Integrated analysis of differently expressed microRNAs and mRNAs at different postnatal stages reveals intramuscular fat deposition regulation in goats (Capra hircus).

Intramuscular fat refers to the adipose tissue distributed in the muscle. It is an important indicator that affects the quality of goat meat, and can directly affect the tenderness and flavor of goat meat. Our previous study revealed the mRNA that may be crucial for intramuscular fat deposition during goat growth; however, how the microRNAs (miRNAs) are involved in the process is largely unclear. In the present study, a total of 401 known miRNAs and 120 goat novel miRNAs, including 110 differentially expressed (DE) miRNAs, were identified among longissimus dorsi from three growth stages (2, 9, and 24 months) by miRNA sequencing. Combining analysis of the DE mRNAs and DE miRNAs was then performed by miRDB and miRwalk, and miR-145-5p and FOXO1, miR-487b-3p, and PPARG coactivator 1 α (PPARGC1A), miR-345-3p, and solute carrier family 2 member 4 (SLC2A4), etc. were shown to closely associate with lipid metabolism, which was then validated by a correlation analysis. The final DE mRNAs were significantly enriched in fatty acid transmembrane transport, fatty acid homeostasis, apelin signaling pathway, glucagon signaling pathway, insulin signaling pathway, and AMPK signaling pathway by gene ontology and Kyoto Encyclopedia of Genes and Genomes analysis. Besides, miR-145-5p showed a certain effect on goat intramuscular fat metabolism by acting on the possible target gene Forkhead Box O1 (FOXO1). These data provide some theoretical support for improving the quality of goat meat.

Multikink quantile regression for longitudinal data with application to progesterone data analysis.

Motivated by investigating the relationship between progesterone and the days in a menstrual cycle in a longitudinal study, we propose a multikink quantile regression model for longitudinal data analysis. It relaxes the linearity condition and assumes different regression forms in different regions of the domain of the threshold covariate. In this paper, we first propose a multikink quantile regression for longitudinal data. Two estimation procedures are proposed to estimate the regression coefficients and the kink points locations: one is a computationally efficient profile estimator under the working independence framework while the other one considers the within-subject correlations by using the unbiased generalized estimation equation approach. The selection consistency of the number of kink points and the asymptotic normality of two proposed estimators are established. Second, we construct a rank score test based on partial subgradients for the existence of the kink effect in longitudinal studies. Both the null distribution and the local alternative distribution of the test statistic have been derived. Simulation studies show that the proposed methods have excellent finite sample performance. In the application to the longitudinal progesterone data, we identify two kink points in the progesterone curves over different quantiles and observe that the progesterone level remains stable before the day of ovulation, then increases quickly in 5 to 6 days after ovulation and then changes to stable again or drops slightly.

Effects of Surgical Timing of Facial Nerve Decompression for Patients With Severe Bell's Palsy.

Context: Bell's palsy is a form of idiopathic, facial nerve palsy. Initial treatment includes the use of oral corticosteroids and/or antiviral agents, but facial paralysis may persist. Some surgeons suggest that surgical decompression of the facial nerve can be a beneficial, but the optimal surgical approach, extent of nerve decompression, and timing of surgery remain unclear. Objective: This study intended to evaluate the efficacy of delayed, facial nerve decompression for severe Bell's palsy (BP) and to explore the relationship of opportunity timing for operations, with postoperative recovery for facial nerve function. Design: The research team performed a retrospective study. Setting: The study took place at Beijing Tiantan Hospital of Capital Medical University in Beijing, China. Participants: Participants were 45 patients who had been diagnosed with BP between 2015 and 2021 and who had undergone facial nerve decompression using the transmastoid approach, between 30 and 180 days after the onset of BP. According to the operation's timing, the research team divided the participants into three groups, consisting of participants who underwent surgery: (1) at 30-60-days after BP onset-19 participants, (2) at 61-90 days after BP onset-18 participants, and (3) at more than 90 days after BP onset-8 participants. Outcome Measures: The research team: (1) analyzed participants' demographic and preoperative and postoperative clinical characteristics, (2) compared the surgical outcomes with participants' House-Brackmann (HB) scales, and (3) analyzed the factors affecting the recovery of facial nerve function using logistic regression. Results: Decompression surgery was effective for 29 participants (64.4%), with similar rates for participants who underwent surgery after 30-60 days (73.7%) and 61-90 days (77.8%), but the surgery' success was significantly higher for those groups than for participants who underwent surgery after >90 days (12.5%), with P = .008 and P = .003, respectively. Multivariate logistic regression analysis showed that disease duration was the only factor significantly associated with the effectiveness of surgery (odds ratio = 120.337; 95% confidence interval 2.997-4832.267, P = .011). Conclusions: For patients with severe Bell's palsy with ineffective conservative treatment, surgery performed 30 to 90 days after the onset of paralysis can have therapeutic benefits, whereas surgery performed after 3 months is relatively ineffective.

