Macroalgae and nutrients promote algal turf growth in the absence of herbivores

Coral Reefs - Closely cropped algal turfs are characteristic of healthy coral reefs, but unchecked growth can cause transitions into long sediment-laden turfs, which may be an alternative degraded...     

Complete cysteine-scanning mutagenesis of the Salmonella typhimurium melibiose permease

The melibiose permease of Salmonella typhimurium (MelB_St) catalyzes the stoichiometric symport of galactopyranoside with a cation (H⁺, Li⁺, or Na⁺) and is a prototype for Na⁺-coupled major facilitator superfamily (MFS) transporters presenting from bacteria to mammals. X-ray crystal structures of MelB_St have revealed the molecular recognition mechanism for sugar binding; however, understanding of the cation site and symport mechanism is still vague. To further investigate the transport mechanism and conformational dynamics of MelB_St, we generated a complete single-Cys library containing 476 unique mutants by placing a Cys at each position on a functional Cys-less background. Surprisingly, 105 mutants (22%) exhibit poor transport activities (<15% of Cys-less transport), although the expression levels of most mutants were comparable to that of the control. The affected positions are distributed throughout the protein. Helices I and X and transmembrane residues Asp and Tyr are most affected by cysteine replacement, while helix IX, the cytoplasmic middle-loop, and C-terminal tail are least affected. Single-Cys replacements at the major sugar-binding positions (K18, D19, D124, W128, R149, and W342) or at positions important for cation binding (D55, N58, D59, and T121) abolished the Na⁺-coupled active transport, as expected. We mapped 50 loss-of-function mutants outside of these substrate-binding sites that suffered from defects in protein expression/stability or conformational dynamics. This complete Cys-scanning mutagenesis study indicates that MelB_St is highly susceptible to single-Cys mutations, and this library will be a useful tool for further structural and functional studies to gain insights into the cation-coupled symport mechanism for Na⁺-coupled MFS transporters.     

A meta-analysis and genome-wide association study of platelet count and mean platelet volume in african americans

Several genetic variants associated with platelet count and mean platelet volume (MPV) were recently reported in people of European ancestry. In this meta-analysis of 7 genome-wide association studies (GWAS) enrolling African Americans, our aim was to identify novel genetic variants associated with platelet count and MPV. For all cohorts, GWAS analysis was performed using additive models after adjusting for age, sex, and population stratification. For both platelet phenotypes, meta-analyses were conducted using inverse-variance weighted fixed-effect models. Platelet aggregation assays in whole blood were performed in the participants of the GeneSTAR cohort. Genetic variants in ten independent regions were associated with platelet count (N?=?16,388) with p<5×10(-8) of which 5 have not been associated with platelet count in previous GWAS. The novel genetic variants associated with platelet count were in the following regions (the most significant SNP, closest gene, and p-value): 6p22 (rs12526480, LRRC16A, p?=?9.1×10(-9)), 7q11 (rs13236689, CD36, p?=?2.8×10(-9)), 10q21 (rs7896518, JMJD1C, p?=?2.3×10(-12)), 11q13 (rs477895, BAD, p?=?4.9×10(-8)), and 20q13 (rs151361, SLMO2, p?=?9.4×10(-9)). Three of these loci (10q21, 11q13, and 20q13) were replicated in European Americans (N?=?14,909) and one (11q13) in Hispanic Americans (N?=?3,462). For MPV (N?=?4,531), genetic variants in 3 regions were significant at p<5×10(-8), two of which were also associated with platelet count. Previously reported regions that were also significant in this study were 6p21, 6q23, 7q22, 12q24, and 19p13 for platelet count and 7q22, 17q11, and 19p13 for MPV. The most significant SNP in 1 region was also associated with ADP-induced maximal platelet aggregation in whole blood (12q24). Thus through a meta-analysis of GWAS enrolling African Americans, we have identified 5 novel regions associated with platelet count of which 3 were replicated in other ethnic groups. In addition, we also found one region associated with platelet aggregation that may play a potential role in atherothrombosis.     

