlife. One of these includes CDV nucleotide sequences that grouped with that of a single CDV isolate previously detected in a raccoon from Rhode Island in 2012. The other lineage is independent and genetically distinct from other CDV strains included in the analysis. Additional genetically variable strains were detected, mainly in raccoons, suggesting that this species may be the host responsible for the genetic variability of newly detected strains in the domestic dog population.

The Dynamics of Circulating Monocyte Subsets and Intra-Plaque Proliferating Macrophages during the Development of Atherosclerosis in ApoE-/- Mice.

To detect the development of monocytes and proliferative macrophages in atherosclerosis of ApoE-/- mice, we randomly assigned 84 ApoE-/- mice fed western diet or chow diet. On weeks 2, 4, 6, 8, 10, and 12 after fed high-fat diet or normal chow diet, animals were euthanized (n = 7 for each group at each time point). Flow cytometry methods were used to analyze the proportions of circulation monocyte subsets. The macrophage and proliferative macrophage accumulation within atherosclerotic plaques was estimated by confocal florescence microscopy. Plasma levels of total cholesterol and triglyceride were measured by ELISA kit. The plaques of aortic sinus were stained with Oil Red O. The percent of Ly6Chi circulation monocyte, the density of proliferation macrophage, the total plasma cholesterol and triglyceride levels, the lesion area of ApoE-/- mice were consistently elevated in chow diet throughout the trial. The total plasma cholesterol and triglyceride levels, the lesion area were elevated in western diet group with age, and they were always higher than the chow diet group. The Ly6Chi monocytes and proliferative macrophages reached a plateau at 8 weeks and 6 weeks; despite continued high-triglyceride high-cholesterol diet the percent did not significantly change. Interestingly, the density of macrophage did not change significantly over age in western and chow diet groups. Our results provide a dynamic view of Ly6Chi monocyte subset, the density of macrophage and proliferation macrophage change during the development and progression of atherosclerosis, which is relevant for designing new treatment strategies targeting mononuclear phagocytes in this model.

Streptococcus troglodytidis sp. nov., isolated from a foot abscess of a chimpanzee (Pan troglodytes).

A facultative anaerobic, non-motile, non-spore-forming, Gram-positive-staining, coccus-shaped bacterium was isolated from an abscess on the right foot of a chimpanzee (Pan troglodytes). The colonies were ß-haemolytic. Catalase and oxidase activities were negative. The Lancefield group B antigen was expressed. On the basis of morphological and biochemical characteristics, the bacterium was tentatively identified as a streptococcal species. 16S rRNA gene sequence analysis indicated that the bacterium shared 96.7 %, 96.4 %, 96.1 %, 95.8 % and 95.7 % sequence similarities with Streptococcus gordonii, S. cristatus, S. intermedius, S. anginosus and S. constellatus, respectively. Phylogenetic analyses based on the sequences of the 16S rRNA gene and housekeeping genes encoding D-alanine : D-alanine ligase (ddl), the ß-subunit of RNA polymerase (rpoB) and manganese-dependent superoxide dismutase (sodA) revealed that the bacterium represented a novel species closely related to, albeit different from, S. gordonii, S. cristatus and the anginosus streptococci. The name Streptococcus troglodytidis sp. nov. is proposed. The type strain is M09-11185(T) ( = ATCC BAA-2337(T) = KCTC 33006(T)).

Reactive arthritis and subacute infective endocarditis caused by Streptococcus gordonii infection: A case report.

Streptococcus gordonii (S. gordonii) belongs to the alpha-hemolytic Streptococcus group. It is a symbiotic bacterium found in the human oral mucosa which is present in large quantities on the surface of the teeth. It is generally considered nonpathogenic or weakly pathogenic and is known to cause subacute endocarditis; however, there are few reports of reactive arthritis (ReA) caused by S. gordonii. Herein, we report a case of ReA complicated by subacute infective endocarditis caused by S. gordonii and explore the possible pathogenic mechanism of ReA caused by S. gordonii.

Alu antisense RNA ameliorates methylglyoxal-induced human lens epithelial cell apoptosis by enhancing antioxidant defense.

