KIF11 UFMylation Maintains Photoreceptor Cilium Integrity and Retinal Homeostasis.

The photoreceptor cilium is vital for maintaining the structure and function of the retina. However, the molecular mechanisms underlying the photoreceptor cilium integrity and retinal homeostasis are largely unknown. Herein, it is shown that kinesin family member 11 (KIF11) localizes at the transition zone (connecting cilium) of the photoreceptor and plays a crucial role in orchestrating the cilium integrity. KIF11 depletion causes malformations of both the photoreceptor ciliary axoneme and membranous discs, resulting in photoreceptor degeneration and the accumulation of drusen-like deposits throughout the retina. Mechanistic studies show that the stability of KIF11 is regulated by an interplay between its UFMylation and ubiquitination; UFMylation of KIF11 at lysine 953 inhibits its ubiquitination by synoviolin 1 and thereby prevents its proteasomal degradation. The lysine 953-to-arginine mutant of KIF11 is more stable than wild-type KIF11 and also more effective in reversing the ciliary and retinal defects induced by KIF11 depletion. These findings identify a critical role for KIF11 UFMylation in the maintenance of photoreceptor cilium integrity and retinal homeostasis.

Controlling Nucleation and Crystallization of CsPbI3 Perovskites for Efficient Inverted Solar Cells.

The unfavorable morphology and high crystallization temperature (Tc ) of inorganic perovskites pose a significant challenge to their widespread application in photovoltaics. In this study, an effective approach is proposed to enhance the morphology of cesium lead triiodide (CsPbI3 ) while lowering its Tc . By introducing dimethylammonium acetate into the perovskite precursor solution, a rapid nucleation stage is facilitated, and significantly enhances the crystal growth of the intermediate phase at low annealing temperatures, followed by a slow crystal growth stage at higher annealing temperatures. This results in a uniform and dense morphology in CsPbI3 perovskite films with enhanced crystallinity, simultaneously reducing the Tc from 200 to 150 °C. Applying this approach in positive-intrinsic-negative (p-i-n) inverted cells yields a high power conversion efficiency of 19.23%. Importantly, these cells exhibit significantly enhanced stability, even under stress at 85 °C.

Constructing Ultra-Shallow Near-Edge States for Efficient and Stable Perovskite Solar Cells.

Electronic band structure engineering of metal-halide perovskites (MHP) lies at the core of fundamental materials research and photovoltaic applications. However, reconfiguring the band structures in MHP for optimized electronic properties remains challenging. This article reports a generic strategy for constructing near-edge states to improve carrier properties, leading to enhanced device performances. The near-edge states are designed around the valence band edge using theoretical prediction and constructed through tailored material engineering. These states are experimentally revealed with activation energies of around 23 milli-electron volts by temperature-dependent time-resolved spectroscopy. Such small activation energies enable prolonged carrier lifetime with efficient carrier transition dynamics and low non-radiative recombination losses, as corroborated by the millisecond lifetimes of microwave conductivity. By constructing near-edge states in positive-intrinsic-negative inverted cells, a champion efficiency of 25.4% (25.0% certified) for a 0.07-cm2 cell and 23.6% (22.7% certified) for a 1-cm2 cell is achieved. The most stable encapsulated cell retains 90% of its initial efficiency after 1100 h of maximum power point tracking under one sun illumination (100 mW cm-2) at 65 °C in ambient air.

EML4-ALK fusions drive lung adeno-to-squamous transition through JAK-STAT activation.

Human lung adenosquamous cell carcinoma (LUAS), containing both adenomatous and squamous pathologies, exhibits strong cancer plasticity. We find that ALK rearrangement is detectable in 5.1-7.5% of human LUAS, and transgenic expression of EML4-ALK drives lung adenocarcinoma (LUAD) formation initially and squamous transition at late stage. We identify club cells as the main cell-of-origin for squamous transition. Through recapitulating lineage transition in organoid system, we identify JAK-STAT signaling, activated by EML4-ALK phase separation, significantly promotes squamous transition. Integrative study with scRNA-seq and immunostaining identify a plastic cell subpopulation in ALK-rearranged human LUAD showing squamous biomarker expression. Moreover, those relapsed ALK-rearranged LUAD show notable upregulation of squamous biomarkers. Consistently, mouse squamous tumors or LUAD with squamous signature display certain resistance to ALK inhibitor, which can be overcome by combined JAK1/2 inhibitor treatment. This study uncovers strong plasticity of ALK-rearranged tumors in orchestrating phenotypic transition and drug resistance and proposes a potentially effective therapeutic strategy.

Multi-scale structures of the mammalian radial spoke and divergence of axonemal complexes in ependymal cilia.