Expression Variation of CPT1A Induces Lipid Reconstruction in Goat Intramuscular Precursor Adipocytes.

Intramuscular fat (IMF) deposition is one of the most important factors affecting meat quality and is closely associated with the expression of carnitine palmitoyl transferase 1A (CPT1A) which facilitates the transfer of long-chain fatty acids (LCFAs) into the mitochondria. However, the role of how CPT1A regulates the IMF formation remains unclear. Herein, we established the temporal expression profile of CPT1A during the differentiation of goat intramuscular precursor adipocytes. Functionally, the knockdown of CPT1A by siRNA treatment significantly increased the mRNA expression of adipogenic genes and promoted lipid deposition in goat intramuscular precursor adipocytes. Meanwhile, a CPT1A deficiency inhibited cell proliferation and promoted cell apoptosis significantly. CPT1A was then supported by the overexpression of CPT1A which significantly suppressed the cellular triglyceride deposition and promoted cell proliferation although the cell apoptosis also was increased. For RNA sequencing, a total of 167 differential expression genes (DEGs), including 125 upregulated DEGs and 42 downregulated DEGs, were observed after the RNA silencing of CPT1A compared to the control, and were predicted to enrich in the focal adhesion pathway, cell cycle, apoptosis and the MAPK signaling pathway by KEGG analysis. Specifically, blocking the MAPK signaling pathway by a specific inhibitor (PD169316) rescued the promotion of cell proliferation in CPT1A overexpression adipocytes. In conclusion, the expression variation of CPT1A may reconstruct the lipid distribution between cellular triglyceride deposition and cell proliferation in goat intramuscular precursor adipocyte. Furthermore, we demonstrate that CPT1A promotes the proliferation of goat adipocytes through the MAPK signaling pathway. This work widened the genetic regulator networks of IMF formation and delivered theoretical support for improving meat quality from the aspect of IMF deposition.

Dissecting the Protein Dynamics Coupled Ligand Binding with Kinetic Models and Single-Molecule FRET.

Protein-ligand interactions are crucial to many biological processes. Ligand binding and dissociation are the basic steps that allow proteins to function. Protein conformational dynamics have been shown to play important roles in ligand binding and dissociation. However, it is challenging to determine the ligand binding kinetics of dynamic proteins. Here, we undertook comprehensive single-molecule FRET (smFRET) measurements and kinetic model analysis to characterize the conformational dynamics coupled ligand binding of glutamine-binding protein (GlnBP). We showed that hinge and T118A mutations of GlnBP affect its conformational dynamics as well as the ligand binding affinity. Based on smFRET measurements, the kinetic model of ligand-GlnBP interactions was constructed. Using experimentally measured parameters, we solved the rate equations of the model and obtained the undetectable parameters of the model which allowed us to understand the ligand binding kinetics fully. Our results demonstrate that modulation of the conformational dynamics of GlnBP affects the ligand binding and dissociation rates. This study provides insights into the binding kinetics of ligands, which are related to the protein function itself.

Electrophoresis of Phosphoproteins on a Tandem Polymerized Gel.

A substrate with n phosphorylated sites may have 2n phosphor-forms for temporal-spatial regulation of biological events. Because phosphates do not significantly change molecular masses but net charges of proteins, those isoforms cannot be separated by regular mass-based sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE). A tandem polymerized gel was developed to resolve phosphor-isoforms with different masses, charges, and posttranslational modifications. Without the usage of SDS, the electrophoresis was primarily performed on three adjacent acidic polyacrylamide gels. After being concentrated on a stacking gel, protonated proteins were then separated on the Zr4+ immobilized gel through the coordination of metal ions with phosphates followed by further charge and mass (z/m)-based electrophoretic separation on a TiO2 containing gel. The presence of TiO2 nanoparticles in the third gel is aimed for the initiation of the polymerization of acrylamide in acidic conditions upon ultraviolet irradiation. Distinct isoforms of α-S1-casein, α-S2-casein, ß-casein, and κ casein model proteins located on 11, 8, 8, and 7 different bands of the tandem gel were unambiguously identified, respectively. With the tandem polymerized gel electrophoresis, new phosphorylation events that may occur simultaneously or sequentially were discovered in not only model proteins but also complex biological samples including human saliva, chicken egg, and sprouting maize. This provides a new tool to dissect complex biological processes that are triggered by dynamic phosphorylation events.

