Acute hepatopancreatic necrosis disease (AHPND) is a severe, newly emergent penaeid shrimp disease caused by Vibrio parahaemolyticus that has already led to tremendous losses in the cultured shrimp industry. Until now, its disease-causing mechanism has remained unclear. Here we show that an AHPND-causing strain of V. parahaemolyticus contains a 70-kbp plasmid (pVA1) with a postsegregational killing system, and that the ability to cause disease is abolished by the natural absence or experimental deletion of the plasmid-encoded homologs of the Photorhabdus insect-related (Pir) toxins PirA and PirB. We determined the crystal structure of the V. parahaemolyticus PirA and PirB (PirAvp and PirBvp) proteins and found that the overall structural topology of PirAvp/PirBvp is very similar to that of the Bacillus Cry insecticidal toxin-like proteins, despite the low sequence identity (<10%). This structural similarity suggests that the putative PirABvp heterodimer might emulate the functional domains of the Cry protein, and in particular its pore-forming activity. The gene organization of pVA1 further suggested that pirABvp may be lost or acquired by horizontal gene transfer via transposition or homologous recombination.

Two-pore channels (TPCs) contain two copies of a Shaker-like six-transmembrane (6-TM) domain in each subunit and are ubiquitously expressed in both animals and plants as organellar cation channels. Here, we present the first crystal structure of a vacuolar two-pore channel from Arabidopsis thaliana, AtTPC1, which functions as a homodimer. AtTPC1 activation requires both voltage and cytosolic Ca2+. Ca2+ binding to the cytosolic EF-hand domain triggers conformational changes coupled to the pair of pore-lining inner helices (IS6 helices) from the first 6-TM domains, whereas membrane potential only activates the second voltage-sensing domain (VSD2) whose conformational changes are coupled to the pair of inner helices (IIS6 helices) from the second 6-TM domains. Luminal Ca2+ or Ba2+ can modulate voltage activation by stabilizing VSD2 in the resting state and shifts voltage activation towards more positive potentials. Our Ba2+ bound AtTPC1 structure reveals a voltage sensor in the resting state, providing hitherto unseen structural insight into the general voltage-gating mechanism among voltage-gated channels.

High-conductance Ca2+-gated K+ (BK) channels are essential for many biological processes such as smooth muscle contraction and neurotransmitter release1-4. This group of channels can be activated synergistically by both voltage and intracellular Ca2+, with the large C-terminal intracellular portion being responsible for Ca2+ sensing5-13. Here we present the crystal structure of the entire cytoplasmic region of the human BK channel in a Ca2+ free state. The structure reveals four intracellular subunits, each comprising two tandem RCK domains, assembled into a gating ring similar to that seen in the MthK channel14 and likely representing its physiological assembly. Three Ca2+ binding sites including the Ca2+ bowl are mapped onto the structure based on mutagenesis data. The Ca2+ bowl, located within the second RCK domain, forms an EF-hand like motif and is strategically positioned close to the assembly interface between two subunits. The other two Ca2+ (or Mg2+) binding sites, Asp367 and Glu374/Glu399, are located on the first RCK domain. The Asp367 site has high Ca2+ sensitivity and is positioned in the groove between the N- and C- terminal subdomains of RCK1, whereas the low affinity Mg2+-binding Glu374/Glu399 site is positioned on the upper plateau of the gating ring and close to the membrane. Our structure also contains the linker connecting the transmembrane and intracellular domains, allowing us to dock a voltage-gated K+ channel pore of known structure onto the gating ring with reasonable accuracy and generate a structural model for the full BK channel.

Mitochondrial calcium uptake is crucial to the regulation of eukaryotic Ca 2+ homeostasis and is mediated by the mitochondrial calcium uniporter (MCU). While MCU alone can transport Ca 2+ in primitive eukaryotes, metazoans require an essential single membrane-spanning auxiliary component called EMRE to form functional channels; however, the molecular mechanism of EMRE regulation remains elusive. Here, we present the cryo-EM structure of the human MCU-EMRE complex, which defines the interactions between MCU and EMRE as well as pinpoints the juxtamembrane loop of MCU and extended linker of EMRE as the crucial elements in the EMRE-dependent gating mechanism among metazoan MCUs. The structure also features the dimerization of two MCU-EMRE complexes along an interface at the N-terminal domain (NTD) of human MCU that is a hotspot for post-translational modifications. Thus, the human MCU-EMRE complex, which constitutes the minimal channel components among metazoans, provides a framework for future mechanistic studies on MCU.

