Bordetella pertussis CyaA-RTX subdomain requires calcium ions for structural stability against proteolytic degradation

Previously, the 126-kDa Bordetella pertussis CyaA pore-forming (CyaA-PF) domain expressed in Escherichia coli was shown to retain its hemolytic activity. Here, a 100-kDa RTX (Repeat-in-ToXin) subcloned fragment (CyaA-RTX) containing a number of putative calcium-binding repeats was further investigated. The recombinant CyaA-RTX protein, although expressed as a soluble form in a protease-deficient E. coli strain BL21(DE3)pLysS, was found to be highly sensitive to proteolytic degradation. Interestingly, the addition of calcium ions in a millimolar range into the CyaA-RTX preparation significantly prevented the degradation. Moreover, levels of proteolytic degradation were dependent on calcium concentrations, implying an important role for calcium-binding sites in the RTX subdomain for structural stability. Homology-based modeling of the repetitive blocks in the CyaA-RTX subdomain supports that this calcium-bound protein folds into a parallel β-roll structure with calcium ions acting as a structural stabilizing bridge.     

Fermentation and quality of yellow pigments from golden brown rice solid culture by a selected Monascus mutant

A single peak (λmax 370) yellow pigment-producing mutant derived from Monascus sp. TISTR 3179 was used for the pigment production in solid rice culture. Various factors affecting yellow tones were investigated. Hom-mali rice variety was the best amongst five Thai local varieties used for fungus culture. It was also better than corn, mungbean, soybean, potato, sweet potato, or cassava tubers. The moisture content and temperature were the key environmental factors affecting the color tones of creamy, tangerine, and golden brown rice solid cultures. The golden brown rice culture gave the highest yellow pigment concentration. Under an optimum room temperature of 28–32 °C, an initial moisture content of 42 %, and 7-day-old inoculum size of 2 % (v/w) the maximum yield at 2,224.63 A₃₇₀U/gdw of yellow pigment was produced. A mellow yellow powder at 550 A₃₇₀U/gdw could be obtained using spray-drying techniques. The powder had a moisture content of 5.15 %, a water activity value of 0.398, a hue angle of 73.70 ° (yellowish orange), high lightness (L*) of 74.63, color saturation (C*) of 28.97, a neutral pH of 7.42, 0.12 % acidity and solubility of 0.211 g/10 ml. It was noteworthy that the Chinese fresh noodle with spray-dried yellow powder showed no discoloration during 8-day storage.     

Fischoederius cobboldi: A scanning electron microscopy investigation of surface morphology of adult rumen fluke

Adults Fischoederius cobboldi are conical-shaped, concave ventrally and convex dorsally, measures about 8-10mm in length and 4-6mm in width across the mid section. Scanning electron microscopy (SEM) of entire body showed that the tegumental surface exhibits highly corrugation and transverse folds alternating with grooves and without spines. At higher magnification, the surface of each fold is further increased with a meshwork of ridges separated by irregular-sized pits. The ventral surface has more complex corrugations and invaginations than those of the dorsal surface of the body. Both anterior and posterior suckers have thick edges covered with transverse folds and appear spineless. The genital pore is located at the anterior one-third of the body. There are two types of sensory papillae on the surface: type 1 is bulbous in shape and nipple-like tips, measuring 10-15 μm in diameter at the base, and also type 2 is a similar shape and has short cilia on tips. These sensory papillae occur in large clusters, each having between 7 and 25 units depending on the region of the body. Clusters of papillae on the ventral surface and around the anterior suckers tend to be more abundant and larger in size. The dorsal side of the body exhibit similar surface features, but papillae appear less numerous and are smaller. Corrugations and invaginations of the dorsal aspect are also less extensive than those on the ventral surface of the body.     

