Guanosine-5′-diphosphate-3′-diphosphate inhibits the in, vitro synthesis of β-lactamase from pBR322 DNA

The $\text{in}\text{,}\text{vitro}$ synthesis of β-lactamase directed by pBR322 DNA is inhibited by guanosine-5′-diphosphate-3′-diphosphate.     

Intra-host mathematical model of chronic wasting disease dynamics in deer (Odocoileus)

Bioassays of native cervid hosts have established the presence of infectious chronic wasting disease (CWD) prions in saliva, blood, urine, and feces of clinically diseased and pre-clinical infected deer. The intra-host trafficking of prions from the time of initial infection to shedding has been less well defined. We created a discrete-time compartmentalized model to simulate the misfolding catalysis, trafficking, and shedding of infectious prions throughout the organ systems of CWD-infected cervids. Using parameter values derived from experimental infections of North American deer (Odocoileus spp.), the exponential-based model predicts prion deposition over time with: 1) nervous tissues containing the highest deposition of prions at 20 months post-infection and 2) excreted fluids containing low levels of prions throughout infection with the highest numbers of prions predicted to be shed in saliva and feces (as high as 10 lethal doses (1.34 × 10²⁹ prions) in 11–15 months). These findings are comparable to prion deposition described in literature as assayed by conventional and ultrasensitive amplification assays. The comparison of our model to published data suggests that highly sensitive assays (sPMCA, RT-QuIC, and bioassay) are appropriate for early prion detection in bodily fluids and secretions. The model provides a view of intra-host prion catalysis leading to pre-clinical shedding and provides a framework for continued development of antemortem diagnostic methods.     

Activation of Akt/protein kinase B overcomes a G(2)/m cell cycle checkpoint induced by DNA damage

Activation of Akt, or protein kinase B, is frequently observed in human cancers. Here we report that Akt activation via overexpression of a constitutively active form or via the loss of PTEN can overcome a G(2)/M cell cycle checkpoint that is induced by DNA damage. Activated Akt also alleviates the reduction in CDC2 activity and mitotic index upon exposure to DNA damage. In addition, we found that PTEN null embryonic stem (ES) cells transit faster from the G(2)/M to the G(1) phase of the cell cycle when compared to wild-type ES cells and that inhibition of phosphoinositol-3-kinase (PI3K) in HEK293 cells elicits G(2) arrest that is alleviated by activated Akt. Furthermore, the transition from the G(2)/M to the G(1) phase of the cell cycle in Akt1 null mouse embryo fibroblasts (MEFs) is attenuated when compared to that of wild-type MEFs. These results indicate that the PI3K/PTEN/Akt pathway plays a role in the regulation of G(2)/M transition. Thus, cells expressing activated Akt continue to divide, without being eliminated by apoptosis, in the presence of continuous exposure to mutagen and accumulate mutations, as measured by inactivation of an exogenously expressed herpes simplex virus thymidine kinase (HSV-tk) gene. This phenotype is independent of p53 status and cannot be reproduced by overexpression of Bcl-2 or Myc and Bcl-2 but seems to counteract a cell cycle checkpoint mediated by DNA mismatch repair (MMR). Accordingly, restoration of the G(2)/M cell cycle checkpoint and apoptosis in MMR-deficient cells, through reintroduction of the missing component of MMR, is alleviated by activated Akt. We suggest that this new activity of Akt in conjunction with its antiapoptotic activity may contribute to genetic instability and could explain its frequent activation in human cancers.     

Widespread Infection with Homologues of Human Parvoviruses B19, PARV4, and Human Bocavirus of Chimpanzees and Gorillas in the Wild

