Various vaccines, like polio, measles, and rotavirus, have been developed by serial-passaging in cell culture. Live oral rotavirus vaccines have been shown to be generally safe, but mechanisms of attenuation are not known. We have used a new, entirely plasmid-based reverse genetics system to artificially generate the novel human rotavirus vaccine strain CDC-9 (G1P[8]) and analyze the effect of the mutations within the VP4 gene on adaptation in vitro and attenuation in vivo. We demonstrated that out of the 6 amino acid mutations that appeared after serial passaging in Vero cells, mutations of wild-type CDC-9 P11 at VP4 AA331 and AA385 each or in combination were associated with increased replication in vitro comparable to cell-culture adapted CDC-9 P45. Neonatal rats infected with the single AA331 or AA385 mutant had reduced viral shedding, comparable to cell-culture passaged CDC-9 P45. We observed additional reduced shedding in neonatal rats that were infected with combination mutants harboring mutations at position AA331_385_388, indicative of a slight additive effect. Our data indicate that mutations in the VP5* region of the VP4 gene, particularly at position AA331 and AA385, are the determining factor for in vitro replication adaptation and in vivo attenuation of a G1P[8] rotavirus vaccine. This information provides great potential for targeted mutation in rotavirus vaccine generation instead of labor-consuming serial passaging in cell culture.IMPORTANCELive oral rotavirus vaccines have been developed through serial passaging in cell culture and found to be generally safe and efficacious in children. Live vaccines are also found to be associated with rare but severe adverse events, such as intussusception, in vaccinated children. Mechanisms for vaccine attenuation and adverse effects are unknown. We have developed a novel human rotavirus vaccine strain (CDC-9) and demonstrated several amino acid mutations in the VP4 gene of cell-passaged virus. In the present study, we identified two key amino acid mutations via reverse genetics technology in VP4 that mediated enhanced growth in cell culture, including a human intestinal cell line, reduced virus shedding, and downregulated inflammatory response in neonatal rats. This study is the first to identify the molecular signatures that define attenuation of human rotavirus vaccine and should help provide guidance for developing new generations of safe and effective vaccines.

We have previously introduced the first generation of C3P3, an artificial system that allows the autonomous in-vivo production of mRNA with m7GpppN-cap. While C3P3-G1 synthesized much larger amounts of capped mRNA in human cells than conventional nuclear expression systems, it produced a proportionately much smaller amount of the corresponding proteins, indicating a clear defect of mRNA translatability. A possible mechanism for this poor translatability could be the rudimentary polyadenylation of the mRNA produced by the C3P3-G1 system. We therefore sought to develop the C3P3-G2 system using an artificial enzyme to post-transcriptionally lengthen the poly(A) tail. This system is based on the mutant mouse poly(A) polymerase alpha fused at its N terminus with an N peptide from the λ virus, which binds to BoxBr sequences placed in the 3′UTR region of the mRNA of interest. The resulting system selectively brings mPAPαm7 to the target mRNA to elongate its poly(A)-tail to a length of few hundred adenosine. Such elongation of the poly(A) tail leads to an increase in protein expression levels of about 2.5–3 times in cultured human cells compared to the C3P3-G1 system. Finally, the coding sequence of the tethered mutant poly(A) polymerase can be efficiently fused to that of the C3P3-G1 enzyme via an F2A sequence, thus constituting the single-ORF C3P3-G2 enzyme. These technical developments constitute an important milestone in improving the performance of the C3P3 system, paving the way for its applications in bioproduction and non-viral human gene therapy.

Rotaviruses pose a significant threat to young children. To identify novel pro- and anti-rotavirus host factors, we performed genome-wide CRISPR/Cas9 screens using rhesus rotavirus and African green monkey cells. Genetic deletion of either SERPINB1 or TMEM236, the top two antiviral factors, in MA104 cells increased virus titers in a rotavirus strain independent manner. Using this information, we optimized the existing rotavirus reverse genetics systems by combining SERPINB1 knockout MA104 cells with a C3P3-G3 helper plasmid. We improved the recovery efficiency and rescued several low-titer rotavirus reporter and mutant strains that prove difficult to rescue otherwise. Furthermore, we demonstrate that TMEM236 knockout in Vero cells supported higher yields of two live-attenuated rotavirus vaccine strains than the parental cell line and represents a more robust vaccine-producing cell substrate. Collectively, we developed a third-generation optimized rotavirus reverse genetics system and generated gene-edited Vero cells as a new substrate for improving rotavirus vaccine production.

