Amerindian and NonAmerindian Autosome Molecular Variability – A Test Analysis

Analysis of 404 microsatellites, and 2–9 site haplotypes obtained considering 17 loci, all of them widely distributed over the human chromosomes and retrieved from two large data banks, yielded basically the same results. Colonization of the Americas may have led to some loss of genetic variability, but the range of differences found among five Native American populations was two times higher than those found between the most variable Amerindian (Maya) and a control Yoruba sample. Differentiation within the continent and the relatively recent history of these groups should be always considered in any discussion of Native American genetic variability.     

A Novel μCT Analysis Reveals Different Responses of Bioerosion and Secondary Accretion to Environmental Variability

Corals build reefs through accretion of calcium carbonate (CaCO₃) skeletons, but net reef growth also depends on bioerosion by grazers and borers and on secondary calcification by crustose coralline algae and other calcifying invertebrates. However, traditional field methods for quantifying secondary accretion and bioerosion confound both processes, do not measure them on the same time-scale, or are restricted to 2D methods. In a prior study, we compared multiple environmental drivers of net erosion using pre- and post-deployment micro-computed tomography scans (μCT; calculated as the % change in volume of experimental CaCO₃ blocks) and found a shift from net accretion to net erosion with increasing ocean acidity. Here, we present a novel μCT method and detail a procedure that aligns and digitally subtracts pre- and post-deployment μCT scans and measures the simultaneous response of secondary accretion and bioerosion on blocks exposed to the same environmental variation over the same time-scale. We tested our method on a dataset from a prior study and show that it can be used to uncover information previously unattainable using traditional methods. We demonstrated that secondary accretion and bioerosion are driven by different environmental parameters, bioerosion is more sensitive to ocean acidity than secondary accretion, and net erosion is driven more by changes in bioerosion than secondary accretion.     

Phytoremediation—A Novel and Promising Approach for Environmental Clean-up

ABSTRACT

Phytoremediation is an eco friendly approach for remediation of contaminated soil and water using plants. Phytoremediation is comprised of two components, one by the root colonizing microbes and the other by plants themselves, which degrade the toxic compounds to further non-toxic metabolites. Various compounds, viz. organic compounds, xenobiotics, pesticides and heavy metals, are among the contaminants that can be effectively remediated by plants. Plant cell cultures, hairy roots and algae have been studied for their ability to degrade a number of contaminants. They exhibit various enzymatic activities for degradation of xenobiotics, viz. dehalogenation, denitrification leading to breakdown of complex compounds to simple and non-toxic products. Plants and algae also have the ability to hyper accumulate various heavy metals by the action of phytochelatins and metallothioneins forming complexes with heavy metals and translocate them into vacuoles. Molecular cloning and expression of heavy metal accumulator genes and xenobiotic degrading enzyme coding genes resulted in enhanced remediation rates, which will be helpful in making the process for large-scale application to remediate vast areas of contaminated soils. A few companies worldwide are also working on this aspect of bioremediation, mainly by transgenic plants to replace expensive physical or chemical remediation techniques. Selection and testing multiple hyperaccumulator plants, protein engineering of phytochelatin and membrane transporter genes and their expression would enhance the rate of phytoremediation, making this process a successful one for bioremediation of environmental contamination. Recent years have seen major investments in the R&D, which have also resulted in competition of filing patents by several companies for economic gains. The details of science & technology related to phytoremediation have been discussed with a focus on future trends and prospects of global relevance.     

A Novel Approach to Solve the “Missing Marker Problem” in Marker-Based Motion Analysis That Exploits the Segment Coordination Patterns in Multi-Limb Motion Data

