The patient experience of ambulatory cancer treatment: a descriptive study.

Background: Patient experience is often measured quantitatively, but that approach has limitations for understanding the entire experience. Qualitative methods can help to understand more complex issues most important to patients and their families. The purpose of the present work was to use a qualitative analysis examining the patient experience of ambulatory cancer care in Ontario to generate a deeper understanding of the patient experience and to lead to solutions for improvement. Methods: Data from the Ambulatory Oncology Patient Satisfaction Survey (aopss) for 2013-2015 were used to conduct a qualitative content analysis. The aopss is a retrospective paper-based survey, mailed to patients who are currently receiving cancer treatment or who have received cancer treatment within the preceding 6 months, that is designed to capture their experiences. Patients who were surveyed were asked, "Is there anything else you would like to tell us about your cancer care services?" The National Research Corporation Canada's patient-centred care framework was used to guide the analysis. Results: From the 5391 patients who responded, 7328 coded responses were generated, of which 3658 (49.9%) were related to the patient-centred care framework. New subthemes were identified: diagnosis sensitivity; emotional support resources; care delivery with care, compassion, and comfort; continuity of care between departments and in the community; access to cancer centre personnel; patient-health care provider communication; confidence in the health care provider; wait times; health care provider and treatment coordination; and parking. Conclusions: The results identify facilitators and barriers to the patient experience in the ambulatory cancer treatment setting from the patient perspective and identify opportunities to improve the patient experience.

Rydberg-Dressed Magneto-optical Trap.

We propose and demonstrate the laser cooling and trapping of Rydberg-dressed Sr atoms. By off-resonantly coupling the excited state of a narrow (7 kHz) cooling transition to a high-lying Rydberg state, we transfer Rydberg properties such as enhanced electric polarizability to a stable magneto-optical trap operating at <1 µK. Simulations show that it is possible to reach a regime where the long-range interaction between Rydberg-dressed atoms becomes comparable to the kinetic energy, opening a route to combining laser cooling with tunable long-range interactions.

Spin squeezing in a Rydberg lattice clock.

We theoretically demonstrate a viable approach to spin squeezing in optical lattice clocks via optical dressing of one clock state to a highly excited Rydberg state, generating switchable atomic interactions. For realistic experimental parameters, these interactions are shown to generate over 10 dB of squeezing in large ensembles within a few microseconds and without degrading the subsequent clock interrogation.

Distribution and dynamics of risk factors associated with highly pathogenic avian influenza H5N1.

Within China's Poyang Lake region, close interactions between wild migratory birds and domestic poultry are common and provide an opportunity for the transmission and subsequent outbreaks of highly pathogenic avian influenza (HPAI) virus. We overlaid a series of ecological factors associated with HPAI to map the risk of HPAI in relation to natural and anthropogenic variables, and we identified two hotspots for potential HPAI outbreaks in the Poyang Lake region as well as three corridors connecting the two hotspot areas. In hotspot I, there is potential for migratory birds to bring new avian influenza (AI) strains that can reassort with existing strains to form new AI viruses. Hotspot II features high-density poultry production where outbreaks of endemic AI viruses are likely. The three communication corridors that link the two hotspots further promote HPAI H5N1 transmission and outbreaks and lead to the persistence of AI viruses in the Poyang Lake region. We speculate that the region's unevenly distributed poultry supply-and-demand system might be a key factor inducing HPAI H5N1 transmission and outbreaks in the Poyang Lake region.

A vapor cell based on dispensers for laser spectroscopy.

We describe a simple strontium vapor cell for laser spectroscopy experiments. Strontium vapor is produced using an electrically heated commercial dispenser source. The sealed cell operates at room temperature, and without a buffer gas or vacuum pump. The cell was characterized using laser spectroscopy, and was found to offer stable and robust operation, with an estimated lifetime of >10 000 h. By changing the dispenser, this technique can be readily extended to other alkali and alkaline earth elements.

Adenovirus late gene expression does not require a Rev-like nuclear RNA export pathway.

Adenovirus late mRNA export is facilitated by viral early proteins of 55 and 34 kDa. The 34-kDa protein contains a leucine-rich nuclear export signal (NES) similar to that of the human immunodeficiency virus Rev protein. It was proposed that the 34-kDa protein might facilitate the export of adenovirus late mRNA through a Rev-like NES-mediated export pathway. We have tested the role of NES-mediated RNA export during adenovirus infection, and we find that it is not essential for the expression of adenovirus late genes.

Organization of splicing factors in adenovirus-infected cells reflects changes in gene expression during the early to late phase transition.

