Susceptibility of Human Placenta Derived Mesenchymal Stromal/Stem Cells to Human Herpesviruses Infection

Fetal membranes (FM) derived mesenchymal stromal/stem cells (MSCs) are higher in number, expansion and differentiation abilities compared with those obtained from adult tissues, including bone marrow. Upon systemic administration, ex vivo expanded FM-MSCs preferentially home to damaged tissues promoting regenerative processes through their unique biological properties. These characteristics together with their immune-privileged nature and immune suppressive activity, a low infection rate and young age of placenta compared to other sources of SCs make FM-MSCs an attractive target for cell-based therapy and a valuable tool in regenerative medicine, currently being evaluated in clinical trials. In the present study we investigated the permissivity of FM-MSCs to all members of the human Herpesviridae family, an issue which is relevant to their purification, propagation, conservation and therapeutic use, as well as to their potential role in the vertical transmission of viral agents to the fetus and to their potential viral vector-mediated genetic modification. We present here evidence that FM-MSCs are fully permissive to infection with Herpes simplex virus 1 and 2 (HSV-1 and HSV-2), Varicella zoster virus (VZV), and Human Cytomegalovirus (HCMV), but not with Epstein-Barr virus (EBV), Human Herpesvirus-6, 7 and 8 (HHV-6, 7, 8) although these viruses are capable of entering FM-MSCs and transient, limited viral gene expression occurs. Our findings therefore strongly suggest that FM-MSCs should be screened for the presence of herpesviruses before xenotransplantation. In addition, they suggest that herpesviruses may be indicated as viral vectors for gene expression in MSCs both in gene therapy applications and in the selective induction of differentiation.     

Contribution of the residue at position 4 within classical nuclear localization signals to modulating interaction with importins and nuclear targeting

Nuclear import involves the recognition by importin (IMP) superfamily members of nuclear localization signals (NLSs) within protein cargoes destined for the nucleus, the best understood being recognition of classical NLSs (cNLSs) by the IMPα/β1 heterodimer. Although the cNLS consensus [K-(K/R)-X-(K/R) for positions P2–P5] is generally accepted, recent studies indicated that the contribution made by different residues at the P4 position can vary. Here, we apply a combination of microscopy, molecular dynamics, crystallography, in vitro binding, and bioinformatics approaches to show that the nature of residues at P4 indeed modulates cNLS function in the context of a prototypical Simian Virus 40 large tumor antigen-derived cNLS (KKRK, P2–5). Indeed, all hydrophobic substitutions in place of R impaired binding to IMPα and nuclear targeting, with the largest effect exerted by a G residue at P4. Substitution of R with neutral hydrophobic residues caused the loss of electrostatic and van der Waals interactions between the P4 residue side chains and IMPα. Detailed bioinformatics analysis confirmed the importance of the P4 residue for cNLS function across the human proteome, with specific residues such as G being associated with low activity. Furthermore, we validate our findings for two additional cNLSs from human cytomegalovirus (HCMV) DNA polymerase catalytic subunit UL54 and processivity factor UL44, where a G residue at P4 results in a 2–3-fold decrease in NLS activity. Our results thus showed that the P4 residue makes a hitherto poorly appreciated contribution to nuclear import efficiency, which is essential to determining the precise nuclear levels of cargoes.     

Interannual Changes in Biomass Affect the Spatial Aggregations of Anchovy and Sardine as Evidenced by Geostatistical and Spatial Indicators

Geostatistical techniques were applied and a series of spatial indicators were calculated (occupation, aggregation, location, dispersion, spatial autocorrelation and overlap) to characterize the spatial distributions of European anchovy and sardine during summer. Two ecosystems were compared for this purpose, both located in the Mediterranean Sea: the Strait of Sicily (upwelling area) and the North Aegean Sea (continental shelf area, influenced by freshwater). Although the biomass of anchovy and sardine presented high interannual variability in both areas, the location of the centres of gravity and the main spatial patches of their populations were very similar between years. The size of the patches representing the dominant part of the abundance (80%) was mostly ecosystem- and species-specific. Occupation (area of presence) appears to be shaped by the extent of suitable habitats in each ecosystem whereas aggregation patterns (how the populations are distributed within the area of presence) were species-specific and related to levels of population biomass. In the upwelling area, both species showed consistently higher occupation values compared to the continental shelf area. Certain characteristics of the spatial distribution of sardine (e.g. spreading area, overlapping with anchovy) differed substantially between the two ecosystems. Principal component analysis of geostatistical and spatial indicators revealed that biomass was significantly related to a suite of, rather than single, spatial indicators. At the spatial scale of our study, strong correlations emerged between biomass and the first principal component axis with highly positive loadings for occupation, aggregation and patchiness, independently of species and ecosystem. Overlapping between anchovy and sardine increased with the increase of sardine biomass but decreased with the increase of anchovy. This contrasting pattern was attributed to the location of the respective major patches combined with the specific occupation patterns of the two species. The potential use of spatial indices as auxiliary stock monitoring indicators is discussed.     