Azaphilone pigments from a yellow mutant of the fungus Monascus kaoliang

Azaphilone pigments, monascusones A (1) and B (2), together with two known azaphilones, monascin (3) and FK17-P2b2 (4), were isolated from the CH2Cl2 extract of a yellow mutant of the fungus M. kaoliang grown on rice. Structures of the isolated compounds were elucidated by analyses of spectroscopic data. Monascusone A (1), the major metabolite of M. kaoliang, showed no antimalarial (against Plasmodium falciparum), antitubercular (against Mycobacterium tuberculosis H37Ra), and antifungal (toward Candida albicans) activities. Compound 1 exhibited no cytotoxicity against BC (breast cancer) and KB (human epidermoid carcinoma of cavity) cell lines.     

Production of vitamin B12 by immobilized cells of a propionic acid bacterium

Summary The ability of immobilized cells of propionic acid bacteria to form vitamin B₁₂ has been investigated. Propionibacterium arl AKU 1251 having a considerable activity to produce the vitamin was selected as a test organism among six strains of propionic acid bacteria tested. The whole cells were entrapped with urethane prepolymers, photo-crosslinkable resin prepolymers or several other materials such as -carrageenan, agar or sodium alginate, and their vitamin B₁₂ productivity was compared. Based on the criteria of the convenience of preparation and the stability of the cell-entrapping gels, a hydrophilic urethane prepolymer, PU-9, was employed as gel material. Satisfactory vitamin B₁₂ production was obtained when 5–10 g of wet cells precultured to the late exponential growth phase were entrapped with 1 g of the prepolymer. Addition of a suitable amount of cobaltous ion and of 5,6-dimethyl benzimidazole to the culture medium was effective for the production of the vitamin by the immobilized cells. The repeated use of the immobilized cells was successfully achieved when a suitable amount of cells were entrapped and allowed the proliferation of cells inside gel matrices.     

The First Rapid Assessment of Avoidable Blindness (RAAB) in Thailand

Background

The majority of vision loss is preventable or treatable. Population surveys are crucial for planning, implementation, and monitoring policies and interventions to eliminate avoidable blindness and visual impairments. This is the first rapid assessment of avoidable blindness (RAAB) study in Thailand.

Methods

A cross-sectional study of a population in Thailand age 50 years old or over aimed to assess the prevalence and causes of blindness and visual impairments. Using the Thailand National Census 2010 as the sampling frame, a stratified four-stage cluster sampling based on a probability proportional to size was conducted in 176 enumeration areas from 11 provinces. Participants received comprehensive eye examination by ophthalmologists.

Results

The age and sex adjusted prevalence of blindness (presenting visual acuity (VA) <20/400), severe visual impairment (VA <20/200 but ≥20/400), and moderate visual impairment (VA <20/70 but ≥20/200) were 0.6% (95% CI: 0.5–0.8), 1.3% (95% CI: 1.0–1.6), 12.6% (95% CI: 10.8–14.5). There was no significant difference among the four regions of Thailand. Cataract was the main cause of vision loss accounted for 69.7% of blindness. Cataract surgical coverage in persons was 95.1% for cut off VA of 20/400. Refractive errors, diabetic retinopathy, glaucoma, and corneal opacities were responsible for 6.0%, 5.1%, 4.0%, and 2.0% of blindness respectively.

Conclusion

Thailand is on track to achieve the goal of VISION 2020. However, there is still much room for improvement. Policy refinements and innovative interventions are recommended to alleviate blindness and visual impairments especially regarding the backlog of blinding cataract, management of non-communicative, chronic, age-related eye diseases such as glaucoma, age-related macular degeneration, and diabetic retinopathy, prevention of childhood blindness, and establishment of a robust eye health information system.     

Effects on haemolytic activity of single proline substitutions in the <Emphasis Type="Italic">Bordetella pertussis</Emphasis> CyaA pore-forming fragment

