Diosgenin from Dioscorea bulbifera: Novel Hit for Treatment of Type II Diabetes Mellitus with Inhibitory Activity against α-Amylase and α-Glucosidase

Diabetes mellitus is a multifactorial metabolic disease characterized by post-prandial hyperglycemia (PPHG). α-amylase and α-glucosidase inhibitors aim to explore novel therapeutic agents. Herein we report the promises of Dioscorea bulbifera and its bioactive principle, diosgenin as novel α-amylase and α-glucosidase inhibitor. Among petroleum ether, ethyl acetate, methanol and 70% ethanol (v/v) extracts of bulbs of D. bulbifera, ethyl acetate extract showed highest inhibition upto 72.06 ± 0.51% and 82.64 ± 2.32% against α-amylase and α-glucosidase respectively. GC-TOF-MS analysis of ethyl acetate extract indicated presence of high diosgenin content. Diosgenin was isolated and identified by FTIR, ¹H NMR and ¹³C NMR and confirmed by HPLC which showed an α-amylase and α-glucosidase inhibition upto 70.94 ± 1.24% and 81.71 ± 3.39%, respectively. Kinetic studies confirmed the uncompetitive mode of binding of diosgenin to α-amylase indicated by lowering of both Km and Vm. Interaction studies revealed the quenching of intrinsic fluorescence of α-amylase in presence of diosgenin. Similarly, circular dichroism spectrometry showed diminished negative humped peaks at 208 nm and 222 nm. Molecular docking indicated hydrogen bonding between carboxyl group of Asp300, while hydrophobic interactions between Tyr62, Trp58, Trp59, Val163, His305 and Gln63 residues of α-amylase. Diosgenin interacted with two catalytic residues (Asp352 and Glu411) from α-glucosidase. This is the first report of its kind that provides an intense scientific rationale for use of diosgenin as novel drug candidate for type II diabetes mellitus.     

The Synthetic Tryptanthrin Analogue Suppresses STAT3 Signaling and Induces Caspase Dependent Apoptosis via ERK Up Regulation in Human Leukemia HL-60 Cells

Tryptanthrin is a natural product which has been reported to have several medicinal properties. In this study, we tried to investigate the detailed molecular mechanism of its bromo analogue (TBr), a potent cytotoxic agent in the induction of cancer cell death. It was found that TBr primarily targets STAT3 and ERK signaling during the induction of apoptosis in several human leukemia cell lines. In HL-60 cells, TBr treatment caused early down regulation of p-STAT3 with concomitant up regulation of p-ERK which led to the activation of intrinsic and extrinsic pathways of apoptosis. The mechanism of TBr mediated inhibition of p-STAT3 was found to be due to the activation of ubiquitin dependent degradation of tyrosine 705 and serine 727 p-STAT3. As IL-6 is the main driver of the STAT3 pathway, the effect of TBr on cell death was subdued when treated in the combination with IL-6 in HL60 cells. Interestingly, PD98059 significantly reduced the apoptotic effects of TBr, thus showing the direct involvement of p-ERK in TBr mediated cell death. It was further shown that apoptotic protein Bax silencing in HL-60 cells resists TBr mediated ERK dependent apoptosis. In summary, for the first time we report the mechanism of TBr mediated cell death in human leukemia cell lines by targeting STAT3 and ERK pathways.     

A Protocol for Genetic Induction and Visualization of Benign and Invasive Tumors in Cephalic Complexes of Drosophila melanogaster

Drosophila has illuminated our understanding of the genetic basis of normal development and disease for the past several decades and today it continues to contribute immensely to our understanding of complex diseases ^1-7. Progression of tumors from a benign to a metastatic state is a complex event ⁸ and has been modeled in Drosophila to help us better understand the genetic basis of this disease ⁹. Here I present a simple protocol to genetically induce, observe and then analyze the progression of tumors in Drosophila larvae. The tumor induction technique is based on the MARCM system ¹⁰ and exploits the cooperation between an activated oncogene, Ras^V12 and loss of cell polarity genes (scribbled, discs large and lethal giant larvae) to generate invasive tumors ⁹. I demonstrate how these tumors can be visualized in the intact larvae and then how these can be dissected out for further analysis. The simplified protocol presented here should make it possible for this technique to be utilized by investigators interested in understanding the role of a gene in tumor invasion.     

Are Subject-Specific Musculoskeletal Models Robust to the Uncertainties in Parameter Identification?

Subject-specific musculoskeletal modeling can be applied to study musculoskeletal disorders, allowing inclusion of personalized anatomy and properties. Independent of the tools used for model creation, there are unavoidable uncertainties associated with parameter identification, whose effect on model predictions is still not fully understood. The aim of the present study was to analyze the sensitivity of subject-specific model predictions (i.e., joint angles, joint moments, muscle and joint contact forces) during walking to the uncertainties in the identification of body landmark positions, maximum muscle tension and musculotendon geometry. To this aim, we created an MRI-based musculoskeletal model of the lower limbs, defined as a 7-segment, 10-degree-of-freedom articulated linkage, actuated by 84 musculotendon units. We then performed a Monte-Carlo probabilistic analysis perturbing model parameters according to their uncertainty, and solving a typical inverse dynamics and static optimization problem using 500 models that included the different sets of perturbed variable values. Model creation and gait simulations were performed by using freely available software that we developed to standardize the process of model creation, integrate with OpenSim and create probabilistic simulations of movement. The uncertainties in input variables had a moderate effect on model predictions, as muscle and joint contact forces showed maximum standard deviation of 0.3 times body-weight and maximum range of 2.1 times body-weight. In addition, the output variables significantly correlated with few input variables (up to 7 out of 312) across the gait cycle, including the geometry definition of larger muscles and the maximum muscle tension in limited gait portions. Although we found subject-specific models not markedly sensitive to parameter identification, researchers should be aware of the model precision in relation to the intended application. In fact, force predictions could be affected by an uncertainty in the same order of magnitude of its value, although this condition has low probability to occur.     

