Structural Insight into CVB3-VLP Non-Adjuvanted Vaccine

Coxsackievirus B (CVB) enteroviruses are common pathogens that can cause acute and chronic myocarditis, dilated cardiomyopathy, aseptic meningitis, and they are hypothesized to be a causal factor in type 1 diabetes. The licensed enterovirus vaccines and those currently in clinical development are traditional inactivated or live attenuated vaccines. Even though these vaccines work well in the prevention of enterovirus diseases, new vaccine technologies, like virus-like particles (VLPs), can offer important advantages in the manufacturing and epitope engineering. We have previously produced VLPs for CVB3 and CVB1 in insect cells. Here, we describe the production of CVB3-VLPs with enhanced production yield and purity using an improved purification method consisting of tangential flow filtration and ion exchange chromatography, which is compatible with industrial scale production. We also resolved the CVB3-VLP structure by Cryo-Electron Microscopy imaging and single particle reconstruction. The VLP diameter is 30.9 nm on average, and it is similar to Coxsackievirus A VLPs and the expanded enterovirus cell-entry intermediate (the 135s particle), which is similar to 2 nm larger than the mature virion. High neutralizing and total IgG antibody levels, the latter being a predominantly Th2 type (IgG1) phenotype, were detected in C57BL/6J mice immunized with non-adjuvanted CVB3-VLP vaccine. The structural and immunogenic data presented here indicate the potential of this improved methodology to produce highly immunogenic enterovirus VLP-vaccines in the future.Peer reviewe

Accuracy of steps measured by smartphones-based WeRun compared with ActiGraph-GT3X accelerometer in free-living conditions

ObjectivesThe purpose of this study was to evaluate the accuracy and reliability of steps tracked by smartphone-based WeChat app compared with Actigraph-GT3X accelerometer in free-living conditions.DesignA cross-sectional study and repeated measures.MethodsA total of 103 employees in the Pudong New Area of Shanghai, China, participated in this study. The participants wore an ActiGraph-GT3X accelerometer during the period of August to September 2019 (Time 1), December 2019 (Time 2) and September 2020 (Time 3). Each time, they wore the ActiGraph-GT3X accelerometer continuously for 7 days to assess their 7-day step counts. The smartphone-based WeRun step counts were collected in the corresponding period when subjects wore accelerometers. The subjects were invited to complete basic demographic characteristics questionnaires and to perform physical examination to obtain health-related results such as height, body weight, body fat percentage, waist circumference, hip circumference, and blood pressure.ResultsBased on 103 participants' 21 days of data, we found that the Spearman correlation coefficient between them was 0.733 (P < 0.01). The average number of WeRun steps measured by smartphones was 8,975 (4,059) per day, which was higher than those measured by accelerometers (8,462 ± 3,486 per day, P < 0.01). Demographic characteristics and different conditions can affect the consistency of measurements. The consistency was higher in those who were male, older, master's degree and above educated, and traveled by walking. Steps measured by smartphone and accelerometer in working days and August showed stronger correlation than other working conditions and time. Mean absolute percent error (MAPE) for step counts ranged from 0.5 to 15.9%. The test-retest reliability coefficients of WeRun steps ranged from 0.392 to 0.646. A multiple regression analysis adjusted for age, gender, and MVPA/step counts measured during Time 1 showed that body composition (body weight, BMI, body fat percentage, waist circumference, and hip circumference) was correlated with moderate-to-vigorous intensity physical activity, but it was not correlated with WeRun step counts.ConclusionsThe smartphone-based WeChat app can be used to assess physical activity step counts and is a reliable tool for measuring steps in free-living conditions. However, WeRun step counts' utilization is potentially limited in predicting body composition

Mouse mammary tumor virus-like gene sequences are present in lung patient specimens

