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The patch clamp technique has become the gold standard for neuron electrophysiology research in brain science. Brain slices have been widely utilized as the targets of the patch clamp technique due to their higher optical transparency compared to a live brain and their intercellular connectivity in comparison to cultured single neurons. However, the narrow working space, small scope, and depth of the field of view make the positioning of the operation’s micropipette to the target neuron a time-consuming task reliant on a high level of experience, significantly slowing down operation of the patch clamp technique in brain slices. Further, the current poor controllability in gigaseal formation, which is the key to electrophysiology signal recording, significantly lowers the patch clamp success rate. In this paper, a stepwise navigation of the micropipette is conducted to accelerate the positioning process of the micropipette tip to the target neuron in the brain slice. Then, a fuzzy proportional–integral–derivative controller is designed to control the gigaseal formation process along a designed resistance curve. The experimental results demonstrate an almost doubled patch clamp technique speed, with a 25% improvement in the success rate compared to the conventional manual method. The above advantages may promote the application of our method in brain science research based on brain slice platforms.

Under live-working conditions in distribution networks, the electric, magnetic, and thermal fields generated by current-carrying conductors inevitably affect the contact pressure between the conductor and the connecting fitting, thereby influencing the bolt preload of the fitting. Therefore, this study focuses on evaluating bolt preload in T-type clamps and investigating the influence of current under live-working conditions on that preload. A multiphysics coupling-based approach is proposed for the accurate determination of bolt preload in T-type clamps. First, the governing equations and boundary conditions for the bolt preload of the T-type clamp are derived based on elastic mechanics theory. Then, an iterative coupled electro–magneto–thermal multiphysics model is established, and the governing equations and boundary conditions for bolt preload, accounting for current-carrying effects, are derived. Finally, the preload equations are solved using the finite element method, enabling accurate evaluation of bolt preload under live-working conditions. A comparative analysis between experimental and simulation results is also conducted. Compared with experimental results from the Zhejiang Shangjian Electric Power Testing Institute, the proposed method has an average error of 5.82%. In comparison with methods that neglect multiphysics coupling, the average error is reduced by 11.41%. The proposed method enables an accurate determination of bolt preload in T-type clamps under live-working conditions in distribution networks.

• We combined the patch-clamp technique with ratiometric fluorescence imaging using the proton-responsive dye BCECF as a luminal probe. • Upon application of a steep cytosol-directed potassium ion (K⁺) gradient in Arabidopsis mesophyll vacuoles, a strong and reversible acidification of the vacuolar lumen was detected, whereas no associated electrical currents were observed, in agreement with electroneutral cation/H⁺ exchange. • Our data show that this acidification was generated by NHX antiport activity, because: it did not distinguish between K⁺ and sodium (Na⁺) ions; it was sensitive to the NHX inhibitor benzamil; and it was completely absent in vacuoles from nhx1 nhx2 double knockout plants. Our data further show that NHX activity could be reversed, was voltage-independent and specifically impaired by the low-abundance signaling lipid PI(3,5)P₂, which may regulate salt accumulation in plants by acting as a common messenger to coordinately shut down secondary active carriers responsible for cation and anion uptake inside the vacuole. • Finally, we developed a theory based on thermodynamics, which supports the data obtained by our novel experimental approach. • This work, therefore, represents a proof-of-principle that can be applied to the study of proton- dependent exchangers from plants and animals, which are barely detectable using conventional techniques.

The metal rubber clamp, serving as a crucial supporting component, often generates non-linear forces and brings complex dynamics to the pipeline system. However, the nonlinearity of metal rubber clamp in pipeline system has been rarely incorporated in open research. Therefore, a novel nonlinear clamp model with four degrees of freedom is proposed based on the genetic algorithm and the finite element method. The nonlinear parameters are identified by the genetic algorithm, where the objective function is defined as the error between experiment and simulation. The proposed nonlinear clamp–pipeline model is verified through modal tests and vibration response experiments conducted under various boundary conditions. Furthermore, the pipeline response tests of different clamp types are conducted to evaluate the supporting performance. The results show that when the excitation amplitude increases from 0.5 g to 3 g, the resonance frequency offsets of the three clamp types are different from each other. The frequency offset of the metal rubber clamp is 8.2 Hz, the copper clamp has a frequency offset of 5.5 Hz, and the clamp without metal rubber exhibits a frequency offset of 5 Hz. The soft nonlinearity of the metal rubber clamp is most obvious, followed by the copper clamp. The clamp without metal rubber exhibits the weakest level of soft nonlinearity. The numerical results are basically consistent with the experimental data. The proposed model effectively simulates the nonlinear effects of the pipeline system under different clamps, enabling more accurate prediction of vibration responses.

To address the issues of excessive observation equipment and discontinuous observation points in tethered discrete profiling observations, a mobile profiling observation platform with mooring and cable-traversing capabilities has been designed. This platform is propelled by electric thrusters and incorporates a mooring mechanism that clamps onto the tether to achieve clinging positioning. A dynamic simulation model of the mobile platform was established to analyse the thrust required for traversing the cable and the clamping force and torque needed during mooring. The study reveals that the magnitude of horizontal flow velocity is the primary factor influencing the thruster thrust and clamping torque, both of which increase with higher flow velocities. The designed cable-traversing mobile observation platform provides a methodological approach for conducting profiling observations on tethered marine observation platforms.

