With a genome-wide approach (Chromatin Immunoprecipitation Sequencing), many phospho-STAT3 binding sites were identified in fatty dHepaRG cells and several STAT3 and/or NAFLD-regulated microRNAs showed increased expression levels, including miR-21. Innovative CARS (Coherent Anti-Stokes Raman Scattering) microscopy revealed that chemical inhibition of STAT3 activity decreased lipid accumulation and deregulated STAT3-responsive microRNAs, including miR-21, in lipid overloaded dHepaRG cells. We were able to show in vivo that reducing phospho-STAT3-miR-21 levels in C57/BL6 mice liver, by long-term treatment with metformin, protected mice from aging-dependent hepatic vesicular steatosis. Our results identified a microRNAs-phosphoSTAT3 pathway involved in the development of hepatic steatosis, which may represent a molecular marker for both diagnosis and therapeutic targeting.Non-alcoholic fatty liver disease (NAFLD) is a leading cause of chronic liver disease. Although genetic predisposition and epigenetic factors contribute to the development of NAFLD, our understanding of the molecular mechanism involved in the pathogenesis of the disease is still emerging. Here we investigated a possible role of a microRNAs-STAT3 pathway in the induction of hepatic steatosis. Differentiated HepaRG cells treated with the fatty acid sodium oleate (fatty dHepaRG) recapitulated features of liver vesicular steatosis and activated a cell-autonomous  The excessive accumulation of triglyceride-containing lipid droplets (LD) within hepatocytes in NAFLD patients is a potentially reversible process, that may evolve into a severe necro-inflammatory form called non-alcoholic steatohepatitis (NASH), which can eventually lead to cirrhosis and hepatocarcinoma (HCC)3. Genetic and epigenetic factors together with multiple risk factors  have been described to promote liver injury4. Intrahepatic activation of pro-inflammatory signaling, such as the IL6/STAT3 pathway, plays a significant role in the development of chronic liver diseases. STAT3 is constitutively activated in HCC by phosphorylation of Tyr705 and Ser727, resulting in STAT3 dimerization, nuclear translocation, DNA binding and gene transcription6. IL6/STAT3-dependent molecular mechanisms driving hepatic steatosis and the pathological processes that cause NAFLD progression are still poorly understood7.Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease in developed countries9. There is a growing body of evidence demonstrating that miRNAs are closely associated with the IL6/STAT3 signaling pathway, supporting the existence of regulatory feedback loops between expression of specific miRNAs and the STAT3 pathway in HCC and other diseases11.MicroRNAs (miRNAs) are a class of noncoding endogenous RNAs that regulate gene expression and specific miRNA expression profiles are strongly associated with several pathological conditions, including NAFLDin vitro model, represented by hepatic differentiated HepaRG cells treated with sodium oleate, fatty dHepaRG. Indeed, lipid overloaded dHepaRG cells showed typical features of mature hepatocytes, as we observed by CARS (Coherent Anti-stokes Raman Scattering) microscopy, an innovative technique that enables lipid droplets visualization and quantification without labeling, by probing their characteristic vibrational properties12.The present study investigates the role of a miRNAs-phospho-STAT3 pathway in the induction of liver vesicular steatosis in an in vivo that reducing both phospho-STAT3 and miR-21 levels in C57BL6 mice liver, by long-term treatment with metformin, a widely used anti-diabetic drug, strongly improved age dependent hepatic steatosis. Activation of a new miRNAs-phospho-STAT3 pathway that is involved in the development of hepatic steatosis may represent a target for novel therapeutic strategies and might also have potential as a biomarker for diagnosis.We identified by Chromatin Immunoprecipitation Sequencing that phospho-STAT3, which is activated in fatty dHepaRG, is able to bind and regulate several miRNAs after lipid overload. Our results showed for the first time that suppression of the miRNAs-phospho-STAT3 pathway by S3I-201, a STAT3 specific inhibitor, decreased hepatic lipid accumulation. We have also observed 13, we decided to investigate whether the effect of lipid droplets and ROS generation were able to trigger gene expression modulation in dHepaRG cells. We performed a qPCR analysis of sodium oleate 250 μM treated dHepaRG transcripts using a Human Fatty Liver Array (Sabiosciences), that profiles the expression of 84 key genes involved in the mechanisms of NAFLD and hepatic insulin resistance . Treatment of differentiated HepaRG cells (dHepaRG) with the fatty acid sodium oleate 250 μM for 2 or 4 days (Supplementary 1c) led to generation of cytoplasmic lipid droplets detected by Oil Red O staining Fig. , and indTo investigate the role of the inflammatory response in the pathogenesis of vesicular steatosis, we analyzed a TaqMan Low Density Array (TLDA Cards) which profiles the expression of 96 key genes involved in the Interferon signaling pathway . We tested Ruxolitinib 1μM and NAC 10 mM activity by IL6 ELISA assay and DHCFDA FACS quantification, respectively  analysis on fatty dHepaRG cells using a phosphoTyr705-STAT3-specific antibody. Raw data for ChIP-seq are available at GEO (GSE89157). Results indicated 82883 total peaks, corresponding to phospho-STAT3 candidate binding sites, in control dHepaRG cells (Ctrl) and 184946 in sodium oleate 250 μM treated dHepaRG cells, which partially overlapped with the control peaks (51332 peaks); among the 184946 peaks in sodium oleate treated dHepaRG cells we found a significant amount of phospho-STAT3 bindings specifically induced by sodium oleate (132994 Oleate-specific) Fig. . Motif e14. We tested S3I-201 inhibitory activity by IL6 ELISA assay and qPCR mRNA levels quantification of a known STAT3-transcritpional target HP (Haptoglobin) . HFD mice showed typical features of NAFLD/NASH, with increased ALT  liver enzyme activity levels, body weight and glucose levels as compared to mice fed normal diet (ND)  showed an evident macro-vesicular liver steatosis due to aging, with well-defined fat droplets occupying the cytoplasm of hepatocytes   versus only 23.8% in metformin treated mice  Fig.  and decrice Fig.  and alsoThese results demonstrate that downregulation of both phospho-STAT3 and miR-21 levels in the liver after long-term treatment with metformin improves age-dependent macro-vesicular liver steatosis in mice.9. NAFLD and NASH are associated with substantial metabolic stress and inflammation, mediated by IL6/STAT3 signaling, that affects both cell survival and determine an increased risk of developing hepatocellular carcinoma22.NAFLD is a complex disease trait whose development and progression are determined by the combination of genetic and epigenetic changesin vitro model of vesicular steatosis, with consequent de-regulation of several STAT3-related miRNAs. Human hepatic HepaRG cell line, a valuable alternative to primary human hepatocytes, has been used as a model of vesicular steatosis23. Indeed, we already showed that addition of sodium oleate to differentiated HepaRG cells induces lipid storage in droplets, that were statistically quantified in terms of numbers, distribution and morphology at single cell level, utilizing a new label-free microscopy technique, coherent anti-Stokes Raman scattering (CARS)12. CARS microscopy it is now recognized as a powerful non-invasiveness tool for the investigation of biological specimens, as it is capable of imaging unstained living cells with a high vibrational sensitivity and chemical selectivity19.The present study demonstrates the activation of the IL6-STAT3 axis in an 24, was not modulated. We found by Chip-seq using an antibody that specifically recognizes phosphorylation at specific Tyrosine 705 residue, that phospho-STAT3-Tyr705 is able to differentially bind many promoters, including miR-21 regulatory region, after the induction of vesicular steatosis in dHepaRG cells (raw data for ChIP-seq are available at GEO GSE89157). Lipid overloading-dependent activation of STAT3 is directly responsible for the transcriptional modulation of miR-21 and several other miRNAs. Indeed, we were able to show for the first time that inhibition of STAT3 activity deregulates miRNA expression and in parallel improved steatohepatitis in vitro and in vivo, reducing neutral lipid accumulation and droplets generation, suggesting STAT3 as a potential therapeutic target for NAFLD.Here, we demonstrated that lipid overloaded dHepaRG cells induced the activation of an intracellular autonomous inflammatory response, with increased STAT3-Tyr705 phosphorylation levels. Contrarily, STAT3-Ser727 phosphorylation, which also enhances STAT3 transcriptional efficacy, but it is not mandatory for STAT3 activation26. Nevertheless, it has been shown that metformin has anti-oxidant, anti-inflammatory and anti-fibrogenic properties in liver diseases and induces reduction in tissue lipid storage with an increase in both fatty acid oxidation and inhibition of lipogenesis, presumably mediated by AMPK activation29. Moreover, metformin is able to induce alterations in STAT3 signaling in many cell types21. However, metformin-induced activation of energy metabolism, via the energy-sensor AMPK, may not account for all benefit effects of the drug on NAFLD and the exact molecular mechanisms of its therapeutic action remain obscure29. Here, we have been able to show that reduction of STAT3 activation and miR-21 levels in C57/BL6 mice liver, by metformin long-term treatment (72 weeks), strongly ameliorated steatohepatitis that spontaneously occurred in control mice due to aging.No drug is currently available as a specific treatment for NAFLD and the combination of diet and lifestyle modifications remain the pillars of NAFLD and NASH managementin vitro and in vivo converge to indicate that miR-21 is involved in NAFLD pathogenesis. Indeed, it has been recently