The high ability group, on the other hand showed significantly larger articulation space in all three conditions. In addition, articulation space also correlated positively with imitation ability . Our results suggest that an expanded articulation space for high ability individuals allows access to a larger repertoire of sounds, thereby providing skilled imitators greater flexibility in pronunciation and language learning.We investigated individual differences in speech imitation ability in late bilinguals using a neuro-acoustic approach. One hundred and thirty-eight German-English bilinguals matched on various behavioral measures were tested for “speech imitation ability” in a foreign language, Hindi, and categorized into “high” and “low ability” groups. Brain activations and speech recordings were obtained from 26 participants from the two extreme groups as they performed a functional neuroimaging experiment which required them to “imitate” sentences in three conditions: (A) German, (B) English, and (C) German with fake English accent. We used recently developed novel acoustic analysis, namely the “articulation space” as a metric to compare speech imitation abilities of the two groups. Across all three conditions, direct comparisons between the two groups, revealed brain activations (FWE corrected,  Speech sound imitation is a pivotal learning mechanism for humans. Vocal imitation provides a basis for acquisition of both languages and musical systems Fitch, . Yet indAlthough the origins of differences in imitation ability—whether neurobiological, genetic, psycho-cognitive, social or environmental are still unknown, recent neuroscientific research has suggested brain structural and functional differences as a partial explanation  at the ends of a normally distributed curve of individual differences in speech imitation capacity. We hypothesized that lower ability speech imitators would engage more widespread, diffuse neural activation around articulation-relevant areas in the brain and that acoustic analyses would be capable of capturing differences in segmental and suprasegmental speech articulation patterns as a function of individual differences in speech imitation ability.SD ± 5.2), 90% right-handers, 85 females/53 males, mixed fields of study backgrounds from natural sciences, business to humanities and languages, mother tongue German, AOA: 10.5 years (SD ± 0.8), 50% language study background, no neurological disorders. They all knew at least one foreign language, which was English, 24% knew only one L2 (English), 30% knew two L2s, 22% knew three, 17.5% knew four, 3.5% five, 2% six, and 1% nine foreign languages. All participants together knew 27 different foreign languages to varying degrees including very low levels of proficiency for some of the mentioned languages: English, French, Italian, Spanish, Dutch, Greek, Portuguese, Hungarian, Turkish, Polish, Russian, Czech, Slowenian, Rumanian, Norwegian, Swedish, Danish, Finnish, Hebrew, Arabic, Persian, Korean, Japanese, Chinese, Africans, Esperanto, and Latin. Mean number of foreign languages was 2.5, minimum was 0  and maximum number was 9. All of these additional languages were acquired between age 10 and 35 at various ages within this range. All participants gave informed written consent for their participation in the study. The study was approved by the local Ethics Committee and was in accordance with the Helsinki declaration.One hundred and thirty-eight German-English second language learners  with varying degrees of proficiency in English, matched for age, handedness, gender, educational status, mother tongue, age of onset of second language acquisition (AOA), linguistic experience, neurological status, verbal and non-verbal IQ were randomly recruited for the study. There were no “true,” “equal” or early bilinguals (those with AOA before puberty) included in the study. Participants were randomly recruited from the urban catchment areas surrounding the universities of Tübingen and Stuttgart. This large pre-search pool was investigated closely with respect to speech imitation/speech production abilities to characterize the participants' behavioral performance in articulation ability to be better able to select participants for the further examinations. Their mean age was 25.9 years . Their verbal  participants of this pre-search pool were asked for willingness to participate in the fMRI experiments. 53 participants, distributed over all “imitation ability” groups were willing to participate again in the MR experiments. 13 needed to be excluded because of various reasons including MR safety issues , so that 40 participants could be scanned and presented with our fMRI task “experiment 2,” described below under “behavioral experiment 2 (“sentence reading and accent faking”).To characterize their basic ability of foreign sound imitation, the 138 participants were recorded in a sound-proof room while they performed different speech imitation, pronunciation or reading tasks in the languages German (L1), English (L2), and Hindi (L0 for unknown language). For details of the different task types and elicitation techniques see Jilka .N = 18) into the database. The speech samples were presented in random order. For the intuitive rating scale  we performed further indirect imitation tasks, namely sentence reading tasks (imitation from memory). 26 participants belonging to the highest and lowest ability groups according to the behavioral experiment 1 (Hindi imitation scoring) were scanned and recorded in the scanner on sentence reading tasks in their L1 German, L2 English, and “faking an accent” (faking L2 in L1). We chose this task to be performed in the scanner, because it was described in the literature as a task which discriminates well between high and low aptitude second language learners in accent imitation because phonemic awareness is especially taxed in the case of the imitations from memory  we selected the two extreme groups within the second and third SD below and above the mean, resulting in 13 participants each group. Details for the high ability group: 9 males/4 females, mean age was 28.38 years (SD ± 5.0), age of onset of L2 English: 10.38 years (SD ± 0.9), mean number of second languages spoken: 2.38 (SD ± 1.1), mixed study background, Hindi score: 6.4 (SD ± 0.7). Details for the low ability group: 8 males/5 females, mean age was 25.46 years (SD ± 5.0), age of onset of L2 English: 10.69 years (SD ± 0.9), mean number of second languages spoken: 2.23 (SD ± 1.3), mixed study background, Hindi score: 3.6 (SD ± 0.7). The two groups were significantly different with regard to the Hindi score (p = 0.000**), but no other significant group differences were found in these behavioral background parameters.Based on the ratings of the Hindi imitation task of our large pre-selection study, participants were categorized along a continuum of the Hindi imitation ability score. From this overall pool of 138 subjects we re-invited a similarly  distributed subsample to our fMRI experiments. 40 participants were scanned. For the fMRI group vs. group analysis  with sentences presented and read during the scanner pauses. Stimuli were randomized for time-onsets and order of presentation. Inter-stimulus baseline trials were inserted alternatingly every second TR accompanied by fixating a white cross on black screen. The produced speech was recorded by a commercially available optical MR-microphone. Before the start of the fMRI scanning session subjects were familiarized with sample stimuli. Examples of each condition are provided below.We scanned these already well described participants  during overt, microphone-monitored speech production (sentence reading). The monitored utterances were quality controlled in two ways: once online during the in-scanner task and once offline for omissions and error rates. Using an event-related, sparse-sampling fMRI paradigm, we administered an overt sentence reading task (30 min), which was subdivided into the three sub-conditions: (A) reading aloud German sentences (L1), (B) reading aloud English sentences (L2) and (C) reading German sentences with a fake English accent (any variety). This task required phonemic awareness of foreign accented speech characteristics. We chose this task to be performed in the scanner, because it was described in the literature as a task which discriminates well between high and low aptitude second language learners in accent imitation because phonemic awareness is especially taxed in the case of the imitations from memory  “The mechanic repaired cars in the garage” (L2 English)“Der Professor präsentiert das Resultat”(L1 with fake English accent) (English translation: the professor presents the result)TR = 12 s, TA = 3 s, delay in TR (pause) = 9 s, TE = 48 ms, slice number = 36 transversal, Flip angle: 90°, Slice thickness = 3 mm + 1 mm gap, Voxel Size: 3 × 3 × 4 mm3, FoV = 192 × 192 × 143 mm3, ma-trix = 64 × 64. EPI images were acquired in transversal orientation. Acquisition was interleaved and descending.A Siemens Vision 1.5 T scanner was used. For functional imaging (fMRI) of the blood oxygen level dependent (BOLD) signal, we used an echo planar imaging (EPI) Gradient Echo sequence with sparse sampling method set at the following parameters: TR) = 7.92 ms; echotime (TE) = 2.48 ms; inversion time (TI) = 910 ms; FA = 16°; voxelsize: 1 mm × 1 mm × 1 mm, FoV = 176 mm × 256 mm × 256 mm, slices per slab = 176 sagittal, matrix = 256 × 256, acquisition orientation: transversal. An eight-channel headcoil was used.For structural  image acquisition, structural MRI scans of all subjects were performed on the same scanner, using a high resolution T1-weighted MDEFT sequence (Modified Driven-Equilibrium Fourier Transform), scantime = 12 min, rep-etition time  described in the following sections.FMRI images were analyzed using the free software packet SPM5 (Statistic Parametric Mapping).Data pre-processing: each fMRI data set underwent spatial realignment by aligning the first scan from each session with the first scan of the first session and aligning the images within sessions with the first image of a particular session. The realigned data were spatially normalized to the standard Montreal neurological institute (MNI) T1 template, with the coregistered individual T1 image as a reference. Volumes were resliced to a voxel size of 3 mm × 3 mm × 3 mm, motion corrected and spatially smoothed using a10-mm full-width-at-half-maximum Gaussian kernel and prepared for later random effects analyses. Temporal correction (slice timing) was not applicable because the sparse sampling acquisition mode was adopted here.x, y, and z). Regressors were defined with onsets at the time of appearance of the corresponding event and convolved with the canonical hemodynamic response function. At the second level, group analysis was performed using analysis of variance (ANOVA), with one between subject factor “ability group” (high vs. low ability group) and one within-subjects factor “language type” . Main effects for group and language type and the interaction effect of group by language type were calculated separately for each session. A statistical threshold of p < 0.05 (whole brain cluster level correction for multiple comparisons) was obtained. Results were overlaid on the mean anatomical image of the group (N = 26) and for the activation areas to be displayed we used a cluster extent threshold of 400 voxels.At the first level, design matrices of individual general linear models incorporated three regressors of language type  for the task sentence reading task. Additional six regressors of movement parameters were added as well , which a probability distribution of the different spectral and temporal modulations in a vocal utterance. It is obtained by calculating the two-dimensional Fourier transform of the autocorrelation matrix of the amplitude envelope of the vocal utterance in its spectrographic representation  are energy fluctuations across a frequency spectrum at particular times, whereas temporal modulations (ωt) are energy fluctuations at a particular frequency over time. Based on the rate of fluctuation, spectro-temporal modulations have been proposed to encode three articulatory features, namely (1) syllabicity or syllabic rhythm (SR) (2–10 Hz), (2) formant transitions (FT) reflecting consonant blends and transitions (20–40 Hz), and (3) place of articulation (POA) reflecting finer, rapid-scale changes in utterance (50–100 Hz).Speech is a signal that involves processing at multiple time scales Rosen, . It is tA 2-D Fourier transform of the spectrogram yields a probability distribution of these different articulatory features and is called the SMS  Indian native speakers living in India, performing the ratings online using earphones, indicating judgments on a 0–10 intuitive rating scale. They were paid for their services of several hours of rating time needed for a sample of 138 subjects (plus 18 native subjects). The inter-rater reliability, Cronbach's Alpha was 0.964. Originally a larger number of raters (36) performed the online ratings, but 6 raters were excluded due to their low inter-rater reliability. For more details on the Hindi speech imitations and ratings procedure also see Jilka  with 70% of subjects ranging within one SD below and above the mean (average ability). The subjects' mean score was 4.62, ee Jilka  and Reitee Jilka .