S-glutathionylation is the formation of disulfide bonds between the tripeptide glutathione and cysteine residues of the protein, protecting them from irreversible oxidation and in some cases causing change in their functions. Regulatory glutathionylation of proteins is a controllable and reversible process associated with cell response to the changing redox status. Prediction of cysteine residues that undergo glutathionylation allows us to find new target proteins, which function can be altered in pathologies associated with impaired redox status. We set out to analyze this issue and create new tool for predicting S-glutathionylated cysteine residues.One hundred forty proteins with experimentally proven S-glutathionylated cysteine residues were found in the literature and the RedoxDB database. These proteins contain 1018 non-S-glutathionylated cysteines and 235 S-glutathionylated ones. Based on 235 S-glutathionylated cysteines, non-redundant positive dataset of 221 heptapeptide sequences of S-glutathionylated cysteines was made. Based on 221 heptapeptide sequences, a position-specific matrix was created by analyzing the protein sequence near the cysteine residue (three amino acid residues before and three after the cysteine). We propose the method for calculating the glutathionylation propensity score, which utilizes the position-specific matrix and a criterion for predicting glutathionylated peptides.Non-S-glutathionylated sites were enriched by cysteines in − 3 and + 3 positions. The proposed prediction method demonstrates 76.6% of correct predictions of S-glutathionylated cysteines. This method can be used for detecting new glutathionylation sites, especially in proteins with an unknown structure. S-glutathionylation is the reversible formation of disulfide bonds between cysteine residues of a protein and glutathione tripeptide associated with cell response to changes in redox conditions, including hypoxia, ischemia and reperfusion. This mechanism is an important part of the co- and post-translational regulation of the function of various proteins –3. GlutaThus, the determination of cysteine residues, which are potential targets for glutathionylation, is important for understanding the mechanisms of glutathionylation effect on proteins functioning and confirmation of targets by mutagenesis. Prediction of glutathionylating cysteine residues allows finding new target proteins that alter function in pathologies associated with impaired redox status.There are a number of studies based on these collected data on the prediction of glutathionylation sites in proteins –13. An ahttp://csb.cse.yzu.edu.tw/GSHSite/index.php).RedoxDB was the first protein glutathionylation database . It inclhprd.org. Sequences of animal and human proteins, where glutathionylation was confirmed by methods of molecular biology (mass spectrometry and site-directed mutagenesis) were selected for further analysis. Some sequences were obtained from uniprot.org, search was performed using the keyword “Glutathionylation [KW-0318]”. Sequences were taken as heptapeptides, containing three aminoacid residues to the left and to the right from the target cysteine residue.S-glutathionylathion sites were obtained from the reviews dedicated to regulatory glutathionylation of proteins , 15, 16 Repeating heptapeptides were excluded from S-glutathionylation positive dataset. Thus, 221 non-redundant S-glutathionylation sites of 140 proteins with experimental verification were found .The distribution of the score for all possible sequences of heptapeptides with cysteine in the central position is calculated by enumerating 20 aminoacids in three positions to the right and three positions to the left from the cysteine residue Fig. . Thus, tPrediction with respect to glutathionylation was done on the basis of S score. Cut off value splits graphs of the distributions for all combinatorially possible heptapeptide sequences and for positive dataset sequences so that areas under the curves are equal Fig. . Сut offTo perform a validity test we removed three randomly selected heptapeptides from non-redundant positive dataset, then we constructed PSM for the remaining 218 heptapeptides from the dataset . Next, using a modified position-specific matrix, we calculated Glutathionylation Propensity Scores for all possible heptapeptide sequences with cysteine in the central position and for new positive dataset of 218 heptapeptides. For excluded peptides, we calculated the Glutathionylation Propensity Score and compared it with a threshold value. In each experiment, it was determined how many of the three heptapeptides were recognized correctly . Jackknife resampling was repeated 200 times. The overall accuracy of the method was determined as the number of correctly defined sequences of 3 * 200 = 600 control points.We selected 140 proteins containing cysteines susceptible to glutathionylation to analyze the effect of short flanking regions .However, we believe that it is possible to find experimental conditions that any cysteine available to the solvent will interact with glutathione. Thus, the set of non-S-glutathionylated cysteines is fundamentally absent. Indeed, it was not possible to find experimentally confirmed cases in the literature that cysteines, being accessible to the solvent and glutathione, do not react with it. In the absence of confirmed data on non-S-glutathionylated cysteines, we used the distribution of S score for all combinatorially possible heptapeptides with cysteine in the central position as the base sample . The Wilcoxon test also gives a similar result . Thus, both tests confirm the formal difference between these distributions.The calculated Mann-Whitney statistics U = 1050 (the number of degrees of freedom df = 51). The probability that these two distributions differ is In order to make the simplest decisive rule for predicting S-glutathionylation of central cysteine of heptapeptide, it is necessary to determine the cut-off value with respect to the areas under the graphs of the two compared distributions that are equal. Having determined the area under the distribution curves, we found that this boundary is S = 294. Thus, the decision on whether the heptapeptide belongs to the class of S-glutathionylated is made if the value of the calculated glutathionylation propensity score S exceeds the threshold value of 294.Conclusions on this decision rule, as when testing any statistical hypothesis, are formulated with varying degrees of confidence. Namely, we make the prediction of cysteine S-glutathionylation with greater confidence than non-S-glutathionylation. This is due to the lack of experimentally proven negative control, i.e. such cysteines on the surface of the protein, which, being accessible to the solvent and glutathione, do not react with it.We applied the proposed decision rule to 1282 cysteines from 140 S-glutathionylated proteins  was correctly recognized by the proposed method. In 25 cases out of 200, one control peptide of the three was correctly recognized, in 102 cases two control peptides were recognized, and in 68 cases all three control peptides were recognized correctly. This indicates a good predictive ability of the proposed method on real data. The percentage of correct classification is 72.2% (Sensitivity).Glutathionylation plays an indispensable role in protecting cell viability during oxidative stress. S-glutathionylation prevents irreversible oxidation of cysteines and protects proteins from subsequent inactivation and degradation. Some glutathionylated proteins significantly change their function, which helps the cell adapt to altered redox conditions. For example, inhibition of Na,K-АТPase under hypoxia prevents ATP depletion and promotes cell survival .The prediction of S-glutathionylated cysteine residues in proteins is very important. It will allow checking the predicted residues by site-directed mutagenesis, analyzing structural changes and understanding how the function of these proteins changes during oxidative stress. It is necessary to have as full as possible prediction of S-glutathionylated cysteine residues. In this case, it is more important not to lose potential candidates than to receive false positive predictions. This requirement corresponds to maximization of the Sensitivity score, which reflects the completeness of prediction of the positive dataset.The interaction of glutathione with cysteine residues of proteins is possible due to at least two critical factors. The first is the accessibility of the cysteine residue for the solvent, and, accordingly, for interaction with glutathione. The flanking regions around cysteine indirectly characterize its position in the protein structure. Thus, the presence of hydrophobic residues may suggest the location of this cysteine inside the protein. The presence of charged, polar or residues with hydrogen bonds may indicate the location of such cysteine on the surface and accessibility to the solvent. However, it is impossible to check the accessibility of cysteine to solvent in the absence of the structure of this protein. Even the presence of an experimentally determined protein structure cannot guarantee the absence of steric difficulties in the interaction of glutathione with cysteine in vitro and in vivo. This is evident, because there are local and even global differences in the three-dimensional structure of protein under different conditions.The second factor is the reactivity of cysteine residue. Reactivity is determined by many factors, such as the properties of the solution  and amino acid context of the sequence, which affects the shift in electron density. Adjacent positively charged amino acids can facilitate deprotonation of cysteines SH group  and increase their reactivity . It is pWe have created S-glutathionylation predictor that does not take into account the negative control Table , Fig. 1.In addition, we suggest that the spatial context may affect the ability to react with glutathione. That is, amino acid residues that are distant in sequence but close in space can, in our opinion, affect the reactivity of this cysteine to glutathionylation. This assumption requires further study.Possibly, recognition indicators of glutathione-cysteine can be improved if not all theoretically possible sequences of seven residues (heptapeptides) with cysteine in the central position were considered for comparison with S-glutathionylated heptapeptide sequences dataset, but only those that really do not react with glutathione when accessible to solvent and glutathione. In addition, it is evident that not all combinatorially possible heptapeptide sequences exist in nature. It can also be expected that the accuracy of the prediction will increase with an increase in the number of known of S-glutathionylation sites.In this article, we have presented simple successful predictor for identifying S-glutathionylated sites from short flanking sequences around cysteine residues. The proposed predictor is based on position-specific matrix, S-glutathionylation propensity score calculation and cut-off value of 294. The performance of the method was measured with a sensitivity of 76.6% and MCC of 0.13. Additionally, the proposed method was evaluated using a Jackknife resampling experiment resulting in a sensitivity of 72.2%. The proposed method may be useful for detecting new glutathionylation sites, especially in proteins with an unknown structure. Sensitivity achieved with our position-specific matrix is greater than when calculations were performed using the method of Pal et al. , is baseHere the new method for prediction of glutathionylation sites in proteins and peptides has been developed. High sensitivity of the method allows not to lose potential glutathionylation sites in proteins. The matrix reflecting the occurrence of various types of amino acid residues in three positions to the right and left of such cysteine was created. Non-S-glutathionylated sites were enriched by cysteines in − 3 and + 3 positions. The proposed method is useful for detecting new glutathionylation sites, especially in proteins with an unknown structure.Additional file 1.Additional file 2.


