CLC family proteins translocate chloride ions across cell membranes to maintain the membrane potential, regulate the transepithelial Cl- transport, and control the intravesicular pH among different organelles. CLC-7/Ostm1 is an electrogenic Cl-/H+ antiporter that mainly resides in lysosomes and osteoclast ruffled membranes. Mutations in human CLC-7/Ostm1 lead to lysosomal storage disorders and severe osteopetrosis. Here, we present the cryo-electron microscopy (cryo-EM) structure of the human CLC-7/Ostm1 complex and reveal that the highly glycosylated Ostm1 functions like a lid positioned above CLC-7 and interacts extensively with CLC-7 within the membrane. Our complex structure reveals a functionally crucial domain interface between the amino terminus, TMD, and CBS domains of CLC-7. Structural analyses and electrophysiology studies suggest that the domain interaction interfaces affect the slow gating kinetics of CLC-7/Ostm1. Thus, our study deepens understanding of CLC-7/Ostm1 transporter and provides insights into the molecular basis of the disease-related mutations.

Using smart photochromic and luminescent tissues in camouflage/cloaking of natural creatures has inspired efforts to develop synthetic stimuli-responsive materials for data encryption and anticounterfeiting. Although many optical data-encryption materials have been reported, they generally require only one or a simple combination of few stimuli for decryptions and rarely offer output corruptibility that prevents trial-and-error attacks. Here, we report a series of multiresponsive donor-acceptor Stenhouse adducts (DASAs) with unprecedented switching behavior and controlled reversibility via diamine conformational locking and substrate free-volume engineering and their capability of sequential logic encryption (SLE). Being analogous to the digital circuits, the output of DASA gel-based data-encryption system depends not only on the present input stimulus but also on the sequence of past inputs. Incorrect inputs/sequences generate substantial fake information and lead attackers to the point of no return. This work offers new design concepts for advanced data-encryption materials that operate via SLE, paving the path toward advanced encryptions beyond digital circuit approaches.

Chromatin topological organization is instrumental in gene transcription. Gene-enhancer interactions are accommodated in the same CTCF-mediated insulated neighborhoods. However, it remains poorly understood whether and how the 3D genome architecture is dynamically restructured by external signals. Here, we report that LATS kinases phosphorylated CTCF in the zinc finger (ZF) linkers and disabled its DNA-binding activity. Cellular stress induced LATS nuclear translocation and CTCF ZF linker phosphorylation, and altered the landscape of CTCF genomic binding partly by dissociating it selectively from a small subset of its genomic binding sites. These sites were highly enriched for the boundaries of chromatin domains containing LATS signaling target genes. The stress-induced CTCF phosphorylation and locus-specific dissociation from DNA were LATS-dependent. Loss of CTCF binding disrupted local chromatin domains and down-regulated genes located within them. The study suggests that external signals may rapidly modulate the 3D genome by affecting CTCF genomic binding through ZF linker phosphorylation.

Non-small cell lung cancer (NSCLC) is the most commonly diagnosed cancer and the leading cause of cancer death worldwide. More than half of patients with NSCLC die after developing distant metastases, so rapid, minimally invasive prognostic biomarkers are needed to reduce mortality. We used proteomics to identify proteins differentially expressed on extracellular vesicles (EVs) of nonmetastatic 393P and metastatic 344SQ NSCLC cell lines and found that tetraspanin-8 (Tspan8) was selectively enriched on 344SQ EVs. NSCLC cell lines treated with EVs overexpressing Tspan8 also exhibited increased Matrigel invasion. Elevated Tspan8 expression on serum EVs of individuals with stage III premetastatic NSCLC tumors was also associated with reduced distant metastasis-free survival, suggesting that Tspan8 levels on serum EVs may predict future metastasis. This result suggests that a minimally invasive blood test to analyze EV expression of Tspan8 may be of potential value to guide therapeutic decisions for patients with NSCLC and merits further study.