Group A rotavirus (RV) remains as the single most important cause of severe acute gastroenteritis among infants and young children worldwide. An entirely plasmid-based reverse genetics (RG) system was recently developed, opening new ways for in-depth molecular study of RV. Despite several improvements to further optimize the RG efficiency, it has been reported that current strategies do not enable the rescue of all cultivatable RV strains. Here, we described a helpful modification to the current strategies and established a tractable RG system for the rescue of the simian RRV strain, the human CDC-9 strain, and a murine-like RV strain, which is suitable for both in vitro and in vivo studies. This improved RV reverse genetics system will facilitate study of RV biology in both in vitro and in vivo systems that will facilitate the improved design of RV vaccines, better antiviral therapies, and expression vectors.

Most eukaryotic expression systems make use of host-cell nuclear transcriptional and post-transcriptional machineries. Here, we present the first generation of the chimeric cytoplasmic capping-prone phage polymerase (C3P3-G1) expression system developed by biological engineering, which generates capped and polyadenylated transcripts in host-cell cytoplasm by means of two components. First, an artificial single-unit chimeric enzyme made by fusing an mRNA capping enzyme and a DNA-dependent RNA polymerase. Second, specific DNA templates designed to operate with the C3P3-G1 enzyme, which encode for the transcripts and their artificial polyadenylation. This system, which can potentially be adapted to any in cellulo or in vivo eukaryotic expression applications, was optimized for transient expression in mammalian cells. C3P3-G1 shows promising results for protein production in Chinese Hamster Ovary (CHO-K1) cells. This work also provides avenues for enhancing the performances for next generation C3P3 systems.

Background: Chronic pancreatitis is the most common cause of exocrine pancreatic insufficiency (EPI) in adults. EPI is a deficiency in exocrine pancreatic enzymes resulting in an inability to maintain normal digestion. This inadequate digestion, especially fat malabsorption, occurs when intraduodenal levels of lipase fall below 5–10% of normal enzyme output. Furthermore, in EPI due to chronic pancreatitis, decreased bicarbonate output leads to impairment of micelle formation of fats. Fat maldigestion is compounded by decreased pancreatic secretion of lipase and colipase, further dampening hydrolysis of intraluminal fat.

segmental hyperalgesia (n=14); and 4) impaired CPM and segmental hyperalgesia (n=16) (Figure).Significant differences in average clinical pain scores, as well as BPI pain and interference scores, were observed across subgroups, with higher pain scores observed in subgroup 4 (Table ).In contrast, anxiety, depression, and pain catastrophizing scores were comparable across subgroups, implying that QST profiles were not associated with psychiatric comorbidity.Conclusion: Patients with segmental hyperalgesia and impaired CPM have significantly more pain compared to patients with normal QST profiles.As psychological profiles were not dependent on pain modulatory phenotypes, QST provides an unbiased mean for characterization of pain on an individual patient level.This information can be used for prognostication and tailoring of management strategies.