So far, the prevalence of human T-lymphotropic virus (HTLV) type 1 and 2 in some highly populated countries such as China is still unknown. In this study, a multi-center nationwide serological survey was designed and performed, to reveal the seroprevalence of HTLV infection among Chinese blood donors.Among 8,411,469 blood donors from 155 blood establishments, 435 were finally confirmed as HTLV carriers. The prevalence of HTLV infection in China varied in different provinces: Fujian had the highest prevalence of 36.240/100,000 (95% CI 31.990–41.050) and eleven provinces did not find HTLV-seropositive donors in the three years. no HTLV-2 infection was found. The overall prevalence of HTLV-1 in China decreased from 2016 to 2018. Female was identified as an independent risk factor of HTLV infection in China. Besides, seroconversion was observed in two of seven seroindeterminate donors 85 and 250 days after their last donation, respectively.The seroprevalence of HTLV infection in most areas of China among blood donors is quite low, but it varies significantly in different geographic areas. Screening anti-HTLV-1/2 antibody and follow-up of serointederminate donors are essential to ensure blood safety especially in areas where we have found HTLV infected donors. Human T-lymphotropic virus type 1 (HTLV-1) was the first found human retrovirus in the early 1980s by Gallo et al.  and HTLVHTLV-1 shows a unique endemic distribution in the world, with high endemic rates of infection in southwestern Japan, the Caribbean islands, Central and South America, Central Australia, sub-Saharan Africa, Romania and parts of the Middle East. It is estimated that there are at least 5–10 million HTLV-1 infected individuals . HoweverSince HTLV was found as a member of the transfusion-transmitted infectious pathogen , blood dIn China, previous regional studies showed that a prevalence ranged from 16.9/100,000 to 99/100,000 in Fujian province –16. WherHTLV-1/2 antibody assay has been introduced in certain blood establishments in China since 2006. In Fujian, Guangdong, and Zhejiang province, which were estimated to have a high prevalence of HTLV-1 infection, all blood donors were tested HTLV-1/2 antibody routinely. While, in other provinces, no less than 10% of donation samples were randomly selected and screened every year. In this study, we reported the national epidemiological data from 8.4 million blood donors screened by HTLV-1/2 antibody among 155 blood establishments from 29 provinces or municipalities from 2016 to 2018.From 2016 to 2018, 8,411,469 blood donors were routinely screened anti-HTLV-1/2 antibodies all over China. In Fujian, Guangdong (except Shenzhen), and Zhejiang province, 4,497,616 blood donors were tested, among which 2629 were repeatedly reactive by screening tests and 370 donors were finally confirmed as HTLV-1 infection by a confirmatory test.In other areas in China, 3,913,853 blood donors were tested and 1650 donors were repeatedly reactive. 1546 samples were sent to the National Center for Clinical Laboratories (NCCL) for further testing and 104 samples weren’t tested because of lack of volume or other reasons. 63.9% (988/1546) samples were non-reactive by all four different supplementary assays, while 558 samples were reactive by at least one assay and then tested by line immunoassay. 63 samples were positive by the confirmatory test, among which 49 donors were discriminated as HTLV-1 infection and 14 were untypeable HTLV infection . While the prevalence of all other areas in China was only 2.432/100,000 , nearly one-fifteenth of that of Fujian  of the screening HTLV-1/2 antibody assays varied from 0–26.30% in different provinces, with an average value of 10.17% over all samples. Among 63 confirmed positive samples in NCCL, the positive detection rates of the four screening assays were 71.43% (R1), 84.13% (R2), 90.00% (R3), and 88.46% (R4), respectively.Demographic Information  from 416 to 435 confirmed HTLV-1/2 infection donors were acquired and analyzed. Compared with all blood donors’ distribution , HTLV-1/Seven from 120 initial indeterminate donors by INNO-LIA were followed once from 85 days to 302 days after their last donation , Fujian (donated blood in Shenzhen), and Hunan (donated blood in Shenzhen), respectively. 12 of 13 donors denied having been to any HTLV-endemic countries, except one who had a short visiting history in Japan. Four of 13 HTLV carriers have been to Fujian: one stayed there for three years, another had annual short visiting, and the information of the other two donors was unknown. 11 of 13 donors have experienced invasive procedures, such as surgery, transfusion, acupuncture, or tooth washing. No epidemiological association was found in those HTLV-1/2 carriers through targeted questionnaires.HTLV-1 infection is still a neglected disease , and alsIn the study, 14 confirmed positive donations couldn’t be discriminated into HTLV-1 or HTLV-2. Since all other HTLV positive donors were HTLV-1 carriers, therefore, we presumed that these untypeable HTLV infections were probably HTLV-1 infection, and their low titer of HTLV antibodies led to untypeable results. According to previous studies in China, only three HTLV-2 carriers were found in 5480 samples, among which two HTLV-2 infections were from the high-risk group and one were from 3548 blood donors in two blood centers from Henan and Hubei , howeverThe HTLV prevalence all over China varies significantly in different geographic areas. In 2019, Li et al.  reportedBesides, we found that PPVs of the repeatedly reactive samples were higher in regions with higher seroprevalence of HTLV infection and different screening assays showed different performance on samples collected from non-epidemic areas. Two assays based on the ELISA method (R1 and R2) showed unsatisfied positive detection rates using confirmed positive samples. Furthermore, our previous study showed remarkable different PPVs of these four assays, varying from 21.0% (R3) to 91.7% (R1) . TherefoFrom this study, compared with information from all blood donor population , limitedA high proportion of indeterminate results of HTLV confirmation has always been a challenge worldwide. In our study, among 558 donations further tested by INNO-LIA, 120 were seroindeterminate. Previous studies reported different rates of seroconversion of indeterminate donations. Martins et al.  made a 4There were several limitations in the study. First of all, due to the large population of donors in China, we could not use a consistency algorithm in the HTLV antibody testing across all of the blood establishments. Therefore, in the study three initial screening assays and two different confirmatory assays were involved, which may influence the true seroprevalence of HTLV-1/2 infection in different regions of China. Secondly, the seroprevalence of our study was derived from data among healthy blood donors, which is a specific population that may have different demographic characteristics to the general population. And due to the limitation of the study population, the seroprevalence of children, teenagers, or the old people (aged > = 60 years old) were unable to be estimated from this study. In the third, only a few donors with indeterminate results and confirmed donors were successfully followed. The true infection status of the others was unknown. In addition, since this study was based on the investigation of seroprevalence of HTLV-1/2 infection, not all the initial positive samples were tested HTLV-1/2 proviral DNA by PCR method, and we did not get the HTLV sequence of the confirmed positive samples either. Therefore, we couldn’t analyze the origin of the virus among blood donors in China.In summary, the HTLV prevalence of most areas among blood donors in China is as low as other non-endemic countries or areas. The prevalence varies significantly in different geographic areas all over China and Fujian has a higher prevalence compared with other areas. Screening anti-HTLV-1/2 antibody and follow-up of serointederminate donors are essential to ensure blood safety, especially in areas where we have found HTLV infected donors. Strategy on screening and confirmation of HTLV infection in Chinese blood donors should be made and applied especially in some provinces with high prevalence.From 2016 to 2018, blood donations from 155 blood establishments all over mainland China except Qinghai and Tibet screened HTLV-1/2 antibody after routinely tested viral markers of Hepatitis B virus, Hepatitis C virus, Human immunodeficiency virus, and syphilis, among which all donations were tested in Fujian, Guangdong and Zhejiang province and no less than 10% of donations from other areas were tested anti-HTLV-1/2 antibody. All donors should meet the basic requirements of blood donation in China. The legal age for blood donation in China is from 18 years old to 55 years old. If repeat donors pass the physical examination, they could donate until 60 years old, or even 65 years old in some regions. All the blood donors who donated samples several times were counted as one case in the study. Donors who were confirmed infection of HTLV-1/2 before or during the study would not donate blood anymore.Blood samples from each blood donor were screened HTLV-1/2 antibody using one of three ELISA assays: Wantai HTLV-1/2 antibody ELISA kits , Murex HTLV I + II , and Foresight HTLV-1/2 antibody ELISA kits , which all detect both HTLV-1 and HTLV-2 antibody. All these three assays were based on a double-antigen sandwich method and approved by China National Medical Products Administration (NMPA). Any initial reactive samples should be retested twice by the same screening assay and those repeatedly reactive samples were sent to confirmatory lab to be further tested. During the period for confirmatory testing, the blood donors could not donated their blood until the confirmatory testing results came out.Samples from Fujian, Guangdong (except Shenzhen), and Zhejiang province were confirmed in one of blood centers in each province  using MP HTLV Blot 2.4 . HTLV-I viral proteins, derived from native, inactivated viral lysate and HTLV-I/II genetically engineered proteins were incorporated into the strips. Five antigens, rgp46-I, rgp46-II, GD21, p19, and p24 were used to confirm the presence of anti-HTLV-1/2 antibody.gag antigens (p19 I/II and p24 I/II) and two env antigens (gp46 I/II and gp21 I/II) were applied as non-type specific antigens to confirm the presence of anti-HTLV I/II. Three type-specific antigens, gag p19-I, env gp46-I, and env gp46-II were aimed to differentiate between HTLV-1 and HTLV-2 infections. All the assays using for supplemental tests were approved by the Food and Drug Administration (FDA) or the Conformite Europeenne (CE) and operated strictly according to the manufactures’ instructions. All the HTLV-1/2 antibody assays used in the study were summarized in Table In other areas, all screening reactive samples were further tested in the National Center for Clinical Laboratories (NCCL). Four different serologic assays were performed on one sample simultaneously. Among them two were ELISA assays: Avioq HTLV-I/II Microelisa System  and Murex HTLV I + II . The other were based on electro-chemiluminescent or chemiluminescent method: Elecsys HTLV-I/II  and Lumipulse G HTLV-I/II . Any reactive sample tested by any of the four assays above were confirmed and discriminated by Innogenetics line immunoassay . Two All the indeterminate donations were tested proviral DNA by an in-house real-time PCR method based on Taqman probe targeting HTLV-1 pol region . Total DAll the information of donor’s age, sex, ethnicity, and donation history were collected from the laboratory system. Donors confirmed positive in NCCL were contacted by staff in blood establishments to complete a further targeted questionnaire. Donors confirmed indeterminate in NCCL were followed once more than 2 months after their last donation.Logistic regression analysis was used to investigate the role of individual demographic factors on the outcome of HTLV infections using SPSS version 21.0 . p < 0.05 was considered statistically significant.Additional file 1: Table S1. Repeatedly reactive donations (n=63) by confirmatory test