** (p = 0.0001). For more details on the scoring systems see also Hu et al. . As shown in Figure A statistical comparison of the articulation areas between the high and low ability groups Figure  showed sr = 0.74, p = 0.0000**  correlated r = 0.62, p = 0.001. Articulation space also correlated with the English pronunciation ability scores, although to a lesser degree and significance level: English score correlated positively with articulation space for condition A: r = 0.4, p = 0.04; condition B: r = 0.5, p = 0.01; with condition C: r = 0.4, p = 0.02. Thus, the English imitation score correlated most strongly with condition B, the “articulation space of reading English sentences.” See also Figure A correlation of the articulation space area scores with the behavioral language imitation scores, showed a positive correlation in all three conditions with the highest positive correlation for condition C (faking the accent): Pearson * Figure , followep < 0.05; two tailed, t test for independent samples). Error counts do not correlate with our imitation/pronunciation ability measure, the Hindi score, only and not surprisingly, the English score correlates negatively with the error rates for reading English sentences .The microphone monitored sentences were recorded and controlled offline for omissions and error rates. We computed a global measure for the errors in sentence reading. Generally there were few errors so that it was not meaningful to split the errors into subtypes of errors. We identified: “partial omissions,” “mispronunciations,” “slips of the tongue,” “repetitions,” “self-corrections,” “hesitations”. Not a single complete omission of a sentence was observed. In all 3 sub-conditions  there were no statistically significant differences between the groups  activated voxel of the left supramarginal gyrus was chosen as ROI. The second reason for choosing this ROI was based on the hypothesis that the left supramarginal gyrus is an important hub area for sensorimotor integration , and not rely on pre-experienced, entrenched categories (rule-based learning). If transfer/interference from L1 had taken place, their speech output would not have been rated as being close to the native speaker range of Hindi speakers.Our behavioral and computational acoustic data point into the same direction. There is higher neuro-cognitive flexibility, reflected by higher articulatory flexibility in the group of the more talented speech imitators. Since the Hindi imitation task did not involve any pre-existing experience with the language, we assume that some individuals must still possess this openness to build new phonetic categories on an With respect to phonological theories of second language learning, critical periods, and interference from L1 our speech imitation data point to a “variable” view. It is difficult to confer an all-or-nothing theory like “all second language speakers show transfer or interference from L1,” or “all late second language learners cannot phonetically imitate native-like speech because they are beyond an assumed critical period.” Instead the emerging picture is differentiated or gradual and could be summarized by saying “it depends on.” Our high ability phonetic imitators produced speech production samples similar to the native speakers of the language as reflected in the native speaker judgments. However, the low ability group was rated significantly different (very low) by native judges. This means that the world of second language speakers is not best described by a unifying theory according to which all speakers act according to either one or another theory, but seem to follow graded distributions of the form “some speakers contradict traditional theories,” “some speakers highly corroborate traditional theories,” and “many speakers are in transitional positions.” Thus, for our high ability speakers we can refute a critical period for phonetic learning and propose instead a successful mastery of native speech where interference from L1 is not at work. Phonetic interference phenomena from L1 do exist, but they depend on the individual level of phonetic aptitude.Our data provide strong evidence that speaker-related individual differences in speech imitation ability/aptitude play a decisive role in speech production, being similarly expressed for L1 and L2, and can be visualized and quantified by various biometric methods of signal analysis. Regarding speech imitation ability on a neurometric scale, we confirmed the theory of cortical processing effort by showing that both greater intensity and extension in activation of cortico-subcortical speech relevant areas can be shown as a function of speech imitation ability even in the case of the mother tongue. Poorer skills were associated with greater consumption of neural workspace. Focal area of the statistical group differences was the left supramarigal gyrus, a hub area in sensorimotor integration, along the dorsal language processing stream. With regard to the newly applied acoustic measures of speech output, we found a larger articulation space to be a marker of high ability in L2 speech imitation skills. Further research is needed to refine the acoustic parameters for discriminating differences in proficiency and ability of the speech imitation skills. We further showed that faking or imitating foreign accents is a valuable task in differentiating speakers with different levels of speech sound imitation and pronunciation ability. It remains to be clarified exactly why faking accents manifests such a considerable obstacle for speakers with low mimicking skills. We conclude from our present experiment that speech-motor flexibility, neurocognitive flexibility, working memory, and auditory long term memory may be amongst the predictors of speech imitation capacities, but the picture is surely more complex and multi-causal. Future research needs to clarify the role of auditory and sensory-motor memory in relation to other factors  more precisely. However, it becomes clear that these individual differences visible in mother tongue as well as foreign speech processing do exist, are detectable, even predictable to a certain degree of specificity, by neurometric and audiometric means of signal analysis.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Huangqi injection is derived from Astragalus membranaceus root. In China, recent reports of Huangqi injection for the treatment of leucopenia have emerged. However, a systematic review of these reports has not been performed. Thus, we conducted a meta-analysis of clinical controlled trials to assess the clinical value of Huangqi injection in the treatment of leucopenia. We searched the Chinese Biomedical Literature Database (CBM), Wanfang Database, China National Knowledge Infrastructure (CNKI), Chinese Scientific Journals Full-text Database (VIP), as well as PubMed and EMBASE to collect the data about trials of Huangqi injection for treating leucopenia. A meta-analysis was performed using RevMan 5.2 software. P < 0.05). No publication bias was evident, according to Egger’s test. A total of 13 studies involving 841 patients were included in this study. The overall study quality was lower according to the Jadad scale. The meta-analysis showed that experimentally treated patients experienced greater therapeutic efficacy and lower white blood cell counts than control groups treated with Western medicine (The validity of this meta-analysis was limited by the overall poor quality of the included studies. Huangqi injection may have potential clinical value in the treatment of leucopenia, but confirmation with rigorously well-designed multi-center trials is needed. Leucopenia is defined by a lower-than-normal peripheral white blood cell (WBC) count. Leucopenia commonly arises from cancer chemotherapy or radiotherapy, viral infection, drug-induced reactions, and certain immune diseases -7. Recen9/L). When total WBCs were greater than 4.0×109/L or elevated more than 1.0×109/L in the peripheral blood arising from a drug intervention, treatments were considered to be effective. Before treatment, the baseline peripheral WBCs were comparable between the experimental group (Huangqi injection) and the control group (Western medicine) (P>0.05). In this study, we restricted no clinical trials based on patient gender, race, or language of the publication.The clinical trials were clinically controlled studies and experimental groups were treated with Huangqi injection. Controls in the studies were treated with Western medicine as described in Duplicated literature, reviews, non-clinical studies, case observations, and non-injection formulae literature were excluded in this study.“Huangqi” or “huang qi” or “astragalus” or “astragali” or “astragalus miltiorrhiza” or “Chinese traditional medicine herb” AND “leucopenia” or “leucocytopenia” or “leucopenia” or “aleucocytosis” or “hypolekocytosis” or “hypoleucocytosis” or “hypoleukemia” or “hypoleukia” or “hypoleukocytosis leucocytopenia” or “leucopenia” or “oligoleukocythemia” or “oligoleukocytosis” or “white blood count” were selected as the free-text terms or MeSH terms. The Chinese Biomedical Literature Database (CBM), Wanfang Database, China National Knowledge Infrastructure (CNKI), Chinese Scientific Journals Full-text Database (VIP), PubMed, and EMBASE were searched. Data extraction and quality assessment were independently performed by two researchers [CTZ and YL] and any discrepancies were resolved by consensus or in consultation with a third reviewer [CSZ]. The lack of information in any study was supplemented by contact with the authors in charge of the clinical trials. The database retrieval process is shown in Statistical analysis was performed using Cochrane RevMan 5.2 and STATA 11.0 software. Categorical variables were compared using odds ratios (OR), and continuous variables were compared using standard mean differences (SMD). A 95% confidence interval (CI) was calculated and the Chi-square test was used for heterogeneity of inclusion trials. Data of heterogeneity were studied using a random effects model; otherwise a fixed effects model was used. A funnel plot and Egger’s test were employed to judge potential publication bias.Thirteen articles -21 involThe Jadad scale was scored by randomization, randomization methodology, double-blinding, withdrawals/dropouts, and allocation concealment -24. The P < 0.05; fixed effects model] , s model] . WBC cous model] . P < 0.05; fixed effects model] , s model] . WBCs ins model] . In this study, five reports ,13,16,19P = 0.264). The funnel plot drawn by Cochrane Revman 5.2 was basically symmetric Click here for additional data file.


