Genome-Wide Diet-Gene Interaction Analyses for Risk of Colorectal Cancer

Dietary factors, including meat, fruits, vegetables and fiber, are associated with colorectal cancer; however, there is limited information as to whether these dietary factors interact with genetic variants to modify risk of colorectal cancer. We tested interactions between these dietary factors and approximately 2.7 million genetic variants for colorectal cancer risk among 9,287 cases and 9,117 controls from ten studies. We used logistic regression to investigate multiplicative gene-diet interactions, as well as our recently developed Cocktail method that involves a screening step based on marginal associations and gene-diet correlations and a testing step for multiplicative interactions, while correcting for multiple testing using weighted hypothesis testing. Per quartile increment in the intake of red and processed meat were associated with statistically significant increased risks of colorectal cancer and vegetable, fruit and fiber intake with lower risks. From the case-control analysis, we detected a significant interaction between rs4143094 (10p14/near GATA3) and processed meat consumption (OR = 1.17; p = 8.7E-09), which was consistently observed across studies (p heterogeneity = 0.78). The risk of colorectal cancer associated with processed meat was increased among individuals with the rs4143094-TG and -TT genotypes (OR = 1.20 and OR = 1.39, respectively) and null among those with the GG genotype (OR = 1.03). Our results identify a novel gene-diet interaction with processed meat for colorectal cancer, highlighting that diet may modify the effect of genetic variants on disease risk, which may have important implications for prevention.     

Rapid Functional Analysis in Xenopus Oocytes of Po Protein Adhesive Interactions

We have developed a coupled Xenopus oocyte expression system for evaluating the functional effects of mutations in known or suspected adhesion molecules, which allows for a very rapid assessment of intercellular adhesion. As a model protein, we first used Protein zero (Po), an adhesion molecule that mediates self-adhesion of the Schwann cell plasma membrane to form compact myelin in the mammalian PNS. A wide variety of mutations in Po cause certain human peripheral neuropathies, such as the Charcot-Marie-Tooth disease (CMT) type 1B and Dejerine-Sottas syndrome (DSS). After wild-type Po mRNA is injected, the protein is synthesized and correctly targeted to the oocyte cell surface. When two oocytes are paired, wild-type Po redistributes and concentrates at the cell-cell apposition region, and by electron microscopy, the oocyte pairs show close cell-cell appositions and are devoid of the microvilli that are observed in uninjected oocyte pairs. These are hallmark features of highly adhesive cell:cell interfaces. Several point mutations in Po were engineered, corresponding to the molecular defects in the CMT type 1B or DSS. The proteins encoded by these mutations reached the cell surface but failed to concentrate at the oocyte interface. Po carrying a point mutation that is found in DSS is not targeted on the plasma membrane and fail to accumulate at the cell-cell contact site.     

Modulatory Interactions of Resting-State Brain Functional Connectivity

The functional brain connectivity studies are generally based on the synchronization of the resting-state functional magnetic resonance imaging (fMRI) signals. Functional connectivity measures usually assume a stable relationship over time; however, accumulating studies have reported time-varying properties of strength and spatial distribution of functional connectivity. The present study explored the modulation of functional connectivity between two regions by a third region using the physiophysiological interaction (PPI) technique. We first identified eight brain networks and two regions of interest (ROIs) representing each of the networks using a spatial independent component analysis. A voxel-wise analysis was conducted to identify regions that showed modulatory interactions (PPI) with the two ROIs of each network. Mostly, positive modulatory interactions were observed within regions involved in the same system. For example, the two regions of the dorsal attention network revealed modulatory interactions with the regions related to attention, while the two regions of the extrastriate network revealed modulatory interactions with the regions in the visual cortex. In contrast, the two regions of the default mode network (DMN) revealed negative modulatory interactions with the regions in the executive network, and vice versa, suggesting that the activities of one network may be associated with smaller within network connectivity of the competing network. These results validate the use of PPI analysis to study modulation of resting-state functional connectivity by a third region. The modulatory effects may provide a better understanding of complex brain functions.     

