TECATOR Fibertec System I. készülék alkalmazása élelmiszerek rosttartalmának meghatározására

A szerzők az AOAC összélelmi-rost meghatározására szolgáló módszert adaptáták a TECATOR Fibertec System I. típusú, svéd gyártmányú készülékre. 14 bébiételből és 5 zöldségféléből végeztek összehasonlító méréseket. Az eredmények alapján a TECATOR készüléken a műszerhez tartozó módszergyűjteményben javasoltakon kívül az összélelmi-rost mérése is elvégezhető. The method for the determination of AOAC total nutritional fiber were adapted for the TECATOR Fibertec System I. Swedish instrument by the authors. Comparative tests were done from 14 baby foods and 5 vegetables. You can do the determination of total nutritional fiber as well with TECATOR instrument based on results. Verfasser adaptierten die AOAC-Methode zur Bestimmung des Gesamtrohfasergehaltes unter Anwendung des schwedischen Meßgerätes TECATOR Fibertec System I. Von 14 Babynahrungproben und 5 Gemüsearten wurden vergleichende Meßungen durchgeführt. Auf der Grundlage der Ergebnisse kann die zum Meßgerät TECATOR gehörende Methodensammlung mit der Messung des Gesamtrohfasergehaltes ergänzt werden

Élelmiszerek egyes rost-frakcióinak vizsgálata

A szerzők kereskedelmi forgalomból származó egyes zöldségfélék és almafajták vízoldhatatlan rost-frakcióit (cellulóz, hemicellulóz, lignin) határozták meg Tecator Fibertec System I. típusú svéd gyártmányú készüléken. The water insoluble fiber fractions (cellulose, hemicellulose, lignin) were determined in vegetables and fruits from trade by a Tecator Fibertec System I type instrument, made in Sweden. Verfasser haben die wasserunlöslichen Rohfaserfraktionen (Zellulose, Hemizellulose, Lignin) einzelner Gemüsearten und Apfelsorten aus dem Handel untersucht. Dafür wurde das schwedische Tecator Fibertec System I. eingesetzt

Recoding of the Vesicular Stomatitis Virus L Gene by Computer-Aided Design Provides a Live, Attenuated Vaccine Candidate

ABSTRACT Codon pair bias (CPB), which has been observed in all organisms, is a neglected genomic phenomenon that affects gene expression. CPB results from synonymous codons that are paired more or less frequently in ORFeomes regardless of codon bias. The effect of an individual codon pair change is usually small, but when it is amplified by large-scale genome recoding, strikingly altered biological phenotypes are observed. The utility of codon pair bias in the development of live attenuated vaccines was recently demonstrated by recodings of poliovirus (a positive-strand RNA virus) and influenza virus (a negative-strand segmented RNA virus). Here, the L gene of vesicular stomatitis virus (VSV), a nonsegmented negative-sense RNA virus, was partially recoded based on codon pair bias. Totals of 858 and 623 silent mutations were introduced into a 5′-terminal segment of the viral L gene (designated L1) to create sequences containing either overrepresented or underrepresented codon pairs, designated L1sdmax and L1min, respectively. Analysis revealed that recombinant VSV containing the L1min sequence could not be recovered, whereas the virus with the sdmax sequence showed a modest level of attenuation in cell culture. More strikingly, in mice the L1sdmax virus was almost as immunogenic as the parental strain but highly attenuated. Taken together, these results open a new road to attain a balance between VSV virulence and immunogenicity, which could serve as an example for the attenuation of other negative-strand, nonsegmented RNA viruses

A Freeze Frame View of Vesicular Stomatitis Virus Transcription Defines a Minimal Length of RNA for 5′ Processing

The RNA synthesis machinery of vesicular stomatitis virus (VSV) comprises the genomic RNA encapsidated by the viral nucleocapsid protein (N) and associated with the RNA dependent RNA polymerase, the viral components of which are a large protein (L) and an accessory phosphoprotein (P). The 241 kDa L protein contains all the enzymatic activities necessary for synthesis of the viral mRNAs, including capping, cap methylation and polyadenylation. Those RNA processing reactions are intimately coordinated with nucleotide polymerization such that failure to cap results in termination of transcription and failure to methylate can result in hyper polyadenylation. The mRNA processing reactions thus serve as a critical check point in viral RNA synthesis which may control the synthesis of incorrectly modified RNAs. Here, we report the length at which viral transcripts first gain access to the capping machinery during synthesis. By reconstitution of transcription in vitro with highly purified recombinant polymerase and engineered templates in which we omitted sites for incorporation of UTP, we found that transcripts that were 30-nucleotides in length were uncapped, whereas those that were 31-nucleotides in length contained a cap structure. The minimal RNA length required for mRNA cap addition was also sufficient for methylation since the 31-nucleotide long transcripts were methylated at both ribose-2′-O and guanine-N-7 positions. This work provides insights into the spatial relationship between the active sites for the RNA dependent RNA polymerase and polyribonucleotidyltransferase responsible for capping of the viral RNA. We combine the present findings with our recently described electron microscopic structure of the VSV polymerase and propose a model of how the spatial arrangement of the capping activities of L may influence nucleotide polymerization

