Evaluation of selected tropical marine microalgal cultures for use in biophotovoltaic platforms.

In this study, the bioelectrical power generation potential of four tropical marine microalgal strains native to Malaysia was investigated using BPV platforms. Chlorella UMACC 258 produced the highest power density (0.108 mW m-2), followed by Halamphora subtropica UMACC 370 (0.090 mW m-2), Synechococcus UMACC 371 (0.065 mW m-2) and Parachlorella UMACC 245 (0.017 mW m-2). The chlorophyll-a (chl-a) content was examined to have a linear positive relationship with the power density (p < 0.05). The photosynthetic performance of strains was studied using the pulse-amplitude modulation (PAM) fluorometer; parameters measured include the following: maximum quantum efficiency (Fv/Fm), alpha (α), maximum relative electron transport rate (rETRmax), photo-adaptive index (Ek) and non-photochemical quenching (NPQ). The Fv/Fm values of all strains, except Synechococcus UMACC 371, ranged between 0.37 and 0.50 during exponential and stationary growth phases, suggesting their general health during those periods. The low Fv/Fm value of Synechococcus UMACC 371 was possibly caused by the presence of background fluorescence from phycobilisomes or phycobiliproteins. Electrochemical studies via cyclic voltammetry (CV) suggest the presence of electrochemically active proteins on the cellular surface of strains on the carbon anode of the BPV platform, while morphological studies via field emission scanning electron microscope (FESEM) imaging verify the biocompatibility of the biofilms on the carbon anode. KEY POINTS: • Maximum power output of 0.108 mW m-2 is recorded by Chlorella UMACC 258 • There is a positive correlation between chl-a content and power output • Proven biocompatibility between biofilms and carbon anode sans exogenous mediators.

Inhibition of lysosomal vacuolar proton pump down-regulates cellular acidification and enhances E. coli bactofection efficiency.

Endosomal escape is considered a crucial barrier that needs to be overcome by integrin-mediated E. coli for gene delivery into mammalian cells. Bafilomycin, a potent inhibitor of the H+ proton pump commonly employed to lower endosomal pH, was evaluated as part of the E. coli protocol during delivery. We found an increase in green fluorescent protein expression up 6.9, 3.2, 5.0, 2.8, and 4.5 fold in HeLa, HEK-293, A549, HT1080, and MCF-7 respectively, compared to untreated cells. Our result showed for the first time that Inhibition of lysosomal V-ATPase enhances E. coli efficiency.

RFP-based method for real-time tracking of invasive bacteria in a heterogeneous population of cells.

Quantification of bacterial invasion into eukaryotic cells is a prerequisite to unfold the molecular mechanisms of this vector's function to obtain insights for improving its efficiency. Invasion is traditionally quantified by antibiotic protection assays that require dilution plating and counting of colony-forming units rescued from infected cells. However, to differentiate between attached and internalized bacteria vector, this assay requires supplementation by a time-consuming and tedious immunofluorescence staining, making it laborious and reduces its reliability and reproducibility. Here we describe a new red fluorescent protein (RFP)-based high-throughput and inexpensive method for tracking bacterial adherence and internalization through flow cytometry to provide a convenient and real-time quantification of bacterial invasiveness in a heterogeneous population of cells. We invaded MCF-7, A549, and HEK-293 cells with the E. coli vector and measured RFP using imaging flow cytometry. We found high cellular infection of up to 70.47% in MCF-7 compared to 27.4% and 26.2% in A549 and HEK-293 cells, respectively. The quantitative evaluation of internalized E. coli is rapid and cell-dependent, and it distinctively differentiates between attached and cytosolic bacteria while showing the degree of cellular invasiveness. This imaging flow cytometry approach can be applied broadly to study host-bacteria interaction.

Influence of elevated river flow on hypoxia occurrence, nutrient concentration and microbial dynamics in a tropical estuary.

We sampled the Klang estuary during the inter-monsoon and northeast monsoon period (July-Nov 2011, Oct-Nov 2012), which coincided with higher rainfall and elevated Klang River flow. The increased freshwater inflow into the estuary resulted in water column stratification that was observed during both sampling periods. Dissolved oxygen (DO) dropped below 63 µM, and hypoxia was observed. Elevated river flow also transported dissolved inorganic nutrients, chlorophyll a and bacteria to the estuary. However, bacterial production did not correlate with DO concentration in this study. As hypoxia was probably not due to in situ heterotrophic processes, deoxygenated waters were probably from upstream. We surmised this as DO correlated with salinity (R2 = 0.664, df = 86, p < 0.001). DO also decreased with increasing flushing time (R2 = 0.556, df = 11, p < 0.01), suggesting that when flushing time (> 6.7 h), hypoxia could occur at the Klang estuary. Here, we presented a model that related riverine flow rate to the post-heavy rainfall hypoxia that explicated the episodic hypoxia at Klang estuary. As Klang estuary supports aquaculture and cockle culture, our results could help protect the aquaculture and cockle culture industry here.

