Speaking Rate, Oro-Laryngeal Timing, and Place of Articulation Effects on Burst Amplitude: Evidence From English and Tamil.

The relationship between speaking rate and burst amplitude was investigated in plosives with differing oro-laryngeal timing: long-lag voice-onset time (VOT) (North American English) and short-lag VOT (Indian Tamil). Burst amplitude (reflecting both intraoral pressure and flow geometry of the oral channel) was hypothesized to decrease in pre-vocalic plosive syllables with the increase in speaking rate, which imposes temporal constraints on both intraoral pressure buildup behind the oral occlusion and respiratory air flow. The results showed that decreased vowel duration (which is associated with increased speaking rate) led to decreased burst amplitude in both short- and long-lag plosives. Aggregate models of bilabial and velar plosives (found in both languages) suggested lower burst amplitudes in short-lag stops. Place-of-articulation effects in both languages were consistent with models of stop consonant acoustics, and place interactions with vowel duration were most apparent with long-lag English stops. The results are discussed in terms of speaking rate and language-internal forces, contributing to burst amplitude variation and their implications for speech perception and potential to affect lenition phenomena.

Optimization of RNA Aptamer SELEX Methods: Improved Aptamer Transcript 3'-End Homogeneity, PAGE Purification Yield, and Target-Bound Aptamer RNA Recovery.

Since its inception in the early 1990s, SELEX remains the gold standard for discovering RNA aptamers specific for proteins and small molecules. The SELEX process has undergone countless modifications and now encompasses a breadth of innovative selection schemes to pare an aptamer library toward target-specific aptamers. Common to all these RNA aptamer SELEX processes are the steps for the preparation of DNA template and in vitro transcription of aptamer RNA. These steps have remained mostly unchanged over the past three decades and would benefit from optimization. We focused on three key areas: improving the homogeneity of in vitro transcribed aptamer RNA, increasing the efficiency of in vitro transcribed aptamer RNA purification by PAGE, and improving the quality of target-bound aptamer RNA recovered during SELEX. Together, these optimizations contribute toward a more efficient SELEX process and are applicable to both protein-based and cell-based RNA aptamer selections.

Overtone focusing in biphonic tuvan throat singing.

Khoomei is a unique singing style originating from the republic of Tuva in central Asia. Singers produce two pitches simultaneously: a booming low-frequency rumble alongside a hovering high-pitched whistle-like tone. The biomechanics of this biphonation are not well-understood. Here, we use sound analysis, dynamic magnetic resonance imaging, and vocal tract modeling to demonstrate how biphonation is achieved by modulating vocal tract morphology. Tuvan singers show remarkable control in shaping their vocal tract to narrowly focus the harmonics (or overtones) emanating from their vocal cords. The biphonic sound is a combination of the fundamental pitch and a focused filter state, which is at the higher pitch (1-2 kHz) and formed by merging two formants, thereby greatly enhancing sound-production in a very narrow frequency range. Most importantly, we demonstrate that this biphonation is a phenomenon arising from linear filtering rather than from a nonlinear source.

The republic of Tuva, a remote territory in southern Russia located on the border with Mongolia, is perhaps best known for its vast mountainous geography and the unique cultural practice of "throat singing". These singers simultaneously create two different pitches: a low-pitched drone, along with a hovering whistle above it. This practice has deep cultural roots and has now been shared more broadly via world music performances and the 1999 documentary Genghis Blues. Despite many scientists being fascinated by throat singing, it was unclear precisely how throat singers could create two unique pitches. Singing and speaking in general involves making sounds by vibrating the vocal cords found deep in the throat, and then shaping those sounds with the tongue, teeth and lips as they move up the vocal tract and out of the body. Previous studies using static images taken with magnetic resonance imaging (MRI) suggested how Tuvan singers might produce the two pitches, but a mechanistic understanding of throat singing was far from complete. Now, Bergevin et al. have better pinpointed how throat singers can produce their unique sound. The analysis involved high quality audio recordings of three Tuvan singers and dynamic MRI recordings of the movements of one of those singers. The images showed changes in the singer's vocal tract as they sang inside an MRI scanner, providing key information needed to create a computer model of the process. This approach revealed that Tuvan singers can create two pitches simultaneously by forming precise constrictions in their vocal tract. One key constriction occurs when tip of the tongue nearly touches a ridge on the roof of the mouth, and a second constriction is formed by the base of the tongue. The computer model helped explain that these two constrictions produce the distinctive sounds of throat singing by selectively amplifying a narrow set of high frequency notes that are made by the vocal cords. Together these discoveries show how very small, targeted movements of the tongue can produce distinctive sounds.