The spatial distribution of splicing factors is temporally regulated during adenovirus (ad) infection. Here we focus on two splicing factor distribution patterns characteristic of ad-infected cells. During the intermediate phase splicing factors surround sites of viral DNA accumulation in regions of high transcriptional activity. This distribution appears as a series of interconnected rings when viewed by microscopy. We refer to cells with this staining pattern as "ring cells." We have previously shown that at late times after infection, splicing factors are present in discrete structures identified as enlarged interchromatin granules (IGs) that also contain spliced viral RNA. We refer to cells with this pattern as "cluster cells." We determined which steps in viral gene expression occurred in ring and cluster cells. We found that transcription and some splicing of viral late genes had occurred in ring cells. Late RNA was present at transcription sites in ring cells. Cluster cells contained spliced viral late RNA in nuclear IGs and in the cytoplasm. The presence of cluster cells in the infected culture was well correlated with the export of viral RNA to the cytoplasm. Cluster cells had synthesized late proteins. Our data show that the dynamic localization of splicing factors reflects changes in gene expression activity of the infected cell as it switches over to late gene expression.

Spliced exons of adenovirus late RNAs colocalize with snRNP in a specific nuclear domain.

Posttranscriptional steps in the production of mRNA include well characterized polyadenylation and splicing reactions, but it is also necessary to understand how RNA is transported within the nucleus from the site of its transcription to the nuclear pore, where it is translocated to the cytoplasmic compartment. Determining the localization of RNA within the nucleus is an important aspect of understanding RNA production and may provide clues for investigating the trafficking of RNA within the nucleus and the mechanism for its export to the cytoplasm. We have previously shown that late phase adenovirus-infected cells contain large clusters of snRNP and non-snRNP splicing factors; the presence of these structures is correlated with high levels of viral late gene transcription. The snRNP clusters correspond to enlarged interchromatin granules present in late phase infected cells. Here we show that polyadenylated RNA and spliced tripartite leader exons from the viral major late transcription unit are present in these same late phase snRNP-containing structures. We find that the majority of the steady state viral RNA present in the nucleus is spliced at the tripartite leader exons. Tripartite leader exons are efficiently exported from the nucleus at a time when we detect their accumulation in interchromatin granule clusters. Since the enlarged interchromatin granules contain spliced and polyadenylated RNA, we suggest that viral RNA may accumulate in this late phase structure during an intranuclear step in RNA transport.

Dynamic organization of splicing factors in adenovirus-infected cells.

Adenovirus infection affects the nuclear distribution of host splicing factors. Late phase-infected cells contain discrete clusters of small nuclear ribonucleoproteins (snRNPs) that are separate from centers containing the viral 72-kilodalton DNA-binding protein (72K protein). In the present study, we demonstrate that these snRNP clusters also contain splicing factors from the SR protein family. We show that a previously described monoclonal antibody, 3C5, detects SR proteins. Furthermore, we demonstrate that late region 3 transcription occurs at a maximal rate in infected cultures in which greater than 90% of the cells contain the snRNP clusters, indicating that such cells are actively transcribing their late genes. During the onset of the late phase, the intranuclear distribution of splicing factors is very different from that seen after the late phase is established. When late viral transcription commences, cells with snRNP clusters are less prevalent than in cultures that are maintaining maximum levels of late transcription. Instead, a cell type which shows snRNPs, concentrated in foci that also contain the viral 72K DNA-binding protein is detected. This cell type disappears from cultures by 18 to 20 h after a high-multiplicity infection. These results suggest a dynamic organization of splicing factors in infected cells that can be correlated to the status of viral gene expression. Our work also provides an explanation for the differing results that have been published concerning the organization of splicing factors in the adenovirus-infected cell nucleus (L. F. Jiménez-García and D. L. Spector, Cell 73:47-59, 1993). During the present study we observed that a monoclonal antibody against the SC-35 protein, which was used by Jiménez-García and Spector to study the localization of the SC-35 splicing factor in adenovirus-infected cells, cross-reacts with the adenovirus 72K DNA-binding protein and is thus unsuitable for this type of study.

Adenovirus replication and transcription sites are spatially separated in the nucleus of infected cells.

We have visualized the intranuclear topography of adenovirus replication and transcription in infected HeLa cells. The results show that viral DNA replication occurs in multiple foci that are highly organized in the nucleoplasm. Pulse-chase experiments indicate that newly synthesized viral double-stranded DNA molecules are displaced from the replication foci and spread throughout the nucleoplasm, while the single-stranded DNA replication intermediates accumulate in adjacent sites. Double-labelling experiments and confocal microscopy show that replication occurs in foci localized at the periphery of the sites where single-stranded DNA accumulates. The simultaneous visualization of viral replication and transcription reveals that the sites of transcription are predominantly separated from the sites of replication. Transcription is detected adjacent to the replication foci and extends around the sites of single-stranded DNA accumulation. These data indicate that newly synthesized double-stranded DNA molecules are displaced from the replication foci and spread in the surrounding nucleoplasm, where they are used as templates for transcription. Splicing snRNPs are shown to co-localize with the sites of transcription and to be excluded from the sites of replication. This provides evidence that splicing of viral RNAs occurs co-transcriptionally and that the sites of viral DNA replication are spatially distinct from the sites of RNA transcription and processing.

