Inferring short tandem repeat variation from paired-end short reads

The advances of high-throughput sequencing offer an unprecedented opportunity to study genetic variation. This is challenged by the difficulty of resolving variant calls in repetitive DNA regions. We present a Bayesian method to estimate repeat-length variation from paired-end sequence read data. The method makes variant calls based on deviations in sequence fragment sizes, allowing the analysis of repeats at lengths of relevance to a range of phenotypes. We demonstrate the method’s ability to detect and quantify changes in repeat lengths from short read genomic sequence data across genotypes. We use the method to estimate repeat variation among 12 strains of Arabidopsis thaliana and demonstrate experimentally that our method compares favourably against existing methods. Using this method, we have identified all repeats across the genome, which are likely to be polymorphic. In addition, our predicted polymorphic repeats also included the only known repeat expansion in A. thaliana, suggesting an ability to discover potential unstable repeats.     

Fucoxanthin induced apoptotic cell death in oral squamous carcinoma (KB) cells

Fucoxanthin (Fx) is an active compound commonly found in the many types of seaweed with numerous biological activities. The main goal of this investigation is to explore the effect of Fx against the cell proliferation, apoptotic induction and oxidative stress in the oral squamous (KB) cell line. Cytotoxicity of Fx was determined by MTT assay. The intracellular ROS production, mitochondrial membrane potential (MMP) and apoptosis induction in KB cells were examined through DCFH-DA, Rhodamine-123 and DAPI, and dual staining techniques. Effect of Fx on the antioxidant enzymes and lipid peroxidation in the KB cells was studied through the standard procedures. Fx treated KB cells showed morphological changes and reduced cell survival, which is exhibited by the cytotoxic activity of 50 µM/ml (IC50) Fx against the KB cells. The Fx treatment considerably induced the apoptotosis cells (EB/AO) and decreased the MMP (Rh-123) in KB cells. Further, it was pointed out that there was an increased lipid peroxidation (LPO) with decreased antioxidants (CAT, SOD and GSH). These results concluded that Fx has the cytotoxic effect against KB cells and has the potential to induce the apoptosis via increased oxidative stress. Hence, the Fx can be a promising agent for the treatment of oral cancer and it may lead to the development of cancer therapeutics.     

Twenty-four well plate miniature bioreactor system as a scale-down model for cell culture process development

Increasing the throughput and efficiency of cell culture process development has become increasingly important to rapidly screen and optimize cell culture media and process parameters. This study describes the application of a miniaturized bioreactor system as a scaled-down model for cell culture process development using a CHO cell line expressing a recombinant protein. The microbioreactor system (M24) provides non-invasive online monitoring and control capability for process parameters such as pH, dissolved oxygen (DO), and temperature at the individual well level. A systematic evaluation of the M24 for cell culture process applications was successfully completed. Several challenges were initially identified. These included uneven gas distribution in the wells due to system design and lot to lot variability, foaming issues caused by sparging required for active DO control, and pH control limitation under conditions of minimal dissolved CO2. A high degree of variability was found which was addressed by changes in the system design. The foaming issue was resolved by addition of anti-foam, reduction of sparge rate, and elimination of DO control. The pH control limitation was overcome by a single manual liquid base addition. Intra-well reproducibility, as indicated by measurements of process parameters, cell growth, metabolite profiles, protein titer, protein quality, and scale-equivalency between the M24 and 2 L bioreactor cultures were very good. This evaluation has shown feasibility of utilizing the M24 as a scale-down tool for cell culture application development under industrially relevant process conditions.     

Cis-regulatory Changes at FLOWERING LOCUS T Mediate Natural Variation in Flowering Responses of Arabidopsis thaliana

