Integrase-deficient lentiviral vectors mediate efficient gene transfer to human vascular smooth muscle cells with minimal genotoxic risk

We have previously shown that injury-induced neointima formation was rescued by adenoviral-Nogo-B gene delivery. Integrase-competent lentiviral vectors (ICLV) are efficient at gene delivery to vascular cells but present a risk of insertional mutagenesis. Conversely, integrase-deficient lentiviral vectors (IDLV) offer additional benefits through reduced mutagenesis risk, but this has not been evaluated in the context of vascular gene transfer. Here, we have investigated the performance and genetic safety of both counterparts in primary human vascular smooth muscle cells (VSMC) and compared gene transfer efficiency and assessed the genotoxic potential of ICLVs and IDLVs based on their integration frequency and insertional profile in the human genome. Expression of enhanced green fluorescent protein (eGFP) mediated by IDLVs (IDLV-eGFP) demonstrated efficient transgene expression in VSMCs. IDLV gene transfer of Nogo-B mediated efficient overexpression of Nogo-B in VSMCs, leading to phenotypic effects on VSMC migration and proliferation, similar to its ICLV version and unlike its eGFP control and uninfected VSMCs. Large-scale integration site analyses in VSMCs indicated that IDLV-mediated gene transfer gave rise to a very low frequency of genomic integration compared to ICLVs, revealing a close-to-random genomic distribution in VSMCs. This study demonstrates for the first time the potential of IDLVs for safe and efficient vascular gene transfer

Phonological Factors Affecting L1 Phonetic Realization of Proficient Polish Users of English

Acoustic phonetic studies examine the L1 of Polish speakers with professional level proficiency in English. The studies include two tasks, a production task carried out entirely in Polish and a phonetic code-switching task in which speakers insert target Polish words or phrases into an English carrier. Additionally, two phonetic parameters are studied: the oft-investigated VOT, as well as glottalization vs. sandhi linking of word-initial vowels. In monolingual Polish mode, L2 interference was observed for the VOT parameter, but not for sandhi linking. It is suggested that this discrepancy may be related to the differing phonological status of the two phonetic parameters. In the code-switching tasks, VOTs were on the whole more English-like than in monolingual mode, but this appeared to be a matter of individual performance. An increase in the rate of sandhi linking in the code-switches, except for the case of one speaker, appeared to be a function of accelerated production of L1 target items

Protein chainmail variants in dsDNA viruses.

First discovered in bacteriophage HK97, biological chainmail is a highly stable system formed by concatenated protein rings. Each subunit of the ring contains the HK97-like fold, which is characterized by its submarine-like shape with a 5-stranded β sheet in the axial (A) domain, spine helix in the peripheral (P) domain, and an extended (E) loop. HK97 capsid consists of covalently-linked copies of just one HK97-like fold protein and represents the most effective strategy to form highly stable chainmail needed for dsDNA genome encapsidation. Recently, near-atomic resolution structures enabled by cryo electron microscopy (cryoEM) have revealed a range of other, more complex variants of this strategy for constructing dsDNA viruses. The first strategy, exemplified by P22-like phages, is the attachment of an insertional (I) domain to the core 5-stranded β sheet of the HK97-like fold. The atomic models of the Bordetella phage BPP-1 showcases an alternative topology of the classic HK97 topology of the HK97-like fold, as well as the second strategy for constructing stable capsids, where an auxiliary jellyroll protein dimer serves to cement the non-covalent chainmail formed by capsid protein subunits. The third strategy, found in lambda-like phages, uses auxiliary protein trimers to stabilize the underlying non-covalent chainmail near the 3-fold axis. Herpesviruses represent highly complex viruses that use a combination of these strategies, resulting in four-level hierarchical organization including a non-covalent chainmail formed by the HK97-like fold domain found in the floor region. A thorough understanding of these structures should help unlock the enigma of the emergence and evolution of dsDNA viruses and inform bioengineering efforts based on these viruses

A latent ability to persist: differentiation in Toxoplasma gondii

A critical factor in the transmission and pathogenesis of Toxoplasma gondii is the ability to convert from an acute disease-causing, proliferative stage (tachyzoite), to a chronic, dormant stage (bradyzoite). The conversion of the tachyzoite-containing parasitophorous vacuole membrane into the less permeable bradyzoite cyst wall allows the parasite to persist for years within the host to maximize transmissibility to both primary (felids) and secondary (virtually all other warm-blooded vertebrates) hosts. This review presents our current understanding of the latent stage, including the factors that are important in bradyzoite induction and maintenance. Also discussed are the recent studies that have begun to unravel the mechanisms behind stage switching

Efficient Gene Targeting Mediated by Adeno-Associated Virus and DNA Double-Strand Breaks