Drosophila larvae. We showed that optoRaf or optoAKT activation not only enhanced axon regeneration in both regeneration-competent and -incompetent sensory neurons in the peripheral nervous system but also allowed temporal tuning and proper guidance of axon regrowth. Furthermore, optoRaf and optoAKT differ in their signaling kinetics during regeneration, showing a gated versus graded response, respectively. Importantly in the central nervous system, their activation promotes axon regrowth and functional recovery of the thermonociceptive behavior. We conclude that non-neuronal optogenetics targets damaged neurons and signaling subcircuits, providing a novel strategy in the intervention of neural damage with improved precision.Neuroregeneration is a dynamic process synergizing the functional outcomes of multiple signaling circuits. Channelrhodopsin-based optogenetics shows the feasibility of stimulating neural repair but does not pin down specific signaling cascades. Here, we utilized optogenetic systems, optoRaf and optoAKT, to delineate the contribution of the ERK and AKT signaling pathways to neuroregeneration in live  Most cells have a built-in regeneration signaling program that allows them to divide and repair. But, in the cells of the central nervous system, which are called neurons, this program is ineffective. This is why accidents and illnesses affecting the brain and spinal cord can cause permanent damage. Reactivating regeneration in neurons could help them repair, but it is not easy. Certain small molecules can switch repair signaling programs back on. Unfortunately, these molecules diffuse easily through tissues, spreading around the body and making it hard to target individual damaged cells. This both hampers research into neuronal repair and makes treatments directed at healing damage to the nervous system more likely to have side-effects. It is unclear whether reactivating regeneration signaling in individual neurons is possible.One way to address this question is to use optogenetics. This technique uses genetic engineering to fuse proteins that are light-sensitive to proteins responsible for relaying signals in the cell. When specific wavelengths of light hit the light-sensitive proteins, the fused signaling proteins switch on, leading to the activation of any proteins they control, for example, those involved in regeneration.Wang et al. used optogenetic tools to determine if light can help repair neurons in fruit fly larvae. First, a strong laser light was used to damage an individual neuron in a fruit fly larva that had been genetically modified so that blue light would activate the regeneration program in its neurons. Then, Wang et al. illuminated the cell with dim blue light, switching on the regeneration program. Not only did this allow the neuron to repair itself, it also allowed the light to guide its regeneration. By focusing the blue light on the damaged end of the neuron, it was possible to guide the direction of the cell's growth as it regenerated.Regeneration programs in flies and mammals involve similar signaling proteins, but blue light does not penetrate well into mammalian tissues. This means that further research into LEDs that can be implanted may be necessary before neuronal repair experiments can be performed in mammals. In any case, the ability to focus treatment on individual neurons paves the way for future work into the regeneration of the nervous system, and the combination of light and genetics could reveal more about how repair signals work. Inadequate neuroregeneration remains a major roadblock toward functional recovery after nervous system damage such as stroke, spinal cord injury (SCI), and multiple sclerosis. Extracellular factors from oligodendrocyte, astroglial, and fibroblastic sources restrict axon regrowth  but elimXenopus laevis embryonic development  injury. We note that most of the previous optogenetic control of neural repair studies were based on channelrhodopsion in C. elegans  and the regeneration-incompetent class III da (C3da) sensory neurons in  elegans , mouse D elegans  or motor elegans . Another elegans . These wTo reversibly control the Raf/MEK/ERK and AKT signaling pathways, we constructed a single-transcript optogenetic system using the p2A bicistronic construct that co-expresses fusion-proteins with the N-terminus of cryptochrome-interacting basic-helix-loop-helix (CIBN) and the photolyase homology region of cryptochrome 2 . Following a similar design of the optimized optoRaf , we imprWestern blot analysis on pERK (activated by optoRaf) in HEK293T cells showed that pERK activity  increaseBinding of neurotrophins to their receptor activates multiple downstream signaling subcircuits, including the Raf/MEK/ERK and AKT pathways. Delineation of signaling outcomes of individual subcircuits remains difficult with pharmacological assays given the unpredictable off-targets of small-molecule drugs. We hypothesized that optoRaf and optoAKT could delineate signaling outcomes because they bypass ligand binding and activate the intracellular signaling pathway. To test this hypothesis, we probed phosphorylated proteins, including pERK and pAKT with WB analysis in response to light-mediated activation of optoRaf and optoAKT. Results show that optoRaf activation does not increase endogenous pAKT . SimilarAlthough activation of both optoRaf and optoAKT bypasses ligand-receptor binding, it remains unclear if other upstream signaling molecules are required to activate optoRaf and optoAKT. Endogenous Raf1 activation requires its membrane translocation mediated by the GTP-bound form of Ras, followed by phosphorylation at several residues, including Ser338, which is located in the junction region between the regulator domain and the kinase domain . ReplaceSimilarly, for optoAKT, it remains unclear if its activation requires upstream PI3K signaling. Full activation of AKT requires phosphorylation on both T308 in the activation loop of the catalytic protein kinase core and S473 in a C-terminal hydrophobic motif . PH-doma2) increased the neuritogenesis ratio from the basal level (0.24 ± 0.04) to 0.52 ± 0.03  and optoAKT (UAS-optoAKT). We induced the expression of the transgenes in a type of fly sensory neurons, the dendritic arborization (da) neurons, which have been used extensively to study dendrite morphogenesis and remolding (pickpocket (ppk)-Gal4, we specifically expressed optoRaf in the class IV da (C4da) neurons, to test whether light stimulation would activate the Raf/MEK/ERK pathway. At 72 hr after egg laying (h AEL), wild-type (WT) and optoRaf-expressing larvae were anesthetized with ether and subjected to whole-field continuous blue light for 5, 10 and 15 min, while as a control, another transgenic group was incubated in the dark (0 min). The larval body walls were then dissected and immunostained with the pERK1/2 antibody, as a readout of the Raf/MEK/ERK pathway activation. We found that 5 min light stimulation was sufficient to significantly increase the pERK signal in the cell body of C4da neurons in optoRaf-expressing larvae, while 15 min illumination enhanced pERK activation and induced ERK translocation into the nucleus (S6K)  , which fus (S6K) . These rS6K in response to blue light was only observed in C4da neurons but not in other classes of da neurons or epithelial cells  with a two-photon laser at 72 hr AEL, verified axon degeneration at 24 hr after injury (AI) and assessed axon regeneration at 48 hr AI. At this time point, about 79% C4da neurons in WT showed obvious axon regrowth, and the regeneration index , while there was no difference between WT and unstimulated transgenic flies   , light s0.02752) . Similar0.02752) . This reWhile C4da neurons are known to possess the regenerative potential, it is unclear whether the regenerating axons navigate correctly. To address this question, we focused on v'ada – the lateral C4da neurons. Uninjured v'ada axons grow ventrally, showing a typical turn and then join the axon bundle with the ventral C4da neurons . We founoptoRaf: −0.6062 ± 0.1453; optoAKT: −0.5530 ± 0.1011) was comparable to that of WT (−0.5786 ± 0.08229) , while this transient stimulation resulted in no difference in WT (−0.6018 ± 0.1290)   on the ventral branch for 5 min at 24 hr AI. The lengths of both the ventral and dorsal branches were measured at 24 hr AI and 48 hr AI. We subtracted the increased dorsal branch length  from the increased ventral branch length , then divided that by the total increased length of these two branches . This va0.08229) , confirm 0.1290) . This re0.09644) , revealioptoRaf: 5.375 ± 0.3391; optoAKT: 4.765 ± 0.4236) were significantly increased compared with the WT control (2.643 ± 0.3050), and the percentage of regenerating commissure segments also exhibited a mild increase in both optoRaf- and optoAKT- expressing larvae  and form a ladder-like structure. Each pair of axon bundles correspond to one body segment in an anterior-posterior pattern . We injug larvae . On the g larvae . This reWe then tested whether the axon regrowth in the CNS induced by optoRaf or optoAKT activation leads to behavioral improvement. We utilized a recently established behavioral recovery paradigm based on larval thermonociception . In brieNeurotrophins are known to activate Trk receptors and trigger the Raf/MEK/ERK, AKT, and PLCγ pathways which are involved in cell survival, neural differentiation, axon and dendrite growth and sensation . Here, wS6K; optoAKT does not activate pERK) may arise from the fact that both optoRaf and optoAKT bypass the ligand-binding, receptor activation, Ras activation, and phospholipid production signaling steps. Activation of optoRaf and optoAKT does require upstream signaling molecules (e.g. kinases).Note that in the canonical growth factor signaling pathways, crosstalk actually occurs between the ERK and AKT signaling pathways, particularly at the upstream signaling node such as Ras. Indeed, the binding of growth factors to their receptors activates the transmembrane receptor tyrosine kinase, which recruits adaptor protein such as Grb2 (growth factor receptor-bound protein) and Sos (son of sevenless), a guanine exchange factor (GEF) for Ras. Sos then transforms the inactive, GDP-bound Ras to an active, GTP-bound Ras, which then recruits multiple proteins, including Raf and PI3K, an upstream kinase for AKT, to the plasma membrane. Thus, Ras serves as a common signaling node and therefore creates possible signaling crosstalk between the PI3K-AKT and Raf-MEK-ERK pathway. Another possible signaling crosstalk arises from the PI3K-mediated production of phospholipids, which could recruit a number of signaling