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Determining high-resolution structures of biological macromolecules amassing less than 100 kilodaltons (kDa) has been a longstanding goal of the cryo-electron microscopy (cryo-EM) community. While the Volta phase plate has enabled visualization of specimens in this size range, this instrumentation is not yet fully automated and can present technical challenges. Here, we show that conventional defocus-based cryo-EM methodologies can be used to determine high-resolution structures of specimens amassing less than 100 kDa using a transmission electron microscope operating at 200 keV coupled with a direct electron detector. Our ~2.7 Å structure of alcohol dehydrogenase (82 kDa) proves that bound ligands can be resolved with high fidelity to enable investigation of drug-target interactions. Our ~2.8 Å and ~3.2 Å structures of methemoglobin demonstrate that distinct conformational states can be identified within a dataset for proteins as small as 64 kDa. Furthermore, we provide the sub-nanometer cryo-EM structure of a sub-50 kDa protein. Despite many recent advances in cryo-EM, imaging smaller macromolecules (below 100 kDa) has remained a challenge. Here the authors show that biological specimens amassing <100 kDa can be resolved to better than 3 Å resolution using conventional defocus-based single-particle analysis methods.

Transient receptor potential melastatin (TRPM) ion channels constitute the largest TRP subfamily and are involved in many physiological processes. TRPM8 is the primary cold and menthol sensor, and TRPM4 is associated with cardiovascular disorders. Yin et al. and Autzen et al. shed light on the general architecture of the TRPM subfamily by solving the structures of TRPM8 and TRPM4, respectively (see the Perspective by Bae et al. ). The three-layered architecture of the TRPM8 channel provides the framework for understanding the mechanisms of cold and menthol sensing. The two distinct closed states of TRPM4, with and without calcium, reveal a calcium-binding site and calcium-binding-induced conformational changes. Science , this issue p. 237 , p. 228 ; see also p. 160 The structure of an ion channel provides a framework for understanding the mechanisms of cold and menthol sensing. Transient receptor potential melastatin (TRPM) cation channels are polymodal sensors that are involved in a variety of physiological processes. Within the TRPM family, member 8 (TRPM8) is the primary cold and menthol sensor in humans. We determined the cryo–electron microscopy structure of the full-length TRPM8 from the collared flycatcher at an overall resolution of ~4.1 ångstroms. Our TRPM8 structure reveals a three-layered architecture. The amino-terminal domain with a fold distinct among known TRP structures, together with the carboxyl-terminal region, forms a large two-layered cytosolic ring that extensively interacts with the transmembrane channel layer. The structure suggests that the menthol-binding site is located within the voltage-sensor–like domain and thus provides a structural glimpse of the design principle of the molecular transducer for cold and menthol sensation.

Maintaining the correct balance of calcium concentrations between the cytosol and the mitochondria is essential for cellular physiology. A calcium-selective channel called the mitochondrial calcium uniporter (MCU) mediates calcium entry into mitochondria. Yoo et al. report the high-resolution structure of MCU from Neurospora crassa. The channel is formed by four MCU protomers with differing symmetry between the soluble and membrane domains. The structure, together with mutagenesis, suggests that two acidic rings inside the channel provide the selectivity for calcium. Science , this issue p. 506 The structure of the mitochondrial calcium uniporter reveals a tetrameric architecture and the molecular framework underlying calcium selectivity. Calcium transport plays an important role in regulating mitochondrial physiology and pathophysiology. The mitochondrial calcium uniporter (MCU) is a calcium-selective ion channel that is the primary mediator for calcium uptake into the mitochondrial matrix. Here, we present the cryo–electron microscopy structure of the full-length MCU from Neurospora crassa to an overall resolution of ~3.7 angstroms. Our structure reveals a tetrameric architecture, with the soluble and transmembrane domains adopting different symmetric arrangements within the channel. The conserved W-D-Φ-Φ-E-P-V-T-Y sequence motif of MCU pore forms a selectivity filter comprising two acidic rings separated by one helical turn along the central axis of the channel pore. The structure combined with mutagenesis gives insight into the basis of calcium recognition.

