Stabilization of Metals in Subsurface by Biopolymers: Laboratory Drainage Flow Studies

Environmental contamination with heavy metals and radionuclides remains a major problem worldwide. The current clean-up methodologies are based on energy-intensive engineering processes, which are disruptive and costly. A new universal technology targeted for the permanent enclosure and fixation of nuclear and other extreme hazardous metallic wastes in subsurface sites is needed. Such technology will be useful in treating contamination at many sites in the U.S., with specific applications to Department of Energy (DOE) sites. Biopolymers are potential tools for such an innovative technology. Biopolymers have repeated sequences, and therefore provide ample opportunity for chemical reactions with metals, soil particles, and other biopolymers. They also have the additional ability of creating cross-linking interpenetrating networks that can encapsulate the contaminants. Based on this concept, in the present work five biopolymers (xanthan, chitosan, polyhydroxy butyrate, guar gum, polyglutamic acid) were investigated for potential use in the stabilization of metals in the subsurface. The effects of these biopolymers (used alone and in combinations) on soil characteristics (permeability, shear strength) and their metal uptake ability have been studied using laboratory drainage flow systems. Biopolymer solutions were run through the experimental sandpack columns, followed by copper solution and leaching agents (distilled water and hydrochloric acid). The permeability and shear strength of sand were evaluated. Copper uptake capacity of each biopolymer and combination of biopolymers was also studied along with subsequent leaching. All biopolymers tested improved sand characteristics (by decreasing permeability and increasing shear strength) and had good metal uptake ability (60–90%) with relatively low leachability (10–22%). While biopolymers used alone were more efficient in metal uptake, the combination of two biopolymers (xanthan and chitosan) had an increasing plugging effect. These results show the potential of using biopolymers in subsurface metal stabilization.     

Potential role of estrogen receptor alpha (ERalpha) phosphorylated at Serine118 in human breast cancer in vivo

Post-translational modifications of proteins are known to be important in protein activity and ERalpha is known to be phosphorylated at multiple sites within the protein. The exact function of site-specific phosphorylation in ERalpha is unknown, although several hypotheses have been developed using site-directed mutagenesis and cell culture models. Targeting the ERalpha at the level of such post-translational modification pathways would be a new and exciting approach to endocrine therapy in breast cancer, but adequate knowledge is lacking with regard to the relevance of site-specific phosphorylation in ERalpha in human breast cancer in vivo. Recently, antibodies to P-Serine(118)-ERalpha and P-Serine(167)-ERalpha, two major sites of phosphorylation in ERalpha, have become available and some in vivo data are now available to complement studies in cells in culture. However, the in vivo data are somewhat contradictory and limited by the small cohorts used and the lack of standard well-characterized reagents and protocols.     

Hell's Gate globin I: an acid and thermostable bacterial hemoglobin resembling mammalian neuroglobin

Hell's Gate globin I (HGbI), a heme-containing protein structurally homologous to mammalian neuroglobins, has been identified from an acidophilic and thermophilic obligate methanotroph, Methylacidiphilum infernorum. HGbI has very high affinity for O(2) and shows barely detectable autoxidation in the pH range of 5.2-8.6 and temperature range of 25-50°C. Examination of the heme pocket by X-ray crystallography and molecular dynamics showed that conformational movements of Tyr29(B10) and Gln50(E7), as well as structural flexibility of the GH loop and H-helix, may play a role in modulating its ligand binding behavior. Bacterial HGbI's unique resistance to the sort of extreme acidity that would extract heme from any other hemoglobin makes it an ideal candidate for comparative structure-function studies of the expanding globin superfamily.     