molecules containing the lipid-binding domain  including Sos, which then affects Ras/Raf activation. Our observation that optoRaf and optoAKT do not crosstalk  that mediate the effects of neural regeneration by optoRaf and optoAKT. While ongoing efforts aim to elucidate these common signaling effectors, evidence from previous literature (some from other cell types) implies several possible candidates such as CREB (cAMP response element-binding protein) and FOXO (forkhead box transcription factors). Activation of Raf leads to phosphorylation of CREB, a family of transcription factors that regulate cell survival . EvidencAfter spinal cord injury, the synthesis of neurotrophins is elevated to support axon regrowth . AKT sigThe regenerative capacity varies significantly among different neuronal subtypes, as well as the PNS and CNS. Although the administration of neurotrophins enhances axon regeneration in peripheral neurons, its capacity to promote functional regeneration in the CNS is limited, in part due to the inaccessibility of neurotrophins to reach injured axons   and innaIneffective functional recovery at least partially results from the inappropriate pathfinding of the regenerative neurons. As shown in this study, the majority of regenerating C4da neuron axons preferentially grew away from their original trajectory. We surprisingly found that delivering a 5 min light stimulation to the ventral branch, which extended toward the correct direction, was sufficient to convey guidance instructions and increase the preferential elongation of the ventral branch against the dorsal branch. Correct guidance cannot be achieved by whole-body administration of pharmacological reagents. Similarly, when casting blue light on the whole transgenic larvae, light stimulation must be given at a high frequency to promote axon regrowth (there is a threshold for the light off-time), and the dorsal branch extension was also dominant in this case. This result highlights the importance and necessity of restricted activation of neurotrophic signaling. Indeed, the strength and location of Raf/MEK/ERK and AKT activation during axon regeneration may be important to the functional consequences. Notably, although the transient restricted stimulation likely affects the decision-making of the growth cone at the branching point, constant light is still required to increase overall axon regeneration.Drosophila C4da neurons. Correspondingly, neurons expressing optoRaf and optoAKT responded differently to intermitted light stimulation after injury, suggesting that the strength and activation duration of optoRaf and optoAKT is differentially gauged during axon regeneration. These collectively suggest that, since Raf could be activated by membrane translocation as well as dimerization, CRY2 oligomerization could further lead to a more potent Raf. This multimodal activation mechanism may render that a threshold of optoRaf can be reached so that a saturated ERK activation could be achieved. On the other hand, AKT activation does not depend on dimerization and may display a graded response. As a result, optoAKT activates the AKT pathway in a dose-dependent manner and may not recapitulate the maximum activation of AKT. This work provides a proof-of-concept to use optogenetics to accelerate and navigate axon regeneration in mammalian injury models. Besides spatiotemporal control of the neurotrophic signaling, optoRaf and optoAKT allow for finetuning of the signaling activity with programmed light pattern during axon regeneration. Follow-up studies are warranted to determine how Raf/MEK/ERK and AKT subcircuits are involved in each process of nerve repair, including lesioned axon degeneration, regenerating axon initiation and extension, and the formation of new synapses and remyelination in mammals. Understanding the machinery will, in turn, allow better utilization and development of the optogenetic systems. Recently, Harris et al. succeeded in directing axon outgrowth with optogenetic tools in zebrafish embryos .The plasmid of FOXO3-EGFP generated by cloning the human FOXO3 gene-containing plasmid (a gift from Prof. Anne Brunet at Stanford University) into the pEGFP-N1 backbone using overlap PCR with the . Randomly selected male and female larvae were used. Analyses were not performed blind to the conditions of the experiments. The experimental procedures have been approved by the Institutional Biosafety Committee (IBC) at the Children's Hospital of Philadelphia.19–12-Gal4 , repo-Gapo-Gal80 , ppk-CD4D4-tdGFP , and ppkDa neuron axon lesion and imaging in the PNS was performed in live fly larvae as previously described . VNC injQuantification was performed as previously described . BrieflyLive imaging was performed as described . EmbryosThe behavioral test was performed to detect functional recovery after VNC injury as described . A7 and Drosophila p70 S6 Kinase (Thr398)  and fluorescence-conjugated secondary antibodies . Larval body walls were mounted in VECTASHIELD Antifade Mounting Medium.Third instar larvae or cultured neurons were fixed according to standard protocols. The following antibodies were used: rabbit anti-Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) , rabbit anti-Phospho-2. For western blots, 800 ng of DNA were combined with 2.4 µL Turbofect in 80 µL of serum-free DMEM. The transfection mixtures were incubated at room temperature for 20 min prior to adding to cells cultured in 35 mm dishes with 2 mL complete medium. The transfection medium was replaced with 2 mL serum-free DMEM supplemented with 1 × Penicillin Streptomycin solution after 3 hr of transfection to starve cells overnight. PC12 cells were cultured in F12K medium supplemented with 15% horse serum, 2.5% FBS, and 1 × Penicillin Streptomycin solution. For PC12 neuritogenesis assays, 2400 ng of DNA were combined with 7.2 mL of Turbofect in 240 mL of serum-free F12K. The transfection medium was replaced with 2 mL complete medium after 3 hr of transfection to recover cells overnight. Twenty-four hours after recovery in high-serum F12K medium (15% horse serum + 2.5% FBS), the cell culture was exchanged to a low-serum medium (1.5% horse serum + 0.25% FBS) to minimize the base-level ERK activation induced by serum.HEK293T cells were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS), and 1 × Penicillin Streptomycin solution (complete medium). Cultures were maintained in a standard humidified incubator at 37°C with 5% CO2. For PC12 cell neuritogenesis assay, PC12 cells were illuminated at 0.2 mW/cm2 for 24 hr with the light box placed in the incubator.For western blot analysis, transfected and serum-starved cells were illuminated for different time using a home-built blue LED light box emitting at 0.5 mW/cmThe whole optogenetics setup is modified from previous work . Larvae Local light stimulation was delivered by a 488 nm argon-ion laser using a Zeiss LSM 880 microscope. At 24 hr AI, larvae were anesthetized and C4da neurons were imaged with a confocal microscope. For lesioned axons that bifurcated and formed two branches, we focused the laser beam (at 15% laser power) on the ventral branch for 5 min. The larva was then returned to grape juice agar plates and imaged again at 48 hr AI to assess the increased length of each branch.For the light-induced membrane recruitment assay, BHK-21 cells were co-transfected with optoRaf or optoAKT. Fluorescence imaging of the transfected cells was carried out using a confocal microscope (Zeiss LSM 700). GFP fluorescence was excited by a 488 nm laser beam; mCherry fluorescence was excited by a 555 nm laser beam. Excitation beams were focused via a 40 × oil objective (Plan-Neofluar NA 1.30). Ten pulsed 488 nm and 555 nm excitation were applied for each membrane recruitment experiment. CRY2-CIBN binding induced by 488 nm light was monitored by membrane recruitment of CRY2-mCherry-Raf1 (for optoRaf) or CRY2-mCherry-AKT (for optoAKT) to the CIBN-CIBN-GFP-CaaX-anchored plasma membrane. The powers after the objective for 488 nm and 555 nm laser beam are approximately 40 µW and 75 µW, respectively. Alternatively, an epi-illumination fluorescence microscope (Leica DMI8) equipped with a 100 × objective (HCX PL FLUOTAR 100×/1.30 oil) and a light-emitting diode illuminator (SOLA SE II 365) was used for the CRY2-mCherry-Raf1 membrane translocation assay. Neurite outgrowth of PC12 cells was imaged using an epi-illumination fluorescence microscope (Leica DMI8) equipped with 10× (PLAN 10×/0.25) and 40× (HCXPL FL L 40×/0.6) objectives. Fluorescence from GFP was detected using the GFP filter cube ; fluorescence from mCherry was detected using the Texas Red filter cube .Cells were washed once with 1 mL cold DPBS and lysed with 100 µL cold lysis buffer (RIPA + protease/phosphatase cocktail). Lysates were centrifuged at 17,000 RCF, 4°C for 10 min to pellet cell debris. Purified lysates were normalized using Bradford reagent. Normalized samples were mixed with LDS buffer and loaded onto 10% or 12% polyacrylamide gels. SDS-PAGE was performed at room temperature with a cold water bath. Samples were transferred to PVDF membranes at 30 V 4°C overnight or 80 V for 90 min. Membranes were blocked in 5% BSA/TBST for 1 hr at room temperature and probed with the primary and secondary antibodies according to company guidelines. Membranes were incubated with ECL substrate and imaged using a Bio-Rad ChemiDoc XRS chemiluminescence detector. Signal intensity analysis was performed by ImageJ. Antibodies used were listed in the Key Resources Table.N’ (sample size) are provided in the figure legends. Data are expressed as mean ± SEM in bar graphs, if not mentioned otherwise. No data points were excluded. Two-tailed unpaired Student's t-test was performed for comparison between two groups of samples. One-way ANOVA followed by multiple comparison test was performed for comparisons among three or more groups of samples. Two-way ANOVA followed by multiple comparison test was performed for comparisons between two or more curves. Fisher's exact test was used to compare the percentage. Statistical significance was assigned, *p<0.05, **p<0.01, ***p<0.001.No statistical methods were used to pre-determine sample sizes but our sample sizes are similar to those reported in previous publications , and the In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:Drosophila peripheral and central nervous systems. Using light-activated signaling allows for the dissection and regulation of dendritic branching and axon regeneration. The manuscript has been strengthened with additional controls and experiments to increase the specificity of Raf and Akt signaling through analysis of mutant versions of these enzymes. Both genetic and biochemical data are employed to support the conclusions of the study.This study cleverly uses ontogenetic probes to stimulate the Raf and Akt pathways in the Decision letter after peer review:Drosophila" for consideration by eLife. Your article has been reviewed by three peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Jonathan Cooper as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Philip JS Stork (Reviewer #2).Thank you for submitting your article "Optical control of ERK and AKT signaling promotes axon regeneration and functional recovery of PNS and CNS in The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.eLife". Please let us know if you would like to pursue this option. As the editors have judged that your manuscript is of