Phage N15 protelomerase resolves its tos recognition site into hairpin telomeres within mammalian cells.

Phage N15 protelomerase (TelN) cleaves double-stranded circular DNA containing a telomerase-occupancy-site (tos) and rejoins the resulting linear-ends to form closed-hairpin-telomeres in Escherichia coli (E. coli). Continued TelN expression is essential to support resolution of the linear structure. In mammalian cells, no enzyme with TelN-like activities has been found. In this work, we show that phage TelN, expressed transiently and stably in human and mouse cells, recapitulates its native activities in these exogenous environments. We found TelN to accurately resolve tos-DNA in vitro and in vivo within human and mouse cells into linear DNA-containing terminal telomeres that are resistant to RecBCD degradation, a hallmark of protelomerase processing. In stable cells, TelN activity was detectable for at least 60 days, which suggests the possibility of limited silencing of its expression. Correspondingly, linear plasmid containing a 100 kb human ß-globin gene expressed for at least 120 h in non-ß-globin-expressing mouse cells with TelN presence. Our results demonstrate TelN is able to cut and heal DNA as hairpin-telomeres within mammalian cells, providing a tool for creating novel structures by DNA resolution in these hosts. The TelN protelomerase may be useful for exploring novel technologies for genome interrogation and chromosome engineering.

Rapid preparation of adherent mammalian cells for basic scanning electron microscopy (SEM) analysis.

Sample preparation for scanning electron microscope analysis involves reagents and equipment that are expensive and often hazardous. Here we demonstrate a circumvention of Osmium tetroxide and critical point drying, greatly reducing the duration, complexity and cost of the process. We captured early stage interactions of invasive-bacteria and HeLa cells during the process of bacteria-mediated gene delivery and illustrate sufficient clarity can be obtained using this procedure to preserve and clearly visualize relevant cellular structures. This protocol is significantly cheaper and easier to adapt compared to conventional methods, and will allow routine preparation/viewing of eukaryotic or bacterial samples for basic morphological studies.

Effects of eutrophication on diatom abundance, biovolume and diversity in tropical coastal waters.

Diatom abundance, biovolume and diversity were measured over a 2-year period along the Straits of Malacca at two stations with upper (Klang) and lower (Port Dickson) states of eutrophication. Diatom abundance, which ranged from 0.2 × 104 to 21.7 × 104 cells L-1 at Klang and 0.9 × 103- 41.3 × 103 cells L-1 at Port Dickson, was influenced partly by nutrient concentrations. At Klang, the diatoms were generally smaller and less diverse (H' = 0.77 ± 0.48) and predominated by Skeletonema spp. (60 ± 32% of total diatom biomass). In contrast, diatoms were larger and more diverse (H' = 1.40 ± 0.67) at Port Dickson. Chaetoceros spp. were the most abundant diatoms at Port Dickson but attributed only 48 ± 30% of total diatom biomass. Comparison of both Klang and Port Dickson showed that their diatom community structure differed and that eutrophication reduced diatom diversity at Klang. We also observed how Si(OH)4 affected the abundance of Skeletonema spp. which in turn influenced the temporal variation of diatom community at Klang. Our results highlighted how eutrophication affects diatom diversity and community structure.

Synechococcus production and grazing loss rates in nearshore tropical waters.

Temporal variation of Synechococcus, its production (µ) and grazing loss (g) rates were studied for 2 years at nearshore stations, i.e. Port Dickson and Port Klang along the Straits of Malacca. Synechococcus abundance at Port Dickson (0.3-2.3 × 105 cell ml-1) was always higher than at Port Klang (0.3-7.1 × 104 cell ml-1) (p < 0.001). µ ranged up to 0.98 day-1 (0.51 ± 0.29 day-1), while g ranged from 0.02 to 0.31 day-1 (0.15 ± 0.07 day-1) at Port Klang. At Port Dickson, µ and g averaged 0.47 ± 0.13 day-1 (0.29-0.82 day-1) and 0.31 ± 0.14 day-1 (0.13-0.63 day-1), respectively. Synechococcus abundance did not correlate with temperature (p > 0.25), but nutrient and light availability were important factors for their distribution. The relationship was modelled as log Synechococcus = 0.37Secchi - 0.01DIN + 4.52 where light availability (as Secchi disc depth) was a more important determinant. From a two-factorial experiment, nutrients were not significant for Synechococcus growth as in situ nutrient concentrations exceeded the threshold for saturated growth. However, light availability was important and elevated Synechococcus growth rates especially at Port Dickson (F = 5.94, p < 0.05). As for grazing loss rates, they were independent of either nutrients or light intensity (p > 0.30). In nearshore tropical waters, an estimated 69 % of Synechococcus production could be grazed.

