Effects of Device Limitations on Acquisition of the /t/-/k/ Contrast in Children With Cochlear Implants.

OBJECTIVES: The present study investigated how development of the /t/-/k/ contrast is affected by the unique perceptual constraints imposed on young children using cochlear implants (CIs). We hypothesized that children with CIs would demonstrate unique patterns of speech acquisition due to device limitations, rather than straightforward delays due to a lack of auditory input in the first year of life before implantation. This study focused on the contrast between /t/ and /k/ because it is acquired early in the sequence of development, requires less advanced motor control than later-acquired place contrasts, is differentiated by spectral cues (which are particularly degraded when processed by CIs), and is not easily differentiated by visual cues alone. Furthermore, perceptual confusability between /t/ and /k/ may be exacerbated in front-vowel contexts, where the spectral energy for /k/ is shifted to higher frequencies, creating more spectral overlap with /t/. DESIGN: Children with CIs (n = 26; ages 31 to 66 mo) who received implants around their first birthdays were matched to peers with normal hearing (NH). Children participated in a picture-prompted auditory word-repetition task that included over 30 tokens of word-initial /t/ and /k/ consonants. Tokens were balanced across front-vowel and back-vowel contexts to assess the effects of coarticulation. Productions were transcribed and coded for accuracy as well as the types of errors produced (manner of articulation, voicing, or place of articulation errors). Centroid frequency was also calculated for /t/ and /k/ tokens that were produced correctly. Mixed-effects models were used to compare accuracy, types of errors, and centroid frequencies across groups, target consonants, and vowel contexts. RESULTS: Children with CIs produced /t/ and /k/ less accurately than their peers in both front- and back-vowel contexts. Children with CIs produced /t/ and /k/ with equal accuracy, and /k/ was produced less accurately in front-vowel contexts than in back-vowel contexts. When they produced errors, children with CIs were more likely to produce manner errors and less likely to produce voicing errors than children with NH. Centroid frequencies for /t/ and /k/ were similar across groups, except for /k/ in front-vowel contexts: children with NH produced /k/ in front-vowel contexts with higher centroid frequency than children with CIs, and they produced /k/ and /t/ with equal centroid frequencies in front-vowel contexts. CONCLUSIONS: Children with CIs not only produced /t/ and /k/ less accurately than peers with NH, they also demonstrated idiosyncratic patterns of acquisition, likely resulting from receiving degraded and distorted spectral information critical for differentiating /t/ and /k/. Speech-language pathologists should consider perceptual confusability of consonants (and their allophonic variations) during their assessment and treatment of this unique population of children.

Quantifying robustness of the /t/-/k/ contrast using a single, static spectral feature.

Dynamic spectral shape features accurately classify /t/ and /k/ productions across speakers and contexts. This paper shows that word-initial /t/ and /k/ tokens produced by 21 adults can be differentiated using a single, static spectral feature when spectral energy concentration is considered relative to expectations within a given speaker and vowel context. Centroid and peak frequency-calculated from both acoustic and psychoacoustic spectra-were compared to determine whether one feature could reliably differentiate /t/ and /k/, and, if so, which feature best differentiated them. Centroid frequency from both acoustic and psychoacoustic spectra accurately classified productions of /t/ and /k/.

Complete genome sequences of mycobacteriophages Ageofdapage, Aubs, BABullseye, CheetoDust, ShaboiShabazz, and TomBrady.

We report genome sequences of six mycobacteriophages. Each virus was isolated from a soil sample and belongs to the siphovirus morphology. Genomes are 41,901-60,613 bp in length, contain between 62 and 103 protein-coding genes, with up to 40% of those genes having a predicted function.

Evidence of a Set of Core-Function Genes in 16 Bacillus Podoviral Genomes with Considerable Genomic Diversity.