interest, but as described below that additional experiments are required before it is published, we would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). First, because many researchers have temporarily lost access to the labs, we will give authors as much time as they need to submit revised manuscripts. We are also offering, if you choose, to post the manuscript to bioRxiv  along with this decision letter and a formal designation that the manuscript is "in revision at This study uses ontogenetic probes to activate the Raf and Akt pathways. This approach represents a powerful system to dissect signaling outcomes of these pivotal enzymes. Light-controlled activation of these two relevant signals is used to assess their ability to mediate axonal pathfinding, branching, and regeneration. The optogenetic tools allow for an examination of the sufficiency of either Akt or ERK pathway activation in these processes, in both PNS and CNS neurons. The result that both pathways play significant yet distinct roles in these processes is important and is of wide interest.Summary:Several major and minor reservations were brought up by the reviewers. The major issues are to define more fully the upstream events that activate Raf-1 and Akt and the need for additional controls for the optogenetic probes. The kinetics of activation, as well as cell localization, requires attention. Another request is to examine if there is convergence in the two pathways. The major concerns are described below--Essential revisions:1) This study assumes that the tools trigger signaling pathways independently of upstream (neurotropic) signaling. However, whether these tools require some upstream signaling remain incompletely addressed. For example, activation of Raf1 requires upstream activation by kinases phosphorylating the N-terminal region (Y341 and S338). The phosphorylation of S338 is a commonly used read-out for Raf-1 activation (and mutants at this position show no activation). It would be very informative to examine the status of pS338 in optoRaf and to compare the optoRaf to a mutant S338A version, at least in Hek293 cells. Because these phosphorylations are linked to Raf dimerization, these studies would provide insight into whether Raf dimerization is required or possible in this context.Drosophila neurons to ensure the signals directly go to cells where the functional assessments are being conducted.2) It would be also helpful to include more specificity controls for Raf vs. Akt signaling in 3) The kinetic experiments are interesting but somewhat incomplete, and it is unclear what the takeaway from these experiments should be. Importantly, it is not known how different pulsed light patterns translate temporally to signaling. It seems that from the data in Figure 1, it is possible that in neurons patterns may maintain a constant activation of the pathway. Additional controls looking at the extent of signaling in neurons with these paradigms would be really helpful. Essential revisions:1) This study assumes that the tools trigger signaling pathways independently of upstream (neurotropic) signaling. However, whether these tools require some upstream signaling remain incompletely addressed. For example, activation of Raf1 requires upstream activation by kinases phosphorylating the N-terminal region (Y341 and S338). The phosphorylation of S338 is a commonly used read-out for Raf-1 activation (and mutants at this position show no activation). It would be very informative to examine the status of pS338 in optoRaf and to compare the optoRaf to a mutant S338A version, at least in Hek293 cells. Because these phosphorylations are linked to Raf dimerization, these studies would provide insight into whether Raf dimerization is required or possible in this context.This is an excellent point. As suggested, we have performed Western blot in HEK293T cells using an antibody against phosphor-S338 of Raf1 under the optogenetic stimulation conditions. As a positive control, we used serum stimulation. As expected, in cells transfected with the optoRaf S338A mutant, optoRaf S338A cannot be phosphorylated at this site regardless of serum and light stimulation . Importantly, unlike wild-type optoRaf, optoRaf S338A significantly abolished the light-dependent activation of ERK . This result indicates that activation of optoRaf should require the upstream activation machinery. Furthermore, as suggested by the reviewer, it is very likely that Raf dimerization is involved in the activation of optoRaf.Drosophila neurons to ensure the signals directly go to cells where the functional assessments are being conducted.2) It would be also helpful to include more specificity controls for Raf vs. Akt signaling in S6K) compared with the neighboring cells, which do not express optoRaf or optoAKT . This result confirms that blue light specifically triggers Raf/MEK/ERK or AKT signaling in optoRaf/AKT expressing C4da neurons.Thanks for the suggestion. To make sure that light-mediated enhancement of Raf and AKT signaling occurs specifically in the target cell, we used DAPI staining to identify da neurons with or without the corresponding optogenetic systems. As shown in Figure 2—figure supplement 2, within the field of view, only C4da neurons marked by ppk-CD4tdGFP express optoRaf or optoAKT, which is under the control of ppk-Gal4, an enhancer driver for C4da neurons. In these cells, blue light-stimulation significantly enhances the level of phospho-ERK or phospho-p70 ribosomal S6 kinase (phospho-p703) The kinetic experiments are interesting but somewhat incomplete, and it is unclear what the takeaway from these experiments should be. Importantly, it is not known how different pulsed light patterns translate temporally to signaling. It seems that from the data in Figure 1, it is possible that in neurons patterns may maintain a constant activation of the pathway. Additional controls looking at the extent of signaling in neurons with these paradigms would be really helpful.S6K, respectively. Consistent with the results from mammalian cell culture, 5-10 min of light stimulation was sufficient to activate optoRaf and optoAKT . After the light was off, the level of phospho-ERK and phospho-p70S6K decreases monotonically. We observed that optoAKT inactivates faster than optoRaf, i.e., level of phospho-p70S6K decreases to the basal level within 15 min, whereas phospho-ERK decreases slower . This difference in decay kinetics may arise from distinct signaling threshold, signaling capacity of Raf and AKT, or both.We really appreciate the insightful comments. As suggested, we carried out a kinetic study in C4da neurons to determine if intermittent light pattern leads to intermittent signaling activity. The level of ERK and AKT signaling activities was probed by immunostaining of phospho-ERK and phospho-p70


In early infancy, melody provides the most salient prosodic element for language acquisition and there is huge evidence for infants’ precocious aptitudes for musical and speech melody perception. Yet, a lack of knowledge remains with respect to melody patterns of infants’ vocalisations. In a search for developmental regularities of cry and non-cry vocalisations and for building blocks of prosody (intonation) over the first 6 months of life, more than 67,500 melodies  of 277 healthy infants from monolingual German families were quantitatively analysed. Based on objective criteria, vocalisations with well-identifiable melodies were grouped into those exhibiting a simple (single-arc) or complex (multiple-arc) melody pattern. Longitudinal analysis using fractional polynomial multi-level mixed effects logistic regression models were applied to these patterns. A significant age (but not sex) dependent developmental pattern towards more complexity was demonstrated in both vocalisation types over the observation period. The theoretical concept of melody development (MD-Model) contends that melody complexification is an important building block on the path towards language. Recognition of this developmental process will considerably improve not only our understanding of early preparatory processes for language acquisition, but most importantly also allow for the creation of clinically robust risk markers for developmental language disorders. Human adults2 and even newborns are able to distinguish different languages using prosodic cues6, particularly melody (intonation). Most, if not all, of the linguistic and paralinguistic functions of intonation systems seem to be shared by languages of even widely different origins, a fact which strongly points to their universal role for language acquisition1.The ability to perceive and produce the time varying vocal fundamental frequency (fo)  is an extremely important component of auditory information and an essential suprasegmental aspect of spoken language. Each language is characterized by specific musical elements in the form of prosody, that is, its intonation system and constituent rhythm14. They do so long before they are capable of perceiving the segmental characteristics of speech such as consonants, vowels, or syllables. Infants apply their knowledge of melody in order to segment the continuous speech stream into meaningful parts (prosodic phrasing)16 and experience how protowords begin to emerge from the melody17.Infants recognize auditory information of the surrounding language on the basis of prosodic cues, mainly melody, the prototypical musical element of language22. A newborn’s cortical structures are active in the processing of prosodic information, particularly melody contour, as typified in the exaggerated melodic expression of infant-directed speech (IDS)24. IDS exemplifies how messages are conveyed to the preverbal human infant by melody variation27. According to Falk29, the special, melodic form of vocal communication that exists between a mother and her infant (IDS) could well have co-evolved in prehistoric time. The essential characteristic of IDS is the emotional content conveyed via melody, rather than words. The well-developed perception of melody and rhythm in the foetus30, newborn33 and young infant37 leaves no doubt that the human infant has a specific sensitivity to musical sound features40.Melody contour is probably the most important entity to be memorized and imitated by infants42. In addition to crying, from about the end of the second month of life onwards, new sound properties emerge within the non-cry vocal repertoire of infants. These properties 43, are the result of maturing supralaryngeal mechanisms that often change the sound to be recognized as seemingly more “speech-like”. In most of these non-cry vocalisations, melody still serves as a kind of scaffolding and forms together with supralaryngeally produced constituents the characteristic overall shape  of the sound45.During the first two months, vocal messages are almost entirely and very effectively coded in the laryngeally produced cry melody, while supralaryngeal mechanisms are still immature48. However, the specific protophone typology of pre-speech sounds is lacking the crucial importance of prosodic development and hence, does not fully conform with or capture a young infant’s entire vocal behavior49.Given the importance of melody for language acquisition, there remains a dramatic lack of knowledge with respect to the starting point of melodic variation and age-related development of melody patterns in infants’ cry and non-cry