Impact of X+Y Scheduling on Pediatric Resident and Faculty Perceptions of Education and Patient Care.

PURPOSE: Traditional half-day per week continuity clinic experiences can lead to fragmented education in both the inpatient and outpatient arenas. Five pediatric residency programs were granted the ability from the ACGME to create X+Y scheduling where residents have continuity clinic in "blocks" rather than half-day per week experiences. The aim of this study is to assess the impact X+Y scheduling has on pediatric resident and faculty perceptions of patient care and other educational experiences. METHODS: Electronic surveys were sent to residents and faculty of the participating programs both prior to and 12 months after implementing X+Y scheduling. Survey questions measured resident and faculty perception of continuity clinic schedule satisfaction and the impact of continuity clinic schedules on inpatient and subspecialty rotation experiences using a 5-point Likert Scale. Data were analyzed using z-tests for proportion differences for those answering Agree or Strongly Agree between baseline and post-implementation respondents. RESULTS: Hundred and twenty-six out of 186 residents (68%) responded preimplementation and 120 out of 259 residents (47%) responded post-implementation. 384 faculty members were sent the survey with 51% response pre-implementation and 26% response at 12 months. Statistically significant (P < .05) improvements were noted in resident and faculty perceptions of ability to have continuity with patients and inpatient workflow affected by clinic scheduling. CONCLUSIONS: From both resident and faculty perspectives, X+Y scheduling may improve several aspects of patient care and education. X+Y scheduling could be considered as a potential option by pediatric residency programs, especially if validated with more objective data.

Mechanisms of adeno-associated virus genome encapsidation.

The defective parvovirus, adeno-associated virus (AAV), is under close scrutiny as a human gene therapy vector. AAV's non-pathogenic character, reliance on helper virus co-infection for replication and wide tissue tropism, make it an appealing vector system. The virus' simplicity and ability to generate high titer vector preparations have contributed to its wide spread use in the gene therapy community. The single stranded AAV DNA genome is encased in a 20-25 nm diameter, icosahedral protein capsid. Assembly of AAV occurs in two distinct phases. First, the three capsid proteins, VP1-3, are rapidly synthesized and assembled into an empty virion in the nucleus. In the second, rate-limiting phase, single-strand genomic DNA is inserted into pre-formed capsids. Our rudimentary knowledge of these two phases comes from radioactive labeling pulse-chase experiments, cellular fractionation and immunocytological analysis of infected cells. Although the overall pattern of virus assembly and encapsidation is known, the biochemical mechanisms involved in these processes are not understood. Elucidation of the processes of capsid assembly and encapsidation may lead to improved vector production. While all of the parvoviruses share the characteristic icosahedral particle, differences in their surface topologies dictate different receptor binding and tissue tropism. Based on the analysis of the molecular structures of the parvoviruses and capsid mutagenesis studies, investigators have manipulated the capsid to change tissue tropism and to target different cell types, thus expanding the targeting potential of AAV vectors.

Adeno-associated virus and adenovirus coinfection induces a cellular DNA damage and repair response via redundant phosphatidylinositol 3-like kinase pathways.

During adeno-associated virus and adenovirus (AAV/Ad) coinfection, accumulation of viral genomes and proteins can alter cellular stress responses. To determine how AAV/Ad coinfection affects the host we screened over 60 cellular proteins for their responses. AAV/Ad coinfections induce a robust DNA damage response (DDR) that is distinct from that induced by Ad infection alone. Using chemical inhibitors, deficient cell lines and siRNA knockdowns of the DDR kinases, ATM, ATR and DNA-PK, we determined that DNA-PK and ATM kinases are the initial transducers of this response. AAV/Ad coinfection induces ATM- and DNA-PK mediated phosphorylation of RPA2, NBS1, H2AX and the checkpoint kinases CHK1/2. Inhibition of one or more of the DDR kinases reduces the level of phosphorylation of downstream targets but does not dramatically reduce Ad or AAV protein expression. However, AAV DNA levels are moderately affected by kinase inhibition. These experiments provide new insights into the cellular responses to AAV/Ad coinfections.

Adeno-associated virus interactions with B23/Nucleophosmin: identification of sub-nucleolar virion regions.

Adeno-associated virus (AAV) is a human parvovirus that normally requires a helper virus such as adenovirus (Ad) for replication. The four replication proteins (Rep78, 68, 52 and 40) encoded by AAV are pleiotropic effectors of virus integration, replication, transcription and virion assembly. Using Rep68 column chromatography and mass spectrometry, we have identified the nucleolar, B23/Nucleophosmin (NPM) protein as an Rep-interacting partner. Rep-NPM interactions were verified by co-immunofluorescence and chemical cross-linking studies. We have found that there is demonstrable, but limited co-localization between Rep and NPM in co-infected cells. In contrast, there was significant co-localization between NPM and AAV Cap proteins. In vitro experiments using purified MBPRep78 and NPM show that NPM stimulates MBPRep78 interactions with the AAV ITR as well as endonuclease activity. These studies suggest that NPM plays a role in AAV amplification affecting Rep function and virion assembly.