Ultrastructural studies of alterations induced by microwaves in Toxocara canis eggs: prophylactic interest

The ultrastructure of Toxocara canis eggs is described before and after exposure to microwaves. The morphology of normal eggs is compared to that of eggs from other helminths. Following treatment, the complete disorganization of the surface structure of the shell and the loss of much turgidity of the egg are observed. The destruction of the internal structure is most marked in the center of the egg and is associated with the disappearance of some layers of the shell. In addition, there is substantial damage to the synthesis apparatus (ribosomes, endoplasmic reticulum, lipid cisternae). An explanation based on the specific action of microwaves and micro-overheating is proposed, and the prophylactic use of this technique is considered.     

A SCA7 CAG/CTG repeat expansion is stable in Drosophila melanogaster despite modulation of genomic context and gene dosage

CAG and CTG repeat expansions are the cause of at least a dozen inherited neurological disorders. In these so-called "dynamic mutation" diseases, the expanded repeats display dramatic genetic instability, changing in size when transmitted through the germline and within somatic tissues. As the molecular basis of the repeat instability process remains poorly understood, modeling of repeat instability in model organisms has provided some insights into potentially involved factors, implicating especially replication and repair pathways. Studies in mice have also shown that the genomic context of the repeat sequence is required for CAG/CTG repeat instability in the case of spinocerebellar ataxia type 7 (SCA7), one of the most unstable of all CAG/CTG repeat disease loci. While most studies of repeat instability have taken a candidate gene approach, unbiased screens for factors involved in trinucleotide repeat instability have been lacking. We therefore attempted to use Drosophila melanogaster to model expanded CAG repeat instability by creating transgenic flies carrying trinucleotide repeat expansions, deriving flies with SCA7 CAG90 repeats in cDNA and genomic context. We found that SCA7 CAG90 repeats are stable in Drosophila, regardless of context. To screen for genes whose reduced function might destabilize expanded CAG repeat tracts in Drosophila, we crossed the SCA7 CAG90 repeat flies with various deficiency stocks, including lines lacking genes encoding the orthologues of flap endonuclease-1, PCNA, and MutS. In all cases, perfect repeat stability was preserved, suggesting that Drosophila may not be a suitable system for determining the molecular basis of SCA7 CAG repeat instability.     

CTCF cis-Regulates Trinucleotide Repeat Instability in an Epigenetic Manner: A Novel Basis for Mutational Hot Spot Determination

At least 25 inherited disorders in humans result from microsatellite repeat expansion. Dramatic variation in repeat instability occurs at different disease loci and between different tissues; however, cis-elements and trans-factors regulating the instability process remain undefined. Genomic fragments from the human spinocerebellar ataxia type 7 (SCA7) locus, containing a highly unstable CAG tract, were previously introduced into mice to localize cis-acting “instability elements,” and revealed that genomic context is required for repeat instability. The critical instability-inducing region contained binding sites for CTCF—a regulatory factor implicated in genomic imprinting, chromatin remodeling, and DNA conformation change. To evaluate the role of CTCF in repeat instability, we derived transgenic mice carrying SCA7 genomic fragments with CTCF binding-site mutations. We found that CTCF binding-site mutation promotes triplet repeat instability both in the germ line and in somatic tissues, and that CpG methylation of CTCF binding sites can further destabilize triplet repeat expansions. As CTCF binding sites are associated with a number of highly unstable repeat loci, our findings suggest a novel basis for demarcation and regulation of mutational hot spots and implicate CTCF in the modulation of genetic repeat instability.     

Bacterial IMPDH gene used for the selection of mammalian cell transfectants

Stable cell transfection is used for the expression of exogenous genes or cDNAs in eukaryotic cells. Selection of these transfectants requires a dominant selectable marker. A variety of such markers has been identified and is currently in use. However, many of these are not suitable for all cell types or require unique conditions. Here we describe a simple and versatile dominant selectable marker that involves bacterial IMP dehydrogenase (IMPDH), an enzyme essential for the replication of mammalian and bacterial cells. Although IMPDH is evolutionarily conserved, the bacterial enzyme is orders of magnitude more resistant to the toxic effect of the drug mycophenolic acid, which is an IMPDH inhibitor. We have demonstrated that transfection of human, monkey or Chinese hamster cell lines with an expression vector containing bacterial IMPDH and mycophenolic acid treatment resulted in the selection of colonies with a strikingly increased resistance to mycophenolic acid toxicity. Analysis of cells derived from these colonies indicated that the acquisition of this resistance was associated with bacterial IMPDH protein expression. As a proof of principle, we showed that mammalian cell transfection with a bicistronic IMPDH/GFP expression vector and mycophenolic acid treatment can be used to successfully select transfectants that express the fluorescent protein. These results indicate that bacterial IMPDH is a practical dominant selectable marker that can be used for the selection of transfectants that express exogenous genes or cDNAs in mammalian cells.     