Nuclear organization of splicing small nuclear ribonucleoproteins in adenovirus-infected cells.

We have studied the effect of adenovirus infection on the nuclear organization of splicing small nuclear ribonucleoproteins (snRNPs) in HeLa cells. In uninfected HeLa cells, snRNPs are widespread throughout the nucleoplasm but also are concentrated in specific nuclear structures, including coiled bodies, interchromatin granules, and perichromatin fibrils. We have used immunofluorescence microscopy to study the localization of splicing snRNPs relative to centers of viral DNA synthesis and accumulation identified with antiserum against the viral 72,000-molecular-weight single-stranded DNA-binding protein (72K protein). Splicing snRNPs were independently detected with both monoclonal and polyclonal antibodies specific for common snRNP antigens, snRNP-specific proteins, and the snRNA-specific 2,2,7-trimethylguanosine 5' cap structure. We have examined infected cells 2 to 24 h after infection, and, in the majority of these cells, we observed no colocalization of the snRNP and 72K-protein staining patterns. In the late phase, snRNPs were found to markedly concentrate in discrete clusters that were distinct from the centers of viral DNA synthesis and accumulation identified with anti-72K protein. We have treated cells with hydroxyurea at various times after infection to inhibit aspects of the virus infectious program. We have found that the accumulation of snRNP clusters is correlated with late gene expression rather than with DNA synthesis or early gene expression. Finally, we show that the late-phase snRNP clusters colocalize with a monoclonal antibody that primarily stains interchromatin granules. These results suggest that the centers of snRNP concentration in late-phase infected cells are likely to correspond to interchromatin granule clusters.

Adenovirus early region 4 and viral DNA synthesis.

Mutants of human adenovirus type 5 (Ad5) lacking early region 4 (E4) display a complex phenotype that includes a delay in the onset of viral DNA replication in low-multiplicity infections. Studies of viral DNA replication in vitro have not revealed a requirement for E4 products in DNA synthesis and, for most E4 mutants, defects in DNA replication are not apparent at high multiplicities of infection. The effects of E4 mutations on DNA replication therefore may reflect a role for E4 in the regulation of replication rather than in the process of DNA synthesis. The E4 mutant H5d/1014 carries two deletion mutations that together destroy all E4 open reading frames (ORFs) except ORF 4. Immunoprecipitation measurements of the level of the ORF 4 product confirm that H5d/1014 accumulates the ORF 4 product in somewhat larger amounts than wild-type Ad5. H5d/1014 is profoundly defective in viral DNA replication at a multiplicity of infection (50 PFU/cell) and time (24 hr after infection) that permit mutants lacking all seven E4 products to accumulate approximately normal amounts of DNA. In contrast, H5d/1019, a derivative of H5d/1014 in which the expression of ORF 4 is prevented by a mutation in the ORF 4 ATG initiator codon, produces DNA normally under these conditions. The product of ORF 4 therefore is necessary for the inhibition of viral DNA replication in H5d/1014-infected cells. H5d/1014 also inhibits, in trans, the synthesis of viral DNA by other E4 mutants that lack both E4 ORFs 3 and 6. Viruses that possess either of those ORFs are not subject to inhibition, indicating that the ORF3 and 6 products antagonize the effect of ORF 4. These observations are consistent with a regulatory role for the E4 ORF 3, 4, and 6 products in viral DNA replication in adenovirus-infected cells.

Differential regulation of adenovirus late transcriptional units by the products of early region.

Normal accumulation of adenoviral late mRNAs derived from the major late promoter, is mediated independently by either the ORF 6 or the ORF 3 product of E4. We have examined the role of E4 products in the expression of three late messages that are derived from outside the major late transcriptional unit: polypeptide IX mRNA, IVa2 mRNA, and messages from the E2 late promoter. We conclude that the late RNA accumulation phenotypes of E4 mutants can be broken down into two components. The effect on nuclear RNA accumulation, which is mediated independently by either the ORF 6 or ORF 3 products, is targeted to late messages derived from the major late transcriptional unit. A second small effect, that is mediated only by ORF 6, is needed for optimal accumulation of mRNA in the cytoplasm, and affects major late promoter derived messages as well as the message for polypeptide IX. The levels of IVa2 and E2 late messages are not reduced in cells infected with E4 negative mutants.

Adenovirus E4-dependent activation of the early E2 promoter is insufficient to promote the early-to-late-phase transition.