ABSTRACT Reoviruses, like many eukaryotic viruses, contain an inverted 7-methylguanosine (m7G) cap linked to the 5′ nucleotide of mRNA. The traditional functions of capping are to promote mRNA stability, protein translation, and concealment from cellular proteins that recognize foreign RNA. To address the role of mRNA capping during reovirus replication, we assessed the benefits of adding the African swine fever virus NP868R capping enzyme during reovirus rescue. C3P3, a fusion protein containing T7 RNA polymerase and NP868R, was found to increase protein expression 5- to 10-fold compared to T7 RNA polymerase alone while enhancing reovirus rescue from the current reverse genetics system by 100-fold. Surprisingly, RNA stability was not increased by C3P3, suggesting a direct effect on protein translation. A time course analysis revealed that C3P3 increased protein synthesis within the first 2 days of a reverse genetics transfection. This analysis also revealed that C3P3 enhanced processing of outer capsid μ1 protein to μ1C, a previously described hallmark of reovirus assembly. Finally, to determine the rate of infectious-RNA incorporation into new virions, we developed a new recombinant reovirus S1 gene that expressed the fluorescent protein UnaG. Following transfection of cells with UnaG and infection with wild-type virus, passage of UnaG through progeny was significantly enhanced by C3P3. These data suggest that capping provides nontraditional functions to reovirus, such as promoting assembly and infectious-RNA incorporation. IMPORTANCE Our findings expand our understanding of how viruses utilize capping, suggesting that capping provides nontraditional functions to reovirus, such as promoting assembly and infectious-RNA incorporation, in addition to enhancing protein translation. Beyond providing mechanistic insight into reovirus replication, our findings also show that reovirus reverse genetics rescue is enhanced 100-fold by the NP868R capping enzyme. Since reovirus shows promise as a cancer therapy, efficient reovirus reverse genetics rescue will accelerate production of recombinant reoviruses as candidates to enhance therapeutic potency. NP868R-assisted reovirus rescue will also expedite production of recombinant reovirus for mechanistic insights into reovirus protein function and structure.

It is regularly thought that human complex disorder susceptibility genes show differences in gene expression between normal and pathologic tissues. Thus, differences of transcript amounts could be indicative of complex disorder susceptibility loci and, therefore, be used for the discovery or the validation of human susceptibility genes to complex disorders/traits. Whether human complex disorder susceptibility genes effectively display differences in transcript amounts was tested by meta-analysis of the published literature comparing transcript amounts of well-validated human susceptibility genes to complex traits/disorders. A total of 94 gene-disease associations, which were studied in at least three independent studies and showed strong evidence of positive association, were analyzed. For 23 out of these 94 well-validated gene-disease associations, 120 gene expression studies comparing normal and pathologic human tissues were found. For 60 out of these 120 gene expression studies, the difference of level expression between normal and pathologic human tissues was statistically significant. This result was highly significant, as only 6 significant results were expected randomly under the null hypothesis ( P < 10 −112 ). A large excess of replication studies were also found, which were in agreement with the original report ( P = 6×10 −4 ). However, the overall level of expression change between normal and pathologic human tissues was relatively moderate, because only 36 (60%) and 19 (31.6%) out of the 62 statistically significant gene expression studies reached 2- or 3-fold changes in expression level, respectively. The present meta-analysis confirms statistical differences of expression levels between normal and pathologic human tissues for human susceptibility genes to complex traits/disorders. However, the levels of differences in transcript amounts appear to be relatively weak. These findings rationalize the use of gene expression for the discovery/validation of human susceptibility genes, but the weak differences of expression typically found should be taken into account for the design of such studies.

Les donnees d'epidemiologie genetique ont de longue date suggere l'existence de facteurs genetiques de susceptibilite dans le cancer de la prostate. Certains de ces genes de susceptibilite ont ete recemment localises ou clones. De plus, d'importants progres ont egalement ete realises dans le domaine de la genetique somatique de ce cancer. Le but de cette revue est de presenter les resultats obtenus dans ces domaines, ainsi que les technologies ayant permis de les obtenir.

Genetic epidemiology studies have suggested the responsibility of genetic factors in prostate cancer susceptibility. The development of genomic and high throughput genetic studies has allowed the recent identification or localization of such susceptibility genes. Furthermore, these technologies have contributed to outstanding advances in somatic genetics of prostate cancer. The goal of the present review is to disclose contributing technologies to and results of genetic studies of prostate cancer.

With insulinoma, Zollinger-Ellison syndrome is one of the most common functional islet-cell tumour. Since it is a life-threatening condition, needing appropriate management, the diagnosis must be accurately established This paper reviews the clinical situations leading to suspect the diagnosis and the biological tests, mainly the secretin test, that confirm the diagnosis. The different ways of performing the secretin test and the respective results are presented. This review will also focus on some aspects of the diagnosis of multiple endocrine neoplasia type 1 in these patients.