Localized Populations of CD8low/? MHC Class I Tetramer+ SIV-Specific T Cells in Lymphoid Follicles and Genital Epithelium

CD8 T cells play an important role in controlling viral infections. We investigated the in situ localization of simian immunodeficiency virus (SIV)-specific T cells in lymph and genital tissues from SIV-infected macaques using MHC-class I tetramers. The majority of tetramer-binding cells localized in T cell zones and were CD8⁺. Curiously, small subpopulations of tetramer-binding cells that had little to no surface CD8 were detected in situ both early and late post-infection, and in both vaginally and rectally inoculated macaques. These tetramer⁺CD8^low/− cells were more often localized in apparent B cell follicles relative to T cell zones and more often found near or within the genital epithelium than the submucosa. Cells analyzed by flow cytometry showed similar populations of cells. Further immunohistological characterization revealed small populations of tetramer⁺CD20⁻ cells inside B cell follicles and that tetramer⁺ cells did not stain with γδ-TCR nor CD4 antibodies. Negative control tetramer staining indicated that tetramer⁺CD8^low/− cells were not likely NK cells non-specifically binding to MHC tetramers. These findings have important implications for SIV-specific and other antigen-specific T cell function in these specific tissue locations, and suggest a model in which antigen-specific CD8+ T cells down modulate CD8 upon entering B cell follicles or the epithelial layer of tissues, or alternatively a model in which only antigen-specific CD8 T cells that down-modulate CD8 can enter B cell follicles or the epithelium.     

Introduction of Exogenous Epitopes in the Variable Regions of the Human Immunodeficiency Virus Type 1 Envelope Glycoprotein: Effect on Viral Infectivity and the Neutralization Phenotype

