Accelerated discovery of CO2 electrocatalysts using active machine learning.

The rapid increase in global energy demand and the need to replace carbon dioxide (CO2)-emitting fossil fuels with renewable sources have driven interest in chemical storage of intermittent solar and wind energy1,2. Particularly attractive is the electrochemical reduction of CO2 to chemical feedstocks, which uses both CO2 and renewable energy3-8. Copper has been the predominant electrocatalyst for this reaction when aiming for more valuable multi-carbon products9-16, and process improvements have been particularly notable when targeting ethylene. However, the energy efficiency and productivity (current density) achieved so far still fall below the values required to produce ethylene at cost-competitive prices. Here we describe Cu-Al electrocatalysts, identified using density functional theory calculations in combination with active machine learning, that efficiently reduce CO2 to ethylene with the highest Faradaic efficiency reported so far. This Faradaic efficiency of over 80 per cent (compared to about 66 per cent for pure Cu) is achieved at a current density of 400 milliamperes per square centimetre (at 1.5 volts versus a reversible hydrogen electrode) and a cathodic-side (half-cell) ethylene power conversion efficiency of 55 ± 2 per cent at 150 milliamperes per square centimetre. We perform computational studies that suggest that the Cu-Al alloys provide multiple sites and surface orientations with near-optimal CO binding for both efficient and selective CO2 reduction17. Furthermore, in situ X-ray absorption measurements reveal that Cu and Al enable a favourable Cu coordination environment that enhances C-C dimerization. These findings illustrate the value of computation and machine learning in guiding the experimental exploration of multi-metallic systems that go beyond the limitations of conventional single-metal electrocatalysts.

Sustainable Electrosynthesis of N,N-Dimethylformamide via Relay Catalysis on Synergistic Active Sites.

Electrified synthesis of high-value organonitrogen chemicals from low-cost carbon- and nitrogen-based feedstocks offers an economically and environmentally appealing alternative to traditional thermocatalytic methods. However, the intricate electrochemical reactions at electrode surfaces pose significant challenges in controlling selectivity and activity, especially for producing complex substances such as N,N-dimethylformamide (DMF). Herein, we tackle this challenge by developing relay catalysis for efficient DMF production using a composite WO2-NiOOH/Ni catalyst with two distinctive active sites. Specifically, WO2 selectively promotes dimethylamine (DMA) electrooxidation to produce strongly surface-bound (CH3)2N*, while nearby NiOOH facilitates methanol electrooxidation to yield more weakly bound *CHO. The disparity in binding energetics of the key C- and N-intermediates expedites C-N coupling at the WO2-NiOOH interface. In situ infrared spectroscopy with isotope-labeling experiments, quasi-in situ electron paramagnetic resonance trapping experiments, and electrochemical operating experiments revealed the C-N coupling mechanism and enhanced DMF-synthesis selectivity and activity. In situ X-ray absorption spectroscopy (XAS) and postreaction transmission electron microscopy (TEM) studies verified the stability of WO2-NiOOH/Ni during extended electrochemical operation. A Faradaic efficiency of ∼50% and a production rate of 438 µmol cm-2 h-1 were achieved at an industrially relevant current density of 100 mA cm-2 over an 80 h DMF production period. This study introduces a new paradigm for developing electrothermo relay catalysis for the sustainable and efficient synthesis of valuable organic chemicals with industrial potential.

Polyphyllin II Induces Apoptosis in Fibrosarcoma Cells via Activating Pyruvate Kinase M2.

Aerobic glycolysis is a metabolic reprogramming of tumor cells that is essential for sustaining their phenotype of fast multiplication by continuously supplying energy and mass. Pyruvate kinase M2 (PKM2) has a vital role in this process, which has given it high interest as a target for anticancer drug development. With potent toxicity to many types of cancer cells, polyphyllin II (PP2), a steroidal saponin isolated from the herbaceous plant Rhizoma paridis, brought to our attention that it might interfere with the PKM2 activity. In this study, we discovered that PP2 was a novel agonist of PKM2. PP2 activated recombinant PKM2 and changed the protein's oligomeric state to activate intracellular PKM2. At the same time, PP2 suppressed its protein kinase function by decreasing the content of nuclear PKM2. The mRNA levels of its downstream genes, such as Glut1, LDHA, and MYC, were inhibited. In addition, PP2 induced oxidative stress by downregulating the expression and activity of antioxidant proteins such as NQO1, TrxR, and Trx in HT-1080 cells, which in turn led to mitochondrial dysfunction and ultimately induced apoptosis. Moreover, PP2 reduced the proliferation and migration of HT-1080 cells. Thus, targeting the glycolysis pathway offers an unprecedented mode of action for comprehending PP2's pharmacological impacts and advances PP2's further development in fibrosarcoma therapy.