Background Br Aims: Long-term survival of patients with Zollinger-Ellison syndrome (ZES) is largely determined by the presence or absence of liver metastases, However, because of the lack of precision of these criteria, development of further indicators is still required. Recent evidence showing that autoantibodies directed against the p53 protein could predict poor survival for some types of cancers prompted us to investigate the presence of such antibodies in sera from patients with ZES and their potential value as survival indicator, Methods: Anti-p53 antibodies were detected in the sera of 44 consecutive patients with ZES using both an enzyme-linked immunosorbent assay (ELISA) and Western blotting. The mean follow-up of these patients was 92 months, Results: Anti-p53 antibodies were detected in 7 of the patients with ZES (16%) by both ELISA and Western blotting. Univariate and multivariate analyses showed that the presence of anti-p53 antibodies (P = 0.0009 and P = 0.017, respectively) and liver metastases (P = 0.0009 and P = 0.012, respectively) was independently associated with shorter survival, Conclusions: These results suggest that anti-p53 antibodies are an indicator of survival and could be used in combination with staging to determine which patients with ZES have poor prognoses and therefore require reinforced therapy.

The human EB1 gene product was recently found, by a yeast two-hybrid screening, to be associated with the carboxy terminus of the APC (adenomatous polyposis coli) protein, the product of a tumour-suppressor gene thought to act as a gatekeeper in colorectal carcinogenesis. Because virtually all of the APC mutations result in the synthesis of carboxy-terminal truncated proteins, mutant APC proteins are expected to lose their ability to interact with EB1 gene product. Thus, the interaction between APC and EB1 proteins may be important for the tumour-suppressor activity of APC protein, and raises the hypothesis that EB1 is also involved in sporadic colorectal tumorigenesis. To investigate this hypothesis, somatic mutations in the entire coding sequence of EB1 cDNA were searched by reverse transcriptase single-strand conformational polymorphism (SSCP) analysis in 21 sporadic colorectal cancers and seven adenomas. None of these tumours contained somatic mutation, whereas a silent cDNA variant was identified in 14% of alleles. Furthermore, to investigate whether EB1 locus was included within a region subjected to losses of heterozygosity, four polymorphism markers surrounding EB1 locus were surveyed. Only one out of 28 colorectal tumours contained a loss of heterozygosity at the D20S107 marker. In conclusion, the present findings strongly suggest that EB1 gene is not involved in somatic colorectal carcinogenesis.

BACKGROUND AND AIMS:Mutations of TP53, a tumor suppressor gene, are found in 60% to 70% of colorectal cancers. These mutations usually induce an overexpression caused by modification of the p53 protein conformation. The aim of this study was to investigate whether stool specimens of patients with colorectal cancer contain increased amounts of p53 protein.METHODS:p53 protein was measured using a sandwich enzyme immunoassay in the stool specimens of 52 patients: 25 with colorectal cancer, 4 with colorectal adenomas and 23 apparently free of gastrointestinal disease. Results were expressed as pg/mg of total protein. The presence of fecal occult-blood was searched using Hemoccult II and Hemolex (an immunochemical assay for human hemoglobin).RESULTS:Median concentrations of stool p53 protein were 16.6 pg/mg (range: 0-591 pg/mg) in patients with colorectal cancers, 39.1 pg/mg (range: 5-72 pg/mg) in patients with adenomas and 5.9 pg/mg (range: 0-65 pg/mg) in control subjects. Resection of colorectal cancers caused a marked decrease of stool p53 protein concentrations. When the cut-off value for stool p53 protein was set at 60 pg/mg of fecal protein (concentrations over the 95th percentile), the positivity of the assay was independent of tumor size and Astler-Coller stage, but weakly associated with rectal location of cancer. The sensitivity of stool p53 protein for colorectal cancer was 44%, and the specificity was 96%. In contrast, the sensitivity of Hemoccult II and Hemolex tests was 48% and 44%, whereas their specificity was 91% and 96%, respectively.CONCLUSION:The detection of p53 protein is achievable in stool, but this assay is not more efficient than fecal occult blood tests for detection of colorectal cancer.