vocalisations. Commonly used models of vocal development describe the emerging speech capacity by focussing on the occurrence of segmental (syllable-like) precursors, sometimes called “protophones” after Oller54. In contrast to melody in non-cry vocalisations, cry melody is often ignored in models of vocal development towards language55. The first universal step towards prosody (intonation and rhythm), and hence language development for the young infant requires coordination of respiratory and laryngeal activity for the production of melody variations (phonation)57. There is compelling evidence for a huge melody repertoire in infants’ spontaneous natural crying, recorded in a relatively ‘relaxed context’ in the presence of the mother 60. This is completely different to the fo pattern of cries elicited by application of a painful stimulus. The medical importance of vocal fo and related parameters was often exclusively documented in the area of infant cry diagnostics by eliciting pain-induced cries67. For many years, this approach hindered the recognition of the melody repertoire typical of spontaneous natural crying, which sets human infants apart from other primates68.Considerable importance is placed on the fo patterns of non-cry vocalisations within the field of pre-speech development and language acquisition71. This view has since been refuted78. The mitigated, melodic cries of human infants are at some level, similar to simple musical melodies  and could provide raw material for prosodic constituents of later language58. The same is true for prosodic properties of non-cry vocalisations.In earlier work and, remarkably even more recently, infant crying has been viewed as essentially stereotypic, similar to primate calls45 postulated that early vocal development is reflected by a sequence from simple to complex melody contours that span across infant crying and non-cry vocalisations providing important building blocks for prosody and language development. Furthermore, this complexity pattern is assumed to be regular, uni-directional for each vocalisation type, and universal in nature (ibid.). Melodic complexity is exhibited as an increase in the number of arc-like substructures of fo contour (melody arcs) for the combinatorial assembly of multiple-arc (complex) melodies of cry and non-cry vocalisations during the first months of life77.The Melody Development Model (MD-Model), initially proposed by Wermke and Mende43) show a melody contour that is still rather simple i.e. single-arc-like 42 examined the non-cry vocalisations in 3, 6 and 9-month-old infants during vocal interaction and play situations. Based on visual inspection of frequency spectrograms, they analysed “simple” and “complex” fo shapes to characterize intonation patterns produced by these age groups. Only 11% of multiple-arc (“complex”) patterns were observed at the age of 3 months . By 6 months, the number of complex melody contours had increased to 22% but reduced to 10% by 9 months. This may point to a developmental course of first increasing-then-decreasing production of complex melody contours in infant non-cry vocalisation between the age of 3 and 9 months. A longitudinal study of “speech quality”  and melodic complexity  in the non-cry vocalisations of infants between 2 and 6 months was reported by Hsu et al. (2000)59. However, melody contour was coded only qualitatively based on perceptual impressions. The researchers found a curvilinear trend that seemed to parallel the pattern reported by Kent and Murray (1982)42 of increasing then decreasing production of complex melodies in non-cry vocalisations. However, the decline in melody complexity was found at an earlier age  compared to Kent and Murray (ibid.). The most comprehensive report on melody complexity development in infant crying was provided by Wermke and Mende45. The spontaneous cries of 270 infants were analysed for melodic complexity across the first five months of life. The authors described three developmental phases of cry melody, (1) birth to 8 weeks: initialization phase (increase of complex pattern from 30 to 52%), (2) 8 to 12 weeks: stabilization phase (no further increase) and (3) 12 to 18 weeks: modification phase  during the third month of life was interpreted to be due to the emerging interaction of melody and resonance frequencies . To the best of our knowledge, there are no further developmental studies of the melody features of infant vocalisations over the first six months of life, especially those capturing both cry and non-cry vocalisations.To date, there is a small number of studies that have investigated melody development in infant vocalisation and few that have focused on melody development during the first six months of lifeand non-cry vocalisations. Based on the MD-Model by Wermke and Mende45, we hypothesised that both cry and non-cry vocalisations produced by infants would show a characteristic developmental increase in complex (multiple-arc) melodies.To establish a more comprehensive model of early vocal development and gain a better understanding of early language (prosody) acquisition, we sought to investigate whether there is a developmental pattern of the production of complex melodies across the range of vocalisations produced during the first six months of life . The aim of the study was to perform an objective developmental analysis of prosodic precursors in the form of melody in healthy infants between 1 and 180 days of life in their cry Longitudinal analysis of complex melody pattern development among healthy young infants, stratified by cry and non-cry vocalisations from the baby sound archive at the Center of Pre-Speech Development and Developmental Disorders (University of Wuerzburg).Healthy, term-born  monolingual German infants from birth to the age of 180 days of life. Exclusion criteria were any kind of hearing disorder or developmental disorders over the observation period. The available database totalled 67,629 vocalisations from 277 infants; comprising of 56,537 spontaneous cry utterances from 227 infants  recorded between 1 and 180 days of life, and 11,092 non-cry vocalisations (cooing/babbling sounds) from 50 infants  recorded between 60 and 180 days. Only one infant appeared in both cry and non-cry data sets (for more details see The archive contains anonymized audio files (wav format) of the original recording sessions  as well as all the individual sounds, which were previously manually segmented using a commercially available system . Here, we used all cry and non-cry sounds available in the archive from our participants. Original recordings were approved by the respective ethical boards  and were carried out in accordance with relevant guidelines and regulations; informed consent signed by parents was given. Finally, all recordings were archived as anonymized data sets. Each parent had a minimum of a high school education and the monthly family income was reflective of a middle class standard of living.Cry vocalisations  were recorded under comparable conditions in a hospital (first week) and at home, respectively . Non-cry-vocalisations were recorded in infants’ homes during joyful mother–infant interactions. All vocalisations were spontaneously uttered by the infants and any elicitation of vocalising was avoided. The length of an individual recording session ranged from about 1 to 3 min (crying) and 1 to 30 min .86 are often caused by strong nonlinearities in the restoring forces resulting from an extremely large amplitude-to-length ratio of the very young infants’ vocal folds85. Based on audio-visual inspections of the spectrograms (cf. previous detailed description86), vocalisations containing broad regions of phonatory noise  and/or subharmonics or a highly unstable pattern caused by sudden frequency shifts (register changes) or marked vibrato-like phenomena were excluded from melody pattern analysis. The fo and its course over time (melody) cannot be reliably determined in those signals. The same was true for most vocalisations shorter than 300 ms to avoid effects from vegetative noises and sounds with background noise . All vocalisations without a well-defined melody structure were identified by audio-visual analyses and categorized as exhibiting “no pattern” and subsequently excluded from the analysis. This means that “no pattern” represented a fuzzy class of vocalisations without a clearly definable melody. The remaining vocalisations were assigned as either containing a “simple” or “complex” melody pattern . Statistical analysis revealed that both cry and non-cry vocalisation types had a decreasing occurrence of “no pattern” over age, but occurrence was lower in the non-cry vocalisations and decreased quicker than that for the cries:A cry or non-cry vocalisation was defined as an utterance produced on the expiratory phase of a single respiratory cycle and identified acoustically as the onset and offset of acoustic energy in the waveform. Frequency spectrograms were automatically calculated for each vocalisation using the CSL 4500 . Based on visual inspections of the spectrograms, phonatory noise phenomena and phenomena like sudden fo shifts or subharmonics were identified. These well-known features of young infants’ vocalisationsp < 0.001), given by the equation:For cries, the median age of cries without a well-defined melody (‘no pattern’) was 51 days, Q1 = 25 days, Q3 = 79 days, range: 1–173 days. Looking at the change over age, using a multi-level linear regression, there was a significant decrease in this pattern with increasing age (p < 0.001), given by the equation:For non-cry vocalisations, the median age of sounds without a well-defined melody (‘no pattern’) was 113 days, Q1 = 96 days, Q3 = 140 days, range: 60–180 days. Looking at the change over age, there was a significant decrease in this pattern with increasing age  was applied with a cut-off frequency of about 40 Hz to eliminate high-frequency modulation noise and artifacts44. This time-consuming, but robust analysis method, applied to each individual vocalisation, guaranteed that the subsequent melody pattern analysis was based on correct fo contours.For determining the melody pattern, an automatic fo measurement  was required using PRAAT v. 6.0.317. In agreement with preceding studies58, a complex melody structure was defined as exhibiting ≥ two arcs and/or intra-melodic breaks between arcs by glottal oscillatory pauses or marked laryngeal constrictions that generate rhythmical variations of the acoustic Gestalt. Examples for rhythmic variations of complex (multiple-arc melodies) are available in several previous publications89.Melody complexity analysis was performed using specific in-lab software , which was implemented as a routine procedure at the Center for Pre-Speech Development and Developmental Disorders. Using the fo data calculated with PRAAT, the CDAP software allows for flexible drawings of melody diagrams and quantitative melody contour analysis. To classify melodies in simple versus complex pattern, for each sound melody the number of single arcs was identified (cf. detailed description in Supplementary information). A melody arc was defined as being longer than 150 ms and as exhibiting a frequency amplitude (FM-amplitude) of at least three (cry) or two (non-cry) semitonesBased on these objective criteria, all cry melodies were analysed and subdivided into those with only a simple (single-arc) melody