Marker-based human motion analysis is an important tool in clinical research and in many practical applications. Missing marker information caused by occlusions or a marker falling off is a common problem impairing data quality. The current paper proposes a conceptually new gap filling algorithm and presents results from a proof-of-principle analysis. The underlying idea of the proposed algorithm was that a multitude of internal and external constraints govern human motion and lead to a highly subject-specific movement pattern in which all motion variables are intercorrelated in a specific way. Two principal component analyses were used to determine how the coordinates of a marker with gaps correlated with the coordinates of the other, gap-free markers. Missing marker data could then be reconstructed through a series of coordinate transformations. The proposed algorithm was tested by reconstructing artificially created gaps in a 20-step walking trial and in an 18-s one-leg balance trial. The measurement accuracy’s dependence on the marker position, the length of the gap, and other parameters were evaluated. Even if only 2 steps of walking or 1.8 s of postural sway (10% of the whole marker data) were provided as input in the current study, the reconstructed marker trajectory differed on average no more than 11 mm from the originally measured trajectory. The reconstructed result improved further, on average, to distances below 5 mm if the marker trajectory was available more than 50% of the trial. The results of this proof-of-principle analysis supported the assumption that missing marker information can be reconstructed from the intercorrelations between marker coordinates, provided that sufficient data with complete marker information is available. Estimating missing information cannot be avoided entirely in many situations in human motion analysis. For some of these situations, the proposed reconstruction method may provide a better solution than what is currently available.     

Detecting Change in Biological Rhythms: A Multivariate Permutation Test Approach to Fourier‐Transformed Data

Treatment‐related changes in neurobiological rhythms are of increasing interest to psychologists, psychiatrists, and biological rhythms researchers. New methods for analyzing change in rhythms are needed, as most common methods disregard the rich complexity of biological processes. Large time series data sets reflect the intricacies of underlying neurobiological processes, but can be difficult to analyze. We propose the use of Fourier methods with multivariate permutation test (MPT) methods for analyzing change in rhythms from time series data. To validate the use of MPT for Fourier‐transformed data, we performed Monte Carlo simulations and compared statistical power and family‐wise error for MPT to Bonferroni‐corrected and uncorrected methods. Results show that MPT provides greater statistical power than Bonferroni‐corrected tests, while appropriately controlling family‐wise error. We applied this method to human, pre‐ and post‐treatment, serially‐sampled neurotransmitter data to confirm the utility of this method using real data. Together, Fourier with MPT methods provides a statistically powerful approach for detecting change in biological rhythms from time series data.     

Bionics and Structural Biology： A Novel Approach for Bio-energy Production

Cellular metabolism is a very complex process. The biochemical pathways are fundamental structures of biology. These pathways possess a number of regeneration steps which facilitate energy shuttling on a massive scale. This facilitates the biochemical pathways to sustain the energy currency of the cells. This concept has been mimicked using electronic circuit components and it has been used to increase the efficiency of bio-energy generation. Six of the carbohydrate biochemical pathways have been chosen in which glycolysis is the principle pathway. All the six pathways are interrelated and coordinated in a complex manner. Mimic circuits have been designed for all the six biochemical pathways. The components of the metabolic pathways such as enzymes, cofactors etc., are substituted by appropriate electronic circuit components. Enzymes are related to the gain of transistors by the bond dissociation energies of enzyme-substrate molecules under consideration. Cofactors and coenzymes are represented by switches and capacitors respectively. Resistors are used for proper orientation of the circuits. The energy obtained from the current methods employed for the decomposition of organic matter is used to trigger the mimic circuits. A similar energy shuttle is observed in the mimic circuits and the percentage rise for each cycle of circuit functioning is found to be 78.90. The theoretical calculations have been made using a sample of domestic waste weighing 1.182 kg. The calculations arrived at finally speak of the efficiency of the novel methodology employed.     

A Novel Quantitative Approach to Women’s Reproductive Strategies

The patterned way in which individuals allocate finite resources to various components of reproduction (e.g. mating effort, reproductive timing and parental investment) is described as a reproductive strategy. As energy is limited, trade-offs between and within aspects of reproductive strategies are expected. The first aim of this study was to derive aspects of reproductive strategies using complete reproductive histories from 718 parous Western Australian women. Factor analysis using a subset of these participants resulted in six factors that represented ‘short-term mating strategy’, ‘early onset of sexual activity’, ‘reproductive output’, ‘timing of childbearing’, ‘breastfeeding’, and ‘child spacing’. This factor structure was internally validated by replication using a second independent subset of the data. The second aim of this study examined trade-offs between aspects of reproductive strategies derived from aim one. Factor scores calculated for each woman were incorporated in generalised linear models and interaction terms were employed to examine the effect of mating behaviour on the relationships between reproductive timing, parental investment and overall reproductive success. Early sexual activity correlates with early reproductive onset for women displaying more long-term mating strategies. Women with more short-term mating strategies exhibit a trade-off between child quantity and child quality not observed in women with a long-term mating strategy. However, women with a short-term mating strategy who delay reproductive timing exhibit levels of parental investment (measured as breastfeeding duration per child) similar to that of women with long-term mating strategies. Reproductive delay has fitness costs (fewer births) for women displaying more short-term mating strategies. We provide empirical evidence that reproductive histories of contemporary women reflect aspects of reproductive strategies, and associations between these strategic elements, as predicted from life history theory.     