Stratified management based on surface antibody for the prevention of hepatitis B virus reactivation in lymphoma patients.

This study aimed to investigate a stratified approach based on hepatitis B virus (HBV) surface antibody (anti-HBs) for managing HBV reactivation (HBVr) in lymphoma patients with serological protection against HBV. A retrospective analysis was conducted on 209 lymphoma patients with a baseline anti-HBs titre of ≥10 iu/L, who were either positive or negative for HBV core antibody (anti-HBc). The results revealed that 15.7% of patients lost serological protection following 6-month anti-lymphoma therapy. With a median follow-up of 28.1 months, the cumulative rates of HBVr at 6 months, 2 years and 4 years were 2.9%, 4.7% and 6.3% respectively. Without intervention, the overall rate of reactivation was 2.0% for patients with isolated anti-HBs and 10.5% for those with positive anti-HBs and anti-HBc. To identify patients at high risk of losing seroprotection and susceptible to HBVr, a predictive model was developed. The high-risk group had significantly higher rates of serological protection loss (27.8% vs. 2.2%) and cumulative incidence of HBVr (22.0% vs. 0%) compared to the low-risk group. Overall, this study highlights the risk of HBVr in lymphoma patients with positive anti-HBs, with or without positive anti-HBc, and recommends periodic monitoring for low-risk patients and early intervention for high-risk patients.

Imaging of Carbonic Anhydrase Level in Epilepsy with an Environment-Sensitive Fluorescent Probe.

Carbonic anhydrases (CAs) participate in various physiological and pathological activities by catalyzing the interconversion between carbon dioxide and bicarbonate ions. Under normal circumstances, they guarantee that the relevant biological reactions in our body occur within an appropriate time scale. Abnormal expression or activity alteration of CAs is closely related to the pathogenesis of diverse diseases. This work reports an inhibitor-directed fluorescent probe FMRs-CA for the detection of CAs. Excellent selectivity, favorable biocompatibility, and desirable blood-brain barrier (BBB) penetration endow the probe with the ability to image the fluctuation of CAs in cells and mice. We achieved in situ visualization of the increased CAs in hypoxic cells with this probe. Additionally, probe FMRs-CA was mainly enriched within the liver and gradually metabolized by the liver. With the help of FMRs-CA, the increase of CAs in epileptic mouse brains was revealed first from the perspective of imaging, providing the mechanism connection between abnormal CA expressions and epilepsy.

Promoted Photothermal Catalytic CO Hydrogenation by Using TiC-Supported Co-Fe5 C2 Catalysts.

Photothermal catalytic CO hydrogenation offers the potential to synthesize light hydrocarbons by using solar energy. However, the selectivity and activity of the reaction are still far below those achieved in conventional thermal catalytic processes. Herein, we report that the Co-modified Fe5 C2 on TiC catalyst promotes photothermal catalytic CO hydrogenation with a 59 % C2+ selectivity in the produced hydrocarbons and a 30 % single-pass CO conversion at a high gas hourly space-time velocity of 12 000 mL g-1 h-1 . Using in-situ-irradiated XPS, we show that light-induced hot electron injection from TiC to Fe5 C2 modulates the chemical state of Fe, thereby increasing the CO-to-C2+ conversion. This work suggests that it is possible for plasmon-mediated surface chemistry to enhance the activity and selectivity of photothermal catalytic reactions.

Disrupted resting-state brain functional network properties in non-neuropsychiatric systemic lupus erythematosus patients.