Temporal variation of phytoplankton growth and grazing loss in the west coast of Peninsular Malaysia.

Phytoplankton growth (µ) and grazing loss (g) rates were measured monthly by the Landry-Hassett dilution method over a 2-year period at both estuarine (Klang) and coastal water (Port Dickson) systems along the Straits of Malacca. Chlorophyll a (Chl a) concentration ranged from 0.20 to 4.47 µg L(-1) at Klang except on two occasions when Chl a spiked above 10 µg L(-1). In contrast, Chl a concentrations were relatively stable at Port Dickson (0.14 to 2.76 µg L(-1)). From the rate measurements, µ was higher (t = 2.01, df = 43, p < 0.05) at Klang (0.30 to 2.26 day(-1)) than at Port Dickson (0.18 to 1.66 day(-1)), but g was not significantly different (p > 0.80). g ranged from 0.30 to 1.50 and 0.21 to 1.51 day(-1) at Klang and Port Dickson, respectively. In this study, grazing loss was coupled to phytoplankton growth, and the ratio of g/µ or grazing pressure which estimates the proportion of primary production grazed was 50% at Klang and lower than at Port Dickson (68%; t = 2.213, df = 36, p < 0.05). We found that the higher growth rates in a eutrophic system, i.e., Klang, were not matched by higher grazing loss, and this may have implications for the biogeochemical cycling in coastal waters.

Short homologies efficiently generate detectable homologous recombination events.

When recombineering bacterial artificial chromosomes (BACs), it is common practice to design the ends of the donor molecule with 50 bp of homology specifying its insertion site. We demonstrate that desired recombinants can be produced using intermolecular homologies as short as 15 bp. Although the use of shorter donor end regions decreases total recombinants by several fold, the frequency of recombinants with correctly inserted donor molecules was high enough for easy detection by simple polymerase chain reaction (PCR) screening. This observation may have important implications for the design of oligonucleotides for recombineering, including significant cost savings, especially for high-throughput projects that use large quantities of primers.

Investigating Escherichia coli habitat transition from sediments to water in tropical urban lakes.

Background: Escherichia coli is a commonly used faecal indicator bacterium to assess the level of faecal contamination in aquatic habitats. However, extensive studies have reported that sediment acts as a natural reservoir of E. coli in the extraintestinal environment. E. coli can be released from the sediment, and this may lead to overestimating the level of faecal contamination during water quality surveillance. Thus, we aimed to investigate the effects of E. coli habitat transition from sediment to water on its abundance in the water column. Methods: This study enumerated the abundance of E. coli in the water and sediment at five urban lakes in the Kuala Lumpur-Petaling Jaya area, state of Selangor, Malaysia. We developed a novel method for measuring habitat transition rate of sediment E. coli to the water column, and evaluated the effects of habitat transition on E. coli abundance in the water column after accounting for its decay in the water column. Results: The abundance of E. coli in the sediment ranged from below detection to 12,000 cfu g-1, and was about one order higher than in the water column (1 to 2,300 cfu mL-1). The habitat transition rates ranged from 0.03 to 0.41 h-1. In contrast, the E. coli decay rates ranged from 0.02 to 0.16 h-1. In most cases (>80%), the habitat transition rates were higher than the decay rates in our study. Discussion: Our study provided a possible explanation for the persistence of E. coli in tropical lakes. To the best of our knowledge, this is the first quantitative study on habitat transition of E. coli from sediments to water column.

Green synthesis of reduced graphene oxide by using tropical microalgae and its application in biophotovoltaic devices.