Tackling feline infectious peritonitis via reverse genetics

Feline infectious peritonitis (FIP) is caused by feline coronaviruses (FCoVs) and represents one of the most important lethal infectious diseases of cats. To date, there is no efficacious prevention and treatment, and our limited knowledge on FIP pathogenesis is mainly based on analysis of experiments with field isolates. In a recent study, we reported a promising approach to study FIP pathogenesis using reverse genetics. We generated a set of recombinant FCoVs and investigated their pathogenicity in vivo. The set included the type I FCoV strain Black, a type I FCoV strain Black with restored accessory gene 7b, two chimeric type I/type II FCoVs and the highly pathogenic type II FCoV strain 79-1146. All recombinant FCoVs and the reference strain isolates were found to establish productive infections in cats. While none of the type I FCoVs and chimeric FCoVs induced FIP, the recombinant type II FCoV strain 79-1146 was as pathogenic as the parental isolate. Interestingly, an intact ORF 3c was confirmed to be restored in all viruses (re)isolated from FIP-diseased animals

Development of Feline Ileum- and Colon-Derived Organoids and Their Potential Use to Support Feline Coronavirus Infection

Feline coronaviruses (FCoVs) infect both wild and domestic cat populations world-wide. FCoVs present as two main biotypes: the mild feline enteric coronavirus (FECV) and the fatal feline infectious peritonitis virus (FIPV). FIPV develops through mutations from FECV during a persistence infection. So far, the molecular mechanism of FECV-persistence and contributing factors for FIPV development may not be studied, since field FECV isolates do not grow in available cell culture models. In this work, we aimed at establishing feline ileum and colon organoids that allow the propagation of field FECVs. We have determined the best methods to isolate, culture and passage feline ileum and colon organoids. Importantly, we have demonstrated using GFP-expressing recombinant field FECV that colon organoids are able to support infection of FECV, which were unable to infect traditional feline cell culture models. These organoids in combination with recombinant FECVs can now open the door to unravel the molecular mechanisms by which FECV can persist in the gut for a longer period of time and how transition to FIPV is achieved

Genome Organization and Reverse Genetic Analysis of a Type I Feline Coronavirus▿

In this study we report the complete sequence and genome organization of the serotype I feline coronavirus (FCoV) strain Black. Furthermore, a reverse genetic system was established for this FCoV strain by cloning a full-length cDNA copy into vaccinia virus. This clone served as basis for the generation of recombinant FCoV (recFCoV) that was shown to bear the same features in vitro as the parental FCoV. Using this system, accessory 3abc genes in the FCoV genome were replaced by green fluorescent protein (recFCoV-GFP) and Renilla luciferase genes (recFCoV-RL). In addition, we showed that feline CD14+ blood monocytes and dendritic cells can be easily detected after infection with recFCoV-GFP. Thus, our established reverse genetic system provides a suitable tool to study the molecular biology of serotype I FCoV

Determination to a single nucleotide the length at which VSV mRNA gets capped.

<p>(A) An autoradiograph of a 6% polyacrylamide gel is shown indicating the products of the IVT reactions with recombinant VSV containing a 60-nt long non-essential gene that lacks sites for UTP incorporation except at positions 31, 32, 33, 34, 35, 36, 37 or 38 respectively. The reaction was performed in the absence of UTP, but containing [α-³²P]-GTP. The identity of the purified viruses used for the IVT reactions are shown at the top panel. The uncapped leader RNA and the different transcripts between 30 and 38 nt are indicated. Asterisks indicate the 1–2 nt longer than anticipated transcripts of the rVSV(A-)-33 and -34 viruses. (B) Recombinant VSV(A-)-30, -31, -33, -34, -35, -36 and -37 were used in <i>in vitro</i> transcription (IVT) reactions lacking UTP, but containing [α-³²P]-GTP. The products were analyzed on 6% polyacrylamide gel and visualized using a phosphorimager. The products of transcription were either untreated (-) or digested with TAP (+) which cleaves the cap-structure. The identity if the purified viruses used for the IVT reactions are shown at the top panel. The uncapped leader RNA and the different transcripts between 30 and 38 nt are indicated. A representative gel from two independent experiments is shown.</p

Capping occurs at a nascent RNA chain length of 31-nt.

<p><i>In vitro</i> transcription reactions were reconstituted with 5 µg of N-RNA template, 2 µg of purified P, and 4 µg of the indicated L protein in the absence of UTP, but in the presence of [α-³²P]-GTP. Purified RNA was analyzed on a 6% polyacrylamide gel and detected using a phosphorimager. The identity of the template and for the reactions used polymerase molecules are shown at the top the panel. The uncapped leader RNA and the different transcripts between 30 and 34 nt are indicated. Asterisks indicate the uncapped transcripts made by H1227A-L with the different templates. A representative gel from two independent experiments is shown.</p