A novel human TPIP splice-variant (TPIP-C2) mRNA, expressed in human and mouse tissues, strongly inhibits cell growth in HeLa cells

Alternative splicing of mRNAs is known to involve a major regulation of gene expression at RNA level in mammalian cells. The PTEN (Phosphatase and TENsin homologue deleted from the human chromosome 10), TPTE (Transmembrane Phosphatase with TEnsin homology) and TPIP (TPTE and PTEN homologous Inositol lipid Phosphatase) belong to a family of dual-specific lipid and protein phosphatases. PTEN is a well characterized tumor suppressor, which plays crucial role in cell survival, cell cycle regulation, cell proliferation as well as adhesion, motility and migration of cells. The C2-domain of PTEN is essential for PTEN-functions. We have isolated a novel 1019 bp human TPIP cDNA (TPIP-C2) from a human testis cDNA library. In silico analysis of the cDNA revealed that it is produced from the TPIP-locus on the human chromosome 13 by alternative RNA-splicing. It has a unique 5'-Alu sequence, a LINE sequence followed by a 582 bp Open Reading Frame (ORF) encoding a 193 aa polypeptide with a partial phosphatase domain and a C2-domain. TPIP-C2 mRNA is expressed in human testis and in mouse tissues. Mouse testis and brain showed higher levels of TPIP-C2 mRNA in comparison to the heart, liver and kidney under normal physiological conditions. TPIP-C2 mRNAs from human and mouse testes show extensive sequence identity. Over-expression of TPIP-C2 cDNA in HeLa cells strongly (up to 85%) inhibited cell growth/proliferation and caused apoptosis in a caspase 3-dependent manner. These findings suggest for the first time that a TPIP splice-variant mRNA with a partial phosphatase domain and a C2-domain is expressed in cells and tissues of human and murine origins under normal physiological conditions. Inhibition of cell growth/proliferation and induction of apoptosis by overexpression of TPIP-C2 mRNA in HeLa cells suggest that it may be involved in negative regulation of cell growth/proliferation.     

Anti-Cancer Efficacy of Silybin Derivatives - A Structure-Activity Relationship

Silybin or silibinin, a flavonolignan isolated from Milk thistle seeds, is one of the popular dietary supplements and has been extensively studied for its antioxidant, hepatoprotective and anti-cancer properties. We have envisioned that potency of silybin could be further enhanced through suitable modification/s in its chemical structure. Accordingly, here, we synthesized and characterized a series of silybin derivatives namely 2,3-dehydrosilybin (DHS), 7-O-methylsilybin (7OM), 7-O-galloylsilybin (7OG), 7,23-disulphatesilybin (DSS), 7-O-palmitoylsilybin (7OP), and 23-O-palmitoylsilybin (23OP); and compared their anti-cancer efficacy using human bladder cancer HTB9, colon cancer HCT116 and prostate carcinoma PC3 cells. In all the 3 cell lines, DHS, 7OM and 7OG demonstrated better growth inhibitory effects and compared to silybin, while other silybin derivatives showed lesser or no efficacy. Next, we prepared the optical isomers (A and B) of silybin, DHS, 7OM and 7OG, and compared their anti-cancer efficacy. Isomers of these three silybin derivatives also showed better efficacy compared with respective silybin isomers, but in each, there was no clear cut silybin A versus B isomer activity preference. Further studies in HTB cells found that DHS, 7OM and 7OG exert better apoptotic activity than silibinin. Clonogenic assays in HTB9 cells further confirmed that both the racemic mixtures as well as pure optical isomers of DHS, 7OM and 7OG were more effective than silybin. Overall, these results clearly suggest that the anti-cancer efficacy of silybin could be significantly enhanced through structural modifications, and identify strong anti-cancer efficacy of silybin derivatives, namely DHS, 7OM, and 7OG, signifying that their efficacy and toxicity should be evaluated in relevant pre-clinical cancer models in rodents.     

A population balance model to modulate shear for the control of aggregation in Taxus suspension cultures

Cellular aggregation in plant suspension cultures directly affects the accumulation of high value products, such as paclitaxel from Taxus. Through application of mechanical shear by repeated, manual pipetting through a 10 ml pipet with a 1.6 mm aperture, the mean aggregate size of a Taxus culture can be reduced without affecting culture growth. When a constant level of mechanical shear was applied over eight generations, the sheared population was maintained at a mean aggregate diameter 194 μm lower than the unsheared control, but the mean aggregate size fluctuated by over 600 μm, indicating unpredictable culture variability. A population balance model was developed to interpret and predict disaggregation dynamics under mechanical shear. Adjustable parameters involved in the breakage frequency function of the population balance model were estimated by nonlinear optimization from experimentally measured size distributions. The optimized model predictions were in strong agreement with measured size distributions. The model was then used to determine the shear requirements to successfully reach a target aggregate size distribution. This model will be utilized in the future to maintain a culture with a constant size distribution with the goal of decreasing culture variability and increasing paclitaxel yields.     