The adenovirus E4 ORF6/7 protein has been shown to activate the cellular transcription factor E2F. E2F activation leads to activation of the adenovirus early E2 promoter which controls the production of viral DNA replication proteins. In the present study an adenovirus type 5 cDNA mutant, H5ilE4L, was constructed. This mutant is capable of making the ORF6/7 polypeptide but lacks the coding sequences for all other E4 products. H5ilE4L trans activates the early E2 promoter to wild-type levels, but still it is defective for viral DNA replication. A mutant expressing ORF6 in addition to ORF6/7, H5ilE4I, is normal for viral DNA replication. This indicates that activation of the early E2 promoter is insufficient to promote efficient viral DNA replication and that another E4-encoded function is necessary. The ORF6 protein seems to provide this function. We suggest that ORF6/7-induced activation of E2F is not necessary for adenovirus growth in HeLa cells. Rather, this activation might be of importance in the normal, growth-arrested host cell, since E2F has been shown to bind to the promoter regions of a number of immediate-early genes involved in regulation of cell proliferation (M. Mudryj, S. W. Hiebert, and J. R. Nevins, EMBO J. 9:2179-2184, 1990).

Interaction of adenoviral E4 and E1b products in late gene expression.

The adenovirus 294R protein of E4 ORF 6 forms a physical and functional complex with the 496R protein product of E1b. The E4 294R ORF 6 protein also functions in parallel with the E4 116R ORF 3 protein in viral late protein synthesis. We have examined the roles of these three proteins and the protein complex in viral late protein synthesis and late message metabolism, by comparing the phenotypes of E4 294R-, E4 116R-, and E1b 496R- mutants to those of a series of double mutants. Our data indicate that the 294R and 116R proteins act in parallel to permit the accumulation of normal levels of unprocessed late viral RNA in the nucleus of infected cells. Both 294R and 496R function in parallel with the 116R protein in viral nuclear RNA accumulation, but do so to different degrees, suggesting that 294R and 496R may have roles apart from the functional complex in mediating accumulation of viral messages in the nucleus, or that they have nonequivalent roles within the complex. Our results are also consistent with a role for the 496R/294R protein complex in mediating efficient transport of late messages from the nucleus to the cytoplasm and/or in maintaining the stability of those messages on reaching the cytoplasm, as suggested previously (S. Pilder, M. Moore, J. Logan, and T. Shenk, 1986, Mol. Cell. Biol. 6, 470-476). Finally the 116R protein seems to act in parallel with the complex to permit normal viral DNA replication.

Complementation of adenovirus E4 mutants by transient expression of E4 cDNA and deletion plasmids.

Human adenovirus mutants that carry a large deletion in early region 4 (E4) are severely defective in the synthesis of viral late proteins. Plasmids that carry intact E4 sequences can complement the late protein synthetic defect of such mutants when introduced into infected cells by transfection, presumably due to the transient expression of E4 products. Cells transfected with cDNA clones capable of expressing E4 open reading frame (ORF) 6, or deletion mutant clones expected to express either E4 ORF 6 or E4 ORF 3, also complement the mutants' defects. Thus, these E4 ORFs can individually satisfy the requirement for E4 products in viral late gene expression, and function effectively in the absence of other E4 products. Some E4 deletion mutants also exhibit a defect in the production of viral DNA. All of the clones that stimulate late gene expression also enhance one such mutant's ability to accumulate viral DNA. Thus, the ORF 3 and ORF 6 products are also individually sufficient to provide an E4 function necessary for normal viral DNA replication in the absence of other E4 products.

Redundant control of adenovirus late gene expression by early region 4.

A series of human adenovirus type 5 derivatives carrying deletion mutations in early region 4 (E4) were constructed and characterized with respect to viral late protein synthesis, viral cytoplasmic late message accumulation, viral DNA accumulation, and plaquing ability. Viral late protein synthesis was essentially normal in cells infected by mutants expected to produce either the E4 open reading frame (ORF) 3 product or the E4 ORF 6 product. In cells infected by mutants lacking both ORF 3 and ORF 6, late protein synthesis was dramatically reduced. The basis for this reduction appears to be a concomitant reduction in cytoplasmic late message levels. Our results suggest that the products of ORFs 3 and 6 are redundant, since they are individually able to satisfy the requirement for E4 in late gene expression. Two of the mutants examined were defective for viral late protein synthesis but showed no measurable defect in viral DNA accumulation. The defect in late gene expression is not, therefore, a reflection of a primary defect in viral DNA synthesis. Finally, mutants expected to express ORF 3 or ORF 6 formed plaques with normal or only modestly reduced efficiency, whereas mutants expected to express neither ORF formed plaques with an efficiency less than 10(-6) that of wild-type virus. Thus, plaque-forming ability reflected late protein synthetic ability, suggesting that among these mutants late protein synthetic proficiency is the principle determinant of plaquing efficiency.