The mitochondrial calcium uniporter (MCU) is a highly selective calcium channel localized to the inner mitochondrial membrane. Here, we describe the structure of an MCU orthologue from the fungus Neosartorya fischeri (NfMCU) determined to 3.8 Å resolution by phase-plate cryo-electron microscopy. The channel is a homotetramer with two-fold symmetry in its amino-terminal domain (NTD) that adopts a similar structure to that of human MCU. The NTD assembles as a dimer of dimers to form a tetrameric ring that connects to the transmembrane domain through an elongated coiled-coil domain. The ion-conducting pore domain maintains four-fold symmetry, with the selectivity filter positioned at the start of the pore-forming TM2 helix. The aspartate and glutamate sidechains of the conserved DIME motif are oriented towards the central axis and separated by one helical turn. The structure of NfMCU offers insights into channel assembly, selective calcium permeation, and inhibitor binding.

Cells and viruses possess several known ‘restart' pathways to overcome lesions during DNA replication. However, these ‘bypass' pathways leave a gap in replicated DNA or require recruitment of accessory proteins, resulting in significant delays to fork movement or even cell division arrest. Using single-molecule and ensemble methods, we demonstrate that the bacteriophage T7 replisome is able to directly replicate through a leading-strand cyclobutane pyrimidine dimer (CPD) lesion. We show that when a replisome encounters the lesion, a substantial fraction of DNA polymerase (DNAP) and helicase stay together at the lesion, the replisome does not dissociate and the helicase does not move forward on its own. The DNAP is able to directly replicate through the lesion by working in conjunction with helicase through specific helicase–DNAP interactions. These observations suggest that the T7 replisome is fundamentally permissive of DNA lesions via pathways that do not require fork adjustment or replisome reassembly.

The bacterial Mfd ATPase is increasingly recognized as a general transcription factor that participates in the resolution of transcription conflicts with other processes/roadblocks. This function stems from Mfd's ability to preferentially act on stalled RNA polymerases (RNAPs). However, the mechanism underlying this preference and the subsequent coordination between Mfd and RNAP have remained elusive. Here, using a novel real-time translocase assay, we unexpectedly discovered that Mfd translocates autonomously on DNA. The speed and processivity of Mfd dictate a "release and catch-up" mechanism to efficiently patrol DNA for frequently stalled RNAPs. Furthermore, we showed that Mfd prevents RNAP backtracking or rescues a severely backtracked RNAP, allowing RNAP to overcome stronger obstacles. However, if an obstacle's resistance is excessive, Mfd dissociates the RNAP, clearing the DNA for other processes. These findings demonstrate a remarkably delicate coordination between Mfd and RNAP, allowing efficient targeting and recycling of Mfd and expedient conflict resolution.

Traditional therapeutics have encountered a bottleneck caused by diagnosis delay and subjective and unreliable assessment. Biomarkers can overcome this bottleneck and guide us toward personalized precision medicine for oral squamous cell carcinoma. To achieve this, it is important to efficiently and accurately screen out specific biomarkers from among the huge number of molecules. Progress in omics-based high-throughput technology has laid a solid foundation for biomarker discovery. With credible and systemic biomarker models, more precise and personalized diagnosis and assessment would be achieved and patients would be more likely to be cured and have a higher quality of life. However, this is not straightforward owing to the complexity of molecules involved in tumorigenesis. In this context, there is a need to focus on tumor heterogeneity and homogeneity, which are discussed in detail. In this review, we aim to provide an understanding of biomarker discovery and application for precision medicine of oral squamous cell carcinoma, and have a strong belief that biomarker will pave the road toward future precision medicine.