Neuron Contact Detection Based on Pipette Precise Positioning for Robotic Brain-Slice Patch Clamps.

A patch clamp is the "gold standard" method for studying ion-channel biophysics and pharmacology. Due to the complexity of the operation and the heavy reliance on experimenter experience, more and more researchers are focusing on patch-clamp automation. The existing automated patch-clamp system focuses on the process of completing the experiment; the detection method in each step is relatively simple, and the robustness of the complex brain film environment is lacking, which will increase the detection error in the microscopic environment, affecting the success rate of the automated patch clamp. To address these problems, we propose a method that is suitable for the contact between pipette tips and neuronal cells in automated patch-clamp systems. It mainly includes two key steps: precise positioning of pipettes and contact judgment. First, to obtain the precise coordinates of the tip of the pipette, we use the Mixture of Gaussian (MOG) algorithm for motion detection to focus on the tip area under the microscope. We use the object detection model to eliminate the encirclement frame of the pipette tip to reduce the influence of different shaped tips, and then use the sweeping line algorithm to accurately locate the pipette tip. We also use the object detection model to obtain a three-dimensional bounding frame of neuronal cells. When the microscope focuses on the maximum plane of the cell, which is the height in the middle of the enclosing frame, we detect the focus of the tip of the pipette to determine whether the contact between the tip and the cell is successful, because the cell and the pipette will be at the same height at this time. We propose a multitasking network CU-net that can judge the focus of pipette tips in complex contexts. Finally, we design an automated contact sensing process in combination with resistance constraints and apply it to our automated patch-clamp system. The experimental results show that our method can increase the success rate of pipette contact with cells in patch-clamp experiments.

Robotic Intracellular Pressure Measurement Using Micropipette Electrode.

Intracellular pressure, a key physical parameter of the intracellular environment, has been found to regulate multiple cell physiological activities and impact cell micromanipulation results. The intracellular pressure may reveal the mechanism of these cells' physiological activities or improve the micro-manipulation accuracy for cells. The involvement of specialized and expensive devices and the significant damage to cell viability that the current intracellular pressure measurement methods cause significantly limit their wide applications. This paper proposes a robotic intracellular pressure measurement method using a traditional micropipette electrode system setup. First, the measured resistance of the micropipette inside the culture medium is modeled to analyze its variation trend when the pressure inside the micropipette increases. Then, the concentration of KCl solution filled inside the micropipette electrode that is suitable for intracellular pressure measurement is determined according to the tested electrode resistance-pressure relationship; 1 mol/L KCl solution is our final choice. Further, the measurement resistance of the micropipette electrode inside the cell is modeled to measure the intracellular pressure through the difference in key pressure before and after the release of the intracellular pressure. Based on the above work, a robotic measurement procedure of the intracellular pressure is established based on a traditional micropipette electrode system. The experimental results on porcine oocytes demonstrate that the proposed method can operate on cells at an average speed of 20~40 cells/day with measurement efficiency comparable to the related work. The average repeated error of the relationship between the measured electrode resistance and the pressure inside the micropipette electrode is less than 5%, and no observable intracellular pressure leakage was found during the measurement process, both guaranteeing the measurement accuracy of intracellular pressure. The measured results of the porcine oocytes are in accordance with those reported in related work. Moreover, a 90% survival rate of operated oocytes was obtained after measurement, proving limited damage to cell viability. Our method does not rely on expensive instruments and is conducive to promotion in daily laboratories.

Mimicking Photosynthesis: A Natural Z-Scheme Photocatalyst Constructed from Red Mud Bauxite Waste for Overall Water Splitting.

All-solid-state Z-Scheme photocatalysts have attracted significant attention due to their great potential for solar fuel production. However, delicately coupling two individual semiconductors with a charge shuttle by a material strategy remains a challenge. Herein, we demonstrate a new protocol of natural Z-Scheme heterostructures by strategically engineering the component and interfacial structure of red mud bauxite waste. Advanced characterizations elucidated that the hydrogen-induced formation of metallic Fe enabled the effective Z-Scheme electron transfer from γ-Fe2 O3 to TiO2 , leading to the significantly boosted spatial separation of photo-generated carriers for overall water splitting. To the best of our knowledge, it is the first Z-Scheme heterojunction based on natural minerals for solar fuel production. Thus our work provides a new avenue toward the utilization of natural minerals for advanced catalysis applications.

The toxic effects and possible mechanisms of decabromodiphenyl ethane on mouse oocyte.