This review focuses on the diversity of immunoglobulin (Ig) genes and Ig isotypes that are expressed in domestic animals. Four livestock species—cattle, sheep, pigs, and horses—express a full range of Ig heavy chains (IgHs), including μ, δ, γ, ϵ, and α. Two poultry species (chickens and ducks) express three IgH isotypes, μ, υ, and α, but not δ. The κ and λ light chains are both utilized in the four livestock species, but only the λ chain is expressed in poultry. V(D)J recombination, somatic hypermutation (SHM), and gene conversion (GC) are three distinct mechanisms by which immunoglobulin variable region diversity is generated. Different domestic animals may use distinct means to diversify rearranged variable regions of Ig genes. These molecules are usually expressed in either a membrane-bound form (B cell receptor) or a secreted form (antibody). Regardless of the form, a typical immunoglobulin molecule contains a heterodimer consisting of two identical heavy chains and two identical light chains. In a very small number of species, such as sharks and camels, antibodies consisting only of heavy chains have been identified ,2. Both ic cells . A thirdic cells . FinallyOver the past few decades, Ig genes in mice and humans have been intensively studied, and these efforts have contributed immensely to our understanding of the generation of antibody diversity and other relevant molecular mechanisms. However, a significant lesson from comparative studies has also taught us that different animals may utilize different mechanisms to accomplish the same aim. In this regard, we have also learned a great deal by studying Ig genes from other species, including domestic animals.Artiodactyla, suborder Ruminantia, and family Bovidae. Ig genes from these two species are very similar in many aspects. Below, we focus on cattle to describe the major findings related to their Ig genes.Both cattle and sheep belong to the order Like humans and mice, cattle express five classes of IgH chains: μ, δ, γ, ϵ, and α. Compared with the presence of four subclasses of IgG  and two subclasses of IgA (encoded by α1 and α2) in humans, only three γ genes  and a single α gene have been identified in cattle ,8. The eIt is particularly interesting that cattle (as well as sheep) appear to have two IgH loci in their genomes: one in BTA21 and one in BTA11 ,11. The Compared with humans and mice, cattle have a very limited number of germline heavy chain V, D, and J segments. Only a single VH family, designated as BoVH1, is expressed at the cDNA level, and it is thought that the bovine genome contains no more than 20 VH segments ,17. OnlyAlthough similar to humans and mice, two types of Ig light chains (λ and κ) are expressed in cattle, though the λ/κ ratio differs significantly among these animals. In mice, approximately 96% of light chains in the serum are the κ type, while the κ type in humans accounts for only 66% of the total population of Ig light chains. In contrast, the light chain repertoire in cattle is dominated by λ chains (96%) . This apArtiodactyla, suborder Suina, and family Suidae. Pig species also express five IgH isotypes , in which the μ, δ, ϵ, and α genes are all present in a single copy at the IgH locus. In contrast, the porcine γ gene is extensively diversified and thus can develop multiple subclass-encoding genes; at least six distinct γ genes and some allelic variants have been defined in pigs [Pigs belong to the order  in pigs ,24. The Mapping of the porcine IgH locus revealed only two functional DH and a single functional JH, suggesting that V(D)J recombination contributes little to the VH repertoire in pigs . PreviouUnlike other species, pigs have a balanced λ/κ ratio (~52% vs. 48%) at the protein level . ConsistPerissodactyla, suborder Equidae, and family Equus. Similar to all artiodactyls, horses also have single copies of the μ, δ, ϵ, and α genes and a large number of γ genes per haploid genome. The genomic organization of the horse IgH locus has been completely mapped, giving the order 5’-VH-DH-JH-μ-δ-γ1-γ2-γ3-γ7-γ4-γ6-γ5-ϵ-α-3’ [Horses belong to the order 5-ϵ-α-3’ . The gen5-ϵ-α-3’ . Phyloge5-ϵ-α-3’ ,31.Based on horse genomic data, 50 VH, 40 DH, and 8 JH segments were identified upstream of the CH locus. All VH segments can be classified into seven subgroups, which are distributed into all three mammalian VH clans. The VH segments from subgroup 2, corresponding to the BoVH1, are predominantly expressed at the cDNA level; however, the other VH subgroups are rarely expressed ,33. BecaSimilar to cattle and sheep, horses are another λ-predominant mammalian species . The preBoth chickens and ducks express only three classes of IgH chains, namely IgM, IgY (encoded by υ gene), and IgA. Although the IgD-encoding gene has now been identified in nearly all groups of jawed vertebrates, no bird species has been found that contains an IgD-encoding gene ,37. As tBird IgY is functionally equivalent but structurally different from mammalian IgG. IgY consists of four CH domains but contains no hinge region . InteresDespite being orthologous to mammalian IgA, bird IgA differs structurally from its mammalian counterpart because it has four CH domains and no hinge region ,39,43. TThe most interesting observation obtained from studies of chicken Ig genes is the mechanism used by this species to generate immunoglobulin diversity. In the chicken IgH locus, only a single functional VH, 16 DH, and a single JH segment have been identified ,45. TherTwo types of IgL chains (λ and κ) are found in both mammals and reptiles, two groups of animals that are phylogenetically close to birds. It is quite surprising that only the λ chains are expressed in both chickens and ducks ,47. In tStudies that have analyzed the Ig genes in domestic animals have generated novel information that we could not learn from research solely focusing on humans and mice, thereby indicating the importance of comparative studies. This new information also emphasizes the fact that different species may use distinct mechanisms to fulfill a principally similar function. Compared with humans and mice, it appears that all of the domestic animals described above have a relatively smaller germline V gene repertoire. Future studies are needed to completely characterize Ig gene loci and the mechanism responsible for diversity generation in these species. A better understanding of the genetic components of the animal immune system, including the Ig genes, would surely be useful in the study of disease control in farm animals.Ig: Immunoglobulin; H: Heavy; L: Light; V: Variable; D: Diversity; J: Joining; C: Constant; RSSs: Recombination signal sequences; SHM: Somatic hypermutation; GC: Gene conversion; EST: Expressed sequence tag; S: Switch region; CSR: Class switch recombination; CDR: Complementarity-determining region; N: Non-templated; P: Palindromic.The authors have no relevant conflict of interests.YS, ZL, LR, ZW, PW, NL and YZ. All authors participated in concept design, data collection and analysis, drafting and revising the manuscript. All authors read and approved the final manuscript.


