Exploring and explaining variation in phrase-final f0 movements in spontaneous Papuan Malay.

This study investigates the variation in phrase-final f0 movements found in dyadic unscripted conversations in Papuan Malay, an Eastern Indonesian language. This is done by a novel combination of exploratory and confirmatory classification techniques. In particular, this study investigates the linguistic factors that potentially drive f0 contour variation in phrase-final words produced in a naturalistic interactive dialogue task. To this end, a cluster analysis, manual labelling and random forest analysis are carried out to reveal the main sources of contour variation. These are: taking conversational interaction into account; turn transition, topic continuation, information structure (givenness and contrast), and context-independent properties of words such as word class, syllable structure, voicing and intrinsic f0. Results indicate that contour variation in Papuan Malay, in particular f0 direction and target level, is best explained by turn transitions between speakers, corroborating similar findings for related languages. The applied methods provide opportunities to further lower the threshold of incorporating intonation and prosody in the early stages of language documentation.

Dog and human neural sensitivity to voicelikeness: A comparative fMRI study.

Voice-sensitivity in the auditory cortex of a range of mammals has been proposed to be determined primarily by tuning to conspecific auditory stimuli, but recent human findings indicate a role for a more general tuning to voicelikeness. Vocal emotional valence, a central characteristic of vocalisations, has been linked to the same basic acoustic parameters across species. Comparative neuroimaging revealed that during voice perception, such acoustic parameters modulate emotional valence-sensitivity in auditory cortical regions in both family dogs and humans. To explore the role of voicelikeness in auditory emotional valence-sensitivity across species, here we constructed artificial emotional sounds in two sound categories: voice-like vs. sine-wave sounds, parametrically modulating two main acoustic parameters, f0 and call length. We hypothesised that if mammalian auditory systems are characterised by a general tuning to voicelikeness, voice-like sounds will be processed preferentially, and acoustic parameters for voice-like sounds will be processed differently than for sine-wave sounds - both in dogs and humans. We found cortical areas in both species that responded stronger to voice-like than to sine-wave stimuli, while there were no regions responding stronger to sine-wave sounds in either species. Additionally, we found that in bilateral primary and emotional valence-sensitive auditory regions of both species, the processing of voice-like and sine-wave sounds are modulated by f0 in opposite ways. These results reveal functional similarities between evolutionarily distant mammals for processing voicelikeness and its effect on processing basic acoustic cues of vocal emotions.

Perceived naturalness of emotional voice morphs.

Research into voice perception benefits from manipulation software to gain experimental control over acoustic expression of social signals such as vocal emotions. Today, parameter-specific voice morphing allows a precise control of the emotional quality expressed by single vocal parameters, such as fundamental frequency (F0) and timbre. However, potential side effects, in particular reduced naturalness, could limit ecological validity of speech stimuli. To address this for the domain of emotion perception, we collected ratings of perceived naturalness and emotionality on voice morphs expressing different emotions either through F0 or Timbre only. In two experiments, we compared two different morphing approaches, using either neutral voices or emotional averages as emotionally non-informative reference stimuli. As expected, parameter-specific voice morphing reduced perceived naturalness. However, perceived naturalness of F0 and Timbre morphs were comparable with averaged emotions as reference, potentially making this approach more suitable for future research. Crucially, there was no relationship between ratings of emotionality and naturalness, suggesting that the perception of emotion was not substantially affected by a reduction of voice naturalness. We hold that while these findings advocate parameter-specific voice morphing as a suitable tool for research on vocal emotion perception, great care should be taken in producing ecologically valid stimuli.

High-Level CNN and Machine Learning Methods for Speaker Recognition.

Speaker Recognition (SR) is a common task in AI-based sound analysis, involving structurally different methodologies such as Deep Learning or "traditional" Machine Learning (ML). In this paper, we compared and explored the two methodologies on the DEMoS dataset consisting of 8869 audio files of 58 speakers in different emotional states. A custom CNN is compared to several pre-trained nets using image inputs of spectrograms and Cepstral-temporal (MFCC) graphs. AML approach based on acoustic feature extraction, selection and multi-class classification by means of a Naïve Bayes model is also considered. Results show how a custom, less deep CNN trained on grayscale spectrogram images obtain the most accurate results, 90.15% on grayscale spectrograms and 83.17% on colored MFCC. AlexNet provides comparable results, reaching 89.28% on spectrograms and 83.43% on MFCC.The Naïve Bayes classifier provides a 87.09% accuracy and a 0.985 average AUC while being faster to train and more interpretable. Feature selection shows how F0, MFCC and voicing-related features are the most characterizing for this SR task. The high amount of training samples and the emotional content of the DEMoS dataset better reflect a real case scenario for speaker recognition, and account for the generalization power of the models.