Infections with human parvoviruses B19 and recently discovered human bocaviruses (HBoVs) are widespread, while PARV4 infections are transmitted parenterally and prevalent specifically in injecting drug users and hemophiliacs. To investigate the exposure and circulation of parvoviruses related to B19 virus, PARV4, and HBoV in nonhuman primates, plasma samples collected from 73 Cameroonian wild-caught chimpanzees and gorillas and 91 Old World monkey (OWM) species were screened for antibodies to recombinant B19 virus, PARV4, and HBoV VP2 antigens by enzyme-linked immunosorbent assay (ELISA). Moderate to high frequencies of seroreactivity to PARV4 (63% and 18% in chimpanzees and gorillas, respectively), HBoV (73% and 36%), and B19 virus (8% and 27%) were recorded for apes, while OWMs were uniformly negative (for PARV4 and B19 virus) or infrequently reactive (3% for HBoV). For genetic characterization, plasma samples and 54 fecal samples from chimpanzees and gorillas collected from Cameroonian forest floors were screened by PCR with primers conserved within Erythrovirus, Bocavirus, and PARV4 genera. Two plasma samples (chimpanzee and baboon) were positive for PARV4, while four fecal samples were positive for HBoV-like viruses. The chimpanzee PARV4 variant showed 18% and 15% nucleotide sequence divergence in NS and VP1/2, respectively, from human variants (9% and 7% amino acid, respectively), while the baboon variant was substantially more divergent, mirroring host phylogeny. Ape HBoV variants showed complex sequence relationships with human viruses, comprising separate divergent homologues of HBoV1 and the recombinant HBoV3 species in chimpanzees and a novel recombinant species in gorillas. This study provides the first evidence for widespread circulation of parvoviruses in primates and enables future investigations of their epidemiology, host specificity, and (co)evolutionary histories.Autonomous parvoviruses known to infect humans comprise parvovirus B19 (18) and the recently discovered PARV4 (22) and human bocavirus (HBoV) (3). Members of the family Parvoviridae are genetically and biologically diverse and are classified into several genera or groups, showing marked differences in host range, pathology, and tissue/cellular tropisms (18). Human parvovirus B19, a member of the Erythrovirus genus, is transmitted primarily by the respiratory route but causes systemic infections. Erythroid progenitor cells are specifically targeted through expression of globoside P antigen, which acts as the B19 virus receptor for entry (5). In common with infections by most parvoviruses, B19 virus infections are acute; a period of intense viremia is followed by seroconversion for antibody to B19 virus and lifelong immunity from reinfection (29). Despite the clearance of viremia and seroconversion for antibody, lifelong persistence of viral DNA in tissues has been shown to occur (12, 20, 26, 28, 43, 58). Three genotypes of B19 virus have been described, differing in nucleotide sequence by approximately 13 to 14% (7, 21, 41, 53); genotypes 1 and 2 have been found in Europe, the United States, and other Western countries, while genotype 3 is restricted to sub-Saharan Africa and South America (7, 47, 49). B19 virus widely circulates in human populations worldwide; in Western countries, several studies have documented increasing frequencies of B19 virus seropositivity with age, rising to approximately 60 to 70% by adulthood (15, 39, 48, 61).Another human parvovirus, PARV4, shows markedly different epidemiology and transmission routes. It was originally detected in plasma from an individual with an “acute infection syndrome” resembling that of primary human immunodeficiency virus (HIV) infection (22). While this clinical presentation has not been observed again, infection with PARV4 is known to be widespread specifically in individuals with a history of parenteral exposure (injecting drug users [IDUs], hemophiliacs, polytransfused individuals), with a strikingly higher incidence in those infected with HIV-1 (13, 14, 30, 35, 54). These observations suggest that PARV4 is primarily transmitted though parenteral routes in Western countries (54, 56). In common with infection with the better-characterized human parvovirus B19, infection with PARV4 is associated with a period of acute viremia, followed by seroconversion for antibody and long-term persistence of viral DNA sequences in lymphoid and other tissue (33, 37, 52). Circulating variants of PARV4 have been classified into three distinct genotypes exhibiting approximately 8% nucleotide sequence divergence from each other. Genotypes 1 and 2 circulate in Western countries, while genotype 3 has to date been recorded only in sub-Saharan Africa (45, 55).The third human parvovirus, HBoV (3), shows a number of epidemiological and clinical attributes different from those of both B19 virus and PARV4. HBoV was originally found in the respiratory tract of young children and has been the subject of intense investigation as a potential cause of human respiratory disease (reviewed in references 1, 51, and 62). Although it is frequently detected by PCR in the nasopharynx of viremic individuals with primary infections with lower respiratory tract disease, other coinfecting respiratory viruses are frequently detected (19). HBoV additionally shows long-term, low-level carriage in the respiratory tract after primary infection, which further complicates PCR-based etiological studies (2, 38) and warrants the use of other diagnostic strategies, such as serology (30, 32, 59). In contrast to the rather minimal genetic diversity of B19 virus and PARV4 genotypes, bocaviruses infecting humans are now known to comprise three to four major genetic variants (termed types or species 1 to 4) (23, 24). HBoV1 and HBoV2 show 22%, 33%, and 20% amino acid sequence divergence from each other in the encoded viral nonstructural (NS), NP-1, and structural VP1/VP2 proteins, respectively, the latter potentially leading to antigenic diversity and some loss of antigenic cross-reactivity. A third type/species of HBoV is a chimeric form with a nonstructural gene region (NS, NP1) most similar to HBoV1, a recombination breakpoint in the intergenic region between NP1 and VP1, and structural genes related to those of HBoV2 (4, 23). Current data suggest that only HBoV1 is capable of infecting the respiratory tract; most published large-scale screening studies have failed to detect HBoV2 (or HBoV3) in respiratory samples (10, 11, 60), while all three types/species are detectable in fecal samples, indicating the existence of an alternative or additional site of virus replication (23). Despite extensive inquiry, the exact role of HBoV1 in respiratory disease remains unclear, as is the proposed etiological role of HBoV2 (and possibly HBoV3) in gastroenteritis (4, 11, 23, 50). Very recently, a fourth species/type, HBoV4, has been detected in fecal samples; genetically it also shows evidence for past recombination, with NS and NP1 region sequences grouping with HBoV2, while VP1/VP2 is more closely related to HBoV3 (23).We have little understanding of the past epidemiology, evolution, and origins of human parvoviruses. For both B19 virus and PARV4, evidence has been obtained for a temporal succession of genotypes over time (37, 43); in Europe, B19 virus genotype 1 largely replaced type 2 in the 1960 and 1970s (43), while current data indicate that a similar replacement of PARV4 genotypes occurred within the last 20 years (37). The highly restricted sequence diversity of currently circulating variants of PARV4 and B19 virus and of HBoV1 variants supports the hypothesis of a relatively recent emergence and spread of these viruses in human populations (36, 42, 64).The existence and evolution of parvoviruses on a much longer time scale is suggested by the observations that members of the Erythrovirus and Parvovirus genera both contain viruses that are highly host species specific and that the molecular phylogenies of both genera are largely congruent with those of their hosts (34). This has led to the hypothesis of long-term coevolution of parvoviruses with their host over the 90 million years of mammalian evolution and perhaps beyond. Among erythroviruses, simian homologues of B19 virus have been found in cynomolgus monkeys (44) and rhesus and pig-tailed macaques (16) and more genetically distant viruses have been characterized in chipmunks and cows (9, 63). Divergent homologues of PARV4 in pigs and cows have been described (31), while the bovine and canine parvoviruses distantly related to HBoV are the originally described members of the Bocavirus genus. However, the process of virus-host codivergence is known to be punctuated by occasional cross-species transmissions, including the well-documented spread of feline parvovirus to dogs (46). Based on serological evidence, the possible transmission of simian erythroviruses to animal handlers has been proposed (6).To gain further insights into the origins and evolution of human parvoviruses, we have performed large-scale serological and PCR-based screening of nonhuman primates (chimpanzees and gorillas) and of several species of Old World monkeys (OWMs) for evidence of infection with parvoviruses that are antigenically related to the human B19, PARV4, and HBoV viruses. By PCR, we have sought to genetically characterize homologues of the three autonomous human parvoviruses in apes and Old World monkey species and to analyze their evolutionary relationship to human and other mammalian homologues of these viruses.     

