Light-activated cytotoxic compounds from Malaysian microorganisms for photodynamic therapy of cancer

Photodynamic therapy (PDT) is a promising cancer treatment which involves activation of a photosensitizing drug with light to produce reactive oxygen species that kill tumors without causing damage to unirradiated normal tissues. To date, only Photofrin^®, Foscan^® and Levulan^® have been approved for clinical treatment of cancer. Tropical habitats such as those found in Malaysia are attractive sources of new therapeutic compounds as tremendous chemical diversity is found in a large number of plants, animals, marine- and micro-organisms. In our screening program for novel photosensitizers from nature, colorful strains of fungi (from Aspergillus and Penicillium genus) and bacteria (including actinomycetes and photosynthetic bacteria) were collected from various habitats in Peninsular Malaysia, such as coastal soil, peat soil, marine sponges and wastewater ponds. Methanolic extracts from a total of 85 different species were evaluated with a short-term cell viability assay for photo-cytotoxicity, where a promyelocytic leukemia cell-line, HL60 incubated with 20 μg/ml of extracts was irradiated with 9.6 J/cm² of a broad spectrum light. Two of these extracts, one from Rhodobacter sphaeroides (PBUM003) and one from Rhodopseudomonas palustris (PBUM001) showed moderate to strong photo-cytotoxicity. Subsequent bioassay guided isolation of the PBUM001 extract yielded known photosensitisers that are based on bacteriochlorophyll-a by comparing their molecular weight data, HPLC profiles and UV–vis absorption spectra with literature values, thereby demonstrating the validity of our screening approach.     

Protocols for nuclei isolation and nuclear protein extraction from the resurrection plant Xerophyta viscosa for proteomic studies

The plant nucleus is an important subcellular organelle but the isolation of pure and enriched nuclei from plants and subsequent extraction of nuclear proteins for proteomic studies is challenging. Here, we present protocols for nuclei isolation and nuclear protein extraction from the resurrection plant, Xerophyta viscosa, and show optimization and modification of the most critical steps.     

A peptide derived from the putative transmembrane domain in the tail region of E. coli toxin hemolysin E assembles in phospholipid membrane and exhibits lytic activity to human red blood cells: Plausible implications in the toxic activity of the protein

Hemolysin E (HlyE), a pore-forming protein-toxin and a potential virulence factor of Escherichia coli, exhibits cytotoxic activity to mammalian cells. However, very little is known about how the different individual segments contribute in the toxic activity of the protein. Toward this end, the role of a 33-residue segment comprising the amino acid region 88 to 120, which contains the putative transmembrane domain in the tail region of HlyE has been addressed in the toxic activity of the protein-toxin by characterizing the related wild type and mutant peptides and the whole protein. Along with the 33-residue wild type peptide, H-88, two mutants of the same size were synthesized; in one mutant a conserved valine at 89^th position was replaced by aspartic acid and in the other both glycine and valine at the 88^th and 89^th positions were substituted by aspartic acid residues. These mutations were also incorporated in the whole toxin HlyE. Results showed that only H-88 but not its mutants permeabilized both lipid vesicles and human red blood cells (hRBCs). Interestingly, while H-88 exhibited a moderate lytic activity to human red blood cells, the mutants were not active. Drastic reduction in the depolarization of hRBCs and hemolytic activity of the whole toxin HlyE was also observed as a result of the same double and single amino acid substitution in it. The results indicate an important role of the amino acid segment 88-120, containing the putative transmembrane domain of the tail region of the toxin in the toxic activity of hemolysin E.     

Fear and everyday life in rural Nepal

This paper analyses fear in a village in central Nepal during the ‘People’s War’. Spanning the years from 1999 to 2008, the paper illustrates how the different phases of the insurgency and individual circumstances resulted in people’s relationship with fear changing over time. By presenting a chronological analysis of fear, the authors draw attention to the interrelationship between fear, temporality and sociality and show that fear is always contextually situated, differently experienced through time and related to personal circumstances. Villagers had strongly developed coping strategies which they drew upon to support themselves and decrease their fear. Some people, however, suffered such a degree of structural violence that experiencing fear was seen as a privilege. Others denied their fear as part of their performance of manliness while others coped by ridiculing fear. Although a certain amount of suspicion and mistrust lingered, most people recovered from the impact of chronic fear. They fully returned to their field and forest work as well as their previous social activities following the peace agreement of 2006.     

