The Dual Role of Neutrophil Extracellular Traps (NETs) in Sepsis and Ischemia-Reperfusion Injury: Comparative Analysis across Murine Models.

A better understanding of the function of neutrophil extracellular traps (NETs) may facilitate the development of interventions for sepsis. The study aims to investigate the formation and degradation of NETs in three murine sepsis models and to analyze the production of reactive oxygen species (ROS) during NET formation. Murine sepsis was induced by midgut volvulus (720° for 15 min), cecal ligation and puncture (CLP), or the application of lipopolysaccharide (LPS) (10 mg/kg body weight i.p.). NET formation and degradation was modulated using mice that were genetically deficient for peptidyl arginine deiminase-4 (PAD4-KO) or DNase1 and 1L3 (DNase1/1L3-DKO). After 48 h, mice were killed. Plasma levels of circulating free DNA (cfDNA) and neutrophil elastase (NE) were quantified to assess NET formation and degradation. Plasma deoxyribonuclease1 (DNase1) protein levels, as well as tissue malondialdehyde (MDA) activity and glutathione peroxidase (GPx) activity, were quantified. DNase1 and DNase1L3 in liver, intestine, spleen, and lung tissues were assessed. The applied sepsis models resulted in a simultaneous increase in NET formation and oxidative stress. NET formation and survival differed in the three models. In contrast to LPS and Volvulus, CLP-induced sepsis showed a decreased and increased 48 h survival in PAD4-KO and DNase1/1L3-DKO mice, when compared to WT mice, respectively. PAD4-KO mice showed decreased formation of NETs and ROS, while DNase1/1L3-DKO mice with impaired NET degradation accumulated ROS and chronicled the septic state. The findings indicate a dual role for NET formation and degradation in sepsis and ischemia-reperfusion (I/R) injury: NETs seem to exhibit a protective capacity in certain sepsis paradigms (CLP model), whereas, collectively, they seem to contribute adversely to scenarios where sepsis is combined with ischemia-reperfusion (volvulus).

Bridging the biochemistry lecture and laboratory courses: Construction and application of the "Innovative Experimental Design" module.

Both lecture and laboratory courses of biochemistry are important professional courses for undergraduates with biology related majors. Course optimization and update is crucial but challenging, especially for the laboratory course. Although taught separately, here we showed a strategy to bridge the two courses and promote the improvement of both. In addition to knowledge teaching, we implanted the "Innovative Experimental Design" module in the lecture course in which students were required to design and present their own experimental ideas. After evaluation by the faculty group, the best idea was supported for further experimental test. Here we described the preliminary experiments and optimization procedures about the idea of microbial fuel cells. This experiment is ready to be included into the laboratory course program in spring 2023.

Development of Improved Spectrophotometric Assays for Biocatalytic Silyl Ether Hydrolysis.

Reported herein is the development of assays for the spectrophotometric quantification of biocatalytic silicon-oxygen bond hydrolysis. Central to these assays are a series of chromogenic substrates that release highly absorbing phenoxy anions upon cleavage of the sessile bond. These substrates were tested with silicatein, an enzyme from a marine sponge that is known to catalyse the hydrolysis and condensation of silyl ethers. It was found that, of the substrates tested, tert-butyldimethyl(2-methyl-4-nitrophenoxy)silane provided the best assay performance, as evidenced by the highest ratio of enzyme catalysed reaction rate compared with the background (uncatalysed) reaction. These substrates were also found to be suitable for detailed enzyme kinetics measurements, as demonstrated by their use to determine the Michaelis-Menten kinetic parameters for silicatein.

Pre-breeding of spontaneous Robertsonian translocations for density planting architecture by transferring Agropyron cristatum chromosome 1P into wheat.

