Welfare Assessment on Pasture: A Review on Animal-Based Measures for Ruminants

Outdoor and extensive farming systems allow animals to behave in a natural way and are often perceived as welfare friendly. Nonetheless, the natural environment poses multiple challenges to the welfare of animals, sometimes hampering their capacity to cope. Welfare assessment in outdoor and extensive systems has been rarely investigated, and little is known about the most appropriate indicators. The aim of this review was to identify animal-based measures of welfare to apply in extensive and pasture-based systems in domestic ruminants. Through the use of a dedicated software for systematic reviews, 810 papers were screened and a total of 52 papers were retained for in-depth analysis. ABM resulting from these papers were initially divided according to the species (cattle and small ruminants, including sheep and goats) and then to four principles: comfort, behavior, feeding and health. The results showed that welfare data were collected applying different methodologies, with an increasing use of sensors in recent years. The need to herd and restrain animals for individual data collection is one of the major constraints to data collection in extensive farming systems. It is suggested that welfare assessment in outdoor/extensive farming systems is carried out by following shared procedures in order to provide evidence of the higher animal welfare claims that these products often imply compared to indoor systems

T Lymphocytes Transduced with a Lentiviral Vector Expressing F12-vif Are Protected from HIV-1 Infection in an APOBEC3G-Independent Manner

The viral infectivity factor (Vif) is an essential component of the HIV-1 infectious cycle. Vif counteracts the action of the cytidine deaminase APOBEC3G (AP3G), which confers nonimmune antiviral defense against HIV-1 to T lymphocytes. Disabling or interfering with the function of Vif could represent an alternative therapeutic approach to AIDS. We have expressed a natural mutant of Vif, F12-Vif, in a VSV-G-pseudotyped lentiviral vector under the Tat-inducible control of the HIV-1 LTR. Conditional expression of F12-Vif prevents replication and spreading of both CXCR4 and CCR5 strains of HIV-1 in human primary T lymphocyte and T cell lines. T cells transduced with F12-Vif release few HIV-1 virions and with reduced infectivity. Several lines of evidence indicate that HIV-1 interference requires the presence of both wild-type and F12-Vif proteins, suggesting a dominant-negative feature of the F12-Vif mutant. Surprisingly, however, the F12-Vif-mediated inhibition does not depend on the reestablishment of the AP3G function

Rapid, comprehensive analysis of the dystrophin transcript by a custom micro-fluidic exome array

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606. Identification of a 45-aa Domain of the F12-Vif Mutant Possessing Anti-HIV Activity

Our previous results have demonstrated that T-cell lines and primary T lymphocytes transduced with a Tat-dependent HIV-based lentiviral vectors expressing the mutant isoform of the vif gene, F12-vif, are protected from HIV-1 infection. F12-Vif is a 192-aa natural variant polypeptide owing 14 unique amino acid substitutions. The substitutions are randomly scattered along the entire sequence with the exception of a 5-aa cluster located at positions 127, 128, and 130|[ndash]|132. None of the 14 aa substitutions is present in the SOCS box that recruits the E3 ubiquitin ligase responsible of APOBEC3G (AP3G) degradation during HIV infection. In line with this notion, we have shown that the antiviral function of F12-Vif is not due to a dominant negative feature of the mutant in regards to the Vif-mediated degradation of AP3G rather to some other unknown means. Therefore, in the effort to elucidate the F12-Vif mechanism of action, we started to identify the protein domain of F12-Vif responsible of HIV-1 inhibition. To this end, we have constructed three chimeric genes (Chim1, Chim2 and Chim3) composed by wild-type and F12-vif regions. T cell lines and cord blood derived CD4+T lymphocytes were transduced with the lentiviral vectors expressing the chimeric genes and then challenged with both X4 and R5 HIV-1 strains. We show that 45 amino acids in the C-terminal domain of the F12-Vif mutant are sufficient to exert anti-viral effect in transduced cells. In contrast to F12-Vif, Chim3 does not allow the rescue of the replication of a vif-deficient HIV-1 in the context of either X4 or R5 tropism in non permissive cells. This specific feature renders Chim3 a truly dominant negative protein more suitable than F12-Vif for an anti-HIV gene therapy approach

Chim3 confers survival advantage to CD4+ T cells upon HIV-1 infection by preventing HIV-1 DNA integration and HIV-1–induced G2 cell-cycle delay