shown, in mice fed a high-fat diet, that liver-specific miR-21 knockout prevents steatosis by altering the expression of several master metabolic regulators30, and in a model of NASH-associated liver damage, miR-21 knockout mice display reduced steatosis, inflammation and lipoapoptosis, with impairment of fibrosis15. MiR-21 is overexpressed in many human cancers and is thought to play a significant role in carcinogenesis32. Circulating and liver tissue miR-21 level are also significantly increased in patients suffering from NASH and in inflammatory states of fatty liver disease17. However, the mechanism by which miR-21 might contribute to NASH/NAFLD development is still unknown. Bio-informatic prediction of miR-21 potential mRNAs target revealed putative genes involved in both HCC and in Fatty Liver disease , supplemented with 10% FBS (Hyclone II GE), 1% penicillin/streptomycin (Sigma), 5 µg/mL insulin (Sigma), 0.5 µM hydrocortisone hemisuccinate (Sigma). After 1 week at confluence, cells were shifted into the same medium supplemented with 50 µM hydrocortisone hemisuccinate and 2% DMSO (Sigma) for 2 more weeks to obtain confluent differentiated cultures. Cells were then treated with different drugs  for 72 weeks (7 mice per group) Fig. . At the 5 μm-thick sections were trimmed from each FFPE histological specimen, and stained with Haematoxylin/Eosin and with phospho-STAT3 (Tyr705)  antibody as described in Supplementary methods.To quantify lipid accumulation Bodipy dye (Sigma) was used . 2′,7′-Dichlorofluorescin diacetate (DHCFDA) dye (Sigma) was used to monitor intracellular ROS production . Cytofluorimetric analysis was performed using a FACS-CANTO (BD).The expression levels of interleukin 6 (IL6) secreted from sodium oleate treated dHepaRG cells were detected by enzyme-linked immunosorbent assay, IL6 ELISA, from Abcam (Ab 46042). Cell culture media was centrifuged at 1,000 g for 10 minutes to remove debris and supernatants were collected to perform standard ELISA as manufacturer’s protocol.See Supplementary methods.Cells were lysed in NET buffer  and immunoblotted with the following antibodies: anti-STAT3 (#9139), anti-phospho-STAT3(Tyr705) (#9131), anti-phospho-STAT3(Ser727) (#9134), anti-IL1β (#12242), anti-phospho-JAK2(Tyr1007/1008) (#3771), anti-PAI-1 (D9C4) (#11907) from Cell Signaling; anti-NFκB p65 (sc-372), anti-USP18 (sc-98431) and anti-Actin (sc-1616) from Santa Cruz Biotechnology. Proteins of interest were detected with HRP-conjugated anti-mouse/rabbit/goat IgG antibodies from Santa Cruz Biotechnology and visualized with the Pierce ECL Western blotting substrate (ThermoScientific), according to the provided protocol. Autoradiography images were developed with a KODAK MIN-R processor. Full-length blots are included in Supplementary Figures 39 was performed as described in Supplementary methods.Chromatin from dHepaRG cells was immuno-precipitated with the following antibodies: anti-phospho-STAT3-Tyr705 , anti-tetra-acetylated-histone-H4-Lys-6-9-13-17 (06-866 Upstate) and non-specific IgG (Santa Cruz Biotechnology Inc.). Chromatine Immunoprecipitated was analyzed by qPCR using fluorescent dye SYBR Green in a Light Cycler 480 instrument (Roche Diagnostics). Deep sequencing analysis−1 as coherent Raman image contrast for lipids, as well as multiphoton auto-fluorescence contrast, as described in Supplementary methods. All data generated by CARS are included in Supplementary Table A multimodal nonlinear microscope was used to record images in formaldehyde fixed dHepaRG cells, using the strong methylene vibration at 2840 cmP-values were determined using the 2-tailed Student’s T-test: *0,01 ≤ P < 0,05; **0,001 ≤ P < 0,01; ***P < 0,001.Supplementary Information


This article presents an overview of the central role of STAT3 in the crosstalk between endothelial cells and cardiac myocytes in the heart. Endothelial cell STAT3 has a key role in inflammation that underlies cardiovascular disease and impacts on cardiac structure and function. STAT3 in endothelial cells contributes to adverse cardiomyocyte genetic reprograming, for instance, during peripartum cardiomyopathy. Conversely, cardiomyocyte STAT3 is important for maintaining endothelial cell function and capillary integrity with aging and hypertension. In addition, STAT3 serves as a sentinel for stress in the heart. Recent evidence has revealed that the redox nature of STAT3 is regulated, and STAT3 is responsive to oxidative stress (ischemia-reperfusion) so as to induce protective genes. At the level of the mitochondrion, STAT3 is important in regulating reactive oxygen species (ROS) formation, metabolism, and mitochondrial integrity. STAT3 may also control calcium release from the ER so as to limit its subsequent uptake by mitochondria and the induction of cell death. Under normal conditions, some STAT3 localizes to intercalated discs of cardiomyocytes and serves to transmit pro-fibrotic gene induction signals in the nucleus with increased blood pressure. Further research is needed to understand how the sentinel role of STAT3 in both endothelial cells and cardiomyocytes is integrated in order to coordinate the response of the heart to both physiological and pathological demands. Signal transducer and activator of transcription 3 (STAT3) was first recognized around 1994 as the acute-phase response factor that is activated by inflammation and couples to enhanced gene expression . STAT3 iCardiac Microvascular Endothelium Contribution to Cardiac Myocyte Growth, Structure, and Contractile Function. Endothelial cell STAT3 plays a key role in inflammation that underlies cardiovascular disease and contributes to adverse cardiomyocyte genetic reprograming during peripartum cardiomyopathy. In addition, recent evidence suggests that endothelial STAT3 may indirectly contribute to cardiomyocyte metabolism and function via its effects on endothelial autophagy. On the other hand, cardiomyocyte STAT3 is important for maintaining endothelial cell function and capillary integrity. We begin with an overview of the increasingly appreciated role of STAT3 as a sentinel for stress in the heart. Accumulating evidence indicates that STAT3 is both redox-sensitive and positioned within endothelial cells and cardiac myocytes at various sites to respond quickly to outside stress stimuli  . Y705 phIschemia or ischemia–reperfusion (IR) is reported to induce the translocation of STAT3 to mitochondria in cardiac myocytes by undefined means , 5, whicSTAT3 has been shown to modulate mitochondrial complex activity and ROS production by several means , 3. STAT2O2 due to a deficiency in a mitochondrial complex I protein, peroxiredoxin-2 expression was elevated and was associated with an increase in STAT3 dimerization and Bcl2 gene expression . This occurs via STAT3-mediated suppression of miR-199a-5p levels and the subsequent upregulation in expression of the UPR-related proteins activating transcription factor 6 (ATF6) and 78 kDa glucose-regulated protein (GRP78)  of certain cancer cells so as to limit Caneration , 15. Thi (GRP78) .IV-spectrin at intercalated discs , with transverse aortic constriction so as to mimic hypertension, caused phosphorylation of βIV-spectrin and the displacement of STAT3. Once displaced, STAT3 translocated to the nucleus and was implicated in the induction of genes for fibrosis and loss of cardiac function, but not cardiac hypertrophy. Thus, STAT3 may serve as a sensor in the heart to assuage increased wall stress through increased extracellular matrix deposition. However, the possible role of STAT3 at intercalated discs in regulating genes of cardiac myocytes that are important for endothelial cell function, such as vascular endothelial growth factor (VEGF), was not reported.In cardiac myocytes, a pool of STAT3 was recently shown to be associated with the cytoskeletal protein βed discs . Activataka CX3CL1), vascular cell adhesion molecule 1 (VCAM1), and intercellular adhesion molecule 1 (ICAM1)  , 32. It  (ICAM1) .On the other hand, STAT3 may exert anti-inflammatory actions involving endothelial cells. STAT3 may suppress iNOS levels by binding to its promoter, and thereby have anti-oxidant and anti-inflammatory properties . In endoEndothelial cell STAT3 has a protective role in post-ischemic myocardial function. In a mouse model of IR injury, ablation of endothelial cell STAT3 worsened capillary integrity and cardiac function, and increased myocardial inflammatory signaling . GreaterIn addition to driving cardiac hypertrophy and fibrosis through the upregulation of IL-6 and IL-8 , endotheTaken together, evidence indicates that endothelial STAT3 can have either a beneficial or detrimental impact on cardiac myocytes depending upon pathophysiological context. Further research is needed to understand whether these actions are mediated by specific sentinel compartments for STAT3 in endothelial cells.The expression of STAT3 in cardiac myocytes is required for myocardial capillary growth postnatally. In the mouse heart, targeted knockout of STAT3 in cardiomyocytes with the Cre-Lox system was associated with a gradual reduction in capillary density after birth that becomes significant at 2–3 months . PreviouUpregulation of miR-199a-5p with STAT3 KO, along with an increase in miR-7a-5p, sensitizes the normal and peripartum heart to chronic β-adrenergic signaling with isoproterenol . CardiomNRAS  illustrates several aspects of the interplay between endothelium and cardiomyocyte communication, including its derangement. This life-threatening pregnancy-associated cardiomyopathy occurs in previously healthy women. Of note, STAT3 protein levels in the left ventricle are decreased in patients with end-stage heart failure due to PPCM . In pregNRAS . In addiNRAS , 54. MorNRAS . Akt actNRAS , PPCM paNRAS .STAT3 serves a critical function on coordinating crosstalk between cardiomyocytes and endothelial cells of the heart . This twAll authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.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.


