Combinatorial treatment with ß-glucanase enzyme and chlorhexidine induces cysticidal effects in Acanthamoeba cyst.

Acanthamoeba cysts have a cellulose cell wall made up of a solid layer of ß-glucan, which confers resistance to the dormant phase of this microorganism. The ability of Acanthamoeba to change to this dormant phase causes difficulties in treating its infection at the cyst stage as compared to the trophozoite stage. Therefore, targeting cyst total mortality can help to prevent re-infection in patients. To ensure cysticidal treatment, a ß-glucanase enzyme was introduced in vitro to the Acanthamoeba cyst, followed by a chlorhexidine solution treatment. ß-glucanase enzyme and chlorhexidine dose-response analysis was performed based on cell wall integrity measurement. The treatment was also performed on human corneal epithelial cells to confirm the safety of the treatment in vitro. The surface morphology of the cysts was observed using scanning electron microscopy (SEM), while the protein alterations were determined using 1D protein analysis. The interaction of the ß-glucanase enzyme with cellulose linkages was investigated based on molecular dosimetry. Incubation of the cyst for 24 h at 8.75 units/ml of ß-glucanase followed by 0.88 µg/ml of chlorhexidine resulted in a substantial reduction in the total chlorhexidine used, which made it safer for human corneal epithelial cells. Ultrastructural changes revealed the reduction of the thickness in ectocyst and endocyst layers with the loss of the internal structure of the cyst. After combination treatment of chlorhexidine and ß-glucanase, a decrease in the cyst protein from the size of 37 to 25 kDa was observed. The enzyme-substrate interaction validated these results based on molecular docking between 1,4-ß-D-glucan and 1,4- ß-D-xylan with the ß-glucanase enzyme. In silico analysis revealed that two catalytic glutamate residues (Glu160 and Glu267) are essential to catalysing the hydrolytic reaction. Molecular dynamic simulation analysis revealed that both ligands formed stable interactions throughout the simulation. This work concludes that the enzymatic approach combined with chlorhexidine is a novel and effective technique for ensuring the cysticidal effects against the Acanthamoeba cyst. The interaction of the chlorhexidine and ß-glucanase enzyme on the surface of the cyst of amoeba resulted in the ecto-and endo cyst layer being damaged and confirmed the cysticidal effects.

Inhibitory effect of thiourea derivatives on the growth of blue-green algae.

High nutrient loading in aquatic environment has become the main causative of harmful algae blooms (HABs) in water resources particularly pond, lake and river. HABs are mostly dominated by microalgae derived from the group of blue-green algae which are capable of releasing harmful toxins. Therefore, this study aims to investigate the inhibitory effects of thiourea derivatives on the growth of such blue-green algae. Thiourea derivatives have been proven to exhibit antifungal and antibacterial effects. However, there is still limited study had been conducted on the effect of thiourea derivatives toward blue-green algae species in recent years. In this research, a species of blue-green algae from Kenyir Lake, Terengganu, Malaysia was successfully isolated using morphological characters and molecularly identified as Synechoccus elongatus. Four new thiourea derivative compounds were also successfully synthesised. The compounds were designed with variation on different R-substitution group and characterised using Nuclear Magnetic Resonance (NMR) to confirm their molecular structure. Those compounds were characterised as 1-Benzyl-3-(3,5-dimethoxy-benzoyl)-thiourea (C1), 1-(3-Chloro-benzyl)-3-(3,5-dimethoxy-benzoyl)-thiourea (C2), 1-(3,5-Dimethoxy-benzoyl)-3-(3-methyl-benzyl)-thiourea (C3) and 1-(3,5-Dimethoxy-benzoyl)-3-(3-trifluoromethyl-benzyl)-thiourea (C4). For the inhibition assessment,S. elongatus were treated with C1-C4 for 5 day at concentration of 2, 5, 10 and 20 µg/ml, respectively. C3 compound showed the highest inhibition percentage with 98% of inhibition after 5 days treatment. By using Bradford method, protein extraction of S. elongatus was conducted at the highest inhibition percentage. Protein concentration of treated species was observed with 3.28 µg/ml as compared to protein concentration of control with 6.48 µg/ml. This result indicated the reduction of protein content after the treatment. Protein band pattern was identified intensed after the treatment SDS PAGE was carried out. The thiourea derivatives compound proved to have successfully inhibited the growth of blue-green algae. Hence, further study should be carried out to ensure the compound can be practically utilized in the pond and in natural environment.

Food Consumption, Developmental Time, and Protein Profile of the Digestive System of the Red Palm Weevil, Rhynchophorus ferrugineus (Coleptera: Dryophthoridae) Larvae Reared on Three Different Diets.

The red palm weevil (RPW), Rhynchophorus ferrugineus Olivier (Coleoptera: Dryophthoridae) is one of the most dangerous pests of major cultivated palms including coconut, oil palm, and sago. The larval stage of the weevil causes the most destruction of the palms as it completely destroys the palm cabbage. In this study, the larvae were given three different diets-coconut cabbage, oil palm cabbage, and sago stem, under laboratory conditions for food consumption and developmental time experiment. The protein profiles of the digestive systems of the larvae fed on these three diets were also determined. Although the coconut diet was the most consumed by RPW larvae compared to oil palm and sago diets, the growth rate of RPW larvae on oil palm diet was however significantly shorter than those on the coconut and sago diets: the RPW only need 1 mo and 9 d to complete the larval duration. Proteins profiling of eight 2-DE gel protein spots that range 50-20 kDa were identified by mass spectrometry sequence analysis. Based on the Matrix Science Software, the most dominant protein was cationic trypsin. However, based on the NCBI BLAST tool, aminopeptidase N was the most dominant enzyme. This finding can lead to the development of pest control strategies based on the antinutritional protease inhibitors as potential biocontrol agents. Urgent action to find effective control methods should be taken seriously as this weevil is presumed to be one of the serious pests of oil palm industry in Malaysia.