Identification of an Active Site-bound Nitrile Hydratase Intermediate through Single Turnover Stopped-flow Spectroscopy

Stopped-flow kinetic data were obtained for the iron-type nitrile hydratase from Rhodococcus equi TG328-2 (ReNHase) using methacrylonitrile as the substrate. Multiple turnover experiments suggest a three-step kinetic model that allows for the reversible binding of substrate, the presence of an intermediate, and the formation of product. Microscopic rate constants determined from these data are in good agreement with steady state data confirming that the stopped-flow method used was appropriate for the reaction. Single turnover stopped-flow experiments were used to identify catalytic intermediates. These data were globally fit confirming a three-step kinetic model. Independent absorption spectra acquired between 0.005 and 0.5 s of the reaction reveal a significant increase in absorbance at 375, 460, and 550 nm along with the hypsochromic shift of an Fe3+←S ligand-to-metal charge transfer band from 700 to 650 nm. The observed UV-visible absorption bands for the Fe3+-nitrile intermediate species are similar to low spin Fe3+-enzyme and model complexes bound by NO or N3−. These data provide spectroscopic evidence for the direct coordination of the nitrile substrate to the nitrile hydratase active site low spin Fe3+ center

The Iron-Type Nitrile Hydratase Activator Protein Is A GTPase

The Fe-type nitrile hydratase activator protein from Rhodococcus equi TG328-2 (ReNHase TG328-2) was successfully expressed and purified. Sequence analysis and homology modeling suggest that it is a G3E P-loop guanosine triphosphatase (GTPase) within the COG0523 subfamily. Kinetic studies revealed that the Fe-type activator protein is capable of hydrolyzing GTP to GDP with a kcat value of 1.2 × 10−3 s−1 and a Km value of 40 μM in the presence of 5 mM MgCl2 in 50 mM 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid at a pH of 8.0. The addition of divalent metal ions, such as Co(II), which binds to the ReNHase TG328-2 activator protein with a Kd of 2.9 μM, accelerated the rate of GTP hydrolysis, suggesting that GTP hydrolysis is potentially connected to the proposed metal chaperone function of the ReNHase TG328-2 activator protein. Circular dichroism data reveal a significant conformational change upon the addition of GTP, which may be linked to the interconnectivity of the cofactor binding sites, resulting in an activator protein that can be recognized and can bind to the NHase α-subunit. A combination of these data establishes, for the first time, that the ReNHase TG328-2 activator protein falls into the COG0523 subfamily of G3E P-loop GTPases, many of which play a role in metal homeostasis processes

The Fe-type Nitrile Hydratase from \u3cem\u3eComamonas testosteroni\u3c/em\u3e Ni1 Does Not Require an Activator Accessory Protein for Expression in \u3cem\u3eEscherichia coli\u3c/em\u3e

We report herein the functional expression of an Fe-type nitrile hydratase (NHase) without the co-expression of an activator protein or the Escherichia coli chaperone proteins GroES/EL. Soluble protein was obtained when the α- and β-subunit genes of the Fe-type NHase Comamonas testosteroni Ni1 (CtNHase) were synthesized with optimized E. coli codon usage and co-expressed. As a control, the Fe-type NHase from Rhodococcus equi TG328–2 (ReNHase) was expressed with (ReNHase+Act) and without (ReNHase−Act) its activator protein, establishing that expression of a fully functional, metallated ReNHase enzyme requires the co-expression of its activator protein, similar to all other Fe-type NHase enzymes reported to date, whereas the CtNHase does not. The X-ray crystal structure of CtNHase was determined to 2.4 Å resolution revealing an αβ heterodimer, similar to other Fe-type NHase enzymes, except for two important differences. First, two His residues reside in the CtNHase active site that are not observed in other Fe-type NHase enzymes and second, the active site Fe(III) ion resides at the bottom of a wide solvent exposed channel. The solvent exposed active site, along with the two active site histidine residues, are hypothesized to play a role in iron incorporation in the absence of an activator protein

The ACM Multimedia 2022 Computational Paralinguistics Challenge: vocalisations, stuttering, activity, & mosquitoes

The ACM Multimedia 2022 Computational Paralinguistics Challenge addresses four different problems for the first time in a research competition under well-defined conditions: In the Vocalisations and Stuttering Sub-Challenges, a classification on human non-verbal vocalisations and speech has to be made; the Activity Sub-Challenge aims at beyond-audio human activity recognition from smartwatch sensor data; and in the Mosquitoes Sub-Challenge, mosquitoes need to be detected. We describe the Sub-Challenges, baseline feature extraction, and classifiers based on the 'usual' ComParE and BoAW features, the auDeep toolkit, and deep feature extraction from pre-trained CNNs using the DeepSpectrum toolkit; in addition, we add end-to-end sequential modelling, and a log-mel-128-BNN

Déficits Primários e Secundários de Funções Executivas Pós-TCE: análise de dissociações

The present study evaluated the presence of associations and dissociations between impairments in episodic memory and executive functions in patients with traumatic brain injury (TBI), and verified whether these deficits were primary or secondary. Eighty-one patients with TBI were assessed using the Rey Auditory Verbal Learning Test and the Hayling Test. The results suggest that impairments in inhibition speed may contribute to deficits in episodic memory, and that initiation and inhibition abilities may be complementary and the first precedes the second. Our findings highlighted that primary executive impairment following TBI may lead to episodic memory deficits.Este estudo avaliou as associações e dissociações encontradas entre déficits de memória episódica em relação aos de funções executivas e verificou se estes déficits encontrados eram primários ou secundários. Os 81 pacientes pós-Traumatismo Cranioencefálico (TCE) foram avaliados por meio do Teste de Aprendizagem Auditivo-Verbal de Rey e do Teste Hayling. Os resultados sugerem que prejuízo na velocidade de controle inibitório pode contribuir para déficit na memória episódica e que as velocidades de iniciação e inibição parecem ser complementares, mas a primeira precede a segunda. Nossos achados ressaltam que os prejuízos executivos provavelmente sejam primários em nossa amostra de pacientes pós-TCE e que estes prejuízos podem causar déficits na memória episódica