KEY MESSAGE: We developed T1AL·1PS and T1AS·1PL Robertsonian translocations by breakage-fusion mechanism based on wheat-A. cristatum 1P(1A) substitution line with smaller leaf area, shorter plant height, and other excellent agronomic traits Agropyron cristatum, a wild relative of wheat, is a valuable germplasm resource for improving wheat genetic diversity and yield. Our previous study confirmed that the A. cristatum chromosome 1P carries alien genes that reduce plant height and leaf size in wheat. Here, we developed T1AL·1PS and T1AS·1PL Robertsonian translocations (RobTs) by breakage-fusion mechanism based on wheat-A. cristatum 1P (1A) substitution line II-3-1c. Combining molecular markers and cytological analysis, we identified 16 spontaneous RobTs from 911 F2 individuals derived from the cross of Jimai22 and II-3-1c. Fluorescence in situ hybridization (FISH) was applied to detect the fusion structures of the centromeres in wheat and A. cristatum chromosomes. Resequencing results indicated that the chromosomal junction point was located at the physical position of Triticum aestivum chromosome 1A (212.5 Mb) and A. cristatum chromosome 1P (230 Mb). Genomic in situ hybridization (GISH) in pollen mother cells showed that the produced translocation lines could form stable ring bivalent. Introducing chromosome 1PS translocation fragment into wheat significantly increased the number of fertile tillers, grain number per spike, and grain weight and reduced the flag leaf area. However, introducing chromosome 1PL translocation fragment into wheat significantly reduced flag leaf area and plant height with a negative effect on yield components. The pre-breeding of two spontaneous RobTs T1AL·1PS and T1AS·1PL was important for wheat architecture improvement.

Mediation Role of Recreational Physical Activity in the Relationship between the Dietary Intake of Live Microbes and the Systemic Immune-Inflammation Index: A Real-World Cross-Sectional Study.

The main topic of this research is the relationship between dietary intake of live microbe-containing (LMC) foods, recreational physical activity (RPA), and the systemic immune-inflammation index (SII). This study presented a cohort of 26,254 individuals in the National Health and Nutrition Examination Survey (NHANES), representing an estimated weighted population of 193,637,615 in the United States. Weighted multivariable linear regression models were used in consideration of the multi-stage sampling design. Results: The study found that medium-LMC foods were negatively associated with the SII [ß (95% CI): -4.807 (-7.752, -1.862), p = 0.002], indicating that their intake was correlated with lower levels of the SII. However, no significant associations were found with low- or high-LMC foods. The study also explored the relationship between RPA and the SII, finding that more time spent in RPA was negatively associated with the SII [ß (95% CI): -0.022 (-0.034, -0.011), p < 0.001]. A mediation analysis was conducted to investigate the role of RPA in the relationship between medium-LMC food intake and the SII. The analysis revealed that RPA had a notable indirect effect, contributing to 6.7% of the overall change in the SII. Overall, this study suggests that medium-LMC food intake and RPA may have beneficial effects on systemic immune inflammation.

Effect of Surface Treatment and Resin Cement on the Bond Strength of an Advanced Lithium Disilicate.

OBJECTIVES: The aim of the study was to evaluate the effect of surface treatment and resin cement on the bond strength of conventional and advanced lithium disilicates (ALDs). MATERIALS AND METHODS: Ceramic slices (2 × 13 × 15 mm) of conventional lithium disilicate (LD) (IPS e.max CAD) and ALD (CEREC Tessera) were sectioned, polished, and divided into 16 groups (n = 10) according to the factors: ceramic, surface treatment, and resin cement (Panavia V5 and Variolink Esthetic DC). Surface treatments consisted of hydrofluoric acid 4.9% etching for 20 seconds (Hf20) or 30 seconds (Hf30), self-etching ceramic primer (Se), and sandblasting (Sb). Then, a resin cement cylinder (Ø = 2.5 mm) was manufactured on each specimen's surface. The specimens were then submitted to a shear bond strength (SBS) test. Surface roughness was evaluated through a contact profilometer, and surface morphology was evaluated under scanning electron microscopy for qualitative analysis. STATISTICAL ANALYSIS: Two-way analysis of variance (ANOVA) was used to analyze the data of SBS and surface roughness. For bond strength, the effects of surface treatment, resin cement, and the interaction were analyzed for each ceramic. For roughness, analyzed factors include ceramic and surface treatment. RESULTS: ANOVA revealed that ceramic (p = 0.047) and surface treatment (p < 0.001) factors affected the bond strength, while the cements performed similarly. Both materials showed adequate bond strength (ALD 19.1 ± 7.7 MPa; LD 17.1 ± 7.9 MPa). Sb protocol showed the lowest mean value (9.6 ± 2.9 MPa) compared with Hf20 (22.0 ± 7.1 MPa), Hf30 (21.7 ± 7.4 MPa), and Se (19.3 ± 6.0 MPa). CONCLUSION: For both ceramics, the highest performance was obtained after applying Se and Hf20 or Hf30. Therefore, longer etching time is unnecessary. Sb protocol must be avoided.