Artificial Intelligence-Based Voice Assessment of Patients with Parkinson's Disease Off and On Treatment: Machine vs. Deep-Learning Comparison.

Parkinson's Disease (PD) is one of the most common non-curable neurodegenerative diseases. Diagnosis is achieved clinically on the basis of different symptoms with considerable delays from the onset of neurodegenerative processes in the central nervous system. In this study, we investigated early and full-blown PD patients based on the analysis of their voice characteristics with the aid of the most commonly employed machine learning (ML) techniques. A custom dataset was made with hi-fi quality recordings of vocal tasks gathered from Italian healthy control subjects and PD patients, divided into early diagnosed, off-medication patients on the one hand, and mid-advanced patients treated with L-Dopa on the other. Following the current state-of-the-art, several ML pipelines were compared usingdifferent feature selection and classification algorithms, and deep learning was also explored with a custom CNN architecture. Results show how feature-based ML and deep learning achieve comparable results in terms of classification, with KNN, SVM and naïve Bayes classifiers performing similarly, with a slight edge for KNN. Much more evident is the predominance of CFS as the best feature selector. The selected features act as relevant vocal biomarkers capable of differentiating healthy subjects, early untreated PD patients and mid-advanced L-Dopa treated patients.

Vowel-internal cues to vowel quality and prominence in speech perception.

This study examines how variation in F0 and intensity impacts the perception of American English vowels. Both properties vary intrinsically as a function of vowel features in the speech production literature, raising the question of the perceptual impact of each. In addition to considering listeners' interpretation of either cue as an intrinsic property of the vowel, the possible prominence-marking function of each is considered. Two patterns of prominence strengthening in vowels, sonority expansion and hyperarticulation, are tested in light of recent findings that contextual prominence impacts vowel perception in line with these effects (i.e. a prominent vowel is expected by listeners to be realized as if it had undergone prominence strengthening). Across four vowel contrasts with different height and frontness features, listeners categorized phonetic continua with variation in formants, F0 and intensity. Results show that variation in level F0 height is interpreted as an intrinsic cue by listeners. Higher F0 cues a higher vowel, following intrinsic F0 effects in the production literature. In comparison, intensity is interpreted as a prominence-lending cue, for which effect directionality is dependent on vowel height. Higher intensity high vowels undergo perceptual re-calibration in line with (acoustic) hyperarticulation, whereas higher intensity non-high vowels undergo perceptual re-calibration in line with sonority expansion.

Questioning questions - the perception of f0 scaling in German questions between categorical function and continuous attitude.

In previous studies comparing the intonation of questions and statements in German, greater f0 excursions of phrase-final rises have been associated with questions in both read speech and spontaneous speech. This holds for production studies as well as perception studies. However, a major question remains whether these differences are perceived categorically or continuously. Furthermore, we ask whether the differences in f0 scaling correspond to categorical linguistic functions or rather an attitudinal continuum. We conducted three different perception experiments: a classical categorical perception task, an imitation task, and a semantic evaluation task. The results suggest that f0 scaling in phrase-final rises is perceived as a phonetic continuum rather than in phonological categories. Furthermore, the gradual increase of the final rise is associated with a gradual increase in perceived questioning. Lastly, the phonetic cues to this degree of questioning are distinct from those to the other investigated meanings surprise and uncertainty. Accordingly, this study supports the assumption that questioning constitutes an attitudinal meaning in its own right.

Effects of near-monotonous speech of persons with Parkinson's disease on listening effort and intelligibility.

