Seeking unique and common biological themes in multiple gene lists or datasets: pathway pattern extraction pipeline for pathway-level comparative analysis

Background  

One of the challenges in the analysis of microarray data is to integrate and compare the selected (e.g., differential) gene lists from multiple experiments for common or unique underlying biological themes. A common way to approach this problem is to extract common genes from these gene lists and then subject these genes to enrichment analysis to reveal the underlying biology. However, the capacity of this approach is largely restricted by the limited number of common genes shared by datasets from multiple experiments, which could be caused by the complexity of the biological system itself.     

Piscidin 4, a novel member of the piscidin family of antimicrobial peptides

Piscidins are linear, amphipathic, antimicrobial peptides (AMPs) with broad, potent, activity spectrum. Piscidins and other members of the piscidin family appear to comprise the most common group of AMPs in teleost fish. All piscidins and related members of the piscidin family described to date are 18–26 amino acids long. We report here the isolation of a novel 5329.25 Da, 44-residue (FFRHLFRGAKAIFRGARQGXRAHKVVSRYRNRDVPETDNNQEEP) antimicrobial peptide from hybrid striped bass (Morone chrysops female x M. saxatilis male). We have named this peptide “piscidin 4” since it has considerable (to > 65%) N-terminal sequence homology to piscidins 1–3 and this distinctive, 10 to 11-residue, N-terminus is characteristic of piscidins. The native peptide has a modified amino acid at position 20 that, based upon mass spectrometry data, is probably a hydroxylated tryptophan. Synthetic piscidin 4 (with an unmodified tryptophan at position 20) has similar antibacterial activity to that of the native peptide. Piscidin 4 demonstrates potent, broad-spectrum, antibacterial activity against a number of fish and human pathogens, including multi-drug resistant bacteria. Its potent antimicrobial activity suggests that piscidin 4 plays a significant role in the innate defense system of hybrid striped bass.     

Subcellular Localization of the Barley Stripe Mosaic Virus Triple Gene Block Proteins