Identification of Nonlinear Soil Properties from Downhole Array Data Using a Bayesian Model Updating Approach.

An accurate seismic response simulation of civil structures requires accounting for the nonlinear soil response behavior. This, in turn, requires understanding the nonlinear material behavior of in situ soils under earthquake excitations. System identification methods applied to data recorded during earthquakes provide an opportunity to identify the nonlinear material properties of in situ soils. In this study, we use a Bayesian inference framework for nonlinear model updating to estimate the nonlinear soil properties from recorded downhole array data. For this purpose, a one-dimensional finite element model of the geotechnical site with nonlinear soil material constitutive model is updated to estimate the parameters of the soil model as well as the input excitations, including incident, bedrock, or within motions. The seismic inversion method is first verified by using several synthetic case studies. It is then validated by using measurements from a centrifuge test and with data recorded at the Lotung experimental site in Taiwan. The site inversion method is then applied to the Benicia-Martinez geotechnical array in California, using the seismic data recorded during the 2014 South Napa earthquake. The results show the promising application of the proposed seismic inversion approach using Bayesian model updating to identify the nonlinear material parameters of in situ soil by using recorded downhole array data.

Nb2CTx MXene as High-Performance Energy Storage Material with Na, K, and Liquid K-Na Alloy Anodes.

Two-dimensional MXenes perform well as hosts in batteries, which are promising for next-generation energy storage materials. With low price and high performance, sodium (Na) and potassium (K) own the potential to replace lithium in energy storage devices, but the larger radii and dendrite growth restrict their commercialization. Herein, we successfully synthesized an accordion-like Nb2CTx MXene, whose crystal structure integrity and lamellar separation have been confirmed by characterization methods like high-resolution transmission electron microscopy (HR-TEM). Combined with solid Na and K and liquid K-Na alloy as anodes, the Nb2CTx MXene shows excellent electrochemical performance, such as high capacity retention after large current shock in tests of rate performance and long time stability for more than 500 cycles, etc. Also, the Nb2CTx MXene coupled with liquid K-Na anode performs better than that coupled with solid K for the dendrite-controlling character of the liquid electrode. The Nb2CTx MXene would boost the exploitation of more suitable host materials for Na/K-ion batteries and promote an in-depth understanding of MXenes.

Raman spectroscopic techniques for detecting structure and quality of frozen foods: principles and applications.

Frozen foods are among the most popular food products and the development of techniques for the rapid detection of their structures and quality is important for the frozen food industry. In the current review, the principles and applications of Raman spectroscopic techniques in detecting and evaluating the structure changes and quality of frozen foods are discussed and merits and drawbacks of each Raman type are presented. These techniques include dispersive, Fourier transform, spatially offset, micro Raman spectroscopies. Raman spectroscopy can assess the compositional, physicochemical, and sensory characteristics of the frozen foods without any pretreatment. It can also be applied to investigate the structural alterations of protein, lipid, water and other components. Further trends of developing the Raman spectroscopy in monitoring the frozen foods should focus on reducing overlapping signals, achieving real noninvasive detection, eliminating misclassification and quantification errors, establishing standardized Raman database, and controlling cost.

Subgroup identification via homogeneity pursuit for dense longitudinal/spatial data.

In the clinical trial community, it is usually not easy to find a treatment that benefits all patients since the reaction to treatment may differ substantially across different patient subgroups. The heterogeneity of treatment effect plays an essential role in personalized medicine. To facilitate the development of tailored therapies and improve the treatment efficacy, it is important to identify subgroups that exhibit different treatment effects. We consider a very general framework for subgroup identification via the homogeneity pursuit methods usually employed in econometric time series analysis. The change point detection algorithm in our procedure is most suitable for analyzing dense longitudinal or spatial data which are quite common for biomedical studies these days. We demonstrate that our proposed method is fast and accurate through extensive numerical studies. In particular, our method is illustrated by analyzing a diffusion tensor imaging data set.