The majority of patients with Parkinson's disease (PD) develop hypokinetic dysarthria with a disturbance of prosody. The most important acoustic characteristic of prosodic impairment in PD is a lack of fundamental frequency (F0)-variability. It is well established that a lack of F0-variability can negatively influence the speech intelligibility of neurotypical speakers in background noise. The purpose of this study was to investigate which aspect of processing speech realised by speakers with PD is affected by lack of intonation: intelligibility as measured by a transcription task, scaled intelligibility and/or perceived listening effort when there is no background noise. F0-flattening of Semantically Unpredictable Sentences (SUS) was achieved, while the natural F0-declination was preserved. This kind of F0-flattening affected scaled intelligibility and perceived listening effort, while transcription performance remained unchanged.

Inorganic Polyphosphate and F0F1-ATP Synthase of Mammalian Mitochondria.

Inorganic polyphosphate is a polymer which plays multiple important roles in yeast and bacteria. In higher organisms the role of polyP has been intensively studied in last decades and involvements of this polymer in signal transduction, cell death mechanisms, energy production, and many other processes were demonstrated. In contrast to yeast and bacteria, where enzymes responsible for synthesis and hydrolysis of polyP were identified, in mammalian cells polyP clearly plays important role in physiology and pathology but enzymes responsible for synthesis of polyP or consumption of this polymer are still not identified. Here, we discuss the role of mitochondrial F0F1-ATP synthase in polyP synthesis with results, which confirm this proposal. We also discuss the role of other enzymes which may play important roles in polyP metabolism.

PERK-mediated induction of microRNA-483 disrupts cellular ATP homeostasis during the unfolded protein response.

Endoplasmic reticulum (ER) stress activates the unfolded protein response (UPR), which reduces levels of misfolded proteins. However, if ER homeostasis is not restored and the UPR remains chronically activated, cells undergo apoptosis. The UPR regulator, PKR-like endoplasmic reticulum kinase (PERK), plays an important role in promoting cell death when persistently activated; however, the underlying mechanisms are poorly understood. Here, we profiled the microRNA (miRNA) transcriptome in human cells exposed to ER stress and identified miRNAs that are selectively induced by PERK signaling. We found that expression of a PERK-induced miRNA, miR-483, promotes apoptosis in human cells. miR-483 induction was mediated by a transcription factor downstream of PERK, activating transcription factor 4 (ATF4), but not by the CHOP transcription factor. We identified the creatine kinase brain-type (CKB) gene, encoding an enzyme that maintains cellular ATP reserves through phosphocreatine production, as being repressed during the UPR and targeted by miR-483. We found that ER stress, selective PERK activation, and CKB knockdown all decrease cellular ATP levels, leading to increased vulnerability to ER stress-induced cell death. Our findings identify miR-483 as a downstream target of the PERK branch of the UPR. We propose that disruption of cellular ATP homeostasis through miR-483-mediated CKB silencing promotes ER stress-induced apoptosis.

Action spectrum of the redox state of the plastoquinone pool defines its function in plant acclimation.

The plastoquinone (PQ) pool mediates electron flow and regulates photoacclimation in plants. Here we report the action spectrum of the redox state of the PQ pool in Arabidopsis thaliana, showing that 470-500, 560 or 650-660 nm light favors Photosystem II (PSII) and reduces the PQ pool, whereas 420-440, 520 or 690 nm light favors Photosystem I (PSI) and oxidizes PQ. These data were used to construct a model predicting the redox state of PQ from the spectrum of any polychromatic light source. Moderate reduction of the PQ pool induced transition to light state 2, whereas state 1 required highly oxidized PQ. In low-intensity PSI light, PQ was more oxidized than in darkness and became gradually reduced with light intensity, while weak PSII light strongly reduced PQ. Natural sunlight was found to favor PSI, which enables plants to use the redox state of the PQ pool as a measure of light intensity.

Cortical compensation for hearing loss, but not age, in neural tracking of the fundamental frequency of the voice.