<p>Abstract</p> <p>Background</p> <p>Previous studies have reported on the presence of Murine Mammary Tumor Virus (MMTV)-like gene sequences in human cancer tissue specimens. Here, we search for MMTV-like gene sequences in lung diseases including carcinomas specimens from a Mexican population. This study was based on our previous study reporting that the INER51 lung cancer cell line, from a pleural effusion of a Mexican patient, contains MMTV-like <it>env </it>gene sequences.</p> <p>Results</p> <p>The MMTV-like <it>env </it>gene sequences have been detected in three out of 18 specimens studied, by PCR using a specific set of MMTV-like primers. The three identified MMTV-like gene sequences, which were assigned as INER6, HZ101, and HZ14, were 99%, 98%, and 97% homologous, respectively, as compared to GenBank sequence accession number <ext-link ext-link-id="AY161347" ext-link-type="gen">AY161347</ext-link>. The INER6 and HZ-101 samples were isolated from lung cancer specimens, and the HZ-14 was isolated from an acute inflammatory lung infiltrate sample. Two of the <it>env </it>sequences exhibited disruption of the reading frame due to mutations.</p> <p>Conclusion</p> <p>In summary, we identified the presence of MMTV-like gene sequences in 2 out of 11 (18%) of the lung carcinomas and 1 out of 7 (14%) of acute inflamatory lung infiltrate specimens studied of a Mexican Population.</p

The DNA/RNA-Dependent RNA Polymerase QDE-1 Generates Aberrant RNA and dsRNA for RNAi in a Process Requiring Replication Protein A and a DNA Helicase

The Neurospora RNA-dependent RNA polymerase QDE-1 is an RNA polymerase that can use both RNA and DNA as templates, suggesting a new mechanism for small RNA production

Differential Influence of Clonal Integration on Morphological and Growth Responses to Light in Two Invasive Herbs

Background and aims: In contrast to seeds, high sensitivity of vegetative fragments to unfavourable environments may limit the expansion of clonal invasive plants. However, clonal integration promotes the establishment of propagules in less suitable habitats and may facilitate the expansion of clonal invaders into intact native communities. Here, we examine the influence of clonal integration on the morphology and growth of ramets in two invasive plants, Alternanthera philoxeroides and Phyla canescens, under varying light conditions. Methods: In a greenhouse experiment, branches, connected ramets and severed ramets of the same mother plant were exposed under full sun and 85 % shade and their morphological and growth responses were assessed. Key results: The influence of clonal integration on the light reaction norm (connection6light interaction) of daughter ramets was species-specific. For A. philoxeroides, clonal integration evened out the light response (total biomass, leaf mass per area, and stem number, diameter and length) displayed in severed ramets, but these connection6light interactions were largely absent for P. canescens. Nevertheless, for both species, clonal integration overwhelmed light effect in promoting the growth of juvenile ramets during early development. Also, vertical growth, as an apparent shade acclimation response, was more prevalent in severed ramets than in connected ramets. Finally, unrooted branches displayed smaller organ size and slower growth than connected ramets, but the pattern of light reaction was similar, suggesting mothe

Small Molecule Amiloride Modulates Oncogenic RNA Alternative Splicing to Devitalize Human Cancer Cells

Alternative splicing involves differential exon selection of a gene transcript to generate mRNA and protein isoforms with structural and functional diversity. Abnormal alternative splicing has been shown to be associated with malignant phenotypes of cancer cells, such as chemo-resistance and invasive activity. Screening small molecules and drugs for modulating RNA splicing in human hepatocellular carcinoma cell line Huh-7, we discovered that amiloride, distinct from four pH-affecting amiloride analogues, could “normalize” the splicing of BCL-X, HIPK3 and RON/MISTR1 transcripts. Our proteomic analyses of amiloride-treated cells detected hypo-phosphorylation of splicing factor SF2/ASF, and decreased levels of SRp20 and two un-identified SR proteins. We further observed decreased phosphorylation of AKT, ERK1/2 and PP1, and increased phosphorylation of p38 and JNK, suggesting that amiloride treatment down-regulates kinases and up-regulates phosphatases in the signal pathways known to affect splicing factor protein phosphorylation. These amiloride effects of “normalized” oncogenic RNA splicing and splicing factor hypo-phosphorylation were both abrogated by pre-treatment with a PP1 inhibitor. Global exon array of amiloride-treated Huh-7 cells detected splicing pattern changes involving 584 exons in 551 gene transcripts, many of which encode proteins playing key roles in ion transport, cellular matrix formation, cytoskeleton remodeling, and genome maintenance. Cellular functional analyses revealed subsequent invasion and migration defects, cell cycle disruption, cytokinesis impairment, and lethal DNA degradation in amiloride-treated Huh-7 cells. Other human solid tumor and leukemic cells, but not a few normal cells, showed similar amiloride-altered RNA splicing with devitalized consequence. This study thus provides mechanistic underpinnings for exploiting small molecule modulation of RNA splicing for cancer therapeutics