The transotic approach for vestibular schwannoma: indications and results.

To analyze retrospectively the indications and the results obtained with the transotic approach in a series of patients with vestibular schwannoma. The study included 36 patients from 2007 to 2013 with a vestibular schwannoma that was removed with a transotic approach. All patients underwent preoperative pure tone audiometry, evaluation of facial function and CT and MR imaging. All patients having (1) a hearing loss of more than 50 dB, (2) an average tumor size of 2.7 cm (range 0.5-5.0 cm) and (3) signs of a contracted mastoid (high jugular bulb, anteriorly located sigmoid sinus, low middle cranial fossa, or reduced pneumatization) were selected. The tumor was totally removed in 34 and near-totally removed in 2 patients. The facial nerve was preserved in all patients. The postoperative facial function after 6 weeks was House-Brackmann grade I in 7, grade II in 27, and grade III in 2 patients. All patients presented postoperatively with unilateral total deafness. Seven patients experienced transitory postoperative imbalance. There were two patients who required revision surgery, one with intracranial hemorrhage and another with a CSF leak. There were no deaths and no severe complications such as hemiplegia or intracranial infections. The transotic approach has proven to be of value for the removal of vestibular schwannomas up to 5.0 cm in the presence of temporal bone contraction. Hearing was not preserved; however, other clinical outcomes were very favorable, including high rates of total tumor removal and facial nerve preservation, and low rates of complications.

Laser Activated Electron Tunneling Based Mass Spectrometric Imaging of Molecular Architectures of Mouse Brain Revealing Regional Specific Lipids.

A comprehensive description of overall brain architecture at the molecular level is essential for understanding behavioral and cognitive processes in health and diseases. Although fluorescent labeling of target proteins has been successfully established to visualize a brain connectome, the molecular basis for diverse neurophysiological phenomena remains largely unknown. Here we report a brain-wide, molecular-level, and microscale imaging of endogenous metabolites, in particular, lipids of mouse brain by using laser activated electron tunneling (LAET) and mass spectrometry. In this approach, atomic electron emission along with finely tuned laser beam size provides high resolution that can be down to the sub-micrometer level to display spatial distribution of lipids in mouse brain slices. Electron-directed soft ionization has been achieved through exothermal capture of tunneling photoelectrons as well as unpaired electron-initiated chemical bond cleavages. Regionally specific lipids including saturated, mono-unsaturated, and poly-unsaturated fatty acids as well as other lipids, which may be implicated in neurological signaling pathways, have been discovered by using this laser activated electron tunneling based mass spectrometric imaging (LAET-MSI) technique.

Semi-varying coefficient multinomial logistic regression for disease progression risk prediction.

This paper proposes a risk prediction model using semi-varying coefficient multinomial logistic regression. We use a penalized local likelihood method to do the model selection and estimate both functional and constant coefficients in the selected model. The model can be used to improve predictive modelling when non-linear interactions between predictors are present. We conduct a simulation study to assess our method's performance, and the results show that the model selection procedure works well with small average numbers of wrong-selection or missing-selection. We illustrate the use of our method by applying it to classify the patients with early rheumatoid arthritis at baseline into different risk groups in future disease progression. We use a leave-one-out cross-validation method to assess its correct prediction rate and propose a recalibration framework to evaluate how reliable are the predicted risks. Copyright © 2016 John Wiley & Sons, Ltd.

Low-dimensional confounder adjustment and high-dimensional penalized estimation for survival analysis.

High-throughput profiling is now common in biomedical research. In this paper we consider the layout of an etiology study composed of a failure time response, and gene expression measurements. In current practice, a widely adopted approach is to select genes according to a preliminary marginal screening and a follow-up penalized regression for model building. Confounders, including for example clinical risk factors and environmental exposures, usually exist and need to be properly accounted for. We propose covariate-adjusted screening and variable selection procedures under the accelerated failure time model. While penalizing the high-dimensional coefficients to achieve parsimonious model forms, our procedure also properly adjust the low-dimensional confounder effects to achieve more accurate estimation of regression coefficients. We establish the asymptotic properties of our proposed methods and carry out simulation studies to assess the finite sample performance. Our methods are illustrated with a real gene expression data analysis where proper adjustment of confounders produces more meaningful results.

Chemical imaging of latent fingerprints by mass spectrometry based on laser activated electron tunneling.