The central auditory system has a crucial role in tinnitus generation and maintenance. Curative treatments for tinnitus do not yet exist. However, recent attempts in the therapeutic application of both acoustic stimulation/training procedures and electric/magnetic brain stimulation techniques have yielded promising results. Here, for the first time we combined tailor-made notched music training (TMNMT) with transcranial direct current stimulation (tDCS) in an effort to modulate TMNMT efficacy in the treatment of 32 patients with tonal tinnitus and without severe hearing loss. TMNMT is characterized by regular listening to so-called notched music, which is generated by digitally removing the frequency band of one octave width centered at the individual tinnitus frequency. TMNMT was applied for 10 subsequent days (2.5 hours of daily treatment). During the initial 5 days of treatment and the initial 30 minutes of TMNMT sessions, tDCS  vs. cathodal (N = 11) vs. sham (N = 11) groups) was applied simultaneously. The active electrode was placed on the head surface over left auditory cortex; the reference electrode was put over right supra-orbital cortex. To evaluate treatment outcome, tinnitus-related distress and perceived tinnitus loudness were assessed using standardized tinnitus questionnaires and a visual analogue scale. The results showed a significant treatment effect reflected in the Tinnitus Handicap Questionnaire that was largest after 5 days of treatment. This effect remained significant at the end of follow-up 31 days after treatment cessation. Crucially, tDCS did not significantly modulate treatment efficacy - it did not make a difference whether anodal, cathodal, or sham tDCS was applied. Possible explanations for the findings and functional modifications of the experimental design for future studies (e.g. the selection of control conditions) are discussed. Chronic tinnitus (i.e. permanent and lasting ringing sensation in the ear(s) in the absence of a physical sound source) is a significant public health concern that impairs the quality of life for millions of patients around the world. Tinnitus incidence and prevalence rates appear to be increasing not only in older people, but also in younger adults, probably due to the exposure to occupational and recreational sounds such as amplified music central auditory pathway. One possible consequence of injury to hair cells (and the subsequently decreased input to tonotopic maps in auditory cortex) is a loss of lateral inhibition from cortical neural populations which would normally code activity from the now damaged and silent receptors. As a result of such and other disturbances of the balance of excitatory and inhibitory neural transmissions, activations of neural plasticity in the central auditory system lead to alterations of neuronal activity. Among them are (i) hyperactivity, (ii) increased synchrony, and (iii) increased burst firing In the majority of cases, tinnitus is probably triggered by inner ear hair cell injury. Nonetheless, the neural generators of tinnitus are most likely located in the Traditional and also the more recently developed tinnitus treatment programs use management strategies like cognitive behavior therapy or sound masking which are aimed at the successful habituation of the effects caused by the tinnitus. However, most patients with tinnitus want more of a relief or a cure Other forms of non-invasive brain-stimulation have also been used to influence perceived tinnitus loudness and/or tinnitus-related distress. Methods such as transcranial magnetic stimulation (TMS) and transcranial direct current stimulation (tDCS) become increasingly popular to examine causal contributions of particular neural structures to defined cognitive processes  and as neuromodulatory tools to treat patients with psychiatric or neurologic diseases  15O]H2O PET studies 18F]deoxyglucose PET found increased left auditory cortex activation in tinnitus patients compared to controls, independent of perceived tinnitus laterality On grounds of studies in animals, it has been proposed that three neuronal mechanisms might underlie tinnitus perception: (i) spontaneous firing rate alterations of central auditory system neurons, (ii) changes in temporal activity patterns of such neurons (increased synchrony), and (iii) plastic reorganization of tonotopic maps However, even though the auditory cortex appears to be an obvious treatment target in tinnitus, it should be noted that the dorsolateral prefrontal cortex (DLPFC) has also been postulated as a possible target for non-invasive brain stimulation, considering that it is important for the integration of sensory and emotional aspects of tinnitus alone could alleviate perceived tinnitus intensity In the recent past, it has become more and more obvious that tinnitus is a system-wide problem modulate the efficacy of short-term combined tDCS + TMNMT treatment for not severely hearing impaired patients suffering from chronic tonal tinnitus. TMNMT (2.5 hours of training per day over 10 subsequent days) and tDCS , cathodal (N = 11), or sham (N = 11) stimulation) were applied simultaneously - the direct current was delivered while the patients were listening to their individually modified training music. Given that TMNMT is a re-training strategy, requiring repeated and regular “exercise”, we decided to also apply tDCS repeatedly (5 subsequent days)  . IteratiThe study was performed in accordance with the Declaration of Helsinki and was approved by the Ethics Commission of the Medical Faculty, University of Münster, Germany. The patients gave written informed consent for the participation in the study.We recruited 34 patients who had (i) chronic (≥3 months) tonal tinnitus, (ii) dominant tinnitus frequencies below 9 kHz, and (iii) reported to have no history of psychiatric or neurologic diseases. All patients reported hearing one single tinnitus percept. Tinnitus could be either uni- or bilateral. In case of bilateral tinnitus, the dominant tinnitus frequency did not differ between ears according to patient’s reports.Two patients dropped out during treatment  due to an inability to comply with the study requirements; patient 2  due to novel tinnitus percepts arising in the treatment phase). 32 patients (94.1%) completed the study. The participants were randomly assigned to one out of three tDCS treatment conditions: (i) anodal group (N = 10), (ii) cathodal group (N = 11), (iii) or sham group (N = 11). Retrospectively, there were no significant differences between groups regarding relevant patient characteristics . The tDCS treatment was administered for 5 consecutive days ; the TMNMT was administered for 10 consecutive days . During the initial 5 days , both treatments were administered simultaneously. In this phase, the patients received tDCS during the initial 30 min of music listening (which took 2.5 hours per day without interruptions). During the last 5 days , only the TMNMT was administered. A waiting phase of 4 days  preceded treatment onset. Treatment offset was followed by an observation phase of 31 days  .2) was horizontally placed over the skull surface representation of left Heschl’s Gyrus  was placed contra-laterally to the active electrode in the supra-orbital region, just above the right eyebrow. The current strength was set to 2 mA. Stimulation duration was 30 min per training day.tDCS was applied using the “DC-STIMULATOR PLUS” . Independent of tinnitus laterality, the active electrode  tDCS conditions, the direct current was faded in to 2 mA over the course of 30 sec. After 29.5 min of stimulation, the current was faded out to 0 mA. Total stimulation duration was 30 min. In the sham tDCS condition, the direct current was faded in to 2.0 mA and then directly faded out to 0 mA, in each case over the course of 30 sec. The same procedure was repeated 29 min after stimulation onset . Thus, i−1/2; high notch edge = tinnitus frequency · 21/2) Each patient provided up to 10 hours of their favorite music in CD quality . The music was modified in two successive steps. First, the energy spectrum of the music was “flattened” by the re-distribution of energy from low to high frequency ranges. Second, the frequency band of one octave width centered at the individual tinnitus frequency (i.e. the most prominent pitch match frequency) was removed from the music energy spectrum using a Butterworth notch filter  was matched once prior to study onset. The matching was performed by audiometrists using a clinical audiometer  and a closed headphone  following a structured protocol. The frequency resolution was 1/12 octave. In case of unilateral tinnitus, the tinnitus ear was tested. In case of bilateral tinnitus, the ear in which tinnitus was perceived as being louder was tested. In case of identical tinnitus loudness in both ears, the better hearing ear was tested.In a first step, seven “tinnitus frequency candidates” were collected. During this procedure, the tinnitus frequency and loudness were matched seven times, starting from seven different anchor frequencies .In a second step, the “winner tinnitus frequency candidate” was determined. During this procedure, in each case two of the previously determined tinnitus frequency candidates (with matched tinnitus loudness) were directly compared in a two-forced-choice procedure, starting with the lowest candidate. The winner of each comparison was tested against the lowest remaining candidate frequency. This procedure was repeated until the winner tinnitus frequency candidate was found.In a third step, an octave confusion test was performed. First, the octaves of the winner tinnitus frequency candidate between 1000 and 16000 Hz were calculated. Second, the tinnitus loudness was matched for each of these octaves. Third, the winner tinnitus frequency candidate and its octaves (with matched tinnitus loudness) were directly compared in a two-forced-choice procedure, according to step two, until the tinnitus frequency was finally determined.To assess treatment outcome, (i) perceived tinnitus-related distress and (ii) perceived tinnitus loudness were monitored throughout the study, i.e. prior to