Replicon rescue: a novel strategy to clone the genomic DNA flanking insertions of integrating shuttle vector DNA.

A novel cloning strategy, replicon rescue, was developed for cloning genes disrupted by plasmid insertions. After ligation to a tetracycline resistance cassette, fragments containing a bacterial origin of replication from the insertion are recovered in Escherichia coli because they replicate autonomously. Restriction enzymes for cloning are so chosen that the only legitimate two fragment ligation yielding TetR clones involves a fragment spanning the boundary of the insertion. Replicon rescue was used successfully firstly in a test system to clone the chromosomal orl from a Klebsiella aerogenes strain, and secondly to recover a disrupted gene from a phototaxis-deficient mutant of Dictyostelium.     

Linkage of cutaneous malignant melanoma/dysplastic nevi to chromosome 9p, and evidence for genetic heterogeneity.

We examined the relationship between cutaneous malignant melanoma/dysplastic nevi (CMM/DN) and chromosome 9p in 13 pedigrees with two or more living cases of invasive melanoma. We used two highly informative (CA)n repeats, D9S126 and IFNA, previously implicated in familial malignant melanoma (MLM), to conduct linkage analysis. Three analyses were performed: (1) CMM alone--all individuals without either confirmed melanoma or borderline lesions were considered unaffected (model A); (2) CMM/DN with both variable age at onset and sporadics (model B); and (3) CMM affecteds only--all individuals either without confirmed melanoma or with borderline lesions were designated "unknown" (model C). There was significant evidence for linkage to IFNA in all three models. For CMM alone, the maximum lod score (Zmax) was 4.36 at theta = .10 for model A and 3.39 at theta = .10 for model C. For CMM/DN (model B), Zmax = 3.05 at theta = .20. There was no significant evidence for linkage between CMM alone or CMM/DN and chromosome 9p marker D9S126. In addition, there was significant evidence for heterogeneity when a homogeneity test allowing for linkage to chromosome 9p or chromosome 1p or neither region was used. These results suggest that there is an MLM susceptibility locus on chromosome 9p but that familial melanoma is heterogeneous and not all families with CMM/DN are linked to a locus in this region.     

Social problems in geriatric dentistry: an overview

Many studies have reported on the dental status of elderly patients. The results demonstrate consistent differences between normative and perceived needs in addition to varying levels of awareness of dental education among carers. This review article attempts to integrate a range of social factors which must be considered if holistic and comprehensive care is to be delivered to elderly patients.     

Regional chromosomal assignment of the Kell blood group locus (KEL) to chromosome 7q33-q35 by fluorescence in situ hybridization: evidence for the polypeptide nature of antigenic variation

The gene encoding the Kell blood group polypeptide has been localized to chromosome 7q33-35 by in situ hybridization using a biotinylated 1.1-kb DNA fragment containing the 3 half of the human cDNA. This assignment is in accord with genetic localization using antigenic variation as a marker, and strongly suggests that Kell antigenic determinants are part of the polypeptide chain rather than the associated sugar molecules.     

Piping plover chick ecology following landscape-level disturbance

Population declines of disturbance-dependent species due to suppression of natural disturbances are realized across ecosystems. The piping plover (Charadrius melodus; plover), a disturbance-dependent and conservation-reliant shorebird that nests on sandy beaches and barrier islands on the Atlantic Coast, was listed under the United States Endangered Species Act in 1986. In 2012, Hurricane Sandy landed on Fire and Westhampton islands, barrier island nesting sites for plovers in New York, USA. Hurricane Sandy was a natural disturbance in this system, creating abundant nesting habitat. The number of chicks produced by a pair, or a population, is a direct measure of reproductive output, and gaining a better understanding of productivity and chick behavior following large-scale habitat creation may improve plover habitat management and potentially species persistence. We evaluated the effects of landscape features on habitat selection, behavior, and survival of plover broods using logistic regression, generalized linear mixed effects models, and survival models. Plover broods selected flatter sites with less dense vegetation than available at random. Chick foraging rates were highest in moist substrates and were lower in areas of higher nesting plover density. Chick survival was greater for broods that hatched earlier in the breeding season and increased as chicks aged. Generally, providing access to sites with flatter, moist substrates will likely result in higher quality brood rearing habitat on the landscape. Ultimately, vegetation removal and habitat management may be needed to reduce plover nesting density and ensure sufficient habitat, which may in turn improve plover chick survival. Moreover, sustaining natural landscape disturbances such as those resulting from storms, and not taking actions to prevent hurricane-created overwash, will allow these landscape features to persist.     

COVID-19 plasma proteome reveals novel temporal and cell-specific signatures for disease severity and high-precision disease management

Coronavirus disease 2019 (COVID-19) is a systemic inflammatory condition with high mortality that may benefit from personalized medicine and high-precision approaches. COVID-19 patient plasma was analysed with targeted proteomics of 1161 proteins. Patients were monitored from Days 1 to 10 of their intensive care unit (ICU) stay. Age- and gender-matched COVID-19-negative sepsis ICU patients and healthy subjects were examined as controls. Proteomic data were resolved using both cell-specific annotation and deep-analysis for functional enrichment. COVID-19 caused extensive remodelling of the plasma microenvironment associated with a relative immunosuppressive milieu between ICU Days 3–7, and characterized by extensive organ damage. COVID-19 resulted in (1) reduced antigen presentation and B/T-cell function, (2) increased repurposed neutrophils and M1-type macrophages, (3) relatively immature or disrupted endothelia and fibroblasts with a defined secretome, and (4) reactive myeloid lines. Extracellular matrix changes identified in COVID-19 plasma could represent impaired immune cell homing and programmed cell death. The major functional modules disrupted in COVID-19 were exaggerated in patients with fatal outcome. Taken together, these findings provide systems-level insight into the mechanisms of COVID-19 inflammation and identify potential prognostic biomarkers. Therapeutic strategies could be tailored to the immune response of severely ill patients.     

Sensitivity of random amplified polymorphic DNA analysis to detect genetic change in sugarcane during tissue culture

Random amplified polymorphic DNA (RAPD) analysis using 10-mer oligonucleotide primers efficiently differentiated sugarcane cultivars and proved suitable for detecting gross genetic change such as that which can occur in sugarcane subjected to prolonged tissue culture, for example in protoplast-derived callus. However, RAPD analysis was not sufficiently sensitive to detect smaller genetic changes that occur during sugarcane genetic transformation. The length of DNA scored for polymorphism per primer averaged 13.2 kb, or 0.0001% of the typical sugarcane genome size of 1.2 × 10₇ kb (2C). RAPD analysis of sugarcane plants regenerated from embryogenic callus revealed very few polymorphisms, indicating that gross genetic change is infrequent during this tissue culture procedure, although epigenetic effects result in transient morphological changes in regenerated plants. More sensitive variations on the RAPD technique may increase the practicality of DNA-based screening of regenerated plant lines to reveal somaclonal variants.