Functional Interactions of the Influenza Virus Glycoproteins

Hemagglutinin (HA) and neuraminidase (NA) are functionally related envelope glycoproteins of the influenza virus (Flu). HA interacts with terminal sialyl residues of oligosaccharides and ensures the binding of the virus particle to the cell surface. NA is a receptor-destroying enzyme that removes sialyl residues from oligosaccharides contained in cell and virus components and thereby prevents aggregation of virus particles. Analysis of reassortants combining low-functional NA of human Flu with HA of avian Flu showed that sialyl residues are not completely removed in some cases. With high HA affinity for sialyl substrates, such virus particles aggregate, aggregates accumulate on the cell surface, and virus yield decreases. Serial passaging of low-yield aggregating reassortants may lead to selection of high-yield variants, which do not aggregate. The loss of aggregation is due to a decrease in HA affinity for high-molecular-weight sialyl substrates. On evidence of sequencing of the HA gene in original reassortants and their nonaggregating variants, for different HA antigenic subtypes (H2, H3, H4, and H13) the affinity is reduced and aggregation lost through a common mechanism: an increase in the negative charge as a result of an amino acid substitution in the vicinity of the receptor-binding pocket of HA. Taken together, these findings suggest a way of postreassortment adaptation, which improves the functional match of HA and NA. The experimental system employed provides a model of natural processes associated with emergence of Flu variants having a pandemic potential.     

A Genetic Analysis of the Functional Interactions within Mycobacterium tuberculosis Single-Stranded DNA Binding Protein

Single-stranded DNA binding proteins (SSBs) are vital in all organisms. SSBs of Escherichia coli (EcoSSB) and Mycobacterium tuberculosis (MtuSSB) are homotetrameric. The N-terminal domains (NTD) of these SSBs (responsible for their tetramerization and DNA binding) are structurally well defined. However, their C-terminal domains (CTD) possess undefined structures. EcoSSB NTD consists of β1-β1′-β2-β3-α-β4-β45₁-β45₂-β5 secondary structure elements. MtuSSB NTD includes an additional β-strand (β6) forming a novel hook-like structure. Recently, we observed that MtuSSB complemented an E. coli Δssb strain. However, a chimeric SSB (mβ4-β5), wherein only the terminal part of NTD (β4-β5 region possessing L₄₅ loop) of EcoSSB was substituted with that from MtuSSB, failed to function in E. coli in spite of its normal DNA binding and oligomerization properties. Here, we designed new chimeras by transplanting selected regions of MtuSSB into EcoSSB to understand the functional significance of the various secondary structure elements within SSB. All chimeric SSBs formed homotetramers and showed normal DNA binding. The mβ4-β6 construct obtained by substitution of the region downstream of β5 in mβ4-β5 SSB with the corresponding region (β6) of MtuSSB complemented the E. coli strain indicating a functional interaction between the L₄₅ loop and the β6 strand of MtuSSB.     

Comparing DNA Extraction Methods for Analysis of Botanical Materials Found in Anti-Diabetic Supplements

A comparative performance evaluation of DNA extraction methods from anti-diabetic botanical supplements using various commercial kits was conducted, to determine which produces the best quality DNA suitable for PCR amplification, sequencing and species identification. All plant materials involved were of suboptimal quality showing various levels of degradation and therefore representing real conditions for testing herbal supplements. Eight different DNA extraction methods were used to isolate genomic DNA from 13 medicinal plant products. Two methods for evaluation, DNA concentration measurements that included absorbance ratios as well as PCR amplifiability, were used to determine quantity and quality of extracted DNA. We found that neither DNA concentrations nor commonly used UV absorbance ratio measurements at A ₂₆₀/A ₂₈₀ between 1.7 and 1.9 are suitable for globally predicting PCR success in these plant samples, and that PCR amplifiablity itself was the best indicator of extracted product quality. However, our results suggest that A ₂₆₀/A ₂₈₀ ratios below about 1.3 and above 2.3 indicated a DNA quality too poor to amplify. Therefore, A ₂₆₀/A ₂₈₀ measurements are not useful to identify samples that likely will amplify but can be used to exclude samples that likely will not amplify reducing the cost for unnecessarily subjecting samples to PCR. The two Nucleospin^® plant II kit extraction methods produced the most pure and amplifiable genomic DNA extracts. Our results suggest that there are clear, discernable differences between extraction methods for low quality plant samples in terms of producing contamination-free, high-quality genomic DNA to be used for further analysis.     