INTRODUCTION: Previous fMRI studies revealed that the abnormal functional connectivity (FC) was related to cognitive impairment in patients with SLE. However, it remains unclear how the disease severity affects the functional topological organization of the whole-brain network in SLE patients without neuropsychiatric symptoms (non-NPSLE). OBJECTIVE: We aim to examine the impairment of the whole-brain functional network in SLE patients without neuropsychiatric symptoms (non-NPSLE), which may improve the understanding of neural mechanism in SLE. METHODS: We acquired resting-state fMRI data from 32 non-NPSLE patients and 32 healthy controls (HC), constructed their whole-brain functional network, and then estimated the topological properties including global and nodal parameters by using graph theory. Meanwhile, we also investigated the differences in intra- and inter-network FC between the non-NPSLE patients and the HC. RESULTS: The non-NPSLE patients showed significantly lower clustering coefficient, global and local efficiency, but higher characteristic path length than the HC. The non-NPSLE patients had significantly lower nodal strength in two regions, ventromedial prefrontal cortex (vmPFC) and anterior PFC (aPFC) than the HC. We found the non-NPSLE patients had significantly lower intra-network FC within frontal-parietal network (FPN) and within default mode network (DMN), and significantly lower inter-network FC between DMN and FPN than the HC. The intra-network FC within DMN was negatively correlated with systemic lupus erythematosus disease activity index (SLEDAI). CONCLUSION: Abnormal whole-brain functional network properties and abnormal intra- and inter-network FC may be related to cognitive impairment and disease degree in the non-NPSLE patients. Our findings provide a network perspective to understand the neural mechanisms of SLE.

Synthesis and discovery of Baylis-Hillman adducts as potent and selective thioredoxin reductase inhibitors for cancer treatment.

The selenoprotein thioredoxin reductase (TrxR) is of paramount importance in maintaining cellular redox homeostasis, and aberrant upregulation of TrxR is frequently observed in various cancers due to their elevated oxidative stress in cells. Thus, it seems promising and feasible to target the ablation of intracellular TrxR for the treatment of cancers. We report herein the design and synthesis of a series of Baylis-Hillman adducts, and identified a typical adduct that possesses the superior cytotoxicity against HepG2 cells over other types of cancer cells. The biological investigation shows the selected typical adduct selectively targets TrxR in HepG2 cells, which thereafter results in the collapse of intracellular redox homeostasis. Further mechanistic studies reveal that the selected typical adduct arrests the cell cycle in G1/G0 phase. Importantly, the malignant metastasis of HepG2 cells is significantly restrained by the selected typical adduct. With well-defined molecular target and mechanism of action, the selected typical adduct, even other Baylis-Hillman skeleton-bearing compounds, merits further development as candidate or ancillary agent for the treatment of various cancers.

Synthesis and evaluation of Piperine analogs as thioredoxin reductase inhibitors to cause oxidative stress-induced cancer cell apoptosis.

Inhibiting thioredoxin reductase (TrxR) to disrupt the redox equilibrium and induce tumor cell apoptosis is a significant tumor therapeutic strategy. Piperine, a natural product from black pepper, has been demonstrated to suppress tumor cell proliferation by enhancing reactive oxygen species (ROS), subsequently leading to cell death. However, the development of Piperine as an active molecule is hampered by its weak cytotoxicity. To develop a compound with higher activity, we synthesized 22 Piperine analogs and evaluated their pharmacological properties. Ultimately, B5 was screened by the results of cytotoxicity and inhibition of TrxR activity. In contrast to Piperine, B5 had significant cytotoxicity with a 4-fold increase. The structure-activity relationship demonstrated that the introduction of an electron-withdrawing group into the benzene ring adjacent to the amino group, particularly in the meta-position, was positive and that shortening the olefin double bond had no appreciable impact on cytotoxicity. Further investigating the physiological activity of B5 in HeLa cells, we found that B5 selectively inhibits the activity of TrxR by binding to Sec residues on TrxR. B5 then induces cellular oxidative stress and finally leads to apoptosis. As a result, the study of B5 paved the way for further investigation into the modification and function of Piperine analogs as TrxR inhibitors.

Achieving High Single-Pass Carbon Conversion Efficiencies in Durable CO2 Electroreduction in Strong Acids via Electrode Structure Engineering.

Acidic CO2 reduction (CO2 R) holds promise for the synthesis of low-carbon-footprint chemicals using renewable electricity. However, the corrosion of catalysts in strong acids causes severe hydrogen evolution and rapid deterioration of CO2 R performance. Here, by coating catalysts with an electrically nonconductive nanoporous SiC-NafionTM layer, a near-neutral pH was stabilized on catalyst surfaces, thereby protecting the catalysts against corrosion for durable CO2 R in strong acids. Electrode microstructures played a critical role in regulating ion diffusion and stabilizing electrohydrodynamic flows near catalyst surfaces. This surface-coating strategy was applied to three catalysts, SnBi, Ag, and Cu, and they exhibited high activity over extended CO2 R operation in strong acids. Using a stratified SiC-NafionTM /SnBi/polytetrafluoroethylene (PTFE) electrode, constant production of formic acid was achieved with a single-pass carbon efficiency of >75 % and Faradaic efficiency of >90 % at 100 mA cm-2 over 125 h at pH 1.

