We introduce a new method for mesoscopic modeling of protein diffusion in an entire cell. This method is based on the construction of a three-dimensional digital model cell from confocal microscopy data. The model cell is segmented into the cytoplasm, nucleus, plasma membrane, and nuclear envelope, in which environment protein motion is modeled by fully numerical mesoscopic methods. Finer cellular structures that cannot be resolved with the imaging technique, which significantly affect protein motion, are accounted for in this method by assigning an effective, position-dependent porosity to the cell. This porosity can also be determined by confocal microscopy using the equilibrium distribution of a non-binding fluorescent protein. Distinction can now be made within this method between diffusion in the liquid phase of the cell (cytosol/nucleosol) and the cytoplasm/nucleoplasm. Here we applied the method to analyze fluorescence recovery after photobleach (FRAP) experiments in which the diffusion coefficient of a freely-diffusing model protein was determined for two different cell lines, and to explain the clear difference typically observed between conventional FRAP results and those of fluorescence correlation spectroscopy (FCS). A large difference was found in the FRAP experiments between diffusion in the cytoplasm/nucleoplasm and in the cytosol/nucleosol, for all of which the diffusion coefficients were determined. The cytosol results were found to be in very good agreement with those by FCS.

f Canine parvovirus (CPV) infection leads to reorganization of nuclear proteinaceous subcompartments. Our studies showed that virus infection causes a time-dependent increase in the amount of viral nonstructural protein NS1 mRNA. Fluorescence recovery after photobleaching showed that the recovery kinetics of nuclear transcription-associated proteins, TATA binding protein (TBP), transcription factor IIB (TFIIB), and poly(A) binding protein nuclear 1 (PABPN1) were different in infected and noninfected cells, pointing to virus-induced alterations in binding dynamics of these proteins. In animals, several DNA viruses depend on host cell nuclear replication and transcription machinery (52). TATA binding protein (TBP) and transcription factor IIB (TFIIB) are key constituents of assembly of the host cell transcription initiation complex. Previous studies have shown that TBP interacts with viral transcription activators, including adenovirus EIA (21, 22, 32), hepatitis B virus pX and NS5A (38), herpes simplex virus 1 (HSV-1) VP16 (24, 34), human cytomegalovirus IE2 (23, 31, 49), and human immunodeficiency virus (HIV) Tat (30,39,48). The poly(A) binding protein nuclear 1 (PABPN1) accumulates to splicing speckles. It binds with high affinity to nascent poly(A) tails, thus stimulating their extension and controlling their length (27). Interaction of viral components with PABPN1 can lead to stimulated transcription (HSV-1 ICP27) (18, 19) or reduced host cell mRNA maturation and export (influenza A virus NS1) (9, 10). TAP and CRM1, essential export factors of nuclear mRNA, are also responsible for the nuclear export of HSV and influenza A virus mRNAs (8,28,40,41). Moreover, promyelocytic leukemia (PML) nuclear bodies, involved in a wide variety of cellular processes, including regulation of transcription, interact with nuclear components of HSV-1 (35), polyomaviruses (29,45) , and parvoviruses (adeno-associated virus [AAV], minute virus of mice [MVM]) (20, 51).Canine parvovirus (CPV) is a single-stranded DNA virus (46). Its nonstructural protein NS1 serves as an initiator and a helicase in viral DNA replication and as an activator of the viral promoters during diversion of the cellular machinery toward viral protein expression (11,13,36,37).We examined CPV infection-induced alterations in distribu-

Highlights  An effective way to decrease perinatal risks is to avoid multiple pregnancies  Elective single frozen embryo transfer may result in surplus thawed good embryos  Recryopreservation cycle with vitrification results in good embryo survival rates  Transfers of twice-frozen embryos result in favorable pregnancy outcomes  Perinatal outcome after recryopreservation with vitrification is uncompromised Pregnancy potential of embryos cryopreserved and thawed twice.

This study presents the reactive self-assembly of isocyanate functional and amphiphilic six-arm, star-shaped polyether prepolymers in water into nanogels. Intrinsic molecular amphiphilicity, mainly driven by the isophorone moiety at the distal endings of the star-shaped molecules, allows for the preparation of spherical particles with an adjustable size of 100-200 nm by self-assembly and subsequent covalent cross-linking without the need for organic solvents or surfactants. Covalent attachment of a fluorescence dye and either the cell-penetrating TAT peptide or a random control peptide sequence shows that only TAT-labeled nanogels are internalized by HeLa cells. The nanogels thus specifically enter the cells and accumulate in the perinuclear area in a time- and concentration-dependent manner.