Virus-based SELEX (viro-SELEX) allows development of aptamers targeting knotty proteins.

It has been 100 years since the worst flu (Spanish flu) mankind has ever experienced. Rapid, accurate diagnosis and subtyping of flu are still an urgent unmet medical need. By using surrogate virus-based SELEX (viro-SELEX), we report here multiple advances incorporated into the field of flu diagnostics: (i) aptamers that can bind to the native virus well even though they cannot bind strongly to a recombinant protein (hemagglutinin); (ii) a couple of aptamers that can target a broad range of strains belonging to the H1N1 subtype and detect only the H1N1 subtype and nothing else; (iii) a highly sensitive lateral flow assay system (limit of detection is 0.08 HAU) using fluorescence-tagged aptamers. The viro-SELEX method of aptamer selection in conjunction with a fluorescent tag on aptamers is a very useful approach to develop highly sensitive, specific, portable, rapid, and quantitative point-of-care testing diagnostic tools for the future.

Development of a Subtype-Specific Diagnostic System for Influenza Virus H3N2 Using a Novel Virus-Based Systematic Evolution of Ligands by Exponential Enrichment (Viro-SELEX).

Aptamers are oligonucleotide molecules that bind to specific target molecules generated by systematic evolution of ligands by exponential enrichment (SELEX). Aptamers have high future potential for use in diagnostics and therapeutics as molecular probes that recognize target molecules. To develop aptamers against a target protein using a SELEX process, it is necessary to purify the target protein. Purifying a membrane protein, however, is usually a challenging task. Here, we report a novel approach to developing aptamers against membrane proteins. Surrogate viruses containing target proteins on the surface of an enveloped virus (e.g., baculovirus), instead of purified proteins, were used in a new SELEX process. We designated this new SELEX process as "surrogate virus-based SELEX (viro-SELEX)." Using viro-SELEX, we developed a pair of aptamers that specifically interact with the hemagglutinin protein of influenza subtype H3N2. Using the aptamer pair and a lateral flow assay system, we developed a very sensitive point-of-care diagnostic system for specifically detecting influenza virus subtype H3N2.

Words Get in the Way: Linguistic Effects on Talker Discrimination.

A speech perception experiment provides evidence that the linguistic relationship between words affects the discrimination of their talkers. Listeners discriminated two talkers' voices with various linguistic relationships between their spoken words. Listeners were asked whether two words were spoken by the same person or not. Word pairs varied with respect to the linguistic relationship between the component words, forming either: phonological rhymes, lexical compounds, reversed compounds, or unrelated pairs. The degree of linguistic relationship between the words affected talker discrimination in a graded fashion, revealing biases listeners have regarding the nature of words and the talkers that speak them. These results indicate that listeners expect a talker's words to be linguistically related, and more generally, indexical processing is affected by linguistic information in a top-down fashion even when listeners are not told to attend to it.

Speech rate and pitch characteristics of infant-directed speech: Longitudinal and cross-linguistic observations.

The speech rate and pitch (F0) characteristics of naturalistic, longitudinally recorded infant- and adult-directed speech are reported for three, genetically diverse languages. Previous research has suggested that the prosodic characteristics of infant-directed speech are slowed speech rate, raised mean pitch, and expanded pitch range relative to adult-directed speech. Sixteen mothers (5 Sri Lankan Tamil, 5 Tagalog, 6 Korean) were recorded in their homes during natural interactions with their young infants, and adults, over the course of 12 months beginning when the infant was 4 months old. Regression models indicated that the difference between infant- and adult-directed speech rates decreased across the first year of infants' development. Models of pitch revealed predicted differences between infant- and adult-directed speech but did not provide evidence for cross-linguistic or longitudinal effects within the time period investigated for the three languages. The universality of slowed speech rate, raised pitch, and expanded pitch range is discussed in light of individuals' highly variable implementation of these prosodic features in infant-directed speech.

Optimization of Compound Plate Preparation to Address Precipitation Issue in Mammalian A549 Cytotoxicity Assay.

This study illustrates the optimization of low-volume dispensing on a liquid handling system (LHS) to overcome the precipitation of compounds in the mammalian cytotoxicity assay with low dimethyl sulfoxide (DMSO) tolerance. All compounds at AstraZeneca Bangalore are tested in the mammalian cytotoxicity assay. In order to maintain the DMSO levels, serially diluted plates were prepared in DMSO/water. It was observed that some of the compounds precipitated. The IC50 data for such compounds were therefore erratic. To circumvent the problem of compound precipitation, the LHS was optimized to dispense low volumes (<1 µL). The plates were serially diluted using neat DMSO. Since the dilution was done using neat DMSO, there were no issues with precipitation. The serially diluted sample (0.5 µL) from the plate was stamped onto the assay plate to give the desired DMSO concentration. No significant differences in IC50 data were observed for 1 µL dispenses made from DMSO/water and 0.5 µL dispenses from neat DMSO for the samples with no precipitation issues. These data therefore gave us the confidence to switch over to 0.5 µL dispenses for the cytotoxicity assay to address the precipitation issue. However, precipitation of samples in the assay buffer is beyond the scope of this discussion.

