Potential role for saccharopine reductase in swainsonine metabolism in endophytic fungus, Undifilum oxytropis

Locoweed plants in the southwestern United States often harbour a slow-growing endophytic fungus, Undifilum oxytropis (Phylum: Ascomycota; Order: Pleosporales), which produces a toxic alkaloid, swainsonine. Consumption of U. oxytropis by grazing animals induces a neurological disorder called locoism for which the toxic alkaloid swainsonine has been reported to be the causal agent. Little is known about the biosynthetic pathway of swainsonine in endophytic fungi, but previous studies on non-endophytic ascomycetous fungi indicate that pipecolic acid and saccharopine are key intermediates. We have used degenerate primers, Rapid amplification of cDNA ends (RACE)-PCR and inverse PCR to identify the gene sequence of U. oxytropis saccharopine reductase. To investigate the role of this gene product in swainsonine metabolism, we have developed a gene deletion system for this slow-growing endophyte based on our recently established transformation protocol. A strain of U. oxytropis lacking saccharopine reductase had decreased levels of saccharopine and lysine along with increased accumulation of pipecolic acid and swainsonine. Thus, saccharopine reductase influences the accumulation of swainsonine and its precursor, pipecolic acid, in U. oxytropis.     

A translation-coupling DNA cassette for monitoring protein translation in Escherichia coli

A major challenge to using heterologous expression in metabolic engineering experiments is the inability to quickly dissect experiments that have failed at the stage of translating mRNA. While many methods of detecting proteins exist, methods that detect untagged proteins at low levels are limited. Here, we describe a method to quickly determine whether Escherichia coli is capable of expressing the product of any target gene by coupling translation of a target gene to a detectable response gene. A translational coupling cassette was designed to encode a mRNA sequence that forms a secondary structure in the absence of translation and contains the translational start sequence of a detectable response gene. The translational coupling method was successfully tested with fluorescent proteins and antibiotic resistance markers. Only when the target gene was fully translated was the response observed. Further characterization demonstrated that translational coupling functions at both low and high levels of expression and that the response signal is proportional to the amount of target gene product. The translational coupling system was used to determine that a large multi-domain enzyme was not actively translated in E. coli, to isolate the translation problems to the C-terminal domains, and to optimize conditions for expressing a codon-optimized sequence variant.     

Starvation causes disturbance in amino acid and fatty acid metabolism in Diporeia

The benthic amphipod Diporeia spp. was once the predominant macroinvertebrate in deep, offshore regions of the Laurentian Great Lakes. However, since the early 1990s, Diporeia populations have steadily declined across the area. It has been hypothesized that this decline is due to starvation from increasing competition for food with invasive dreissenid mussels. In order to gain a better understanding of the changes in Diporeia physiology during starvation, we applied two-dimensional gas chromatography coupled with time of flight mass spectrometry (GCXGC/TOF-MS) for investigating the responses in Diporeia metabolome during starvation. We starved Diporeia for 60 days and collected five organisms every 12 days for metabolome analyses. Upon arrival to the laboratory, organisms were flash frozen and served as control (day 0). We observed an increase in lipid oxidation and protein catabolism with subsequent declines of essential amino acids (proline, glutamine, and phenylalanine), down-regulation of glycerophospholipid and sphingolipid metabolism, and decreased polyunsaturated fatty acid abundance in nutritionally stressed Diporeia. Abundance of 1-Iodo-2-methylundecane, a metabolite closely related to insect pheromones, also declined with starvation. This research has further substantiated the applicability of GCXGC/TOF-MS as a research tool in the field of environmental metabolomics. The next step is to apply this new knowledge for evaluating nutritional status of feral Diporeia to elucidate the underlying cause(s) responsible for their decline in the Great Lakes.     

Exposure to heavy ion radiation induces persistent oxidative stress in mouse intestine