AbstractThe long-term expression and the ability of a therapeutic gene to confer survival advantage to transduced cells are mandatory requirements for successful anti-HIV gene therapy. In this context, we developed lentiviral vectors (LVs) expressing the F12–viral infectivity factor (Vif) derivative Chim3. We recently showed that Chim3 inhibits HIV-1 replication in primary cells by both blocking the accumulation of retrotranscripts, independently of either human APOBEC3G (hA3G) or Vif, and by preserving the antiviral function of hA3G. These results were predictive of long-lasting survival of Chim3+ cells after HIV-1 infection. Furthermore, Vif, like Vpr, deregulates cell-cycle progression by inducing a delay in G2 phase. Thus, the aim of this study was to investigate the role of Chim3 on both cell survival and cell-cycle regulation after HIV-1 infection. Here, we provide evidence that infected Chim3+ T cells prevail over either mock- or empty-LV engineered cells, show reduced G2 accumulation, and, as a consequence, ultimately extend their lifespan. Based on these findings, Chim3 rightly belongs to the most efficacious class of antiviral genes. In conclusion, Chim3 usage in anti-HIV gene therapy based on hematopoietic stem cell (HSC) modification has to be considered as a promising therapeutic intervention to eventually cope with HIV-1 infection

Heterogeneity of Signal Transducer and Activator of Transcription Binding Sites in the Long-Terminal Repeats of Distinct HIV-1 Subtypes

HIV-1 can be subdivided into distinct subtypes; the consequences of such a genomic variability remain largely speculative. The long terminal repeats (LTR) control HIV transcription and reflect the major differences of distinct viral subtypes. Three regions in the HIV-1 subtype B LTR are close matches to the Signal Transducer and Activator of Transcription (STAT) consensus sequence. Here, we show heterogeneity in these putative STAT binding sites among HIV-1 LTR subtypes A through G. Transfection of constitutively activated STAT5 lead to transcriptional activation of HIV-1 expression in 293T cells transfected with a reporter assay driven by HIV-1 LTR subtype B. Constitutively activated STAT5 transactivated the LTR of various subtypes in U937 cells with different potency. These findings support and expand the potential relevance of STAT5 activation in HIV infection and may bear relevance for a differential regulation of latency and expression of different subtypes of HIV-1

C6orf10 low-frequency and rare variants in italian multiple sclerosis patients

In light of the complex nature of multiple sclerosis (MS) and the recently estimated contribution of low-frequency variants into disease, decoding its genetic risk components requires novel variant prioritization strategies. We selected, by reviewing MS Genome Wide Association Studies (GWAS), 107 candidate loci marked by intragenic single nucleotide polymorphisms (SNPs) with a remarkable association (p-value <= 5 x 10(-6)). A whole exome sequencing (WES)-based pilot study of SNPs with minor allele frequency (MAF) <= 0.04, conducted in three Italian families, revealed 15 exonic low-frequency SNPs with affected parent-child transmission. These variants were detected in 65/120 Italian unrelated MS patients, also in combination (22 patients). Compared with databases (controls gnomAD, dbSNP150, ExAC, Tuscany-1000 Genome), the allelic frequencies of C6orf10 rs 16870005 and IL2RA rs12722600 were significantly higher (i.e., controls gnomAD, p = 9.89 x 10(-7) and p < 1 x 10(-20)). TET2 rs61744960 and TRAF3 rs138943371 frequencies were also significantly higher, except in Tuscany-1000 Genome. Interestingly, the association of C6orf10 rs16870005 (Ala431Thr) with MS did not depend on its linkage disequilibrium with the HLA-DRB1 locus. Sequencing in the MS cohort of the C6orf10 3' region revealed 14 rare mutations (10 not previously reported). Four variants were null, and significantly more frequent than in the databases. Further, the C6orf10 rare variants were observed in combinations, both intra-locus and with other low-frequency SNPs. The C6orf10 Ser389Xfr was found homozygous in a patient with early onset of the MS. Taking into account the potentially functional impact of the identified exonic variants, their expression in combination at the protein level could provide functional insights in the heterogeneous pathogenetic mechanisms contributing to MS.In light of the complex nature of multiple sclerosis (MS) and the recently estimated contribution of low-frequency variants into disease, decoding its genetic risk components requires novel variant prioritization strategies. We selected, by reviewing MS Genome Wide Association Studies (GWAS), 107 candidate loci marked by intragenic single nucleotide polymorphisms (SNPs) with a remarkable association (p-value ≤ 5 × 10−6). A whole exome sequencing (WES)-based pilot study of SNPs with minor allele frequency (MAF) ≤ 0.04, conducted in three Italian families, revealed 15 exonic low-frequency SNPs with affected parent-child transmission. These variants were detected in 65/120 Italian unrelated MS patients, also in combination (22 patients). Compared with databases (controls gnomAD, dbSNP150, ExAC, Tuscany-1000 Genome), the allelic frequencies of C6orf10 rs16870005 and IL2RA rs12722600 were significantly higher (i.e., controls gnomAD, p = 9.89 × 10−7 and p < 1 × 10−20). TET2 rs61744960 and TRAF3 rs138943371 frequencies were also significantly higher, except in Tuscany-1000 Genome. Interestingly, the association of C6orf10 rs16870005 (Ala431Thr) with MS did not depend on its linkage disequilibrium with the HLA-DRB1 locus. Sequencing in the MS cohort of the C6orf10 3′ region revealed 14 rare mutations (10 not previously reported). Four variants were null, and significantly more frequent than in the databases. Further, the C6orf10 rare variants were observed in combinations, both intra-locus and with other low-frequency SNPs. The C6orf10 Ser389Xfr was found homozygous in a patient with early onset of the MS. Taking into account the potentially functional impact of the identified exonic variants, their expression in combination at the protein level could provide functional insights in the heterogeneous pathogenetic mechanisms contributing to MS