Effect of printing layer orientation and polishing on the fatigue strength of 3D-printed dental zirconia.

OBJECTIVE: The aim of the study was to evaluate the influence of surface polishing and printing layer orientation on the fatigue behaviour of 3 mol% yttria-stabilized zirconia (3Y-TZP) by stereolithography (SLA) in comparison with subtractive manufacturing. MATERIALS AND METHODS: 60 experimental zirconia bar-shaped specimens were 3D-printed (P) via SLA, and 30 specimens were milled (M) from commercial zirconia block (Lava™ Frame, 3 M ESPE AG). All specimens had the same dimensions (1 mm × 1 mm x 12 mm) after sintering. The 3D-printed specimens were randomly divided according to printing orientations: parallel or perpendicular to the tensile surface in the fatigue test. The specimens were subsequently submitted to two surface finishing protocols (n = 15/gr): unpolished or polished. Their phase compositions were analysed by X-ray diffraction. The fatigue behaviour was evaluated by a stepwise approach. RESULTS: The milled and both 3D-printed groups showed similar phase compositions for the as-sintered condition. Considerable amounts of rhombohedral phase were detected after polishing. Milled unpolished samples presented significantly higher fatigue strength than 3D-printed unpolished samples. Polishing did not improve the fatigue strength for milled zirconia but was advantageous for the 3D-printed specimens. 3D-printed specimens with parallel printing-layer orientation were significantly stronger than specimens with perpendicular layers regardless of surface finishing. CONCLUSION: The manufacturing techniques had a significant influence on the fatigue strength of 3Y-TZP, but not on the phase compositions of the surface. The polishing protocol showed different effects on 3Y-TZP fatigue strength and induced phase transition of the 3Y-TZP from Tetragonal to Rhombohedral. The best fatigue strength was achieved through milling using an unpolished surface and SLA-printed layers that were parallel to the tensile surface, followed by polishing.

Does glaze firing affect the strength of advanced lithium disilicate after simulated defects?

OBJECTIVE: To study the influence of glazing on strength repair of lithium disilicate glass-ceramics after defect incorporation in different production processing phases. MATERIALS AND METHODS: Bar-shaped specimens (1 × 1 × 12 mm, n = 280; 20/group) made from different lithium disilicate ceramics (IPS e.max CAD, Ivoclar, "LD" or advanced lithium disilicate CEREC Tessera, Dentsply Sirona, "ALD") were exposed to 7 different protocols: crystallized without (c) and with glaze layer (cg), with a defect incorporated before crystallization without (ic) and with glaze layer (icg), with a defect after crystallization without (ci) or with glaze layer (cig), and defect incorporated after the glaze layer (cgi). The flexural strength was determined using the three-point bending test. Analysis of indented areas and fractured specimens was performed by scanning electron microscopy. Flexural strength data were evaluated by two-way ANOVA followed by Tukey tests (α = 5%). RESULTS: Two-way ANOVA revealed a significant influence of ceramic (p < 0.001; F = 55.45), protocol (p < 0.001; F = 56.94), and the interaction protocol*ceramic (p < 0.001; F = 13.86). Regardless of ceramics, defect incorporation as final step resulted in the worst strength, while defects introduced before crystallization did not reduce strength. Glaze firing after defect incorporation led to strength repair for ALD, whereas such an effect was not evident for LD. CONCLUSIONS: The advanced lithium disilicate must receive a glaze layer to achieve its highest strength. Defects incorporated in the pre-crystallized stage can be healed during crystallization. Defects should not be incorporated after glazing. CLINICAL RELEVANCE: Clinical adjustments should be performed on pre-crystallized or crystalized restorations that receive a glazer layer afterwards.

A digital chairside repair protocol for removable partial dentures with polyetheretherketone frameworks.

Polyetheretherketone (PEEK) has become popular for removable partial denture (RPD) frameworks but reports on their clinical follow-up and repair are lacking. Two defective PEEK-framework RPDs were repaired with computer-aided design and manufacturing technology, saving costs and time and simplifying the treatment process.

Emerging Pharmacotherapeutic Strategies to Overcome Undruggable Proteins in Cancer.