Gene targeting is the in situ manipulation of the sequence of an endogenous gene by the introduction of homologous exogenous DNA. Presently, the rate of gene targeting is too low for it to be broadly used in mammalian somatic cell genetics or to cure genetic diseases. Recently, it has been demonstrated that infection with recombinant adeno-associated virus (rAAV) vectors can mediate gene targeting in somatic cells, but the mechanism is unclear. This paper explores the balance between random integration and gene targeting with rAAV. Both random integration and spontaneous gene targeting are dependent on the multiplicity of infection (MOI) of rAAV. It has previously been shown that the introduction of a DNA double-stranded break (DSB) in a target gene can stimulate gene targeting by several-thousand-fold in somatic cells. Creation of a DSB stimulates the frequency of rAAV-mediated gene targeting by over 100-fold, suggesting that the mechanism of rAAV-mediated gene targeting involves, at least in part, the repair of DSBs by homologous recombination. Absolute gene targeting frequencies reach 0.8% with a dual vector system in which one rAAV vector provides a gene targeting substrate and a second vector expresses the nuclease that creates a DSB in the target gene. The frequencies of gene targeting that we achieved with relatively low MOIs suggest that combining rAAV vectors with DSBs is a promising strategy to broaden the application of gene targeting

Transcription activator like effector (TALE)-directed piggyBac transposition in human cells.

Insertional therapies have shown great potential for combating genetic disease and safer methods would undoubtedly broaden the variety of possible illness that can be treated. A major challenge that remains is reducing the risk of insertional mutagenesis due to random insertion by both viral and non-viral vectors. Targetable nucleases are capable of inducing double-stranded breaks to enhance homologous recombination for the introduction of transgenes at specific sequences. However, off-target DNA cleavages at unknown sites can lead to mutations that are difficult to detect. Alternatively, the piggyBac transposase is able perform all of the steps required for integration; therefore, cells confirmed to contain a single copy of a targeted transposon, for which its location is known, are likely to be devoid of aberrant genomic modifications. We aimed to retarget transposon insertions by comparing a series of novel hyperactive piggyBac constructs tethered to a custom transcription activator like effector DNA-binding domain designed to bind the first intron of the human CCR5 gene. Multiple targeting strategies were evaluated using combinations of both plasmid-DNA and transposase-protein relocalization to the target sequence. We demonstrated user-defined directed transposition to the CCR5 genomic safe harbor and isolated single-copy clones harboring targeted integrations

Human endogenous retrovirus K106 (HERV-K106) was infectious after the emergence of anatomically modern humans.

HERV-K113 and HERV-K115 have been considered to be among the youngest HERVs because they are the only known full-length proviruses that are insertionally polymorphic and maintain the open reading frames of their coding genes. However, recent data suggest that HERV-K113 is at least 800,000 years old, and HERV-K115 even older. A systematic study of HERV-K HML2 members to identify HERVs that may have infected the human genome in the more recent evolutionary past is lacking. Therefore, we sought to determine how recently HERVs were exogenous and infectious by examining sequence variation in the long terminal repeat (LTR) regions of all full-length HERV-K loci. We used the traditional method of inter-LTR comparison to analyze all full length HERV-Ks and determined that two insertions, HERV-K106 and HERV-K116 have no differences between their 5' and 3' LTR sequences, suggesting that these insertions were endogenized in the recent evolutionary past. Among these insertions with no sequence differences between their LTR regions, HERV-K106 had the most intact viral sequence structure. Coalescent analysis of HERV-K106 3' LTR sequences representing 51 ethnically diverse individuals suggests that HERV-K106 integrated into the human germ line approximately 150,000 years ago, after the emergence of anatomically modern humans

Lattice congruences, fans and Hopf algebras

We give a unified explanation of the geometric and algebraic properties of two well-known maps, one from permutations to triangulations, and another from permutations to subsets. Furthermore we give a broad generalization of the maps. Specifically, for any lattice congruence of the weak order on a Coxeter group we construct a complete fan of convex cones with strong properties relative to the corresponding lattice quotient of the weak order. We show that if a family of lattice congruences on the symmetric groups satisfies certain compatibility conditions then the family defines a sub Hopf algebra of the Malvenuto-Reutenauer Hopf algebra of permutations. Such a sub Hopf algebra has a basis which is described by a type of pattern-avoidance. Applying these results, we build the Malvenuto-Reutenauer algebra as the limit of an infinite sequence of smaller algebras, where the second algebra in the sequence is the Hopf algebra of non-commutative symmetric functions. We also associate both a fan and a Hopf algebra to a set of permutations which appears to be equinumerous with the Baxter permutations.Comment: 34 pages, 1 figur