Flowering time, a critical adaptive trait, is modulated by several environmental cues. These external signals converge on a small set of genes that in turn mediate the flowering response. Mutant analysis and subsequent molecular studies have revealed that one of these integrator genes, FLOWERING LOCUS T (FT), responds to photoperiod and temperature cues, two environmental parameters that greatly influence flowering time. As the central player in the transition to flowering, the protein coding sequence of FT and its function are highly conserved across species. Using QTL mapping with a new advanced intercross-recombinant inbred line (AI-RIL) population, we show that a QTL tightly linked to FT contributes to natural variation in the flowering response to the combined effects of photoperiod and ambient temperature. Using heterogeneous inbred families (HIF) and introgression lines, we fine map the QTL to a 6.7 kb fragment in the FT promoter. We confirm by quantitative complementation that FT has differential activity in the two parental strains. Further support for FT underlying the QTL comes from a new approach, quantitative knockdown with artificial microRNAs (amiRNAs). Consistent with the causal sequence polymorphism being in the promoter, we find that the QTL affects FT expression. Taken together, these results indicate that allelic variation at pathway integrator genes such as FT can underlie phenotypic variability and that this may be achieved through cis-regulatory changes.MOLECULAR analysis of the phenotypic variation in life history traits is key to understanding how plants evolve in diverse natural environments. Among such traits, flowering time is critical for the reproductive success of the plant and is highly variable among natural Arabidopsis thaliana strains, providing an attractive paradigm for studying adaptive evolution (Johanson et al. 2000; Hagenblad and Nordborg 2002; Stinchcombe et al. 2004; Lempe et al. 2005; Shindo et al. 2005; Werner et al. 2005a). Two major environmental parameters that modulate flowering time are light and temperature (Koornneef et al. 1998). Temperature and light conditions vary substantially within the geographical range of A. thaliana, and natural populations presumably need to adapt to the local environment to ensure reproductive success. Flowering in A. thaliana is generally accelerated by long photoperiods, vernalization (exposure to winter-like conditions), and elevated ambient temperatures (Bäurle and Dean 2006). All these cues favor flowering of A. thaliana during spring or early summer, although the contribution from each individual cue and the interactions among them vary depending on the local environmental conditions (Wilczek et al. 2009).Flowering time is controlled through several genetic cascades that converge on a set of integrator genes including FLOWERING LOCUS T (FT), which encodes a protein that is highly conserved in flowering plants (Kardailsky et al. 1999; Kobayashi et al. 1999; Ahn et al. 2006). FT and its homologs are very likely an integral part of the mobile signal (florigen) that is produced in leaves and travels to the shoot apex to induce flowering (Abe et al. 2005; Wigge et al. 2005; Lifschitz et al. 2006; Corbesier et al. 2007; Jaeger and Wigge 2007; Lin et al. 2007; Mathieu et al. 2007; Tamaki et al. 2007; Notaguchi et al. 2008). In A. thaliana, FT expression is controlled by photoperiod, vernalization, and ambient growth temperature. Photoperiod in conjunction with the circadian clock promotes daily oscillations in FT RNA levels, which are greatly elevated at the end of long days. The central role of FT in determining the timing of flowering appears to be conserved in many species, making FT an attractive target for altering flowering time in cereals and other plants of economic importance (recently reviewed by Kobayashi and Weigel 2007; Turck et al. 2008).Wild strains of A. thaliana show extensive variation in flowering time and much of this is due to variation in the activity of the floral repressor FLOWERING LOCUS C (FLC). While some of this variation maps to FLC itself, much of it is due to differential activity at the epistatically acting FRIGIDA (FRI) locus (Michaels and Amasino 1999; Sheldon et al. 1999; Johanson et al. 2000; Michaels et al. 2003; Lempe et al. 2005; Shindo et al. 2005, 2006). Flowering is typically substantially delayed when the FRI/FLC system is active, unless these plants are first vernalized. However, FRI and FLC do not explain all of the flowering time variation seen in wild strains, and functionally divergent alleles of several additional flowering regulators, including CRYPTOCHROME 2 (CRY2), HUA2, FLOWERING LOCUS M (FLM), PHYTOCHROME C (PHYC), and PHYTOCHROME D (PHYD), have been identified in different strains of A. thaliana (Aukerman et al. 1997; Alonso-Blanco et al. 1998; El-Assal et al. 2001; Werner et al. 2005b; Balasubramanian et al. 2006a; Wang et al. 2007). Finally, there are many genotype-by-environment interactions that dramatically affect the contribution of a specific locus to the overall phenotype.The study of natural variation in A. thaliana has been greatly facilitated through the use of recombinant inbred line (RIL) populations (Koornneef et al. 2004). We have recently established two advanced intercross (AI)-RIL sets, in which the genetic map is greatly expanded, allowing for high-resolution QTL mapping (Balasubramanian et al. 2009). Here we use one of the new AI-RIL populations along with an independent F₂ population to identify the molecular basis of a light and temperature-sensitive flowering time QTL that mapped to the promoter of the FT gene. We show that FT is likely the causal gene for variation in light and temperature-sensitive flowering. Our results, in combination with those from other species, suggest that cis-regulatory variation rather than structural variation at FT contributes to phenotypic variation in natural populations.     