Rhizophora mangle at regional and local scales in the Yucatan Peninsula. Thirteen mangrove populations , located in seven sites, were sampled, and their morphological variability and relationship with the availability of phosphorus and salinity were analyzed. The diversity and genetic structure were estimated at different hierarchical levels with nine microsatellites, also Bayesian inference and Principal Coordinates Analysis were used. We found a great morphological variability of R. mangle that responded to local environmental variability and not to the precipitation gradient of the peninsula. The genetic diversity found in the peninsula was greater than that reported for other populations in Mexico and was grouped into two regions: the Gulf of Mexico and the Caribbean Sea. At a local scale, tall and scrub mangroves had significant genetic differentiation suggesting that ecological barriers promote genetic differentiation within sites. These results need to be considered in future population genetic studies and for mangrove management and conservation.The environmental variability at local scale results in different physiognomic types of mangrove forest. However, this variability has never been considered in studies of mangrove genetic variability. This study analyzed the genetic and morphological variability and structure of  Also, the tall and scrub R. mangle will show genetic differentiation within a site.Accordingly, the principal aim of this work was to evaluate the morphological and genetic structure and variability of 22.1This study was made along the northern and eastern coasts of the Yucatan Peninsula, which is a low elevation karstic platform (slope <1%) with irregular topography Pannier, . The cliRhizophora mangle (Rhizophoraceae) forests along the coast of the Yucatan Peninsula were selected  were randomly selected. We measured the following: (a) leaf length and width ; (b) length of ten propagules with a graduated ruler (cm); (c) height (m), measured with a Vertex Laser ; and (d) stem diameter . At each site, interstitial salinity (~30 cm depth) was measured with an YSI Pro 2030 portable conductivity meter . Also, a soil core with a depth of 30 cm was obtained and transported to the laboratory to measure total phosphorus (P) and extractable P. Total P was determined as orthophosphate after Aspila, Agemian, and Chau  and Pars2.2.1r1 × r2; r1: leaf width, r2: leaf length); and the environmental variables: salinity and extractable P. Analyzes were realized in XLSTAT v.7.5.2. .A Canonical Discriminant Analysis was performed to know the morphological differences between populations of Yucatan Peninsula and the most discriminant variables. Also, to distinguish a pattern among the environmental variables to the mangrove type, a Principal Component Analysis was used. Finally, a Canonical Correlation Analysis was applied to identify the correlation among the morphological variables: height, length of the propagule and to include the foliar characters, leaf area was calculated considering the leaf as an oval  per population were chosen for leaf sampling. Polyvinylpyrrolidone (PVP) was applied to each leaf in the abscission zone to avoid oxidation; leaf samples were stored at −20°C for further analysis.For the molecular analysis, leaf samples (young leaves without apparent damage) from 130 individuals of g for 20 min, 700 μl of the supernatant were transferred and extracted with 650 μl of 24:1 (v/v) chloroform‐isoamyl alcohol, and centrifuged at 18,000 g for 10 min to separate the phases. This process was repeated twice. The resulting supernatant DNA was precipitated with 700 μl of cold isopropanol and 35 μl of 3 M sodium acetate, left overnight incubating at −20°C and centrifuged for 20 min at 18,000 g. The pellet was washed with 200 μl of cold absolute ethanol and centrifuged for 6 min at 18,000 g. The DNA obtained was resuspended with 70 μl of 1X TE and stored at −20°C for further analysis. The quality of the extracted DNA was verified by electrophoresis in 1% agarose gels stained with ethidium bromide (10 mg/ml). The DNA was quantified on a NanoDrop 2000 UV‐Vis spectrophotometer (Thermo Scientific), and subsequently, the concentration of each sample was homogenized to 25 μg/μl and stored at −20°C until further use.DNA was isolated by the modified CTAB/PVP method , 1 u of Taqpolimerase, and 2 μl of DNA (50 ng). Amplification was performed on an Applied Biosystems Gene Amp PCR SYSTEM 9,700 thermocycler  at the following temperatures: initial denaturation of 94°C for 2 min, followed by 37 cycles of 15 s at 94°C, 15 s at 50°C  An individual assignment test was done with a Bayesian approach implemented in the Program STRUCTURE v.2.3.4  and checked the consistency of results. The optimal K value was calculated using the ΔK method described by Evanno, Regnaut, and Oudet . A Pnd Oudet ; this annd Oudet . The Neind Oudet . Also, tnd Oudet , with 1,nd Oudet .R. mangle was evaluated at different organizational levels: Yucatan Peninsula, main groups defined by STRUCTURE and PCoA, sites, populations and by physiognomic type of mangrove within regions . The evaluated estimators were expected (HE) and observed heterozygosity (HO) and allelic richness (Na), all estimators were obtained with GenAlex V6 program  was estimated for all populations, regions, and mangrove types, as well as for all the Yucatan Peninsula. The evaluated estimators were compared among populations, regions, and type of mangrove, using 1,000 permutations with FSTAT program ver. 2.9.3.2 .The study populations had high morphological variability within and among sites Table  and, accp < 0.05). However, the λ Wilks showed a poor predictive power (0.85). This analysis also showed that salinity was the variable that contributed the most to the variability in morphology and had a negative correlation with tree height and leaf area, but EP had a positive correlation with these latter variables ; sites with low salinity and high EP values had tall mangroves ; and sites with low salinity and EP values had scrub mangrove  Figure . Two sit3.2R. mangle sampled in the Yucatan Peninsula were divided into four groups according to the optimal K obtained with the Evanno method . Moreover, the STRUCTURE analysis based in K = 4 presented two main groups: one group composed of the populations from the Gulf of Mexico coast: Celestún (Cel‐T and Cel‐S), Rio Lagartos , and Progreso ; and another group integrated by the populations from the Caribbean coast: Nichupté (Nic‐S), Puerto Morelos (Pue‐S), Sian Ka'an , and Mahahual (Mah‐S) . This reinforces the existence of the two main groups, the Gulf of Mexico and the Caribbean Sea  Figure a. Also, s Figure a,c. Altha Figure b. BecausThe grouping pattern obtained with STRUCTURE was partially supported by the three‐dimensional Principal Coordinate Analysis . Also, the genetic differentiation between regions was 6.5%  than by populations. However, the differentiation between regions was similar  (Table p < 0.05). The lowest genetic differentiation was observed among tall and scrub populations of Rio Lagartos . With exception of Celestún, sites from the Gulf of Mexico had less genetic differentiation between physiognomic types than those from the Caribbean Sea ; while the analysis made by populations presented a weak but significant relation . Within regions, the Mantel test did not present significant relationship.The genetic structure of 5) Table . A largeea Table . The rel3.3HE) and observed heterozygosity (HO) of 0.37 ± 0.003 and 0.27 ± 0.003, respectively, and an allelic richness (Na) of 2.41 ± 0.02. Also, the HO was higher for the Caribbean Sea region (0.37 ± 0.01) than for the Gulf of Mexico region (0.23 ± 0.02), but their differences between HE and HO were lower , while the scrub populations showed the highest values of genetic diversity in the Caribbean Sea region . The population with the highest HO was tall‐Nichupté while scrub‐Celestún had the lowest HO;Na varied from 1.89 for tall‐Sian Ka'an to 2.77 from tall‐Rio Lagartos, scrub‐Nichupté and scrub‐Sianka'an1. The HE was higher than HO in all populations; however, the populations in the Gulf of Mexico had the highest difference, especially in the site of Celestún  for the populations showed positive significantly values in all populations (from 0.06 ± 0.02 in Nic‐T to 0.48 ± 0.05 in Cel‐S). The FIS for the Yucatan Peninsula was 0.26 ± 0.01. The Caribbean Sea region had a significantly lower FIS (p < 0.05) than that of the Gulf of Mexico  than scrub populations (0.31 ± 0.02), but values were not significantly different.The inbreeding coefficient  are two of the main factors that have been related to mangrove forest structure, especially in scrub mangroves  was greater than that reported for the Baja California Peninsula in Mexico  and Puerto Morelos , which are similar to our values for the same sites . Future studies should include more individuals per physiognomic type of mangrove within a site to validate the sample size.The genetic diversity of o et al.  reportedR. mangle in the Yucatan Peninsula is clearly arranged in two regions: the Gulf of Mexico and the Caribbean Sea coasts  due to currents that interrupt continuous genetic flow along the Brazilian coast  was found; the upwelling of deep waters at Cabo Catoche in the northeastern of the Peninsula interrupts the continuous flow of the Yucatan current, which drives to northeast Gulf of México as the loop current (Figure R. mangle propagules along the coast from the Caribbean Sea to the Gulf of Mexico, preventing the genetic flow and keeping genetic differentiation between both regions (Martínez & Pares, At the most recent glacial period, an extreme geographic isolation took place for many species, with subsequent species‐specific patterns of postglacial expansion (Kennedy et al., t Figure . This cuIn our study, no morphological differences between regions were found, which reflect that the genetic variability did not respond to historical differences between populations, but to local adaptation to environment. For instance, European oaks do not show any association between genetic divergence connected to colonization events and those associated with local selection pressures (Kremer et al., 4.3R. mangle showed significant genetic differences that could be caused by their contrasting environments within a site (Table R. mangle, especially in the scrub mangrove, represents a physical barrier for propagules dispersion (Tonné, Beeckman, Robert, & Koedam, Tall and scrub populations of te Table . These ete Table . Thus, tte Table . On the FIS found in the scrub than in the tall mangrove populations in the Caribbean Sea indicate that the autogamous nature of the R. mangle has accentuated the differences between populations at local scale by genetic drift. The main adaptive advantage of gene drift, despite the low diversity associated with inbreeding, denotes reproductive safety and adaptation to a particular niche (Loveless & Hamrick, hitch‐hiking effect (Smith & Haigh, The environmental and ecological differences between tall and scrub mangroves, which coexist at short distances, could be generating breeding barriers that limit the genetic flow between them and promoting a greater genetic differentiation even more than among sites Tables  and 4. TAvicennia species between populations at a fine scale have been found using RAPDs markers and microsatellites (Dahdouh‐Guebas et al., Laguncularia racemosa. Also, genetic differences using AFLP markers were reported among salt marsh and riverside A. schaueriana (Lira‐Medeiros, Cardoso, Fernandes, & Gomes‐Ferreira, R. mangle mangrove, environmental variables, or ecological characteristics of our study. Generally, the majority of population genetic studies focus on the classic pattern of isolation by distance, and studies on mangroves are not the exception; however, the idea that adaptive responses to divergent natural selection may impact genomewide population structure has gained momentum and needs to be considered in future studies (Nosil et al., The genetic variability at local scale driven by environmental variables has also been reported before for other species (DeWoody et al., R. mangle, observed at local scale in the Yucatan Peninsula, suggests that each site is probably composed of demes, where the ecological limits could be generating reproductive barriers. In continuous populations, recurrent processes, such as gene flow, genetic drift, and selection, act in concert to shape the genetic structure (Latta, The genetic structure of e Latta, . Fine‐sce Latta, , even une Latta, .R. mangle considered in our study could probably allow us to find a greater genetic structure in the Yucatan Peninsula in comparison with the reported studies that include a greater number of populations distributed in larger geographic areas (Cerón‐Souza et al., The two different types of D.J.C.delaC., J.M.C., J.H.S., and J.L.A. conceived and designed the study; D.J.C.delaC., M.O.G., and R.U.S. collected the data; D.J.C.delaC., L.Y.E., and J.M.C. analyzed the data; D.J.C.delaC, J.M.C., and J.L.A. wrote the text; all authors provided editorial advice and approved the final version.https://doi.org/10.5061/dryad.1578ks0.Morphological and environmental data and microsatellite genotypes are available at