AIM: To determine whether an antisense RNA corresponding to the human Alu transposable element (Aluas RNA) can protect human lens epithelial cells (HLECs) from methylglyoxal-induced apoptosis. METHODS: Cell counting kit-8 (CCK-8) and 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays were used to assess HLEC viability. HLEC viability/death was detected using a Calcein-AM/PI double staining kit; the annexin V-FITC method was used to detect HLEC apoptosis. The cytosolic reactive oxygen species (ROS) levels in HLECs were determined using a reactive species assay kit. The levels of malondialdehyde (MDA) and the antioxidant activities of total-superoxide dismutase (T-SOD) and glutathione peroxidase (GSH-Px) were assessed in HLECs using their respective kits. RT-qPCR and Western blotting were used to measure mRNA and protein expression levels of the genes. RESULTS: Aluas RNA rescued methylglyoxal-induced apoptosis in HLECs and ameliorated both the methylglyoxal-induced decrease in Bcl-2 mRNA and the methylglyoxal-induced increase in Bax mRNA. In addition, Aluas RNA inhibited the methylglyoxal-induced increase in Alu sense RNA expression. Aluas RNA inhibited the production of ROS induced by methylglyoxal, restored T-SOD and GSH-Px activity, and moderated the increase in MDA content after treatment with methylglyoxal. Aluas RNA significantly restored the methylglyoxal-induced down-regulation of Nrf2 gene and antioxidant defense genes, including glutathione peroxidase, heme oxygenase 1, γ-glutamylcysteine synthetase and quinone oxidoreductase 1. Aluas RNA ameliorated methylglyoxal-induced increases of the mRNA and protein expression of Keap1 that is the negative regulator of Nrf2. CONCLUSION: Aluas RNA reduces apoptosis induced by methylglyoxal by enhancing antioxidant defense.

Optimized conditions for Listeria, Salmonella and Escherichia whole genome sequencing using the Illumina iSeq100 platform with point-and-click bioinformatic analysis.

Whole-genome sequencing (WGS) data have become an integral component of public health investigations and clinical diagnostics. Still, many veterinary diagnostic laboratories cannot afford to implement next generation sequencing (NGS) due to its high cost and the lack of bioinformatic knowledge of the personnel to analyze NGS data. Trying to overcome these problems, and make NGS accessible to every diagnostic laboratory, thirteen veterinary diagnostic laboratories across the United States (US) initiated the assessment of Illumina iSeq100 sequencing platform for whole genome sequencing of important zoonotic foodborne pathogens Escherichia coli, Listeria monocytogenes, and Salmonella enterica. The work presented in this manuscript is a continuation of this multi-laboratory effort. Here, seven AAVLD accredited diagnostic laboratories explored a further reduction in sequencing costs and the usage of user-friendly platforms for genomic data analysis. Our investigation showed that the same genomic library quality could be achieved by using a quarter of the recommended reagent volume and, therefore a fraction of the actual price, and confirmed that Illumina iSeq100 is the most affordable sequencing technology for laboratories with low WGS demand. Furthermore, we prepared step-by-step protocols for genomic data analysis in three popular user-friendly software (BaseSpace, Geneious, and GalaxyTrakr), and we compared the outcomes in terms of genome assembly quality, and species and antimicrobial resistance gene (AMR) identification. No significant differences were found in assembly quality, and the three analysis methods could identify the target bacteria species. However, antimicrobial resistance genes were only identified using BaseSpace and GalaxyTrakr; and GalaxyTrakr was the best tool for this task.

Multi-laboratory evaluation of the Illumina iSeq platform for whole genome sequencing of Salmonella, Escherichia coli and Listeria.

There is a growing need for public health and veterinary laboratories to perform whole genome sequencing (WGS) for monitoring antimicrobial resistance (AMR) and protecting the safety of people and animals. With the availability of smaller and more affordable sequencing platforms coupled with well-defined bioinformatic protocols, the technological capability to incorporate this technique for real-time surveillance and genomic epidemiology has greatly expanded. There is a need, however, to ensure that data are of high quality. The goal of this study was to assess the utility of a small benchtop sequencing platform using a multi-laboratory verification approach. Thirteen laboratories were provided the same equipment, reagents, protocols and bacterial reference strains. The Illumina DNA Prep and Nextera XT library preparation kits were compared, and 2×150 bp iSeq i100 chemistry was used for sequencing. Analyses comparing the sequences produced from this study with closed genomes from the provided strains were performed using open-source programs. A detailed, step-by-step protocol is publicly available via protocols.io (https://www.protocols.io/view/iseq-bacterial-wgs-protocol-bij8kcrw). The throughput for this method is approximately 4-6 bacterial isolates per sequencing run (20-26 Mb total load). The Illumina DNA Prep library preparation kit produced high-quality assemblies and nearly complete AMR gene annotations. The Prep method produced more consistent coverage compared to XT, and when coverage benchmarks were met, nearly all AMR, virulence and subtyping gene targets were correctly identified. Because it reduces the technical and financial barriers to generating WGS data, the iSeq platform is a viable option for small laboratories interested in genomic surveillance of microbial pathogens.