The recombinant Bordetella pertussis CyaA pore-forming (CyaA-PF) fragment was previously shown to be expressed separately in Escherichia coli as a soluble precursor that can be in vivo palmitoylated to exert haemolytic activity. In this study, PCR-based mutagenesis was employed to investigate the contributions to haemolysis of five predicted helices within the N-terminal hydrophobic region of the CyaA-PF fragment. Single proline substitutions were made for alanine near the centre of each predicted helix as a means of disrupting local secondary structure. All mutant proteins were over-expressed in E. coli as a 126-kDa soluble protein at levels comparable to the wild-type. Marked reductions in haemolytic activity against sheep erythrocytes of mutants, A510P, A538P, A583P and A687P pertaining to the putative helices 1_500–522, 2_529–550, 3_571–593 and 5_678–698, respectively, were observed. However, a slight decrease in haemolytic activity was found for the proline replacement in the predicted helix 4_602–627 (A616P). MALDI–TOF–MS and LC–MS–MS analyses verified the palmitoylation at Lys⁹⁸³ of all five mutants as identical to that of the CyaA-PF wild-type protein, indicating that toxin modification via this acylation was not affected by the mutations. Altogether, these results suggest that structural integrity of the predicted helices 1, 2, 3 and 5, but not helix 4, is important for haemolytic activity, particularly for the putative transmembrane helices 2 and 3 that might conceivably be involved in pore formation of the CyaA-PF fragment.     

Development of Mannosylated Liposomes Using Synthesized N-Octadecyl-d-Mannopyranosylamine to Enhance Gastrointestinal Permeability for Protein Delivery

The lysozyme (LZ)-entrapped mannosylated liposomes were prepared in this study by the use of N-octadecyl-D-mannopyranosylamine (SAMAN), which had been synthesized in-house and confirmed by characterization with FTIR and NMR. The reactant residues of synthesized SAMAN were found to be less than 1%. The mean sizes, zeta potentials, drug entrapment efficiencies, and loading capacities of all liposomal formulations were in the ranges of 234.7 to 431.0 nm, -10.97 to -25.80 mV, 7.52 to 14.10%, and 1.44 to 2.77%, respectively. The permeability of mannosylated LZ liposomes across Caco-2 cell monolayers was significantly enhanced to about 2.5- and 7-folds over those of conventional liposomes and solution, respectively, which might be due to the role of mannose receptor or mannose-binding protein on the intestinal enterocytes.     

Characterization of red sternum syndrome in mud crab farms from Thailand

Samples of abnormal mud crabs, Scylla serrata (Forsk?l, 1755) (Decapoda: Portunidae), were collected from crab farms in Samutsongkhram Province, Thailand. These crabs had hard carapaces, red chelipeds and joints, pale hepatopancreas, gills, and soft muscles. They were almost immobile and finally died. The haemolymph revealed three stages of the syndrome, namely orange, orange-white, and milky-white in colors. The haemolymph, integument, hepatopancreas, gills, abdominal and claw muscle, stomach, and heart were dissected and histologically examined using transmission and scanning electron microscopy. Closer examinations found infection with rod-, curve rod-, or coccus-shape bacteria with thin and thick cell walls in all investigated organs and haemolymph. Isolation of the microorganisms from the infected tissues of red sternum syndrome crabs resulted in five types of bacteria. No microorganism growth was observed in normal crabs. Interestingly, the types of isolated bacteria can be classified according to the severity of the disease. Additionally, the degree of bacterial infection found was consistent with the stages of the disease. It was postulated that the bacteria entered the crabs via the gills, and then migrated through circulating haemocytes, before reaching the internal organs.     

High level of soluble expression in <Emphasis Type="Italic">Escherichia coli</Emphasis> and characterisation of the CyaA pore-forming fragment from a <Emphasis Type="Italic">Bordetella pertussis</Emphasis> Thai clinical isolate

Bordetella pertussis adenylate cyclase toxin-haemolysin (CyaA) can permeabilise erythrocytes by forming lytic pores. Here, a gene segment encoding CyaA pore-forming (CyaA-PF) domain cloned from genomic DNA of B. pertussis Thai isolate was over-expressed in Escherichia coli as a 126-kDa soluble protein which cross-reacted with anti-RTX monoclonal antibody. By co-expressing with acyltransferase CyaC, the CyaA-PF protein was found palmitoylated at Lys⁹⁸³. Unlike E. coli lysate with the non-acylated form, the lysate containing acylated CyaA-PF exhibited high haemolytic activity against sheep erythrocytes. This study presents that the recombinant CyaA-PF protein comprising pore-forming domain can be expressed separately as soluble native-folded precursor that conserves at least part of its functionality.