Radial spokes (RS) transmit mechanochemical signals between the central pair (CP) and axonemal dynein arms to coordinate ciliary motility. Atomic-resolution structures of metazoan RS and structures of axonemal complexes in ependymal cilia, whose rhythmic beating drives the circulation of cerebrospinal fluid, however, remain obscure. Here, we present near-atomic resolution cryo-EM structures of mouse RS head-neck complex in both monomer and dimer forms and reveal the intrinsic flexibility of the dimer. We also map the genetic mutations related to primary ciliary dyskinesia and asthenospermia on the head-neck complex. Moreover, we present the cryo-ET and sub-tomogram averaging map of mouse ependymal cilia and build the models for RS1-3, IDAs, and N-DRC. Contrary to the conserved RS structure, our cryo-ET map reveals the lack of IDA-b/c/e and the absence of Tektin filaments within the A-tubule of doublet microtubules in ependymal cilia compared with mammalian respiratory cilia and sperm flagella, further exemplifying the structural diversity of mammalian motile cilia. Our findings shed light on the stepwise mammalian RS assembly mechanism, the coordinated rigid and elastic RS-CP interaction modes beneficial for the regulation of asymmetric ciliary beating, and also facilitate understanding on the etiology of ciliary dyskinesia-related ciliopathies and on the ependymal cilia in the development of hydrocephalus.

Correction to: Identification of TAZ as the essential molecular switch in orchestrating SCLC phenotypic transition and metastasis.

[This corrects the article DOI: 10.1093/nsr/nwab232.].

O-GlcNAcylation Regulates Centrosome Behavior and Cell Polarity to Reduce Pulmonary Fibrosis and Maintain the Epithelial Phenotype.

O-GlcNAcylation functions as a cellular nutrient and stress sensor and participates in almost all cellular processes. However, it remains unclear whether O-GlcNAcylation plays a role in the establishment and maintenance of cell polarity, because mice lacking O-GlcNAc transferase (OGT) are embryonically lethal. Here, a mild Ogt knockout mouse model is constructed and the important role of O-GlcNAcylation in establishing and maintaining cell polarity is demonstrated. Ogt knockout leads to severe pulmonary fibrosis and dramatically promotes epithelial-to-mesenchymal transition. Mechanistic studies reveal that OGT interacts with pericentriolar material 1 (PCM1) and centrosomal protein 131 (CEP131), components of centriolar satellites required for anchoring microtubules to the centrosome. These data further show that O-GlcNAcylation of PCM1 and CEP131 promotes their centrosomal localization through phase separation. Decrease in O-GlcNAcylation prevents PCM1 and CEP131 from localizing to the centrosome, instead dispersing these proteins throughout the cell and impairing the microtubule-centrosome interaction to disrupt centrosome positioning and cell polarity. These findings identify a previously unrecognized role for protein O-GlcNAcylation in establishing and maintaining cell polarity with important implications for the pathogenesis of pulmonary fibrosis.

Effectiveness of the treatment of depression associated with cancer and neuroimaging changes in depression-related brain regions in patients treated with the mediator-deuterium acupuncture method.

Cancer patients should be concerned about depression, which can negatively impact their mental health. To develop efficient therapies, it is essential to comprehend the connection between cancer and depression. This study used neuroimaging to investigate the use of mediator-deuterium acupuncture (MDA) for people with cancer-induced depression and its effects on brain regions associated with depression. Resting-state functional magnetic resonance imaging and neurocognitive testing were conducted on the participants, and statistical package for the social sciences was utilized to analyze the behavioral data. Clinical and theoretical data were analyzed to evaluate acupuncture's effectiveness against gynecological cancer. In the research, there were 40 participants, 20 in each group. Except for psychomotor speed, there was no discernible difference in pre-chemotherapy cognitive test results between patients and healthy controls (HCs). However, there were substantial differences in post-treatment cognition test results, showing that the patient group had progressed. According to longitudinal graph analysis, the patient group's local and global brain efficiency significantly declined, and lower local efficiency was associated with lower raw Trail Making Test-A results. Furthermore, poorer verbal memory scores were associated with lower overall performance in the sick group but not in the HC group. According to the research, MDA has potential as a supplemental therapy since it may improve brain function and address depression-related neurological abnormalities in cancer patients. More research is required to fully comprehend the variations between cancer and depression-related brain areas during patient therapy, maybe incorporating MDA.

Quantitative analysis of acetylation in peste des petits ruminants virus-infected Vero cells.

