A comparative study on effective cell disruption methods for lipid extraction from microalgae

Aims: To compare effective cell disruption methods for lipid extraction from fresh water microalgae. Methods and Results: Chlorella sp., Nostoc sp. and Tolypothrix sp. were isolated from fresh water ponds in and around Gandhigram, Dindigul District, Tamilnadu, India, and used for lipid extraction. Different methods, including autoclaving, bead beating, microwave, sonication and a 10% NaCl solution treatments, were tested to identify the most effective cell disruption method. The total lipids from three microalgal species were extracted using a mixture of chloroform and methanol. Fatty acid composition was detected by gas chromatography (GC). Nostoc sp. and Tolypothrix sp. showed higher oleic acid content of 13·27 mg g^?1 dw and 17·75 mg g^?1 dw, respectively, whereas Chlorella sp. had high linoleic acid content of 17·61 mg g^?1 dw when the cells were disrupted using the sonication method. Conclusions: Finally, the sonication method was found to be the most applicable and efficient method of lipid extraction from microalgae. The highest lipid content was extracted from Chlorella sp. Significance and Impact of the Study: In biodiesel production from microalgae, lipid extraction is a crucial step and important as cell disruption comes in this step. Therefore, the appropriate cell disruption method and device is a key to increase the lipid extraction efficiency.     

Association of interleukin 1 receptor antagonist (IL1RN) gene polymorphism with recurrent pregnancy loss risk in the North Indian Population and a meta-analysis

An appropriate ratio of interleukin 1 beta to interleukin 1 receptor antagonist (IL1Ra) is required for successful pregnancy. Our objective was to study the genetic association between IL1RN variable numbers of tandem repeat (VNTR) polymorphism and recurrent pregnancy loss (RPL). To analyze the association between IL1RN VNTR allele and RPL, we investigated the IL1RN VNTR polymorphism in 136 RPL patients and in 200 healthy control women. Meta-analysis on this polymorphism was conducted to support our findings. PCR based approach was used to analyze IL1RN VNTR polymorphism and it was further confirmed by sequencing. Systematic review and meta-analysis was done using electronic database (Pub-Med, Google Scholar and Ovid) up to February 27, 2013. This meta-analysis was assessed by comprehensive meta-analysis software version 2. For meta-analysis 549 cases and 1,450 controls were included. The frequency of IL1RN genotype 2/2 was significantly higher in RPL compared to control group (AORs 3.10, 95 % CI 1.58–6.11, p = 0.001). The presence of rare allele also increased the risk of RPL significantly (ORs 1.63, 95 % CI 1.16–2.29, p = 0.004). The meta-analysis stratified by ethnicity showed that individuals with allele 2 had increased risk of RPL (OR 1.29, 95 % CI 1.04–1.61, p = 0.01), in Asians population by using fixed model. However the data of the present study clearly suggests that IL1RN VNTR polymorphism is a genetic risk factor for pregnancy loss in the study population.     

Targeting Lipid Esterases in Mycobacteria Grown Under Different Physiological Conditions Using Activity-based Profiling with Tetrahydrolipstatin (THL)

Tetrahydrolipstatin (THL) is bactericidal but its precise target spectrum is poorly characterized. Here, we used a THL analog and activity-based protein profiling to identify target proteins after enrichment from whole cell lysates of Mycobacterium bovis Bacillus Calmette-Guérin cultured under replicating and non-replicating conditions. THL targets α/β-hydrolases, including many lipid esterases (LipD, G, H, I, M, N, O, V, W, and TesA). Target protein concentrations and total esterase activity correlated inversely with cellular triacylglycerol upon entry into and exit from non-replicating conditions. Cellular overexpression of lipH and tesA led to decreased THL susceptibility thus providing functional validation. Our results define the target spectrum of THL in a biological species with particularly diverse lipid metabolic pathways. We furthermore derive a conceptual approach that demonstrates the use of such THL probes for the characterization of substrate recognition by lipases and related enzymes.Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is responsible for nearly 2 million deaths each year. The host immune response toward aerosol infection is to quarantine tubercle bacilli in a granulomatous structure (1, 2). However, granuloma-associated mycobacteria can switch to a non-replicative, “dormant” state and successfully evade immune response for decades after infection (3, 4). The metabolic events that permit tubercle bacilli to enter host cells and revive from states of persistence suggest that lipids are utilized as a carbon source (5–7). During times of oxygen deprivation and in the absence of host cells, cultivated mycobacteria store fatty acids (FAs) in the form of triacylglycerol (TAG)¹-enriched lipid droplets (8–10). Upon resuscitation (by the re-introduction of oxygen), these lipid droplets vanish and TAGs are hydrolyzed (11). Unfortunately, the molecular mechanisms for TAG build-up and breakdown are far less well understood in bacteria when compared with those processes in eukaryotes.Comparative sequence analysis of the Mtb genome has revealed that it contains 250 genes encoding enzymes involved in lipid metabolism compared with only 50 enzymes in Escherichia coli, which has a genome of comparable size. Among these genes, 150 are predicted to encode proteins involved in lipid catabolism (12, 13). A family of 24 carboxyl ester hydrolases called “lip” genes (lipC to Z, except K and S) has been predicted to play a role in lipid catabolism (14). Among these, only a few have been functionally characterized and related to mycobacterial dormancy and resuscitation (15–18).Tetrahydrolipstatin, a serine esterase inhibitor, covalently binds to and inhibits mammalian lipases and fatty acid synthase (FAS) and is marketed as “Orlistat” for the treatment of severe forms of obesity (19). THL was previously shown to inhibit both active and latent forms of mycobacteria (11, 20–22) but the bacterial target spectrum remains poorly characterized. Therefore, to (1) define the THL target spectrum in a mycobacterial species and (2) to obtain biochemical insights into regulation of lipases and esterases in different metabolic states, we employed a chemical-proteomics approach using activity-based protein profiling (ABPP) with a bait that has been described to bind to lipolytic enzymes (23–25). We identified several known lipases (as anticipated), putative lipase and esterases, and hypothetical proteins of unknown functions, thereby providing a comprehensive resource of experimentally determined THL targets in mycobacteria. Importantly, we systematically compared readouts of fluorescently tagged THL-proteins (7 bands on one-dimensional SDS-PAGE) with those of mass spectrometry-based peptide identification of enriched protein fractions (247 in growing cells). This comparison led to the identification of 14 THL targets, two of which were further validated experimentally. We furthermore provide a conceptual framework for the evaluation of this target list using both experimental as well as bioinformatics approaches in two examples, lipH and tesA. Overall, our data indicate that THL is an anti-mycobacterial drug because of its potential to (1) bind to a relatively wide range of lipolytic enzymes and (2) prevent bacilli from resuscitating from a nonreplicating persistent (NRP) state when lipid metabolism is particularly important.     