Purification of lipase derived from Burkholderia pseudomallei with alcohol/salt-based aqueous two-phase systems

Alcohol/salt-based aqueous two-phase systems (ATPSs) were used to recover lipase derived from Burkholderia pseudomallei (B. pseudomallei). Nine biphasic systems, comprised of an alcohol-based top phase (ethanol, 2-propanol and 1-propanol) and a salt-based bottom phase (ammonium sulfate, potassium phosphate and sodium citrate), were evaluated for their effectiveness in lipase recovery. The stability of lipase in each of the solutions was tested, and phase diagrams were constructed for each system. The optimum partition efficiency for the purification of lipase was obtained in an ATPS of 16% (w/w) 2-propanol and 16% (w/w) phosphate in the presence of 4.5% (w/v) NaCl. The purified lipase had a purification factor of 13.5 and a yield of 99%.     

Probabilistic Risk Assessment of Polycyclic Aromatic Hydrocarbons (PAHs) in Produced Water

The produced water extracted during oil and gas production includes formation water, injected water, small volumes of condensed water, and any chemical added during the oil/water separation process. Produced water contains both organic and inorganic constituents, and several studies have been conducted in the past to assess their risk. The toxicity and persistence of polycyclic aromatic hydrocarbons (PAHs) in produced water is of particular environmental concern, but there are very few studies on human health risk assessment from PAHs of produced water. This article summarizes the results of a conservative human health risk assessment approach for PAHs in produced water discharges to the marine environment. Due to the absence of available toxicity data for PAHs, the cancer slope factors were determined by using the relative potency factors (RPF) and Toxicity Equivalency Factors (TEF). Using the concentration distribution factors, the maximum cancer risks to humans were predicted in the range of 4.07 × 10^?7 to 2.95 × 10^?6. The 95th-percentile values show that the risks are well within the acceptable limits.     

Usefulness and efficiency of formalin-fixed paraffin-embedded specimens from laryngeal squamous cell carcinoma in HPV detection by IHC and PCR/DEIA

The use of formalin-fixed paraffin-embedded (FFPE) tissues for HPV DNA detection by PCR from biopsy materials is not entirely clear in retrospective studies. The aim of our study was to evaluate the usefulness and efficiency of FFPE tissues from laryngeal cancer (LSCC) in HPV detection by immunohistochemistry reaction (IHC) and PCR-DNA enzyme immunoassay method (PCR/DEIA) and to compare with HPV detection from DFT. HPV-DNA was amplified from 54 FFPE tissues from LSCC specimens by the short PCR fragment (SPF10) primer set using PCR/DNA method and monoclonal anti Human Papillomavirus antibodies in IHC. In the same patients 54 specimens were collected and immediately deep-frozen and stored at (-70°C) to (-80°C). All the FFPE and deep-frozen tissue (DFT) specimens were positive for β-globin amplification. HPV was detected by two methods (SPF10 PCR/DEIA and IHC) in 14 (25.92%) out of 54 specimens from FFPE. Significant differences were found between the HPV detection using PCR/DEIA method and IHC method in FFPE tissues. The comparative analysis of the 54 samples after assuming PCR method in FFPE tissues showed accuracy of 92.6%, sensitivity of 90.5% and specificity of 93.9%. The FFPE tissues method has high sensitivity, specificity and accuracy when used to detect HPV DNA by PCR reaction and it is comparable to DFT results. DNA quality of FFPE samples is adequate and it can be used in HPV-DNA detection and in retrospective studies on LSCC.     

A highly sensitive and specific multiplex PCR assay for simultaneous detection of Vibrio cholerae,Vibrio parahaemolyticus and Vibrio vulnificus

Aims: To develop an effective multiplex PCR for simultaneous and rapid detection of Vibrio cholerae, Vibrio vulnificus and Vibrio parahaemolyticus, the three most important Vibrio species that can cause devastating health hazards among human. Methods and Results: Species‐specific PCR primers were designed based on toxR gene for V. cholerae and V. parahaemolyticus, and vvhA gene for V. vulnificus. The multiplex PCR was validated with 488 Vibrio strains including 322 V. cholerae, 12 V. vulnificus, and 82 V. parahaemolyticus, 20 other Vibrio species and 17 other bacterial species associated with human diseases. It could detect the three target bacteria without any ambiguity even among closely related species. It showed good efficiency in detection of co‐existing target species in the same sample. The detection limit of all the target species was ten cells per PCR tube. Conclusions: Specificity and sensitivity of the multiplex PCR is 100% each and sufficient for simultaneous detection of these potentially pathogenic Vibrio species in clinical and environmental samples. Significance and Impact of the Study: This simple, rapid and cost‐effective method can be applicable in a prediction system to prevent disease outbreak by these Vibrio species and can be considered as an effective tool for both epidemiologist and ecologist.