Auditory processing is affected by advancing age and hearing loss, but the underlying mechanisms are still unclear. We investigated the effects of age and hearing loss on temporal processing of naturalistic stimuli in the auditory system. We used a recently developed objective measure for neural phase-locking to the fundamental frequency of the voice (f0) which uses continuous natural speech as a stimulus, that is, "f0-tracking." The f0-tracking responses from 54 normal-hearing and 14 hearing-impaired adults of varying ages were analyzed. The responses were evoked by a Flemish story with a male talker and contained contributions from both subcortical and cortical sources. Results indicated that advancing age was related to smaller responses with less cortical response contributions. This is consistent with an age-related decrease in neural phase-locking ability at frequencies in the range of the f0, possibly due to decreased inhibition in the auditory system. Conversely, hearing-impaired subjects displayed larger responses compared with age-matched normal-hearing controls. This was due to additional cortical response contributions in the 38- to 50-ms latency range, which were stronger for participants with more severe hearing loss. This is consistent with hearing-loss-induced cortical reorganization and recruitment of additional neural resources to aid in speech perception.NEW & NOTEWORTHY Previous studies disagree on the effects of age and hearing loss on the neurophysiological processing of the fundamental frequency of the voice (f0), in part due to confounding effects. Using a novel electrophysiological technique, natural speech stimuli, and controlled study design, we quantified and disentangled the effects of age and hearing loss on neural f0 processing. We uncovered evidence for underlying neurophysiological mechanisms, including a cortical compensation mechanism for hearing loss, but not for age.

Neural tracking of the fundamental frequency of the voice: The effect of voice characteristics.

Traditional electrophysiological methods to study temporal auditory processing of the fundamental frequency of the voice (f0) often use unnaturally repetitive stimuli. In this study, we investigated f0 processing of meaningful continuous speech. EEG responses evoked by stories in quiet were analysed with a novel method based on linear modelling that characterizes the neural tracking of the f0. We studied both the strength and the spatio-temporal properties of the f0-tracking response. Moreover, different samples of continuous speech (six stories by four speakers: two male and two female) were used to investigate the effect of voice characteristics on the f0 response. The results indicated that response strength is inversely related to f0 frequency and rate of f0 change throughout the story. As a result, the male-narrated stories in this study (low and steady f0) evoked stronger f0-tracking compared to female-narrated stories (high and variable f0), for which many responses were not significant. The spatio-temporal analysis revealed that f0-tracking response generators were not fixed in the brainstem but were voice-dependent as well. Voices with high and variable f0 evoked subcortically dominated responses with a latency between 7 and 12 ms. Voices with low and steady f0 evoked responses that are both subcortically (latency of 13-15 ms) and cortically (latency of 23-26 ms) generated, with the right primary auditory cortex as a likely cortical source. Finally, additional experiments revealed that response strength greatly improves for voices with strong higher harmonics, which is particularly useful to boost the small responses evoked by voices with high f0.

Inorganic polyphosphate is produced and hydrolyzed in F0F1-ATP synthase of mammalian mitochondria.

Inorganic polyphosphate (polyP) is a polymer present in all living organisms. Although polyP is found to be involved in a variety of functions in cells of higher organisms, the enzyme responsible for polyP production and consumption has not yet been identified. Here, we studied the effect of polyP on mitochondrial respiration, oxidative phosphorylation and activity of F0F1-ATPsynthase. We have found that polyP activates mitochondrial respiration which does not coupled with ATP production (V2) but inhibits ADP-dependent respiration (V3). Moreover, PolyP can stimulate F0F1-ATPase activity in the presence of ATP and, importantly, can be hydrolyzed in this enzyme instead of ATP. Furthermore, PolyP can be produced in mitochondria in the presence of substrates for respiration and phosphate by the F0F1-ATPsynthase. Thus, polyP is an energy molecule in mammalian cells which can be produced and hydrolyzed in the mitochondrial F0F1-ATPsynthase.

Impaired mitochondrial functions and energy metabolism in MPTP-induced Parkinson's disease: comparison of mice strains and dose regimens.