An oxidative and salinity stress induced peroxisomal ascorbate peroxidase from Avicennia marina: Molecular and functional characterization

APX (EC, 1.11.1.11) has a key role in scavenging ROS and in protecting cells against their toxic effects in algae and higher plants. A cDNA encoding a peroxisomal ascorbate peroxidase, Am-pAPX1, was isolated from salt stressed leaves of Avicennia marina (Forsk.) Vierh. by EST library screening and its expression in the context of various environmental stresses was investigated. Am-pAPX1 contains an ORF of 286 amino acids coding for a 31.4kDa protein. The C-terminal region of the Am-pAPX1 ORF has a putative transmembrane domain and a peroxisomal targeting signal (RKKMK), suggesting peroxisomal localization. The peroxisomal localization of Am-pAPX1 was confirmed by stable transformation of the GFP-(Ala)(10)-Am-pAPX1 fusion in tobacco. RNA blot analysis revealed that Am-pAPX1 is expressed in response to salinity (NaCl) and oxidative stress (high intensity light, hydrogen peroxide application and excess iron). The isolated genomic clone of Am-pAPX1 was found to contain nine exons. A fragment of 1616bp corresponding to the 5' upstream region of Am-pAPX1 was isolated by TAIL-PCR. In silico analysis of this sequence reveals the presence of putative light and abiotic stress regulatory elements.     

Genetic characterization of antibiotic-resistant Staphylococcus aureus from milk in the North-West Province,South Africa

Food borne diseases are a major public health concern worldwide. Staphylococcus aureus is one of the potential food borne pathogens which causes nosocomial and community acquired infections. In the present study, 74 representative strains of S. aureus isolated and characterized in previous study from different milk samples were subjected to random amplified polymorphic DNA (RAPD) polymerase chain reaction (PCR) and enterobacterial repetitive intergenic consensus (ERIC)-PCR to generate fingerprints to determine the genetic relationships of the isolated strains. A total of 20 RAPD patterns were generated and the number of amplified fragments obtained ranged from 0 to 8 with molecular weight ranging from 250 to 2000 bp. A dendrogram based on fingerprinting pattern grouped isolates into twelve major clusters (I–XII). In the case of ERIC-PCR 9 banding patterns were obtained with amplicons ranging from 1 to 8 and band sizes ranging from 250 to 2000 bp. A total of four major clusters (I–IV) were observed in the dendrogram based on ERIC fingerprints. The discrete banding patterns obtained both from ERIC-PCR and RAPD-PCR showed remarkably the genetic diversity of S. aureus. The findings of this study indicate that raw, bulk and pasteurized milk in the North-West Province was contaminated with toxigenic and multi-drug resistant S. aureus strains. This emphasizes the need to implement appropriate control measures to reduce contamination as well as the spread of virulent S. aureus strains to reduce the burden of disease in humans.     

The relationship between the dominant Additional vein mutant in Drosophila melanogaster and engrailed.

Additional vein (Adv) is a dominant mutation that affects the first wing vein in Drosophila. It also manifests a recessive lethal phenotype and is associated with a large inversion. Using a combination of genetic and cytogenetic techniques, we show that Adv interacts with engrailed (en), likely because one of the inversion breakpoints interferes with en function. Genetic interaction studies reveal that Adv is lethal in trans with various lethal alleles of en and gives an engrailed-like wing phenotype with weak alleles of en. In situ hybridization to polytene chromosomes using en cDNA demonstrates that one of the inversion breakpoints lies within the en coding region. Although the cause of the wing phenotype is not determined herein, it likely is caused by the other inversion breakpoint interfering with a different function. The characterization of this mutation could expedite studies to understand what molecular events result in the Adv phenotype and thereby provide insight into the development of the first wing vein in Drosophila.     

Investigating spatial structure in specific tree species in ancient semi-natural woodland using remote sensing and marked point pattern analysis

Remote sensing classification has the potential to provide important information, such as tree species distribution maps, to ecologists, at a range of spatial and temporal scales. However, standard classification procedures often fail to provide the high accuracies required for many ecological applications. Previously, a modified remote sensing classification technique was used to provide very high classification accuracies for one or two classes (e.g. species) of interest. The aim of this paper was to demonstrate that the output from the method can be suitable for spatial ecological analyses, and to provide a generic simulation framework for assessing the adequacy of any given remote sensing classification for such analyses. Marked point pattern analysis (MPPA) was applied to tree species distribution data obtained for sycamore Acer pseudoplatanus and ash Fraxinus excelsior from a 400 ha ancient semi-natural woodland in southern England using the modified remote sensing classification method to test several hypotheses of ecological interest relating to the spatial distribution and interaction of these species. Monte Carlo simulation methods were then used to evaluate the data and data quality requirements of the MPPA to check that the classified tree species maps for sycamore and ash were adequate. Using the combined method the spatial distributions for sycamore and ash were found to be aggregated and inter-dependent at a range of spatial scales. Together, the remote sensing classification and simulation approaches provide the basis for exploiting more fully the potential of remote sensing to provide information of value to ecologists.