Bacteriophage genomes represent an enormous level of genetic diversity and provide considerable potential to acquire new insights about viral genome evolution. In this study, the genome sequences of sixteen Bacillus-infecting bacteriophages were explored through comparative genomics approaches to reveal shared and unique characteristics. These bacteriophages are in the Salasmaviridae family with small (18,548-27,206 bp) double-stranded DNA genomes encoding 25-46 predicted open reading frames. We observe extensive nucleotide and amino acid sequence divergence among a set of core-function genes that present clear synteny. We identify two examples of sequence directed recombination within essential genes, as well as explore the expansion of gene content in these genomes through the introduction of novel open reading frames. Together, these findings highlight the complex evolutionary relationships of phage genomes that include old, common origins as well as new components introduced through mosaicism.

Instructional Models for Course-Based Research Experience (CRE) Teaching.

The course-based research experience (CRE) with its documented educational benefits is increasingly being implemented in science, technology, engineering, and mathematics education. This article reports on a study that was done over a period of 3 years to explicate the instructional processes involved in teaching an undergraduate CRE. One hundred and two instructors from the established and large multi-institutional SEA-PHAGES program were surveyed for their understanding of the aims and practices of CRE teaching. This was followed by large-scale feedback sessions with the cohort of instructors at the annual SEA Faculty Meeting and subsequently with a small focus group of expert CRE instructors. Using a qualitative content analysis approach, the survey data were analyzed for the aims of inquiry instruction and pedagogical practices used to achieve these goals. The results characterize CRE inquiry teaching as involving three instructional models: 1) being a scientist and generating data; 2) teaching procedural knowledge; and 3) fostering project ownership. Each of these models is explicated and visualized in terms of the specific pedagogical practices and their relationships. The models present a complex picture of the ways in which CRE instruction is conducted on a daily basis and can inform instructors and institutions new to CRE teaching.

Complete Genome Sequences of Bacillus cereus Group Phages AaronPhadgers, ALPS, Beyonphe, Bubs, KamFam, OmnioDeoPrimus, Phireball, PPIsBest, YungSlug, and Zainny.

Here, we report genome sequences of 10 Bacillus cereus group-infecting bacteriophages. Each virus was isolated from an environmental sample, contained a double-stranded DNA genome, and belonged to the Myoviridae family. Nine phages exhibit a conserved genome structure, and one phage appears novel in genome structure, sequence, and protein content.

Covalent binding of the natural antimicrobial peptide indolicidin to DNA abasic sites.

Indolicidin is a host defense tridecapeptide that inhibits the catalytic activity of HIV-1 integrase in vitro. Here we have elucidated its mechanism of integrase inhibition. Using crosslinking and mass spectrometric footprinting approaches, we found that indolicidin interferes with formation of the catalytic integrase-DNA complex by directly binding DNA. Further characterization revealed that the peptide forms covalent links with abasic sites. Indolicidin crosslinks single- or double-stranded DNAs and various positions of the viral cDNA with comparable efficiency. Using truncated and chemically modified peptides, we show that abasic site crosslinking is independent of the PWWP motif but involves the indolicidin unique lysine residue and the N- and C- terminal NH2 groups. Because indolicidin can also inhibit topoisomerase I, we believe that multiple actions at the level of DNA might be a common property of antimicrobial peptides.

Integrase inhibitors to treat HIV/AIDS.

HIV integrase is a rational target for treating HIV infection and preventing AIDS. It took approximately 12 years to develop clinically usable inhibitors of integrase, and Phase I clinical trials of integrase inhibitors have just begun. This review focuses on the molecular basis and rationale for developing integrase inhibitors. The main classes of lead compounds are also described, as well as the concept of interfacial inhibitors of protein-nucleic-acid interactions that might apply to the clinically used strand-transfer inhibitors.

Complete Genome Sequences of Bacillus Phages Janet and OTooleKemple52.

We report here the genome sequences of two novel Bacillus cereus group-infecting bacteriophages, Janet and OTooleKemple52. These bacteriophages are double-stranded DNA-containing Myoviridae isolated from soil samples. While their genomes share a high degree of sequence identity with one another, their host preferences are unique.