Heterogenous diseases such as Parkinson's disease (PD) needs an efficient animal model to enhance understanding of the underlying mechanisms and to develop therapeutics. MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine), a neurotoxin, has been widely used to replicate the pathophysiology of PD in rodents, however, the knowledge about its effects on energy metabolism is limited. Moreover, susceptibility to different dose regimens of MPTP also varies among mice strains. Thus, the present study compares the effect of acute and sub-acute MPTP administration on mitochondrial functions in C57BL/6 and BALB/c mice. In addition, activity of enzymes involved in energy metabolism was also studied along with behavioural alterations. The findings show that acute dose of MPTP in C57BL/6 mice had more profound effect on the activity of electron transport chain complexes. Further, the activity of MAO-B was increased following acute and sub-acute MPTP administration in C57BL/6 mice. However, no significant change was observed in BALB/c mice. Acute MPTP treatment resulted in decreased mitochondrial membrane potential along with increased swelling of mitochondria in C57BL/6 mice. In addition, perturbations were observed in hexokinase, the rate limiting enzyme of glycolysis and pyruvate dehydrogenase, the enzymes that connects glycolysis and TCA cycle. The activity of TCA cycle enzymes; citrate synthase, aconitase, isocitrate dehydrogenase and fumarase were also altered following MPTP intoxication. Furthermore, acute MPTP administration led to drastic reduction in dopamine levels in striatum of C57BL/6 as compared to BALB/c mice. Behavioral tests such as open field, narrow beam walk and footprint analysis revealed severe impairment in locomotor activity in C57BL/6 mice. These results clearly demonstrate that C57BL/6 strain is more vulnerable to MPTP-induced mitochondrial dysfunctions, perturbations in energy metabolism and motor defects as compared to BALB/c strain. Thus, the findings suggest that the dose and strain of mice need to be considered for pre-clinical studies using MPTP-induced model of Parkinson's disease.

Seven traditional Chinese herbal extracts fermented by Lactobacillus rhamnosus provide anti-pigmentation effects by regulating the CREB/MITF/tyrosinase pathway.

Skin pigmentation is resulted from several processes, such as melanin synthesis transportation and abnormal melanin accumulation in keratinocytes. Various studies have suggested that seven traditional Chinese herbal extracts from Atractylodes macrocephala, Paeonia lactiflora, Bletilla striata, Poria cocos, Dictamnus dasycarpus, Ampelopsis japonica and Tribulus terrestris (which we collectively named ChiBai), show several protective effects toward skin-related diseases. Lactobacillus rhamnosus, a lactic acid bacterium, has been reported to treat skin inflammation and atopic dermatitis. In this study, the broth produced by the cofermentation of ChiBai with Lactobacillus rhamnosus was studied for its effects on skin pigmentation through in vitro and in vitro experiments. In the in vitro experiments, we found that the fermented broth of ChiBai (FB-ChiBai) suppressed alpha-melanocyte stimulating hormone (α-MSH)-induced melanogenesis in B16F0 murine melanoma cells without any cytotoxicity at a concentration of 0.5%. FB-ChiBai significantly attenuated melanin production, tyrosinase activities and melanogenesis-related signaling pathways. Treatment with FB-ChiBai also reduced the nuclear translocation and promoter binding activities of MITF. In the in vivo experiments, FB-ChiBai was topically applied to the dorsal skin of C57BL/6J nude mice and concurrently irradiated with UVB, three times a week for 8 weeks. The results indicated that FB-ChiBai alleviated UVB-induced hyperpigmentation by reducing epidermal hyperplasia and inhibiting the CREB/MITF/tyrosinase pathway. In conclusion, our data indicated that the anti-melanogenic effects of FB-ChiBai are mediated by the inhibition of CREB/MITF/tyrosinase signaling pathway. The findings suggest that FB-ChiBai can protect against UV-B irradiation and that it might be used as an agent in cosmetic products to protect against UVB-induced hyperpigmentation.

Deciphering Repertoire of B16 Melanoma Reactive TCRs by Immunization, In Vitro Restimulation and Sequencing of IFNγ-Secreting T Cells.

Recent advances in cancer immunotherapy have great promise for the treatment of solid tumors. One of the key limiting factors that hamper the decoding of physiological responses to these therapies is the inability to distinguish between specific and nonspecific responses. The identification of tumor-specific lymphocytes is also the most challenging step in cancer cell therapies such as adoptive cell transfer and T cell receptor (TCR) cloning. Here, we have elaborated a protocol for the identification of tumor-specific T lymphocytes and the deciphering of their repertoires. B16 melanoma engraftment following anti-PD1 checkpoint therapy provides better antitumor immunity compared to repetitive immunization with heat-shocked tumor cells. We have also revealed that the most error-prone part of dendritic cell (DC) generation, i.e., their maturation step, can be omitted if DCs are cultured at a sufficiently high density. Using this optimized protocol, we have achieved a robust IFNγ response to B16F0 antigens, but only within CD4+ T helper cells. A comparison of the repertoires of IFNγ-positive and -negative cells shows a prominent enrichment of certain clones with putative tumor specificity among the IFNγ+ fraction. In summary, our optimized protocol and the data provided here will aid in the acquisition of broad statistical data and the creation of a meaningful database of B16-specific TCRs.