[Inhibition of three pentacyclic triterpenoids on calcium-induced liver mitochondrial permeability transition in mice].

OBJECTIVE: To study effects of three pentacyclic triterpenoids, oleanolic acid (OA), ursolic acid (UA) and asiatic acid (AA) on Ca(2+)-induced liver mitochondrial permeability transition (MPT). METHOD: Effects of three compounds on liver MPT induced by Ca2+ were assessed by measuring the change in mitochondrial swelling, mitochondrial membrane potential and release of matrix Ca2+ in vitro. RESULT: Obvious mitochondrial swelling, loss of mitochondrial membrane potential and release of matrix Ca2+ occurred after the addition of 50 micromol x L(-1) Ca2+. However, preincubation with 50 mg x L(-1) OA, UA or AA significantly blocked the above changes. In addition, it was also found that there are differences in the inhibitions of three compounds on liver MPT induced by Ca2+. CONCLUSION: Three pentacyclic triterpenoids, OA, UA and AA, have significant mitochondrial protection through blocking on liver MPT and the inhibition on liver MPT of AA is stronger than that of UA and OA.

Thermodynamic Assessment of Bio-Oriented Ti-Ta-Sn System.

The alloying elements Ta and Sn can effectively increase the stability of ß-bcc phase, reduce Young's modulus and improve the shape-memory property of Ti-based biomedical alloys. The development of the thermodynamic database for Ti-based biomedical alloys promises thermodynamic predictions in composition design and process optimization. In this work, one key sub-ternary Ti-Ta-Sn system has been thermodynamically assessed based on critical evaluation of experimental phase equilibria. A self-consistent thermodynamic description for the Ti-Ta-Sn system including one ternary compound Ti36Ta28Sn36 and six binary compounds considering the solubility of the third element has been obtained. Two isothermal sections at 973 and 1173 K and the liquidus projection have been calculated. Comparisons between the calculated and experimental phase equilibria validate the reliability of the present thermodynamic description. The influence of Ta and Sn contents on the transformation temperature and amount of α_hcp-Ti phase in ß_bcc-(Ti,Ta) phase has been investigated based on thermodynamic calculations. The solidified phases in Ti-20Ta-xSn (x = 5, 15 and 25 at.%) as-cast alloys have been thermodynamically calculated based on Scheil solidification simulations. The presently developed thermodynamic description of the Ti-Ta-Sn system would promote the establishment of muti-component Ti-based thermodynamic database and guide the development of Ti-based alloys.

Anti-aging and anti-oxidant activities of murine short interspersed nuclear element antisense RNA.

Short interspersed nuclear elements (SINEs) play a key role in regulating gene expression, and SINE RNAs are involved in age-related diseases. We investigated the anti-aging effects of a genetically engineered murine SINE B1 antisense RNA (B1as RNA) and explored its mechanism of action in naturally senescent BALB/c (≥14 months) and moderately senscent C57BL/6N (≥9 months) mice. After tail vein injection, B1as RNA was available in the blood of mice for approximately 30 min, persisted for approximately 2-4 h in most detected tissues and persisted approximately 48 h in lungs. We found that treatment with B1as RNA improved stamina and promoted hair re-growth in aged mice. Treatment with B1as RNA also partially rescued the increase in mitochondrial DNA copy number in liver and spleen tissues observed in aged and moderately senescent mice. Finally, treatment with B1as RNA increased the activities of superoxide dismutase and glutathione peroxidase in aged and moderately senescent mice, reduced these animals' malondialdehyde and reactive oxygen species levels, and modulated the expression of several aging-associated genes, including Sirtuin 1, p21, p16Ink4a, p15Ink4b and p19Arf, and anti-oxidant genes (Sesn1 and Sesn 2). These data suggest that B1as RNA inhibits the aging process by enhancing antioxidant activity, promoting the scavenging of free radicals, and modulating the expression of aging-associated genes. This is the first report describing the anti-aging activity of SINE antisense RNA, which may serve as an effective nucleic acid drug for the treatment of age-related diseases.

A recyclable heterogeneous-homogeneous-heterogeneous NiO/AlOOH catalysis system for hydrocarboxylation of acetylene to acrylic acid.