Lung cancer is the second cancer and the leading cause of tumor-related mortality worldwide. Angiogenesis is a crucial hallmark of cancer development and a promising target in lung cancer. However, the anti-angiogenic drugs currently used in the clinic do not achieve long-term efficacy and are accompanied by severe adverse reactions. Therefore, the development of novel anti-angiogenic therapeutic approaches for lung cancer is urgently needed. Non-coding RNAs (ncRNAs) participate in multiple biological processes in cancers, including tumor angiogenesis. Many studies have demonstrated that ncRNAs play crucial roles in tumor angiogenesis. This review discusses the regulatory functions of different ncRNAs in lung cancer angiogenesis, focusing on the downstream targets and signaling pathways regulated by these ncRNAs. Additionally, given the recent trend towards utilizing ncRNAs as cancer therapeutics, we also discuss the tremendous potential applications of ncRNAs as biomarkers or novel anti-angiogenic tools in lung cancer.

Multipotent mesenchymal stem cells (MSCs) have high self-renewal and multi-lineage differentiation potentials and low immunogenicity, so they have attracted much attention in the field of regenerative medicine and have a promising clinical application. MSCs originate from the mesoderm and can differentiate not only into osteoblasts, cartilage, adipocytes, and muscle cells but also into ectodermal and endodermal cell lineages across embryonic layers. To design cell therapy for replacement of damaged tissues, it is essential to understand the signaling pathways, which have a major impact on MSC differentiation, as this will help to integrate the signaling inputs to initiate a specific lineage. Hedgehog (Hh) signaling plays a vital role in the development of various tissues and organs in the embryo. As a morphogen, Hh not only regulates the survival and proliferation of tissue progenitor and stem populations but also is a critical moderator of MSC differentiation, involving tri-lineage and across embryonic layer differentiation of MSCs. This review summarizes the role of Hh signaling pathway in the differentiation of MSCs to mesodermal, endodermal, and ectodermal cells.

Transient receptor potential vanilloid channel 3 (TRPV3), a member of the thermosensitive TRP (thermoTRPV) channels, is activated by warm temperatures and serves as a key regulator of normal skin physiology through the release of pro-inflammatory messengers. Mutations in trpv3 have been identified as the cause of the congenital skin disorder, Olmsted syndrome. Unlike other members of the thermoTRPV channel family, TRPV3 sensitizes upon repeated stimulation, yet a lack of structural information about the channel precludes a molecular-level understanding of TRPV3 sensitization and gating. Here, we present the cryo-electron microscopy structures of apo and sensitized human TRPV3, as well as several structures of TRPV3 in the presence of the common thermoTRPV agonist 2-aminoethoxydiphenyl borate (2-APB). Our results show α-to-π-helix transitions in the S6 during sensitization, and suggest a critical role for the S4-S5 linker π-helix during ligand-dependent gating. Transient receptor potential vanilloid channel 3 (TRPV3) responds to temperature and sensitizes upon repeated stimulation with either heat or agonists. Here authors present the cryo-EM structures of apo and sensitized human TRPV3 and describe the structural basis of sensitization.

The transient receptor potential melastatin 2 (TRPM2) channel plays a key role in redox sensation in many cell types. Channel activation requires binding of both ADP-ribose (ADPR) and Ca 2+ . The recently published TRPM2 structures from Danio rerio in the ligand-free and the ADPR/Ca 2+ -bound conditions represent the channel in closed and open states, which uncovered substantial tertiary and quaternary conformational rearrangements. However, it is unclear how these rearrangements are achieved within the tetrameric channel during channel gating. Here we report the cryo-electron microscopy structures of Danio rerio TRPM2 in the absence of ligands, in complex with Ca 2+ alone, and with both ADPR and Ca 2+ , resolved to ~4.3 Å, ~3.8 Å, and ~4.2 Å, respectively. In contrast to the published results, our studies capture ligand-bound TRPM2 structures in two-fold symmetric intermediate states, offering a glimpse of the structural transitions that bridge the closed and open conformations. The transient receptor potential channel member 2 (TRPM2) ion channel has a function in redox-dependent signaling. Here the authors present the cryo-EM structures of zebrafish TRPM2 in the ligand-free form, with Ca 2+ and both ADP-ribose/Ca 2+ and observe two-fold symmetric quaternary structure rearrangements in the ligand-bound structures that likely represent intermediate gating states.