γ-Tocotrienol Protects against Mitochondrial Dysfunction, Energy Deficits, Morphological Damage, and Decreases in Renal Functions after Renal Ischemia.

Ischemia-induced mitochondrial dysfunction and ATP depletion in the kidney result in disruption of primary functions and acute injury of the kidney. This study tested whether γ-tocotrienol (GTT), a member of the vitamin E family, protects mitochondrial function, reduces ATP deficits, and improves renal functions and survival after ischemia/reperfusion injury. Vehicle or GTT (200 mg/kg) were administered to mice 12 h before bilateral kidney ischemia, and endpoints were assessed at different timepoints of reperfusion. GTT treatment reduced decreases in state 3 respiration and accelerated recovery of this function after ischemia. GTT prevented decreases in activities of complexes I and III of the respiratory chain, and blocked ischemia-induced decreases in F0F1-ATPase activity and ATP content in renal cortical tissue. GTT improved renal morphology at 72 h after ischemia, reduced numbers of necrotic proximal tubular and inflammatory cells, and enhanced tubular regeneration. GTT treatment ameliorated increases in plasma creatinine levels and accelerated recovery of creatinine levels after ischemia. Lastly, 89% of mice receiving GTT and 70% of those receiving vehicle survived ischemia. Conclusions: Our data show novel observations that GTT administration improves mitochondrial respiration, prevents ATP deficits, promotes tubular regeneration, ameliorates decreases in renal functions, and increases survival after acute kidney injury in mice.

Lysine methylation by the mitochondrial methyltransferase FAM173B optimizes the function of mitochondrial ATP synthase.

Lysine methylation is an important post-translational modification that is also present on mitochondrial proteins, but the mitochondrial lysine-specific methyltransferases (KMTs) responsible for modification are in most cases unknown. Here, we set out to determine the function of human family with sequence similarity 173 member B (FAM173B), a mitochondrial methyltransferase (MTase) reported to promote chronic pain. Using bioinformatics analyses and biochemical assays, we found that FAM173B contains an atypical, noncleavable mitochondrial targeting sequence responsible for its localization to mitochondria. Interestingly, CRISPR/Cas9-mediated KO of FAM173B in mammalian cells abrogated trimethylation of Lys-43 in ATP synthase c-subunit (ATPSc), a modification previously reported as ubiquitous among metazoans. ATPSc methylation was restored by complementing the KO cells with enzymatically active human FAM173B or with a putative FAM173B orthologue from the nematode Caenorhabditis elegans Interestingly, lack of Lys-43 methylation caused aberrant incorporation of ATPSc into the ATP synthase complex and resulted in decreased ATP-generating ability of the complex, as well as decreased mitochondrial respiration. In summary, we have identified FAM173B as the long-sought KMT responsible for methylation of ATPSc, a key protein in cellular ATP production, and have demonstrated functional significance of ATPSc methylation. We suggest renaming FAM173B to ATPSc-KMT (gene name ATPSCKMT).

High-efficiency non-mosaic CRISPR-mediated knock-in and indel mutation in F0 Xenopus.

The revolution in CRISPR-mediated genome editing has enabled the mutation and insertion of virtually any DNA sequence, particularly in cell culture where selection can be used to recover relatively rare homologous recombination events. The efficient use of this technology in animal models still presents a number of challenges, including the time to establish mutant lines, mosaic gene editing in founder animals, and low homologous recombination rates. Here we report a method for CRISPR-mediated genome editing in Xenopus oocytes with homology-directed repair (HDR) that provides efficient non-mosaic targeted insertion of small DNA fragments (40-50 nucleotides) in 4.4-25.7% of F0 tadpoles, with germline transmission. For both CRISPR/Cas9-mediated HDR gene editing and indel mutation, the gene-edited F0 embryos are uniformly heterozygous, consistent with a mutation in only the maternal genome. In addition to efficient tagging of proteins in vivo, this HDR methodology will allow researchers to create patient-specific mutations for human disease modeling in Xenopus.