Discovery and Biochemical Characterization of PlyP56, PlyN74, and PlyTB40-Bacillus Specific Endolysins.

Three Bacillus bacteriophage-derived endolysins, designated PlyP56, PlyN74, and PlyTB40, were identified, cloned, purified, and characterized for their antimicrobial properties. Sequence alignment reveals these endolysins have an N-terminal enzymatically active domain (EAD) linked to a C-terminal cell wall binding domain (CBD). PlyP56 has a Peptidase_M15_4/VanY superfamily EAD with a conserved metal binding motif and displays biological dependence on divalent ions for activity. In contrast, PlyN74 and PlyTB40 have T7 lysozyme-type Amidase_2 and carboxypeptidase T-type Amidase_3 EADs, respectively, which are members of the MurNAc-LAA superfamily, but are not homologs and thus do not have a shared protein fold. All three endolysins contain similar SH3-family CBDs. Although minor host range differences were noted, all three endolysins show relatively broad antimicrobial activity against members of the Bacillus cereus sensu lato group with the highest lytic activity against B. cereus ATCC 4342. Characterization studies determined the optimal lytic activity for these enzymes was at physiological pH (pH 7.0⁻8.0), over a broad temperature range (4⁻55 °C), and at low concentrations of NaCl (<50 mM). Direct comparison of lytic activity shows the PlyP56 enzyme to be twice as effective at lysing the cell wall peptidoglycan as PlyN74 or PlyTB40, suggesting PlyP56 is a good candidate for further antimicrobial development as well as bioengineering studies.

Prophage-mediated defence against viral attack and viral counter-defence.

Temperate phages are common, and prophages are abundant residents of sequenced bacterial genomes. Mycobacteriophages are viruses that infect mycobacterial hosts including Mycobacterium tuberculosis and Mycobacterium smegmatis, encompass substantial genetic diversity and are commonly temperate. Characterization of ten Cluster N temperate mycobacteriophages revealed at least five distinct prophage-expressed viral defence systems that interfere with the infection of lytic and temperate phages that are either closely related (homotypic defence) or unrelated (heterotypic defence) to the prophage. Target specificity is unpredictable, ranging from a single target phage to one-third of those tested. The defence systems include a single-subunit restriction system, a heterotypic exclusion system and a predicted (p)ppGpp synthetase, which blocks lytic phage growth, promotes bacterial survival and enables efficient lysogeny. The predicted (p)ppGpp synthetase coded by the Phrann prophage defends against phage Tweety infection, but Tweety codes for a tetrapeptide repeat protein, gp54, which acts as a highly effective counter-defence system. Prophage-mediated viral defence offers an efficient mechanism for bacterial success in host-virus dynamics, and counter-defence promotes phage co-evolution.

Mechanisms and inhibition of HIV integration.

HIV integrase is required for viral replication and a rationale target for antiretroviral therapies. Integrase inhibitors are potentially complementary to current treatments. This review focuses on the mechanisms of HIV integration. The roles of viral and cellular co-factors during pre-integration complex (PIC) formation and integration are reviewed. The biochemical mechanisms of integration, integrase structures and approaches to inhibit integration are described.

Complete Genome Sequences of Nine Bacillus cereus Group Phages.

We report the sequences of nine novel Bacillus cereus group bacteriophages: DIGNKC, Juglone, Nemo, Nigalana, NotTheCreek, Phrodo, SageFayge, Vinny, and Zuko. These bacteriophages are double-stranded DNA-containing Myoviridae isolated from soil samples using B. thuringiensis subsp. kurstaki as the host bacterium.

HIV-1 integrase inhibitors: a decade of research and two drugs in clinical trial.

AIDS is currently treated with a combination therapy of reverse transcriptase and protease inhibitors. Recently, the FDA approved a drug targeting HIV-1 entry into cells. There are currently no FDA approved drugs targeting HIV-1 integrase, though many scientists and drug companies are actively in pursuit of clinically useful integrase inhibitors. The objective of this review is to provide an update on integrase inhibitors reported in the last two years, including two novel inhibitors in early clinical trials, recently developed hydroxylated aromatics, natural products, peptide, antibody and oligonucleotide inhibitors. Additionally, the proposed mechanism of diketo acid inhibition is reviewed.