The successful commercialization of algal biophotovoltaics (BPV) technology hinges upon a multifaceted approach, encompassing factors such as the development of a cost-efficient and highly conductive anode material. To address this issue, we developed an environmentally benign method of producing reduced graphene oxide (rGO), using concentrated Chlorella sp. UMACC 313 suspensions as the reducing agent. The produced rGO was subsequently coated on the carbon paper (rGO-CP) and used as the BPV device's anode. As a result, maximum power density was increased by 950% for Chlorella sp. UMACC 258 (0.210 mW m-2) and 781% for Synechococcus sp. UMACC 371 (0.555 mW m-2) compared to bare CP. The improved microalgae adhesion to the anode and improved electrical conductivity of rGO brought on by the effective removal of oxygen functional groups may be the causes of this. This study has demonstrated how microalgal-reduced GO may improve the efficiency of algal BPV for producing bioelectricity.

Storage and dynamics of soil organic carbon in allochthonous-dominated and nitrogen-limited natural and planted mangrove forests in southern Thailand.

Mangrove forests can help to mitigate climate change by storing a significant amount of carbon (C) in soils. Planted mangrove forests have been established to combat anthropogenic threats posed by climate change. However, the efficiency of planted forests in terms of soil organic carbon (SOC) storage and dynamics relative to that of natural forests is unclear. We assessed SOC and nutrient storage, SOC sources and drivers in a natural and a planted forest in southern Thailand. Although the planted forest stored more C and nutrients than the natural forest, the early-stage planted forest was not a strong sink relative to mudflat. Both forests were predominated by allochthonous organic C and nitrogen limited, with total nitrogen being a major driver of SOC in both cases. SOC showed a significant decline along land-to-sea and depth gradients as a result of soil texture, nutrient availability, and pH in the natural forest.

Escherichia coli bactofection using Lipofectamine.

Successful gene delivery into mammalian cells using bactofection requires entry of the bacterial vector via cell surface integrin receptors followed by release of plasmid DNA into the cellular environment. We show, for the first time, that addition of the DNA transfection reagent Lipofectamine improves entry of invasive Escherichia coli into HeLa cells and enhances up to 2.8-fold green fluorescent protein (GFP) expression from a reporter plasmid. The addition of Lipofectamine may be applicable to other bacterial vectors to increase their DNA delivery efficiency into mammalian cells.

Investigating the spatial distribution of phototrophic picoplankton in a tropical estuary.

We sampled extensively (29 stations) at the Klang estuarine system over a 3-day scientific expedition. We measured physical and chemical variables (temperature, salinity, dissolved oxygen, total suspended solids, dissolved inorganic nutrients) and related them to the spatial distribution of phototrophic picoplankton (Ppico). Multivariate analysis of variance of the physicochemical variables showed the heterogeneity of the Klang estuarine system where the stations at each transect were significantly different (Rao's F18, 36 = 8.401, p < 0.001). Correlation analyses also showed that variables related to Ppico abundance and growth were mutually exclusive. Distribution of Ppico was best explained by the physical mixing between freshwater and seawater whereas Ppico growth was correlated with temperature.

Phage N15-Based Vectors for Gene Cloning and Expression in Bacteria and Mammalian Cells.

Bacteriophage N15 is the first virus known to deliver linear prophage into Escherichia coli. During its lysogenic cycle, N15 protelomerase (TelN) resolves its telomerase occupancy site (tos) into hairpin telomeres. This protects the N15 prophage from bacterial exonuclease degradation, enabling it to stably replicate as a linear plasmid in E. coli. Interestingly, purely proteinaceous TelN can retain phage DNA linearization and hairpin formation without involving host- or phage-derived intermediates or cofactors in the heterologous environment. This unique feature has led to the advent of synthetic linear DNA vector systems derived from the TelN-tos module for the genetic engineering of bacterial and mammalian cells. This review will focus on the development and advantages of N15-based novel cloning and expression vectors in the bacterial and mammalian environments. To date, N15 is the most widely exploited molecular tool for the development of linear vector systems, especially the production of therapeutically useful miniDNA vectors without a bacterial backbone. Compared to typical circular plasmids, linear N15-based plasmids display remarkable cloning fidelity in propagating unstable repetitive DNA sequences and large genomic fragments. Additionally, TelN-linearized vectors with the relevant origin of replication can replicate extrachromosomally and retain transgenes functionality in bacterial and mammalian cells without compromising host cell viability. Currently, this DNA linearization system has shown robust results in the development of gene delivery vehicles, DNA vaccines and engineering mammalian cells against infectious diseases or cancers, highlighting its multifaceted importance in genetic studies and gene medicine.

Prevalence and Diversity of Antibiotic Resistant Escherichia coli From Anthropogenic-Impacted Larut River.