a,b, thoshttps://www.strobe-statement.org/)90. Analyses were stratified by melodies of cry and non-cry vocalisations. Summary statistics of infant’s sex, age and vocalisation signals were recorded, and the overall signal classification distributions were reported. Bubble plots 95 degree-2 fractional polynomial powers of infant age were considered from the set . The best models were then selected by minimising the deviance statistic, and the Χ2 test used to investigate deviance differences between models. These best fractional polynomial models defined the functional relationship of age (measured in days) to the binary complexity data for pursuant investigations. Once specified, both random intercept and random coefficient models were next investigated, and the Bayesian Information Criterion (BIC) used to select between these competing models96. The BIC rewards for goodness-of-fit to the data but penalises for model complexity, with the preferred model balancing these opposing demands and yielding the lowest BIC statistic. A sex difference was then tested, treating sex as a fixed effect. All statistical analyses and associated graphs were performed using Stata SE version 16.0 , and α = 0.05 defined significance.The reporting of analyses was informed by the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) guidelines; an international, collaborative initiative of epidemiologists, methodologists, statisticians, researchers and journal editors involved in the conduct and dissemination of observational studies , with 21,789 (38.5%) signals defined to complex, 20,917 (37.0%) as simple, and 13,831 (24.5%) had ‘no pattern’ (and were subsequently set to missing). Overall, 226 infants had at least one recorded spontaneous cry utterances classified as simple or complex, with an average of 189 such recordings . Figure −1 and age1 was significantly better than the best model containing one power of age (p < 0.001) or the model with a linear function of age (p < 0.001). This preferred random intercept model resulted in a BIC = 55,597. Extending the multi-level mixed effects logistic regression model to include both random intercept and random slopes for children yielded BIC = 54,815, a value superior to the random intercept only model. In this model, the fixed effects components were given by:p < 0.001); as were the random effects terms, with variability given by: constant SD = 1.13 ; age−1 SD = 5.94 ; age1 SD = 0.013 ; corr = 0.56 ; corr = − 0.55 ; and, corr = − 0.82 . No sex difference was identified (p = 0.13).Fitting fractional polynomial multi-level mixed effects logistic regression models, the best model containing two powers of age, namely ageentclass1pt{minimathe mean recorded infant age of vocalisations was 125 days , with 4527 (40.8%) signals defined to complex, 5428 (48.9%) as simple, and 1137 (10.3%) as having ‘no pattern’ (and were subsequently set to missing). Simple or complex cooing/babbling sounds were recorded from 50 infants; with an average of 199 such sounds . Figure 2 and ln(age) × age2, and was significantly better than the best models containing one power (p < 0.001) or a linear function of age (p < 0.001). This preferred random intercept model yielded a BIC = 13,235. Extending the multi-level mixed effects logistic regression model to also include random slopes for children, the algorithm failed to converge when both age2 and ln(age) × age2 terms were simultaneously considered. However, the model including a random intercept and a random slope component for age2 yielded BIC = 13,040, a value superior to the random intercept only model. In this superior model, the fixed effects components were given by:p < 0.001); as were the children’s random effects terms, with variability given by: constant SD = 1.066 ; age2 SD = 5.177E−05 ; and, corr = − 0.813 . Again, no differences between boys and girls was identified (p = 0.64).This non-linear pattern was confirmed by the application of fractional polynomial multi-level mixed effects logistic regression models. The best model contained two powers of age, namely ageBased on longitudinal analysis of melody structure (simple vs. complex pattern), we found that human infants acquire a repertoire of complex vocal melodies over the first six months of life, with rapid gains early on. Particularly impressive was the rapid increase of the prediction curve for melody complexity development derived from the final multi-level mixed effects logistic regression model in crying Fig. . This be98. For example, Holland et al.97 found that brain development is most rapid during the neonatal period with 64% of whole brain growth occurring within the first 90 days” (ibid. p. 6).The curve predicts a cry repertoire with about 53% of the vocalisations exhibiting a complex melody at the end of the first month and a further increase thereafter. This rapid gain requires mature functioning of neuro-physiological mechanisms underlying melody production, which corresponds to the fast brain growth at this early age99. From birth, newborns are capable of a highly developed laryngeal-respiratory control that serves breathing and phonation57. This aptitude facilitates survival, but at the same time enables the newborn to play with his laryngeal options for vocalising and hence, to quickly acquire complex melody patterns.The early occurrence of complex cry melodies would further suggest a ‘preparatory’ intrauterine development. The muscles of the larynx are an integral part of the respiratory system. Like other respiratory muscles, they undergo considerable use prior to birth101. These findings suggest that fetuses encode suprasegmental characteristics of speech  rather than segmental features, which is due to segmental information being filtered out by tissue and fluid before it reaches the fetus. Prenatal sensitivity to prosodic features was demonstrated in newborns, who exhibited a preference for a low-pass filtered maternal voice and language (focused on melody) in contrast to natural voice during their first days of life5.Intrauterine preparation also involves perceptive components. Indeed, there is evidence that late-term fetuses demonstrate the ability to discriminate their native language, to which they were exposed in utero, from an unknown language102. From both, a phylogenetic and ontogenetic perspective on spoken language evolution, it seems essential that the rapid gain in producing crying characterized by complex melodies occurs long before supralaryngeal maturation  allows for pre-articulatory activities in sound production.The non-cry vocalisations produced by the infants were found to parallel a similar developmental path observed earlier for cry vocalisations. Specifically, the model curve for the non-cry vocalisations initially also shows a low percentage of complex melodies about 30%) and a fast increase in their share of complex melodies over the next 2.5 months up to about 50%  during the developmental process. In contrast to past simplistic suggestions that human infant crying is a fixed and monotonous reflex response comparable to animal vocalizations71, we see a developmental progression of vocal growth. Our study further elucidates the prominence of melody variation in cry development. The early occurrence of complex melodies also confirms a previous report suggesting that by approximately two months of age, a majority of spontaneous cry vocalisations should contain complex melodies. Otherwise, the infant may be at risk for an early language impairment89. This hypothesis is supported by a recent study of Francois et al. (2017)103. The authors demonstrated that neonatal brain responses for sung streams predicted expressive vocabulary at 18 months. These findings further corroborate the importance of melody production and perception for language development long before “speech-like” vocalisations emerge.Our observation that melody development in both cry and non-cry vocalisations is characterized by an increase in complexity points to a similar strategy in the acquisition and rehearsal of prosodic building blocks as postulated by the MD-Model, introduced by Wermke and MendeThere is major agreement between scientists of several disciplines, that the first universal steps taken by an infant on his way to spoken language include melodic-rhythmic aptitudes, with respect to both perceptive and productive performances. A systematic increase in melody complexity in cry and non-cry vocalisations, as demonstrated here, provides the raw material for later language prosody. This takes place to an extent that has been often underestimated in spontaneous crying and is continued in non-cry vocalisations. Consequently, infants’ melodic sound characteristics, i.e. early building blocks of prosody, are crucial in order to characterize their path to spoken language in the first months of life.44. The tuning in non-cry vocalisations is additionally challenged by an increasing articulatory activity required to produce syllabic combinations in babbling from about five to six months on. This new developmental period requires a temporary “regression” in melody development to establish vocal development on a higher hierarchical level17. Thereafter, the infant begins to intentionally imitate intonation patterns of the surrounding language(s) in consonant–vowel syllable sequences in babbling45. The identification of primitive precursors of later articulatory speech elements , observed in early cry and non-cry vocalisations49, show the close interaction of suprasegmental and segmental phonatory activity by the infant at an early age. A logical next step in our line of research is to examine the possible interaction between vocant and closant articulations and melodic complexity.While the model curve of complex melodies among the cry repertoire did not decline at the end of the observation period, occurrence of complex melodies in non-cry vocalisations slightly decreased from about 4.5 to 5 months. This is most likely due to the new constituents of non-cry vocalisations that emerged to interact with the overall melodic contour, namely vowel-like (vocants) and consonant-like elements (closants). During the process of spoken language acquisition, the human infant must modify his laryngeally produced melodies repeatedly and tune them to the resonance frequencies of a vocal tract that continues to grow and changeThe study provides the first statistical model to demonstrate a systematic melody development in cry and non-cry vocalisations of infants. Our data revealed a strong developmental continuity in spontaneous crying with respect to melody complexity across the first 180 days. Additionally, there was a continuous increase in complex melodies in non-cry development with a slight reduction occurring at approximately 140 days. Recognition of this developmental process will considerably improve not only our understanding of early preparatory processes for spoken language acquisition, but most importantly also allow for the creation of clinically robust risk markers for developmental language disorders. This is the crucial prerequisite to enable us to develop innovative therapies for infants at-risk for developing language disorders. This developmental model could help to better understand why the human infant acquires so quickly and seemingly effortlessly such a complex faculty as language.Supplementary Information.Supplementary Audio 1.Supplementary Audio 2.Supplementary Audio 3.Supplementary Audio 4.


