Analysis of the N-terminal region of the murine leukemia virus nucleocapsid protein

Lentiviruses such as the human immunodeficiency virus (HIV-1) and alpharetroviruses such as Rous Sarcoma virus encode an element that spans the precursor Gag (PrGag) protein capsid (CA) C-terminus, a spacer peptide (SP), and the N-terminus of nucleocapsid (NC). Perturbation of this element causes the assembly of aberrant, non-infectious virus particles. To determine whether this element is conserved in gammaretroviruses such as the Moloney murine leukemia virus (MLV), we examined the effects of insertion mutations in the N-terminal portion of the MLV NC coding region. Interestingly, we found that insertions of as many as twenty residues after the twelfth residue of MLV NC yielded proteins that directed the efficient assembly of virus particles. Virus morphologies and crosslink profiles appeared normal, and assembled viruses retained significant levels of infectivity in single cycle infection assays. Two variants were examined in the context of replicating virus constructs, and the mutations were found to be maintained during multiple rounds of infection in a cell culture system. These results suggest that the alpharetrovirus and lentivirus assembly elements either are not needed for gammaretroviruses, or are replaced by an alternative assembly element. Our results also indicate that the N-terminal region of MLV NC is amenable to genetic manipulation.     

Determinants of the HIV-1 core assembly pathway

Based on structural information, we have analyzed the mechanism of mature HIV-1 core assembly and the contributions of structural elements to the assembly process. Through the use of several in vitro assembly assay systems, we have examined details of how capsid (CA) protein helix 1, ß-hairpin and cyclophilin loop elements impact assembly-dependent protein interactions, and we present evidence for a contribution of CA helix 6 to the mature assembly-competent conformation of CA. Additional experiments with mixtures of proteins in assembly reactions provide novel analyses of the mature core assembly mechanism. Our results support a model in which initial assembly products serve as scaffolds for further assembly by converting incoming subunits to assembly proficient conformations, while mutant subunits increase the probability of assembly termination events.     

The 2005 Italian Quality Control Study for the evaluation of CD4 cells in centers involved in the treatment of HIV type 1 patients

We report the results of an external quality control program, including 17 Italian centers involved in the care of patients infected by HIV, to evaluate CD4 T cell count proficiency and reproducibility. The centers received two commercial stabilized blood preparations, one with "normal" and one with "low" CD4 T cell content. The centers were asked to process the samples two times, 1 week apart, with the same procedure used for samples from HIV patients. Most centers showed a good performance of CD4 frequency and absolute count determinations. In particular, the "low" sample was correctly analyzed by all centers; only two underestimated the "normal" sample CD4 frequency, and only one underestimated the CD4 absolute count by >100 CD4 cells/microl. Overall, our data suggest that most Italian laboratories provide reliable and reproducible results in evaluating CD4 T cells in HIV(+) samples.     

A generic consensus assessment of undergraduate competence in forceps exodontia in the United Kingdom

The assessment of competence in clinical skills has become more frequent in published healthcare curricula and syllabuses recently. There are agreed mechanisms for the assessment of competence in the post‐graduate environment, but no consensus within the undergraduate curriculum. This paper seeks to develop an agreed generic checklist for the assessment of competence in forceps exodontia. Materials and methods:  A modified Delphi process was undertaken with representatives from all UK dental schools (n = 13) to develop a generic checklist for the assessment of competence in forceps exodontia. A content analysis of the assessments employed by each school was used to help discussion and inform the Delphi process. Results:  Seven schools currently employ a summative assessment of competence in forceps exodontia, with the majority employing a structured clinical objective test (n = 6). From the seven assessments, there were a total of 29 putative items and 10 putative domains identified for a generic checklist. These were reduced to five domains and 19 items through the content analysis and Delphi process, and a generic overarching checklist was created. Conclusion:  Using this generic checklist, it may now be possible to pool data inter‐institution to perform more powerful analyses on how our students obtain, or fail to obtain competence in forceps exodontia.     

Forest responses to increasing aridity and warmth in the southwestern United States

In recent decades, intense droughts, insect outbreaks, and wildfires have led to decreasing tree growth and increasing mortality in many temperate forests. We compared annual tree-ring width data from 1,097 populations in the coterminous United States to climate data and evaluated site-specific tree responses to climate variations throughout the 20th century. For each population, we developed a climate-driven growth equation by using climate records to predict annual ring widths. Forests within the southwestern United States appear particularly sensitive to drought and warmth. We input 21st century climate projections to the equations to predict growth responses. Our results suggest that if temperature and aridity rise as they are projected to, southwestern trees will experience substantially reduced growth during this century. As tree growth declines, mortality rates may increase at many sites. Increases in wildfires and bark-beetle outbreaks in the most recent decade are likely related to extreme drought and high temperatures during this period. Using satellite imagery and aerial survey data, we conservatively calculate that ≈ 2.7% of southwestern forest and woodland area experienced substantial mortality due to wildfires from 1984 to 2006, and ≈ 7.6% experienced mortality associated with bark beetles from 1997 to 2008. We estimate that up to ≈ 18% of southwestern forest area (excluding woodlands) experienced mortality due to bark beetles or wildfire during this period. Expected climatic changes will alter future forest productivity, disturbance regimes, and species ranges throughout the Southwest. Emerging knowledge of these impending transitions informs efforts to adaptively manage southwestern forests.