A high proportion of distributed generators (DGs) connected to the distribution network causes a significant change in the normal and fault currents of the system as well as in the linearization of the characteristics. It is difficult to adapt to conventional protection. This paper theoretically analyzes the possible impact of fault current characteristics on traditional protection based on single-ended informativeness after connecting to DGs. From the perspective of protection action, the evaluation index system of DG protection is established by considering the maximum short-circuit current output from DG. Combined with the relay protection requirements, the calculation method of evaluation indexes is given concerning the protection characteristics and expert experience. An analytic hierarchy process (AHP) and a CRITIC combination assignment method based on the principle of minimum information identification are proposed. The scores of different types of protection before and after DG access are calculated using the proposed methodology employing a typical distribution network example. The proposed method can quantitatively obtain the distribution network protection adaptability boundary. In the actual calculation example selected in this paper, a DG can reasonably improve the adaptability of the three-stage current protection when it increases the current amplitude at a penetration rate of 50%; the DG needs to adjust the three-stage current protection rectification value when it decreases the current amplitude at a penetration rate of 20%; and adaptive overcurrent protection and inverse time limit current protection need to be adjusted when the penetration rate of DG is 50%. Compared with the traditional protection evaluation method, the method adopted in this paper can intuitively derive the weak link between protection handling faults after DG access as well as the appropriate capacity of DG to improve protection performance. It can provide a powerful reference for the optimization of protection schemes after the high percentage of DG access.

Previous studies attribute asymmetries across the East Pacific Rise to horizontal temperature or pressure gradients in the deep asthenosphere caused by the Pacific Superswell, which, however, cannot explain asymmetries observed across the Juan de Fuca Ridge. Here, we provide seismic evidence that the asymmetric seafloor depth across the Juan de Fuca Ridge is primarily caused by thermal buoyancy due to lithospheric heating and thinning. Based on a seismic model generated from Rayleigh wave measurements, we demonstrate that the seafloor depth on the western flank of the ridge, which is shallower ( > 150 m) than the prediction from the plate age, agrees with the relatively younger apparent thermal age inferred from the seismic data, whereas the buoyancy of the deeper asthenosphere alone can only account for <25% of the rise. On the eastern flank, both plate age and apparent thermal age are consistent with observed seafloor depth.

PurposeTo evaluate the effects of different mechanical ventilation modes on critical patients.MethodsPubMed, Embase, Web of science, and Cochrane Library databases were searched from their inception to November 15, 2022 for randomized controlled trials on the application of different mechanical ventilation modes in critical patients. Two researchers independently screened the literature, extracted data, and assessed the risk of bias in the included studies. R4.2.1 was used for this network meta-analysis.ResultsTwenty-eight RCTs involving 3,189 patients were included. The interventions in these RCTs included NAVA (neurally adjusted ventilatory assist), PAV (proportional assist ventilation), ASV (adaptive support ventilation), Smartcare/PS (Smartcare/pressure support), PSV (pressure support ventilation), PSV_ATC (pressure support ventilation_automatic tube compensation), and SIMV (synchronized intermittent mandatory ventilation). The network meta-analysis showed that, compared with the PSV group, there was no significant difference in duration of mechanical ventilation, duration of ICU stay, and hospital stay between NAVA, SIMV, AVS, PAV, Smartcare/PS, and PSV_ATC groups. Compared with PSV, PAV improved the success rate of withdrawal of ventilator [OR = 3.07, 95%CI (1.21, 8.52)]. Compared with PSV and PAV, NAVA reduced mortality in the ICU [OR = 0.63, 95%CI (0.43, 0.93); OR = 0.45, 95%CI (0.21, 0.97)].ConclusionNAVA can reduce mortality in ICU, and PAV may increase the risk of withdrawal of the ventilator. There was no significant difference between PSV and other mechanical ventilation modes (NAVA, SIMV, AVS, PAV, Smartcare/PS, and PSV_ATC) in the duration of mechanical ventilation, duration of ICU stay, or hospital stay. Due to the limitations, more high-quality studies are needed to verify these findings.