A Novel Approach to Delayed-Start Analyses for Demonstrating Disease-Modifying Effects in Alzheimer’s Disease

One method for demonstrating disease modification is a delayed-start design, consisting of a placebo-controlled period followed by a delayed-start period wherein all patients receive active treatment. To address methodological issues in previous delayed-start approaches, we propose a new method that is robust across conditions of drug effect, discontinuation rates, and missing data mechanisms. We propose a modeling approach and test procedure to test the hypothesis of noninferiority, comparing the treatment difference at the end of the delayed-start period with that at the end of the placebo-controlled period. We conducted simulations to identify the optimal noninferiority testing procedure to ensure the method was robust across scenarios and assumptions, and to evaluate the appropriate modeling approach for analyzing the delayed-start period. We then applied this methodology to Phase 3 solanezumab clinical trial data for mild Alzheimer’s disease patients. Simulation results showed a testing procedure using a proportional noninferiority margin was robust for detecting disease-modifying effects; conditions of high and moderate discontinuations; and with various missing data mechanisms. Using all data from all randomized patients in a single model over both the placebo-controlled and delayed-start study periods demonstrated good statistical performance. In analysis of solanezumab data using this methodology, the noninferiority criterion was met, indicating the treatment difference at the end of the placebo-controlled studies was preserved at the end of the delayed-start period within a pre-defined margin. The proposed noninferiority method for delayed-start analysis controls Type I error rate well and addresses many challenges posed by previous approaches. Delayed-start studies employing the proposed analysis approach could be used to provide evidence of a disease-modifying effect. This method has been communicated with FDA and has been successfully applied to actual clinical trial data accrued from the Phase 3 clinical trials of solanezumab.     

Golden GATEway Cloning – A Combinatorial Approach to Generate Fusion and Recombination Constructs

The design and generation of DNA constructs is among the necessary but generally tedious tasks for molecular biologists and, typically, the cloning strategy is restricted by available restriction sites. However, increasingly sophisticated experiments require increasingly complex DNA constructs, with an intricacy that exceeds what is achievable using standard cloning procedures. Many transgenes such as inducible gene cassettes or recombination elements consist of multiple components that often require precise in-frame fusions. Here, we present an efficient protocol that facilitates the generation of these complex constructs. The golden GATEway cloning approach presented here combines two established cloning methods, namely golden Gate cloning and Multisite Gateway^TM cloning. This allows efficient and seamless assembly as well as reuse of predefined DNA elements. The golden Gate cloning procedure follows clear and simple design rules and allows the assembly of multiple fragments with different sizes into one open reading frame. The final product can be directly integrated into the widely used Multisite Gateway^TM cloning system, granting more flexibility when using a transgene in the context of multiple species. This adaptable and streamlined cloning procedure overcomes restrictions of “classical construct generation” and allows focusing on construct design.     

Histological Criteria for Encapsulating Peritoneal Sclerosis – A Standardized Approach

Background

The two most relevant pathologies of long-term peritoneal dialysis (PD) are simple sclerosis and encapsulating peritoneal sclerosis (EPS). The histological differentiation of those two entities is difficult. The Aim of the study was to establish a method to standardize and facilitate the differentiation between simple sclerosis and EPS

Methods

We investigated 58 peritoneal biopsies - 31 EPS patients and 27 PD patients. Two blinded investigators analyzed 20 histological characteristics in EPS and PD patients.

Results

The following findings were significantly more common in EPS than in patients on PD without EPS: fibroblast like cells (FLC) (p<0.0001), mesothelial denudation (p<0.0001), decreased cellularity (p = 0.008), fibrin deposits (p<0.03), Fe deposits (p = 0.05), podoplanin vascular (p<0.0001), podoplanin avascular (p<0.0001). Using all predictor variables we trained the classification method Random Forest to categorize future cases. Podoplanin vascular and avascular were taken together (p<0.0001), FLC (p<0.0001), mesothelial denudation (p = 0.0005), calcification (p = 0.0026), acellular areas (p = 0.0094), and fibrin deposits (p = 0.0336) showed up as significantly important predictor variables. Estimated misclassification error rate when classifying new cases turned out to be 14%.