130 purification of large scale mrna encoding zfn nucleases by dhplc technology

A novel strategy of targeted gene correction of the interleukin-2 receptor common gamma chain (IL2RG) gene for the treatment of X-linked Severe Combined Immunodeficiency (SCID-X1) is achieved by the combination of a pair of IL2RG-specific Zinc Finger Nucleases (ZFN) and the correct-gene template DNA delivered by integration-defective lentiviral vector (IDLV).The transient expression of the ZFN pair targeting the disease-causing gene is obtained by the electroporation of the two corresponding mRNAs, produced by in vitro transcription starting from plasmid DNA template. A major limitation of the mRNA transcribed in vitro is the presence of residual contaminants such as short RNAs and double stranded (ds)RNAs that may affect the function and spectrophotometric quantification of the product hampering therefore the delivery of high quality and precise amount of mRNA to target cells. Moreover, dsRNA contaminants represent a possible risk in terms of immunogenicity of the product, leading to activation of unwanted innate immune response with consequent reduction/abrogation of mRNA translation as well as potential alteration of the properties of the transfected cells. To improve nuclease expression while decreasing cellular innate response to mRNA transfection we combined different strategies: (i) inclusion of UTRs and polyA tails in the DNA template used for mRNA production; (ii) use of modified nucleotides during mRNA production and (iii) purification of the mRNAs by dHPLC with a reverse phase column made of non-porous matrix consisting of polystyrene-divinylbenzene copolymer beads alkylated with C-18 chains (Transgenomic, LTD.). In particular, the purification of in vitro transcribed mRNAs by means of dHPLC has been shown to strongly improve the translation of mRNA and significantly reduce the contaminant presence thus preventing innate immunity and eventually increasing modified cells persistence in vivo. We have developed feasible and reproducible, small and large scale mRNA production and downstream purification processes of the ZFN pairs obtaining accurate RNA quantification and reduced risk of immunogenicity. The full process achieved a 60% yield, loading with a 500µg RNA for each run with a single clean chromatographic peak. Furthermore, the level of residual organic solvent (i.e. Acetonitrile) used in the purification process is compatible with that applicable into clinic. The highly translatable non-immunogenic dHPLC-purified mRNA can be delivered without toxicity and represents a powerful and safe tool for the application of gene therapy protocols

The combination of transcriptomics and informatics identifies pathways targeted by miR-204 during neurogenesis and axon guidance

Vertebrate organogenesis is critically sensitive to gene dosage and even subtle variations in the expression levels of key genes may result in a variety of tissue anomalies. MicroRNAs (miRNAs) are fundamental regulators of gene expression and their role in vertebrate tissue patterning is just beginning to be elucidated. To gain further insight into this issue, we analysed the transcriptomic consequences of manipulating the expression of miR-204 in the Medaka fish model system. We used RNA-Seq and an innovative bioinformatics approach, which combines conventional differential expression analysis with the behavior expected by miR-204 targets after its overexpression and knockdown. With this approach combined with a correlative analysis of the putative targets, we identified a wider set of miR-204 target genes belonging to different pathways. Together, these approaches confirmed that miR-204 has a key role in eye development and further highlighted its putative function in neural differentiation processes, including axon guidance as supported by in vivo functional studies. Together, our results demonstrate the advantage of integrating next-generation sequencing and bioinformatics approaches to investigate miRNA biology and provide new important information on the role of miRNAs in the control of axon guidance and more broadly in nervous system development. \uc2\ua9 The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research