Mutation Size Optimizes Speciation in an Evolutionary Model

The role of mutation rate in optimizing key features of evolutionary dynamics has recently been investigated in various computational models. Here, we address the related question of how maximum mutation size affects the formation of species in a simple computational evolutionary model. We find that the number of species is maximized for intermediate values of a mutation size parameter μ; the result is observed for evolving organisms on a randomly changing landscape as well as in a version of the model where negative feedback exists between the local population size and the fitness provided by the landscape. The same result is observed for various distributions of mutation values within the limits set by μ. When organisms with various values of μ compete against each other, those with intermediate μ values are found to survive. The surviving values of μ from these competition simulations, however, do not necessarily coincide with the values that maximize the number of species. These results suggest that various complex factors are involved in determining optimal mutation parameters for any population, and may also suggest approaches for building a computational bridge between the (micro) dynamics of mutations at the level of individual organisms and (macro) evolutionary dynamics at the species level.     

Structural determinants of tissue tropism and in vivo pathogenicity for the parvovirus minute virus of mice

Two strains of the parvovirus minute virus of mice (MVM), the immunosuppressive (MVMi) and the prototype (MVMp) strains, display disparate in vitro tropism and in vivo pathogenicity. We report the crystal structures of MVMp virus-like particles (MVMp(b)) and native wild-type (wt) empty capsids (MVMp(e)), determined and refined to 3.25 and 3.75 A resolution, respectively, and their comparison to the structure of MVMi, also refined to 3.5 A resolution in this study. A comparison of the MVMp(b) and MVMp(e) capsids showed their structures to be the same, providing structural verification that some heterologously expressed parvovirus capsids are indistinguishable from wt capsids produced in host cells. The structures of MVMi and MVMp capsids were almost identical, but local surface conformational differences clustered from symmetry-related capsid proteins at three specific domains: (i) the icosahedral fivefold axis, (ii) the "shoulder" of the protrusion at the icosahedral threefold axis, and (iii) the area surrounding the depression at the icosahedral twofold axis. The latter two domains contain important determinants of MVM in vitro tropism (residues 317 and 321) and forward mutation residues (residues 399, 460, 553, and 558) conferring fibrotropism on MVMi. Furthermore, these structural differences between the MVM strains colocalize with tropism and pathogenicity determinants mapped for other autonomous parvovirus capsids, highlighting the importance of common parvovirus capsid regions in the control of virus-host interactions.     