Role of homodimerization of human cytomegalovirus DNA polymerase accessory protein UL44 in origin-dependent DNA replication in cells

The presumed processivity subunit of human cytomegalovirus (HCMV) DNA polymerase, UL44, forms homodimers. The dimerization of UL44 is important for binding to DNA in vitro; however, whether it is also important for DNA replication in a cellular context is unknown. Here we show that UL44 point mutants that are impaired for dimerization, but not for nuclear localization or interaction with the C terminus of the polymerase catalytic subunit, are not capable of supporting HCMV oriLyt-dependent DNA replication in cells. These data suggest that the disruption of UL44 homodimers could represent a novel anti-HCMV strategy.     

An importin alpha/beta-recognized bipartite nuclear localization signal mediates targeting of the human herpes simplex virus type 1 DNA polymerase catalytic subunit pUL30 to the nucleus

Although the 1235 amino acids human herpes simplex virus type 1 (HSV-1) DNA polymerase catalytic subunit, pUL30, is essential for HSV-1 replication in the nucleus of host cells, little information is available regarding its nuclear import mechanism. The present study addresses this issue directly, characterizing pUL30's nuclear import pathway for the first time using quantitative confocal laser scanning microscopy (CLSM) on living cells, and fluorescent binding assays. In addition to a previously described nuclear localization signal (NLS) located within the pUL30 binding site for the polymerase accessory protein (PAP) pUL42, that appears to be dispensable for nuclear targeting, pUL30 possesses three putative basic NLSs. Intriguingly, the core of pUL30-NLS2 (residues 1114-1120) is highly homologous to that of the recently described NLS, similarly located upstream of the PAP binding site, of the human cytomegalovirus (HCMV) DNA polymerase catalytic subunit, pUL54. Here we show for the first time that pUL30-NLS2 itself is only partially functional in terms of nuclear import due to residue P1118 present in position 3 of the NLS core. Intriguingly, pUL30-NLS2 together with pUL30-NLS3 (residues 1133-1136) represents a fully functional bipartite NLS (pUL30-NLSbip), required for nuclear targeting of pUL30, and able to confer nuclear localization on heterologous proteins by conferring high-affinity interaction with the importin (IMP) alpha/beta heterodimer. Since nuclear targeting of HSV-1 proteins forming the replication fork is crucial for viral replication, the pUL30-NLSbip emerges for the first time as a viable therapeutic target.     

Habitat Selection Response of Small Pelagic Fish in Different Environments. Two Examples from the Oligotrophic Mediterranean Sea

A number of scientific papers in the last few years singled out the influence of environmental conditions on the spatial distribution of fish species, highlighting the need for the fisheries scientific community to investigate, besides biomass estimates, also the habitat selection of commercially important fish species. The Mediterranean Sea, although generally oligotrophic, is characterized by high habitat variability and represents an ideal study area to investigate the adaptive behavior of small pelagics under different environmental conditions. In this study the habitat selection of European anchovy Engraulis encrasicolus and European sardine Sardina pilchardus is analyzed in two areas of the Mediterranean Sea that largely differentiate in terms of environmental regimes: the Strait of Sicily and the North Aegean Sea. A number of environmental parameters were used to investigate factors influencing anchovy and sardine habitat selection. Acoustic surveys data, collected during the summer period 2002–2010, were used for this purpose. The quotient analysis was used to identify the association between high density values and environmental variables; it was applied to the entire dataset in each area in order to identify similarities or differences in the “mean” spatial behavioral pattern for each species. Principal component analysis was applied to selected environmental variables in order to identify those environmental regimes which drive each of the two ecosystems. The analysis revealed the effect of food availability along with bottom depth selection on the spatial distribution of both species. Furthermore PCA results highlighted that observed selectivity for shallower waters is mainly associated to specific environmental processes that locally increase productivity. The common trends in habitat selection of the two species, as observed in the two regions although they present marked differences in hydrodynamics, seem to be driven by the oligotrophic character of the study areas, highlighting the role of areas where the local environmental regimes meet ‘the ocean triad hypothesis’.     

