Resolving individuals contributing trace amounts of DNA to highly complex mixtures using high-density SNP genotyping microarrays

We use high-density single nucleotide polymorphism (SNP) genotyping microarrays to demonstrate the ability to accurately and robustly determine whether individuals are in a complex genomic DNA mixture. We first develop a theoretical framework for detecting an individual's presence within a mixture, then show, through simulations, the limits associated with our method, and finally demonstrate experimentally the identification of the presence of genomic DNA of specific individuals within a series of highly complex genomic mixtures, including mixtures where an individual contributes less than 0.1% of the total genomic DNA. These findings shift the perceived utility of SNPs for identifying individual trace contributors within a forensics mixture, and suggest future research efforts into assessing the viability of previously sub-optimal DNA sources due to sample contamination. These findings also suggest that composite statistics across cohorts, such as allele frequency or genotype counts, do not mask identity within genome-wide association studies. The implications of these findings are discussed.     

Comparing biology majors from large lecture classes with TA-facilitated laboratories to those from small lecture classes with faculty-facilitated laboratories

The teaching faculty for this course sought to address their own concerns about the quality of student learning in an impersonal large lecture biology class for majors, the difficulties in getting to know each student by name, and difficulties in soliciting answers and reactions from the students during the lecture. Questions addressed by this study were, Do active-learning activities in a small and personal lecture setting enhance student learning more than active-learning activities in large impersonal lectures? and Are students more satisfied with an educational experience in a small and personal lecture setting? Based on faculty perceptions of how they best relate to their students, the prediction was that the students in the experimental group with small lecture classes and increased direct contact with the teaching faculty would learn physiological principles better than the students in the control group in the large impersonal lecture portion of the course. One of the laboratory sections of this large enrollment biology course was randomly selected to be taught with separate small lectures by the teaching faculty. In addition, the teaching faculty participated in the laboratory with these students during their experiments correlated with the lecture material. The students in both groups were compared by pre- and posttests of physiological principles, final course grades, and class satisfaction surveys.     

Synthesis and SAR of highly potent dual 5-HT1A and 5-HT1B antagonists as potential antidepressant drugs

Novel 5-HT₁ autoreceptor ligands based on the N-4-aryl-piperazinyl-N′-ethyl-5,6,7,8-tetrahydropyrido[4′, 3′:4,5]thieno[2,3-d]pyrimidin-4(3H)-one core are described. Aiming at antidepressants with a novel mode of action our objective was to identify potent antagonists showing balanced affinities and high selectivity for the 5-HT_1A and 5-HT_1B receptors. Strategies for the development of dual 5-HT_1A and 5-HT_1B antagonists based on 1 and 2 as leads and the corresponding results are discussed. Isoquinoline analogue 33 displayed high affinity and an antagonistic mode of action for the 5-HT_1A and the 5-HT_1B receptors and was characterized further with respect to selectivity, electrically stimulated [³H]5-HT release and in vivo efficacy.     

Comprehensive proteomic analysis of Saccharomyces cerevisiae cell walls: identification of proteins covalently attached via glycosylphosphatidylinositol remnants or mild alkali-sensitive linkages

The cell wall of yeast contains proteins that are covalently bound to the glycan network. These cell wall proteins (CWPs) mediate cell-cell interactions and may be involved in cell wall biosynthesis. Using tandem mass spectrometry, we have identified 19 covalently bound CWPs of Saccharomyces cerevisiae. Twelve of them are shown for the first time to be covalently incorporated into the cell wall. The identified proteins include 12 predicted glycosylphosphatidylinositol-modified CWPs, all four members of the Pir protein family, and three additional proteins (Scw4p, Scw10p, and Tos1p) that are, like Pir proteins, connected to the cell wall glycan network via an alkali-sensitive linkage. However, Scw4p, Scw10p, and Tos1p do not contain internal repeat sequences shown to be essential for Pir protein incorporation and may represent a separate class of CWPs. Strikingly, seven of the identified proteins (Gas1p, Gas3p, Gas5p, Crh1p, Utr2p, Scw4p, and Scw10p) are classified as glycoside hydrolases. Phenotypic analysis of deletion mutants lacking the corresponding CWP-encoding genes indicated that most of them have altered cell wall properties, which reinforces the importance of the identified proteins for proper cell wall formation. In particular, gas1Delta and ecm33Delta were highly sensitive to Calcofluor White and high temperature, whereas gas1Delta, scw4Delta, and tos1Delta were highly resistant to incubation with beta-1,3-glucanase. The CWP identification method developed here relies on directly generating tryptic peptides from isolated cell walls and is independent of the nature of the covalent linkages between CWPs and cell wall glycans. Therefore, it will probably be equally effective in many other fungi.     