training onset (waiting phase), during training (treatment phase), and after training completion (observation phase) .E+C) was analyzed total).Tinnitus-related distress was assessed with (i) the Tinnitus Handicap Questionnaire  Tinnitus loudness was estimated by visual analog scale (VAS) twice per day  vs. 100  The data were analyzed with “Statistica 9”. The significance level was set to α = .05 (two-tailed). If the sphericity assumption of repeated measures was violated, degrees of freedom were Greenhouse-Geisser corrected. Significant main effects or interactions were further explored by means of least significant difference (Fisher LSD) post-hoc tests (family-wise error rate controlled). = 0.38, p = 0.69) nor were there significant interactions tDCS-CONDITION×EAR (left vs. right)  = 0.29, p = 0.75), tDCS-CONDITION×FREQUENCY (.125 vs.25 vs.5 vs. 1 vs. 2 vs. 3 vs. 4 vs. 5 vs. 6 vs. 7.1 vs. 8 vs. 10 vs. 12.5 vs. 16 kHz)  = 0.31, p = 0.91), or tDCS-CONDITION×EAR×FREQUENCY  = 0.71, p = 0.73))  = 0.11, p = 0.90), (logarithmized) tinnitus frequency  = 0.18, p = 0.84), and tinnitus duration  = 1.07, p = 0.36) did not significantly differ between tDCS conditions  = 0.22, p = 0.81), depression (“ADS-L”)  = 1.55, p = 0.23), and state anxiety (“STAI”)  = 0.05, p = 0.95) between tDCS conditions  (F= 0.73)) . Moreovenditions . Furthernditions . = 0.996, p = 0.381) as well as degree of relaxation experienced during listening to the training music  = 0.112, p = 0.895) did not significantly differ between tDCS conditions /Vbaseline)−1].In order to assess treatment outcome, we calculated relative change values for the time points (i) after the tDCS treatment offset at day 5 (“after tDCS + TMNMT”), (ii) after the TMNMT treatment offset at day 10 (“after TMNMT only”), (iii) three days after treatment completion at day 13 (“obs3”), and (iv) 31 days after treatment completion at day 41 (“obs31”). The baseline values were sampled directly before treatment onset. The following formula was used to calculate relative change values: [, the data were analyzed by ANOVA including TIME (baseline vs. after tDCS + TMNMT vs. after TMNMT only vs. obs3 vs. obs31) as repeated measure, and tDCS-CONDITION  as between subjects measure. There was a significant main effect of TIME  = 3.44, p = 0.042) for THQ change. Post-hoc tests revealed significant differences between “baseline” and “after tDCS + TMNMT” (p = 0.0007), “baseline” and “TMNMT only” (p = 0.009), “baseline” and “obs3” (p = 0.007), and “baseline” and “obs31” (p = 0.017)  . There w = 0.071, p = 0.932).Given that longer lasting treatment effects would be most relevant for the efficacy of the intervention, we calculated one additional, explorative statistical test at the last time point of measurement (i.e. obs31). The one-way ANOVA using tDCS-CONDITION  as between-subjects factor did not show a significant main effect  over left auditory cortex would modulate the efficacy of a combined tDCS + TMNMT short-term treatment for not severely hearing impaired patients suffering from chronic tonal tinnitus. To the best of our knowledge, this study was the first to repeatedly apply tDCS over auditory cortex in chronic tinnitus patients, and it was also the first study to combine tDCS with an acoustic neuromodulation strategy. The results indicate that, under the prevailing circumstances, there was no significant modulating effect of tDCS polarity: significant main effects or interactions of tDCS condition were neither found in the primary outcome measure THQ; ; Table 3tDCS appears to be a promising tinnitus treatment strategy, because this technique allows to modulate cortical excitability through anodal and cathodal stimulation, and the combination of external sensory stimulation with non-invasive brain-stimulation might enhance the effect of each stimulation by itself and increase the possibility of synaptic plasticity to occur A study in healthy probands showed that auditory discrimination abilities can indeed decline when cathodal tDCS is applied over the surface representation of auditory cortex The absence of significant tDCS effects in the present study should be evaluated in the context of the specific tDCS settings that were applied. One aspect to consider is the potentially functional role of the reference electrode over right supraorbital cortex. Interactions between auditory and orbitofrontal regions could have been modulated by stimulation at either nodal point and could have had an effect on tinnitus perception. Nevertheless, there were large differences in the electrode size, making it less likely that there was a biologically meaningful effect over the orbito-frontal region. A second aspect to consider is the functional anatomy of the auditory cortex, more precisely the tonotopic organization of Heschl’s gyrus; given the usually high tinnitus frequencies, it appears conceivable that the tinnitus percept is elicited more medial than lateral on Heschl’s gyrus. A more medial location would make it harder for tDCS to have an effect on the corresponding cortex. Further, it is possible and likely that tDCS effects were not limited to the auditory cortex; rather, neural activity in associated and more remote nodes of the tinnitus network could have been enhanced or suppressed. The effects of such modulations on treatment efficacy are difficult to assess. Obviously, these points should be carefully considered in subsequent studies combining tDCS and TMNMT. Crucially, a  measure of cortical activity should be included in order to assess whether intended modulations of neural activity are actually achieved.Relevant tDCS settings include for instance DC strength, stimulation duration, repetition scheme, electrode locations, and electrode sizes. In this context it appears at least unlikely that current strength (2 mA) and/or stimulation duration (30 min) were too weak or too short to evoke effects; on the contrary, compared to previous studies these parameters were rather exhausted to the limits of safe stimulation. However, optimal electrode positioning is an important point in transcranial stimulation designs. On the one hand, there are electrode location differences between different studies targeting identical cortex regions, including auditory cortex. On the other hand, modeling studies indicate that traditional electrode positioning schemes  are probably amendable when the goal is to maximize current density in the region of interest. Moreover, differential conductivities of different brain tissues  and corresponding local current flow differences might play an important role First, during the initial 5 days and in each case the initial 30 min of treatment, tDCS and TMNMT were applied simultaneously  were applied; beyond that, the TMNMT treatment was continued for another 5 days, while the tDCS treatment (which could have had lasting and accumulative effects) was stopped  tDCS globally decreases/increases (depending on polarity) overall neural activity in an additive manner. (iv) The activity difference/ratio between involved (“tinnitus-related peak”) and non-involved (“baseline”) neurons would be critical for perceived tinnitus intensity, while the magnitude of baseline neural activity  does not influence tinnitus perception. Under these conditions, a tDCS polarity-independent reduction in tinnitus intensity under combined tDCS and TMNMT treatments would be expected. In future studies, sequential combination of TMNMT and anodal/cathodal tDCS would possibly be a valuable option to treat tinnitus.However, although it remains unresolved whether and how tDCS and TMNMT effects may have interacted, the significant post-hoc test in the main outcome measure THQ; ; Table 3The present findings should be reviewed in the context of our previous, “pure” TMNMT studies The present pilot study is the first attempt to simultaneously apply TMNMT and tDCS in order to treat chronic tinnitus. Our results are difficult to understand, since (i) we do not know how and where in the brain TMNMT and tDCS interact, and since (ii) the study design could have been further optimized by including additional control conditions . Nevertheless, the effects in our primary outcome measure, THQ, suggest that a short-term combined treatment of tDCS + TMNMT may have reduced tinnitus-related distress. Thus, the stimulation and treatment parameters of combined tDCS + TMNMT treatment for chronic tinnitus should be further explored in future studies.


Saccharomyces cerevisiae. We investigated here the ability of isolated mitochondria to degrade externally added FAD and NAD (in both its reduced and oxidized forms). A set of kinetic experiments demonstrated that mitochondrial FAD and NAD(H) destroying enzymes are different from each other and from the already characterized NUDIX hydrolases. We studied here, in some detail, FAD pyrophosphatase (EC 3.6.1.18), which is inhibited by NAD+ and NADH according to a noncompetitive inhibition, with Ki values that differ from each other by an order of magnitude. These findings, together with the ability of mitochondrial FAD pyrophosphatase to metabolize endogenous FAD, presumably deriving from mitochondrial holoflavoproteins destined to degradation, allow for proposing a novel possible role of mitochondrial NAD redox status in regulating FAD homeostasis and/or flavoprotein degradation in S. cerevisiae.Flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide (NAD) are two redox cofactors of pivotal importance for mitochondrial functionality and cellular redox balance. Despite their relevance, the mechanism by which intramitochondrial NAD(H) and FAD levels are maintained remains quite unclear in  Flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide (NAD) are two molecules of pivotal importance for mitochondrial functionality, given their role as redox cofactors of a large number of dehydrogenases, reductases, and oxidases mainly involved in energy production and redox homeostasis –4. AdditSaccharomyces cerevisiae, as the premier model to understand the biochemistry and molecular biology of mammalian cells and to decipher molecular mechanisms underlying human diseases [The relevance of such processes merits further research aimed to better describe NAD and FAD homeostasis and flavoenzyme biogenesis, especially in those organisms that can be simple and suitable model for human diseases. The conserved biological processes with all eukaryotic cells, together with the possibility of simple and quick genetic manipulation, allowed for proposing the budding yeast, diseases –17.de novo synthesis of NAD, the regeneration of NAD from its nicotinamide degradation products has been described in some detail [+-dependent signaling processes which, differently from those in which NAD works as redox cofactor, require constant replenishment of cellular NAD pools. NAD+ salvage pathway takes place in the nucleus [+ is not synthesized in yeast mitochondria; consistently, two mitochondrial carriers (NDT1/2) seem to be responsible for replenishing mitochondrial NAD+ level in yeasts [In yeast and most other organisms, besides e detail , 18, 19. nucleus . Differe nucleus , 22, NADn yeasts , 24.