Cloning and comparative protein modelling of two MADS-box genes, HsMADS1 and HsMADS2 isolated from Hibiscus sabdariffa L. var. UMKL (roselle)

Hibiscus sabdariffa' L. var. UMKL or commonly known as roselle is cultivated in Malaysia mainly for its calyx, which is high in vitamin C and anthocyanin. Unfortunately, the genetic information regarding the flowering pathway of roselle is very scarce. It is essential to understand the genetics underlying roselle's flower developmental process by studying MADS-box transcription factor genes that play crucial roles in controlling the development of calyx in flowering plants. Designated as 'HsMADS1' and 'HsMADS2', two MADS-box genes were isolated from the calyx tissues of roselle from different developmental stages using 3'- RACE PCR and primer walking approaches. The different motifs in the C domain region of 'HsMADS1' and 'HsMADS2' deduced amino acid sequences suggested that both genes probably originated from 'SEP' and 'AGL6' subfamilies of MADS-box gene respectively. The putative functions of the genes based on BLAST searches and phylogenetic analyses suggested that 'HsMADS1' possibly involves in the expression of SEP gene in stem, leaf, bud and flower organs of roselle, whereas 'HsMADS2' may probably involve in the late expression of floral tissue for stem branching. The alpha helix rich structures of SRF-TF identified in the deduced amino acid sequences of HsMADS1 and HsMADS2 supported the involvement of both proteins in DNA binding and dimerisation

Poly lactic-co-glycolic acid nanoparticles: flow rates and gauge sizes influence the droplet surface tension and particle sizing

Electrospray is a novel and versatile approach to the synthesis of nanosized particles. In this paper, the effects of electrospray process parameters such as gauge size (18 G-25 G) and flow rate (0.9-1.5 ml hr-1) on droplet surface tension and electrospray particle size were evaluated. 25 mg Poly Lactic-co-Glycolic Acid (PLGA) were dissolved in 100 ml acetone before being subjected to electrospray. Tate’s Law was used to calculate the surface tension while the Malvern nanosizer was used to measure the particle size. Based on Tate’s Law calculation, when the gauze size increased from 18 G-25 G, the droplet surface tension increased from 10.07 Nm-1 to 18.17 Nm-1 showing a direct pattern. At the same time, the flowrate is inversely proportional to droplet surface tension. When the flow rate increased from 0.9-1.5 ml hr-1, the droplet surface tension was reduced. This is due to the increasing ratio of vicious force and surface tension. For particle size, as surface tension increased from 10.07 Nm-1 to 18.17 Nm-1, particle size increased from 205.57 nm to 612 nm owing to the corona discharge producing larger progeny droplets that chain into smaller particles through coulomb fission as more charge is required to overcome the surface tension. In conclusion, flow rate and gauge size influenced the surface tension thus affecting the nanosized particles

Culture and characterization of Microcystis spp. and their effects on cladoceran population growth