Design of a functionally equivalent nonglycosylated analog of the glycopeptide antibiotic formaecin I

Various nonglycosylated analogs were designed in order to explore the role of glycosylation in formaecin I, an antibacterial glycopeptide of insect origin. The functional behavior of a designed nonglycosylated analog (P(7),endo P(8a),DeltaT(11))formaecin I was found to be similar to that of native glycosylated peptide. Both the peptides showed similar antibacterial activities against Escherichia coli and Salmonella strains. The designed nonglycosylated analog (P(7),endo P(8a),DeltaT(11))formaecin I has low binding affinity to LPS identical to that of native glycopeptide, formaecin I. Both the peptides have similar killing kinetics and are nontoxic to erythrocytes. Formaecin I and designed nonglycosylated (P(7),endo P(8a),DeltaT(11))formaecin I have no definite conformational features associated with them. The glycosylated residue of threonine in formaecin I and proline residues in designed peptide [(P(7),endo P(8a),DeltaT(11))formaecin I], possibly help in stabilizing the correct conformation that facilitates presentation of the peptide to its receptor. It is evident that a functionally equivalent nonglycosylated analog of native glycosylated antibacterial peptide can be designed by strategically modifying the sequence.     

DNA barcoding reveals the occurrence of cryptic species in host-associated population of Conogethes punctiferalis (Lepidoptera: Crambidae)

Conogethes punctiferalis (Guénee) is a critical pest that commonly infests castor (Ricinus communis Linnaeus) and cardamom (Elettaria cardamomum Maton) in India. The moths of both castor and cardamom appear to be similar in wing pattern and color. However, the results of behavioral studies elicited a doubt that there may be differences in terms of host specialization. In the present study, we conducted morphological studies and DNA barcode analyses using cytochrome oxidase I gene, which unraveled the mystery of C. punctiferalis breeding on castor and cardamom. The differences in male aedeagus and female bursae were prominent, yet, not sufficient enough to say that they are different species. The results showed high haplotype diversity (0.817 ± 0.073) and nucleotide diversity (0.0285 ± 0.002) in C. punctiferalis. In addition, topologies of neighbor-joining trees indicate that Conogethes sp. breeding on castor belongs to C. punctiferalis while those on cardamom are of a separate clade. Further genetic analysis revealed significant genetic differentiations among the two sampled populations, reflecting limited gene flow. Neutrality tests and mismatch distributions showed population expansion in C. punctiferalis, while the results of an analysis of molecular variance (AMOVA) indicated the existence of significant genetic variation among the examined host races. Conclusively, analysis using mitochondrial DNA showed an amount of genetic divergence between the two host-associated populations compatible with cryptic species rather than host races.     

MTHFR C677T Predisposes to POAG but Not to PACG in a North Indian Population: A Case Control Study

Hyperhomocysteinemia induced by the C677T genetic variant in MTHFR (methylenetetrahydrofolate reductase) has been implicated in neuronal cell death of retinal ganglion cells (RGC), which is a characteristic feature of glaucoma. However, association of MTHFR C677T with glaucoma has been controversial because of inconsistent results across association studies. Association between MTHFR C677T and glaucoma has not been reported in Indian population. Therefore, with a focus on neurodegenerative death of RGC in glaucoma, the current study aimed to investigate association of MTHFR C677T with Primary Open Angle Glaucoma (POAG) and Primary Angle Closure Glaucoma (PACG) in a North Indian population. A total of 404 participants (231 patients and 173 controls) were included in this study. Genotyping was performed by Polymerase Chain Reaction-Restriction Fragment Length Polymorphism. A few random samples were also tested by direct sequencing. Genotypic and allelic distributions of the POAG and PACG cohorts were compared to that of controls by chi-square test and odds ratios were reported with 95% confidence intervals. Genotypic and allelic distributions between POAG cases and controls were significantly different (p = 0.03 and p = 0.01 respectively). Unlike POAG, we did not find significant difference in the genotypic and allelic distributions of C677T between PACG cases and controls (p>0.05). We also observed a higher proportion of TT associated POAG in females than that in males. However, this is a preliminary indication of gender specific risk of C677T that needs to be replicated in a larger cohort of males and females. The present investigation on MTHFR C677T and glaucoma reveals that the TT genotype and T allele of this polymorphism are significant risk factors for POAG but not for PACG in North Indian population. Ours is the first report demonstrating association of MTHFR C677T with POAG but not PACG in individuals from North India.     

Cyanea capillata Bell Kinematics Analysis through Corrected In Situ Imaging and Modeling Using Strategic Discretization Techniques

Obtaining accurate kinematic data of animals is essential for many biological studies and bio-inspired engineering. Many animals, however, are either too large or too delicate to transport to controlled environments where accurate kinematic data can be easily obtained. Often, in situ recordings are the only means available but are often subject to multi-axis motion and relative magnification changes with time leading to large discrepancies in the animal kinematics. Techniques to compensate for these artifacts were applied to a large jellyfish, Cyanea capillata, freely swimming in ocean waters. The bell kinematics were captured by digitizing exumbrella profiles for two full swimming cycles. Magnification was accounted for by tracking a reference point on the ocean floor and by observing the C. capillata exumbrella arclength in order to have a constant scale through the swimming cycles. A linear fit of the top bell section was used to find the body angle with respect to the camera coordinate system. Bell margin trajectories over two swimming cycles confirmed the accuracy of the correction techniques. The corrected profiles were filtered and interpolated to provide a set of time-dependent points along the bell. Discrete models of the exumbrella were used to analyze the bell kinematics. Exumbrella discretization was conducted using three different methods. Fourier series were fitted to the discretized models and subsequently used to analyze the bell kinematics of the C. capillata. The analysis showed that the bell did not deform uniformly over time with different segments lagging behind each other. Looping of the bell trajectory between contraction and relaxation was also present through most of the exumbrella. The bell margin had the largest looping with an outer path during contraction and inner path during relaxation. The subumbrella volume was approximated based on the exumbrella kinematics and was found to increase during contraction.