+ into nicotinamide degradation products conversion, which occurs in many NAD+-dependent signaling processes, the NAD+ glycoside bound is potentially cleaved via reactions catalyzed by transferases (EC 2.4.2.-), like poly(ADP-ribose)polymerase; deacetylases (EC 3.5.1.-), like sirtuins; or hydrolases (EC 3.2.2.5) to produce nicotinamide and a variety of ADP-ribosyl products [+ consuming enzymes, only sirtuins (SIRTs) have been identified in yeasts [S. cerevisiae sirtuins, seems to be localized into mitochondria.As regards NADses EC 3..1.-, likproducts . From thn yeasts . No membYGL067W, is able to catalyze NAD+ hydrolysis and also very weakly FAD hydrolysis; it was reported to be located in peroxisomes, active at alkaline pH, and strongly inhibited by F−. The existence of a mitochondrial isoenzyme has not been reported so far [Another way to cleave the pyridine nucleotide molecule is at the level of pyrophosphate bond via hydrolytic enzymes. A diphosphatase (pyrophosphatase), performing an enzymatic activity towards NADH, as preferred substrate, and giving NMNH and AMP as products, has been characterized in yeast as belonging to the NUDIX hydrolase family (EC 3.6.1.-) . This end so far .2) via the sequential action of riboflavin kinase or ATP: riboflavin 5′-phosphotransferase  and FAD synthase or FMN: ATP adenylyltransferase . The first eukaryotic genes encoding for RFK [S. cerevisiae. Besides in the cytosol, FAD synthesis occurs also in yeast mitochondria [Turning to FAD, it is synthesized starting from riboflavin  [Arabidopsis and named AtNUDX23. It belongs to the NUDIX hydrolase family and is distributed in plastids [S. cerevisiae NUDIX hydrolase family were able to hydrolyze FAD remains to be investigated, in the frame of characterization of a putative mitochondrial FADppase [The knowledge of FAD cleavage events in yeast is rather poor, as opposed to its biosynthesis. The first investigation on FAD hydrolysis was carried out on cellular extracts obtained by the flavinogenic yeast ermondii , 37. Folermondii , furtheria (SCM) , 40. Theplastids , 42. TheS. cerevisiae.Here we studied the ability of SCM to catalyze NAD and FAD hydrolysis via enzymatic activities which are different from the already characterized NUDIX hydrolases. The differential inhibition by the oxidized and reduced form of NAD, together with the ability of mitochondrial FADppase to metabolize endogenous FAD, presumably deriving from mitochondrial holoflavoproteins destined to degradation, allows for proposing a novel possible role of mitochondrial NAD redox status in regulating FAD homeostasis and, possibly, flavoprotein degradation in All reagents and enzymes were from Sigma-Aldrich , Zymolyase was from ICN , and Bacto Yeast Extract was from Difco . Mitochondrial substrates were used as Tris salts at pH 7.0. Solvents and salts used for HPLC were from J.T.Baker .S. cerevisiae strain  used in this work was derived from the CEN.PK series of yeast strain and was obtained from P. Kotter , as already described in [2PO4, 1 g/L NH4Cl, 0.5 g/L NaCl, 0.5 g/L CaCl2·2H2O, MgCl2·6H2O, 20 mg/L uracil, 0.05% glucose) supplemented with 2% ethanol as carbon source. The pH of the medium was adjusted to 5.5 with HCl.The wild-type ribed in . Cells wS. cerevisiae mitochondria (SCM) were isolated according to [Crude rding to . For purrding to . The intrding to , 44 by trding to . The SCMrding to , 45. Mitrding to .S. cerevisiae was monitored by means of fluorimetric and HPLC measurements, essentially as in [2 by means of a LS50B Perkin Elmer spectrofluorimeter. Flavin fluorescence emission spectra were corrected as in [FAD externally added or endogenous FAD metabolism in ly as in , 47. In ed as in  by addinε450 of 12.2 mM−1 · cm−1 for FMN and Rf, and 11.3 mM−1 · cm−1 for FAD. Under the experimental conditions used here, FAD fluorescence constant (KFAD) proved to be about ten times lower than those of FMN and Rf (KFMN/Rf) which did not differ significantly from each other [−1 · mg−1 protein, was calculated from the rate of fluorescence increase, measured as tangent to the initial part of the experimental curve by applying the following equation:F is expressed in fluorescence arbitrary units, Vf is expressed in mL, ΔK = KFMN/RF − KFAD is expressed as μM−1, Δt is expressed in min, and m is the mass of protein in mg.In each experiment, FAD, FMN, and Rf fluorescence was calibrated individually using standard solutions whose concentrations were calculated by using ch other , 49. ThuμL), taken at various times, perchloric extracts were obtained and neutralized with KOH [o via HPLC showed no significant difference, these experimental approaches were used in this work indifferently.In the case of HPLC measurements, from aliquots of the subcellular suspension  and time drive measurements  were carried out at 25°C in 2 mL of a standard medium.NADH metabolism in + can be revealed (when it is the sole process responsible for NADH disappearance) as a fluorescence decrease at 260/456 nm and 340/456 nm as tangent to the linear part of the experimental curve asFecc/em is expressed in fluorescence arbitrary units, Vf is expressed in mL, KNADH260 and KNADH340 are expressed as μM−1, and Δt is expressed in min.NADH oxidation to give the nonfluorescence NADF260/456/Δt higher than ΔF340/456/Δt.Differently from oxidation, NADH hydrolysis results in products which are not fluorescent at 260/456 nm but still fluorescent at 340/456 nm . Therefore, when hydrolysis is the sole process responsible for NADH disappearance, it can be revealed as a fluorescence decrease at 260/456 nm and 340/456 nm as tangent to the linear part of the experimental curve asRearranging  and 3),,3), it i−1 · mg−1 protein, can be calculated by applying the following equation:F260/456/Δt higher or equal to ΔF340/456/Δt.The rate of NADH hydrolysis in the presence of residual NADH oxidation, expressed as nmol NADH hydrolyzed · minThe rate of NADH oxidation in the mixed system can be calculated asKNADH260 and KNADH340 values were determined by calibrating NADH fluorescence with standard solutions, whose concentrations were spectrophotometrically defined (ε340 was 6.2 mM−1 · cm−1). NMNH fluorescence was calibrated using a standard curve produced by incubating NADH (at concentrations ranging from 1 to 10 μM) with excess amounts of commercial nucleotide pyrophosphatase (EC 3.6.1.9) to determine the ΔK340 value.In each experiment, the + hydrolysis was measured by fluorimetrically monitoring the hydrolysis of nicotinamide 1,N6-ethenoadenine dinucleotide (ε-NAD+), essentially as described in [ε-NAD+ (at concentrations ranging from 1 to 6 μM) with excess amounts of commercial NADase [ε-NAD+ was determined by the conversion of ε-NAD+ to ε-NADH using the commercial alcohol dehydrogenase reaction and assuming a molar extinction coefficient for ε-NADH of 6.2 mM−1 · cm−1 at 340 nm.NADribed in , 51. Flul NADase . The conData fitting was performed according to the Michaelis-Menten equation:t-test. Values of P < 0.05 were considered statistically significant.All experiments were repeated at least 3 times with different mitochondrial preparations. Results are presented as mean ± standard deviation. Statistical significance was evaluated by Student's −1 · mg−1 protein. When ADP (0.1 mM) was added, the oxygen uptake rate increased up to 1240 ngatoms O · min−1 · mg−1 protein, with a respiratory control index (RCI) value equal to 1.8. Under this ADP limiting concentration a P/O value equal to 1.4 was measured. Addition of ADP at higher concentration (1 mM) increased the oxygen consumption rate up to 1730 ngatoms O · min−1 · mg−1 protein, with a RCI value equal to 2.5. In three experiments, performed with different mitochondrial preparations, SCM showed RCI values ranging from 2.0 to 3.0. As expected, NADH-induced respiration was almost totally inhibited by KCN (1 mM), thus excluding a significant contribution by alternative oxidase pathway [In order to study NAD(H) mitochondrial metabolism we isolated functionally active SCM, as described before , 32, and pathway .