Toxic cyanobacterial species such as Microcystis spp. can form harmful blooms that cause water quality deterioration and negatively impact aquatic life in addition to triggering health risks towards human. This study aimed to isolate Microcystis spp. that produce a toxin, microcystin, and assess their impacts on the growth and reproductive capacity of a cladoceran zooplankton which feeds on microalgae as its main diet. Two Microcystis spp. were isolated and identified with both conventional and molecular methods. Species and toxicity identification for both species were done by using polymerase chain reaction (PCR) with the use of 16S rRNA and mcyB gene sequence. Apart from molecular approach, nuclear magnetic resonance (NMR) was used to detect the presence of microcystin in both isolates. Samples were obtained during the exponential phase, freeze dried and kept in -80˚C freezer prior to toxin extraction. Lyophilized cells were extracted using 75% methanol and dried in vacuo at 40˚C. Each sample was transferred to 1.5 ml amber vial before analysis. 10% of both Microcystis culture (at exponential phase) was transferred into the culture medium with limited nutrient availability (25% reduction = N75 and P75; 50% reduction = N50 and P50; 75% reduction = N25 and P25 from initial concentration (15g L-1). Growth was determined by cell density, optical density and dry weight measurements. Moina micrura was used in population growth study and chronic bioassays. For the population growth study, M. micrura was exposed to three different species of microalgae; Microcystis aeruginosa, Microcystis viridis, and Chlorella vulgaris as a control. For chronic bioassay, 20 neonates (< 24h) were individually reared in glass vials. All the glass vials were checked daily (at 12h intervals) to determine age at first reproduction (day), fecundity (no of eggs female-1), total offsprings (no. of offsprings female-1) and longevity (no. of days). The chronic bioassays were terminated when all the cladocerans died (13 days). Based on 16S rRNA and mcyB genes sequences, two potential microcystin producer Microcystis spp. were successfully isolated, purified and identified as Microcystis aeruginosa (UPMC-A0038, GenBank ID number KX447651.1) and Microcystis viridis (UPMC-A0039, GenBank ID number KY009735.1). Both isolates varied substantially in terms of morphological features such as cell size, colonial formation and cell arrangement. In addition, 1H NMR results showed the presence of Adda group had confirmed microcystin in both Microcystis species. Both Microcystis spp. growth decreased under low nutrient concentrations. Nitrogen and phosphorus play an equal roles in the growth of Microcystis. Compared to M. aeruginosa, the growth of M. viridis was severely affected under low phosphorus level. In addition, M. viridis responded differently toward nitrogen limitation and exhibited adaptive mechanism in low nutrient environment. Both Microcystis spp. were toxic to M. micrura. The mortality rates of M. micrura subjected to M. aeruginosa and M. viridis were significantly higher (p<0.05) than the control treatment. Moina micrura exposed to M. aeruginosa did not reach maturity as their mean body size only reached 627.80±31.4 μm compared to M. micrura fed with C. vulgaris (814.94 ±21.84 μm) and M. viridis (914.21±12.64 μm). The population growth rate of M. micrura fed with C. vulgaris was 0.28 day-1 while growth rates were negative when fed with M. aeruginosa (-0.23 day-1) and M. viridis (-0.20 day-1). Longer exposure of M. micrura to M. aeruginosa resulted in delayed production of M. micrura’s first offspring, which only occurred on day 6 compared to M. micrura fed with C. vulgaris which produced their first offspring on day 3. In conclusion, both Microcystis spp. were microcystin producer species and nutrients play an important role in promoting Microcystis growth. This study also indicated that toxicity of both Microcystis spp. negatively affected M. micrura growth, survival as well as their reproductive capacity

Reduced reproductive capacity in Moina micrura Kurz, 1875 exposed to toxic Microcystis spp.

Toxic cyanobacterial blooms are considered harmful to all consuming organisms along the aquatic food chain and top consumers, including humans. Hence this study was conducted to assess the impacts of two toxic Microcystis spp. on a tropical cladoceran, Moina micrura Kurz, 1875. Population growth studies and chronic bioassays were conducted using Microcystis aeruginosa (Kützing) Kützing, 1846, Microcystis viridis f. viridis (A. Braun) Elenkin, 1938, and a green alga, Chlorella vulgaris f. tertia Fott et Novakova, 1972 (as the control). Both Microcystis spp. negatively affected M. micrura. The population growth rate of M. micrura fed with C. vulgaris was 0.51 day-1 , while growth rates were negative when fed with M. aeruginosa (- 0.33 day-1 ) and M. viridis (- 0.13 day-1 ). In the chronic bioassay, the exposure of M. micrura to M. aeruginosa resulted in delayed production of M. micrura’s first batch of offsprings, which only occurred on day 6 compared to M. micrura fed with C. vulgaris which produced their first batch of offsprings earlier on day 3. This study showed that exposure of M. micrura to both toxic Microcystis spp. reduced the population density, fecundity, total offspring production and longevity of M. micrura compared to those fed with C. vulgaris