Molecular, forensic and haplotypic inconsistencies regarding the identity of the Ekaterinburg remains.

BACKGROUND: A set of human remains unearthed near Ekaterinburg, Russia has been attributed to the Romanov Imperial Family of Russia and their physician and servants. That conclusion was officially accepted by the Russian government following publication of DNA tests that were widely publicized. The published study included no discussion of major forensic discrepancies and the information regarding the burial site and remains included irregularities. Furthermore, its conclusion of Romanov identity was based on molecular behaviour that indicates contamination rather than endogenous DNA. The published claim to have amplified by PCR a 1223 bp region of degraded DNA in a single segment for nine individuals and then to have obtained sequence of PCR products derived from that segment without cloning indicates that the Ekaterinburg samples were contaminated with non-degraded, high molecular weight, 'fresh' DNA. AIM: Noting major violations of standard forensic practices, factual inconsistencies, and molecular behaviours that invalidate the claimed identity, we attempted to replicate the findings of the original DNA study. SUBJECT: We analysed mtDNA extracted from a sample of the relic of Grand Duchess Elisabeth, sister of Empress Alexandra. RESULTS: Among clones of multiple PCR targets and products, we observed no complete mtDNA haplotype matching that reported for Alexandra. The consensus haplotype of Elisabeth differs from that reported for Alexandra at four sites. CONCLUSION: Considering molecular and forensic inconsistencies, the identity of the Ekaterinburg remains has not been established. Our mtDNA haplotype results for Elisabeth provide yet another line of conflicting evidence regarding the identity of the Ekaterinburg remains.

Chemical mutagenesis of the parasitic nematode Strongyloides ratti to isolate ivermectin resistant mutants.

We describe a strategy for the mutagenesis of the free-living adult generation of Strongyloides ratti and selection of worms carrying new mutations in the subsequent F2 generation of infective larvae. We demonstrate that this strategy is successful via the selection of infective larvae that are resistant to the anthelmintic ivermectin at a concentration of 10 ng/ml. The majority of these larvae were unable to give rise to patent infections when used to infect parasite naive rats, implying that the majority of the ivermectin resistance mutations confer pleiotropic defects on parasitic, but not on free-living, development.

mtDNA affinities of the peoples of North-Central Mexico.

mtDNA haplotypes of representatives of the cosmopolitan peoples of north-central Mexico were studied. Two hundred twenty-three samples from individuals residing in vicinities of two localities in north-central Mexico were analyzed. A combination of strategies was employed to identify the origin of each haplotype, including length variation analysis of the COII and tRNALYS intergenic region, nucleotide sequence analysis of control region hypervariable segment 1, and RFLP analysis of PCR products spanning diagnostic sites. Analysis of these data revealed that the majority of the mtDNA haplotypes were of Native American origin, belonging to one of four primary Native American haplogroups. Others were of European or African origin, and the frequency of African haplotypes was equivalent to that of haplotypes of European derivation. These results provide diagnostic, discrete character, molecular genetic evidence that, together with results of previous studies of classical genetic systems, is informative with regard to both the magnitude of African admixture and the relative maternal contribution of African, European, and Native American peoples to the genetic heritage of Mexico. Phylogenetic analysis revealed that African sequences formed a basal, paraphyletic group.

Hypomethylation of an expanded FMR1 allele is not associated with a global DNA methylation defect.

The vast majority of fragile-X full mutations are heavily methylated throughout the expanded CGG repeat and the surrounding CpG island. Hypermethylation initiates and/or stabilizes transcriptional inactivation of the FMR1 gene, which causes the fragile X-syndrome phenotype characterized, primarily, by mental retardation. The relation between repeat expansion and hypermethylation is not well understood nor is it absolute, as demonstrated by the identification of nonretarded males who carry hypomethylated full mutations. To better characterize the methylation pattern in a patient who carries a hypomethylated full mutation of approximately 60-700 repeats, we have evaluated methylation with the McrBC endonuclease, which allows analysis of numerous sites in the FMR1 CpG island, including those located within the CGG repeat. We report that the expanded-repeat region is completely free of methylation in this full-mutation male. Significantly, this lack of methylation appears to be specific to the expanded FMR1 CGG-repeat region, because various linked and unlinked repetitive-element loci are methylated normally. This finding demonstrates that the lack of methylation in the expanded CGG-repeat region is not associated with a global defect in methylation of highly repeated DNA sequences. We also report that de novo methylation of the expanded CGG-repeat region does not occur when it is moved via microcell-mediated chromosome transfer into a de novo methylation-competent mouse embryonal carcinoma cell line.

From long range mapping to sequence-ready contigs on human chromosome 6.

Our aim is to construct physical clone maps covering those regions of chromosome 6 that are not currently extensively mapped, and use these to determine the DNA sequence of the whole chromosome. The strategy we are following involves establishing a high density framework map of the order of 15 markers per Megabase using radiation hybrid (RH) mapping. The markers are then used to identify large-insert genomic bacterial clones covering the chromosome, which are assembled into sequence-ready contigs by restriction enzyme fingerprinting and sequence tagged site (STS) content analysis. Contig gap closure is performed by walking experiments using STSs developed from the end sequences of the clone inserts.

Assessing cross-media impacts: a comparative risk approach.

The Oregon Department of Environmental Quality has developed a Cross-Media Comparative Risk Assessment model to address certain regulatory concerns. The model generates a Human and Ecological Risk Index for a facility releasing toxins into the environment. The risk indices are based on chemical fate and transport predictions, toxicity, population density, and ecological sensitive areas. The model can be used to rank facilities for inspection or as a tool to assess the progress of pollution prevention programs. Regulatory permitting departments can use the model to address the cross-media transfer of pollutants from one environmental compartment to another. The versatility of the model allows adaptation to each specific users needs.

Phase II trial of thymosin fraction 5 in advanced renal cancer.

Partially purified thymosin fraction 5 ( TF5 ) was administered to 21 patients with advanced renal cancer. Two different loading dose schedules and doses (60 and 120 mg/m2) of SQ TF5 were employed in 10 patients each. Of 20 evaluable patients, three exhibited partial responses and two exhibited stable disease. All five of these patients had had prior nephrectomies and lung metastases as the dominant site of disease. Toxicity was minimal but included one probable systemic allergic reaction. We could not identify any specific relationship between TF5 dose/schedule or pretreatment immune abnormalities and tumor responsiveness. Our results indicate that the administration of TF5 alone can induce regressions of renal cancer. Additional trials with larger numbers of patients appear to be justified.

Multiple myeloma: presenting as a neurological disorder.

A case is reported of multiple myeloma presenting with signs and symptoms of paraplegia in a patient with a history of hypertension and remote cerebral vascular accident. The laboratory findings of hyperproteinemia and uricemia suggest a protein synthesizing abnormality. This case emphasizes that most patients with protein abnormality should be investigated by protein electrophoresis and immunoelectrophoresis.Unusual clinical presentation of multiple myeloma may result in an erroneous diagnosis unless proper investigation in the appropriate line is made.

An approach to the evaluation of hemostasis.

Effective hemostasis may be adequately and quickly evaluated using tests that are readily available in the usual hospital laboratory. This evaluation includes peripheral smear for platelet morphology and number, platelet count, bleeding time, activated partial thromboplastin time (aPTT), prothrombin time (PT), and clot retraction. Normal results of these tests exclude all but the mildest disturbances of hemostasis.