μM) in the absence and in the presence of KCN (1 mM)  . Fluores−1 · mg−1 protein was calculated by applying . Similar results were obtained when the fluorescence decrease was continuously measured at both the wavelength pairs 260/456 nm and 340/456 nm, respectively. Typical traces are reported in A possible explanation for this asymmetry derived from the hypothesis that oxidation is not the sole process responsible for NADH disappearance in the suspension, with a possible contribution due to putative hydrolytic processes. In this case a NADH hydrolysis rate equal to 14 nmol NADH hydrolyzed · min−1 · mg−1 protein); this allows us to propose a mitochondrial localization for NADH destroying enzyme, even if not exclusive. The already characterized hydrolase which is known to cleave NADH, namely, Npy1p , powerful inhibitors of the peroxisomal enzyme, were tested  and CaCle tested . Since t+ fluorescent analog, namely, nicotinamide 1,N6-ethenoadenine dinucleotide (ε-NAD+) [ε-NAD+ (50 μM) emission spectrum is reported, as revealed at pH 7.5, in the 340–500 nm range, with excitation wavelength set a 310 nm. As expected, it reveals a characteristic peak at 410 nm, whose intensity linearly depends on ε-NAD+ concentration. Addition of SCM (previously solubilized with TX100) induced an increase in fluorescence emission at 410 nm, as a linear function of time up to 20 min. Since the ε-adenine ring specific fluorescence constant is about ten-fold higher than that of the ε-NAD+, the observed fluorescence increase strongly suggests that ε-NAD+ was cleaved with a rate equal to 0.23 nmol ε-NAD+ cleaved · min−1 · mg−1 protein. In ε-NAD+ (50 μM) suspensions, following the addition of solubilized SCM, were continuously measured, at the fixed excitation and emission wavelengths , corresponding to an initial rate of hydrolysis equal to 0.25 nmol · min−1 · mg−1 protein (+TX100). No increase was observed when freshly isolated intact SCM were used (−TX100), thus suggesting an intramitochondrial localization for NAD+-cleaving activity.A simpler investigation of NAD-destroying activities makes use of a NAD(ε-NAD+) , 51. Thiε-NAD+ catalyzed by solubilized SCM was determined in the pH range 4.5–10, using 100 mM acetate/acetic acid and 50 mM Tris/HCl buffering mixtures .To gain some insights into the substrate affinity, the dependence of the rate of ntration . Hyperboware see . Thus, Kε-NAD+ hydrolytic activity was also measured in solubilized sphero, under the same experimental conditions. As reported in ε-NAD+ hydrolytic activity in sphero is similar to that measured in SCM. This finding, as previously observed for NADH hydrolytic activity, suggests a preferential, but not exclusive, mitochondrial localization for this enzyme.The specific ε-NAD+ hydrolytic activity was not inhibited by both NaF (1 mM) and CaCl2 (1 mM)  but also significantly inhibited by nicotinamide, with an IC50 of about 5 mM  , thus agout 5 mM .This last experiment strongly supports the proposal that a nicotinamide-sensitive pyridine nucleotide-destroying enzyme is enriched in SCM, even if, unfortunately, it did not allow to precisely identify the product of mitochondrial NAD(H) hydrolysis. Nevertheless, the strong inhibition by AMP allows us to postulate that the dinucleotide was broken at the level of pyrophosphate bond, as already demonstrated for certain animal and plants FAD pyrophosphatases  , 55. + degradation, as well as FAD degradation (see below), strictly depends on pH, which in yeast is highly dynamic [Particular attention merits the finding that NAD dynamic ; thus, w dynamic , presuma+, as well as FAD degradation (see below), which in turn may regulate redox homeostasis, cell growth, and defense responses [Mild uncoupling  or varioesponses , 62.Since the contribution of mitochondrial NAD(H) degrading enzyme activity on the economy of NAD(H) homeostasis and, in turn, on cell longevity and stress resistance is still unexplored, our future goal will be to identify acidic mitochondrial NAD pyrophosphatase in yeast.S. cerevisiae [μM) was added to solubilized SCM and flavin fluorescence emission spectra (with excitation wavelength set at 450 nm) registered at pH 7.5, as a function of time. As expected, an increase in the 500–600 nm wavelength range with a peak at 520 nm was observed in −1 · mg−1 protein.As far as FAD degradation is concerned, the occurrence of AMP-inhibited FAD hydrolysis in isolated intact mitochondria was for the first time demonstrated in rat liver  and, morrevisiae . In orderevisiae , could aμM) fluorescence was continuously measured at the fixed excitation and emission wavelengths . No fluorescence increase was observed after addition of intact and coupled SCM (−TX100). When FAD was added to solubilized SCM an increase was observed with a rate equal to 0.30 nmol FAD hydrolyzed · min−1 · mg−1 protein (+TX100). In seven experiments performed with different mitochondrial preparations, a mean rate of 0.32 ± 0.05 nmol FAD hydrolyzed · min−1 · mg−1 protein was measured. FAD hydrolysis could be revealed only when SCM were solubilized by TX100 and in this aspect SCM are different from rat liver mitochondria.In In order to characterize the products of FAD hydrolysis catalyzed by solubilized mitochondria, under the same experimental conditions described in μM) were incubated either in the absence (panel (A)) or in the presence of AMP (panel (B)). In a parallel assay the effect of NaPPi (panel (C)), which was proved to prevent FMN hydrolysis in SCM [Solubilized SCM added with FAD . At longer incubation time, Rf, that was not initially detectable, appeared in the suspension (see inset) and it increased linearly up to 60 min (with a rate of 0.10 · min−1 · mg−1 protein), thus prevailing over FMN (panel (A)) which remained relatively constant (at about 0.06 μM). Since FAD hydrolysis rate does not depend on the rate of FMN into Rf conversion in the first 10 min , kinetics measurements of FADppase reaction were performed consistently by fluorimetric method.As shown in −1 · mg−1 proteins), which was slowly accompanied by Rf appearance with an 85% reduced rate, in comparison to the rate measured in the absence of NaPPi (panel (C)). As expected, the rate of FAD hydrolysis was totally inhibited by AMP (1 mM); neither FMN nor Rf was found to appear during the incubation time (panel (B)). Conversely, in the presence of NaPPi (1 mM), the rate of FAD hydrolysis resulted unchanged, with a notable increase in FMN  hydrolysis was studied at pH 6 , FAD disappearance was found to occur with a 2.5-fold higher rate and to be still in a good agreement with a stoichiometric ratio of 1 : 1 with the appearance of FMN, that is the preeminent species at this pH value, whereas the rate of Rf appearance is extremely low .When the mitochondrial FAD . This pH profile is quite different from that described in [GraFit software .Consistently, the rate of FAD cleavage catalyzed by solubilized SCM, fluorimetrically determined in the pH range 4.0–10 in ribed in , whose oware see . Thus, KThese results confirm that in SCM, a FADppase exists and it is able to catalyze the cleavage of the diphosphate bound in dinucleotides. This enzyme is inhibited by AMP and stimulated by acidic pH value. A putative mitochondrial FMN phosphohydrolase activity is involved in FMN dephosphorylation; it is specifically inhibited by NaPPi and its activity is significantly reduced at acidic pH value.2 (1 mM), differently from AMP (1 mM), did not reduce FAD hydrolytic activity in SCM  and CaCly in SCM , as alreε-NAD+ hydrolysis, nicotinamide (as well as NMN) did not affect FAD hydrolysis rate. Therefore, we may conclude that mitochondrial NAD(H) and FAD destroying activities are different. Another difference between mitochondrial NAD(H) and FAD destroying activities resides in the observation that specific FAD hydrolytic activity catalyzed by solubilized SCM corresponded to about only 27% of that measured in the sphero . This strongly suggests that endogenous FAD has been hydrolyzed to FMN/Rf. Endogenous flavin fluorescence was found to increase as a linear function of time up to 30 min with a rate equal to 7 pmol endogenous FAD hydrolyzed · min−1 · mg−1 protein (see inset). It should be noted that under our experimental conditions less that 20% of total mitochondrial FAD amount was hydrolyzed, presumably due to an inhibition by FAD hydrolysis product(s) or because the remaining FAD was bound to holoflavoprotein and thus protected by hydrolytic activity.In a first attempt to determine the physiological role for the intramitochondrial FADppase, the possibility that this enzyme can play a major role in the intramitochondrial FAD degradation was studied by both fluorescence and HPLC experiments. A typical fluorimetric experiment is reported in + (1 mM), which was found to completely inhibit endogenous FAD to FMN conversion (+NAD+). Since FADppase is located inside mitochondria the stimulatory effect of the detergent could not be easy explained, unless some internal metabolite, whose concentration is high in the matrix of intact organelles and diluted following membrane rupture, is able to inhibit FADppase. In this hypothesis we tested a possible inhibitory effect by NAD−1 protein, respectively, and no Rf was detected (panel (A)). As a result of TX100 addition, FAD and FMN were found to decrease, with significant appearance of Rf (panel (B)). Rf content was found to increase up to 20 min in a fairly good stoichiometric ratio (1 : 1) with FAD decrease (data not shown). The rate of FAD hydrolysis  was equal to 7.2 pmol · min−1 · mg−1 protein, in good agreement with fluorescence measurements. Rf appearance was strongly prevented by NAD+ (panel (C)).In the same experiment, the hydrolysis of endogenous FAD was confirmed by HPLC measurements of FAD, FMN, and Rf amounts before and after TX100 addition . In the + was confirmed on exogenous FAD hydrolysis by HPLC (data not shown) and the nature of the inhibition studied fluorimetrically  and guanosine nucleotides, but not nicotinamide and NMN, are able to reduce FAD hydrolysis rate (data not shown); therefore we feel that the purine moiety is relevant for inhibitor recognition by FADppase. Consistently, NADH is also able to inhibit in a noncompetitive way the rate of FAD hydrolysis, but, interestingly, the Ki differed for an order magnitude, being equal to 100 μM for NADH, as calculated from the data reported in Adenosine and derived compounds  and by defense systems (some of which are flavoenzymes) [+ to NADH is expected to favor hydrolysis of free FAD, since the oxidized form of the pyridine cofactor is more powerful as inhibitor (lower Ki) than the reduced form (higher Ki), at least in ruptured SCM. Thus, we can speculate that reducing conditions are accompanied by a more rapid local “recycling” of FAD, presumably deriving from holoflavoprotein turnover. This situation could allow a more rapid intramitochondrial assembly of novel flavoproteins  . Accordilike SDH , 63) andin vivo by pyridine nucleotide redox status inside SCM as postulated and summarized in the scheme in Whether or not flavin-coenzymes homeostasis is modulated Here we proved the ability of SCM to catalyze NAD(H) and FAD hydrolysis, via enzymatic activities which are different from the already characterized NUDIX hydrolases. S. cerevisiae.The differential inhibition by the oxidized and reduced form of NAD toward the mitochondrial FADppase, together with the ability of mitochondrial FADppase to metabolize endogenous FAD, presumably deriving from mitochondrial holoflavoproteins destined to degradation, allows for proposing a novel possible role of mitochondrial NAD redox status in regulating FAD homeostasis and, possibly, flavoprotein degradation in