In the first part of this paper we review the evidence that suggests that both genetic and non-genetic information contribute to inheritance in livestock  and discuss how the environment may influence non-genetic inherited factors. Then, in a second step, we consider methods for favoring the transmission of non-genetic inherited factors by estimating and selecting animals on their extended transmissible value and/or introducing favorable non-genetic factors via the animals’ environment.Evolutionary biologists studying wild species have demonstrated that genetic and non-genetic sources of information are inherited across generations and are therefore responsible for phenotypic resemblance between relatives. Although it has been postulated that non-genetic sources of inheritance are important in natural selection, they are not taken into account for livestock selection that is based on genetic inheritance only. According to the natural selection theory, the contribution of non-genetic inheritance may be significant for the transmission of characters. If this theory is confirmed in livestock, not considering non-genetic means of transmission in selection schemes might prevent achieving maximum progress in the livestock populations being selected. The present discussion paper reviews the different mechanisms of genetic and non-genetic inheritance reported in the literature as occurring in livestock species. Non-genetic sources of inheritance comprise information transmitted  Learning from conspecifics or adaptation to the environment may occur through observation, imitation, teaching or interactions in the form of play behavior, aggressive encounters, cooperation, or competition to access a resource  and that causes phenotypic differences between individuals of a population  leading It is well established that DNA is transmitted from one generation to the next through the sexual reproduction process (meiosis). Offspring receive one half of their DNA from each parent apart from mitochondrial DNA that is transmitted by the dam only . Based oDGAT1 gene induces a modification of its expression which has a major effect on the milk fat content and other milk characteristics  and heterozygous individuals for the mutated allele carried by the paternal chromosome (patIGF2mut|matIGF2wt) show hypermuscularity (DLK1 mutation in sheep (mut|matDLK1wt) express the phenotype of muscular hypertrophy  in order to better comprehend the relationship between genotype and phenotype . Numeroueristics . Nonetheeristics . In live in pigs  and a muin sheep . The IGFcularity . A more in sheep . In thisertrophy .To sum up, genes are inherited by the transmission of genetic material, DNA, and their expression plays an important role in the development of all phenotypes. Genetic inheritance thus contributes to phenotypic resemblance between relatives.in utero exposure of developing embryos may directly impact phenotypes at birth and later on of individuals of the first generation (F1); it is called intergenerational transmission. Secondly, the prenatal exposure of fetuses may alter their gametes and affect performances of offspring produced in the second generation (F2); it corresponds to a multigenerational transmission. Thirdly, a phenotypic effect persists in individuals of the third generation (F3) while a modification of the environment occurred only on F1 embryos and gametes; this is a true transgenerational inheritance. In addition, if a male or non-pregnant female adult animal is subjected to an environmental exposure, then changes seen in the F2 generation or later are considered transgenerational.The epigenome consists of all the molecular processes that interact with the genome of an organism and contribute to the regulation of gene expression without modifying the DNA sequence. Epigenetic modifications include DNA methylation or hydroxymethylation of CG dinucleotides, chemical modifications of histones, interactions between the DNA and small RNAs, and different states of chromatin condensation . Epigenevia epigenetic mechanisms such as histone modifications and microRNA expression  they imply to wait until the third generation, ii) germ cells are more heterogeneous, and iii) gametes sampling is much more difficult. In addition, the idea that the paternal experience may have direct implications for the fitness of the offspring is quite recent. Besides the nuclear genome, sperm and semen contain a range of epigenetic elements  that are delivered to the zygote upon fertilization  that reside in and on the bodies of animals.In utero, the mammal digestive tract is practically sterile and the main microbial taxa that reside within the mammalian gut do not develop outside their host (vs. Cesarean section) and suckling type  impact greatly on the gut microbiota colonization process (vs. building vs. in an isolator with antibiotic treatment) results in differences in microbiota composition that persist until after mother-piglet separation  resemblance between parents and offspring . Inherite exists . Moreovee exists . Thus, tA focal individual can benefit from social learning acquired during social interactions and will be influenced by the  behavior of the conspecifics it is raised with, i.e. reciprocal influences of individuals sharing a common environment over short or long periods of time. This transmission of social skills and innovations at the group level refers to cultural inheritance  in its  studies . The infffspring . Maternavia different means of transmission.To sum up, we described four different sources of inheritance that differ according to their mean of transmission. It is important not to confuse the mean of transmission with the mean of action. Such distinction is particularly relevant in the case of indirect effects . For insIn livestock, selection is aimed at improving numerous phenotypes in order to increase animal production, facilitate breeding, reduce animal mortality and morbidity, increase animal welfare, etc. Selecting, as parent of the next generation, animals that will transmit their good skills to their offspring for the characters of interest proved to be an efficient way to enhance the above characters. Only genetic values are accounted for selection in livestock. The inclusion of the other sources of inheritance, by predicting the extended (genetic and non-genetic) transmissible value of an individual in the selection of the future reproducers, would be an added value. In addition, since non-genetic inherited factors are sensitive to the environment, amending the animals’ environment to promote non-genetic inherited factors that are transmitted to the next generations is key for improving performances.yi of individual, is decomposed into its different components by: For selection in livestock species, knowing the potential that an animal can transmit to its offspring is crucial to select the best animals  as parents for the next generation. This potential can be predicted by analyzing the phenotypic resemblance between relatives using a linear mixed model. The phenotype β is the vector of fixed effects and xi the vector that links the fixed effects to the observation of animal i. ai, iepi, and imic are the additive genetic, epigenetic, cultural/behavioral, and microbiotal transmitted values of individual i, respectively. Distribution of the different transmitted values, under the assumption that the variance of the transmitted effect is constant over generations, is written t = a,epi,cult or mic and t of different individuals. To predict the transmitted values of an individual, the variance components of the aforementioned model have to be estimated. To do so, phenotypic records recorded in a proper design, genealogic information and the distributions , i.e. the average relationships assuming infinite loci. Using SNP markers, it is possible to compute the “real” IBD relationships matrix (genomic relationship matrix G) which is slightly different given the finite genome size  in order to increase the reliability of the predictions of the transmittable genetic value of individuals  that reside in and on the bodies of animals  influenced by the environment  and ii) dynamic and modulated during the animal’s lifetime. This means that breeders can initiate favorable epigenetic marks, microbiota, or behavior that will be later transmitted across generations by controlling the animal’s environment. It is too early to clearly define the breeding practices to be implemented to influence such factors positively and research is still underway on the subject. However, given current knowledge on non-genetic inherited factors, it is possible to identify different key moments in the lives of the future reproducers when non-genetic inherited factors may be influenced  for species in which it is possible . Indeed,Withdrawal is a critical period for the young animal, especially in terms of stress. The benefits of early social separation and socialization to the development of social skills have been reported in pigs and related to less damage later in life . But earIn several livestock species , offspring are separated from their mother. Nonetheless, it has been shown that after birth, the mother's absence disrupts microbiota transmission to the offspring and the intestine is colonized by microorganisms from environmental metacommunities . DisruptEarly life is also the time when interactions with the breeder begin. The effect of early experience on temperament, learning ability, and cognition is a complex phenomenon that requires further investigation, and depends upon the major role of stressors. In our opinion, breeders should ensure that first experiences are positive no matter when the handling of animals begins. At a higher level of complexity, sex-specific differences in the intergenerational transmission of non-genetic effects should be considered. Later in the career of the animal, it has been shown that epigenetic mechanisms are key regulators underlying the neuroendocrine control of puberty . Howevervia other animals, and actions that modulate non-genetic inherited factors via favorable environmental conditions  selection will remain the main lever for improving livestock characters. However, recent knowledge about the other sources of inheritance in livestock offer the possibility to favor the transmission of non-genetic inherited factors across generations. Combining genetic and non-genetic inheritance will surely improve the benefit of selection.ID, LC, SC, and JD wrote the manuscript.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.