Decabromodiphenyl ethane (DBDPE), a widely used new brominated flame retardant, is added into flammable materials to achieve fire retardation. As it is continuously detected in the environment, it has become an emerging environmental pollutant. However, the effects of DBDPE exposure on oocyte maturation and its underlying mechanisms remain unknown. This study found that DBDPE exposure inhibited the rate of germinal vesicle breakdown (GVBD), first polar body extrusion (PBE) and fertilization of mouse oocytes. After 14 h of exposure to DBDPE, metaphase II (MII) oocytes showed that the hardness of zona pellucida (ZP) markedly increased and that the spindle morphology was abnormal. Moreover, DBDPE exposure induced abnormal mitochondrial distribution, mitochondrial dysfunction, and ATP deficiency. Simultaneously, DBDPE exposure down-regulated the expression of antioxidant-related genes (Sod2, Gpx1) and increased the level of reactive oxygen species (ROS) in oocytes. The results of immunofluorescence and qRT-PCR revealed that autophagy occurred in DBDPE-treated oocytes with high expression of autophagy-related protein (LC3) and genes (Lc3, Beclin1). Meanwhile, DBDPE significantly up-regulated the protein (Bax) and mRNA (Bax, Caspase3) levels of pro-apoptosis genes. However, the protein and mRNA expression of anti-apoptosis genes Bcl-2 was dramatically down-regulated in DBDPE-exposed oocytes. Collectively, DBDPE exposure impaired mitochondrial function, causing oxidative damage, autophagy and apoptosis in oocytes.

Ion migration and dark current suppression in quasi-2D perovskite-based X-ray detectors.

Cesium-based lead-free double perovskite materials (Cs2AgBiBr6) have garnered significant attention in the X-ray detection field due to their environment friendly characteristics. However, their substantial ion migration properties lead to large dark currents and detection limits in Cs2AgBiBr6-based X-ray detectors, restricting the detection performance of the device. In terms of process technology, ultrasonic spraying is more suitable than a spin-coating method for fabricating large-area, micron-scale perovskite thick films, with higher cost-effectiveness, which is crucial for X-ray detection. This work introduces a BA+ (BA+ = CH3CH2CH2CH2NH3 +, n-butyl) source into the precursor solution and employs ultrasonic spraying to fabricate quasi-two-dimensional structured polycrystalline (BA)2Cs9Ag5Bi5Br31 perovskite thick films, developing a low-cost, eco-friendly X-ray detector with low dark current density and low detection limit. Characterization results reveal that the ion migration activation energy of (BA)2Cs9Ag5Bi5Br31 reaches 419 meV, approximately 17% higher than that of traditional three-dimensional perovskites, effectively suppressing perovskite ion migration and subsequently reducing the dark current. The (BA)2Cs9Ag5Bi5Br31-based X-ray detectors exhibit high resistivity (about 1.75 × 1010 Ω cm), low dark current density (66 nA cm-2), minimal dark current drift (0.016 pA cm-1 s-1 V-1), and detection limit (138 nGyair s-1), holding considerable promise for applications in low-noise, low-dose X-ray detection.

TiO2/CsPbBr3 S-scheme heterojunctions with highly improved CO2 photoreduction activity through facet-induced Fermi level modulation.

Photocatalytic CO2 reduction is a promising method to resolve the energy shortage problem. Developing photocatalysts with strong redox capabilities is urgently needed to achieve high photocatalytic activity. Herein, we synthesized TiO2/CsPbBr3 S-scheme heterojunctions with modulated internal electric field by facet engineering of TiO2 to control charge transfer for improved photocatalytic activity. Density functional theory (DFT) calculation reveals that there is a wider Fermi level difference between TiO2-(101) and CsPbBr3 than that between TiO2-(001) and CsPbBr3, which will induce more obvious band bending. Subsequently, more efficient spatial separation will occur around the interface. Thus, TiO2-(101)/CsPbBr3 heterostructures effectively reduce CO2 into CO with the selectivity of 90.2 % and reduction rate of 12.5 µmol h-1, 15.6 and 5.6 times improvement than that of 101-TiO2 and TiO2-(001)/CsPbBr3, respectively. This report proposes a feasible idea of employing facet engineering to take the advantage of S-scheme heterojunction.

Integrated transcriptomic and metabolomic analysis sheds new light on adaptation of Pinctada fucata martensii to short-term hypoxic stress.