Conclusion

The introduced statistical method allows discriminating between simple sclerosis and EPS. The misclassification error will likely improve with every new case added to the database.     

A Novel Approach for Characterization of Cathepsin D Protease and Its Effect on Tau and ��-Amyloid Proteins

Cathepsin D is the lysosomal protease abundantly expressed in the brain. It plays an important role in the regulation of cellular apoptosis. In addition, cathepsin D has been shown to be involved in the pathogenesis of Alzheimer disease and autism. In this study, we developed a novel approach for the preparation of highly purified cathepsin D from the calf brain. This high grade purification is achieved by using DEAE-Sephacel Chromatography before the final step of applying to the Pepstatin-Sepharose 4B column. The properties of cathepsin D have also been studied. We show that cathepsin D cleaves both tau and β-amyloid precursor protein (APP). Both tau and APP are involved in the pathogenesis of Alzheimer's disease. Our findings strongly suggest a link between the lysosomal dysfunction of cathepsin D and the etiology of Alzheimer's disease. Our findings also indicate that cathepsin D could be a new approach to treating Alzheimer's disease.     

A Click Approach to Novel D-Ring-Substituted 16α-Triazolylestrone Derivatives and Characterization of Their Antiproliferative Properties

A simple and efficient synthesis of novel, D-ring substituted estrone derivatives containing a 16α-triazolyl moiety is described. Two epimeric azido alcohols (16α-azido-17α-hydroxy and 16α-azido-17β-hydroxy) of estra-1,3,5(10)-triene-3-methyl ether were prepared, followed by copper(I)-catalyzed azide-alkyne cycloaddition with various terminal alkynes. The steroidal triazoles were obtained in high yields and their activities against three human cancer cell lines (HeLa, MCF7 and A431) were screened. The most effective analogs were submitted to additional experiments in order to characterize their antiproliferative properties. As evidenced by flow cytometry, the selected steroids induced a disturbance in the HeLa cell cycle in a concentration- and exposure-dependent manner, through an increase of the hypodiploid population (subG1) and a cell cycle arrest in the G2/M phase. A noncancerous human fibroblast cell line (MRC5) was used to determine the selectivities of these compounds. Fluorescent microscopy after Hoechst 33258 - propidium iodide (HOPI) double staining revealed nuclear condensation and disturbed cell membrane integrity. The enhanced activities of caspase-3 and caspase-9 without activation of caspase-8 in the treated cells indicated the activation of the intrinsic pathway of apoptosis. The levels of cell cycle regulators (CDK1, cyclin B1/B2 and cdc25B) were decreased and the ratio Bax/Bcl-2 was increased 24 h after the treatment of HeLa cells (determined at an mRNA level by means of an RT-PCR technique). Under the same conditions, two agents elicited substantially increased degrees of phosphorylation of stathmin, as evidenced by Western blotting. The presented results demonstrate that these steroids can be regarded as appropriate structural scaffolds for the design and synthesis of further steroid analogs as innovative drug candidates with good efficacy.     

Life or Death? A Physiogenomic Approach to Understand Individual Variation in Responses to Hemorrhagic Shock

Severe hemorrhage due to trauma is a major cause of death throughout the world. It has often been observed that some victims are able to withstand hemorrhage better than others. For decades investigators have attempted to identify physiological mechanisms that distinguish survivors from nonsurvivors for the purpose of providing more informed therapies. As an alternative approach to address this issue, we have initiated a research program to identify genes and genetic mechanisms that contribute to this phenotype of survival time after controlled hemorrhage. From physiogenomic studies using inbred rat strains, we have demonstrated that this phenotype is a heritable quantitative trait, and is therefore a complex trait regulated by multiple genes. Our work continues to identify quantitative trait loci as well as potential epigenetic mechanisms that might influence survival time after severe hemorrhage. Our ultimate goal is to improve survival to traumatic hemorrhage and attendant shock via regulation of genetic mechanisms and to provide knowledge that will lead to genetically-informed personalized treatments.     