The Flexible Loop of the Human Cytomegalovirus DNA Polymerase Processivity Factor ppUL44 Is Required for Efficient DNA Binding and Replication in Cells

Phosphoprotein ppUL44 of the human cytomegalovirus (HCMV) DNA polymerase plays an essential role in viral replication, conferring processivity to the DNA polymerase catalytic subunit pUL54 by tethering it to the DNA. Here, for the first time, we examine in living cells the function of the highly flexible loop of ppUL44 (UL44-FL; residues 162 to 174 [PHTRVKRNVKKAP¹⁷⁴]), which has been proposed to be directly involved in ppUL44''s interaction with DNA. In particular, we use a variety of approaches in transfected cells to characterize in detail the behavior of ppUL44Δloop, a mutant derivative in which three of the five basic residues within UL44-FL are replaced by nonbasic amino acids. Our results indicate that ppUL44Δloop is functional in dimerization and binding to pUL54 but strongly impaired in binding nuclear structures within the nucleus, as shown by its inability to form nuclear speckles, reduced nuclear accumulation, and increased intranuclear mobility compared to wild-type ppUL44. Moreover, analysis of cellular fractions after detergent and DNase treatment indicates that ppUL44Δloop is strongly reduced in DNA-binding ability, in similar fashion to ppUL44-L86A/L87A, a point mutant derivative impaired in dimerization. Finally, ppUL44Δloop fails to transcomplement HCMV oriLyt-dependent DNA replication in cells and also inhibits replication in the presence of wild-type ppUL44, possibly via formation of heterodimers defective for double-stranded DNA binding. UL44-FL thus emerges for the first time as an important determinant for HCMV replication in cells, with potential implications for the development of novel antiviral approaches by targeting HCMV replication.The Betaherpesviridae subfamily member human cytomegalovirus (HCMV) is a major human pathogen, causing serious disease in newborns following congenital infection and in immunocompromised individuals (28, 42). Replication of its double-stranded DNA (dsDNA) genome occurs in the nuclei of infected cells via a rolling-circle process mediated by 11 virally encoded proteins (32, 33), including a viral DNA polymerase holoenzyme, comprising a catalytic subunit, pUL54, and a proposed processivity factor, ppUL44 (14). ppUL44 is readily detectable in virus-infected cells as a 52-kDa phosphoprotein of 433 amino acids with strong dsDNA-binding ability (30, 45). Defined as a “polymerase accessory protein” (PAP) whose function is highly conserved among herpesviruses, ppUL44 is an essential factor for viral replication in cultured cells and hence represents a potential therapeutic target to combat HCMV infection (39). It is a multifunctional protein capable of self-associating (5, 10), as well as interacting with a plethora of viral and host cell proteins, including the viral kinase pUL97 (29), the viral transactivating protein pUL84 (15), the viral uracil DNA glycosylase ppUL114 (37), and the host cell importin α/β (IMPα/β) heterodimer, which is responsible for its transport into the nucleus (4). The activities of ppUL44 as a processivity factor, including the ability to dimerize, as well as bind to, pUL54 and DNA, reside in the N-terminal portion (26, 45), whereas the C terminus is essential for phosphorylation-regulated, IMPα/β-dependent nuclear targeting of ppUL44 monomers and dimers (4-6). Once within the nucleus, ppUL44 is thought to tether the DNA polymerase holoenzyme to the DNA, thus increasing its processivity (14).Recent studies have identified specific residues responsible for ppUL44 interaction with pUL54, as well as for the interaction with IMPα/β and homodimerization (4, 10, 27, 41). The crystal structure of ppUL44''s N-terminal domain (Fig. ​(Fig.1A)1A) reveals striking similarity to that of other processivity factors, such as proliferating cell nuclear antigen (PCNA) and its herpes simplex virus type 1 (HSV-1) homologue UL42 (10, 46). Unlike the PCNA trimeric ring, however, both ppUL44 and UL42, which bind to dsDNA as dimers and monomers, respectively, have an open structure, which is believed to be the basis for their ability to bind to dsDNA in the absence of clamp loaders and ATP (9, 10, 46). Both ppUL44 and UL42 share a very basic “back” face, which appears to be directly involved in DNA binding via electrostatic interactions (19, 22, 23, 38, 46). One striking difference between ppUL44 and UL42 is the presence on the former of an extremely basic flexible loop (UL44-FL, PHTRVKRNVKKAP¹⁷⁴) protruding from the basic back face of the protein (Fig. ​(Fig.1A).1A). Comparison of ppUL44 homologues from different betaherpesviruses, including human herpesvirus 6 (HHV-6) and 7 (HHV-7), showed that all possess similar sequences in the same position (44) (Fig. ​(Fig.1B),1B), implying functional significance.Open in a separate windowFIG. 