A systematic assessment of deep learning methods for drug response prediction: from in vitro to clinical applications.

Drug response prediction is an important problem in personalized cancer therapy. Among various newly developed models, significant improvement in prediction performance has been reported using deep learning methods. However, systematic comparisons of deep learning methods, especially of the transferability from preclinical models to clinical cohorts, are currently lacking. To provide a more rigorous assessment, the performance of six representative deep learning methods for drug response prediction using nine evaluation metrics, including the overall prediction accuracy, predictability of each drug, potential associated factors and transferability to clinical cohorts, in multiple application scenarios was benchmarked. Most methods show promising prediction within cell line datasets, and TGSA, with its lower time cost and better performance, is recommended. Although the performance metrics decrease when applying models trained on cell lines to patients, a certain amount of power to distinguish clinical response on some drugs can be maintained using CRDNN and TGSA. With these assessments, we provide a guidance for researchers to choose appropriate methods, as well as insights into future directions for the development of more effective methods in clinical scenarios.

NAFLDkb: A Knowledge Base and Platform for Drug Development against Nonalcoholic Fatty Liver Disease.

Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease with a broad spectrum of histologic manifestations. The rapidly growing prevalence and the complex pathologic mechanisms of NAFLD pose great challenges for treatment development. Despite tremendous efforts devoted to drug development, there are no FDA-approved medicines yet. Here, we present NAFLDkb, a specialized knowledge base and platform for computer-aided drug design against NAFLD. With multiperspective information curated from diverse source materials and public databases, NAFLDkb presents the associations of drug-related entities as individual knowledge graphs. Practical drug discovery tools that facilitate the utilization and expansion of NAFLDkb have also been implemented in the web interface, including chemical structure search, drug-likeness screening, knowledge-based repositioning, and research article annotation. Moreover, case studies of a knowledge graph repositioning model and a generative neural network model are presented herein, where three repositioning drug candidates and 137 novel lead-like compounds were newly established as NAFLD pharmacotherapy options reusing data records and machine learning tools in NAFLDkb, suggesting its clinical reliability and great potential in identifying novel drug-disease associations of NAFLD and generating new insights to accelerate NAFLD drug development. NAFLDkb is freely accessible at https://www.biosino.org/nafldkb and will be updated periodically with the latest findings.

Revealing modifier variations characterizations for elucidating the genetic basis of human phenotypic variations.

Epistatic interactions complicate the identification of variants involved in phenotypic effect. In-depth knowledge in modifiers and in pathogenic variants would benefit the mechanistic studies on the genetic basis of complex traits. We systematically compared the modifier variants which have evidence of modifier effect with the pathogenic variants from multiple angles. Our study found that genomic loci of modifier variations differ from pathogenic loci in many aspects, such as population genetics statistics, epigenetic features, evolutionary characteristics and functional properties of the variations. Genes containing modifier variation(s) exhibit higher probability of being haploinsufficient and higher probability of recessive disease causation, and they are relatively more important in network communication. Furthermore, we reinforced that co-expression analysis is an effective methodology to predict functional associations between modifier genes and their potential target genes. In many aspects, we detected statistically significant differences between modifier variants/genes and pathogenic variants/genes, and investigated relationships between modifiers and their potential targets. Our results offer some actionable insights that may provide appropriate guidelines to clinical genetics and researchers to elucidate the molecular mechanism underlying the human phenotypic variation.

EpiDISH web server: Epigenetic Dissection of Intra-Sample-Heterogeneity with online GUI.

SUMMARY: It is well recognized that cell-type heterogeneity hampers the interpretation of Epigenome-Wide Association Studies (EWAS). Many tools have emerged to address this issue, including several R/Bioconductor packages that infer cell-type composition. Here we present a web application for cell-type deconvolution, which offers the functionality of our EpiDISH Bioconductor/R package in a user-friendly GUI environment. Users can upload their data to infer cell-type composition and differentially methylated cytosines in individual cell-types (DMCTs) for a range of different tissues. AVAILABILITY AND IMPLEMENTATION: EpiDISH web server is implemented with Shiny in R, and is freely available at https://www.biosino.org/EpiDISH/.

mTOR Signaling in X/A-Like Cells Contributes to Lipid Homeostasis in Mice.