A Bayesian Approach to Pathway Analysis by Integrating Gene–Gene Functional Directions and Microarray Data

Many statistical methods have been developed to screen for differentially expressed genes associated with specific phenotypes in the microarray data. However, it remains a major challenge to synthesize the observed expression patterns with abundant biological knowledge for more complete understanding of the biological functions among genes. Various methods including clustering analysis on genes, neural network, Bayesian network and pathway analysis have been developed toward this goal. In most of these procedures, the activation and inhibition relationships among genes have hardly been utilized in the modeling steps. We propose two novel Bayesian models to integrate the microarray data with the putative pathway structures obtained from the KEGG database and the directional gene–gene interactions in the medical literature. We define the symmetric Kullback–Leibler divergence of a pathway, and use it to identify the pathway(s) most supported by the microarray data. Monte Carlo Markov Chain sampling algorithm is given for posterior computation in the hierarchical model. The proposed method is shown to select the most supported pathway in an illustrative example. Finally, we apply the methodology to a real microarray data set to understand the gene expression profile of osteoblast lineage at defined stages of differentiation. We observe that our method correctly identifies the pathways that are reported to play essential roles in modulating bone mass.     

A Novel Two-Tag System for Monitoring Transport and Cleavage through the Classical Secretory Pathway – Adaptation to HIV Envelope Processing

The classical secretory pathway is essential for the transport of a host of proteins to the cell surface and/or extracellular matrix. While the pathway is well-established, many factors still remain to be elucidated. One of the most relevant biological processes that occur during transport involves the cleavage of pro-proteins by enzymes residing in the endoplasmic reticulum/Golgi/TransGolgi Network compartment. Teasing out the requirements involved in the classical secretory pathway and cleavage during transport would shed new light into mis-regulation leading to disease. Current methodologies fail to link transport and cleavage at the single cell level. Here, we describe a cell-based assay that relies on an engineered protein scaffold that can discriminate between transport to the cell surface, in the absence or presence of cleavage. Our novel two-tag system works in a robust and quantitative manner and distinguishes between cleaved and non-cleaved events based on cell surface expression of one or two epitope tags, respectively. Here, we have used the HIV-1 envelope as a substrate, which is cleaved during transport, as proof of principle. Importantly, this assay can be easily coupled to existing siRNA-based screens to identify novel regulators and effectors involved in transport and/or cleavage of cell surface proteins. In addition, unlike other in vivo based assays, the assay described here can also be easily adapted to drug discovery purposes.     

A Novel Two-Stage Approach for Epistasis Detection in Genome-Wide Case–Control Studies

A significant challenge in epistasis detection is the huge amount of data, which leads to combinatorial explosion. This study focuses on a two-stage approach for detecting epistasis only among single nucleotide polymorphisms (SNPs) that show some marginal effect. We present this two-stage approach based on the fusion of two criteria (TwoFC) to detect epistatic interactions. We fuse the G ² test and absolute probability difference function as a scoring function to measure the strength of association between SNPs and disease status. The fused scoring function is an excellent measure of the strength of such an association. The two-stage strategy greatly reduces the computation load on epistasis detection. We use both simulated data sets and a real disease data set to evaluate our method. The results of an experiment on the simulated data sets show that TwoFC exhibits high power and sample efficiency. The results of an experiment on the real disease data set show that our method performs well even with large-scale data sets.     

Genopal?: A Novel Hollow Fibre Array for Focused Microarray Analysis

Expression profiling of target genes in patient blood is a powerful tool for RNA diagnosis. Here, we describe Genopal™, a novel platform ideal for efficient focused microarray analysis. Genopal™, which consists of gel-filled fibres, is advantageous for high-quality mass production via large-scale slicing of the Genopal™ block. We prepared two arrays, infectant and autoimmunity, that provided highly reliable data in terms of repetitive scanning of the same and/or distinct microarrays. Moreover, we demonstrated that Genopal™ had sensitivity sufficient to yield signals in short hybridization times (0.5 h). Application of the autoimmunity array to blood samples allowed us to identify an expression pattern specific to Takayasu arteritis based on the Spearman rank correlation by comparing the reference profile with those of several autoimmune diseases and healthy volunteers (HVs). The comparison of these data with those obtained by other methods revealed that they exhibited similar expression profiles of many target genes. Taken together, these data demonstrate that Genopal™ is an advantageous platform for focused microarrays with regard to its low cost, rapid results and reliable quality.