Concerns about the high-valued utilization of coal- and natural gas-based acetylene has provided particular impetus for exploration of acrylic acid (AA) production via one-step hydrocarboxylation reaction. Motivated by simple recovery, recycling and reuse of the catalyst, we report a high-performance NiO/AlOOH catalyst with AA space-time-yield of 412 gAA gcat. -1 h-1, obtainable by a simple incipient wetness impregnation method. Detailed kinetic and controlled experiments confirmed that nickel species on such a solid catalyst provide a heterogeneous-homogeneous-heterogeneous catalytic cycle where the chelates formed between CO and leached nickel act as the active species. The thorough recovery of leached nickel species improves the catalyst stability greatly. These preliminary findings indicate further prospects for new heterogeneous catalyst design in traditional homogeneous catalytic systems.

Inhibition monitoring in veterinary molecular testing.

Many of the sample matrices typically used for veterinary molecular testing contain inhibitory factors that can potentially reduce analytic sensitivity or produce false-negative results by masking the signal produced by the nucleic acid target. Inclusion of internal controls in PCR-based assays is a valuable strategy not only for monitoring for PCR inhibitors, but also for monitoring nucleic acid extraction efficiency, and for identifying technology errors that may interfere with the ability of an assay to detect the intended target. The Laboratory Technology Committee of the American Association of Veterinary Laboratory Diagnosticians reviewed the different types of internal controls related to monitoring inhibition of PCR-based assays, and provides information here to encourage veterinary diagnostic laboratories to incorporate PCR internal control strategies as a routine quality management component of their molecular testing.

Admission Low-Density Lipoprotein Cholesterol Stratified by Circulating CD14++CD16+ Monocytes and Risk for Recurrent Cardiovascular Events Following ST Elevation Myocardial Infarction: Lipid Paradox Revised.

Lower level of low-density lipoprotein cholesterol (LDL-C) is paradoxically associated with increased mortality in ST elevation myocardial infarction (STEMI) patients. The underlying mechanism remains unclear. In a cohort of 220 de novo STEMI patients receiving timely primary percutaneous coronary intervention, admission LDL-C was negatively associated with circulating CD14++CD16+ monocyte counts. Moreover, admission LDL-C < 85 mg/dL was associated with increased risk for major adverse cardiovascular events (MACE) during a median follow-up of 2.7 years. After categorizing the patients according to the cutoff values of 85 mg/dL for LDL-C and the median for CD14++CD16+ monocytes, low LDL-C-associated MACE risk was only observed in those with high CD14++CD16+ monocyte counts (low LDL-C/high CD14++CD16+ monocytes vs. low LDL-C/low CD14++CD16+ monocytes: hazard ratio 5.38, 95% confidence interval 1.52 to 19.06, P = 0.009). This work provided the proof-of-principle evidence indicating a role of CD14++CD16+ monocytes in risk stratification of STEMI patients presenting with low LDL-C level. Graphical abstract.

Preparation of genetically engineered murine SINE RNA without endotoxin contamination.

RNAs have been elucidated to play the critical role in regulating gene expression and to be expected as effective drugs in the treatment of cancer and age-related diseases. RNAs are extracted by SDS-NaCl centrifugation after transformation of E.coli by expression vectors, which is a method to obtain genetically engineered RNAs. But the prepared RNAs by this method contain endotoxin, which limits their application in vivo and in cell experments. Here we improved SDS-NaCl filtration method based on SDS-NaCl centrifugation method. Endotoxin removal efficiency of SDS-NaCl filtration was nearly 4.2 times more than did SDS-NaCl centrifugation. Triton X-114 phase separation was used to reduce futher the endotoxin content of SDS-NaCI filtration-extracted RNA (from 11.25 EU/µg RNA/ml to 0.08 EU/µg RNA/ml). RNA prepared using the methods established in this paper meets the requirements for in vivo and cell culture experiments. Here we describe the process of preparing endotoxin-free B1as RNA from pET-B1as-DE3 E. coli (DE3 transformed by pET-B1as expression vector which containing a tandem SINE B1 elements) using SDS-NaCl filtration incorporating Triton X-114 phase separation.•The endotoxin removal efficiency of SDS-NaCl filtration is higher than that of SDS-NaCl centrifugation.•RNA prepared by SDS-NaCl filtration incorporating Triton X-114 meets the requirements for in vivo experiments on animals.

Induction of CXC chemokine messenger-RNA expression in chicken oviduct epithelial cells by Salmonella enterica serovar enteritidis via the type three secretion system-1.