Gold nanoparticles–conjugated quercetin induces apoptosis via inhibition of EGFR/PI3K/Akt–mediated pathway in breast cancer cell lines (MCF‐7 and MDA‐MB‐231)

Epidermal growth factor plays a major role in breast cancer cell proliferation, survival, and metastasis. Quercetin, a bioactive flavonoid, is shown to exhibit anticarcinogenic effects against various cancers including breast cancer. Hence, the present study was designed to evaluate the effects of gold nanoparticles–conjugated quercetin (AuNPs‐Qu‐5) in MCF‐7 and MDA‐MB‐231 breast cancer cell lines. Borohydride reduced AuNPs were synthesized and conjugated with quercetin to yield AuNPs‐Qu‐5. Both were thoroughly characterized by several physicochemical techniques, and their cytotoxic effects were assessed by MTT assay. Apoptotic studies such as DAPI, AO/EtBr dual staining, and annexin V‐FITC staining were performed. AuNPs and AuNPs‐Qu‐5 were spherical with crystalline nature, and the size of particles range from 3.0 to 4.5 nm. AuNPs‐Qu‐5 exhibited lower IC₅₀ value compared to free Qu. There was a considerable increase in apoptotic population with increased nuclear condensation seen upon treatment with AuNPs‐Qu‐5. To delineate the molecular mechanism behind its apoptotic role, we analysed the proteins involved in apoptosis and epidermal growth factor receptor (EGFR)–mediated PI3K/Akt/GSK‐3β signalling by immunoblotting and immunocytochemistry. The pro‐apoptotic proteins (Bax, Caspase‐3) were found to be up regulated and anti‐apoptotic protein (Bcl‐2) was down regulated on treatment with AuNPs‐Qu‐5. Additionally, AuNPs‐Qu‐5 treatment inhibited the EGFR and its downstream signalling molecules PI3K/Akt/mTOR/GSK‐3β. In conclusion, administration of AuNPs‐Qu‐5 in breast cancer cell lines curtails cell proliferation through induction of apoptosis and also suppresses EGFR signalling. AuNPs‐Qu‐5 is more potent than free quercetin in causing cancer cell death, and hence, this could be a potential drug delivery system in breast cancer therapy.     

Electrospray Ionization-Mass Spectrometry and Tandem Mass Spectrometry Reveal Self-Association and Metal-Ion Binding of Hydrophobic Peptides: A Study of the Gramicidin Dimer

Gramicidin is a membrane pentadecapeptide that acts as a channel, allowing the passage of monovalent metal ions and assisting in bacterial cell death. The active form is a noncovalently bound dimer. One means to study the self-assembly of this peptide has been to compare the state of the peptide in various solvents ranging from hydrophilic (e.g., trifluoroethanol) to hydrophobic (e.g., n-propanol). In this article, we report the use of electrospray mass spectrometry to study the self-association of gramicidin in various organic and mixed solvents that are introduced directly into the mass spectrometer. The dimer (both homo and hetero) can survive the introduction into the gas phase, and the amount in the gas phase increases with the decreasing dielectric constant of the solvent, reflecting solution-phase behavior. Tandem mass spectrometry data reveal that the stability of dimer in the gas phase decreases with increasing metal ion size, strongly suggesting that the metal ion binds inside the dimer between the monomers.     

Biosequestration of carbon dioxide using a silicified carbonic anhydrase catalyst

Using recombinant DNA technology, we constructed a dual fusion gene expression plasmid, pRCAH-30, encoding carbonic anhydrase (CA) from the cyanobacterium Synechocystis sp. PCC6803, an R5 peptide sequence, and an affinity (His)₆ tag, to allow the simultaneous purification and immobilization of the encoded fusion enzyme, termed RCAH. The expressed fusion protein was approximately 30 kDa, and could be rapidly purified using affinity resins. To enhance enzyme activity, the R5 peptide facilitated immobilization by means of silicification with tetramethoxysilane; the aggregated particles were approximately 300 nm in diameter. Activity tests revealed that the enzyme functioned optimally between pH 7.0 and 7.5; maximum stability was achieved between 25 and 45°C, at pH 6.0 ～ 8.0. Activity of the fusion enzyme persisted, even after encapsulation by biomimetic silicification. In fact, silicone embedding stabilized the enzyme structure, thereby increasing its stability and reusability rate under different environmental conditions. In addition, the silicified enzyme reduced waste CO₂ gas from 800 to 42 ppm, resulting in a gas capture rate of 94.7% after conversion. Thus, the construct developed in this study can be effectively utilized for the sequestration of industrial waste CO₂ gas.