Dalbergia odorifera T. Chen (Fabaceae) is a woody tree species indigenous to Hainan Island in China. Due to its high medicinal and commercial value, this tree species has been planted over 3500 ha2 in southern China. There is an urgent need for improvement of the D. odorifera germplasm, however, limited information on germplasm collection, conservation, and assessment of genetic resources is available. Therefore, we have built a database of 251 individuals collected across the whole of southern China, which included 42 wild trees and 210 cultivated trees, with the following objectives. (1) Evaluate genetic diversity and population structure of the database using 19 microsatellite markers and (2) develop a core collection for improvement and breeding programs. Totally, the 19 microsatellite markers harbored 77 alleles across the database with the polymorphic information content (PIC) ranging from 0.03 to 0.66. Medium genetic diversity level was inferred by Nei’s gene diversity (0.38), Shannon’s information index (0.65), and observed (0.33) and expected heterozygosity (0.38). Structure analysis showed that four was the optimum cluster size using the model-based Bayesian procedure, and the 251 D. odorifera individuals were grouped into five populations including four pure ones (RP1-4) and one mixed one (MIX) based on their maximum membership coefficients. Among these populations, the expected heterozygosity varied from 0.30 (RP3) to 0.38 (RP4). Analysis of molecular variance (AMOVA) showed 11% genetic variation existed among populations, and moderate population differentiation was inferred by the matrix of pairwise Fst (genetic differentiation among populations), which was in the range of 0.031 to 0.095. Moreover, a core collection of 31 D. odorifera individuals including six wild and 25 cultivated trees was developed, which was only 12.4% of the database but conserved the whole genetic diversity. The results of this study provided additional insight into the genetic structure of the large D. odorifera germplasm, and the core collection will be useful for the efficient and sustainable utilization of genetic resources, as well as efficient improvement in breeding programs. Dalbergia odorifera T. Chen, formerly named Dalbergia hainanensis Merr. et Chun, is a semideciduous perennial woody tree species of high medicinal and commercial value. The heartwood of its root and stem is a traditional Chinese medicine, named “Jiangxiang”, which plays crucial roles in cardiovascular treatments [D. odorifera—an indigenous to Hainan Island in southern China and is confined to a relatively narrow tropical geographic area [eatments , diabeteeatments , blood deatments ,4 as it eatments ,6,7,8,9.eatments . For exahic area —that hashic area . The reshic area ,13.D. odorifera was introduced to subtropical areas of Guangdong, Guangxi, and Fujian provinces in China in the 1950s. After several decades, the introduced trees exhibited a satisfactory growth performance, and, at most sites, has even formed valuable heartwoods, which take approximately 50 years to mature in the wild [D. odorifera trees because of its ecological and economic importance. Moreover, the plantation area of this species has exceeded 3500 ha2 in southern China. There is an urgent need for improvements of the D. odorifera germplasm, particularly for higher yield of heartwood in a shorter harvest cycle. Such improvements are based on the germplasm collection, conservation, and assessment of genetic resources.Due to its high medicinal and commercial value, the wild . RecentlD. odorifera germplasm collection [Germplasm is the basis of plant breeding programs . During llection ,17,18. EMalus domestica Borkh) [Persea americana Mill.) [Castanea sativa Mill.) [D. odorifera yet. Additionally, information on the genetic diversity and genetic variation of plant species has been well demonstrated using molecular markers [D. odorifera individuals using a set of 19 microsatellite markers, and developed a core collection using PowerCore software [D. odorifera germplasm and (2) develop a core collection conserving the entire diversity for improvement and breeding programs. The findings of this study will provide a useful resource as well as guidance for better germplasm utilization in genetic improvement, and serve as a database for identification, parentage, and traceability purposes.A core collection is a subset of the whole germplasm collection, and aims to create a uniform representation of the original genetic space with equal weights across this space . A good a Borkh) ,22, avoca Mill.) , and chea Mill.) . However markers ,26,27,28 markers . Therefosoftware . The maiD. odorifera across the southern China , 0.3 µl dNTPs (10 mM each), 0.15 µL of each primer at 10 µM, 0.3 µL Taq polymerase at 5 units/ µL (TaqUBA), and 1 µL of genomic DNA (40–50 ng). Then, 35 cycles of 94 °C for 15 s, appropriate annealing temperature for 15 s, and 72 °C for 30 s were performed, following the predenaturation at 94 °C for 3 min. All the PCR reactions were repeated at least once. Their diluted PCR products were mixed with 12.5 Hi-Di formamide and 0.25 µL of size standard , and were then separated by capillary electrophoresis and genotyped with an ABI 3730 Genetic Analyzer  at Shanghai Generay Biotech Co., Ltd., Shanghai, China. Peak identification and fragment sizing were done using Gene Mapper v4.0 (Applied Biosystems) with the default settings.A set of 19 microsatellite markers was developed in our previous study  and usedThe genetic structure of the investigated database was analyzed using STRUCTURE 2.0 . The numIS), genetic differentiation among populations (FST), and the pairwise Fst, were computed using GenAlEx version 6.5, which was also conducted the hierarchical analyses of molecular variance (AMOVA) [The frequency of null alleles (FNA) and scoring errors were estimated using the Microchecker software 2.2.3 . Genetic (AMOVA) . The Ewe (AMOVA) .p < 0.05); (3) validating the core collection with unweighted neighbor-joining dissimilarity trees using DARwin software (version 6.0.9) [A core collection—a reduced number of samples which represents the greatest diversity of the initial collection—was generated using PowerCore (v. 1.0) with a heuristic search . The repn 6.0.9) ; (4) andn 6.0.9)  was consn 6.0.9) , based on 6.0.9) .D. odorifera individuals, and the number of alleles detected per locus varied from two to seven -derived Δ K (log probability of data derived delta K) value (An admixture model-based approach was implemented to investigate the population structure of 251 K) value a–c. SubsK) value . Among tK) value , MIX conNeighbor-joining (NJ) phylogenetic analysis and principal component analysis (PCA) were used to detect the genetic relationship across the 251 individuals based on the dissimilarity matrix calculated with Manhattan index. Four clusters were clearly distinguished by both the NJ dendrogram tree and the PCA plot . MoreoveD. odorifera individuals.Among the five populations , the numBoth analysis of molecular variance (AMOVA) and pairwise Fst analysis were performed to investigate the genetic variations among these populations. The results showed that 11% of the total genetic variation occurred among populations . ModeratD. odorifera individuals was constructed using the PowerCore software with the advanced M-strategy  markers and Liu et al. [D. odorifera individuals using 19 microsatellite markers. The difference in genetic diversity may be mainly resulted from the larger population size investigated in the present study [Genetic diversity plays an important role in genetic improvement through breeding programs . Howeverg et al.  evaluateu et al.  assessednt study ,46, or ant study  or typesnt study  used in D. odorifera individuals collected from its whole native habitat and introduced sites covering five provinces in southern China. In total, 19 microsatellite markers harbored 77 alleles across the whole database with the mean polymorphic information content (PIC) of 0.32  [Dalbergia monticola Bosser & R. Rabev. (He 0.83) [Dalbergia nigra (Vell.) Benth.  [Dalbergia cochinchinensis Pierre ex Laness , and Dalbergia oliveri Prain  [D. odorifera is an endemic tree species with original distribution restricted to the small regions of Hainan Island, which is concordant with the general trend that distribution-restricted plant species are associated with relatively low genetic diversity [Assessment on the genetic diversity and population structure of a species is essential to evaluate the applicable potential of a new germplasm resource, and the prior knowledge of genetic diversity and pairwise relatedness can provide beneficial clues for efficient utilization in large collections of genetic resources ,49,50. T of 0.32 . Medium ectively . The resIC 0.30) , but mucHe 0.83) , DalbergHe 0.75) , DalbergHe 0.73) . This diess Ho 0., He 0.55iversity ,58.D. odorifera individuals (D. oliveri (12.6%) [D. sissoo (14.6%) [The Bayesian model-based structure analysis is widely used for the inference of hidden population structure in plant species . In thisividuals . Both neividuals . The res (12.6%)  and D. s (14.6%) , which m (14.6%) .D. odorifera database  are severely fragmented but relationships among individuals are badly influenced by human activities. It is no wonder then that the core collection comprised only six wild individuals compared to 25 cultivated ones.As a whole, the 31 genotypes selected for the core collection are representative samples of the diversity retained from the whole southern China that covering its native habitat and the whole first introduced sites. Notably, the wild resource of D. odorifera. Therefore, this core collection can serve a basic reference on similar research for D. odorifera and other Dalbergia species. Furthermore, it can be considered as a powerful tool for exploring the genetic diversity, as well as a new source for efficient conservation and breeding programs of D. odorifera in the future.Prior to the present study, no information has been reported on core collection of D. odorifera germplasm. A medium level of genetic diversity and genetic variation was presented within the species. Based on the assessment, a core collection was first established using the PowerCore software with the M-strategy. The core collection contained 12.4% (31) out of the 251 germplasm, possessing the intact genetic diversity of the whole germplasm collection. This core collection will serve as a primary source for further genetic association, functional analyses, and function to improve breeding programs in future studies.The present study provides an overall assessment on genetic diversity and structure of 251