The X and Y chromosomes of channel catfish have the same gene contents. Here, we report allelic hypermethylation of the X chromosome within the sex determination region (SDR). Accordingly, the X-borne hydin-1 gene was silenced, whereas the Y-borne hydin-1 gene was expressed, making monoallelic expression of hydin-1 responsible for sex determination, much like genomic imprinting. Treatment with a methylation inhibitor, 5-aza-dC, erased the epigenetic marks within the SDR and caused sex reversal of genetic females into phenotypic males. After the treatment, hydin-1 and six other genes related to cell cycle control and proliferative growth were up-regulated, while three genes related to female sex differentiation were down-regulated in genetic females, providing additional support for epigenetic sex determination in catfish. This mechanism of sex determination provides insights into the plasticity of genetic sex determination in lower vertebrates and its connection with temperature sex determination where DNA methylation is broadly involved.

Imaging chromatin organization at the molecular-scale resolution remains an important endeavor in basic and translational research. Stochastic optical reconstruction microscopy (STORM) is a powerful superresolution imaging technique to visualize nanoscale molecular organization down to the resolution of ~20 to 30 nm. Despite the substantial progress in imaging chromatin organization in cells and model systems, its routine application on assessing pathological tissue remains limited. It is, in part, hampered by the lack of simple labels that consistently generates high-quality STORM images on the highly processed clinical tissue. We developed a fast, simple, and robust small-molecule fluorescent probe-cyanine 5-conjugated Hoechst-for routine superresolution imaging of nanoscale nuclear architecture on clinical tissue. We demonstrated the biological and clinical significance of imaging superresolved chromatin structure in cancer development and its potential clinical utility for cancer risk stratification.

Eukaryotic cells contain numerous membraneless organelles that are made from liquid droplets of proteins and nucleic acids and that provide spatiotemporal control of various cellular processes. However, the molecular mechanisms underlying the formation and rapid stress-induced alterations of these organelles are relatively uncharacterized. Here, we investigated the roles of DEAD-box helicases in the formation and alteration of membraneless nuclear dicing bodies (D-bodies) in Arabidopsis thaliana We uncovered that RNA helicase 6 (RH6), RH8, and RH12 are previously unidentified D-body components. These helicases interact with and promote the phase separation of SERRATE, a key component of D-bodies, and drive the formation of D-bodies through liquid-liquid phase separations (LLPSs). The accumulation of these helicases in the nuclei decreases upon Turnip mosaic virus infections, which couples with the decrease of D-bodies. Our results thus reveal the key roles of RH6, RH8, and RH12 in modulating D-body formation via LLPSs.

High-throughput nanoscopy becomes increasingly important for unraveling complex biological processes from a large heterogeneous cell population at a nanoscale resolution. High-density emitter localization combined with a large field of view and fast imaging frame rate is commonly used to achieve a high imaging throughput, but the image processing speed and the presence of heterogeneous background in the dense emitter scenario remain a bottleneck. Here, we present a simple non-iterative approach, referred to as WindSTORM, to achieve high-speed high-density emitter localization with robust performance for various image characteristics. We demonstrate that WindSTORM improves the computation speed by two orders of magnitude on CPU and three orders of magnitude upon GPU acceleration to realize online image processing, without compromising localization accuracy. Further, WindSTORM is highly robust to maximize the localization accuracy and minimize the image artifacts in the presence of nonuniform background. WindSTORM paves the way for next generation high-throughput nanoscopy.

Posttraumatic stress disorder (PTSD) is a highly prevalent and debilitating psychiatric disease often accompanied by severe defensive behaviors, preventing individuals from integrating into society. However, the neural mechanisms of defensiveness in PTSD remain largely unknown. Here, we identified that the higher-order thalamus, the posteromedial complex of the thalamus (PoM), was overactivated in a mouse model of PTSD, and suppressing PoM activity alleviated excessive defensive behaviors. Moreover, we found that diminished thalamic inhibition derived from the thalamic reticular nucleus was the major cause of thalamic hyperactivity in PTSD mice. Overloaded thalamic innervation to the downstream cortical area, frontal association cortex, drove abnormal defensiveness. Overall, our study revealed that the malfunction of the higher-order thalamus mediates defensive behaviors and highlighted the thalamocortical circuit as a potential target for treating PTSD-related overreactivity symptoms.