Wendy K. Pogozelski

Dietary carbohydrate restriction as the first approach in diabetes management: Critical review and evidence base

The inability of current recommendations to control the epidemic of diabetes, the specific failure of the prevailing low-fat diets to improve obesity, cardiovascular risk, or general health and the persistent reports of some serious side effects of commonly prescribed diabetic medications, in combination with the continued success of low-carbohydrate diets in the treatment of diabetes and metabolic syndrome without significant side effects, point to the need for a reappraisal of dietary guidelines. The benefits of carbohydrate restriction in diabetes are immediate and well documented. Concerns about the efficacy and safety are long term and conjectural rather than data driven. Dietary carbohydrate restriction reliably reduces high blood glucose, does not require weight loss (although is still best for weight loss), and leads to the reduction or elimination of medication. It has never shown side effects comparable with those seen in many drugs. Here we present 12 points of evidence supporting the use of low-carbohydrate diets as the first approach to treating type 2 diabetes and as the most effective adjunct to pharmacology in type 1. They represent the best-documented, least controversial results. The insistence on long-term randomized controlled trials as the only kind of data that will be accepted is without precedent in science. The seriousness of diabetes requires that we evaluate all of the evidence that is available. The 12 points are sufficiently compelling that we feel that the burden of proof rests with those who are opposed.

A weighted insertion-deletion stacked pair thermodynamic metric for DNA codes

Thermodynamic distance functions are important components in the construction of DNA codes and DNA codewords are structural and information building blocks in biomolecular computing and other biotechnical applications that employ DNA hybridization assays. We introduce new metrics for DNA code design that capture key aspects of the nearest neighbor thermodynamic model for hybridized DNA duplexes. One version of our metric gives the maximum number of stacked pairs of hydrogen bonded nucleotide base pairs that can be present in any secondary structure in a hybridized DNA duplex without pseudoknots. We introduce the concept of (t-gap) block isomorphic subsequences to describe new string metrics that are similar to the weighted Levenshtein insertion-deletion metric. We show how our new distances can be calculated by a generalization of the folklore longest common subsequence dynamic programming algorithm. We give a Varshamov-Gilbert like lower bound on the size of some of codes using our distance functions as constraints. We also discuss software implementation of our DNA code design methods.

Development and initial characterization of xenomitochondrial mice

Xenomitochondrial mice harboring trans-species mitochondria on a Mus musculus domesticus (MD) nuclear background were produced. We created xenomitochondrial ES cell cybrids by fusing Mus spretus (MS), Mus caroli (MC), Mus dunni (Mdu), or Mus pahari(MP) mitochondrial donor cytoplasts and rhodamine 6-G treated CC9.3.1 or PC4 ES cells. The selected donor backgrounds reflected increasing evolutionary divergence from MD mice and the resultant mitochondrial–nuclear mismatch targeted a graded respiratory chain defect. Homoplasmic (MS, MC, Mdu, and MP) and heteroplasmic (MC) cell lines were injected into MD ova, and liveborn chimeric mice were obtained (MS/MD 18 of 87, MC/MD 6 of 46, Mdu/MD 31 of 140, and MP/MD l of 9 founder chimeras, respectively). Seven MS/MD, 1 MC/MD, and 11 Mdu/MD chimeric founder females were mated with wild-type MD males, and 18 of 19 (95%) were fertile. Of fertile females, only one chimeric MS/MD (1% coat color chimerism) and four chimeric Mdu/MD females (80–90% coat color chimerism) produced homoplasmic offspring with low efficiency (7 of 135; 5%). Four male and three female offspring were homoplasmic for the introduced mitochondrial backgrounds. Three male and one female offspring proved viable. Generation of mouse lines using additional female ES cell lineages is underway. We hypothesize that these mice, when crossbred with neurodegenerative-disease mouse models, will show accelerated age-related neuronal loss, because of their suboptimal capacity for oxidative phosphorylation and putatively increased oxidative stress.

The mitochondrial genome sequence of Mus terricolor: Comparison with Mus musculus domesticus and implications for xenomitochondrial mouse modeling