Barley stripe mosaic virus (BSMV) spreads from cell to cell through the coordinated actions of three triple gene block (TGB) proteins (TGB1, TGB2, and TGB3) arranged in overlapping open reading frames (ORFs). Our previous studies (D. M. Lawrence and A. O. Jackson, J. Virol. 75:8712-8723, 2001; D. M. Lawrence and A. O. Jackson, Mol. Plant Pathol. 2:65-75, 2001) have shown that each of these proteins is required for cell-to-cell movement in monocot and dicot hosts. We recently found (H.-S. Lim, J. N. Bragg, U. Ganesan, D. M. Lawrence, J. Yu, M. Isogai, J. Hammond, and A. O. Jackson, J. Virol. 82:4991-5006, 2008) that TGB1 engages in homologous interactions leading to the formation of a ribonucleoprotein complex containing viral genomic and messenger RNAs, and we have also demonstrated that TGB3 functions in heterologous interactions with TGB1 and TGB2. We have now used Agrobacterium tumefaciens-mediated protein expression in Nicotiana benthamiana leaf cells and site-specific mutagenesis to determine how TGB protein interactions influence their subcellular localization and virus spread. Confocal microscopy revealed that the TGB3 protein localizes at the cell wall (CW) in close association with plasmodesmata and that the deletion or mutagenesis of a single amino acid at the immediate C terminus can affect CW targeting. TGB3 also directed the localization of TGB2 from the endoplasmic reticulum to the CW, and this targeting was shown to be dependent on interactions between the TGB2 and TGB3 proteins. The optimal localization of the TGB1 protein at the CW also required TGB2 and TGB3 interactions, but in this context, site-specific TGB1 helicase motif mutants varied in their localization patterns. The results suggest that the ability of TGB1 to engage in homologous binding interactions is not essential for targeting to the CW. However, the relative expression levels of TGB2 and TGB3 influenced the cytosolic and CW distributions of TGB1 and TGB2. Moreover, in both cases, localization at the CW was optimal at the 10:1 TGB2-to-TGB3 ratios occurring in virus infections, and mutations reducing CW localization had corresponding effects on BSMV movement phenotypes. These data support a model whereby TGB protein interactions function in the subcellular targeting of movement protein complexes and the ability of BSMV to move from cell to cell.Plants use macromolecular trafficking pathways through plasmodesmata (PD) as a means to regulate developmental processes and physiological functions, and they also rely on these channels as avenues to communicate and mount defense responses to pathogen challenge (2, 37, 55). Local and systemic plant virus invasion depends on the abilities of viruses to use these pathways to spread from initially infected cells to the vascular tissue and distal regions of the plant. To this end, viruses infecting plants have evolved movement proteins (MPs) that coopt host trafficking pathways to target virus genomes to the PD and to facilitate the cell-to-cell transit of infectious entities (4, 13, 36, 48, 55). Virus MPs vary in size, number, and genome organization, but they share a number of functional characteristics including localization to PD, an ability to increase the size exclusion limits of PD, and RNA binding activities (3, 7, 8, 24, 27, 58).Viruses containing triple gene block (TGB) MPs have been the subjects of a number of investigations (4, 6, 39, 53, 54). Interestingly, viruses with a range of diverse genome structures encode MPs in a TGB, but these proteins fall into two major TGB classes that have substantial differences in protein structure and variations in their physical, functional, and cellular interactions (19, 30, 39, 45, 48). For example, the hordeivirus-like TGB1 proteins contain substantial N-terminal extensions that are lacking in the potexvirus-like TGB1 proteins, but the two classes of proteins share a conserved helicase domain at their C termini (39). The available evidence also indicates that hordeivirus-like and potexvirus-like TGB1 proteins share common biochemical features, including RNA binding abilities (3, 13, 23, 35, 44, 56), RNA helicase activities (22), associated NTPase activities (3, 13, 23, 33, 35, 44), and the capacity to form homologous interactions (29, 30, 45). However, the potexvirus-like TGB1 proteins localize at the CW when expressed autonomously and also facilitate increases in PD size exclusion limits, whereas the hordeivirus-like TGB1 proteins lack both these activities (39, 53). Major differences are also evident in the organizations of the potexvirus-like and hordeivirus-like TGB3 proteins, which share no discernible relatedness, differ in the numbers of their transmembrane domains, and indeed appear to have a polyphyletic origin (39).In both TGB classes, the movement strategy employs the coordinated actions of all three proteins. However, the coat protein is dispensable for one or more phases of movement of benyvirus, hordeivirus, pecluvirus, and pomovirus, encoding hordeivirus-like (class I) MPs, but is absolutely required for cell-to-cell movement of potexvirus-like (class II) MPs encoded by allexivirus, carlavirus, foveavirus, and potexvirus (6, 19, 39, 54). These variations clearly demonstrate that the two classes of TGB proteins have profound differences in their functional properties and in their associations with other virus and host proteins. Hence, comparative analyses of the functional and biological properties of the two classes of proteins in their common hosts may reveal important activities relevant to viral pathogenesis. To provide more information about the hordeivirus-like movement mechanisms, we are investigating the TGB interactions of Barley stripe mosaic virus (BSMV).BSMV is the type member of the genus Hordeivirus, which includes Poa semilatent virus (PSLV), Lychnis ringspot virus, and Anthoxanthum latent blanching virus (6, 19). Hordeiviruses have positive-sense, single-stranded RNA genomes consisting of three segments, designated α, β, and γ. The RNAβ segment encodes the coat protein, which is translated directly from genomic RNAβ (gRNAβ), and the TGB proteins, which are expressed from two subgenomic RNAs (sgRNAs), designated sgRNAβ1 and sgRNAβ2 (60). The coat protein is dispensable for the systemic movement of BSMV (41), and mutational analyses indicate that the TGB1, TGB2, and TGB3 proteins are each essential for cell-to-cell movement in monocot and dicot hosts (28). The BSMV TGB1 (58-kDa) protein is expressed from sgRNAβ1 at higher levels than the smaller hydrophobic TGB2 (14-kDa) and TGB3 (17-kDa) proteins, which are coexpressed from the bicistronic sgRNAβ2 during replication (14, 60). BSMV TGB1 has binding activity for both single-stranded and double-stranded RNAs (13) and forms nucleoprotein complexes with each of the BSMV gRNAs and sgRNAs (30). The hordeivirus-like TGB1 proteins differ from the potexvirus-like TGB1 proteins in having longer N-terminal domains with positively charged amino acids, but both classes of proteins have conserved C-terminal NTPase/helicase domains (13, 39, 49). In BSMV, mutations of conserved amino acids within the TGB1 helicase motif abrogate cell-to-cell movement and alter subcellular localization in infected protoplasts (27). Plants infected with a BSMV β-green fluorescent protein-TGB1 (β-GFP-TGB1) reporter virus also exhibited paired foci on both sides of the CW, and the plasma membranes of infected protoplasts developed punctate foci (27). TGB1 and TGB2 are also essential for plasma membrane targeting because β-GFP-TGB1 reporter derivatives that were unable to express TGB2 or TGB3 fluoresce at perinuclear membranes of protoplasts (27). Particle bombardment studies with the related hordeivirus PSLV also suggested that the expression of TGB3 is required to shift the localization of TGB2 from the endoplasmic reticulum (ER) to the peripheral membranes (50), and transgenically expressed PSLV TGB3 appears to be associated with PD due to its colocalization with callose markers (17).We have recently shown that TGB2 and TGB3 interact physically and have identified single amino acids in each protein that are required for these interactions (19, 30). TGB3 also interacts with TGB1, and we have proposed that these interactions facilitate the transport of ribonucleoprotein (RNP) complexes to the PD (30). However, the effects of TGB protein interactions on subcellular localization have not been defined. Moreover, because of possible convergent evolution of the hordeivirus-like and potexvirus-like TGB-containing viruses (39), the mechanisms of action resulting in transport may differ among different genera or even among different virus species within a genus. To obtain more refined information about these processes, we have now expressed fluorescent TGB fusion proteins transiently in Nicotiana benthamiana leaf cells by Agrobacterium tumefaciens infiltration and have assessed the subcellular localization patterns of BSMV wild-type (wt) and mutant TGB derivatives that differ in their interactions. We also have carried out reverse genetic experiments with selected BSMV TGB mutants to provide a biological context for the localization patterns appearing during ectopic Agrobacterium expression. These findings are elaborated in a model for TGB interactions required for the cell-to-cell movement of BSMV.     