Identification of endogenous and exogenous chemicals contained in latent fingerprints is important for forensic science in order to acquire evidence of criminal identities and contacts with specific chemicals. Mass spectrometry has emerged as a powerful technique for such applications without any derivatization or fluorescent tags. Among these techniques, MALDI (Matrix Assisted Laser Desorption Ionization) provides small beam size but has interferences with MALDI matrix materials, which cause ion suppressions as well as limited spatial resolution resulting from uneven distribution of MALDI matrix crystals with different sizes. LAET (Laser Activated Electron Tunneling) described in this work offers capabilities for chemical imaging through electron-directed soft ionization. A special film of semiconductors has been designed for collection of fingerprints. Nanoparticles of bismuth cobalt zinc oxide were compressed on a conductive metal substrate (Al or Cu sticky tape) under 10 MPa pressure. Resultant uniform thin films provide tight and shining surfaces on which fingers are impressed. Irradiation of ultraviolet laser pulses (355 nm) on the thin film instantly generates photoelectrons that can be captured by adsorbed organic molecules and subsequently cause electron-directed ionization and fragmentation. Imaging of latent fingerprints is achieved by visualization of the spatial distribution of these molecular ions and structural information-rich fragment ions. Atomic electron emission together with finely tuned laser beam size improve spatial resolution. With the LAET technique, imaging analysis not only can identify physical shapes but also reveal endogenous metabolites present in females and males, detect contacts with prohibited substances, and resolve overlapped latent fingerprints.

Molecular Characterization, Polymorphism, and Association of Porcine GADD45G Gene.

Growth arrest and DNA-damage-inducible gamma (GADD45G) is a reproduction related gene. In this study, the full-length cDNA sequence of porcine GADD45G gene was cloned through rapid amplification of cDNA ends (RACE) method. The porcine GADD45G gene encodes a protein of 159 amino acids that shares high homology with the GADD45G of nine species: chimpanzee (97%), sumatran orangutan (97%), white-tufted-ear marmoset (97%), northern white-cheeked gibbon (97%), cattle (97%), human (97%), rhesus monkey (97%), rat (96%), and mouse (95%). This novel porcine gene was assigned to GeneID: 100152997. Phylogenetic analysis revealed that the porcine GADD45G gene has a closer genetic relationship with the GADD45G gene of cattle. Computer-assisted analysis indicated that porcine GADD45G gene is structured in four exons and three introns. PCR-Rsa I-RFLP was established to detect an A/G mutation on the position of 294-bp of coding sequence and eight pig breeds display obvious genotype and allele frequency differences at this mutation locus. Association of this SNP with litter size traits was assessed in Large White (n = 100) and Landrace (n = 100) pig populations, and result demonstrated that this polymorphic locus was significantly associated with the litter size of all parities in Large White and Landrace sows (P < 0.01). Therefore, porcine GADD45G gene could be a useful candidate gene in selection for increasing the litter size. These data serve as a foundation for further insight into this novel porcine gene.

A smoothing dynamic model for irregularly time-spaced longitudinal data.

In nondesigned longitudinal observational studies, irregularly spaced measurements are commonly present over a period of follow-up time. We propose a smoothing dynamic model, based on the idea of varying coefficients, to analyze this highly unbalanced longitudinal data. The estimate of model parameters can be obtained by implementing a well-developed B-splines technique. Our method is illustrated with data from a primary care based longitudinal cohort of rheumatoid arthritis patients. The results show that the effects of some risk factors might be underestimated by an intention-to-treat analysis using a last-value-carried-forward method.

[Effect analysis of facial nerve decompression surgery in the treatment of Bell's palsy and Hunt syndrome].