Hemin as a General Static Dark Quencher for Constructing Heme Oxygenase-1 Fluorescent Probes.

The development of small-molecule probes suitable for live-cell applications remains challenging yet highly desirable. We report the first fluorescent probe, RBH, for imaging the heme oxygenase-1 (HO-1) activity in live cells after discovering hemin as a universal dark quencher. Hemin works via a static quenching mechanism and shows high quenching efficiency (>97 %) with fluorophores across a broad spectrum (λex =400-700 nm). The favorable properties of RBH (e.g. long excitation/emission wavelengths, fast response rate and high magnitude of signal increase) enable its use for determining HO-1 activity in complex biological samples. As HO-1 is involved in regulating antioxidant defence, iron homeostasis and gasotransmitter carbon monoxide production, we expect RBH to be a powerful tool for dissecting its functions. Also, the discovery of hemin as a general static dark quencher provides a straightforward strategy for constructing novel fluorescent probes for diverse biological species.

Effects of transition metal doping on electronic structure of metastable ß-Fe2O3 photocatalyst for solar-to-hydrogen conversion.

Metastable ß-Fe2O3 is a promising photocatalyst with a band gap of approximately 1.9 eV, while its intrinsic material properties remain rarely studied by theoretical calculations. Here, using density functional theory, we studied the electronic band structure and effective mass of carriers in Zr, Sn, and Ti doped ß-Fe2O3. The calculation results show that, through the doping of Zr, Sn, or Ti, the dipole moment of FeO6 octahedra in ß-Fe2O3 increases, which favors the separation of photo-excited electron-hole pairs. The electron and hole effective masses in the close-packed orientation [111] in cubic ß-Fe2O3 have the smallest absolute values. After doping with Zr, Sn, and Ti, the absolute values of electron and hole effective masses in the [111] orientation are further reduced. Furthermore, the relative ratio (D) mostly became larger after doping with Zr, Sn, and Ti, which indicates that the photoexcited carriers in the doped structure are effectively separated. Construction of Zr, Sn, and Ti doped ß-Fe2O3 in the [111] orientation may be effective to improve the photocatalytic efficiency.

Synthesis and biological evaluation of disulfides as anticancer agents with thioredoxin inhibition.

Altered redox homeostasis as a hallmark of cancer cells is exploited by cancer cells for growth and survival. The thioredoxin (Trx), an important regulator in maintaining the intracellular redox homeostasis, is cumulatively recognized as a promising target for the development of anticancer drugs. Herein, we synthesized 72 disulfides and evaluated theirinhibition for Trx and antitumor activity. First, we established an efficient and fast method to screen Trx inhibitors by using the probe NBL-SS that was developed by our group to detect Trx function in living cells. After an initial screening of the Trx inhibitory activity of these compounds, 8 compounds showed significant inhibition activity against Trx. We then evaluated the cytotoxicity of these 8 disulfides, compounds 68 and 69 displayed high cytotoxicity to HeLa cells, but less sensitive to normal cell lines. Next, we performed kinetic studies of both two disulfides, 68 had faster inhibition of Trx than 69. Further studies revealed that 68 led to the accumulation of reactive oxygen species and eventually induced apoptosis of Hela cells via inhibiting Trx. The establishment of a method for screening Trx inhibitors and the discovery of 68 with remarkable Trx inhibition provide support for the development of anticancer candidates with Trx inhibition.

LncRNA NEAT1 accelerates renal mesangial cell injury via modulating the miR-146b/TRAF6/NF-κB axis in lupus nephritis.

Although growing advances have been made in the regulation of lupus nephritis recently, lupus nephritis is still one of the major causes of death in SLE patients and the pathogenesis remains largely unknown. Therefore, exploring the pathological mechanisms is urgently needed for designing and developing novel therapeutic strategies for lupus nephritis. Human renal mesangial cells (HRMCs) were transfected with sh-NEAT1, miR-146b mimic, pcDNA-NEAT1, miR-146b inhibitor, or sh-TRAF6 to modify their expression. Lipopolysaccharide (LPS) was used to induce inflammatory injury. Cell viability was examined with CCK8. Apoptosis was determined by flow cytometry and Hoechst staining. qRT-PCR and western blot were used to analyze gene expression. The secretion of inflammatory cytokines was examined with ELISA. The bindings of NEAT1 with miR-146b and miR-146b with TRAF6 were tested by dual-luciferase reporter assay. NEAT1 was upregulated in LPS-treated HRMCs. Both the knockdown of NEAT1 and TRAF6 suppressed the LPS-induced inflammatory injury in HRMCs. NEAT1 directly targeted miR-146b to control miR-146b-mediated regulation of TRAF6 expression in HRMCs. NEAT1 promoted the expression of TRAF6 via targeting miR-146b to accelerate the LPS-mediated renal mesangial cell injury in HRMCs. Moreover, TRAF6 activated the NF-κB signaling in HRMCs. NEAT1 accelerated renal mesangial cell injury via directly targeting miR-146b, promoting the expression of TRAF6, and activating the NF-κB signaling in lupus nephritis. Our investigation elucidated novel pathological mechanisms and provided potential therapeutic targets for lupus nephritis.