Gastric mechanistic target of rapamycin (mTOR) signaling is inversely associated with the expression and secretion of ghrelin, a 28-aa peptide hormone produced by gastric X/A-like cells. Ghrelin contributes to obesity and hepatic steatosis. We sought to control global lipid metabolism via the manipulation of gastric mTOR signaling in X/A-like cells. We established a ghrl-cre transgene in which the Cre enzyme is expressed in X/A-like cells under the control of the ghrelin-promoter. mTORflox/flox and tuberous sclerosis 1 (TSC1)flox/flox mice were separately bred with ghrl-cre mice to generate mTOR-ghrl-cre or TSC1-ghrl-cre mice, within which mTOR signaling was suppressed or activated, respectively. Lipid metabolism in liver and adipose depots was analyzed. Under the control of the ghrelin-promoter, the Cre enzyme was exclusively expressed in stomach X/A-like cells in adult animals. Knockout of mTOR in X/A-like cells increased circulating acyl-ghrelin and promoted hepatic lipogenesis with effects on adipose depots. Activation of mTOR signaling by deletion of its upstream inhibitor, TSC1, decreased ghrelin expression and secretion, altering lipid metabolism as evidenced by resistance to high-fat diet-induced obesity and hepatic steatosis. Both ghrelin administration and injection of rapamycin, an inhibitor of mTOR, altered the phenotypes of TSC1-ghrl-cre mice. Conclusion: Gastric mTOR signaling in X/A-like cells contributes to organism lipid homeostasis by regulating hepatic and adipose lipid metabolism. Gastric mTOR signaling may provide an alternative strategy for intervention in lipid disorders.

Comparative study on skin temperature response to menstruation at acupuncture points in healthy volunteers and primary dysmenorrhea patients.

OBJECTIVE: To assess skin temperature response to menstruation at acupuncture points in primary dysmenorrhea (PD) patients and healthy volunteers so as to explore acupuncture point specificity in reflecting diseases in the light of skin temperature. METHODS: Fifty-two PD patients and 49 healthy volunteers were recruited. Skin temperature measurements were performed with a skin temperature assessment device at 10 points. Absolute difference between skin temperature of the same point on the left and right side is used as main outcome measure. RESULTS: On the first day of menstruation, when menstrual pain attacking in PD patients, a significant increase in skin temperature difference was detected at Taixi (KI 3) compared with the healthy group (P < 0.01). A significant reduction in skin temperature difference was detected at Taixi (KI 3) in the first day of menstruation compared with those values in the third day after menstruation (P < 0.01) in the healthy group. On the third day after menstruation, a significant reduction in skin temperature difference was found at Zhongdu (LR 6) in PD group compared with the healthy group (P < 0.05). No significant differences of skin temperature were detected at other points (P > 0.05). CONCLUSION: The skin temperature difference at menstruation-relevant points in PD patients did not all change significantly more than those in women without PD. Significant difference was only found in Taixi (KI 3), the Yuan-source point of Kidney meridian.

Immediate analgesic effect of needling acupoints (bilateral De Qi vs unilateral De Qi) on primary dysmenorrhea: a multi-center, randomized, controlled trail.

OBJECTIVE: To analyze the effect of needling acupoints (bilateral vs unilateral) with De Qi using data collected from 501 primary dysmenorrhea (PD) patients participating in multi-center, randomized, controlled trail. METHODS: De Qi was defined as at least one of the feelings in soreness, numbness, fullness or heaviness at the acupoints when stimulated with needles. The 501 patients were grouped in 3 groups in terms of De Qi or not De Qi in one side (unilateral) or both sides (bilateral) of the body: bilateral De Qi group, unilateral De Qi group, and non-De Qi group. The abdominal pains were measured using visual analog scale (VAS). RESULTS: In 501 patients, 472 acquired De Qi at unilateral acupoints, 24 De Qi at bilateral acupoint, and 5 had no De Qi at any acupoint. The data of non-De Qi group was excluded as the sample was less than 5% of that in the bilateral De Qi group. There was significant difference in the VAS before and after treatment between unilateral and bilateral De Qi group (P < 0.01). After stratified by acupoints, for the patients needled at Sanyinjiao (SP 6) and Xuanzhong (GB 39), VAS scores in the bilateral De Qi group were larger than those in the unilateral De Qi group (P < 0.05). CONCLUSION: Bilateral De Qi was possibly superior to unilateral De Qi in enhancing the immediate analgesic effect of needling the acupoints, but no statistical significance was observed on the patients of needling at non acupoint, which preliminarily suggested this immediate analgesic effect was perhaps along meridians.