On April 24, 2018, a suspect in California’s notorious Golden State Killer cases was arrested after decades of eluding the police. Using a novel forensic approach, investigators identified the suspect by first identifying his relatives using a free, online genetic database populated by individuals researching their family trees. In the wake of the case, media outlets reported privacy concerns with police access to personal genetic data generated by or shared with genealogy services. Recent data from 1,587 survey respondents, however, provide preliminary reason to question whether such concerns have been overstated. Still, limitations on police access to genetic genealogy databases in particular may be desirable for reasons other than current public demand for them. On April 24, 2018, a suspect in California’s notorious Golden State Killer cases was arrested after decades of eluding the police. The capture of Joseph James DeAngelo, a former police officer, was a critical step toward closing the books on 12 unsolved murders and at least 45 rapes that were committed throughout California from 1976 to 1986 [For decades, California police tracked the Golden State Killer using traditional detective work, but all leads came to dead ends. Six years ago, however, investigators tried a different tactic. Instead of trying to identify the killer—a needle in an enormous haystack—they sought first to reduce the size of the haystack by identifying the killer’s family. Investigators did so by uploading a genetic profile generated from DNA that had been collected from one of the crime scenes to genealogy websites that match users to their genetic relatives .After years of searching and at least one failed attempt , investiHaving substantially reduced the haystack, investigators were able to identify the needle by constructing family trees and scouring them for potential suspects. Investigators eventually zeroed in on DeAngelo and matched his genetic data, which was collected from an object that he discarded while under surveillance, to crime scene DNA .DeAngelo’s arrest was widely celebrated as a clever investigative coup that may have finally brought to justice a man who had terrorized California residents for years. Yet as details of his arrest emerged, privacy concerns were quickly raised about police searches of personal genetic data of the kind used to capture DeAngelo. Law enforcement officers routinely query the “offender database” of the Federal Bureau of Investigation (FBI)-maintained Combined DNA Index System (CODIS), which holds the genetic profiles of known felons, misdemeanants, and arrestees, for matches to crime scene data . But in If there ever was any question, it is now clear that these data are vulnerable to police access—and are increasingly so as investigators turn to them to solve other crimes. Efforts to apply the same technique that identified DeAngelo to other criminals are already underway and have helped identify suspects in at least four other cold murder cases and one recent rape case , 6. MeanSome have suggested that individuals who contribute to genetic genealogy databases have an expectation of privacy in their genetic data that may be violated by familial genetic searches . AlthougTo date, public opinion on this issue has not been assessed, although it is a critical input to policy discussions regarding whether police should be permitted to access data held by personal genetic service providers, including but not limited to searching genetic genealogy databases for the purpose of generating investigative leads. To begin filling this knowledge gap, in May 2018, we distributed a 20-item survey to assess individual perspectives on police access to genetic genealogy websites and customer information from DTC genetic testing companies. We conducted the survey online using Amazon Mechanical Turk (MTurk), a well-known recruiter for survey research reported in scientific journals , 11. We p < 0.05) when the purpose is to identify perpetrators of violent crimes (80%), perpetrators of crimes against children (78%), or missing persons (77%) than when the purpose is to identify perpetrators of nonviolent crimes (39%). Notably, a similar line was drawn by GEDmatch in its updated privacy policy, adopted after the survey was closed, which explicitly permits law enforcement to search GEDmatch for matches to DNA left at scenes of violent crimes, defined by the site as homicide and sexual assault [Among the 1,587 respondents , the maj assault .p < 0.001). However, age; race and ethnicity; annual household income; purchase of DTC genetic testing services; use of genealogy websites to research relatives; personal or relative’s victimization, arrest, or criminal conviction; and personal or relative’s employment in law enforcement were not independent predictors of support in a multivariate model   .Carpenter versus US, which was pending before the US Supreme Court when the survey was conducted [For comparison, we also assessed respondents’ perspectives on police access to cell phone records and social media accounts in the possession of communications service providers. Warrantless seizures and searches of historical cell phone location information was at issue in onducted . We founThese data provide preliminary evidence that individuals may not be particularly concerned about police searches of personal genetic data that populate genetic genealogy databases when the purpose is considered justified. Notably, although arrestees and convicted criminals are disproportionately low income and black  and the Although MTurk samples have been shown to be more diverse than convenience samples , the resIn addition, the majority of respondents (59%) reported that they or family members had been victims of crimes. By comparison, in 2016, the National Crime Victimization Survey (NCVS) administered by the US Bureau of Justice Statistics found that 1.3% of persons aged 12 or older were victims of violent crimes and 8.8% of households were victims of property crimes . HoweverLikewise, nearly a quarter of our survey respondents (24%) reported that they or family members had ever been employed in law enforcement, which was broadly defined to include security guards and bailiffs. As of May 2017, the US Department of Labor’s Bureau of Labor Statistics reports that approximately 1% of the US population is employed in the protective service occupation category, which includes police and correctional officers, security guards, and bailiffs [Finally, survey respondents may have differed from the general population in relevant ways that our survey did not capture. For example, survey participants recruited by MTurk may spend more time online and therefore may be more comfortable with third party access to personal data. However, US respondents of other MTurk surveys reported spending similar periods of time online as the US general public  and beinIn light of these limitations, additional research is needed to assess whether our survey findings are generalizable. But even if they are, and it is shown that public support for law enforcement access to genetic genealogy databases is high, restrictions on police access could be desirable for reasons that are not yet widely appreciated. Forensic use of genetic genealogy databases makes potential suspects of large populations of individuals for the sole reason that they chose to participate in such databases. All of these individuals, except perhaps one, are actually innocent of the specific crime under investigation. But the odds are such that investigators sometimes target the wrong person. This occurred earlier in the hunt for the Golden State Killer, when investigators honed in on an elderly man living in an Oregon nursing home, whose daughter had uploaded her genetic data to a genealogy service called Ysearch.org .Although the man was later cleared, such missteps can have enormous emotional and reputational impacts on those thrust unfairly into the investigative spotlight. While a suspect, an individual might be subjected to a range of invasive detective techniques, including personal surveillance, searches of financial records, and extensive interviews of family, friends, employers, and neighbors. Yet as noted by law professor Erin Murphy, “no law mandates that, once a name is formally cleared, the officer return and assure the suspect’s family and coworkers that he is truly as innocent as he was the day before the investigation began” . MoreoveAll of Us Research Program, which will conduct genetic analyses for a subset of the 1 million anticipated participants [Yet they could become more common as the universe of genetic services intended to facilitate personal discovery expands and more people participate in it. The DTC genetic genealogy testing market more than doubled in 2017, reaching over 12 million customers by year end . All of icipants , 26. SomMeanwhile, the kind and number of online services available to individuals in possession of their genetic data are growing. Some, including GEDmatch, help individuals understand their ancestry or identify genetic relatives. Others, like Promethease and DNA.Land, generate health, wellness, or trait reports interpreting personal genetic data; provide links to scientific literature or curated archives relevant to those data; or connect users to individuals with similar genetic variants or researchers studying them . As the Although the landscape of personal genetic services is diverse and ever changing, a common practice among these services is to retain copies of the data that users share with them. Yet there is no guarantee that data shared by users actually belong to them. With the notable (and new) exception of GEDmatch , most peCarpenter versus US, which held that individuals have a legitimate expectation of privacy in historical cell phone information that bars police from accessing those data without a warrant [As more people become familiar with the vulnerabilities of personal genetic services, opinions may shift regarding the acceptability of police access to data that are generated by and shared with these services. In the meantime, however, policy discussions about whether to place limits on access should at least take into account the various purposes for which access may be sought, as some purposes may be more socially acceptable than others. While perceived invasions of privacy appear to be tolerable when the purpose is to catch violent or particularly depraved offenders, it seems that many would draw a line at searching their data to solve more ordinary crimes. Notably, this conclusion extends to communications providers with whom individuals share their social media and cell phone information, although it may be at odds with the US Supreme Court’s recent decision in  warrant . To the  warrant , 16. Yet warrant .Far from being a forensic anomaly, the public genetic search that led to the arrest of the Golden State Killer suspect is quickly on its way to becoming routine procedure. What limits, if any, to place on police access to genetic genealogy databases must be thoughtfully considered and soon, with robust input from the public.S1 Text(PDF)Click here for additional data file.S2 Text(PDF)Click here for additional data file.S1 Data(XLSX)Click here for additional data file.