Functional Impact of ABCB1 Variants on Interactions between P-Glycoprotein and Methadone

Methadone is a widely used substitution therapy for opioid addiction. Large inter-individual variability has been observed in methadone maintenance dosages and P-glycoprotein (P-gp) was considered to be one of the major contributors. To investigate the mechanism of P-gp’s interaction with methadone, as well as the effect of genetic variants on the interaction, Flp-In™-293 cells stably transfected with various genotypes of human P-gp were established in the present study. The RNA and protein expression levels of human P-gp were confirmed by real-time quantitative RT-PCR and western blot, respectively. Utilizing rhodamine 123 efflux assay and calcein-AM uptake study, methadone was demonstrated to be an inhibitor of wild-type human P-gp via non-competitive kinetic (IC₅₀ = 2.17±0.10 µM), while the variant-type human P-gp, P-gp with 1236T-2677T-3435T genotype and P-gp with 1236T-2677A-3435T genotype, showed less inhibition potency (IC₅₀ = 2.97±0.09 µM and 4.43±1.10 µM, respectively) via uncompetitive kinetics. Methadone also stimulated P-gp ATPase and inhibited verapamil-stimulated P-gp ATPase activity under therapeutic concentrations. These results may provide a possible explanation for higher methadone dosage requirements in patients carrying variant-type of P-gp and revealed the possible drug-drug interactions in patients who receive concomitant drugs which are also P-gp substrates.     

Use of Herbal Supplements for Overactive Bladder

Anticholinergics, specifically antimuscarinic agents, are the most common medications prescribed for overactive bladder (OAB). The most common side effects of these agents are dry mouth and constipation, although other more concerning effects include changes in blood pressure, pulse rate, or heart rhythm when treatment is initiated. Herbal treatments are an increasingly popular alternative for treating OAB. A 2002 survey of US adults aged ≥ 18 years conducted by the Centers for Disease Control and Prevention indicated that 74.6% of those with OAB had used some form of complementary and alternative medicine. The World Health Organization estimates that 80% of the world’s population presently uses herbal medicine for some aspect of primary health care. Women were more likely than men to use complementary and alternative medicine. The authors review the most commonly used herbal medications for OAB.Key words: Overactive bladder, Herbal medicine, Gosha-jinki-gan, Hachi-mi-jio-gan, Buchu (Barosma betuline), Cleavers (Galium aparine), Cornsilk (Zea mays), Horsetail (Equisetum), Ganoderma lucidum, Resinferatoxin, CapsaicinOveractive bladder (OAB) is defined by the International Continence Society as a syndrome that includes urgency, with or without urge incontinence, frequency, and nocturia. The prevalence of OAB is estimated to range between 9% and 16%, depending on the population studied.^1–3 As symptoms of OAB increase with age, they can negatively impact quality of life (QoL).The cost of treating OAB is estimated to be approximately $12 billion annually in the United States.⁴ This estimate accounts for the direct cost of management, including protective undergarments, bedside commodes, and medical treatment, as well as indirect costs, such as those resulting from urinary tract infections and falls due to urgency and nocturia. There are also additional intangible costs that cannot be estimated such as pain, suffering, and poor QoL.The negative impact on health and the sense of well-being as well as the impairment in the ability to perform activities of daily living, has been well-documented. For example, elderly patients with OAB and subsequent incontinence are more likely to be admitted to nursing homes. Thom and colleagues reported a twofold increased risk of admission to a nursing facility for patients with incontinence.⁵ Urinary incontinence can also lead to anxiety, negative self-image, and isolation.⁴ Other problems associated with OAB include skin ulcerations and urinary tract infections. Nocturia is common with OAB and ranks among the most bothersome of lower urinary tract symptoms.⁶ In addition to sleep interruption and resulting fatigue, patients with nocturia may be more likely to suffer from falls and fractures, which are associated with high mortality in elderly patients. Approximately 33% of elderly people do not survive beyond 1 year after a hip fracture.⁷The impact of OAB was clearly reported in the National Overactive Bladder Evaluation (NOBLE) study.⁸ The NOBLE study represented a computer-assisted telephone interview survey that used health-related QoL (HRQoL) questionnaires to compare continent OAB patients, with incontinent OAB patients, and control groups in a nested case-control fashion. In this study, OAB was associated with lower QoL scores, higher scores on depression, and poorer sleep quality when compared with control subjects.⁸ Note that, although the prevalence of OAB increases with age, it should not be considered a normal consequence of aging.Anticholinergics, specifically antimuscarinic agents, represent the most common medications prescribed for OAB. The most common side effects are dry mouth and constipation.^9,10 Other more concerning side effects include changes in blood pressure, pulse rate, or heart rhythm when treatment is initiated. Additional adverse events (AEs) include memory loss, cognitive impairment, and balance problems. Thus, alternative therapies not involving standard medications and their associated risks are sought by patients to alleviate symptoms of OAB.Herbal treatments represent an increasingly popular alternative for treating OAB. A 2002 survey of US adults aged ≥ 18 years conducted by the Centers for Disease Control and Prevention indicated 74.6% of those with OAB had used some form of complementary and alternative medicine. The World Health Organization estimates that 80% of the world’s population presently uses herbal medicine for some aspect of primary health care. Women were more likely than men to use complementary and alternative medicine.^10,11 We review the most commonly used herbal medications used for OAB.     