This Technical Report describes new methods of transcranial magnetic stimulation (TMS) in non-human primates. By combining single neuron recording with a modified TMS coil with focused stimulation in alert macaques, the authors show that this method can reduce stimulation artifact and allow investigation into the neuronal mechanisms of TMS. Transcranial magnetic stimulation (TMS) is a widely used, noninvasive method for stimulating nervous tissue, yet its mechanisms of effect are poorly understood. Here we report new methods for studying the influence of TMS on single neurons in the brain of alert non-human primates. We designed a TMS coil that focuses its effect near the tip of a recording electrode and recording electronics that enable direct acquisition of neuronal signals at the site of peak stimulus strength minimally perturbed by stimulation artifact in awake monkeys ( Macaca mulatta ). We recorded action potentials within ∼1 ms after 0.4-ms TMS pulses and observed changes in activity that differed significantly for active stimulation as compared with sham stimulation. This methodology is compatible with standard equipment in primate laboratories, allowing easy implementation. Application of these tools will facilitate the refinement of next generation TMS devices, experiments and treatment protocols.

Structural maintenance of chromosomes (SMC) complexes are essential for chromatin organization and functions throughout the cell cycle. The cohesin and condensin SMCs fold and tether DNA, while Smc5/6 directly promotes DNA replication and repair. The functions of SMCs rely on their abilities to engage DNA, but how Smc5/6 binds and translocates on DNA remains largely unknown. Here, we present a 3.8 Å cryogenic electron microscopy (cryo-EM) structure of DNA-bound Saccharomyces cerevisiae Smc5/6 complex containing five of its core subunits, including Smc5, Smc6, and the Nse1-3-4 subcomplex. Intricate interactions among these subunits support the formation of a clamp that encircles the DNA double helix. The positively charged inner surface of the clamp contacts DNA in a nonsequence-specific manner involving numerous DNA binding residues from four subunits. The DNA duplex is held up by Smc5 and 6 head regions and positioned between their coiled-coil arm regions, reflecting an engaged-head and open-arm configuration. The Nse3 subunit secures the DNA from above, while the hook-shaped Nse4 kleisin forms a scaffold connecting DNA and all other subunits. The Smc5/6 DNA clamp shares similarities with DNA-clamps formed by other SMCs but also exhibits differences that reflect its unique functions. Mapping cross-linking mass spectrometry data derived from DNA-free Smc5/6 to the DNA-bound Smc5/6 structure identifies multi-subunit conformational changes that enable DNA capture. Finally, mutational data from cells reveal distinct DNA binding contributions from each subunit to Smc5/6 chromatin association and cell fitness. In summary, our integrative study illuminates how a unique SMC complex engages DNA in supporting genome regulation.

Fruiting body development in  Agaricomycetes  represents the most complex and unclear process in the fungi. Mating type pathways ( A and B ) and transcription factors are important regulators in the sexual development of mushrooms. It is known that clampless1 ( clp1 ) is an additional gene that participate under the homeodomain ( HD ) genes in the mat A pathway and clp1 inactivation blocks clamps formation in Coprinopsis cinerea . In this study we identified and analyzed a homologous Fvclp1 gene in the edible mushroom Flammulina velutipes . The coding sequence of the Fvclp1 was 1011 bp without intron interruption, encoding a protein of 336 amino acids. To exhibit the role of Fvclp1 in clamp development and fruiting body formation, knockdown and overexpression mutants were prepared. No significant difference was observed in the monokaryotic hyphal morphology of overexpression and knockdown transformants. In the dikaryotic hyphae from the compatible crossings between the wild-type L22 strain and Fvclp1 knockdown or overexpression mutants, clamp connections developed. However, knockdown mutants could generate fewer fruiting bodies than the wild-type strain. On the contrary, reduced mycelial growth rate but improved fruiting ability was observed in the dikaryotic Fvclp1 overexpression mutants as compared to the wild-type strain. These results indicate that Fvclp1 is necessary and actively involved in fruiting body development in F. velutipes . Overall, these findings suggest that further studies on the function of Fvclp1 would advance our understanding of sexual reproduction and fruiting body development in edible mushrooms.

Synaptotagmin 1 (Syt1) synchronizes neurotransmitter release to action potentials (APs) acting as the fast Ca2+ release sensor and as the inhibitor (clamp) of spontaneous and delayed asynchronous release. While the Syt1 Ca2+ activation mechanism has been wellcharacterized, how Syt1 clamps transmitter release remains enigmatic. Here we show that C2B domain-dependent oligomerization provides the molecular basis for the Syt1 clamping function. This follows from the investigation of a designed mutation (F349A), which selectively destabilizes Syt1 oligomerization. Using a combination of fluorescence imaging and electrophysiology in neocortical synapses, we show that Syt1F349A is more efficient than wild-type Syt1 (Syt1WT) in triggering synchronous transmitter release but fails to clamp spontaneous and synaptotagmin 7 (Syt7)-mediated asynchronous release components both in rescue (Syt1−/− knockout background) and dominant-interference (Syt1+/+ background) conditions. Thus, we conclude that Ca2+-sensitive Syt1 oligomers, acting as an exocytosis clamp, are critical for maintaining the balance among the different modes of neurotransmitter release.

A distinct set of channels and transporters regulates the ion fluxes across the lysosomal membrane. Malfunctioning of these transport proteins and the resulting ionic imbalance is involved in various human diseases, such as lysosomal storage disorders, cancer, as well as metabolic and neurodegenerative diseases. As a consequence, these proteins have stimulated strong interest for their suitability as possible drug targets. A detailed functional characterization of many lysosomal channels and transporters is lacking, mainly due to technical difficulties in applying the standard patch-clamp technique to these small intracellular compartments. In this review, we focus on current methods used to unravel the functional properties of lysosomal ion channels and transporters, stressing their advantages and disadvantages and evaluating their fields of applicability.