Neural representation can be induced without external stimulation, such as in mental imagery. Our previous study found that imagined speaking and imagined hearing modulated perceptual neural responses in opposite directions, suggesting motor-to-sensory transformation and memory retrieval as two separate routes that induce auditory representation . We hypo Our brain processes sensory information that we receive from the environment and mediates mental activity such as imagination. How do mental and perceptual processes interact to shape our cognition? We constructed a computational model that simulated how two types of imagery, imagined speaking and imagined hearing, differentially modulated perception via two distinct neural pathways. This model further predicted a choice shift in perceptual responses to ambiguous auditory stimuli, which was confirmed in a follow-up imagery-adaptation experiment. These results suggest that parallel neural pathways for mental imagery provide distinct functions to influence perception. These findings may implicate multiple strategies for constructing the brain-computer interface.Perception results from interactions between bottom-up and top-down processes . One of Mental imagery has been hypothesized to be a predictive process . It has The dual-stream for generating prediction has been supported by previous studies . In a maTo provide a parsimonious account, we hypothesized that the precision of induced representation was different . BecauseTo test this hypothesis, we built a neural network model with parameters of postsynaptic gain that were differentially modulated by two types of imagery . The modWe first provide an overview of our methods. A two-layer neural network model was built with free parameters (modulations of postsynaptic gain) that were differently tuned by two types of speech imagery. We used this model to fit our previous MEG data  and compo, is the product of firing probability, p, and the postsynaptic firing amplitude, a, as in We built a two-layer neural network model to simulate the MEG adaptation results. The structure of this model was similar to the one in the published study . Each noD is driven by the output, o. The recovery rate, R, is governed by the current status of depression (1-a), so that the recovery term drives the firing amplitude, a, back to 1.The update of postsynaptic firing amplitude follows :(2)dadt=p, as in θ, as in The firing probability, Substitute v, updates according to τ is a time constant indicating the speed of integration. The variables iCand iErepresent the conductance and reversal potential of three channels, excitation, inhibition, and leak.The membrane potential, s”), and the second layer provides a more abstract phonological analysis . In each layer are six nodes representing the processing of different syllables. Based on the common features of syllables, three nodes were grouped together to represent syllables /ba/, /da/, and /ga/. The other group of three nodes represent syllables /pi/, /ti/, and /ki/. The nodes in the first and second layers within a group are fully connected. The connection strength value ijw(from neuron j to i) is 1 for the connections between the same syllable nodes in different layers, and 0.22 for connections between different syllable nodes. There are no connections between nodes across groups.In this two-layer neural network , the firo, with the connection strength between the two neurons. These signals are summed over all input with other sources such as inhibition and leaking. By specifying the summation between and within layers and expanding from dvin. The second input is the leak with the fixed term L. The third input is the lateral inhibition, which is the strength of I multiplied by the sum of output t from k units at level n. The combination of the leak and lateral inhibition drive the membrane potential toward 0 because the reversal potential of iE and lE are set to 0 (as the term in the bracket is multiplied by -v). The fixed parameters are similar to those used in a previous study  and attentional modulation .Moreover, the imagery tasks were assumed to increase attention to the subsequent sound, and different types of imagery may induce the attentional effect differently. Therefore, we model the attentional effect by adding another free parameter, The free parameters of gain modulation in the model were fitted using the MEG results of imagery-induced repetition effects . The simThe output from all neurons at the phonological layer was summed to obtain the dynamics of simulated neural responses. Five waveforms were obtained, separately for auditory responses without repetition (BL), as well as responses after AI or HI in either repeated (AI repeated or HI repeated) or novel (AI novel or AI repeated) conditions. A temporally averaged measure was obtained in a 25-ms time window centered around the peak latency for each waveform. The simulation of repetition effects was calculated separately for the repeated and novel conditions in AI and HI, and it was quantified by the percentage change = (task-condition – BL)/BL. The six free parameters of imagery modulation and attentional modulation were fitted by minimizing the distance between the four simulated percentage changes and the empirical MEG results.After fitting the MEG results, we fixed the free parameters of modulation gain. We further derived specific predictions concerning how the top-down processes could modulate behavior. A behavioral experiment of syllable identification was conducted to test the model prediction. In this experiment, participants were asked to identify syllables in the ambiguous auditory stimuli of /ba/-/da/ continuum, with or without performing a preceding imagined speaking or imagined hearing task . The perceptual response changes as a function of imagery types were quantified and compared to the model prediction, hence further testing the mechanisms of differential gain modulation for different types of imagery tasks.To link the simulated neural output with behavioral responses, a decision rule was applied as in N, was assumed to reduce the performance. Another parameter of decision bias toward the choice, That is, the percentage of choice, N, and bias, The two free parameters, decision noise, N, and decision bias, The neural output waveform was obtained by running the simulation using the fixed parameters without the modulation gains in the neural network model. The peak latencies of output from the phonological layer /ba/ and /da/ neurons were identified for each level of input. The two free parameters, decision noise, N, and decision bias, After fixing the free parameters in the decision rule, we generated the prediction of choice shifts to the /ba/-/da/ continuum after different types of imagery. The neural output waveform to different levels of the /ba/-/da/ continuum after either the AI or HI tasks was obtained by running the input of corresponding ambiguous levels through the neural network with fixed modulation gains for a given imagery task. The percentage of choice for each level in each task was obtained by the identified peak latencies using Twenty-two participants were recruited from East China Normal University, and three of them were excluded. Two participants were excluded because of abnormal perceptual boundaries in the pretest (no differences among responses to different levels of /ba/-da/ continuum stimuli), and the third participant was excluded because this participant could not follow instructions and confused the AI task with the HI task (self-reporting of having difficulty separating two types of speech imagery). In total, nineteen participants were included in this study . All participants were right-handed and received monetary incentives for their participation. This experiment was approved by the local institutional review board at New York University Shanghai. Written consent was obtained from each participant.Pictures of a mouth and an ear were used as visual cues to indicate two different imagery conditions. Each image was presented foveally against a white background. The mouth picture indicated the AI condition and the ear the HI condition. One of two syllables, /ba/ and /da/, was placed at the center of the pictures to indicate the content of the imagery task in each condition.A seven-level /ba/-/da/ continuum, synthesized using Praat software , was useThree conditions were included in this experiment, AI, HI, and BL conditions. A sample trial is depicted in Each condition was presented in a separate block. The block order was randomized. In each block, 252 trials were included with 36 trials for each of the seven levels of the /ba/-/da/ continuum stimuli. In the imagery conditions (AI and HI), participants were required to generate a mental imagery of /ba/ in half of the trials, and /da/ in another half. The presentation order of imagery content and auditory stimuli were randomized.In the AI condition, participants were told to imagine saying /ba/ or /da/ without moving any articulators or producing any sounds. Instead, they were asked to generate the kinesthetic feeling of the articulator movement for a specific pronunciation and to generate the experience of their own voice loud and clear in their mind. In the HI condition, participants were asked to imagine hearing another person say the /ba/ or /da/ sound to them, while minimizing any kinesthetic feeling of movement. We did not obtain EMG recordings from around the larynx to objectively control for possible subvocalizations. Instead, we placed a microphone close to the participants’ mouth throughout the experiment and did not find any noticeable pronunciation. As long as no overt sound was produced and it was consistent across conditions, the perceptual changes observed in the study were not contaminated by the overt production.To strengthen the memory process in HI, a familiarization phase was conducted only in the HI condition. Participants saw a picture of an average male or female face and listened to the /ba/ and /da/ sounds that were opposite to their gender three times each. Participants were told that the person on the screen produced those sounds. The familiarization phase was repeated after every 42 trials to help participants retain their memories during the HI phase. The opposite gender was used to better distinguish the internal voice in HI from their own voice. These manipulations aimed to selectively elicit the motor-induced auditory representation in AI and auditory memory in HI.post hoc one-way repeated measures ANOVA with factors of task conditions at each sound level. To directly test the main hypothesis on the differential modulation effects of AI and HI, a two-way repeated measures ANOVA with factors of task conditions (two levels: AI and HI) and sound levels (seven levels) was conducted out. Post hoc t tests between imagery conditions were conducted at each level of sound.Both response choice and reaction time (RT) were subject to a two-way repeated measures ANOVA with factors of task conditions (three levels) and sound levels (seven levels), followed by 2 Goodness-of-fit tests were conducted to statistically test the model fittings to the MEG repetition effects, the modeling fitting to the BL perceptual responses, and the similarity between the model predictions and behavioral results of imagery modulation.