The messenger-RNA (mRNA) expression of selected cytokines and chemokines in primary chicken oviduct epithelial cells (COEC) was determined following in vitro infections with wild-type or type three secretion system (T3SS)-mutant Salmonella enterica serovar Enteritidis (SE) strains. All SE strains examined in this study elicited the expression of proinflammatory immune mediators including inducible nitric oxide synthase (iNOS), CXCLi1 (K60), CXCLi2 (IL-8), CCLi3 (K203), and CCLi4 (MIP-1beta). SE also triggered the expression of an anti-inflammatory cytokine, IL-10, but repressed TGF-beta3 transcription. Both T3SS-1 (sipA and sipB) and T3SS-2 (pipB and ssaV) mutants showed reduced capacity, compared to the wild-type SE, to stimulate iNOS mRNA expression in COEC. T3SS-1 (sipA and sipB) mutants were significantly impaired in their ability to induce the expression of CXCLi1 and CXCLi2. T3SS-2 mutants displayed a wild-type phenotype in terms of modulating the expression of chemokines and cytokines in COEC. The expression of iNOS, but not CXC chemokines, correlated with the number of intracellular bacteria in COEC. Genetic complementation of the sipA mutation restored a wild-type phenotype. Thus, SE induction of CXCLi1 and CXCLi2 was sipA-dependent. These results provide enhanced insights into the complex interplay between local host innate immune system and bacterial virulence factors.

Trajectories of Circulating Monocyte Subsets After ST-Elevation Myocardial Infarction During Hospitalization: Latent Class Growth Modeling for High-Risk Patient Identification.

It remains unclear if the developmental trajectories of a specific inflammatory biomarker during the acute phase of ST-elevation myocardial infarction (STEMI) provide outcome prediction. By applying latent class growth modeling (LCGM), we identified three distinctive trajectories of CD14++CD16+ monocytes using serial flow cytometry assays from day 1 to day 7 of symptom onset in 96 de novo STEMI patients underwent primary percutaneous coronary intervention. Membership in the high-hump-shaped trajectory (16.8%) independently predicted adverse cardiovascular outcomes during a median follow-up of 2.5 years. Moreover, inclusion of CD14++CD16+ monocyte trajectories significantly improved area under the curve (AUC) when added to left ventricular ejection fraction-based prediction model (ΔAUC = 0.093, P = 0.013). Therefore, CD14++CD16+ monocyte trajectories during STEMI hospitalization are a novel risk factor for post-STEMI adverse outcomes. These results provide the first proof-of-principle evidence in support of the risk stratification role of LCGM-based longitudinal modeling of specific inflammatory markers during acute STEMI.

Failed detection of Bovine viral diarrhea virus 2 subgenotype a (BVDV-2a) by direct fluorescent antibody test on tissue samples due to reduced reactivity of field isolates to raw anti-BVDV antibody.

Bovine viral diarrhea virus 1 (BVDV-1) is associated with mild or subclinical infections, whereas BVDV-2 is frequently implicated in outbreaks of severe thrombocytopenia and acute fatal disease. In the present study, the carcass of a beef breed cow and tissue samples of a beef calf were received for laboratory diagnosis. Both animals exhibited severe clinical signs compatible with thrombocytopenia or hemorrhagic syndrome. Direct fluorescent antibody test (DFAT) failed to detect BVDV antigen in the tissue specimens of both cases. However, immunohistochemistry (IHC) revealed the presence of BVDV antigen in oral and esophageal mucosa and Peyer patches of the beef breed cow. Real-time reverse transcription polymerase chain reaction (real-time RT-PCR) detected BVDV-2 in selected tissues of both animals. Subsequently, BVDV was isolated from both cases and subjected to genetic and serologic characterizations. Mutations in the 5'-untranslated genomic region (5'-UTR) primer and probe binding sites and the E2 gene were associated with reduced efficiency of an established real-time RT-PCR assay and amino acid alterations in the E2 glycoprotein, respectively. Both viral isolates were classified by real-time RT-PCR and phylogenetic analysis as BVDV-2 subgenotype a. Unlike BVDV reference strains Singer and 125c, the isolates cross-reacted with anti-BVDV-1 and anti-BVDV-2 reference sera, indicating antigenic variations in field isolates. The isolates also showed reduced reactivity to porcine anti-BVDV antiserum (the raw serum used to produce BVDV DFA conjugate). In summary, data from the present investigation indicated that genetic and antigenic variations affected the performance of detection assays, especially DFAT, highlighting the need for regular evaluation and modification of BVDV tests.