Whole cell screen based identification of spiropiperidines with potent antitubercular properties.

Whole cell based screens to identify hits against Mycobacterium tuberculosis (Mtb), carried out under replicating and non-replicating (NRP) conditions, resulted in the identification of multiple, novel but structurally related spiropiperidines with potent antitubercular properties. These compounds could be further classified into three classes namely 3-(3-aryl-1,2,4-oxadiazol-5-yl)-1'-alkylspiro[indene-1,4'-piperidine] (abbr. spiroindenes), 4-(3-aryl-1,2,4-oxadiazol-5-yl)-1'-alkylspiro[chromene-2,4'-piperidine] (abbr. spirochromenes) and 1'-benzylspiro[indole-1,4'-piperidin]-2(1H)-one (abbr. spiroindolones). Spiroindenes showed ⩾ 4 log10 kill (at 2-12 µM) on replicating Mtb, but were moderately active under non replicating conditions. Whole genome sequencing efforts of spiroindene resistant mutants resulted in the identification of I292L mutation in MmpL3 (Mycobacterial membrane protein Large), required for the assembly of mycolic acid into the cell wall core of Mtb. MIC modulation studies demonstrated that the mutants were cross-resistant to spirochromenes but not to spiroindolones. This Letter describes lead identification efforts to improve potency while reducing the lipophilicity and hERG liabilities of spiroindenes. Additionally, as deduced from the SAR studies, we provide insights regarding the new chemical opportunities that the spiroindolones can offer to the TB drug discovery initiatives.

Simple and rapid method to determine antimycobacterial potency of compounds by using autoluminescent Mycobacterium tuberculosis.

A major obstacle in the process of discovery of drugs against Mycobacterium tuberculosis is its extremely slow growth rate and long generation time (∼20 to 24 h). Consequently, determination of MICs and minimum bactericidal concentrations (MBCs) of potential drug candidates using current methods requires 7 days (resazurin-based MIC assay [REMA]) and 1 month (CFU enumeration), respectively. We employed a synthetic luciferase operon optimized for expression in high-GC-content bacteria and adapted it for use in mycobacteria. Using luminescence-based readouts, we were able to determine the MICs and bactericidal activities of approved tuberculosis (TB) drugs, which correlated well with currently used methods. Although luminescence-based readouts have been used previously to determine the MICs and bactericidal activities of approved TB drugs, in this study we adapted this assay to carry out a pilot screen using a library of 1,114 compounds belonging to diverse chemical scaffolds. We found that MICs derived from a 3-day luminescence assay matched well with REMA-based MIC values. To determine the bactericidal potencies of compounds, a 1:10 dilution of the cultures from the MIC plate was carried out on day 7, and the bactericidal concentrations determined based on time to positivity in 2 weeks were found to be comparable with MBC values determined by the conventional CFU approach. Thus, the luminescent mycobacterium-based approach not only is very simple and inexpensive but also allowed us to generate the information in half the time required by conventional methods.

Aminoazabenzimidazoles, a novel class of orally active antimalarial agents.

Whole-cell high-throughput screening of the AstraZeneca compound library against the asexual blood stage of Plasmodium falciparum (Pf) led to the identification of amino imidazoles, a robust starting point for initiating a hit-to-lead medicinal chemistry effort. Structure-activity relationship studies followed by pharmacokinetics optimization resulted in the identification of 23 as an attractive lead with good oral bioavailability. Compound 23 was found to be efficacious (ED90 of 28.6 mg·kg(-1)) in the humanized P. falciparum mouse model of malaria (Pf/SCID model). Representative compounds displayed a moderate to fast killing profile that is comparable to that of chloroquine. This series demonstrates no cross-resistance against a panel of Pf strains with mutations to known antimalarial drugs, thereby suggesting a novel mechanism of action for this chemical class.

Novel N-linked aminopiperidine-based gyrase inhibitors with improved hERG and in vivo efficacy against Mycobacterium tuberculosis.

