Helical integrity and microsolvation of transmembrane domains from Flaviviridae envelope glycoproteins

Charged and polar amino acids in the transmembrane domains of integral membrane proteins can be crucial for protein function and also promote helix-helix association or protein oligomerization. Yet, our current understanding is still limited on how these hydrophilic amino acids are efficiently translocated from the Sec61/SecY translocon into the cell membrane during the biogenesis of membrane proteins. In hepatitis C virus, the putative transmembrane segments of envelope glycoproteins E1 and E2 were suggested to heterodimerize via a Lys-Asp ion-pair in the host endoplasmic reticulum. Therefore in this work, we carried out molecular dynamic simulations in explicit lipid bilayer and solvent environment to explore the stability of all possible bridging ion-pairs using the model of H-segment helix dimers. We observed that, frequently, several water molecules penetrated from the interface into the membrane core to stabilize the charged and polar pairs. The hydration time and amount of water molecules in the membrane core depended on the position of the charged residues as well as on the type of ion-pairs. Similar microsolvation events were observed in simulations of the putative E1-E2 transmembrane helix dimers. Simulations of helix monomers from other members of the Flaviviridae family suggest that these systems show similar behaviors. Thus this study illustrates the important contribution of water microsolvation to overcome the unfavorable energetic cost of burying charged and polar amino acids in membrane lipid bilayers. Also, it emphasizes the novel role of bridging charged or polar interactions stabilized by water molecules in the hydrophobic lipid bilayer core that has an important biological function for helix dimerization in several envelope glycoproteins from the family of Flaviviridae viruses.     

Expression Analysis of Fatty Acid Biosynthetic Pathway Genes during Interactions of Oil Palm (Elaeis guineensis Jacq.) with the Pathogenic Ganoderma boninense and Symbiotic Trichoderma harzianum Fungal Organisms

The fatty acid (FA) signaling pathway is emerging as an important mechanism in plant responses during interactions with microbial organisms. For a comprehensive evaluation of key FA biosynthetic pathway genes during interactions of oil palm (Elaeis guineensis Jacq.) with the pathogenic Ganoderma boninense and symbiotic Trichoderma harzianum fungal organisms, a lane-based array analysis of gene expression in artificially inoculated oil palm seedlings was performed. The results obtained demonstrated that acetyl-CoA carboxylase (ACC), β-ketoacyl-ACP synthases (KAS) II and III, palmitoyl-ACP thioesterase (PTE), oleoyl-ACP thioesterase (OTE) and glycerol-3-phosphate acyltransferase (ACT) showed identical responses in root and leaf tissues for the same fungi. The expression of these genes was up-regulated in both root and leaf tissues at 21 days post-inoculation (dpi) during interaction of oil palm with G. boninense. Thereafter, production of physical symptoms occurred at 42 and 63 dpi concomitantly with suppression of expression of these genes. An increase in the expression level of these genes was observed in both tissues at 3–63 dpi, which correlated with the colonization of roots and promotion of plant growth by T. harzianum. These data suggest that FA biosynthetic pathway genes are involved in the defense response of oil palm to infection. Identical plant responses by FA biosynthetic pathway genes may lead to enhanced resistance against G. boninense and could be a useful marker to contribute towards early detection of infection. The distinct expression profile during symbiotic interaction demonstrated its role in plant resistance mechanisms and growth promotion by T. harzianum.     

A comparative genomic analysis of the alkalitolerant soil bacterium Bacillus lehensis G1

Bacillus lehensis G1 is a Gram-positive, moderately alkalitolerant bacterium isolated from soil samples. B. lehensis produces cyclodextrin glucanotransferase (CGTase), an enzyme that has enabled the extensive use of cyclodextrin in foodstuffs, chemicals, and pharmaceuticals. The genome sequence of B. lehensis G1 consists of a single circular 3.99 Mb chromosome containing 4017 protein-coding sequences (CDSs), of which 2818 (70.15%) have assigned biological roles, 936 (23.30%) have conserved domains with unknown functions, and 263 (6.55%) have no match with any protein database. Bacillus clausii KSM-K16 was established as the closest relative to B. lehensis G1 based on gene content similarity and 16S rRNA phylogenetic analysis. A total of 2820 proteins from B. lehensis G1 were found to have orthologues in B. clausii, including sodium–proton antiporters, transport proteins, and proteins involved in ATP synthesis. A comparative analysis of these proteins and those in B. clausii and other alkaliphilic Bacillus species was carried out to investigate their contributions towards the alkalitolerance of the microorganism. The similarities and differences in alkalitolerance-related genes among alkalitolerant/alkaliphilic Bacillus species highlight the complex mechanism of pH homeostasis. The B. lehensis G1 genome was also mined for proteins and enzymes with potential viability for industrial and commercial purposes.     