Construction and Deciphering of Human Phosphorylation-Mediated Signaling Transduction Networks.

Protein phosphorylation is the most abundant reversible covalent modification. Human protein kinases participate in almost all biological pathways, and approximately half of the kinases are associated with disease. PhoSigNet was designed to store and display human phosphorylation-mediated signal transduction networks, with additional information related to cancer. It contains 11 976 experimentally validated directed edges and 216 871 phosphorylation sites. Moreover, 3491 differentially expressed proteins in human cancer from dbDEPC, 18 907 human cancer variation sites from CanProVar, and 388 hyperphosphorylation sites from PhosphoSitePlus were collected as annotation information. Compared with other phosphorylation-related databases, PhoSigNet not only takes the kinase-substrate regulatory relationship pairs into account, but also extends regulatory relationships up- and downstream (e.g., from ligand to receptor, from G protein to kinase, and from transcription factor to targets). Furthermore, PhoSigNet allows the user to investigate the impact of phosphorylation modifications on cancer. By using one set of in-house time series phosphoproteomics data, the reconstruction of a conditional and dynamic phosphorylation-mediated signaling network was exemplified. We expect PhoSigNet to be a useful database and analysis platform benefiting both proteomics and cancer studies.

Applications of pain-related evoked potentials and short-latency somatosensory evoked potentials in acupuncture research: a narrative review.

OBJECTIVE: To review and discuss the Chinese and English literature on the use of pain-related evoked potentials (PREP) and short-latency somatosensory EP (SLSEP) in acupuncture research. METHODS: China National Knowledge Infrastructure Database and MEDLINE were searched for the following key words: acupuncture and PREP or SLSEP. RESULTS: Thirty-seven articles were included in the review. Researchers usually use PREPs to study the analgesic effect of acupuncture, observe influential factors, or for mechanistic exploration. In the SLSEP studies, researchers focused on response characteristics of acupuncture, acupoint specificity, and influential factors of the treatment. There were some problems with the study design and conclusions. CONCLUSION: Researchers could use PREP and SLSEP to objectively validate the effects of acupuncture and explore its mechanisms using nerve electrophysiology. Further studies can benefit from observing more acupoints' effects using PREPs or SLSEPs and investigating the placebo effect of acupuncture.

A tripeptide (NSK) inhibits Japanese encephalitis virus infection in vitro and in vivo.

Japanese encephalitis virus (JEV) is a major pathogen that can cause acute viral encephalitis in both humans and animals. Domain III of the viral envelope protein (EDIII) is involved in binding to host cell receptor(s) to facilitate virus entry. Our previous study showed that the loop3 peptide of EDIII possesses antiviral activity against JEV infection. In this paper, we demonstrate that three residues (NSK) in loop3 are responsible for the antiviral activity of loop3 peptide. In vitro experiments showed that the tripeptide NSK could inhibit JEV infection in both BHK-21 and Neuro-2A cells by inhibiting attachment of JEV to the cells, with IC50 values of 8 µM and 6.5 µM, respectively. In vivo experiments showed that the tripeptide could increase the survival of mice challenged with JEV to 75 % when administrated intracerebrally. Therefore, this tripeptide may serve as the basis for the development of novel antiviral agents against Japanese encephalitis virus infection.

A randomized controlled trial of single point acupuncture in primary dysmenorrhea.