Probing HIV-1 integrase inhibitor binding sites with position-specific integrase-DNA cross-linking assays.

HIV-1 integrase binds site-specifically to the ends of the viral cDNA. We used two HIV-1 integrase-DNA cross-linking assays to probe the binding sites of integrase inhibitors from different chemical families and with different strand transfer selectivities. The disulfide assay probes cross-linking between the integrase residue 148 and the 5'-terminal cytosine of the viral cDNA, and the Schiff base assay probes cross-linking between an integrase lysine residue and an abasic site placed at selected positions in the viral cDNA. Cross-linking interference by eight integrase inhibitors shows that the most potent cross-linking inhibitors are 3'-processing inhibitors, indicating that cross-linking assays probe the donor viral cDNA (donor binding site). In contrast, strand transfer-selective inhibitors provide weak cross-linking interference, consistent with their binding to a specific acceptor (cellular DNA) site. Docking and crystal structure studies illustrate specific integrase-inhibitor contacts that prevent cross-linking formation. Four inhibitors that prevented Schiff base cross-linking to the conserved 3'-terminal adenine position were examined for inhibition at various positions within the terminal 21 bases of the viral cDNA. Two of them selectively inhibited upper strand cross-linking, whereas the other two had a more global effect on integrase-DNA binding. These findings have implications for elucidating inhibitor binding sites and mechanisms of action. The cross-linking assays also provide clues to the molecular interactions between integrase and the viral cDNA.

Synthesis and HIV-1 integrase inhibitory activity of spiroundecane(ene) derivatives.

Fifteen 2,4-dioxaspiro[5.5]undecane ketone and 2,4-dioxa-spiro[5.5]undec-8-ene (spiroundecane(ene)) derivatives were synthesized using the Diels-Alder reaction. Inhibition of human immunodeficiency virus integrase (IN) was examined. Eight spiroundecane(ene) derivatives inhibited both 3'-processing and strand transfer reactions catalyzed by IN. SAR studies showed that the undecane core with at least one furan moiety is preferred for IN inhibition. Moreover, crosslinking experiments showed that spiroundecane derivatives did not affect IN-DNA binding at concentrations that block IN catalytic activity, indicating spiroundecane derivatives inhibit preformed IN-DNA complex. The moderate toxicity of spiroundecane(ene) derivatives encourages the further design of therapeutically relevant analogues based on this novel chemotype of IN inhibitors.

Integration of human immunodeficiency virus as a target for antiretroviral therapy.

PURPOSE OF REVIEW: Most of the studies investigating inhibition of human immunodeficiency virus integration have focused on blocking the enzymatic functions of HIV integrase, with the predominant judgment that integration inhibitors need to block at least one of the integrase-catalyzed reactions. Recent studies, however, have highlighted the importance of other proteins and their contacts with integrase in the preintegration complex, and their involvement in chromosomal integration of the viral DNA. RECENT FINDINGS: Promising results of clinical trials for two new integrase inhibitors were announced recently, providing the proof of the concept for using HIV-1 integrase inhibitors as antiretroviral therapy. Two strategies are currently employed for the development of novel inhibitors of HIV integrase: synthesis of hybrid molecules comprising core structures of two or more known inhibitors, and three-dimensional pharmacophore searches based on previously discovered compounds. By highlighting the role of the cellular cofactor LEDGF/p75 in HIV integration, novel approaches are indicated that aim to develop compounds altering contact between HIV integrase and integration cofactors. SUMMARY: By the discovery of novel inhibitors and targets for HIV integration, coupled with recent studies in characterizing preintegration complex formation, new insight is provided for the rational design of anti-HIV integration inhibitors.

Preferential inhibition of the magnesium-dependent strand transfer reaction of HIV-1 integrase by alpha-hydroxytropolones.