Tumor-Endothelial Cell Three-dimensional Spheroids: New Aspects to Enhance Radiation and Drug Therapeutics

Classic cancer research for several decades has focused on understanding the biology of tumor cells in vitro. However, extending these findings to in vivo settings has been impeded owing to limited insights on the impact of microenvironment on tumor cells. We hypothesized that tumor cell biology and treatment response would be more informative when done in the presence of stromal components, like endothelial cells, which exist in the tumor microenvironment. To that end, we have developed a system to grow three-dimensional cultures of GFP-4T1 mouse mammary tumor and 2H11 murine endothelial cells in hanging drops of medium in vitro. The presence of 2H11 endothelial cells in these three-dimensional cocultures was found to sensitize 4T1-GFP tumor cells to chemotherapy (Taxol) and, at the same time, protect cells from ionizing radiation. These spheroidal cultures can also be implanted into the dorsal skinfold window chamber of mice for fluorescence imaging of vascularization and disease progression/treatment response. We observed rapid neovascularization of the tumor-endothelial spheroids in comparison to tumor spheroids grown in nude mice. Molecular analysis revealed pronounced up-regulation of several proangiogenic factors in the tumor tissue derived from the tumor-endothelial spheroids compared with tumor-only spheroids. Furthermore, the rate of tumor growth from tumor-endothelial spheroids in mice was faster than the tumor cell-only spheroids, resulting in greater metastasis to the lung. This three-dimensional coculture model presents an improved way to investigate more pertinent aspects of the therapeutic potential for radiation and/or chemotherapy alone and in combination with antiangiogenic agents.     

Dynamics of auxin-dependent Ca2+ and pH signaling in root growth revealed by integrating high-resolution imaging with automated computer vision-based analysis

Plants adapt to a changing environment by entraining their growth and development to prevailing conditions. Such 'plastic' development requires a highly dynamic integration of growth phenomena with signal perception and transduction systems, such as occurs during tropic growth. The plant hormone auxin has been shown to play a key role in regulating these directional growth responses of plant organs to environmental cues. However, we are still lacking a cellular and molecular understanding of how auxin-dependent signaling cascades link stimulus perception to the rapid modulation of growth patterns. Here, we report that in root gravitropism of Arabidopsis thaliana, auxin regulates root curvature and associated apoplastic, growth-related pH changes through a Ca2+-dependent signaling pathway. Using an approach that integrates confocal microscopy and automated computer vision-based image analysis, we demonstrate highly dynamic root surface pH patterns during vertical growth and after gravistimulation. These pH dynamics are shown to be dependent on auxin, and specifically on auxin transport mediated by the auxin influx carrier AUX1 in cells of the lateral root cap and root epidermis. Our results further indicate that these pH responses require auxin-dependent changes in cytosolic Ca2+ levels that operate independently of the TIR1 auxin perception system. These results demonstrate a methodology that can be used to visualize vectorial auxin responses in a manner that can be integrated with the rapid plant growth responses to environmental stimuli.     

A novel mechanism of G protein-coupled receptor functional selectivity. Muscarinic partial agonist McN-A-343 as a bitopic orthosteric/allosteric ligand

Many G protein-coupled receptors (GPCRs) possess allosteric binding sites distinct from the orthosteric site utilized by their cognate ligands, but most GPCR allosteric modulators reported to date lack signaling efficacy in their own right. McN-A-343 (4-(N-(3-chlorophenyl)carbamoyloxy)-2-butynyltrimethylammonium chloride) is a functionally selective muscarinic acetylcholine receptor (mAChR) partial agonist that can also interact allosterically at the M(2) mAChR. We hypothesized that this molecule simultaneously utilizes both an allosteric and the orthosteric site on the M(2) mAChR to mediate these effects. By synthesizing progressively truncated McN-A-343 derivatives, we identified two, which minimally contain 3-chlorophenylcarbamate, as pure allosteric modulators. These compounds were positive modulators of the orthosteric antagonist N-[(3)H]methylscopolamine, but in functional assays of M(2) mAChR-mediated ERK1/2 phosphorylation and guanosine 5'-3-O-([(35)S]thio)triphosphate binding, they were negative modulators of agonist efficacy. This negative allosteric effect was diminished upon mutation of Y177A in the second extracellular (E2) loop of the M(2) mAChR that is known to reduce prototypical allosteric modulator potency. Our results are consistent with McN-A-343 being a bitopic orthosteric/allosteric ligand with the allosteric moiety engendering partial agonism and functional selectivity. This finding suggests a novel and largely unappreciated mechanism of "directed efficacy" whereby functional selectivity may be engendered in a GPCR by utilizing an allosteric ligand to direct the signaling of an orthosteric ligand encoded within the same molecule.