BACKGROUND: Acupuncture is often used for primary dysmenorrhea. But there is no convincing evidence due to low methodological quality. We aim to assess immediate effect of acupuncture at specific acupoint compared with unrelated acupoint and nonacupoint on primary dysmenorrhea. METHODS: The Acupuncture Analgesia Effect in Primary Dysmenorrhoea-II is a multicenter controlled trial conducted in six large hospitals of China. Patients who met inclusion criteria were randomly assigned to classic acupoint (N = 167), unrelated acupoint (N = 167), or non-acupoint (N = 167) group on a 1:1:1 basis. They received three sessions with electro-acupuncture at a classic acupoint (Sanyinjiao, SP6), or an unrelated acupoint (Xuanzhong, GB39), or nonacupoint location, respectively. The primary outcome was subjective pain as measured by a 100-mm visual analog scale (VAS). Measurements were obtained at 0, 5, 10, 30, and 60 minutes following the first intervention. In addition, patients scored changes of general complaints using Cox retrospective symptom scales (RSS-Cox) and 7-point verbal rating scale (VRS) during three menstrual cycles. Secondary outcomes included VAS score for average pain, pain total time, additional in-bed time, and proportion of participants using analgesics during three menstrual cycles. FINDINGS: Five hundred and one people underwent random assignment. The primary comparison of VAS scores following the first intervention demonstrated that classic acupoint group was more effective both than unrelated acupoint (-4.0 mm, 95% CI -7.1 to -0.9, P = 0.010) and nonacupoint (-4.0 mm, 95% CI -7.0 to -0.9, P = 0.012) groups. However, no significant differences were detected among the three acupuncture groups for RSS-Cox or VRS outcomes. The per-protocol analysis showed similar pattern. No serious adverse events were noted. CONCLUSION: Specific acupoint acupuncture produced a statistically, but not clinically, significant effect compared with unrelated acupoint and nonacupoint acupuncture in primary dysmenorrhea patients. Future studies should focus on effects of multiple points acupuncture on primary dysmenorrhea.

Prospect of value and application of transcutaneous electrical acupoint stimulation to control blood pressure in proactive healthcare perspective. / 主动健康视域下经皮穴位电刺激调控血压的价值与应用展望.

Hypertension is a global problem threatening human health and life. Although there are many antihypertensive drugs, the low compliance of medication affects its efficacy, and the effect in regulating hypertension has become increasingly prominent. Focusing on the new trend of proactive healthcare management, in the present paper, we made a summary about the status and existing problems of transcutaneous electrical acupoint stimulation (TEAS) in the regulation of blood pressure, and put forward some suggestions, such as selecting acupoints based on classical acupuncture theory to highlight the advantages of TEAS to control blood pressure as a whole, optimizing and screening the parameters of TEAS in the regulation of blood pressure, expanding the research observation indexes etc. We also made a prospect about its future application, hoping to provide new ideas for the proactive regulation, whole-process regulation and integrated regulation of blood pressure.

New insights into acupuncture techniques for poststroke spasticity.

With the trend of aging population getting more obvious, stroke has already been a major public health problem worldwide. As a main disabling motor impairment after stroke, spasticity has unexpected negative impacts on the quality of life and social participation in patients. Moreover, it brings heavy economic burden to the family and society. Previous researches indicated that abnormality of neural modulation and muscle property corelates with the pathogenesis of poststroke spasticity (PSS). So far, there still lacks golden standardized treatment regimen for PSS; furthermore, certain potential adverse-events of the mainstream therapy, for example, drug-induced generalized muscle weakness or high risk related surgery somehow decrease patient preference and compliance, which brings challenges to disease treatment and follow-up care. As an essential non-pharmacological therapy, acupuncture has long been used for PSS in China and shows favorable effects on improvements of spastic hypertonia and motor function. Notably, previous studies focused mainly on the research of antispastic acupoints. In comparison, few studies lay special stress on the other significant factor impacting on acupuncture efficacy, that is acupuncture technique. Based on current evidences from the clinic and laboratory, we will discuss certain new insights into acupuncture technique, in particular the antispastic needling technique, for PSS management in light of its potential effects on central modulations as well as peripheral adjustments, and attempt to provide some suggestions for future studies with respect to the intervention timing and course, application of acupuncture techniques, acupoint selection, predictive and aggravating factors of PSS, aiming at optimization of antispastic acupuncture regimen and improvement of quality of life in stroke patients. More innovations including rigorous study design, valid objective assessments for spasticity, and related experimental studies are worthy to be expected in the years ahead.

Effect of enzyme-assisted fermentation on quality, safety, and microbial community of black soldier fly larvae (Hermetia illucens L.) as a novel protein source.