Tools for Single-Cell Kinetic Analysis of Virus-Host Interactions

Measures of cellular gene expression or behavior, when performed on individual cells, inevitably reveal a diversity of behaviors and outcomes that can correlate with normal or diseased states. For virus infections, the potential diversity of outcomes are pushed to an extreme, where measures of infection reflect features of the specific infecting virus particle, the individual host cell, as well as interactions between viral and cellular components. Single-cell measures, while revealing, still often rely on specialized fluid handling capabilities, employ end-point measures, and remain labor-intensive to perform. To address these limitations, we consider a new microwell-based device that uses simple pipette-based fluid handling to isolate individual cells. Our design allows different experimental conditions to be implemented in a single device, permitting easier and more standardized protocols. Further, we utilize a recently reported dual-color fluorescent reporter system that provides dynamic readouts of viral and cellular gene expression during single-cell infections by vesicular stomatitis virus. In addition, we develop and show how free, open-source software can enable streamlined data management and batch image analysis. Here we validate the integration of the device and software using the reporter system to demonstrate unique single-cell dynamic measures of cellular responses to viral infection.     

Functional Analysis of the Autographa californica Multiple Nucleopolyhedrovirus GP64 Terminal Fusion Loops and Interactions with Membranes

The Autographa californica multiple nucleopolyhedrovirus (AcMNPV) glycoprotein GP64 is the major envelope protein of the budded virus (BV). GP64 is a class III fusion protein that mediates BV attachment to the cell surface and low-pH-triggered membrane fusion between the BV envelope and the endosome membrane during entry. Class III fusion proteins contain terminal looped structures that are believed to interact with membranes. To examine the functions of 3 loops found at the apex of the GP64 postfusion structure, we generated 2-alanine substitutions that scanned the two so-called fusion loops (loop 1 and loop 2) plus an adjacent loop structure (loop 3) that is closely attached to loop 2 and is also found at the apex of the GP64 postfusion structure. We identified essential residues from Y75 to T86 (loop 1) and N149 to H156 (loop 2) that are required for fusion activity, but no essential residues in loop 3. Further analysis revealed that critical fusion loop residues fall within two groups that are associated with either membrane merger (hemifusion) or fusion pore expansion. We next examined the interactions of soluble GP64 proteins and BV with membranes composed of various phospholipids. BV interacted directly with small unilamellar vesicles (SUVs) comprised of phospholipids phosphatidylcholine and phosphatidic acid (PC/PA) or phosphatidylcholine and phosphatidylserine (PC/PS) under neutral and acidic pH. We also examined the interactions of soluble GP64 constructs containing substitutions of the most hydrophobic residues within each of the two fusion loops. We found that a 2-residue substitution in either single loop (loop 1 [positions 81 and 82] or loop 2 [positions 153 and 154]) was not sufficient to substantially reduce the GP64-liposome interaction, but the same substitutions in both fusion loops severely reduced the GP64-liposome association at neutral pH. These results suggest that critical hydrophobic residues in both fusion loops may be involved in the interaction of GP64 with host cellular membranes and direct GP64-membrane interactions may represent a receptor-binding step prior to a low-pH-triggered conformational change.     

Functional Analysis of Variance for Association Studies

While progress has been made in identifying common genetic variants associated with human diseases, for most of common complex diseases, the identified genetic variants only account for a small proportion of heritability. Challenges remain in finding additional unknown genetic variants predisposing to complex diseases. With the advance in next-generation sequencing technologies, sequencing studies have become commonplace in genetic research. The ongoing exome-sequencing and whole-genome-sequencing studies generate a massive amount of sequencing variants and allow researchers to comprehensively investigate their role in human diseases. The discovery of new disease-associated variants can be enhanced by utilizing powerful and computationally efficient statistical methods. In this paper, we propose a functional analysis of variance (FANOVA) method for testing an association of sequence variants in a genomic region with a qualitative trait. The FANOVA has a number of advantages: (1) it tests for a joint effect of gene variants, including both common and rare; (2) it fully utilizes linkage disequilibrium and genetic position information; and (3) allows for either protective or risk-increasing causal variants. Through simulations, we show that FANOVA outperform two popularly used methods – SKAT and a previously proposed method based on functional linear models (FLM), – especially if a sample size of a study is small and/or sequence variants have low to moderate effects. We conduct an empirical study by applying three methods (FANOVA, SKAT and FLM) to sequencing data from Dallas Heart Study. While SKAT and FLM respectively detected ANGPTL 4 and ANGPTL 3 associated with obesity, FANOVA was able to identify both genes associated with obesity.     