In this study we examined whether human immunodeficiency virus type 1 (HIV-1) is equally susceptible to neutralization by a given antibody when the epitope of this antibody is introduced at different positions within the viral envelope glycoprotein (Env). To this end, we introduced two exogenous “epitope tags” at different locations within three major Env regions in two distinct HIV-1 isolates. We examined how the introduction of the exogenous epitopes affects Env expression, Env incorporation into virions, Env fusogenic potential, and viral susceptibility to neutralization. Our data indicate that even within the same Env region, the exact positioning of the epitope impacts the susceptibility of the virus to neutralization by the antibody that binds to that epitope. Our data also indicate that even if the same epitope is introduced in the exact same position on two different Envs, its exposure and, as a result, the neutralization susceptibility of the virus, can be very different. In contrast to the findings of previous studies conducted with HIV-1 isolates other than those used here, but in agreement with results obtained with simian immunodeficiency virus, we observed that tagging of the fourth variable region of Env (V4) did not result in neutralization by the anti-tag antibodies. Our data indicate that epitopes in V4 are not properly exposed within the functional HIV-1 trimeric Env spike, suggesting that V4 may not be a good target for vaccine-elicited neutralizing antibodies.The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) is expressed as a heavily glycosylated peptide of approximately 160 kDa (gp160), which is cleaved intracellularly into two noncovalently associated subunits: an extracellular subunit (gp120), responsible for CD4 and coreceptor (primarily CCR5 and/or CXCR4) binding, and a transmembrane subunit (gp41) that mediates fusion between viral and host cell membranes. Based on amino acid sequence homology analysis of gp120s derived from diverse HIV-1 isolates, gp120 is divided into five “constant” regions (C1 to C5) and five “variable” regions (also called “loops,” because most of them have cysteines in the N and C termini that form disulfide bonds). Despite their extensive amino acid variability, the variable loops of gp120 play central roles during the entry of the virus into the cell, for instance, by directly or indirectly modulating the interaction of Env with coreceptor molecules on the target surfaces during virus-cell fusion. They also offer protection from neutralizing antibodies (NAbs) by various mechanisms. The variable loops themselves are targets of NAbs, and during infection, the replicating virus accumulates mutations in the variable regions that allow it to escape the action of anti-variable loop-directed NAbs, while at the same time the variable loops are positioned within the Env trimer so that they prevent, or minimize, the binding of NAbs to more-conserved epitopes, such as the receptor and coreceptor binding sites (4, 5, 12, 15, 20, 23, 25, 27, 31).HIV-1 strains display distinct neutralization phenotypes. Some isolates, such as SF162, are generally susceptible to NAbs that bind to many distinct regions of Env, including the variable regions, while other isolates, such as YU2 or JRFL, are generally resistant to neutralization by the same NAbs (1). It has been proposed that irrespective of the overall neutralizing phenotype of HIV-1 isolates, the binding of only a single antibody per Env trimer on the virion surface can lead to neutralization, when all Env trimers present on the virion surface are bound by at least one antibody (32). This important observation also implies that the epitope specificity of an antibody may not be as important for neutralization as its ability to bind to its target within the trimeric Env structure. In fact, antibodies to diverse regions of Env, such as V1, V2, V3, and the receptor and coreceptor binding sites, can all neutralize HIV-1 (1, 3, 6, 8, 10, 18, 20, 23, 25, 27, 29, 30).In many cases, a given isolate will not be equally susceptible to neutralization by NAbs that bind to different Env regions, for example, the V3 loop and the CD4-binding site (CD4-BS). Whether differences in the neutralizing potentials of two antibodies that bind to distinct epitopes on HIV-1 Env are due to differences in the binding affinities of the two antibodies or whether they occur because the viruses are intrinsically more susceptible to NAbs that bind certain epitopes and not others (i.e., the relative importance of the various regions of Env in Env function and virus neutralization sensitivity differs) is not yet fully understood. One way to address these issues is to introduce small non-HIV Env amino acid sequences (tags) that are targets of known monoclonal antibodies (MAbs) at various positions within the viral Env and to examine how the placement of the same epitope at different positions within Env affects the neutralization phenotype of the virus.Foreign epitopes have been introduced into the variable regions of HIV and simian immunodeficiency virus (SIV) Envs, and their effects on viral neutralization potential have been examined (14, 19, 22, 33). Yang and colleagues (33) introduced the FLAG epitope into the V4 regions of three HIV-1 isolates (YU2, JRFL, and HxB2) displaying distinct neutralization phenotypes in response to anti-HIV NAbs; they found that all three pseudotyped viruses were equivalently neutralized by an anti-FLAG MAb. One important implication of that study is that neutralization-resistant isolates, such as YU2 or JRFL, are not intrinsically more resistant to neutralization than more-susceptible isolates, such as HxB2, so long as the antibody binds to its epitope on the functional virion-associated Env spike. A second implication is that since the FLAG epitope was exposed in the V4 loops of all three isolates, the V4 loop could theoretically be a good target for vaccine-elicited antibodies. In contrast, Pantophlet et al. (19) introduced the HA tag into various regions of the JRCSF (neutralization-resistant) and HxB2 (neutralization-sensitive) isolates and reported that JRCSF was intrinsically more resistant than HxB2 to anti-HA antibodies. This observation implies, therefore, that some HIV-1 strains (primary, neutralization-resistant strains) have developed mechanisms that limit the accessibility of multiple Env regions, including variable regions, to antibodies developed during infection. Laird and Desrosiers (14) introduced the FLAG epitope into two positions within each of the V1, V2, and V4 loops of SIV239 and SIV316. They reported that the functionality of Env was differentially affected by the precise location of the exogenous tag sequence within the variable loops examined. Importantly, and in contrast to what was reported for the HIV-1 isolates mentioned above, the SIV239 variants containing a V4 FLAG epitope were not neutralized by an anti-FLAG MAb. It appeared, however, that the FLAG epitope was not well exposed on the trimeric Env when introduced into the V4 loop of SIV but was exposed when introduced into the V1 loop of the same virus. Potentially, this means that the V4 loop is differentially exposed in the context of the HIV-1 and SIV Envs.The FLAG epitope (DYKDDDDK) is highly charged. Therefore, it is possible that the effect on Env function and epitope exposure could differ if a different exogenous epitope were inserted instead of FLAG. Here we examined the effect of variable loop tagging on the Env functions and viral neutralization phenotypes of two primary HIV-1 clade B isolates, SF162 (CCR5 tropic) and SF33 (CXCR4 tropic), using two exogenous epitopes (FLAG and hemagglutinin [HA] tags) positioned at multiple locations within the V1, V2, and V4 loops. By placing the same tag in several regions within each loop, we investigated the accessibilities of various parts of the same loop to a given NAb. By using two tags that differ significantly in amino acid composition (FLAG tag, DYKDDDDK; HA tag, YPYDVPDYA), we aimed at distinguishing between the effects of amino acid composition and the positioning of the tag on Env function and overall epitope exposure. Finally, identical evaluations of R5 and X4 Envs may provide information about the relative roles played in neutralization by variable loops in Envs displaying distinct coreceptor usage. We report that both the amino acid sequence and the position of the tag within and among the variable loops greatly affected the functionality of Env. In contrast to previous observations made with other HIV-1 Envs (33) but in agreement with what was reported for the SIV239 Env (14), we observed that tagging of the V4 loops of SF162 and SF33 did not render these isolates susceptible to neutralization by the corresponding anti-tag MAbs.     