Analyses of the transcriptome and metabolome were conducted to clarify alterations of key genes and metabolites in pearl oysters following exposure to short-term hypoxic treatment. We totally detected 209 DEGs between the control and hypoxia groups. Enrichment analysis indicated the enrichment of GO terms including "oxidation-reduction process", "ECM organization", "chaperone cofactor-dependent protein refolding", and "ECM-receptor interaction" KEGG pathway by the DEGs. In addition, between the two groups, a total of 28 SDMs were identified, which were implicated in 13 metabolic pathways, such as "phenylalanine metabolism", "D-amino acid metabolism", and "aminoacyl-tRNA biosynthesis". Results suggest that pearl oysters are exposed to oxidative stress and apoptosis under short-term hypoxia. Also, pearl oysters might adapt to short-term hypoxic treatment by increasing antioxidant activity, modulating immune and biomineralization activities, maintaining protein homeostasis, and reorganizing the cytoskeleton. The results of our study help unveil the mechanisms by which pearl oysters respond adaptively to short-term hypoxia.

Exogenous melatonin protects preimplantation embryo development from decabromodiphenyl ethane-induced circadian rhythm disorder and endogenous melatonin reduction.

Decabromodiphenyl ethane (DBDPE) is a novel flame retardant that is widely used in plastics, electronic products, building materials and textiles. Our previous studies have revealed the oocyte toxicity of DBDPE, but the effect of DBDPE on preimplantation embryo development has not been reported. Here, we investigated whether and how DBDPE exposure affects preimplantation embryo development. Adult female mice were orally exposed to DBDPE (0, 5, 50, 500 µg/kg bw/day) for 14 days. First, we found that after DBDPE exposure, mice showed obvious circadian rhythm disorder. Moreover, the development of preimplantation embryos was inhibited in DBDPE-exposed mice after pregnancy. Then, we further explored and revealed that DBDPE exposure reduced the endogenous melatonin (MLT) level during pregnancy, thereby inhibiting the development of preimplantation embryos. Furthermore, we discovered that exogenous MLT supplementation (15 mg/kg bw/day) rescued the inhibition of preimplantation embryo development induced by DBDPE, and a mechanistic study demonstrated that exogenous MLT inhibited the overexpression of ROS and DNA methylation at the 5-position of cytosine (5-mC) in DBDPE-exposed preimplantation embryos. Simultaneously, MLT ameliorated the DBDPE-induced mitochondrial dysfunction by increasing the mitochondrial membrane potential (MMP), ATP, and Trp1 expression. Additionally, MLT restored DBDPE-induced changes in zona pellucida (ZP) hardness and trophectoderm (TE) cortical tension. Finally, the protective effect of MLT on embryos ameliorated the adverse reproductive outcomes (dead fetus, fetus with abnormal liver, fetal weight loss) induced by DBDPE. Collectively, DBDPE induced preimplantation embryo damage leading to adverse reproductive outcomes, and MLT has emerged as a potential tool to rescue adverse reproductive outcomes induced by DBDPE.

6-benzylaminopurine exposure induced development toxicity and behaviour alteration in zebrafish (Danio rerio).

6-benzylaminopurine (6-BA) is one of the first synthetic hormones and has been widely used in fruit cultivation, gardening and agriculture. However, excessive use of 6-BA will cause potential harm to the environment and humans. Therefore, our research focused on assessing the impact of 6-BA on the development and neurobehavior of zebrafish. The results showed that 6-BA had little effect on the embryos from 2 hpf to 10 hpf. However, delayed development, decreased survival and hatchability were observed under 30 and 40 mg/L 6-BA from 24 hpf. 6-BA also reduced surface tension of embryonic chorions at 24 hpf. In addition, 6-BA caused abnormal morphology and promoted the accumulation of oxidative stress. Transcription of genes in connection with development and oxidative stress was also strikingly altered. Results of movement assay showed that zebrafish were less active and their behavior was significantly inhibited under the 20 and 30 mg/L 6-BA treatments. Locomotion-related genes th and mao were down-regulated by gradient, while the transcription of dbh was upregulated at a low concentration (2 mg/L) but decreased as the concentration increased. Moreover, 6-BA exposure caused increased arousal and decreased sleep. Sleep/wake related genes hcrt and hcrtr2 were upregulated, but decreased at 30 mg/L, while the mRNA level of aanat2 was reduced in a concentration-dependent manner. To sum up, our results showed that 6-BA induced developmental toxicity, promoted the accumulation of oxidative stress, and damaged locomotion and sleep/wake behavior.

Robotic Label-Free Precise Oocyte Enucleation for Improving Developmental Competence of Cloned Embryos.