Diversity of the cassiicolin gene in Corynespora cassiicola and relation with the pathogenicity in Hevea brasiliensis

Corynespora cassiicola is an important plant pathogenic Ascomycete causing the damaging Corynespora Leaf Fall (CLF) disease in rubber tree (Hevea brasiliensis). A small secreted glycoprotein named cassiicolin was previously described as an important effector of C. cassiicola. In this study, the diversity of the cassiicolin-encoding gene was analysed in C. cassiicola isolates sampled from various hosts and geographical origins. A cassiicolin gene was detected in 47 % of the isolates, encoding up to six distinct protein isoforms. In three isolates, two gene variants encoding cassiicolin isoforms Cas2 and Cas6 were found in the same isolate. A phylogenetic tree based on four combined loci and elucidating the diversity of the whole collection was strongly structured by the toxin class, as defined by the cassiicolin isoform. The isolates carrying the Cas1 gene (toxin class Cas1), all grouped in the same highly supported clade, were found the most aggressive on two rubber tree cultivars. Some isolates in which no Cas gene was detected could nevertheless generate moderate symptoms, suggesting the existence of other yet uncharacterized effectors. This study provides a useful base for future studies of C. cassiicola population biology and epidemiological surveys in various host plants.     

CD4+CD25hiCD127low Regulatory T Cells Are Increased in Oral Squamous Cell Carcinoma Patients

Regulatory T cells (Tregs), a subset of CD4⁺ T cells plays a pivotal role in regulating the immune system. An increase in Treg numbers enables cancer progression by dampening the immune system and allowing tumor cells to evade immune detection and destruction. An increase in Treg numbers and expression of inhibitory cytokines including TGF-β and IL-10 are mechanisms by which Tregs exert their immune suppressive function. However, the presence of Tregs and inhibitory cytokines in oral cancer patients is still unclear. In this study, the presence of circulating Tregs in 39 oral cancer patients and 24 healthy donors was examined by studying the presence of the CD4⁺CD25^hiCD127^low cell population in their peripheral blood mononuclear cells using flow cytometry. Serum levels of TGF-β and IL-10 were measured by ELISA. T cell subsets of OSCC patients were found to differ significantly from healthy donors where a decrease in CD8⁺ cytotoxic T cells and an increase in Tregs (CD4⁺CD25^hiCD127^low) were observed. Further, the ratio of CD8⁺ T cells/Tregs was also decreased in patients compared to healthy donors. The presence of Tregs was accompanied by a decrease in IL-10 but not TGF-β secretion in OSCC patients when compared to donors; in addition, the analysis also revealed that an increased presence of Tregs was accompanied by better patient survival. Amongst OSCC patients, smokers had significantly higher levels of TGF-β. It is apparent that the immune system is compromised in OSCC patients and the characterization of the Treg subpopulation could form a basis for improving our understanding of the perturbations in the immune system that occur during OSCC tumorigenesis.     

A Case-Control Study of Risk Factors for Bovine Brucellosis Seropositivity in Peninsular Malaysia

Bovine brucellosis was first reported in Peninsular Malaysia in 1950. A subsequent survey conducted in the country revealed that the disease was widespread. Current knowledge on the potential risk factors for brucellosis occurrence on cattle farms in Malaysia is lacking. Therefore, we conducted a case-control study to identify the potential herd-level risk factors for bovine brucellosis occurrence in four states in the country, namely Kelantan, Pahang, Selangor and Negeri Sembilan. Thirty-five cases and 36 controls of herds were selected where data on farm management, biosecurity, medical history and public health were collected. Multivariable logistic regression identified that Brucella seropositive herds were more likely to; have some interaction with wildlife (OR 8.9, 95% CI = 1.59–50.05); originated from farms where multiple species such as buffalo/others (OR 41.8, 95% CI = 3.94–443.19) and goat/sheep (OR 8.9, 95%Cl = 1.10–71.83) were reared, practice extensive production system (OR 13.6, 95% CI 1.31–140.24) and have had episodes of abortion in the past (OR 51.8, 95% CI = 4.54–590.90) when compared to seronegative herds. Considering the lack of information on the epidemiology of bovine brucellosis in peninsular Malaysia and absence of information on preventing the inception or spread of the disease, this report could contribute to the on-going area-wise national brucellosis eradication program.     