Closing gaps in the human genome using sequencing by synthesis

The most recent release of the finished human genome contains 260 euchromatic gaps (excluding chromosome Y). Recent work has helped explain a large number of these unresolved regions as 'structural' in nature. Another class of gaps is likely to be refractory to clone-based approaches, and cannot be approached in ways previously described. We present an approach for closing these gaps using 454 sequencing. As a proof of principle, we closed all three remaining non-structural gaps in chromosome 15.     

Anisotropic atomic motions in high-resolution protein crystallography molecular dynamics simulations

Molecular dynamics (MD) simulations using empirical force fields are popular for the study of proteins. In this work, we compare anisotropic atomic fluctuations in nanosecond-timescale MD simulations with those observed in an ultra-high-resolution crystal structure of crambin. In order to make our comparisons, we have developed a compact graphical technique for assessing agreement between spatial atomic distributions determined by MD simulations and observed anisotropic temperature factors.     

Spontaneously arising mutL mutators in evolving Escherichia coli populations are the result of changes in repeat length

Over the course of thousands of generations of growth in a glucose-limited environment, 3 of 12 experimental populations of Escherichia coli spontaneously and independently evolved greatly increased mutation rates. In two of the populations, the mutations responsible for this increased mutation rate lie in the same region of the mismatch repair gene mutL. In this region, a 6-bp repeat is present in three copies in the gene of the wild-type ancestor of the experimental populations but is present in four copies in one of the experimental populations and two copies in the other. These in-frame mutations either add or delete the amino acid sequence LA in the MutL protein. We determined that the replacement of the wild-type sequence with either of these mutations was sufficient to increase the mutation rate of the wild-type strain to a level comparable to that of the mutator strains. Complementation of strains bearing the mutator mutations with wild-type copies of either mutL or the mismatch repair gene uvrD rescued the wild-type mutation rate. The position of the mutator mutations-in the region of MutL known as the ATP lid-suggests a possible deficiency in MutL's ATPase activity as the cause of the mutator phenotype. The similarity of the two mutator mutations (despite the independent evolutionary histories of the populations that gave rise to them) leads to a discussion of the potential adaptive role of DNA repeats.     

Effects of i and i+3 residue identity on cis-trans isomerism of the aromatic(i+1)-prolyl(i+2) amide bond: implications for type VI beta-turn formation

Cis-trans isomerization of amide bonds plays critical roles in protein molecular recognition, protein folding, protein misfolding, and disease. Aromatic-proline sequences are particularly prone to exhibit cis amide bonds. The roles of residues adjacent to a tyrosine-proline residue pair on cis-trans isomerism were examined. A short series of peptides XYPZ was synthesized and cis-trans isomerism was analyzed. Based on these initial studies, a series of peptides XYPN, X = all 20 canonical amino acids, was synthesized and analyzed by NMR for i residue effects on cis-trans isomerization. The following effects were observed: (a) aromatic residues immediately preceding Tyr-Pro disfavor cis amide bonds, with K(trans/cis)= 5.7-8.0, W > Y > F; (b) proline residues preceding Tyr-Pro lead to multiple species, exhibiting cis-trans isomerization of either or both X-Pro amide bonds; and (c) other residues exhibit similar values of K(trans/cis) (= 2.9-4.2), with Thr and protonated His exhibiting the highest fraction cis. beta-Branched and short polar residues were somewhat more favorable in stabilizing the cis conformation. Phosphorylation of serine at the i position modestly increases the stability of the cis conformer. In addition, the effect of the i+3 residue was examined in a limited series of peptides TYPZ. NMR data indicated that aromatic residues, Pro, Asn, Ala, and Val at the i+3 residue all favor cis amide bonds, with aromatic residues and Asn favoring more compact phi at Tyr(cis) and Ala and Pro favoring more extended phi at Tyr(cis). D-Alanine at the i+3 position particularly disfavors cis amide bonds.