A myriad of signaling molecules in a heuristic network of the tumor microenvironment (TME) pose a challenge and an opportunity for novel therapeutic target identification in human cancers. MicroRNAs (miRs), due to their ability to affect signaling pathways at various levels, take a prominent space in the quest of novel cancer therapeutics. The role of miRs in cancer initiation, progression, as well as in chemoresistance, is being increasingly investigated. The canonical function of miRs is to target mRNAs for post-transcriptional gene silencing, which has a great implication in first-order regulation of signaling pathways. However, several reports suggest that miRs also perform non-canonical functions, partly due to their characteristic non-coding small RNA nature. Examples emerge when they act as ligands for toll-like receptors or perform second-order functions, e.g., to regulate protein translation and interactions. This review is a compendium of recent advancements in understanding the role of miRs in cancer signaling and focuses on the role of miRs as novel regulators of the signaling pathway in the TME. Ancient human civilization is based on orderly chaos and an ethos of heterogeneity. Solid tumors are heterogeneous in terms of their cellular composition and genetic integrity, and their existence dates back to early human civilization ,2,3,4. TThe landscape of signaling molecules in the TME ranges from soluble peptides, such as chemoattracts and cytokines, to metabolites, such as ATP and lactate ,6,7,8,9.In higher eukaryotes, miRs emerged as an important class of regulatory genes that affect transcription and protein expression ,14. ThesTo appreciate the role of miR as a regulator of signaling in the TME, it is essential to understand the genesis and migration of these entities within the TME. Apart from intracellular miRs, extracellular miRs occur in different compositions and molecular assembly in the TME , which uInterestingly, EVs mediated miR exchange and signaling only accounts for a minor portion of the total TME ‘miRpool’. A major portion of extracellular miRs have been detected as free-floating complexes with RNA-binding proteins (RNPs), especially AGO2 ,51 or nuHighly abundant high- and low-density lipoproteins are some of the most critical miR transporters in the TME ,54 as suThis extra layer of complexity in miR biogenesis and cross-cell effector functions  make theVirtually all mRNAs can be targeted by miRs, at least in vitro, and every miR can target more than one mRNA based on their seed sequence ,58,59,60Since, there are specific interactions between the miR seed sequence and MREs, the presence and availability of MREs on target mRNAs determine the miR action. The majority of MREs are in the 3′ UTRs, an inert region of a transcript with respect to the protein-coding ability. However, 3′ UTRs frequently undergo alternative cleavage and polyadenylation (APA), which results in the loss or gain of multiple MREs. Genome-wide studies revealed that about 50–70% of human mRNAs possess multiple 3′ UTR isoforms ,68. APA-The expression of the RISC complex is one of the most critical factors that regulate the silencing efficacy of miRs in the TME. RISC is a key enzyme assembly that pilots and navigates mature miRs binding with complementary 3′ UTR of target mRNA to bring about target gene suppression. However, in the TME under the influence of hypoxia, the activity of the RISC complex is downregulated. Therefore, the gene suppressing ability of miRs is compromised . Even inMultiple MREs on a particular mRNA might create a regulatory network of mRNA–miR cross-communication ,95. Thismodus operandi of miRs is post-transcriptional gene silencing, however miRs are also known to perform functions such as activation of transcription. There are several examples in the literature that describe this contrary role of miRs on gene transcription by targeting promoter elements, a phenomenon known as RNA activation (RNAa) . S. S78]. SAnother interesting, probably less explored, mechanism of how miRs act as a regulator of signaling is by acting as an RNA decoy that regulates gene expression of various genes by binding either to complementary mRNA or regulatory RNA-binding proteins. Eiring et al. first reported that through interference with the mRNA-regulatory function of RNA-binding proteins in a sequence-dependent manner, i.e., decoy activity and by base pairing with complementary mRNAs, miRs exert posttranslational control of biological processes . Since mMiRs possess another arm in their arsenal due to their chemical nature. Being a nucleic acid species, such as single-stranded RNA in the form of mature miRs, having specific antigenic secondary structure and the fact that miR can be exchanged intracellularly, miRs exert immunogenicity, especially in the TME. Large studies focused on a narrow aspect of miR such as the specific mRNA target by in silico prediction without taking into consideration ‘other’ effects. There are examples in the literature on the non-canonical aspect of miRs. These functions overlap with the antigenic single-stranded viral RNA on a host cell as well as activation of endogenous receptors. Although there is a vivid picture of the immunogenicity of either single-stranded or double-stranded RNAs, the immune response triggered by miRs still requires further interrogation. Fabbri et al. have demonstrated for the first time that miR-21 and miR-29a can act as specific agonists for TLR 7 and TLR 8, respectively see . They doIn essence, for the effective use of miR as regulators of signaling in human cancer, factors such as MRE redundancy, the non-canonical target landscape, decoy activity, and bystander effects on translation should be taken into consideration. These multiple features of miRs exert extra weightage on their utility as a potential therapeutic target in human cancers.MIR29B [In the TME and other pathophysiological situations, regulatory functions of miRs are not only confined to transcriptional suppression, rather they are also integrated into signaling cascades. In fact, owing to their promiscuous targetome, miRs are ideal regulators of signaling since they can interfere at various levels of signaling cascades. One of the best-studied signaling cascades in cancer and inflammation is the NF-κB pathway. NF-κB is an important inducible carcinogenesis mediator and activated in various human cancer subtypes and program malignant tumor cells to evade apoptosis . There aMIR29B .Several surface receptor signaling cascades culminate in NF-κB activation, some of them are under the radar of miRs in tumor progression and inflammation. Tumor necrosis factor receptor-associated factors (TRAF) are a class of multi-functional intracellular signaling adaptor proteins that activate NF-κB through miRs such as miR-146a/b, which inhibits TRAF6 and interleukin-1 receptor-associated kinase (IRAK1) in breast cancer cells . FurtherIn an inflammatory tumor milieu, miRs actively participate in intra- and inter-cellular signaling. In this dynamic context, miRs fluctuate from regulator to being regulated, depending on local environmental cues, which makes them one of the most versatile regulators of signaling in cancer. As important transcriptional regulators, miRs can up- or down-regulate many target genes involved in signaling pathways via negative or positive feedback loops, which are attributed to tumor initiation, development, progression, and metastasis.FOXP1) and BCL2 but was withdrawn due to immune-related adverse effects. Surely, in the future, these predictions may be more accurate with the advent of artificial intelligence [In this review, we highlighted several studies that may serendipitously explain the function of ‘non-seed’ base sequence in mature miRs and may provide a rationale to their structural complexity. Now the picture is becoming clearer about atypical miRs functions, based on their chemical structure and sequences. Even small RNA species like miRs can have a secondary structure. Therefore, they may serve as a scaffold for other regulatory RNAs such as competitive endogenous RNAs (ceRNA) ,95, whiclligence  and growlligence  but the Signaling cascades not only determine the cellular response to extracellular triggers but also shape the local milieu such as the TME. Pharmacological interference with intra- or inter-cellular signaling is the central theme of anti-tumor therapy. To be able to target signaling events, it is incumbent that we understand the fine details and complexity of individual signaling cascades. Classical signals such as cytokines and growth factors exert cascading effects that generally culminate in gene expression. However, in pathophysiological situations such as cancer, miRs also exact their effects without a high degree of cascading by not only repressing, and in some cases activating, gene expression but also targeting the classical signaling cascade triggered by cytokines and growth factors. These multifactorial and Janus-faced traits of miRs make them a double-edged sword (a) as attractive targets for anti-tumor therapy and at the same time (b) one of the trickiest targets due to the collateral effects of miRs regulation of cellular events. Effective RNA therapeutics need to address this dichotomy for regulating cell signaling in human cancers. Nevertheless, the ability of miRs to target multiple genes in the TME is attractive, as this feature may facilitate the targeting of multiple compensatory pathways. However, an miR targetome might include both tumor-promoting and tumor-suppressing genes, as well as several targets not related to cancer signaling, which may complicate the development of selective miR-directed therapeutics. The fact that no miR-directed drug candidates have been entered into phase III clinical trials underlines the complex nature of these molecules for achieving the desired therapeutic outcome.Another aspect of miR therapy, which needs considerable attention, especially with antagomirs, is the influence on the expression of neighboring genes. The extent to which this is due to transcriptional ‘rippling’ effects  vs. bona