Allelopathic Interactions and Allelochemicals: New Possibilities for Sustainable Weed Management

Weeds are known to cause enormous losses due to their interference in agroecosystems. Because of environmental and human health concerns, worldwide efforts are being made to reduce the heavy reliance on synthetic herbicides that are used to control weeds. In this regard the phenomenon of allelopathy, which is expressed through the release of chemicals by a plant, has been suggested to be one of the possible alternatives for achieving sustainable weed management. The use of allelopathy for controlling weeds could be either through directly utilizing natural allelopathic interactions, particularly of crop plants, or by using allelochemicals as natural herbicides. In the former case, a number of crop plants with allelopathic potential can be used as cover, smother, and green manure crops for managing weeds by making desired manipulations in the cultural practices and cropping patterns. These can be suitably rotated or intercropped with main crops to manage the target weeds (including parasitic ones) selectively. Even the crop mulch/residues can also give desirable benefits. Not only the terrestrial weeds, even allelopathy can be suitably manipulated for the management of aquatic weeds. The allelochemicals present in the higher plants as well as in the microbes can be directly used for weed management on the pattern of herbicides. Their bioefficacy can be enhanced by structural changes or the synthesis of chemical analogues based on them. Further, in order to enhance the potential of allelopathic crops, several improvements can be made with the use of biotechnology or genomics and proteomics. In this context either the production of allelochemicals can be enhanced or the transgenics with foreign genes encoding for a particular weed-suppressing allelochemical could be produced. In the former, both conventional breeding and molecular genetical techniques are useful. However, with conventional breeding being slow and difficult, more emphasis is laid on the use of modern techniques such as molecular markers and the selection aided by them. Although the progress in this regard is slow, nevertheless some promising results are coming and more are expected in future. This review attempts to discuss all these aspects of allelopathy for the sustainable management of weeds. Referee: Dr. Amrjits S. Basra, Central Plains Crop Technology, 5912 North Meridian Avenue, Wichita, KS 67204     

Functional Significance and Weight Properties of the Shell in Some Mollusks

Functional, morphological, and weight properties of the shell are discussed for many gastropods and bivalves living under various conditions.__________Translated from Izvestiya Akademii Nauk, Seriya Biologicheskaya, No. 4, 2005, pp. 479–505.Original Russian Text Copyright © 2005 by Alyakrinskaya.     

exo1-Dependent Mutator Mutations: Model System for Studying Functional Interactions in Mismatch Repair

EXO1 interacts with MSH2 and MLH1 and has been proposed to be a redundant exonuclease that functions in mismatch repair (MMR). To better understand the role of EXO1 in mismatch repair, a genetic screen was performed to identify mutations that increase the mutation rates caused by weak mutator mutations such as exo1Delta and pms1-A130V mutations. In a screen starting with an exo1 mutation, exo1-dependent mutator mutations were obtained in MLH1, PMS1, MSH2, MSH3, POL30 (PCNA), POL32, and RNR1, whereas starting with the weak pms1 allele pms1-A130V, pms1-dependent mutator mutations were identified in MLH1, MSH2, MSH3, MSH6, and EXO1. These mutations only cause weak MMR defects as single mutants but cause strong MMR defects when combined with each other. Most of the mutations obtained caused amino acid substitutions in MLH1 or PMS1, and these clustered in either the ATP-binding region or the MLH1-PMS1 interaction regions of these proteins. The mutations showed two other types of interactions: specific pairs of mutations showed unlinked noncomplementation in diploid strains, and the defect caused by pairs of mutations could be suppressed by high-copy-number expression of a third gene, an effect that showed allele and overexpressed gene specificity. These results support a model in which EXO1 plays a structural role in MMR and stabilizes multiprotein complexes containing a number of MMR proteins. A similar role is proposed for PCNA based on the data presented.