Dynamic Properties of Human Default Mode Network in Eyes-Closed and Eyes-Open.

The default mode network (DMN) reflects spontaneous activity in the resting human brain. Previous studies examined the difference in static functional connectivity (sFC) of the DMN between eyes-closed (EC) and eyes-open (EO) using the resting-state functional magnetic resonance imaging (rs-fMRI) data. However, it remains unclear about the difference in dynamic FC (dFC) of the DMN between EC and EO. To this end, we acquired rs-fMRI data from 19 subjects in two different statues (EC and EO) and selected a seed region-of-interest (ROI) at the posterior cingulate cortex (PCC) to generate the sFC map. We identified the DMN consisting of ten clusters that were significantly correlated with the PCC. By using a sliding-window approach, we analyzed the dFC of the DMN. Then, the Newman's modularity algorithm was applied to identify dFC states based on nodal total connectivity strength in each sliding-window. In addition, graph-theory based network analysis was applied to detect dynamic topological properties of the DMN. We identified three group-level dFC states (State1, 2 and 3) that reflects the strength of dFC within the DMN between EC and EO in different time. The following results were reached: (1) no significant difference in sFC between EC and EO, (2) dFC was lower in State2 but higher in State3 in EC than in EO, (3) lower clustering coefficient, local efficiency, and global efficiency, but higher characteristic path length in State2 in EC than in EO, and (4) lower nodal strength in the precuneus (PCUN), PCC, angular gyrus (ANG), middle temporal gyrus (MTG) and medial prefrontal cortex (MPFC) in State3 in EC. These results suggested different resting statuses, EC and EO, may induce different time-varying neural activity in the DMN.

Spatially Overlapping Regions Show Abnormal Thalamo-frontal Circuit and Abnormal Precuneus in Disorders of Consciousness.

Understanding the neural mechanisms of disorders of consciousness (DOC) is essential for estimating the conscious level and diagnosing DOC patients. Although previous studies reported brain functional connectivity (FC) and spontaneous neural activity patterns associated with consciousness, the relationship between them remains unclear. In this study, we identified the abnormal brain regions in DOC patients by performing voxel-wise FC strength (FCS) and fractional amplitude of low-frequency fluctuations (fALFF) analyses on resting-state functional magnetic resonance imaging data of 15 DOC patients and 24 healthy controls. Furthermore, we detected spatial intersections between two measures and estimated the correlations between either the FCS or the fALFF and the subscales of the Coma Recovery Scale-Revised (CRS-R). We found that the right superior frontal gyrus, left thalamus and right precuneus in which the DOC patients had a lower local FCS and fALFF than healthy controls, are coincident with regions of the mesocircuit model. In the right precuneus, the local FCS/fALFF was significantly positively correlated with the oromotor and motor scores/motor score of the CRS-R. Our findings may indicate that the co-occurrent pattern of spontaneous neural activity and functional connectivity in the thalamo-frontal circuit and the precuneus are associated with motor function in DOC patients.

Au nanocluster-embedded chitosan nanocapsules as labels for the ultrasensitive fluorescence immunoassay of Escherichia coli O157:H7.

Herein, Au nanocluster (AuNC)-embedded chitosan (CS) nanocapsules were prepared and used as a novel signal amplification system for the sensitive fluorescence immunoassay of Escherichia coli O157:H7 (E. coli O157:H7). AuNCs were embedded in CS to form AuNCs@CS nanocapsules, where a large number of AuNCs were embedded in each nanocapsule. A rapid, ultrasensitive, and selective method for the fluorescence detection of E. coli O157:H7 was developed by coupling the nanocapsule-amplification strategy with immunological recognition and magnetic separation. Compared with a conventional immunoassay using AuNCs as labels, the fluorescence signals of the proposed immunoassay were greatly amplified, because much more fluorescent AuNCs are linked to each bacterial cell. As a result, a linear range was obtained from 3 to 700 CFU mL-1, with a detection limit of 1 CFU mL-1. This method was successfully used to detect E. coli O157:H7 in drinking water and milk samples.