Aquatic environments, under frequent anthropogenic pressure, could serve as reservoirs that provide an ideal condition for the acquisition and dissemination of antibiotic resistance genetic determinants. We investigated the prevalence and diversity of antibiotic-resistant Escherichia coli by focusing on their genetic diversity, virulence, and resistance genes in anthropogenic-impacted Larut River. The abundance of E. coli ranged from (estimated count) Est 1 to 4.7 × 105 (colony-forming units per 100 ml) CFU 100 ml-1 to Est 1 to 4.1 × 105 CFU 100 ml-1 with phylogenetic group B1 (46.72%), and A (34.39%) being the most predominant. The prevalence of multiple antibiotic resistance phenotypes of E. coli, with the presence of tet and sul resistance genes, was higher in wastewater effluents than in the river waters. These findings suggested that E. coli could be an important carrier of the resistance genes in freshwater river environments. The phylogenetic composition of E. coli and resistance genes was associated with physicochemical properties and antibiotic residues. These findings indicated that the anthropogenic inputs exerted an effect on the E. coli phylogroup composition, diversification of multiple antibiotic resistance phenotypes, and the distribution of resistance genes in the Larut River.

Distribution of faecal indicator bacteria in tropical waters of Peninsular Malaysia and their decay rates in tropical seawater.

We investigated the appropriateness of faecal indicator bacteria in tropical waters. We compared total coliform (undetectable to 7.2 × 105 cfu 100 mL-1), faecal coliform (undetectable to 6.1 × 105 cfu 100 mL-1) and enterococci (undetectable to 3.1 × 104 cfu 100 mL-1) distribution in Peninsular Malaysia. Faecal indicator bacteria was highest in freshwater, and lowest in seawater (q > 4.18, p < 0.01). We also measured the decay rates of Escherichia coli and Enterococcus faecium in microcosms. In seawater, average decay rate for E. coli was 0.084 ± 0.029 h-1, and higher than E. faecium (0.048 ± 0.024 h-1) (t = 2.527, p < 0.05). Grazing accounted for 54 % of both E. coli and E. faecium decay. E. coli decayed in the <0.02 µm seawater fraction (0.023 ± 0.012 h-1) but E. faecium sometimes grew. Seawater warming further uncoupled the response from both E. coli and E. faecium as E. faecium grew and E. coli decayed with warming. Our results suggested that the prevalence of faecal indicator bacteria in tropical waters was not due to faecal pollution alone, and this will have serious implications towards the use of these faecal indicator bacteria.

The impact of eutrophication towards selected bacterial process rates in tropical coastal waters.

The dissolved organic nutrient conditions and bacterial process rates at two tropical coastal sites in Peninsular Malaysia (Port Klang and Port Dickson) were initially studied in 2004-2005 period and later revisited in 2010-2011. We observed that dissolved organic nitrogen (DON) increased about two- and ten-fold at Port Klang and Port Dickson, respectively and resulted in a significant change in DOC:DON ratio (t ≥ 2.077, p < 0.05). Among the bacterial processes measured, bacterial respiration (BR) was lower in the 2010-2011 period at both stations (t ≥ 3.390, p < 0.01). BR also correlated to the DOC:DON ratio (R2 ≥ 0.259, p < 0.01). The increase in substrate quality enabled the bacteria to respire less in the dissolved organic matter degradation. As a result, the average bacterial growth efficiency increased slightly in the 2010-2011 period.

The roles of ribosomal proteins in nasopharyngeal cancer: culprits, sentinels or both.

Ribosomal protein genes encode products that are essential for cellular protein biosynthesis and are major components of ribosomes. Canonically, they are involved in the complex system of ribosome biogenesis pivotal to the catalysis of protein translation. Amid this tightly organised process, some ribosomal proteins have unique spatial and temporal physiological activity giving rise to their extra-ribosomal functions. Many of these extra-ribosomal roles pertain to cellular growth and differentiation, thus implicating the involvement of some ribosomal proteins in organogenesis. Consequently, dysregulated functions of these ribosomal proteins could be linked to oncogenesis or neoplastic transformation of human cells. Their suspected roles in carcinogenesis have been reported but not specifically explained for malignancy of the nasopharynx. This is despite the fact that literature since one and half decade ago have documented the association of ribosomal proteins to nasopharyngeal cancer. In this review, we explain the association and contribution of dysregulated expression among a subset of ribosomal proteins to nasopharyngeal oncogenesis. The relationship of these ribosomal proteins with the cancer are explained. We provide information to indicate that the dysfunctional extra-ribosomal activities of specific ribosomal proteins are tightly involved with the molecular pathogenesis of nasopharyngeal cancer albeit mechanisms yet to be precisely defined. The complete knowledge of this will impact future applications in the effective management of nasopharyngeal cancer.