Optimization of pyrene degradation by white-rot fungus Pleurotus pulmonarius F043 and characterization of its metabolites

Pleurotus pulmonarius F043, a fungus collected from tropical rain forest, was used to degrade pyrene, a four-rings polycyclic aromatic hydrocarbons (PAHs), in a mineral medium broth. A maximum degradation rate of pyrene (90 %) was occurred at pH 3 and the lowest degradation rate was found in the culture at pH 10 (2 %). More than 90 % pyrene degradation was achieved at pH ranged from 3 to 5, whereas the degradation rate significantly declined when the pH was >5. The degradation of pyrene increased from 2 to 96 % when the temperature rose from 4 to 25 °C. When the temperature was increased to 60 °C resulting the lowest degradation rate into 7 %. Among the agitation rates tested, 120 rpm was the best with 95 % degradation, followed by 100 rpm (90 %). The optimum agitation range for pyrene degradation by P. pulmonarius F043 was 100–120 rpm. Among all the concentrations tested, 0.5 % Tween 80 was the best with 98 % degradation, followed by 1 % Tween 80 (90 %). The optimum concentration of Tween 80 for pyrene degradation by P. pulmonarius F043 was 0.5–1 %. The degradation rate decreased, while the concentration of Tween 80 was increased. The metabolic product was found during degradation process through the identification of gentisic acid by TLC, UV-Spectrophotometer, and GC–MS.     

Characterization of VRC01, a potent and broadly neutralizing anti‐HIV mAb,produced in transiently and stably transformed tobacco

The proposed clinical trial in Africa of VRC01, a potent broadly neutralizing antibody (bNAb) capable of neutralizing 91% of known HIV‐1 isolates, raises concerns about testing a treatment which will be too expensive to be accessible by the most important target population, the poor in under‐developed regions such as sub‐Saharan Africa. Here, we report the expression of VRC01 in plants as an economic alternative to conventional mammalian‐cell‐based production platforms. The heavy and light chain genes of VRC01 were cloned onto a single vector, pTRAk.2, which was transformed into Nicotiana benthamiana or Nicotiana tabacum using transient and stable expression production systems respectively. VRC01 has been successfully expressed transiently in plants with expression level of approximately 80 mg antibody/kg; stable transgenic lines expressing up to 100 mg antibody/kg were also obtained. Plant‐produced VRC01 from both systems showed a largely homogeneous N‐glycosylation profile with a single dominant glycoform. The binding kinetics to gp120 IIIB (approximately 1 nm ), neutralization of HIV‐1 BaL or a panel of 10 VRC01‐sensitive HIV‐1 Env pseudoviruses of VRC01 produced in transient and stable plants were also consistent with VRC01 from HEK cells.     

Metabotropic glutamate receptor 1 mediates melanocyte transformation via transactivation of insulin‐like growth factor 1 receptor

Our laboratory previously described the oncogenic properties of metabotropic glutamate receptor 1 (mGluR1) in melanocytes. mGluR1 transformed immortalized mouse melanocytes in vitro and induced vigorous tumor formation in vivo. Subsequently, we observed the activation of PI3K/AKT in mGluR1‐mediated melanocytic tumorigenesis in vivo. In particular, we identified AKT2 being the predominant isoform contributing to the activation of AKT. Suppression of Grm1 or AKT2 using an inducible Tet‐R siRNA system resulted in a 60 or 30% reduction, respectively, in in vivo tumorigenesis. We show that simultaneous downregulation of Grm1 plus AKT2 results in a reduction of approximately 80% in tumor volumes, suggesting that both mGluR1 and AKT2 contribute to the tumorigenic phenotype in vivo. The discrepancy between the mild in vitro transformation characteristics and the aggressive in vivo tumorigenic phenotypes of these stable mGluR1‐melanocytic clones led us to investigate the possible involvement of other growth factors. Here, we highlight a potential crosstalk network between mGluR1 and tyrosine kinase, insulin‐like growth factor 1 receptor (IGF‐1R).     

Long-Term Sorption of Metals Is Similar among Plastic Types: Implications for Plastic Debris in Aquatic Environments