The Gravity Collective: A Search for the Electromagnetic Counterpart to the Neutron Star-Black Hole Merger GW190814

We present optical follow-up imaging obtained with the Katzman Automatic Imaging Telescope, Las Cumbres Observatory Global Telescope Network, Nickel Telescope, Swope Telescope, and Thacher Telescope of the LIGO/Virgo gravitational wave (GW) signal from the neutron star-black hole (NSBH) merger GW190814. We searched the GW190814 localization region (19 deg$^{2}$ for the 90th percentile best localization), covering a total of 51 deg$^{2}$ and 94.6% of the two-dimensional localization region. Analyzing the properties of 189 transients that we consider as candidate counterparts to the NSBH merger, including their localizations, discovery times from merger, optical spectra, likely host-galaxy redshifts, and photometric evolution, we conclude that none of these objects are likely to be associated with GW190814. Based on this finding, we consider the likely optical properties of an electromagnetic counterpart to GW190814, including possible kilonovae and short gamma-ray burst afterglows. Using the joint limits from our follow-up imaging, we conclude that a counterpart with an $r$-band decline rate of 0.68 mag day$^{-1}$, similar to the kilonova AT 2017gfo, could peak at an absolute magnitude of at most $-17.8$ mag (50% confidence). Our data are not constraining for ''red'' kilonovae and rule out ''blue'' kilonovae with $M>0.5 M_{\odot}$ (30% confidence). We strongly rule out all known types of short gamma-ray burst afterglows with viewing angles $<$17$^{\circ}$ assuming an initial jet opening angle of $\sim$$5.2^{\circ}$ and explosion energies and circumburst densities similar to afterglows explored in the literature. Finally, we explore the possibility that GW190814 merged in the disk of an active galactic nucleus, of which we find four in the localization region, but we do not find any candidate counterparts among these sources.Comment: 86 pages, 9 figure

Treatment with 670 nm light up regulates cytochrome C oxidase expression and reduces inflammation in an age-related macular degeneration model.

Inflammation is an umbrella feature of ageing. It is present in the aged retina and many retinal diseases including age-related macular degeneration (AMD). In ageing and in AMD mitochondrial function declines. In normal ageing this can be manipulated by brief exposure to 670 nm light on the retina, which increases mitochondrial membrane potential and reduces inflammation. Here we ask if 670 nm exposure has the same ability in an aged mouse model of AMD, the complement factor H knockout (CFH(-/-)) where inflammation is a key feature. Further, we ask whether this occurs when 670 nm is delivered briefly in environmental lighting rather than directly focussed on the retina. Mice were exposed to 670 nm for 6 minutes twice a day for 14 days in the form of supplemented environmental light. Exposed animals had significant increase in cytochrome c oxidase (COX), which is a mitochondrial enzyme regulating oxidative phosphorylation.There was a significant reduction in complement component C3, an inflammatory marker in the outer retina. Vimetin and glial fibrillary acidic protein (GFAP) expression, which reflect retinal stress in Muller glia, were also significantly down regulated. There were also significant changes in outer retinal macrophage morphology. However, amyloid beta (Aβ) load, which also increases with age in the outer retina and is pro-inflammatory, did not change. Hence, 670 nm is effective in reducing inflammation probably via COX activation in mice with a genotype similar to that in 50% of AMD patients even when brief exposures are delivered via environmental lighting. Further, inflammation can be reduced independent of Aβ. The efficacy revealed here supports current early stage clinical trials of 670 nm in AMD patients

The Variably Intense Vocalizations of Affect and Emotion Corpus (VIVAE)

The Variably Intense Vocalizations of Affect and Emotion Corpus (VIVAE) consists of a set of human non-speech emotion vocalizations. The full set, comprising 1085 audio files, features eleven speakers expressing three positive (achievement/ triumph, sexual pleasure, and surprise) and three negative (anger, fear, physical pain) affective states, each parametrically varied from low to peak emotion intensity. The smaller core set of 480 files represents a fully crossed subsample of the full set (6 emotions x 4 intensities x 10 speakers x 2 items) selected based on judged authenticity

The Paradoxical Role of Emotional Intensity in the Perception of Vocal Affect

Published in Scientific Reports on 06 May 2021: https://www.nature.com/articles/s41598-021-88431-

Vocal expressions differentially transmit emotion categories and affective intensity

The distinctiveness of emotion expressions in faces and voices is thought to increase with increasing emotion intensity, but recent work on human nonverbal vocalizations challenges this commonplace assumption: peak emotions actually reveal maximal confusions. Whether this perceptual pattern reflects changing physical stimulus attributes or varying listener ability to use available information is not known. To adjudicate between these alternatives, we tested intensity effects on objective stimulus properties using supervised learning models and information-theoretic analyses. We show that ambiguity is not a mere perceptual phenomenon but is instead reflected in a tradeoff between emotion category and emotion intensity signal information, available in vocalizations’ low-level acoustic structure. The componential information about emotion is weighted differently: the composition of signal parts serving classification, intensification, or both, varies substantially with the expressed emotional intensity. Maximally intense vocal expressions primarily signal intensity, less so emotion categories, suggesting that the communicative function of vocalizations shifts with their social or biological relevance