OBJECTIVE: The invisibility of domestic oocyte nucleus in bright field currently forces operators to blindly aspirate nucleus out in oocyte enucleation, usually causing large cytoplasm losses and poor developmental competences of cloned embryos. Although fluorescent labeling of nucleus allows for nucleus localization, the involved photobleaching problems and barriers to the execution of enucleation process limit its online-application in oocyte enucleation. This paper reports a novel label-free oocyte enucleation method for precise removal of the nucleus with less cytoplasm loss. METHODS: The relative positions between the injection pipette and nucleus for complete removal of nucleus with less cytoplasm loss were determined through a finite element modeling of nucleus aspiration. To position injection pipette to the above positions relative to nucleus, the appropriate oocyte orientation and trajectory of injection pipette inside oocyte were derived according to the offline-calibrated 3-D distribution of nucleus and the simulated dynamic drift of nucleus that occurs as injection pipette is maneuvered inside oocyte. Finally, a robotic label-free precise enucleation procedure was established. RESULTS: The experimental results on more than 1000 porcine oocytes proved that this system is capable of reducing cytoplasm loss by 60% at the same level of enucleation success rate and almost doubling the cleavage rate of clone embryos in comparison to blind aspiration method. CONCLUSIONS: The results prove that our method significantly improves the developmental competence of cloned embryos in comparison to manual enucleation method. SIGNIFICANCE: Our method is expected to improve the extremely low success rate of animal cloning in the future.

Sequence analysis of digestion-resistant peptides may be an efficient strategy for studying the linear epitopes of Jug r 1, the major walnut allergen.

Jug r 1, the major allergen of walnut, triggers severe allergic reactions through epitopes. Hence, research on the efficient strategy for analyzing the linear epitopes of Jug r 1 are necessary. In this work, bioinformatics analysis was used to predict the linear epitopes of Jug r 1. Overlapping peptide synthesis was used to map linear epitopes. In vitro simulated gastrointestinal digestion and HPLC-MS/MS were used to identify digestion-resistant peptides. The results showed that six predicted linear epitopes were AA28-35, AA42-49, AA55-62, AA65-73, AA97-104, and AA109-121. AA16-30 and AA125-139 were identified by the sera of walnut allergic patients. Five digestion-resistant peptides were AA19-33, AA40-45, AA54-74, AA96-106, and AA117-137. The predicted results only included one of the linear epitopes identified by sera, while the digestion-resistant peptides covered all. Therefore, the digestion-resistant property of food allergens may be a promising direction for studying the linear epitopes of Jug r 1.

Low-grade myofibroblastic sarcomas of the maxilla.

Low-grade myofibroblastic sarcoma (LGMS) is a distinct mesenchymal myofibroblastic malignancy. The tumor may occur at a variety of sites, but is particularly associated with the head and neck. Of the two maxillary sarcomas that were analyzed in the present study, one was misdiagnosed as an inflammatory myofibroblastic tumor during pre-operative excision biopsy, and later presented with a different immunophenotype upon recurrence. Representative paraffin blocks from formalin-fixed tissues were selected from each patient and designated as case 1 and case 2. Immunohistochemical studies were performed on 3-µm thick sections using primary antibodies against α-smooth muscle actin (α-SMA), muscle-specific actin (MSA), desmin, vimentin, calponin, h-caldesmon, fibronectin, cytokeratin, cluster of differentiation 34 (CD34), S-100 protein, anaplastic lymphoma kinase (ALK), epithelial membrane antigen (EMA) and Ki-67. Immunohistochemistry was performed using the streptavidin-biotin-peroxidase complex method. The tumor cells from the two maxillary LGMSs, including the recurrent lesion, were positive for vimentin and fibronectin, and negative for S-100 protein, CD34, EMA, h-caldesmon, ALK, MSA and calponin. The tumor cells from case 1 demonstrated positive staining for α-SMA protein and negative staining for desmin. By contrast, the tumor cells from the primary lesion in case 2 presented with negative staining for α-SMA and positive staining for desmin, while the cells of the recurrent lesion were α-SMA-positive and desmin-negative. The present study concluded that cases of LGMS with immunoprofile alterations are predictive of relatively poor prognoses.

[Study on nutritional characteristics of Pogostemon cablin].

This paper studied the soil nutrient status, the nutrient contents of Pogostemon cablin nutritional organs, the relationship between soil available nutrients and nutritional elements contents of Pogostemon cablin, the nutritional elements contents of leaves and their proportions in different growth periods of the plant, the reuse of the nutritional elements of leaves in Pogostemon cablin base of Guangdong Institute of Chinese Medicine. The results provided scientific basis for the research on the fertilization and special fertilizers of Pogostemon cablin.