Unsteady Boundary Layer Flow and Heat Transfer of a Casson Fluid past an Oscillating Vertical Plate with Newtonian Heating

In this paper, the heat transfer effect on the unsteady boundary layer flow of a Casson fluid past an infinite oscillating vertical plate with Newtonian heating is investigated. The governing equations are transformed to a systems of linear partial differential equations using appropriate non-dimensional variables. The resulting equations are solved analytically by using the Laplace transform method and the expressions for velocity and temperature are obtained. They satisfy all imposed initial and boundary conditions and reduce to some well-known solutions for Newtonian fluids. Numerical results for velocity, temperature, skin friction and Nusselt number are shown in various graphs and discussed for embedded flow parameters. It is found that velocity decreases as Casson parameters increases and thermal boundary layer thickness increases with increasing Newtonian heating parameter.     

Medium optimization for chitinase production from Trichoderma virens using central composite design

Medium development for chitinase production by Trichoderma virens was first carried out using conventional method of one-factor-at-a-time. The medium was further optimized using Central Composite Design in which response surface was generated later from the derived model. An experimental design of four variables including various initial pH values, chitin, ammonium sulphate, and methanol concentrations were created using Design Expert® Software, Version 6.0. The design consists of 30 experiments, which include 6 replicates at center points. The optimal value for each variable are 3.0 g/L, chitin; 0.1 g/L, ammonium sulphate; 0.4% (v/v), methanol; and initial pH, 4.0 with predicted chitinase activity of 0.1495 U/mL. These predicted parameters were tested in the laboratory and the final chitinase activity obtained was 0.1471 U/mL, which is almost reaching the predicted value. The optimal medium design showed an improvement of chitinase activity of 80.9% compared to activity obtained from the original Absidia medium composition.     

In-vitro antioxidant,in-vivo anti-inflammatory,and acute toxicity study of Indonesian propolis capsule from Tetragonula sapiens

Propolis is widely used as traditional medicine since ancient times. It was necessary to conduct the pre-clinical study because of its relevant curative properties. This study aimed to investigate in-vitro antioxidant, standardize quality parameters, study acute toxicity, and determine in-vivo anti-inflammatory. Three spectrophotometric methods were used to determine antioxidant activity. The standardization includes physical, chemical, and microbiological evaluation. Furthermore, an acute toxicity test was conducted using 20 female Sprague Dawley (SD) strain rats divided into 4 groups with different dose of propolis. The in vivo anti-inflammatory test was carried out using the carrageenan induction method on rats' soles. A total of 36 female SD rats were classified into 6 groups as follows, Group normal, negative control, diclofenac sodium, and three propolis groups (72; 144; and 288 mg/kg BW). The results demonstrated the IC₅₀ values of the DPPH and ABTS scavenging activity 9.694 ppm and 2.213 ppm, respectively. The FRAP reducing power was 189.05 mg AaE/g. The physical appearance of propolis capsule was vegicaps as white – white, size 0, with light brown granule. Moreover, the content weight was 418.88 mg with a disintegration time of 7 min 53 s, while the water, flavonoid, and polyphenol contents were 9.07%, 1.59%, and 98.0821 mg GAE/g respectively. The content of heavy metal and microbial contamination were not detected. The acute toxicity results showed LD₅₀ ≥ 5 g/kg BW, no toxicity symptoms, and no abnormalities in all rats. The anti-inflammatory inhibition percentage for groups III, IV, V, and VI was 11.86%, 6.53%, 7.81%, and 6.63% respectively, while the anti-inflammatory drugs effectiveness percentage compared to positive controls were 55.00%, 65.83%, and 55.83% respectively. Based on these results, it can be concluded that propolis capsules fulfilled the standardization requirements, and it is likely to be non-toxic, and effective as antioxidant and anti-inflammatory.