Scalable gene synthesis by selective amplification of DNA pools from high-fidelity microchips

Development of cheap, high-throughput and reliable gene synthesis methods will broadly stimulate progress in biology and biotechnology. Currently, the reliance on column-synthesized oligonucleotides as a source of DNA limits further cost reductions in gene synthesis. Oligonucleotides from DNA microchips can reduce costs by at least an order of magnitude, yet efforts to scale their use have been largely unsuccessful owing to the high error rates and complexity of the oligonucleotide mixtures. Here we use high-fidelity DNA microchips, selective oligonucleotide pool amplification, optimized gene assembly protocols and enzymatic error correction to develop a method for highly parallel gene synthesis. We tested our approach by assembling 47 genes, including 42 challenging therapeutic antibody sequences, encoding a total of ～35 kilobase pairs of DNA. These assemblies were performed from a complex background containing 13,000 oligonucleotides encoding ～2.5 megabases of DNA, which is at least 50 times larger than in previously published attempts.     

Interaction modes at protein hetero-dimer interfaces

Hetero dimer (different monomers) interfaces are involved in catalysis and regulation through the formation of interface active sites. This is critical in cell and molecular biology events. The physical and chemical factors determining the formation of the interface active sites is often large in numbers. The combined role of interacting features is frequently combinatorial and additive in nature. Therefore, it is important to determine the physical and chemical features of such interactions. A number of such features have been documented in literature since 1975. However, the use of such interaction features in the prediction of interaction partners and sites given their sequences is still a challenge. In a non-redundant dataset of 156 hetero-dimer structures determined by X-ray crystallography, the interacting partners are often varying in size and thus, size variation between subunits is an important factor in determining the mode of interface formation. The size of protein subunits interacting are either small-small, largelarge, medium-medium, large-small, large-medium and small-medium. It should also be noted that the interface formed between subunits have physical interactions at N terminal (N), C terminal (C) and middle (M) region of the protein with reference to their sequences in one dimension. These features are believed to have application in the prediction of interaction partners and sites from sequences. However, the use of such features for interaction prediction from sequence is not currently clear.     

Alternative Strategies to Reduce Maternal Mortality in India: A Cost-Effectiveness Analysis

Background

Approximately one-quarter of all pregnancy- and delivery-related maternal deaths worldwide occur in India. Taking into account the costs, feasibility, and operational complexity of alternative interventions, we estimate the clinical and population-level benefits associated with strategies to improve the safety of pregnancy and childbirth in India.