Human Mutation34(4):546–556 (DOI 10.1002/humu.22273).The original article to which this Erratum refers was published in ikM andIn the article cited above, the symbols for The Publisher sincerely regrets this error.u,v, the values of π need to bedetermined. The naïve evaluation of Eq. (7) can be replaced by a recursive scheme . Consider sequence positions k <i <l and denote by ikM andilN, the probabilities that i has a pairing partner inthe interval  and the interval, respectively. Clearly, theseauxiliary variables satisfy ikM =Mi(k − 1) +kiP and ilN =Ni(l + 1) +ilP. Obviously, for all k < i< l, we have M and N thus allows the evaluation of distances andcorrelation coefficients in linear time for each sequence interval.At face value, this minimization is rather expensive because for each index pair


Thermal conductivity in non-metallic crystalline materials results from cumulative contributions of phonons that have a broad range of mean free paths. Here we use high frequency surface temperature modulation that generates non-diffusive phonon transport to probe the phonon mean free path spectra of GaAs, GaN, AlN, and 4H-SiC at temperatures near 80 K, 150 K, 300 K, and 400 K. We find that phonons with MFPs greater than 230 ± 120 nm, 1000 ± 200 nm, 2500 ± 800 nm, and 4200 ± 850 nm contribute 50% of the bulk thermal conductivity of GaAs, GaN, AlN, and 4H-SiC near room temperature. By non-dimensionalizing the data based on Umklapp scattering rates of phonons, we identified a universal phonon mean free path spectrum in small unit cell crystalline semiconductors at high temperature. By non-dimensionalizing the data based on Umklapp scattering rates of phonons, which is the dominant resistive scattering mechanism at high temperature , we discovered a universal thermal conductivity accumulation function .The physics of heat transport in condensed matter is critical to thermal management in a diverse range of technologies as well as thermoelectric energy conversion. The dominant carriers of heat in non-metallic crystalline materials are phonons, defined as quantized lattice vibrations. Specific heat, phonon group velocity, and phonon mean free path (MFP)–the average distance a phonon travels between scattering events–determine a material's thermal conductivity. Recent studies have shown that it is possible to experimentally measure the MFP dependent contributions of phonons to thermal conductivity–the phonon MFP spectrum1234T = 300 K are 501214−1K−117kaccum of GaAs, though the predictions have not been experimentally confirmed20213kaccum are required to understand these effects, which are prevalent in GaAs, GaN, AlN, and SiC technologies.GaAs, GaN, AlN, and SiC are critical to the high power electronics and optoelectronics industries9k, as C is the volumetric heat capacity, RMSv is the root-mean-squared velocity, and l is the phonon MFP, v is the phonon group velocity, CMFP is the volumetric heat capacity per unit phonon MFP, and s indexes the polarization of phonons. Since the integral is taken from 0 to l*, kaccum quantifies the contribution to bulk thermal conductivity of phonons with a MFP less than or equal to l*.Kinetic theory can be used to derive an approximate expression for thermal conductivity, kaccum have been reported using BB-FDTR, time domain thermoreflectance (TDTR), and transient grating techniques1234LP, where f was the modulation frequency of the pump laser. Further studies using TDTR have equated l* to the laser spot size on SiExperimental measurements of kaccum is shown in f1, which upon absorption in the Au transducer film induces a temperature response in the sample at frequency f1. The continuous wave probe laser beam then reflects off the sample with a modulated intensity at frequency f1, as a result of Au's thermoreflectance. The reflected pump and probe have identical frequencies with a phase difference representing the phase-lag of temperature to heat flux at the sample surface. The pump and probe signals are then heterodyned by a second EOM at frequency f2 to reduce coherent and ambient noise as described in the The BB-FDTR apparatus used to measure 1f ≤ 200 MHz) accurately identifies the expected bulk values of SiO2 and platinum samples with little deviation between the measured data and analytical fits1LP. On the other hand, a constant thermal conductivity over all heating frequencies was found to under predict thermal conductivity in single crystal Si−1K−1 for 4H-SiC at T = 304 K (40% of kbulk). This suppression is expected when LP is shorter than the MFPs of some phonons1kj, is plotted in −1K−1 to 74 Wm−1K−1 over the frequency range (200 kHz to 200 MHz).BB-FDTR and TDTR measurements have demonstrated that a constant value of thermal conductivity over the range of measured heating frequencies T = 330 K. As the bulk thermal conductivity of the material increases, phonons with longer MFPs contribute more significantly to the bulk thermal conductivity. Phonons with MFPs less than 1000 ± 200 nm, 2500 ± 800 nm, and 4200 ± 850 nm contribute 50% to the bulk thermal conductivity of GaN, AlN, and 4H-SiC at T = 309 K, 308 K, and 304 K, respectively.The in-planek = cross-planek, was assumed in GaAs, GaN, AlN, and 4H-SiC across all heating frequencies. This assumption is valid for GaAs as its cubic (zinc-blende) crystal structure contains high-order symmetry and its thermal conductivity tensor is isotropic3117LP ~ r  for much of the frequency range in 4H-SiC, heat spreading is not purely one-dimensional. Yet the data must be normalized to a single value. Therefore, we have chosen the [1000] axis bulk values because they are reported over a wide range of temperatures. To examine the effects of anisotropy in 4H-SiC, a heat diffusion model considering in-plane and cross-plane thermal conductivities separately was used to fit phase-lag data as a function of heating frequencycross-planek = 0.8in-planek was held constant. The observed thermal conductivity difference between the anisotropic and isotropic data interpretations varied by less than 11% of kbulk at T = 407 K and 304 K and by less than 4% of kbulk at T = 151 K and 81 K  for a single polarizationn is the Bose-Einstein distribution, and BZEω. Classical occupation leads to kB is the Boltzmann constant. This assumption is accurate for phonons having T < BZEθ, as it is for our materials. Therefore, the normalized kaccum, including acoustic longitudinal and transverse modes is , Lp,nondimensional . The scale of the LP,nondimensional-axis in lmin). For comparison the projections of kuniversal based on truncated Debye and Born-von Karman Slack models are shown in The assumption of Umklapp dominated scattering is adequately met by Si, GaN, AlN, and 4H-SiC at kuniversal indicates that isotope scattering did not strongly affect the τ−1 ∝ ω2 scattering of long MFP phonons probed by BB-FDTR.Some debate exists on the importance of isotope scattering in gallium-based semiconductors, though authors agree that it is diminished at high temperatures313839kaccum enables mapping of thermal conductivity suppression in nanostructures and devices based on their characteristic size and is therefore critical to the thermal design of electronic, photonic, and thermoelectric technologies. The existence of a material independent kuniversal in GaAs, Si, GaN, AlN, and SiC suggests that the phonon mean free path spectrum is a universal feature of intrinsic crystalline semiconductors. This is useful for the projection of kaccum in intrinsic crystalline semiconductors where thermal resistance is dominated by Umklapp processes based on parameters that can be obtained from historical thermal conductivity vs. temperature data. Because our data is based on small unit cell, high thermal conductivity materials, additional experiments are needed to determine whether more complex materials such as alloys and large unit cell crystals adhere to a different form of kuniveral.Knowledge of 20 (1:9) solution for 10 minutes at room temperature to remove the native oxide layer and blown dry in ultra high purity nitrogen. Next, a chromium adhesion layer and a gold transducer layer of thickness reported in Undoped, semiconductor grade (100) GaAs, (0001) GaN, (0001) AlN, and (0001) 4H-SiC bulk wafer samples were purchased and provided by University Wafer Inc., Kyma Technologies Inc., HexaTech Inc., and Cree Inc., respectively. Preparation of bulk wafer samples for BB-FDTR measurements included two steps. 4H-SiC was dipped into an HF:Hkaccum, BB-FDTR heterodynes the pump and probe signals present at 1f with a second modulation at frequency f2 producing signals at f2 − f1 and f1 + f2. This allows for heating frequencies up to 200 MHzf1 + f2 is filtered out and the lower frequency component, f2 − f1, is recorded using an SR-830 lock-in amplifier. An optical band pass filter is used to attenuate the pump or probe beam to permit for measurement of each beam individually. Frequencies f1 and f2 are concurrently swept between 200 kHz and 200 MHz while maintaining f2 − f1 = 86 kHz. A constant 1/e2 spot size radius of 2.65 ± 0.13 μm, measured using a knife-edge technique, was used for all data presented.To measure a large range of To fit thermal conductivity and thermal interface conductance two assumptions were made. 1) The thermal interface conductance between the sample and the chromium-gold layer was frequency independent. This was reasonable as the heating frequency was five orders of magnitude smaller than the relaxation rate of electrons in the gold layer at room temperature The therJ.P.F. prepared samples and performed all measurements. J.H.L., E.A.P., Z.S. and R.F.D. provided samples. J.P.F. and J.A.M. wrote the manuscript. All authors discussed the data and edited the manuscript.Supplementary Information