Concerns regarding plastic debris and its ability to accumulate large concentrations of priority pollutants in the aquatic environment led us to quantify relationships between different types of mass-produced plastic and metals in seawater. At three locations in San Diego Bay, we measured the accumulation of nine targeted metals (aluminum, chromium, manganese, iron, cobalt, nickel, zinc, cadmium and lead) sampling at 1, 3, 6, 9 and 12 months, to five plastic types: polyethylene terephthalate (PET), high-density polyethylene (HDPE), polyvinyl chloride (PVC), low-density polyethylene (LDPE), and polypropylene (PP). Accumulation patterns were not consistent over space and time, and in general all types of plastic tended to accumulate similar concentrations of metals. When we did observe significant differences among concentrations of metals at a single sampling period or location in San Diego Bay, we found that HDPE typically accumulated lesser concentrations of metals than the other four polymers. Furthermore, over the 12-month study period, concentrations of all metals increased over time, and chromium, manganese, cobalt, nickel, zinc and lead did not reach saturation on at least one plastic type during the entire 12-month exposure. This suggests that plastic debris may accumulate greater concentrations of metals the longer it remains at sea. Overall, our work shows that a complex mixture of metals, including those listed as priority pollutants by the US EPA (Cd, Ni, Zn and Pb), can be found on plastic debris composed of various plastic types.     

Functional and Molecular Surveillance of Helicobacter pylori Antibiotic Resistance in Kuala Lumpur

Background

Helicobacter pylori is the etiological agent for diseases ranging from chronic gastritis and peptic ulcer disease to gastric adenocarcinoma and primary gastric B-cell lymphoma. Emergence of resistance to antibiotics possesses a challenge to the effort to eradicate H. pylori using conventional antibiotic-based therapies. The molecular mechanisms that contribute to the resistance of these strains have yet to be identified and are important for understanding the evolutional pattern and selective pressure imposed by the environment.

Methods and Findings

H. pylori was isolated from 102 patients diagnosed with gastrointestinal diseases, who underwent endoscopy at University Malaya Medical Centre (UMMC). The isolates were tested for their susceptibility on eleven antibiotics using Etest. Based on susceptibility test, 32.3% of the isolates were found to have primary metronidazole resistance; followed by clarithromycin (6.8%) and fluoroquinolones (6.8%). To further investigate the resistant strains, mutational patterns of gene rdxA, frxA, gyrA, gyrB, and 23S rRNA were studied. Consistent with the previous reports, metronidazole resistance was prevalent in the local population. However, clarithromycin, fluoroquinolone and multi-drug resistance were shown to be emerging. Molecular patterns correlated well with phenotypic data. Interestingly, multi-drug resistant (MDR) strains were found to be associated with higher minimum inhibitory concentration (MIC) than their single-drug resistant (SDR) counterparts. Most importantly, clarithromycin-resistant strains were suggested to have a higher incidence for developing multi-drug resistance.

Conclusion

Data from this study highlighted the urgency to monitor closely the prevalence of antibiotic resistance in the Malaysian population; especially that of clarithromycin and multi-drug resistance. Further study is needed to understand the molecular association between clarithromycin resistance and multi-drug resistance in H. pylori. The report serves a reminder that a strict antibiotic usage policy is needed in Malaysia and other developing countries (especially those where H. pylori prevalence remained high).     

Determination of the effect of acetylation of specific lysine residues in human growth hormone on its affinity for somatogenic receptors by an affinity selection technique

A technique is described to study the effect of acetylation of individual lysine residues in peptide hormones on the affinity for their receptors, and is illustrated for the case of human growth hormone (hGH) binding to somatogenic receptors. The hGH was partially acetylated with high specific activity [3H]-acetic anhydride and the product ([3H]-Ac-hGH) was incubated with solubilised affinity-purified somatogenic receptors (from male rat liver) in the presence and absence of excess unlabelled hGH. The receptor-bound and unbound labelled hormone were separated by gel filtration and subjected to HPLC tryptic peptide mapping after the addition of cold carrier Ac-hGH. Peaks of [3H] radioactivity were assigned to peptides corresponding to the acetylation of specific lysine residues in the hGH sequence by amino acid analysis and sequencing. Comparison of the relative intensities of corresponding [3H] peaks in the peptide maps of added receptor, bound and unbound [3H]-Ac-hGH, enabled the relative receptor-binding potencies of different acetylated hGH species to be determined. Acetylation of lysine 168 or 172 in hGH greatly decreases its receptor-binding affinity, acetylation of lysine 115 probably causes a minor decrease, whereas acetylation of lysines 38, 70, and the N-terminal amino group have no appreciable effect. Acetylation of lysine 140 causes a significant increase in receptor-binding affinity.