Integration is a crucial step in the life cycle of human immunodeficiency virus type 1 (HIV-1); therefore, inhibitors of HIV-1 integrase are candidates for antiretroviral therapy. Two 7-hydroxytropolone derivatives (alpha-hydroxytropolones) were found to inhibit HIV-1 integrase. A structure-activity relationship investigation with several tropolone derivatives from The National Cancer Institute compound repository demonstrated that the 7-hydroxy group is essential for integrase inhibition. alpha-Hydroxytropolones preferentially inhibit strand transfer and are inhibitory both in the presence of magnesium or manganese. Lack of inhibition of disintegration in the presence of magnesium coupled with results from different cross-linking assays suggests alpha-hydroxytropolones as interfacial inhibitors. We propose that alpha-hydroxytropolones chelate the divalent metal (Mg2+ or Mn2+) in the enzyme active site. The most active compound against HIV-1 integrase in biochemical assays [2,4,6-cycloheptatrien-1-one, 2,7-dihydroxy-4-isopropyl (NSC 18806) IC50 = 4.8 +/- 2.5 microM] exhibits weak cytoprotective activity against HIV-1(IIIB) in a cell-based assay. alpha-Hydroxytropolones represent a new family of inhibitors for the development of novel drugs against HIV infection.

Integration requires a specific interaction of the donor DNA terminal 5'-cytosine with glutamine 148 of the HIV-1 integrase flexible loop.

Integration is essential for retroviral replication and gene therapy using retroviral vectors. Human immunodeficiency virus, type 1 (HIV-1), integrase specifically recognizes the terminal sequences of each long terminal repeat (LTR) and cleaves the 3'-end terminal dinucleotide 5'-GT. The exposed 3'-hydroxyl is then positioned for nucleophilic attack and subsequent strand transfer into another DNA duplex (target or chromosomal DNA). We report that both the terminal cytosine at the protruding 5'-end of the long terminal repeats (5'-C) and the integrase residue Gln-148 are critical for strand transfer. Proximity of the 5'-C and Gln-148 was demonstrated by disulfide cross-linking. Cross-linking is inhibited by the inhibitor 5CITEP 1-(5-chloroindol-3-yl)-3-hydroxy-3-(2H-tetrazol-5-yl)-propenone. We propose that strand transfer requires a conformational change of the integrase-viral (donor) DNA complex with formation of an H-bond between the N-3 of the 5'-C and the amine group of Gln-148. These findings have implications for the molecular mechanisms coupling 3'-processing and strand transfer as well as for the molecular pharmacology of integrase inhibitors.

Effect of DNA modifications on DNA processing by HIV-1 integrase and inhibitor binding: role of DNA backbone flexibility and an open catalytic site.

Integration of the viral cDNA into host chromosomes is required for viral replication. Human immunodeficiency virus integrase catalyzes two sequential reactions, 3'-processing (3'-P) and strand transfer (ST). The first integrase inhibitors are undergoing clinical trial, but interactions of inhibitors with integrase and DNA are not well understood in the absence of a co-crystal structure. To increase our understanding of integrase interactions with DNA, we examined integrase catalysis with oligonucleotides containing DNA backbone, base, and groove modifications placed at unique positions surrounding the 3'-processing site. 3'-Processing was blocked with substrates containing constrained sugars and alpha-anomeric residues, suggesting that integrase requires flexibility of the phosphodiester backbone at the 3'-P site. Of several benzo[a]pyrene 7,8-diol 9,10-epoxide (BaP DE) adducts tested, only the adduct in the minor groove at the 3'-P site inhibited 3'-P, suggesting the importance of the minor groove contacts for 3'-P. ST occurred in the presence of bulky BaP DE DNA adducts attached to the end of the viral DNA suggesting opening of the active site for ST. Position-specific effects of these BaP DE DNA adducts were found for inhibition of integrase by diketo acids. Together, these results demonstrate the importance of DNA structure and specific contacts with the viral DNA processing site for inhibition by integrase inhibitors.