Methods and Findings

Country- and region-specific data were synthesized using a computer-based model that simulates the natural history of pregnancy (both planned and unintended) and pregnancy- and childbirth-associated complications in individual women; and considers delivery location, attendant, and facility level. Model outcomes included clinical events, population measures, costs, and cost-effectiveness ratios. Separate models were adapted to urban and rural India using survey-based data (e.g., unmet need for birth spacing/limiting, facility births, skilled birth attendants). Model validation compared projected maternal indicators with empiric data. Strategies consisted of improving coverage of effective interventions that could be provided individually or packaged as integrated services, could reduce the incidence of a complication or its case fatality rate, and could include improved logistics such as reliable transport to an appropriate referral facility as well as recognition of referral need and quality of care. Increasing family planning was the most effective individual intervention to reduce pregnancy-related mortality. If over the next 5 y the unmet need for spacing and limiting births was met, more than 150,000 maternal deaths would be prevented; more than US$1 billion saved; and at least one of every two abortion-related deaths averted. Still, reductions in maternal mortality reached a threshold (∼23%–35%) without including strategies that ensured reliable access to intrapartum and emergency obstetrical care (EmOC). An integrated and stepwise approach was identified that would ultimately prevent four of five maternal deaths; this approach coupled stepwise improvements in family planning and safe abortion with consecutively implemented strategies that incrementally increased skilled attendants, improved antenatal/postpartum care, shifted births away from home, and improved recognition of referral need, transport, and availability/quality of EmOC. The strategies in this approach ranged from being cost-saving to having incremental cost-effectiveness ratios less than US$500 per year of life saved (YLS), well below India''s per capita gross domestic product (GDP), a common benchmark for cost-effectiveness.

Conclusions

Early intensive efforts to improve family planning and control of fertility choices and to provide safe abortion, accompanied by a paced systematic and stepwise effort to scale up capacity for integrated maternal health services over several years, is as cost-effective as childhood immunization or treatment of malaria, tuberculosis, or HIV. In just 5 y, more than 150,000 maternal deaths would be averted through increasing contraception rates to meet women''s needs for spacing and limiting births; nearly US$1.5 billion would be saved by coupling safe abortion to aggressive family planning efforts; and with stepwise investments to improve access to pregnancy-related health services and to high-quality facility-based intrapartum care, more than 75% of maternal deaths could be prevented. If accomplished over the next decade, the lives of more than one million women would be saved. Please see later in the article for the Editors'' Summary     

Molecular architectures of trimeric SIV and HIV-1 envelope glycoproteins on intact viruses: strain-dependent variation in quaternary structure

The initial step in target cell infection by human, and the closely related simian immunodeficiency viruses (HIV and SIV, respectively) occurs with the binding of trimeric envelope glycoproteins (Env), composed of heterodimers of the viral transmembrane glycoprotein (gp41) and surface glycoprotein (gp120) to target T-cells. Knowledge of the molecular structure of trimeric Env on intact viruses is important both for understanding the molecular mechanisms underlying virus-cell interactions and for the design of effective immunogen-based vaccines to combat HIV/AIDS. Previous analyses of intact HIV-1 BaL virions have already resulted in structures of trimeric Env in unliganded and CD4-liganded states at ∼20 Å resolution. Here, we show that the molecular architectures of trimeric Env from SIVmneE11S, SIVmac239 and HIV-1 R3A strains are closely comparable to that previously determined for HIV-1 BaL, with the V1 and V2 variable loops located at the apex of the spike, close to the contact zone between virus and cell. The location of the V1/V2 loops in trimeric Env was definitively confirmed by structural analysis of HIV-1 R3A virions engineered to express Env with deletion of these loops. Strikingly, in SIV CP-MAC, a CD4-independent strain, trimeric Env is in a constitutively “open” conformation with gp120 trimers splayed out in a conformation similar to that seen for HIV-1 BaL Env when it is complexed with sCD4 and the CD4i antibody 17b. Our findings suggest a structural explanation for the molecular mechanism of CD4-independent viral entry and further establish that cryo-electron tomography can be used to discover distinct, functionally relevant quaternary structures of Env displayed on intact viruses.