Targeted therapies in cancer treatment can improve in vivo efficacy and reduce adverse effects by altering the tissue exposure of specific biomolecules. However, there are still large number of target proteins in cancer are still undruggable, owing to the following factors including (1) lack of ligand-binding pockets, (2) function based on protein-protein interactions (PPIs), (3) the highly specific conserved active sites among protein family members, and (4) the variability of tertiary docking structures. The current status of undruggable targets proteins such as KRAS, TP53, C-MYC, PTP, are carefully introduced in this review. Some novel techniques and drug designing strategies have been applicated for overcoming these undruggable proteins, and the most classic and well-known technology is proteolysis targeting chimeras (PROTACs). In this review, the novel drug development strategies including targeting protein degradation, targeting PPI, targeting intrinsically disordered regions, as well as targeting protein-DNA binding are described, and we also discuss the potential of these strategies for overcoming the undruggable targets. Besides, intelligence-assisted technologies like Alpha-Fold help us a lot to predict the protein structure, which is beneficial for drug development. The discovery of new targets and the development of drugs targeting them, especially those undruggable targets, remain a huge challenge. New drug development strategies, better extraction processes that do not disrupt protein-protein interactions, and more precise artificial intelligence technologies may provide significant assistance in overcoming these undruggable targets.

Internal electric field-assisted copper ions chelated polydopamine/titanium dioxide nano-thin film heterojunctions activate peroxymonosulfate under visible light to catalyze degradation of gatifloxacin: Theoretical calculations and biotoxicity analysis.

The combination of photocatalysis and peroxymonosulfate (PMS) activation is considered effective in treating organic pollutants in water; however, the photocatalysts currently used to activate PMS are primarily in powder form, which cause secondary contamination because they are difficult to recycle. In this study, copper-ion-chelated polydopamine/titanium dioxide (Cu-PDA/TiO2) nanofilm were prepared for PMS activation on fluorine-doped tin oxide substrates using hydrothermal and in-situ self-polymerization methods. The results showed that Cu-PDA/TiO2 + PMS + Vis degraded 94.8% of gatifloxacin (GAT) within 60 min, and the reaction rate constant reached 4.928 × 10-2 min-1, which was 6.25 and 4.04 folds higher than that of TiO2 + PMS + Vis (0.789 × 10-2 min-1) and PDA/TiO2 + PMS + Vis (1.219 × 10-2 min-1), respectively. The Cu-PDA/TiO2 nanofilm is easily recyclable and activates PMS to degrade GAT with no inferior performance, unlike the powder-based photocatalysts, and simultaneously maintains outstanding stability, which is highly suitable for applications in real aqueous environments. Biotoxicity experiments were conducted using E. coli, S. aureus, and mung bean sprouts as experimental subjects, and the results showed that the Cu-PDA/TiO2 + PMS + Vis system had excellent detoxification ability. In addition, a detailed investigation of the formation mechanism of step-scheme (S-scheme) Cu-PDA/TiO2 nanofilm heterojunctions was conducted by density functional theory (DFT) calculations and in-situ X-ray photoelectron spectroscopy (XPS). Finally, a specific process for activating PMS to degrade GAT was proposed, which provides a novel photocatalysts for practical applications in aqueous pollution.

Development of a nomogram based on serum cytokine-related riskscore in breast cancer.

Background: Cytokines are involved in many inflammatory diseases and thus play an important role in tumor immune regulation. In recent years, researchers have found that breast cancer is not only related to genetic and environmental factors, but also to the chronic inflammation and immunity. However, the correlation between serum cytokines and blood tests indicators remain unclear. Methods: A total of 84 serum samples and clinicopathological data of breast cancer patients from Tianjin Cancer Institute & Hospital, Tianjin Medical University, Tianjin, P. R. China were collected. The expression levels of the 12 cytokines were detected by immunofluorescence method. Blood tests results were obtained from medical records. By stepwise Cox regression analysis, a cytokine-related gene signature was generated. Univariate and multivariate Cox regression were used to analyze the influence on the prognosis of patients. A nomogram was constructed to illustrate the cytokine-related riskscore predicting 5-year OS, which was further evaluated and validated by C-index and ROC curve. The correlation between the expression of cytokines in serum and other blood indicators was studied by using Spearman's test. Results: The riskscore was calculated as IL-4×0.99069 + TNF-α×0.03683. Patients were divided into high and low risk groups according to the median riskscore, with the high-risk group has a shorter survival time by log-rank test (training set, P=0.017; validation set, P=0.013). Combined with the clinical characteristics, the riskscore was found to be an independent factor for predicting the OS of breast cancer patients in both training cohort (HR=1.2, P<0.01) and validation cohort (HR=1.6, P=0.023). The 5-year C-index and AUC of the nomogram were 0.78 and 0.68, respectively. IL-4 was further found to be negatively correlated with ALB. Conclusion: In summary, we have developed a nomogram based on two cytokines including IL-4 and TNF-α to predict OS of breast cancer and investigated their correlation with blood test indicators.