High resolution chromosome 3p, 8p, 9q and 22q allelotyping analysis in the pathogenesis of gallbladder carcinoma

Our recent genome-wide allelotyping analysis of gallbladder carcinoma identified 3p, 8p, 9q and 22q as chromosomal regions with frequent loss of heterozygosity. The present study was undertaken to more precisely identify the presence and location of regions of frequent allele loss involving those chromosomes in gallbladder carcinoma. Microdissected tissue from 24 gallbladder carcinoma were analysed for PCR-based loss of heterozygosity using 81 microsatellite markers spanning chromosome 3p (n=26), 8p (n=14), 9q (n=29) and 22q (n=12) regions. We also studied the role of those allele losses in gallbladder carcinoma pathogenesis by examining 45 microdissected normal and dysplastic gallbladder epithelia accompanying gallbladder carcinoma, using 17 microsatellite markers. Overall frequencies of loss of heterozygosity at 3p (100%), 8p (100%), 9q (88%), and 22q (92%) sites were very high in gallbladder carcinoma, and we identified 13 distinct regions undergoing frequent loss of heterozygosity in tumours. Allele losses were frequently detected in normal and dysplastic gallbladder epithelia. There was a progressive increase of the overall loss of heterozygosity frequency with increasing severity of histopathological changes. Allele losses were not random and followed a sequence. This study refines several distinct chromosome 3p, 8p, 9q and 22q regions undergoing frequent allele loss in gallbladder carcinoma that will aid in the positional identification of tumour suppressor genes involved in gallbladder carcinoma pathogenesis

Mechanisms Establishing TLR4-Responsive Activation States of Inflammatory Response Genes

Precise control of the innate immune response is required for resistance to microbial infections and maintenance of normal tissue homeostasis. Because this response involves coordinate regulation of hundreds of genes, it provides a powerful biological system to elucidate the molecular strategies that underlie signal- and time-dependent transitions of gene expression. Comprehensive genome-wide analysis of the epigenetic and transcription status of the TLR4-induced transcriptional program in macrophages suggests that Toll-like receptor 4 (TLR4)-dependent activation of nearly all immediate/early- (I/E) and late-response genes results from a sequential process in which signal-independent factors initially establish basal levels of gene expression that are then amplified by signal-dependent transcription factors. Promoters of I/E genes are distinguished from those of late genes by encoding a distinct set of signal-dependent transcription factor elements, including TATA boxes, which lead to preferential binding of TBP and basal enrichment for RNA polymerase II immediately downstream of transcriptional start sites. Global nuclear run-on (GRO) sequencing and total RNA sequencing further indicates that TLR4 signaling markedly increases the overall rates of both transcriptional initiation and the efficiency of transcriptional elongation of nearly all I/E genes, while RNA splicing is largely unaffected. Collectively, these findings reveal broadly utilized mechanisms underlying temporally distinct patterns of TLR4-dependent gene activation required for homeostasis and effective immune responses

Homologs of genes and anonymous loci on human Chromosome 13 map to mouse Chromosomes 8 and 14

To enhance the comparative map for human Chromosome (Chr) 13, we identified clones for human genes and anonymous loci that cross-hybridized with their mouse homologs and then used linkage crosses for mapping. Of the clones for four genes and twelve anonymous loci tested, cross-hybridization was found for six, COL4A1, COL4A2, D13S26, D13S35, F10, and PCCA. Strong evidence for homology was found for COL4A1, COL4A2, D13S26, D13S35, and F10, but only circumstantial homology evidence was obtained for PCCA. To genetically map these mouse homologs ( Cf10, Col4a1, Col4a2, D14H13S26, D8H13S35 , and Pcca-rs ), we used interspecific and intersubspecific mapping panels. D14H13S26 and Pcca-rs were located on the distal portion of mouse Chr 14 extending by ∼30 cM the conserved linkage between human Chr 13 and mouse Chr 14, assuming that Pcca-rs is the mouse homolog of PCCA. By contrast, Cf10, Col4a1, Col4a2 , and D8H13S35 mapped near the centromere of mouse Chr 8, defining a new conserved linkage. Finally, we identified either a closely linked sequence related to Col4a2 , or a recombination hot-spot between Col4a1 and Col4a2 that has been conserved in humans and mice.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/47022/1/335_2004_Article_BF00352413.pd