Objective:To summarize and analyze the effect of facial nerve decompression surgery for the treatment of Bell's palsy and Hunt syndrome. Methods:The clinical data of 65 patients with facial nerve palsy who underwent facial nerve decompression in our center from October 2015 to October 2022 were retrospectively analyzed, including 54 patients with Bell's palsy and 11 patients with Hunter syndrome. The degree of facial paralysis（HB grade） was evaluated before surgery, and ENoG, pure tone audiometry, temporal bone CT and other examinations were completed. All patients had facial palsy with HB grade V or above after conservative treatment for at least 1 month, and ENoG decreased by more than 90%. All patients underwent facial nerve decompression surgery through the transmastoid approach within 3 months after onset of symptoms. The recovery effect of facial nerve function after surgery in patients with Bell's palsy and Hunter syndrome was summarized and analyzed. In addition, 15 cases in group A（operated within 30-60 days after onset） and 50 cases in group B（operated within 61-90 days after onset） were grouped according to the course of the disease（the interval between onset of symptoms and surgery） to explore the effect of surgical timing on postoperative effect. Results:There was no significant difference between the two groups of patients with Chi-square test（P=0.54） in 42 patients（77.8%, 42/54） with Bell's palsy and 7 patients（63.6%, 7/11） in patients with Hunter syndrome who recovered to grade Ⅰ-Ⅱ. According to the course of the disease, 10 cases（66.7%, 10/15） in group A recovered to grade Ⅰ-Ⅱ after surgery. In group B, 39 patients（78.0%, 39/50） recovered to grade Ⅰ-Ⅱ after surgery, and there was no statistically significant difference between the two groups by Chi-square test（P=0.58）. Conclusion:Patients with Bell's palsy and Hunter syndrome can achieve good results after facial nerve decompression within 3 months of onset, and there is no significant difference in the surgical effect between the two types of patients.

Prevalence and influencing factors of chronic diseases among the elderly in Southwest China: A cross-sectional study based on community in urban and rural areas.

Objective: To explore the influencing factors affecting chronic diseases of elderly in Kunming. Methods: Data were collected from November 2020 to August 2021.The crosssectional study based on community was adopted. And hierarchical random sampling was used. A face to face questionnaire survey was conducted among the respondents or family caregivers. The contents we collected mainly include general demographic characteristics and other related influencing factors, self-reported chronic diseases and disability status. Results: 1161 elderly were investigated in total. The percentage of non-communicable chronic disease among the rural elderly was higher than that of urban elderly. Binary logistic regression analysis showed that in urban areas, female (OR: 0.592;95 %CI:0.396 âˆ¼ 0.885), not in marriage (OR:1.643;95 %CI:1.093 âˆ¼ 2.470)and not very satisfied with family support (OR:1.858;95 %CI:1.115 âˆ¼ 3.096) are the influencing factors of chronic disease, while in rural areas are not in marriage (OR:1.961;95 %CI:1.021 âˆ¼ 3.763), more health-promoting behavior (OR:0.582;95%CI:0.350 âˆ¼ 0.970), not very satisfied with family support (OR:1.858;95 %CI:1.115 âˆ¼ 3.096), age 70-79 (OR:1.805;95 %CI:1.705 âˆ¼ 3.031), age 80 and above (OR:2.081;95 %CI:1.010 âˆ¼ 4.288), empty nest family (OR:0.389;95 %CI:0.186 âˆ¼ 0.811)and personal monthly income 2001-3000 (OR:0.353;95CI%:0.180 âˆ¼ 0.693). The influencing factors of urban-rural multimorbidity and non-communicable chronic disease with disability also exist differences at individual, family and social levels. Conclusions: The prevalence rate of non-communicable chronic diseases among the elderly in Yunnan Province is not optimistic. Personal, family and social factors would affect the non-communicable chronic diseases of the elderly and there exist difference in influencing factor of non-communicable chronic disease between urban and rural areas.

By Carrot or by Stick: The Influence of Encouraging and Discouraging Facial Feedback on Implicit Rule Learning.

Implicit learning refers to the process of unconsciously learning complex knowledge through feedback. Previous studies investigated the influences of different types of feedback (e.g., social and non-social feedback) on implicit learning. This study focused on the social information presented in the learning situation and tried to explore the effects of different social feedback on implicit rule learning. We assigned participants randomly into an encouraging facial feedback group (happy expression for correct answer, neutral but not negative expression for incorrect answer) and a discouraging facial feedback group (neutral but not happy expression for correct answer, negative expression for incorrect answer). The implicit learning task included four difficulty levels, and social feedback was presented in the learning phase but not the testing phase in two experiments. The only difference between the two experiments was that the sad face used as negative feedback in Experiment 1 was replaced with an angry face in Experiment 2 to enhance the ecological validity of the discouraging facial feedback group. These two experiments yielded consistent results: the performances in the encouraging facial feedback group were more accurate in both the learning and the testing phases at all difficulty levels. These findings indicated that the influence of encouraging social feedback for a better implicit learning achievement was stable and established a new groundwork for future research on incentive-based education, making it critical to investigate the impact of various forms of encouraging-based education on learning.