χhttps://github.com/xtian0628/ImgMod.git. The stimuli, behavioral data, and code are available as The code described in the paper is freely available online at 10.1523/ENEURO.0261-19.2019.ed1Extended Data 1Extended Data 1, ZIP file.Stimuli, behavioral data, and scripts. Download The output from the phonological layer was obtained in different imagery conditions, as shown in 2 goodness of fit test, χ2(3) = 0.024, p > 0.99; the insignificance suggested no differences between the empirical and model results and hence a good model fit; The model simulation results were fitted with the actual MEG results and captured the repetition enhancement and suppression in AI and HI, respectively  = 100.23, p < 0.001). These results indicated that a standard psychometric curve that reflected the perceptual categorization was obtained. Moreover, the model, after adding a decision rule, could fit the psychometric curve (χ2(6) = 1.38, p = 0.97; the insignificance suggested no differences between the empirical and model results and hence a good model fit; N = 0.0193, and decision bias to the choice, cB = 0.0469.To generate predictions about behavioral responses from the neural network, we first examined the behavioral responses to the /ba/-/da/ continuum in the BL condition and fitted the decision rule using these behavioral responses without modulation of imagery. A psychometric curve was obtained for the categorization responses to the /ba/-/da/ continuum A. A one-More importantly, by taking the neural output after imagery modulation, predictions of behavioral responses to the /ba/-/da/ continuum were generated. Specifically, the model predicted that the psychometric curve would shift upwards for both types of speech imagery, but the positive shift would be greater after imagined speaking (AI) than after imagined hearing HI; A.F = 197.18, p < 0.001), imagery task  = 10.65, p = 0.004), and imagery token  = 13.61, p = 0.002). More importantly, the two-way interaction between the imagery token and imagery task was significant  = 5.66, p = 0.029), as was the interaction between imagery task and stimulus-level  = 3.53, p = 0.003). These significant interactions suggest that the content in the preceding imagery task greatly influenced the modulation effects of different types of imagery on perception. Therefore, we separated the imagery of /ba/ and /da/ in further analyses.Next, we tested the predicted imagery modulation effects on perceptual categorization. Because motor involvement can affect the strength of the representation established in speech imagery , the imaF = 9.558, p < 0.001, η2 = 0.347) and sound levels  = 169.963, p < 0.001, η2 = 0.904). More importantly, the interaction was also significant  = 2.610, p = 0.003, η2 = 0.127). To assess the interaction, post hoc one-way repeated measures ANOVA was conducted among the task conditions for each level of auditory stimuli. Significant differences among task conditions were found at sound levels 2  = 4.676, p = 0.016), 3  = 4.808, p = 0.014), 4  = 8.154, p = 0.001), 5  = 6.250, p = 0.005), and 7  = 3.394, p = 0.045).We first tested the modulation effects in the imagery tasks with the imagery content of /ba/. A two-way repeated measures ANOVA was conducted with two factors of task conditions (three levels) and sound levels (seven levels). The results showed significant main effects both for task conditions  = 15.820, p < 0.001, η2 = 0.048), but not for the task conditions  = 0.630, p = 0.538, η2 = 0.014). The interaction between them was significant  = 1.954, p = 0.03, η2 = 0.009). A post hoc test did not reveal any significance among task conditions at any level of sound stimuli, suggesting that the RT for selection of choice in different conditions did not differ.A similar two-way repeated measures ANOVA was conducted for the RT with two factors of task conditions (three levels) and sound levels (seven levels). The results showed that the main effect of sound levels was significant  = 14.13, p = 0.001, η2 = 0.01) and sound levels  = 138.51, p < 0.001, η2 = 0.753). The interaction was also significant  = 3.52, p = 0.003, η2 = 0.007). Post hoc comparisons showed significant differences between the AI and HI condition at sound level 5 (t(19) = 4.236, p = 0.004). That is, participants who imagined speaking the syllable /ba/ had a higher chance of perceiving the following ambiguous stimuli as /ba/ than that when imagining hearing the same syllable. More importantly, the behavioral results of imagery adaptation were consistent with the model prediction  = 2.02, p = 0.92; for HI χ2(6) = 5.92, p = 0.43; To directly test the model prediction on modulation differences between AI and HI B, we conF = 13.04, p < 0.001, η2 = 0.079), but neither the main effect of task conditions  = 2.34, p = 0.144, η2 = 0.019) nor the significant interaction  = 1.24, p = 0.292, η2 = 0.005), which suggested that the observed choice results could not be explained by the speed-accuracy trade-off.A similar two-way repeated measures ANOVA was conducted for RT with two factors of task conditions  and sound levels (seven levels). The results showed a significant main effect of sound levels  = 2.06, p = 0.142, η2 = 0.003). The main effect of sound level was still significant  = 220.126, p < 0.001, η2 = 0.809), and there was a significant interaction between task conditions and sound levels  = 1.924, p = 0.033, η2 = 0.005). However, post hoc tests did not reveal any reliable differences among conditions at any of the seven sound levels. For the RT results, only a main effect of sound levels  = 5.527, p < 0.001, η2 = 0.023) was found. The main effect of task conditions  = 0.519, p = 0.600, η2 = 0.0142) and the interaction  = 0.851, p = 0.598, η2 = 0.004) were not significant.When the content of imagery tasks was the syllable /da/, the results showed that the perception /ba/-/da/ continuum was not different among the AI, HI, and BL conditions C, as the2 goodness-of-fit test revealed significant differences between the model results and the empirical results  = 12.87, p = 0.045; for HI χ2(6) = 16.09, p = 0.013). These results suggested that the fitted parameters for imagery /ba/ could not automatically capture the imagery /da/ results, which was consistent with our hypothesis that the inhibition of different degrees of motor involvement in imagery /ba/ and /da/ influences the precision of induced representation.We first used fixed parameters to fit the empirical results of imagery /da/. The χiSg to scale the modulation gain and ASG to scale the attentional gain. The iSg proportionally scales the gain for both imagined and neighboring syllables [Δig = – iSg * (ig – 1)]. In this way, the tuning of the modulation can be changed. ASG is a multiplier that changes the attentional gain (A’G = ASG*AG). By scaling the modulation gain, the model can fit the behavioral results of imagery /da/  = 4.483, p = 0.612; for HI χ2(6) = 7.034, p = 0.318; AISg = 0.9056 and A,AISG = 1.5328, whereas for HI, HiSg = 0.1063, A,HISG = 1.4344. These results showed that the modulation gain for the imagery /da/ was scaled down from the fitted values of imagery /ba/. The scaling down was greater in the AI task than the HI task. These results were consistent with our hypothesis that imagery /da/ would induce a less precise representation because of the inhibition of greater motor involvement, compared to imagery /ba/.Next, we fitted the empirical results of imagery /da/ by adding two free parameters, A neural network model with a built-in gain modulation from different types of imagery successfully captured the different directions of repetition effects observed in a previous MEG study. The simulation results showed that the AI sharpened the gain more than HI. In the behavioral experiment of categorizing /ba/-/da/ continuum sounds, AI induced a greater choice shift in the preceding imagined syllable than HI. This positive perceptual shift was predicted by the model. Both the simulation and behavioral results were consistent with our hypothesis that motor-based speech imagery can induce a more precise auditory representation than memory-based imagery, which resulted in a differential influence on speech perception.Our results provide evidence suggesting the origins and representational format of prediction. Mental imagery is assumed to be a predictive process by inducing representation without external stimulation . The modMore importantly, our results shed light on how the prediction operates and shapes perception. Previous studies of mental imagery have been mostly focused on the representational question, how similar are the representations induced by imagery compared to perception . This stThe distinct behavioral results in conditions of different imagery content (/ba/ or /da/) further suggest that motor engagement constrains the precision of induced auditory representation. The pronunciation of /ba/ is physically realized by the movement of lips and jaw, whereas the articulation of /da/ involves the movement of tongue in addition to the jaw. The lip movement in /ba/ is arguably more correlated with the jaw movement, compared with the correlation between the jaw movement and the tongue movement in /da/. During imagery, one requirement is for the articulators to refrain from moving. Therefore, more efforts of movement inhibition would be in the imagery of /da/ compared with the imagery of /ba/. This greater inhibition of motor system during imagery of /da/ may induce a less precise representation than that of /ba/, and hence, a less positive shift was observed in the imagery of /da/. Our behavioral and simulation results B,C were The model successfully explained the MEG results of distinct modulation directions caused by two types of imagery, which established the proposed mechanisms in a quantitative way. More importantly, it generated novel predictions that were more informative to test hypotheses than explaining existing data. After fixing all free parameters, we tested this model again by using a procedure that was orthogonal to the MEG experiment. We used new acoustic input to generate prediction at a new level , and we tested this novel prediction using an independent dataset (distinct types of imagery modulate the psychometric curves of perception). In this way, our study provides strong evidence that supports a new computational account on how different types of speech imagery work and how they interact with and shape perception, the crucial aspect of computation in addition to the representation of mental imagery. Moreover, our study provides a mechanistic connection between neural-level and psychophysics, contrasting with previous studies on mental imagery.The model trained on the neural data could predict the shifts in the psychometric curve after imagery /ba/. However, the fitted parameters could not reproduce the behavioral results in conditions of imagery /da/, suggesting that fitting the modulation in neural responses does not automatically lead to the results of the behavioral modulation. After adding a scaling parameter that adjusted the modulation gain, the model could fit the behavioral results of imagery /da/. The fitting results for imagery /da/ revealed that the modulation gain was scaled down compared with that under conditions of imagery /ba/. The scaling down was more prominent for the AI task compared with the HI task. These results suggest that in the AI task where motor-to-sensory transformation is required, the inhibition of motor involvement in the imagery task limits the precision of the induced representation. In contrast, in the HI task, which relies more on memory retrieval, the differences in motor involvement between syllables did not influence the precision of induced representation.Attention could be a factor that mediates the observed perceptual shift differences between imagery tasks. However, two reasons make the attention less likely to be a confounding variable in our study. First, we informed participants that the auditory stimuli were randomly presented. There was no relation between what was imagined and the subsequent sound. They were asked to judge the sound solely based on what they heard. In contrast, a sound always followed the imagery task. Therefore, the potential attentional effect was more likely to be a temporal one, facilitating the initiation and processing of all sounds. We modeled the attention effect as a general modulation gain to all nodes. Second, an equal amount of attention would be induced in both imagined contents of /ba/ and /da/. However, we observed differences between the behavioral results when participants imagined different syllables, which highlights that motor involvement constrains the precision of the internally generated representation. Therefore, the observed results cannot be easily explained by different attentional levels between imagery tasks.One of the difficulties in imagery studies is the lack of objective measures, especially in the auditory domain. In our previous studies, we attempted various designs, including a chronometric procedure  and repeOur model simulation and behavioral results demonstrate that different types of mental imagery can distinctively modulate the neural and behavioral perceptual responses. This converging evidence suggests that mental and neural representation can be predicted and constructed internally without external stimulation via motor-based and memory-based pathways. The precision of internally constructed representation is determined by the nature of induction processes, a more precise representation can be induced by the motor-based process compared with the memory-based process. The differential precision levels of internally constructed representations can distinctively tune the sensory gain and shape perception.