RECQL4 is not critical for firing of human DNA replication origins.

Human RECQL4, a member of the RecQ helicase family, plays a role in maintaining genomic stability, but its precise function remains unclear. The N-terminus of RECQL4 has similarity to Sld2, a protein required for the firing of DNA replication origins in budding yeast. Consistent with this sequence similarity, the Xenopus laevis homolog of RECQL4 has been implicated in initiating DNA replication in egg extracts. To determine whether human RECQL4 is required for firing of DNA replication origins, we generated cells in which both RECQL4 alleles were targeted, resulting in either lack of protein expression (knock-out; KO) or expression of a full-length, mutant protein lacking helicase activity (helicase-dead; HD). Interestingly, both the RECQL4 KO and HD cells were viable and exhibited essentially identical origin firing profiles as the parental cells. Analysis of the rate of fork progression revealed increased rates in the RECQL4 KO cells, which might be indicative of decreased origin firing efficiency. Our results are consistent with human RECQL4 having a less critical role in firing of DNA replication origins, than its budding yeast homolog Sld2.

The sequence [EKRKI(E/R)(K/L/R/S/T)] is a nuclear localization signal for importin 7 binding (NLS7).

BACKGROUND: Nuclear translocation of large proteins is mediated through specific protein carriers, collectively named karyopherins (importins, exportins and adaptor proteins). Cargo proteins are recognized by importins through specific motifs, known as nuclear localization signals (NLS). However, only the NLS recognized by importin α and transportin (M9 NLS) have been identified so far METHODS: An unsupervised in silico approach was used, followed by experimental validation. RESULTS: We identified the sequence EKRKI(E/R)(K/L/R/S/T) as an NLS signal for importin 7 recognition. This sequence was validated in the breast cancer cell line T47D, which expresses importin 7. Finally, we verified that importin 7-mediated nuclear protein transport is affected by cargo protein phosphorylation. CONCLUSIONS: The NLS sequence for importin 7 was identified and we propose this approach as an identification method of novel specific NLS sequences for ß-karyopherin family members. GENERAL SIGNIFICANCE: Elucidating the complex relationships of the nuclear transporters and their cargo proteins may help in laying the foundation for the development of novel therapeutics, targeting specific importins, with an immediate translational impact.

Glycerosome of Melissa officinalis L. Essential Oil for Effective Anti-HSV Type 1.

Essential oils are complex mixtures of strongly active compounds, very volatile and sensitive to light, oxygen, moisture and temperature. Loading inside nanocarriers can be a strategy to increase their stability and successfully use them in therapy. In the present study, a commercial Melissa officinalis L. (Lamiaceae) essential oil (MEO) was analyzed by gas chromatography-mass spectrometry, loaded inside glycerosomes (MEO-GS) and evaluated for its anti-herpetic activity against HSV type 1. MEO-GS analyses were prepared by the thin layer evaporation method and they were characterized by light scattering techniques, determining average diameter, polydispersity index and ζ-potential. By transmission electron microscopy, MEO-GS appeared as small nano-sized vesicles with a spherical shape. MEO encapsulation efficiency inside glycerosomes, in terms of citral and ß-caryophyllene, was found to be ca. 63% and 76% respectively, and MEO release from glycerosomes, performed by dialysis bag method, resulted in less than 10% within 24h. In addition, MEO-GS had high chemical and physical stability during 4 months of storage. Finally, MEO-GS were very active in inhibiting HSV type 1 infection of mammalian cells in vitro, without producing cytotoxic effects. Thus, MEO-GS could be a promising tool in order to provide a suitable anti-herpetic formulation.