Changing desiccation tolerance of pea embryo protoplasts during germination

Protoplasts were isolated from pea (Pisum sativum L. cv. Alaska) embryonic axes during and after germination to determine whether the loss of desiccation tolerance in the embryos also occurs in the protoplasts. At all times studied, protoplast survival decreased as water content decreased; however, the sensitivity to dehydration was less when the protoplasts were isolated from embryos that were still desiccation-tolerant (12 h and 18 h of imbibition) than when protoplasts were derived from axes that were sensitive (24 h and 36 h of imbibition). The water content at which 50% of the population was killed (WC50) increased throughout germination and early seedling growth for both the intact tissue and the protoplasts derived from them. Prior to radicle emergence, protoplasts were less desiccation-tolerant than the intact axes; however, protoplasts isolated from radicles shortly after emergence had lower WC50s than the intact radicles. A comparison of protoplast survival after isolation and dehydration in either 500 mM sucrose/raffinose or 700 mM sucrose revealed no difference in tolerance except at 24 h of imbibition, when protoplasts treated in the more concentrated solution had improved tolerance of dehydration. Although intact epicotyls are generally more desiccation-tolerant than radicles, protoplasts isolated separately from epicotyls and radicles did not differ in tolerance. Collectively, these data suggest that protoplasts gradually lose desiccation tolerance during germination, as do the orthodox embryos from which they were derived. However, even prior to radicle emergence, protoplasts display a sensitivity to progressive dehydration that is similar to that shown by recalcitrant and ageing embryos.     

Electron microscopy in cell biology: integrating structure and function

Electron microscopy (EM) is at the highest-resolution limit of a spectrum of complementary morphological techniques. When combined with molecular detection methods, EM is the only technique with sufficient resolution to localize proteins to small membrane subdomains in the context of the cell. Recent procedural and technical developments have increasingly improved the power of EM as a cell-biological tool.     

Production of the bullous pemphigoid antigen 230 (BP230) by Saccharomyces cerevisiae and Pichia pastoris

BP230 is a cytoskeletal linker protein of 2649 amino acids originally identified as the target autoantigen in bullous pemphigoid, a potentially devastating autoimmune skin blistering disorder. To better define its function, we sought to generate recombinant forms of BP230 in both Saccharomyces cerevisiae and Pichia pastoris after cloning its entire cDNA. By immunoblot analysis, full-length BP230 was not found in extracts of P. pastoris, whereas minor amounts of degraded BP230 were detected in extracts of S. cerevisiae. In contrast, both S. cerevisiae and P. pastoris were able to produce the 770-amino acid COOH-terminal domain of BP230. Furthermore, the production level of the recombinant BP230 tail in S. cerevisiae was significantly higher than that observed in P. pastoris and that of endogenous BP230 in cultured human keratinocytes. Finally, 12 of 17 (71%) BP sera recognized the recombinant BP230 protein in yeast extracts. Our results indicate that S. cerevisiae occasionally constitutes a better tool for recombinant protein production than P. pastoris. Although both its large size and poor solubility limit production of BP230, the developed yeast system provides cellular fractions enriched in BP230 recombinant proteins that constitute useful tools for the diagnosis of bullous pemphigoid.