Increasing productivity of Spirulina platensis in photobioreactors using artificial neural network modeling

Although production of microalgae in open ponds is conventionally practiced due to its economy, exposure of the algae to uncontrollable elements impedes achievement of quality and it is desirable to develop closed reactor cultivation methods for the production of high value products. Nevertheless, there are several constraints which affect growth of in closed reactors, some of which this study aims to address for the production of Spirulina. Periodic introduction of fresh medium resulted in increased trichome numbers and improved algal growth compared to growth in medium that was older than 4 weeks in 20 L polycarbonate bottles. Mixing of the cultures by bubbling air and use of draft tube reduced the damage to the growing cells and permitted increased growth. However, there was better growth in inclined cylindrical reactors mixed with bubbling air. The oxygen production rates were very similar irrespective differences in the maintained cultures densities. The uniformity in oxygen production rate suggested a tendency towards homeostasis in Spirulina cultures. The frequency of biomass harvest on the productivity of Spirulina showed that maintenance of moderate culture density between 0.16 and 0.32 g/L resulted in about 14% more productivity than maintaining the cell density between 0.16 and 0.53 g/L or 48% more than by daily harvest above 0.16 g/L. An artificial neural network based predictive model was developed, and the variables useful for predicting biomass output were identified. The model could predict the growth of Spirulina up to 3 days in advance with a coefficient of determination >0.94.     

Aldimine to ketoamine isomerization (Amadori rearrangement) potential at the individual nonenzymic glycation sites of hemoglobin a: Preferential inhibition of glycation by nucleophiles at sites of low isomerization potential

The relative roles of the two structural aspects of nonenzymic glycation sites of hemoglobin A, namely the ease with which the amino groups could form the aldimine adducts and the propensity of the microenvironments of the respective aldimines to facilitate the Amadori rearrangement, in dictating the site selectivity of nonenzymic glycation with aldotriose has been investigated. The chemical reactivity of the amino groups of hemoglobin A forin vitro reductive glycation with aldotriose is distinct from that in the nonreductive mode. The reactivity of amino groups of hemoglobin A toward reductive glycation (i.e., propensity for aldimine formation) decreases in the order Val-1(), Val-1(), Lys-66(), Lys-61(), and Lys-16(). The overall reactivity of hemoglobin A toward nonreductive glycation decreased in the order Lys-16(), Val-1(), Lys-66(), Lys-82(), Lys-61(), and Val-1(). Since the aldimine is the common intermediate for both the reductive and nonreductive modification, the differential selectivity of protein for the two modes of glycation is clearly a reflection of the propensity of the microenvironments of nonenzymic glycation sites to facilitate the isomerization reaction (i.e., Amadori rearrangement). A semiquantitative estimate of this propensity of the microenvironment of the nonenzymic glycation sites has been obtained by comparing the nonreductive (nonenzymic) and reductive modification at individual glycation sites. The microenvironment of Lys-16() is very efficient in facilitating the rearrangement and the relative efficiency decreases in the order Lys-16(), Lys-82(), Lys-66(), Lys-61(), Val-1(), and Val-1(). The propensity of the microenvironment of Lys-16() to facilitate the Amadori rearrangement of the aldimine is about three orders of magnitude higher than that of Val-1() and is about 50 times higher than that of Val-1(). The extent of nonenzymic glycation at the individual sites is modulated by various factors, such as thepH, concentration of aldotriose, and the concentration of the protein. The nucleophiles—such as tris, glycine ethyl ester, and amino guanidine—inhibit the glycation by trapping the aldotriose. The nonenzymic glycation inhibitory power of nucleophile is directly related to its propensity to form aldimine. Thus, the extent of inhibition of nonenzymic glycation at a given site by a nucleophile directly reflects the relative role ofpK _a of the site in dictating the glycation at that site. The nonenzymic glycation of an amino group of a protein is an additive/synergestic consequence of the propensity of the site to form aldimine adducts on one hand, and the propensity of its microenvironment to facilitate the isomerization of the aldimines to ketoamines on the other. The isomerization potential of microenvironment plays the dominant role in dictating the site specificity of the nonenzymic glycation of proteins.     

N-terminal and Central Segments of the Type 1 Ryanodine Receptor Mediate Its Interaction with FK506-binding Proteins

We used site-directed labeling of the type 1 ryanodine receptor (RyR1) and fluorescence resonance energy transfer (FRET) measurements to map RyR1 sequence elements forming the binding site of the 12-kDa binding protein for the immunosuppressant drug, FK506. This protein, FKBP12, promotes the RyR1 closed state, thereby inhibiting Ca²⁺ leakage in resting muscle. Although FKBP12 function is well established, its binding determinants within the RyR1 protein sequence remain unresolved. To identify these sequence determinants using FRET, we created five single-Cys FKBP variants labeled with Alexa Fluor 488 (denoted D-FKBP) and then targeted these D-FKBPs to full-length RyR1 constructs containing decahistidine (His₁₀) “tags” placed within N-terminal (amino acid residues 76–619) or central (residues 2157–2777) regions of RyR1. The FRET acceptor Cy3NTA bound specifically and saturably to these His tags, allowing distance analysis of FRET measured from each D-FKBP variant to Cy3NTA bound to each His tag. Results indicate that D-FKBP binds proximal to both N-terminal and central domains of RyR1, thus suggesting that the FKBP binding site is composed of determinants from both regions. These findings further imply that the RyR1 N-terminal and central domains are proximal to one another, a core premise of the domain-switch hypothesis of RyR function. We observed FRET from GFP fused at position 620 within the N-terminal domain to central domain His-tagged sites, thus further supporting this hypothesis. Taken together, these results support the conclusion that N-terminal and central domain elements are closely apposed near the FKBP binding site within the RyR1 three-dimensional structure.     