Considering the significance and scarcity of quality protein, this study aims to obtain a novel safe protein source through fermenting the black soldier fly larvae (BSFL). Lactobacillus crispatus M1027 and Pichia kudriavzevii DHX19 were added as starters together with neutral protease for enzymolysis during fermentation. The results showed that the low pH value (from 6.60 to 3.99), generated by lactic acid accumulation, created an environment where the pathogen could hardly grow. During fermentation, the flavor compound ethyl acetate content reached up to 406.55 mg/L, and the melanization was effectively inhibited by the starters. The increase of trichloroacetic acid-soluble protein content (from 8.73 % to 17.96 %) contributed to improving the absorbability of product by animals after feeding. Notably, the contents of detrimental substances, including total volatile basic nitrogen and histamine, were both below specified limits after fermentation. Simultaneously, the malonic dialdehyde content remained stable during fermentation. Relative abundance of Lactobacillus and Pichia gradually increased and finally dominated in the culture during fermentation, accompanied by pathogens decline below detection limit (1.0 Log cfu/g). Moreover, there was a close relationship between the dynamics of physicochemical indices and microbial succession. Overall, our studies explored a new process to ferment the BSFL paste which would improve the quality and safety of fermented BSFL paste. This research provided theoretical support for fermented insect as a novel protein source.

Influence of Psychological Factors in Primary Dysmenorrhea Patients on De qi: a Secondary Analysis of a Randomized Controlled Trial.

Background: De qi , the needling sensation, is important in acupuncture treatment. Almost all studies believe that deep needling and manipulation could achieve a significant de qi sensation. However, relatively few studies have examined the effect of psychological factors on de qi, and those that did often reached different conclusions. Objectives: To explore the influence of psychologic factors on de qi in patients with primary dysmenorrhea (PD). Methods: Sixty-eight PD patients with cold and dampness stagnation were randomly allocated to de qi (deep insertion using thick needles, with manipulation, n=17) and non-de qi groups (shallow insertion using thin needles, without manipulation, n=51). Both groups received bilateral needling at Sanyinjiao (SP6) for 30 min. De qi was assessed using the Acupuncture De qi Clinical Assessment Scale (ADCAS). The patients' acupuncture-related anxiety and their expectations of the relationship between needle sensation and curative effect were evaluated using a five-point and four-point scale, respectively. Results: Within the de qi group, all patients experienced the de qi sensation, although anxiety levels were unrelated to de qi. Patients' expectations correlated negatively with de qi timing, and positively with electric sensation. Within the non-de qi group, 59.5% of patients experienced de qi. Between those who experienced it and those who did not, no significant differences were found in anxiety levels, although patients' expectations differed significantly. Among patients who experienced de qi sensations in the non-de qi group, anxiety and throbbing were positively correlated. Additionally, patients' expectations correlated positively with de qi intensity, as well as coldness, and numbness. Conclusion: Psychological factors should be considered when studying de qi since PD patients' expectations could influence the de qi sensation at SP6.

The factors affecting neurogenesis after stroke and the role of acupuncture.

Stroke induces a state of neuroplasticity in the central nervous system, which can lead to neurogenesis phenomena such as axonal growth and synapse formation, thus affecting stroke outcomes. The brain has a limited ability to repair ischemic damage and requires a favorable microenvironment. Acupuncture is considered a feasible and effective neural regulation strategy to improve functional recovery following stroke via the benign modulation of neuroplasticity. Therefore, we summarized the current research progress on the key factors and signaling pathways affecting neurogenesis, and we also briefly reviewed the research progress of acupuncture to improve functional recovery after stroke by promoting neurogenesis. This study aims to provide new therapeutic perspectives and strategies for the recovery of motor function after stroke based on neurogenesis.

Biofeedback physical regulation of hypertension based on acupoints: A clinical trial.