Knowledge of the mitochondrial DNA (mtDNA) sequence of divergent murine species is critical from both a phylogenetic perspective and in understanding nuclear-mitochondrial interactions, particularly as the latter influences our xenocybrid models of mitochondrial disease. To this end, the sequence of the mitochondrial genome of the murine species Mus terricolor (formerly Mus dunni) is reported and compared with the published sequence for the common laboratory mouse Mus musculus domesticus strain C57BL/6J. These species are of interest because xenomitochondrial cybrid mice were created that harbor M. terricolor mtDNA in a M. m. domesticus nuclear background. Although the total of 1763 nucleotide substitutions represents striking heterogeneity, the majority of these are silent, leading to highly conserved protein sequences with only 159 amino acid differences. Moreover, 58% of these amino acid differences represented conservative substitutions. All of the tRNA genes and rRNA genes have homology of 91% or greater. The control region shows the greatest heterogeneity, as expected, with 85% homology overall. Regions of 100% homology were found for Conserved Sequence Block I, Conserved Sequence Block III and the L-strand origin of replication. Complex I genes showed the greatest degree of difference among protein-coding genes with amino acid homology of 91-97% among the seven mitochondrial genes. Complexes III and IV genes show high homology ranging from 98-100%. From these data, complex I differences appear most critical for the viability of M. m. domesticus: M. terricolor cybrids. Moreover, the sequence information reported here should be useful in identifying critical regions for mitochondrial transfer between species, for furthering the understanding of mitochondrial dynamics and pathology in transmitochondrial organisms, and for the study of Mus genus origins.

Quantitative Assessment of the Contribution of Clustered Damage to DNA Double-Strand Breaks Induced by 60Co Gamma Rays and Fission Neutrons

The induction of DNA strand breaks by fission neutrons was studied in aqueous plasmid (pBR322) DNA under aerobic conditions for a wide range of hydroxyl radical (*OH) scavenger concentrations and was compared to the induction of strand breaks by 6OCo gamma rays. Strand breaks were measured using agarose gel electrophoresis coupled with sensitive 32P-based phosphor imaging. Yields are reported for DNA single-strand breaks (SSBs) and double-strand breaks formed linearly with dose (alphaDSBs). The fraction of alphaDSBs that were dependent on the multiply damaged site (MDS) or clustered damage mechanism was also calculated using a model. G values for SSBs and alphaDSBs declined with increasing *OH scavenging capacity. However, with increasing *OH scavenging capacities, the decrease in yields of strand breaks for fission neutrons was not as pronounced as for gamma rays. The percentage of alphaDSBs for gamma rays was dependent on *OH scavenging capacity, appearing negligible at low scavenging capacities but increasing at higher scavenging capacities. In contrast, fission neutrons induced high percentages of alphaDSBs that were approximately independent of *OH scavenging capacity. The levels of alphaDSBs formed by the MDS mechanism after exposure to fission neutrons are consistent with the expected distinctive features of high-LET energy deposition events and track structure. The results also confirm observations made by others that even for low-LET radiation, the MDS mechanism contributes significantly to DNA damage at cell-like scavenging conditions.

New t-Gap Insertion-Deletion-Like Metrics for DNA Hybridization Thermodynamic Modeling

We discuss the concept of t-gap block isomorphic subsequences and use it to describe new abstract string metrics that are similar to the Levenshtein insertion-deletion metric. Some of the metrics that we define can be used to model a thermodynamic distance function on single-stranded DNA sequences. Our model captures a key aspect of the nearest neighbor thermodynamic model for hybridized DNA duplexes. One version of our metric gives the maximum number of stacked pairs of hydrogen bonded nucleotide base pairs that can be present in any secondary structure in a hybridized DNA duplex without pseudoknots. Thermodynamic distance functions are important components in the construction of DNA codes, and DNA codes are important components in biomolecular computing, nanotechnology, and other biotechnical applications that employ DNA hybridization assays. We show how our new distances can be calculated by using a dynamic programming method, and we derive a Varshamov-Gilbert-like lower bound on the size of some of codes using these distance functions as constraints. We also discuss software implementation of our DNA code design methods.

Experimental validation of DNA sequences for DNA computing: use of a SYBR green I assay

In developing hybridization-based DNA computing methods, DNA codes must be created that behave as predicted; otherwise computing errors can result. Here we describe the experimental validation of two DNA codes, each containing 16-nucleotide strands designed to hybridize only with their complements and not with themselves or with any other strands in the set. Code I was constructed simply to restrict potential for cross-hybridized (CH) secondary structure. Code II was constructed using the software SynDCode, incorporating nearest-neighbor thermodynamics and generalizations of the Levenshtein edit distance. Every combination of strands was tested for potential to mispair, both in individual pairings and in pools. Since the strands are designed to be linked together in a long bit string, we also tested end-to-end junctions of Code II strands. Hybridization was examined by measuring fluorescence as a function of temperature in the presence of SYBR Green I, a dye whose fluorescence increases exponentially when bound to double-stranded DNA. This method shows promise as a means for rapid experimental validation of large numbers of sequences.

PCR Optimization for Detection of Transgene Integration

The use of the polymerase chain reaction (PCR) for the identification of transgenic founder mice or their offspring is outlined. Numerous sources of information are available dealing with the theory, performance, and optimization of PCR analysis in a variety of applications. This brief overview is intended to address some of the general considerations for PCR analysis and some of the particular concerns when using this technique to detect transgene integration in experimental animals (especially laboratory mice).