Role of law enforcement response and microbial forensics in investigation of bioterrorism

The risk and threat of bioterrorism and biocrime have become a large concern and challenge for governments and society to enhance biosecurity. Law enforcement plays an important role in assessing and investigating activities involved in an event of bioterrorism or biocrime. Key to a successful biosecurity program is increased awareness and early detection of threats facilitated by an integrated network of responsibilities and capabilities from government, academic, private, and public assets. To support an investigation, microbial forensic sciences are employed to analyze and characterize forensic evidence with the goal of attribution or crime scene reconstruction. Two different molecular biology-based assays – real time polymerase chain reaction (PCR) and repetitive element PCR – are described and demonstrate how molecular biology tools may be utilized to aid in the investigative process. Technologies relied on by microbial forensic scientists need to be properly validated so that the methods used are understood and so that interpretation of results is carried out within the limitations of the assays. The three types of validation are preliminary, developmental, and internal. The first is necessary for rapid response when a threat is imminent or an attack has recently occurred. The latter two apply to implementation of routinely used procedures.     

Root-associated fungi of Arabidopsis thaliana and Microthlaspi perfoliatum

Root-associated fungi, with the focus on endophytic species, were isolated from healthy Arabidopsis thaliana and Microthlaspi perfoliatum plants collected at different locations in Germany. A large number of fungal taxa were discovered with a small-scale approach. This provides additional evidence that root-associated and endophytic fungi are common in Brassicaceae. The most prevalent genera associated with A. thaliana roots were Trichoderma and Fusarium, while the roots of M. perfoliatum were dominated by different species of Fusarium and Penicillium. Differences in species composition and richness might be due to preferences and life-cycle of the two plant species. Strains of endophyte species that did not have closely related species in GenBank searches and those already known as root endophytes were chosen for preliminary co-cultivation experiments using germinating host plants on agar medium to observe effects on plant growth and health. Under these conditions several fungal isolates had an adverse effect on plant growth and health, especially on Arabidopsis thaliana. Some isolates did not adversely affect biomass during initial plant growth, while they altered the shoot-root ratio in favour of the shoot, especially in Microthlaspi perfoliatum. These strains are promising candidates for future research on endophytes as they might have some effects in Brassicaceae that are similar to mycorrhizal fungi. They are also promising candidates for investigating interactions with their host plants.     

Whole-Genome Analysis of Exserohilum rostratum from an Outbreak of Fungal Meningitis and Other Infections

Exserohilum rostratum was the cause of most cases of fungal meningitis and other infections associated with the injection of contaminated methylprednisolone acetate produced by the New England Compounding Center (NECC). Until this outbreak, very few human cases of Exserohilum infection had been reported, and very little was known about this dematiaceous fungus, which usually infects plants. Here, we report using whole-genome sequencing (WGS) for the detection of single nucleotide polymorphisms (SNPs) and phylogenetic analysis to investigate the molecular origin of the outbreak using 22 isolates of E. rostratum retrieved from 19 case patients with meningitis or epidural/spinal abscesses, 6 isolates from contaminated NECC vials, and 7 isolates unrelated to the outbreak. Our analysis indicates that all 28 isolates associated with the outbreak had nearly identical genomes of 33.8 Mb. A total of 8 SNPs were detected among the outbreak genomes, with no more than 2 SNPs separating any 2 of the 28 genomes. The outbreak genomes were separated from the next most closely related control strain by ∼136,000 SNPs. We also observed significant genomic variability among strains unrelated to the outbreak, which may suggest the possibility of cryptic speciation in E. rostratum.     

The Dose-Response Effects of Consuming High Fructose Corn Syrup-Sweetened Beverages on Hepatic Lipid Content and Insulin Sensitivity in Young Adults

Increased hepatic lipid content and decreased insulin sensitivity have critical roles in the development of cardiometabolic diseases. Therefore, our objective was to investigate the dose-response effects of consuming high fructose corn syrup (HFCS)-sweetened beverages for two weeks on hepatic lipid content and insulin sensitivity in young (18–40 years) adults (BMI 18–35 kg/m²). In a parallel, double-blinded study, participants consumed three beverages/day providing 0% (aspartame: n = 23), 10% (n = 18), 17.5% (n = 16), or 25% (n = 28) daily energy requirements from HFCS. Magnetic resonance imaging for hepatic lipid content and oral glucose tolerance tests (OGTT) were conducted during 3.5-day inpatient visits at baseline and again at the end of a 15-day intervention. During the 12 intervening outpatient days participants consumed their usual diets with their assigned beverages. Significant linear dose-response effects were observed for increases of hepatic lipid content (p = 0.015) and glucose and insulin AUCs during OGTT (both p = 0.0004), and for decreases in the Matsuda (p = 0.0087) and Predicted M (p = 0.0027) indices of insulin sensitivity. These dose-response effects strengthen the mechanistic evidence implicating consumption of HFCS-sweetened beverages as a contributor to the metabolic dysregulation that increases risk for nonalcoholic fatty liver disease and type 2 diabetes.     

Genetics of pancreatitis.

There was some recent progress in the understanding of genetic risk factors in chronic pancreatitis. Due to this progress some of the traditional views of the subject will change. Today, genetic risk factors are attributed a much more important role that in the past. The frequency and strength of mutations were higher than expected. Strong variants were the rare autosomal-dominant mutations N29I and R122H of PRSS1 (cationic trypsinogen) and homozygous N34S of SPINK1 (pancreatic secretory trypsin inhibitor). Other mutations (heterozygous N34S, CFTR) were of lower relevance but still mediate a higher risk than alcohol consumption. The course of genetically determined pancreatitis is rather mild. In the long term pancreas cancer was found in some patients but apart from non-smoking no adequate prophylactic strategy is available up to now.     

A novel exocrine protein associated with pancreas transplantation in humans.

After pancreas transplantation, signs of acute pancreatitis are found in the grafted tissue. Pancreatic juice secreted from this organ was analyzed by gel electrophoresis. Initially, the pattern of secretory proteins was similar to that of the juice collected from normal individuals, but high levels of albumin were present. Within 2 days after reperfusion of the grafted pancreas, proteins of molecular weights 17,000-20,000 increased remarkably. Separation by two-dimensional gel electrophoresis showed that this was largely due to the appearance of new a protein, present neither in the juice collected immediately after reperfusion nor in normal pancreatic juice. After transfer onto nitrocellulose, this additional protein was detected by antibodies directed against the recently described rat "pancreatitis-associated protein." Maximal amounts of approximately 7.5% of total secretory protein were found 5 days after transplantation. The concentration of the protein decreased in the further course but was still detectable after 45 days. The isoelectric point (7.1) and the molecular weight (17,500) were similar to those of the rat protein. It is concluded that after inflammation induced by pancreatic transplantation, the human pancreas secretes high amounts of a protein not present in normal juice. Because of its similarities to the rat pancreatitis-associated protein it is designated human pancreatitis-associated protein.