The Genomic Variation and Differentially Expressed Genes on the 6P Chromosomes in Wheat-Agropyron cristatum Addition Lines 5113 and II-30-5 Confer Different Desirable Traits.

Wild relatives of wheat are essential gene pools for broadening the genetic basis of wheat. Chromosome rearrangements and genomic variation in alien chromosomes are widespread. Knowledge of the genetic variation between alien homologous chromosomes is valuable for discovering and utilizing alien genes. In this study, we found that 5113 and II-30-5, two wheat-A. cristatum 6P addition lines, exhibited considerable differences in heading date, grain number per spike, and grain weight. Genome resequencing and transcriptome analysis revealed significant differences in the 6P chromosomes of the two addition lines, including 143,511 single-nucleotide polymorphisms, 62,103 insertion/deletion polymorphisms, and 757 differentially expressed genes. Intriguingly, genomic variations were mainly distributed in the middle of the chromosome arms and the proximal centromere region. GO and KEGG analyses of the variant genes and differentially expressed genes showed the enrichment of genes involved in the circadian rhythm, carbon metabolism, carbon fixation, and lipid metabolism, suggesting that the differential genes on the 6P chromosome are closely related to the phenotypic differences. For example, the photosynthesis-related genes PsbA, PsbT, and YCF48 were upregulated in II-30-5 compared with 5113. ACS and FabG are related to carbon fixation and fatty acid biosynthesis, respectively, and both carried modification variations and were upregulated in 5113 relative to II-30-5. Therefore, this study provides important guidance for cloning desirable genes from alien homologous chromosomes and for their effective utilization in wheat improvement.

Membrane Dynamics Regulated by Cytoskeleton in Plant Immunity.

The plasma membrane (PM), which is composed of a lipid layer implanted with proteins, has diverse functions in plant responses to environmental triggers. The heterogenous dynamics of lipids and proteins in the plasma membrane play important roles in regulating cellular activities with an intricate pathway that orchestrates reception, signal transduction and appropriate response in the plant immune system. In the process of the plasma membrane participating in defense responses, the cytoskeletal elements have important functions in a variety of ways, including regulation of protein and lipid dynamics as well as vesicle trafficking. In this review, we summarized how the plasma membrane contributed to plant immunity and focused on the dynamic process of cytoskeleton regulation of endocytosis and exocytosis and propose future research directions.

Exosomes from tubular epithelial cells undergoing epithelial-to-mesenchymal transition promote renal fibrosis by M1 macrophage activation.

Kidney fibrosis is the common final pathway of chronic kidney disease (CKD), and it is distinguished by inflammation, mesenchymal transition with myofibroblast formation, and epithelial-to-mesenchymal transition (EMT). Macrophages are protuberant inflammatory cells in the kidney, and their roles are dependent on their phenotypes. However, it remains unclear whether tubular epithelial cells (TECs) undergoing EMT can influence the phenotypes of macrophages and the underlying mechanisms during the development of kidney fibrosis. Here, we investigated the characteristics of TECs and macrophages during kidney fibrosis with a focus on EMT and inflammation. We found that the coculture of exosomes from transforming growth factor-beta (TGF-ß)-induced TECs with macrophages induced macrophage M1 polarization, while exosomes from TECs without TGF-ß stimulation or stimulation with TGF-ß alone did not induce an increase in M1 macrophage-related markers. Notably, TECs induced to undergo EMT by TGF-ß treatment released more exosomes than the other groups. Furthermore, it is noteworthy that when we injected exosomes from TECs undergoing EMT into mice, in addition to the high level of inflammatory response and the activation of M1 macrophages, the indicators of EMT and renal fibrosis in mouse kidney tissue were correspondingly elevated. In summary, exosomes from TECs undergoing EMT by TGF-ß treatment induced M1 polarization and led to a positive feedback effect for further EMT and the development of renal fibrosis. Therefore, the obstacle to the release of such exosomes may be a novel therapeutic strategy for CKD.

