Development of a single probe for documentation of chimerism following bone marrow transplantation.

Although numerous genetic markers are available for studying chimerism after bone marrow transplantation (BMT), there remains a need for a practical and highly informative method that is applicable in the early posttransplantation period. Using DNA restriction-fragment-length polymorphisms (RFLPs), we have evaluated the feasibility of developing a single synthetic oligonucleotide probe to study post-BMT chimerism. We have thus tested three candidate probes, termed O-3315-32, O-3315-80, and O-AY-29, that are homologous to tandemly repetitive sequences. Our results demonstrated donor-specific and recipient-specific fragments in 11 of 11 HLA-matched sibling pairs tested using probes O-3315-32 and O-3315-80. When probe O-AY-29 was used, 14 of 17 sibling pairs showed both donor and recipient markers, one had only a recipient marker, and two were identical. We showed that each of the three synthetic probes was effective in documenting donor marrow engraftment, mixed hematopoietic chimerism, the patient's pre-BMT phenotype (by using cultured skin fibroblasts obtained after BMT), and the origin of the malignant hematopoietic cells (i.e., of donor or recipient origin) in patients who developed recurrent hematologic malignancy following BMT. Compared with the use of cloned genomic probes, there are several important advantages to the use of synthetic oligonucleotide probes in studying post-BMT chimerism. Synthetic probes have absolute hybridization specificity and can be designed to suit the purposes of an individual study, since they have adjustable specificity that can be altered by changes in the length of the probe and by changes in the hybridization temperature. A single synthetic probe analogous to several highly polymorphic loci can have a polymorphism information content sufficiently high so that all but a small percentage of BMT patients could be followed easily; for example, if a probe were complementary to three highly polymorphic unlinked loci, it would discriminate approximately 98% of sibling donor/recipient pairs. This would be accomplished using only one restriction-endonuclease digestion and only one gel electrophoresis. Since other genetic markers, e.g., red blood cell antigens, immunoglobulin allotypes, and chromosome analysis, are not uniformly informative and, in some cases, cannot be used in the early posttransplantation period, the use of synthetic oligonucleotide probes for analysis of DNA RFLP is emerging as the method of choice for studies of post-BMT chimerism. This method will allow for the development of new knowledge that has not been possible with previous methods.     

Plant regeneration in vitro of South Pacific taro (Colocasia esculenta var. esculenta cv. Akalomamale,Aracea)

Summary Axillary bud expiants from South Pacific (Solomon Islands) taro, Colocasia esculenta var. esculenta cv. Akalomamale (Araceae) cultured on a modified Murashige-Skoog medium containing 1 mg NAA 1^–1 and TE formed callus and produced multiple plantlets. Explants died if NAA was present at levels lower than 0.1 mg 1^–1. BA was not required and may have been inhibitory. Plantlets developed faster and became larger following transfer to a hormone-free medium two weeks after the start of culture. Fully grown plants were established in a potting mix and are growing well in a greenhouse.Abbreviations BA benzyladenine - BM basal medium - Ca Colocasia esculenta var. antiquorum - Ce Colocasia esculenta var. esculenta - Ck cytokinin(s) - CW coconut water - HSMSM half strength Murashige Skoog macroelements - HSMS half strength Murashige and Skoog medium - IM initial medium(ia) - MS Murashige and Skoog medium - NAA naphthaleneacetic acid - SM second medium - TE taro corm extract - UCI University of California, Irvine     

Immunocytochemical diagnosis of acute leukemia with pleural involvement

Cytologic examination of the pleural effusion from a patient with acute leukemia, leukocytosis and bleeding revealed the presence of many leukemic cells, "lymphocytes" and erythrocytes. The significance of these cellular changes was investigated by simultaneous study of blood and effusion leukocytes by morphologic, cytochemical and immunochemical methods. Both the leukemic blasts and the "lymphocytes" in the effusion and the blood were found to be neoplastic and contained antigens characteristic of both myeloid cells (OKM-1) and lymphoblasts (C-ALLA, common acute lymphoblastic leukemia antigen). These results, when analyzed in the context of the clinical findings, were indicative of acute leukemia with pleural involvement. Such a clinically oriented approach may further enhance the potential of cytodiagnosis in patients with serous effusions.     

Excess amino acid polymorphism in mitochondrial DNA: contrasts among genes from Drosophila, mice, and humans

Recent studies of mitochondrial DNA (mtDNA) variation in mammals and Drosophila have shown an excess of amino acid variation within species (replacement polymorphism) relative to the number of silent and replacement differences fixed between species. To examine further this pattern of nonneutral mtDNA evolution, we present sequence data for the ND3 and ND5 genes from 59 lines of Drosophila melanogaster and 29 lines of D. simulans. Of interest are the frequency spectra of silent and replacement polymorphisms, and potential variation among genes and taxa in the departures from neutral expectations. The Drosophila ND3 and ND5 data show no significant excess of replacement polymorphism using the McDonald-Kreitman test. These data are in contrast to significant departures from neutrality for the ND3 gene in mammals and other genes in Drosophila mtDNA (cytochrome b and ATPase 6). Pooled across genes, however, both Drosophila and human mtDNA show very significant excesses of amino acid polymorphism. Silent polymorphisms at ND5 show a significantly higher variance in frequency than replacement polymorphisms, and the latter show a significant skew toward low frequencies (Tajima's D = -1.954). These patterns are interpreted in light of the nearly neutral theory where mildly deleterious amino acid haplotypes are observed as ephemeral variants within species but do not contribute to divergence. The patterns of polymorphism and divergence at charge-altering amino acid sites are presented for the Drosophila ND5 gene to examine the evolution of functionally distinct mutations. Excess charge-altering polymorphism is observed at the carboxyl terminal and excess charge-altering divergence is detected at the amino terminal. While the mildly deleterious model fits as a net effect in the evolution of nonrecombining mitochondrial genomes, these data suggest that opposing evolutionary pressures may act on different regions of mitochondrial genes and genomes.      

Associations of physical activity with gut microbiota in pre-adolescent children

[Purpose] To determine whether physical activity (PA), primarily the recommended 60 minutes of moderate-to-vigorous PA, is associated with gut bacterial microbiota in 10-year-old children.[Methods] The Block Physical Activity Screener, which provides minutes/day PA variables, was used to determine whether the child met the PA recommendations. 16S rRNA sequencing was performed on stool samples from the children to profile the composition of their gut bacterial microbiota. Differences in alpha diversity metrics (richness, Pielou’s evenness, and Faith’s phylogenetic diversity) by PA were determined using linear regression, whereas beta diversity (unweighted and weighted UniFrac) relationships were assessed using PERMANOVA. Taxon relative abundance differentials were determined using DESeq2.[Results] The analytic sample included 321 children with both PA and 16S rRNA sequencing data (mean age [SD] =10.2 [0.8] years; 54.2% male; 62.9% African American), where 189 (58.9%) met the PA recommendations. After adjusting for covariates, meeting the PA recommendations as well as minutes/day PA variables were not significantly associated with gut richness, evenness, or diversity (p ≥ 0.19). However, meeting the PA recommendations (weighted UniFrac R² = 0.014, p = 0.001) was significantly associated with distinct gut bacterial composition. These compositional differences were partly characterized by increased abundance of Megamonas and Anaerovorax as well as specific Christensenellaceae_R-7_group taxa in children with higher PA.[Conclusion] Children who met the recommendations of PA had altered gut microbiota compositions. Whether this translates to a reduced risk of obesity or associated metabolic diseases is still unclear.