Recent Trends in Drug Delivery System Using Protein Nanoparticles

Engineered nanoparticles that can facilitate drug formulation and passively target tumours have been under extensive research in recent years. These successes have driven a new wave of significant innovation in the generation of advanced particles. The fate and transport of diagnostic nanoparticles would significantly depend on nonselective drug delivery, and hence the use of high drug dosage is implemented. In this perspective, nanocarrier-based drug targeting strategies can be used which improve the selective delivery of drugs to the site of action, i.e. drug targeting. Pharmaceutical industries majorly focus on reducing the toxicity and side effects of drugs but only recently it has been realised that carrier systems themselves may pose risks to the patient. Proteins are compatible with biological systems and they are biodegradable. They offer a multitude of moieties for modifications to tailor drug binding, imaging or targeting entities. Thus, protein nanoparticles provide outstanding contributions as a carrier for drug delivery systems. This review summarises recent progress in particle-based therapeutic delivery and discusses important concepts in particle design and biological barriers for developing the next generation of particles drug delivery systems.     

Encapsulation of Pt-labelled DNA Molecules inside Carbon Nanotubes

Experiments on encapsulating Pt--labelled DNA molecules inside multiwalled carbon nanotubes (MWCNT) were performed under temperature and pressure conditions of 400K and 3 Bar. The DNA-CNT hybrids were purified via agarose gel electrophoresis and analyzed via high resolution transmission electron microscopy (HR-TEM) and energy dispersive X-ray spectroscopy (EDX). The results showed that the Pt-labelled DNA molecules attached to the outside walls of CNTs could be removed by electrophoresis. The HR-TEM and EDX results demonstrated that 2-3% of the Pt-labelled DNA molecules were successfully encapsulated inside the MWCNTs. The experimental study complements our previous molecular dynamics simulations on encapsulation of single stranded DNA oligonucleotides inside single wall carbon nanotubes under similar conditions in water. The van der Waals interaction between CNT and Pt-labelled DNA is believed to be the main driving force for this phenomenon. The DNA-CNT molecular complex could be further explored for potential applications in bio-nanotechnology.     

<Emphasis Type="Italic">In vitro</Emphasis> adventitious shoot organogenesis and plant regeneration from seedling explants of <Emphasis Type="Italic">Albizia odoratissima</Emphasis> L.f. (Benth.)

Epicotyl, petiole, and cotyledon explants derived from 14-d-old seedlings of Albizia odoratissima were cultured on Murashige and Skoog (MS) basal medium supplemented with different concentrations of either 6-benzylaminopurine (BAP) solely or in combination with 0.5 μM naphthalene-3-acetic acid (NAA). The percentage of shoot regeneration and the number of shoots regenerated varied significantly depending on the type of explants used, the concentration of plant growth regulators, and the orientation of explants on the culture medium. The best response in terms of the percentage of shoot regeneration was obtained from epicotyls cultured horizontally on MS medium supplemented with 5 μM BAP, whereas the highest number of shoots per responding explant was recorded on medium containing 2.5 μM BAP and 0.5 μM NAA. Successful rooting was achieved by placing the microshoots onto MS medium containing 25 μM indole-3-butyric acid (IBA) for 24 h first, then transferring to the same medium without IBA. Of the various substrates tested, vermiculite was the best for plant acclimatization, as 75% of the plants survived and became established.     

Advances in polymeric systems for tissue engineering and biomedical applications

The characteristics of tissue engineered scaffolds are major concerns in the quest to fabricate ideal scaffolds for tissue engineering applications. The polymer scaffolds employed for tissue engineering applications should possess multifunctional properties such as biocompatibility, biodegradability and favorable mechanical properties as it comes in direct contact with the body fluids in vivo. Additionally, the polymer system should also possess biomimetic architecture and should support stem cell adhesion, proliferation and differentiation. As the progress in polymer technology continues, polymeric biomaterials have taken characteristics more closely related to that desired for tissue engineering and clinical needs. Stimuli responsive polymers also termed as smart biomaterials respond to stimuli such as pH, temperature, enzyme, antigen, glucose and electrical stimuli that are inherently present in living systems. This review highlights the exciting advancements in these polymeric systems that relate to biological and tissue engineering applications. Additionally, several aspects of technology namely scaffold fabrication methods and surface modifications to confer biological functionality to the polymers have also been discussed. The ultimate objective is to emphasize on these underutilized adaptive behaviors of the polymers so that novel applications and new generations of smart polymeric materials can be realized for biomedical and tissue engineering applications.