Kinetics of influenza A virus infection in humans

Currently, little is known about the viral kinetics of influenza A during infection within an individual. We utilize a series of mathematical models of increasing complexity, which incorporate target cell limitation and the innate interferon response, to examine influenza A virus kinetics in the upper respiratory tracts of experimentally infected adults. The models were fit to data from an experimental H1N1 influenza A/Hong Kong/123/77 infection and suggest that it is important to include the eclipse phase of the viral life cycle in viral dynamic models. Doing so, we estimate that after a delay of approximately 6 h, infected cells begin producing influenza virus and continue to do so for approximately 5 h. The average lifetime of infected cells is approximately 11 h, and the half-life of free infectious virus is approximately 3 h. We calculated the basic reproductive number, R(0), which indicated that a single infected cell could produce approximately 22 new productive infections. This suggests that antiviral treatments have a large hurdle to overcome in moderating symptoms and limiting infectiousness and that treatment has to be initiated as early as possible. For about 50% of patients, the curve of viral titer versus time has two peaks. This bimodal behavior can be explained by incorporating the antiviral effects of interferon into the model. Our model also compared well to an additional data set on viral titer after experimental infection and treatment with the neuraminidase inhibitor zanamivir, which suggests that such models may prove useful in estimating the efficacies of different antiviral therapies for influenza A infection.     

PAQ: Partition Analysis of Quasispecies

MOTIVATION: The complexities of genetic data may not be accurately described by any single analytical tool. Phylogenetic analysis is often used to study the genetic relationship among different sequences. Evolutionary models and assumptions are invoked to reconstruct trees that describe the phylogenetic relationship among sequences. Genetic databases are rapidly accumulating large amounts of sequences. Newly acquired sequences, which have not yet been characterized, may require preliminary genetic exploration in order to build models describing the evolutionary relationship among sequences. There are clustering techniques that rely less on models of evolution, and thus may provide nice exploratory tools for identifying genetic similarities. Some of the more commonly used clustering methods perform better when data can be grouped into mutually exclusive groups. Genetic data from viral quasispecies, which consist of closely related variants that differ by small changes, however, may best be partitioned by overlapping groups. RESULTS: We have developed an intuitive exploratory program, Partition Analysis of Quasispecies (PAQ), which utilizes a non-hierarchical technique to partition sequences that are genetically similar. PAQ was used to analyze a data set of human immunodeficiency virus type 1 (HIV-1) envelope sequences isolated from different regions of the brain and another data set consisting of the equine infectious anemia virus (EIAV) regulatory gene rev. Analysis of the HIV-1 data set by PAQ was consistent with phylogenetic analysis of the same data, and the EIAV rev variants were partitioned into two overlapping groups. PAQ provides an additional tool which can be used to glean information from genetic data and can be used in conjunction with other tools to study genetic similarities and genetic evolution of viral quasispecies.     

Snakebites in Two Rural Districts in Lao PDR: Community-Based Surveys Disclose High Incidence of an Invisible Public Health Problem

BackgroundThe Lao PDR (Laos) is one of the least developed countries in Asia with an estimated 25% of the population living in poverty. It is the habitat of some highly venomous snakes and the majority of the population earns their living from agricultural activities. Under these circumstances the incidence of snakebites is expected to be high.MethodsTwo cross-sectional, community-based surveys were performed in Champone and Phin district, Savannakhet province, Lao PDR to estimate snakebite incidence. Multistage random sampling was used. In the first stage approximately 40% of all villages in each district were randomly selected. In the second stage 33% of all households in each village were randomly chosen. Members of the selected households were interviewed about snakebites during the previous 12 months.ResultsThirty-five of 9856 interviewees reported a snakebite in a 12 month period in Champone district and 79 of 7150 interviewees in Phin district. The estimated incidence is 355 snakebites per 100,000 persons per year and 1105 per 100,000 in Champone and Phin district respectively. All snakebite victims received treatment by traditional healers or self-treatment at home and nobody went to a hospital. Incidence of snakebites, calculated on the basis of hospital records of 14 district hospitals and Savannakhet provincial hospital, ranged from 3 to 14 cases per 100,000 persons per year between 2012 and 2014.ConclusionIncidence of snakebites is high in rural communities in Laos with significant regional differences. Poverty most likely contributes significantly to the higher number of snakebites in Phin district. Hospital statistics profoundly underestimates snakebite incidence, because the majority of snakebite victims receive only treatment by traditional healers or self-treatment in their village. There is an urgent need to train medical staff and students in management of snakebite patients and make snake antivenom available to cope effectively with this important public health problem in order to prevent fatalities and disabilities.     

Public health response to an anthrax attack: an evaluation of vaccination policy options

A discrete-time, deterministic, compartmental model was developed and analyzed to provide insight into how the use of anthrax vaccine before or after a large-scale attack can reduce casualties. The model accounts for important response and protection factors such as antibiotic and vaccine efficacy, the protective effects of buildings, the timing of emergency response, and antibiotic adherence and vaccine coverage in the population prior to the attack. The relative benefit of pre- versus post-exposure vaccination is influenced by the timing of the post-exposure antibiotic distribution campaign as well as assumptions of antibiotic adherence. The results indicate that, regardless of which vaccination policy is adopted, a rapid and effective post-attack medical response has a large impact on the number of lives that can be saved by a post-exposure prophylaxis (PEP) campaign. A sensitivity analysis of the model indicates that uncertainty in medical efficacy and the time to initiate a PEP campaign are the model parameters that have the greatest impact on the number of predicted deaths. It is shown that for each day that a mass prophylaxis campaign is delayed, more casualties and deaths result than for each day that the completion of the campaign is delayed.