DNA gyrase is a clinically validated target for developing drugs against Mycobacterium tuberculosis (Mtb). Despite the promise of fluoroquinolones (FQs) as anti-tuberculosis drugs, the prevalence of pre-existing resistance to FQs is likely to restrict their clinical value. We describe a novel class of N-linked aminopiperidinyl alkyl quinolones and naphthyridones that kills Mtb by inhibiting the DNA gyrase activity. The mechanism of inhibition of DNA gyrase was distinct from the fluoroquinolones, as shown by their ability to inhibit the growth of fluoroquinolone-resistant Mtb. Biochemical studies demonstrated this class to exert its action via single-strand cleavage rather than double-strand cleavage, as seen with fluoroquinolones. The compounds are highly bactericidal against extracellular as well as intracellular Mtb. Lead optimization resulted in the identification of potent compounds with improved oral bioavailability and reduced cardiac ion channel liability. Compounds from this series are efficacious in various murine models of tuberculosis.

4-aminoquinolone piperidine amides: noncovalent inhibitors of DprE1 with long residence time and potent antimycobacterial activity.

4-Aminoquinolone piperidine amides (AQs) were identified as a novel scaffold starting from a whole cell screen, with potent cidality on Mycobacterium tuberculosis (Mtb). Evaluation of the minimum inhibitory concentrations, followed by whole genome sequencing of mutants raised against AQs, identified decaprenylphosphoryl-ß-d-ribose 2'-epimerase (DprE1) as the primary target responsible for the antitubercular activity. Mass spectrometry and enzyme kinetic studies indicated that AQs are noncovalent, reversible inhibitors of DprE1 with slow on rates and long residence times of ∼100 min on the enzyme. In general, AQs have excellent leadlike properties and good in vitro secondary pharmacology profile. Although the scaffold started off as a single active compound with moderate potency from the whole cell screen, structure-activity relationship optimization of the scaffold led to compounds with potent DprE1 inhibition (IC50 < 10 nM) along with potent cellular activity (MIC = 60 nM) against Mtb.

Azaindoles: noncovalent DprE1 inhibitors from scaffold morphing efforts, kill Mycobacterium tuberculosis and are efficacious in vivo.

We report 1,4-azaindoles as a new inhibitor class that kills Mycobacterium tuberculosis in vitro and demonstrates efficacy in mouse tuberculosis models. The series emerged from scaffold morphing efforts and was demonstrated to noncovalently inhibit decaprenylphosphoryl-ß-D-ribose2'-epimerase (DprE1). With "drug-like" properties and no expectation of pre-existing resistance in the clinic, this chemical class has the potential to be developed as a therapy for drug-sensitive and drug-resistant tuberculosis.

Aminopyrazinamides: novel and specific GyrB inhibitors that kill replicating and nonreplicating Mycobacterium tuberculosis.

Aminopyrazinamides originated from a high throughput screen targeting the Mycobacterium smegmatis (Msm) GyrB ATPase. This series displays chemical tractability, robust structure-activity relationship, and potent antitubercular activity. The crystal structure of Msm GyrB in complex with one of the aminopyrazinamides revealed promising attributes of specificity against other broad spectrum pathogens and selectivity against eukaryotic kinases due to novel interactions at hydrophobic pocket, unlike other known GyrB inhibitors. The aminopyrazinamides display excellent mycobacterial kill under in vitro, intracellular, and hypoxic conditions.

The interaction between acoustic salience and language experience in developmental speech perception: evidence from nasal place discrimination.

Previous research suggests that infant speech perception reorganizes in the first year: young infants discriminate both native and non-native phonetic contrasts, but by 10-12 months difficult non-native contrasts are less discriminable whereas performance improves on native contrasts. In the current study, four experiments tested the hypothesis that, in addition to the influence of native language experience, acoustic salience also affects the perceptual reorganization that takes place in infancy. Using a visual habituation paradigm, two nasal place distinctions that differ in relative acoustic salience, acoustically robust labial-alveolar [ma]-[na] and acoustically less salient alveolar-velar [na]-[ enga], were presented to infants in a cross-language design. English-learning infants at 6-8 and 10-12 months showed discrimination of the native and acoustically robust [ma]-[na] (Experiment 1), but not the non-native (in initial position) and acoustically less salient [na]-[ enga] (Experiment 2). Very young (4-5-month-old) English-learning infants tested on the same native and non-native contrasts also showed discrimination of only the [ma]-[na] distinction (Experiment 3). Filipino-learning infants, whose ambient language includes the syllable-initial alveolar (/n/)-velar (/ eng/) contrast, showed discrimination of native [na]-[ enga] at 10-12 months, but not at 6-8 months (Experiment 4). These results support the hypothesis that acoustic salience affects speech perception in infancy, with native language experience facilitating discrimination of an acoustically similar phonetic distinction [na]-[ enga]. We discuss the implications of this developmental profile for a comprehensive theory of speech perception in infancy.