Ionizing radiation-induced oxidative stress is attributed to generation of reactive oxygen species (ROS) due to radiolysis of water molecules and is short lived. Persistent oxidative stress has also been observed after radiation exposure and is implicated in the late effects of radiation. The goal of this study was to determine if long-term oxidative stress in freshly isolated mouse intestinal epithelial cells (IEC) is dependent on radiation quality at a dose relevant to fractionated radiotherapy. Mice (C57BL/6J; 6 to 8 weeks; female) were irradiated with 2 Gy of γ-rays, a low-linear energy transfer (LET) radiation, and intestinal tissues and IEC were collected 1 year after radiation exposure. Intracellular ROS, mitochondrial function, and antioxidant activity in IEC were studied by flow cytometry and biochemical assays. Oxidative DNA damage, cell death, and mitogenic activity in IEC were assessed by immunohistochemistry. Effects of γ radiation were compared to (56)Fe radiation (iso-toxic dose: 1.6 Gy; energy: 1000 MeV/nucleon; LET: 148 keV/μm), we used as representative of high-LET radiation, since it's one of the important sources of high Z and high energy (HZE) radiation in cosmic rays. Radiation quality affected the level of persistent oxidative stress with higher elevation of intracellular ROS and mitochondrial superoxide in high-LET (56)Fe radiation compared to unirradiated controls and γ radiation. NADPH oxidase activity, mitochondrial membrane damage, and loss of mitochondrial membrane potential were greater in (56)Fe-irradiated mice. Compared to γ radiation oxidative DNA damage was higher, cell death ratio was unchanged, and mitotic activity was increased after (56)Fe radiation. Taken together our results indicate that long-term functional dysregulation of mitochondria and increased NADPH oxidase activity are major contributing factors towards heavy ion radiation-induced persistent oxidative stress in IEC with potential for neoplastic transformation.     

Biomarkers for clinical and incipient tuberculosis: performance in a TB-endemic country

Background

Simple biomarkers are required to identify TB in both HIV⁻TB⁺ and HIV⁺TB⁺ patients. Earlier studies have identified the M. tuberculosis Malate Synthase (MS) and MPT51 as immunodominant antigens in TB patients. One goal of these investigations was to evaluate the sensitivity and specificity of anti-MS and –MPT51 antibodies as biomarkers for TB in HIV⁻TB⁺ and HIV⁺TB⁺ patients from a TB-endemic setting. Earlier studies also demonstrated the presence of these biomarkers during incipient subclinical TB. If these biomarkers correlate with incipient TB, their prevalence should be higher in asymptomatic HIV⁺ subjects who are at a high-risk for TB. The second goal was to compare the prevalence of these biomarkers in asymptomatic, CD4⁺ T cell-matched HIV⁺TB⁻ subjects from India who are at high-risk for TB with similar subjects from US who are at low-risk for TB.

Methods and Results

Anti-MS and -MPT51 antibodies were assessed in sera from 480 subjects including PPD⁺ or PPD⁻ healthy subjects, healthy community members, and HIV⁻TB⁺ and HIV⁺TB⁺ patients from India. Results demonstrate high sensitivity (∼80%) of detection of smear-positive HIV⁻TB⁺ and HIV⁺TB⁺ patients, and high specificity (>97%) with PPD⁺ subjects and endemic controls. While ∼45% of the asymptomatic HIV⁺TB⁻ patients at high-risk for TB tested biomarker-positive, >97% of the HIV⁺TB⁻ subjects at low risk for TB tested negative. Although the current studies are hampered by lack of knowledge of the outcome, these results provide strong support for the potential of these biomarkers to detect incipient, subclinical TB in HIV⁺ subjects.

Conclusions

These biomarkers provide high sensitivity and specificity for TB diagnosis in a TB endemic setting. Their performance is not compromised by concurrent HIV infection, site of TB and absence of pulmonary manifestations in HIV⁺TB⁺ patients. Results also demonstrate the potential of these biomarkers for identifying incipient subclinical TB in HIV⁺TB⁻ subjects at high-risk for TB.     

Biological pretreatment of sugarcane trash for its conversion to fermentable sugars

Pretreatment of lignocellulosic biomasses, the first step in their conversion to utilizable molecules requires very high energy (steam and electricity), corrosion resistant high-pressure reactors and high temperatures. These severe conditions not only add to the cost component of the entire process but also lead to the loss of sugars to the side reactions. Microbial pretreatments have been reported to be associated with reducing the cost factors as well as the severities of the reactions. Eight bioagents, including fungi and bacteria, were screened for their pretreatment effects on sugarcane trash. They narrowed down the C:N ratio of trash from 108:1 to a varying range of approximately 42:1 to 60:1.The maximum drop in C:N ratio of 61% was observed using Aspergillus terreus followed by Cellulomonas uda (52%) and Trichoderma reesei and Zymomonas mobilis (49%). The bioagents helped in degradation of sugarcane trash by production of cellulases, the maximum being produced by A. terreus, (12 fold) followed by C. uda (10 fold), Cellulomonas cartae (9 fold) and Bacillus macerans (8 fold). The microbial pretreatment of trash rendered the easy accessibility of sugars for enzymatic hydrolysis, which can be directed for production of alcohol.