A non-canonical transferable signal mediates nuclear import of simian immunodeficiency virus Vpx protein

Protein transport into the nucleus is generally considered to involve specific nuclear localization signals (NLS) though it is becoming increasingly evident that efficient and well controlled import of proteins which lack a canonical NLS also occurs in cells. Vpx, a 112 amino acid protein from human immunodeficiency virus type 2 (HIV-2) and the closely related simian immunodeficiency virus (SIV) is one such protein, which does not have an identifiable canonical NLS and is yet efficiently imported to the nuclear compartment. Here we report that Vpx protein is imported to the nucleus independently of virus-encoded cofactors. When fusions of truncated versions of Vpx with full-length beta-galactosidase (beta-Gal) were tested, the region from Vpx 61 to 80 was found to be sufficient to mediate the import of the heterologous cytoplasmic protein to the nucleus. Inactivation of Vpx NLS precluded nuclear import of Vpx and reduced virus replication in non-dividing macrophage cultures, even when functional integrase and Gag matrix proteins implicated in viral nuclear import were present. Importantly, we identified and characterized a novel type of 20 amino acid transferable nuclear import signal in Vpx that is distinct from other import signals described. In addition, we show that the minimal nuclear targeting domain identified here overlaps with helical domain III (amino acid (aa) 64-82) and the structural integrity of this helical motif is critical for the nuclear import of Vpx. Taken together, these data suggest that Vpx is imported to the nucleus via a novel import pathway that is dependent on its 20 amino acid unique nuclear targeting signal, and that the nuclear import property of Vpx is critical for the optimal virus replication in non-dividing cells such as macrophages.     

Structural and kinetic analysis of drug resistant mutants of HIV-1 protease.

Mutants of HIV-1 protease that are commonly selected on exposure to different drugs, V82S, G48V, N88D and L90M, showed reduced catalytic activity compared to the wild-type protease on cleavage site peptides, CA-p2, p6pol-PR and PR-RT, critical for viral maturation. Mutant V82S is the least active (2-20% of wild-type protease), mutants N88D, R8Q, and L90M exhibit activities ranging from 20 to 40% and G48V from 50 to 80% of the wild-type activity. In contrast, D30N is variable in its activity on different substrates (10-110% of wild-type), with the PR-RT site being the most affected. Mutants K45I and M46L, usually selected in combination with other mutations, showed activities that are similar to (60-110%) or greater than (110-530%) wild-type, respectively. No direct relationship was observed between catalytic activity, inhibition, and structural stability. The mutants D30N and V82S were similar to wild-type protease in their stability toward urea denaturation, while R8Q, G48V, and L90M showed 1.5 to 2.7-fold decreased stability, and N88D and K45I showed 1.6 to 1.7-fold increased stability. The crystal structures of R8Q, K45I and L90M mutants complexed with a CA-p2 analog inhibitor were determined at 2.0, 1.55 and 1.88 A resolution, respectively, and compared to the wild-type structure. The intersubunit hydrophobic contacts observed in the crystal structures are in good agreement with the relative structural stability of the mutant proteases. All these results suggest that viral resistance does not arise by a single mechanism.     

Pseudomonas aeruginosa RRALC3 Enhances the Biomass,Nutrient and Carbon Contents of Pongamia pinnata Seedlings in Degraded Forest Soil

The study was aimed at assessing the effects of indigenous Plant Growth Promoting Bacterium (PGPB) on the legume Pongamia pinnata in the degraded soil of the Nanmangalam Reserve Forest (NRF) under nursery conditions. In total, 160 diazotrophs were isolated from three different nitrogen-free semi-solid media (LGI, Nfb, and JMV). Amongst these isolates, Pseudomonas aeruginosa RRALC3 exhibited the maximum ammonia production and hence was selected for further studies. RRALC3 was found to possess multiple plant growth promoting traits such as nitrogen accumulation (120.6ppm); it yielded a positive amplicon with nifH specific primers, tested positive for Indole Acetic Acid (IAA; 18.3μg/ml) and siderophore production, tested negative for HCN production and was observed to promote solubilization of phosphate, silicate and zinc in the plate assay. The 16S rDNA sequence of RRALC3 exhibited 99% sequence similarity to Pseudomonas aeruginosa JCM5962. Absence of virulence genes and non-hemolytic activity indicated that RRALC3 is unlikely to be a human pathogen. When the effects of RRALC3 on promotion of plant growth was tested in Pongamia pinnata, it was observed that in Pongamia seedlings treated with a combination of RRALC3 and chemical fertilizer, the dry matter increased by 30.75%. Nitrogen, phosphorus and potassium uptake increased by 34.1%, 27.08%, and 31.84%, respectively, when compared to control. Significant enhancement of total sugar, amino acids and organic acids content, by 23.4%, 29.39%, and 26.53% respectively, was seen in the root exudates of P. pinnata. The carbon content appreciated by 4-fold, when fertilized seedlings were treated with RRALC3. From the logistic equation, the rapid C accumulation time of Pongamia was computed as 43 days longer than the control when a combination of native PGPB and inorganic fertilizer was applied. The rapid accumulation time of N, P and K in Pongamia when treated with the same combination as above was 15, 40 and 33 days longer, respectively, as compared to the control.