Comparison of a microsphere-based platform with a multiplex flow cytometric assay for determination of circulating cytokines in the mouse.

BACKGROUND: Measuring expression profiles of inflammatory biomarkers is important in monitoring the polarization of immune responses; therefore, results should be independent of quantitation methods if they are to be accepted as validated clinical pathology biomarkers. To evaluate effects of differing quantitation methods, the seven major circulating Th1/Th2/Th17 cytokines interleukin 2 (IL-2), interferon γ (IFN-γ), tumor necrosis factor α (TNF-α), IL-4, IL-6, IL-10 and IL-17A were quantified in plasma of lipopolysaccharide (LPS)-treated mice with two different multiplex platforms. METHODS: Female C57BL6 mice were treated orally with vehicle or dexamethasone, followed by LPS intravenously. Plasma samples were analyzed 0.5, 1, 2, 4, and 6 h post-LPS challenge with assays at Myriad-RBM and compared to assays performed on a BD Accuri C6 flow cytometer. RESULTS: IL-17A response to LPS was limited but sustained, and the response for the remaining cytokines were early and transient; dexamethasone reduced expression of all 7 cytokines. TNF-α and IL-6 levels were similar across both assays, and IL-4 levels were generally very low. Plasma levels of remaining cytokines were variably lower with BD assays than Myriad-RBM assays. CONCLUSIONS: The present findings demonstrate that quantitation of circulating biomarkers of inflammation can be achieved using multiplexed flow cytometry, but careful consideration must be taken for assay validation when cross-referencing with another multiplexed assay.

Scalable water splitting on particulate photocatalyst sheets with a solar-to-hydrogen energy conversion efficiency exceeding 1.

Photocatalytic water splitting using particulate semiconductors is a potentially scalable and economically feasible technology for converting solar energy into hydrogen. Z-scheme systems based on two-step photoexcitation of a hydrogen evolution photocatalyst (HEP) and an oxygen evolution photocatalyst (OEP) are suited to harvesting of sunlight because semiconductors with either water reduction or oxidation activity can be applied to the water splitting reaction. However, it is challenging to achieve efficient transfer of electrons between HEP and OEP particles. Here, we present photocatalyst sheets based on La- and Rh-codoped SrTiO3 (SrTiO3:La, Rh; ref. ) and Mo-doped BiVO4 (BiVO4:Mo) powders embedded into a gold (Au) layer. Enhancement of the electron relay by annealing and suppression of undesirable reactions through surface modification allow pure water (pH 6.8) splitting with a solar-to-hydrogen energy conversion efficiency of 1.1% and an apparent quantum yield of over 30% at 419 nm. The photocatalyst sheet design enables efficient and scalable water splitting using particulate semiconductors.

Development of a Functional Observational Battery in the Minipig for Regulatory Neurotoxicity Assessments.

A functional observational battery (FOB) is recommended as the first-tier neurotoxicity screening in the preclinical safety pharmacology testing guidelines. Minipigs have increasingly been used in regulatory toxicology studies; however, no current FOB protocol is available for neurotoxicity testing in these species. Hence, a minipig FOB instrument was developed. A complete crossover study with Sinclair minipigs was performed to evaluate physiologic, neurologic, and behavioral effects of amphetamine, ketamine, and diazepam. The treated minipigs were first observed in their home cage, were video-recorded for 10 minutes in an open field, and then went through a complete neurologic examination. Both ketamine and diazepam were shown to reduce the freezing and behavior shifts of treated minipigs, while increasing their exploratory behaviors. Both drugs also caused muscular and gait impairment. The effects of ketamine and diazepam were consistent with their roles as central nervous system (CNS) suppressants. Unique effects were also observed with ketamine and diazepam treatments, which may reflect their unique mechanisms of action. Consistent with its role as a CNS stimulant, amphetamine caused the treated minipigs to be hyperactive and to display increased freezing and behavior shifts and reduced exploring activities. These effects of amphetamine were opposite to those observed with ketamine and diazepam. Amphetamine also increased locomotion in the treated minipigs. The present effects of amphetamine, ketamine, and diazepam are in agreement with observations by others. In conclusion, the minipig is a suitable species for FOB evaluation of pharmaceuticals in preclinical safety pharmacology testing.