BACKGROUND: Hypertension is a predominant risk factor for cardiovascular disease and has imposed a global disease burden. Poor medication compliance is the major obstacle to antihypertensive drug therapy, and negative mood status is also detrimental to blood pressure (BP) management. While transcutaneous electrical acupoint stimulation (TEAS), as an electrical stimulation modality for biofeedback physical regulation based on acupoints, offers a such nondrug alternative option that is noninvasive, safe, and effective with high adherence. However, the optimal stimulation parameters of TEAS for hypertension remain unclear, especially the frequency, which needs further exploration. OBJECTIVE: The study aims to investigate the efficacy of TEAS for hypertension, and to screen the optimal electrical stimulation frequency. METHODS: This is an 8-week, randomized, controlled pilot trial with 3 parallel groups. In a ratio of 1:1:1, 120 patients with stage 1 hypertension will be divided into the TEAS-2Hz group, TEAS-10Hz group, or usual care group. All patients will receive the usual care for hypertension including lifestyle education, etc. Additionally, the 2 TEAS groups will receive 12 sessions of TEAS interventions at 2 Hz or 10 Hz, 3 times weekly for 30 minutes each, with 4 weeks of follow-up. The main outcome will be the change from baseline to week 4 in systolic BP among the groups. Secondary outcomes consist of changes in diastolic BP, mean arterial pressure, heart rate, heart rate variability, medication adherence, and quality of life. The safety outcomes will be any adverse event during the treatment. DISCUSSION: As a pre-study for the next large clinical trial of TEAS for hypertension, this study will offer references for optimized frequency of biofeedback electrical devices and promote more consciousness of the benefits of body-mind holistic regulation of BP, thereby achieving proactive and overall process management of BP.

Effects of acupoints-based TENS combined with tDCS on spasticity and motor function in ischemic stroke with spastic hemiplegia: study protocol for a randomized controlled trial.

Background: Spastic hemiplegia following ischemic strokes seriously impedes the recovery of motor function posing a vast rehabilitation challenge. As the uncertain effects of recommended conventional treatments such as botulinum toxin injections on active functional improvement and potential adverse effects cannot be bypassed, there is an increasing need in alternative, more effective and safer modalities. Acupoints-based transcutaneous electrical nerve stimulation (Acu-TENS) and transcranial direct current stimulation (tDCS) are effective non-invasive modalities for stroke rehabilitation, particularly showing anti-spastic effect and functional improvements as well. However, the optimal stimulation frequency of Acu-TENS and whether combination of Acu-TENS and tDCS exert synergistic effect remain to be investigated. Objective: To evaluate the effects of Acu-TENS combined with tDCS on spasticity and motor function in ischemic stroke patients with spastic hemiplegia and screen the optimal frequency of Acu-TENS. Methods: A total of 90 post-ischemic stroke patients with spastic hemiplegia will be intervened for 4 weeks and followed up for 4 weeks. They will be randomly assigned to three groups including two observation groups and a standard care control group in a 1:1:1 ratio. All patients will receive standard care and regular rehabilitation accordingly. In addition, the two observation groups will receive 12 sessions of Acu-TENS at 20 Hz or 100 Hz for 30 min combined with 1 mA tDCS for 20 min, three times a week, for 4 weeks. The primary outcome is the change in total modified Ashworth scale (MAS) score from baseline to week 4. Secondary outcomes include changes in surface electromyography (SEMG), Fugl-Meyer Motor Function Scale, Modified Barthel Index (MBI), and 10-meter walk test from baseline to week 4. MAS score will also be measured after 4 weeks of follow-up. Adverse events throughout the study will be recorded. Discussion: This trial will evaluate, for the first time, the therapeutic potentials and safety of Acu-TENS combined with tDCS on spasticity and motor function in stroke patients. It will provide evidence for frequency-dependent anti-spastic effect of Acu-TENS, and a reference for rated parameter setting of new mixed transcutaneous and transcranial stimulation system for stroke rehabilitation, thereby promoting proactive healthcare consequently. Trial registration: Chinese Clinical Trials Register ChiCTR2200067186.

Noncoding transcripts are linked to brain resting-state activity in non-human primates.

Brain-derived transcriptomes are known to correlate with resting-state brain activity in humans. Whether this association holds in nonhuman primates remains uncertain. Here, we search for such molecular correlates by integrating 757 transcriptomes derived from 100 macaque cortical regions with resting-state activity in separate conspecifics. We observe that 150 noncoding genes explain variations in resting-state activity at a comparable level with protein-coding genes. In-depth analysis of these noncoding genes reveals that they are connected to the function of nonneuronal cells such as oligodendrocytes. Co-expression network analysis finds that the modules of noncoding genes are linked to both autism and schizophrenia risk genes. Moreover, genes associated with resting-state noncoding genes are highly enriched in human resting-state functional genes and memory-effect genes, and their links with resting-state functional magnetic resonance imaging (fMRI) signals are altered in the brains of patients with autism. Our results highlight the potential for noncoding RNAs to explain resting-state activity in the nonhuman primate brain.