A knowledge-driven decision support system for remote medical management.

Residential medical digital technology is an emerging discipline combining computer network technology and medical research. Based on the idea of knowledge discovery, this study was designed to construct a decision support system for remote medical management, analyze the need for utilization rate calculations and obtain relevant modeling elements for system design. Specifically, the model constructs a design method for a decision support system for the healthcare management of elderly residents through the use of a utilization rate modeling method based on digital information extraction. In the simulation process, the utilization rate modeling and system design intent analysis are combined to obtain the relevant functions and morphological characteristics that are essential to the system. Using regular usage slices, a higher-precision non-uniform rational B-spline (NURBS) usage rate can be fitted and a surface model with better continuity can be constructed. The experimental results show that the deviation of the NURBS usage rate generated by the boundary division from the original data model can reach test accuracies of 83, 87 and 89%, respectively. It is shown that the method can effectively reduce the modeling error caused by the irregular feature model in the process of modeling the utilization rate of digital information, and that it can ensure the accuracy of the model.

Group I Metabotropic Glutamate Receptors Modulate Motility and Enteric Neural Activity in the Mouse Colon.

Glutamate is the major excitatory neurotransmitter in the central nervous system, and there is evidence that Group-I metabotropic glutamate receptors (mGlu1 and mGlu5) have established roles in excitatory neurotransmission and synaptic plasticity. While glutamate is abundantly present in the gut, it plays a smaller role in neurotransmission in the enteric nervous system. In this study, we examined the roles of Group-I mGlu receptors in gastrointestinal function. We investigated the expression of Grm1 (mGlu1) and Grm5 (mGlu5) in the mouse myenteric plexus using RNAscope in situ hybridization. Live calcium imaging and motility analysis were performed on ex vivo preparations of the mouse colon. mGlu5 was found to play a role in excitatory enteric neurotransmission, as electrically-evoked calcium transients were sensitive to the mGlu5 antagonist MPEP. However, inhibition of mGlu5 activity did not affect colonic motor complexes (CMCs). Instead, inhibition of mGlu1 using BAY 36-7620 reduced CMC frequency but did not affect enteric neurotransmission. These data highlight complex roles for Group-I mGlu receptors in myenteric neuron activity and colonic function.

CAR modulates plasma membrane nano-organization and immune signaling downstream of RALF1-FERONIA signaling pathway.

In Arabidopsis, the receptor-like kinase (RLK) FERONIA (FER) senses peptide ligands in the plasma membrane (PM), modulates plant growth and development, and integrates biotic and abiotic stress signaling for downstream adaptive responses. However, the molecular interplay of these diverse processes is largely unknown. Here, we show that FER, the receptor of Rapid Alkalinization Factor 1 (RALF1), physically interacts with C2 domain ABA-related (CAR) proteins to control the nano-organization of the PM. During this process, the RALF1-FER pathway upregulates CAR protein translation, and then more CAR proteins are recruited to the PM. This acts as a rapid feedforward loop that stabilizes the PM liquid-ordered phase. FER interacts with and phosphorylates CARs, thereby reducing their lipid-binding ability and breaking the feedback regulation at later time points. The formation of the flg22-induced FLS2-BAK1 immune complex, which depends on the integrity of FER-containing nanodomains, is impaired in fer and pentuple car14569 mutant. Together, we propose that the FER-CAR module controls the formation of PM nano-organization during RALF signaling through a self-contained amplifying loop including both positive and negative feedback.

Large-volume and deep brain imaging in rabbits and monkeys using COMPACT two-photon microscopy.

In vivo imaging has been widely used for investigating the structure and function of neurons typically located within ~ 800 µm below the cortical surface. Due to light scattering and absorption, it has been difficult to perform in-vivo imaging of neurons in deep cortical and subcortical regions of large animals with two-photon microscopy. Here, we combined a thin-wall quartz capillary with a GRIN lens attached to a prism for large-volume structural and calcium imaging of neurons located 2 mm below the surface of rabbit and monkey brains. The field of view was greatly expanded by rotating and changing the depth of the imaging probe inside a quartz capillary. Calcium imaging of layer 5/6 neurons in the rabbit motor cortex revealed differential activity of these neurons between quiet wakefulness and slow wave sleep. The method described here provides an important tool for studying the structure and function of neurons located deep in the brains of large animals.