1.The highly conserved flexible loop (residues 162 to 174) within ppUL44 protrudes from ppUL44 basic face and is important for efficient nuclear accumulation and localization in nuclear speckles. (A) Schematic representation of ppUL44 N-terminal domain (residues 9 to 270, protein data bank accession no. 1T6L) generated using the Chimera software based on the published crystal structure (10, 35). Color: yellow, β-sheets; red, α-helices. Residues involved in ppUL44 dimerization (P85, L86, L87, L93, F121, and M123), as well as basic residues potentially involved in DNA binding (K21, R28, K32, K35, K128, K158, K224, and K237), are represented as spacefill in orange and green, respectively. Residues P162 and C175, in black, are indicated by arrowheads, while residues 163 to 174 are not visible in the electron density maps and could potentially extend in the cavity formed by ppUL44''s basic face to directly contact DNA. Residues forming ppUL44 connector loop (128-142) are in blue. (B) Sequence alignment between HCMVUL44-FL and the corresponding region of several betaherpesvirus ppUL44 homologues. The single-letter amino acid code is used, with basic residues in boldface. (C) COS-7 cells were transfected to express the indicated GFP fusion proteins and imaged live 16 h after transfection using CLSM and a 40× water immersion objective lens. (D) Quantitative results for the Fn/c and speckle formation for GFP-UL44 fusion proteins. The data for the Fn/c ratios represent the mean Fn/c relative to each protein indicated as a percentage of the mean Fn/c relative to GFP-UL44wt ± the standard error of the mean, with the number of analyzed cells in parentheses. (E) HEK 293 cells expressing the indicated GFP-UL44 fusion proteins were lysed, separated by PAGE, and analyzed by Western blotting as described in Materials and Methods, using either the anti-GFP or the anti-α-tubulin MAbs.A recent study revealed that substitution of UL44-FL basic residues with alanine residues strongly impairs the ability of a bacterially expressed N-terminal fragment of UL44 to bind 30-bp dsDNA oligonucleotides in vitro, suggesting that UL44-FL could be involved in dsDNA-binding during viral replication (22). However, the role of UL44-FL in mediating the binding of full-length UL44 to dsDNA in cells and its role in DNA replication have not been investigated. We use here a variety of approaches to delineate the role of UL44-FL in living cells, our data revealing that UL44-FL is not required for ppUL44 dimerization or binding to the catalytic subunit pUL54 but is crucial for HCMV oriLyt-dependent DNA replication, being required for the formation of nuclear aggregates, nuclear accumulation/retention, and DNA binding of ppUL44. Importantly, ppUL44Δloop exhibits a transdominant-negative phenotype, inhibiting HCMV oriLyt-dependent DNA replication in the presence of wild-type ppUL44, possibly via formation of heterodimers defective for dsDNA binding. This underlines ppUL44-FL as an important determinant for HCMV replication in a cellular context for the first time, with potential implications for the development of novel antiviral approaches.     

Whispering gallery mode aptasensors for detection of blood proteins

Whispering gallery mode resonators (WGMR), as silica microspheres, have been recently proposed as an efficient tool for the realisation of optical biosensors. In this work we present a functionalization procedure based on the DNA‐aptamer sequence immobilization on WGMR, able to recognize specifically thrombin or VEGF protein, preserving a high Q factor. The protein binding was optically characterized in terms of specificity in buffer solution or in 10% diluted human serum. Simulation of the protein flow was found in good agreement with experimental data. The aptasensor was also chemically regenerated and tested again, demonstrating the reusability of our system. (© 2013 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)