BACKGROUND: Peste des petits ruminants virus (PPRV) is a highly contagious pathogen that strongly influences the productivity of small ruminants worldwide. Acetylation is an important post-translational modification involved in regulation of multiple biological functions. However, the extent and function of acetylation in host cells during PPRV infection remains unknown. METHODS: Dimethylation-labeling-based quantitative proteomic analysis of the acetylome of PPRV-infected Vero cells was performed. RESULTS: In total, 1068 proteins with 2641 modification sites were detected in response to PPRV infection, of which 304 differentially acetylated proteins (DAcPs) with 410 acetylated sites were identified (fold change < 0.83 or > 1.2 and P < 0.05), including 109 up-regulated and 195 down-regulated proteins. Gene Ontology (GO) classification indicated that DAcPs were mostly located in the cytoplasm (43%) and participated in cellular and metabolic processes related to binding and catalytic activity. Functional enrichment indicated that the DAcPs were involved in the minichromosome maintenance complex, unfolded protein binding, helicase activity. Only protein processing in endoplasmic reticulum pathway was enriched. A protein-protein interaction (PPI) network of the identified proteins further indicated that a various chaperone and ribosome processes were modulated by acetylation. CONCLUSIONS: To the best of our knowledge, this is the first study on acetylome in PPRV-infected host cell. Our findings establish an important baseline for future study on the roles of acetylation in the host response to PPRV replication and provide novel insights for understanding the molecular pathological mechanism of PPRV infection.

The Bacillus cereus toxin alveolysin disrupts the intestinal epithelial barrier by inducing microtubule disorganization through CFAP100.

Bacillus cereus is a Gram-positive bacterium that mainly causes self-limiting emetic or diarrheal illness but can also cause skin infections and bacteremia. Symptoms of B. cereus ingestion depend on the production of various toxins that target the gastric and intestinal epithelia. From a screen of bacterial isolates from human stool samples that compromised intestinal barrier function in mice, we identified a strain of B. cereus that disrupted tight and adherens junctions in the intestinal epithelium. This activity was mediated by the pore-forming exotoxin alveolysin, which increased the production of the membrane-anchored protein CD59 and of cilia- and flagella-associated protein 100 (CFAP100) in intestinal epithelial cells. In vitro, CFAP100 interacted with microtubules and promoted microtubule polymerization. CFAP100 overexpression stabilized microtubules in intestinal epithelial cells, leading to disorganization of the microtubule network and perturbation of tight and adherens junctions. The disruption of cell junctions by alveolysin depended on the increase in CFAP100, which in turn depended on CD59 and the activation of PI3K-AKT signaling. These findings demonstrate that, in addition to forming membrane pores, B. cereus alveolysin can permeabilize the intestinal epithelium by disrupting epithelial cell junctions in a manner that is consistent with intestinal symptoms and may allow the bacteria to escape the intestine and cause systemic infections. Our results suggest the potential value of targeting alveolysin or CFAP100 to prevent B. cereus-associated intestinal diseases and systemic infections.

Rationalized deep learning super-resolution microscopy for sustained live imaging of rapid subcellular processes.

The goal when imaging bioprocesses with optical microscopy is to acquire the most spatiotemporal information with the least invasiveness. Deep neural networks have substantially improved optical microscopy, including image super-resolution and restoration, but still have substantial potential for artifacts. In this study, we developed rationalized deep learning (rDL) for structured illumination microscopy and lattice light sheet microscopy (LLSM) by incorporating prior knowledge of illumination patterns and, thereby, rationally guiding the network to denoise raw images. Here we demonstrate that rDL structured illumination microscopy eliminates spectral bias-induced resolution degradation and reduces model uncertainty by five-fold, improving the super-resolution information by more than ten-fold over other computational approaches. Moreover, rDL applied to LLSM enables self-supervised training by using the spatial or temporal continuity of noisy data itself, yielding results similar to those of supervised methods. We demonstrate the utility of rDL by imaging the rapid kinetics of motile cilia, nucleolar protein condensation during light-sensitive mitosis and long-term interactions between membranous and membrane-less organelles.

Creation of poxvirus expressing foot-and-mouth and peste des petits ruminant disease virus proteins.

OBJECTIVE: Foot-and-mouth disease (FMD) and Peste des petits ruminant disease (PPR) are acute and severe infectious diseases of sheep and are listed as animal diseases for compulsory immunization. However, there is no dual vaccine to prevent these two diseases. The Modified Vaccinia virus Ankara strain (MVA) has been widely used in the construction of recombinant live vector vaccine because of its large capacity of foreign gene, wide host range, high safety, and immunogenicity. In this study, MVA-GFP recombinant virus skeleton was used to construct dual live vector vaccines against FMD and PPR. METHODS: The recombinant plasmid pUC57-FMDV P1-2A3CPPRV FH was synthesized and transfected into MVA-GFP infected CEF cells for homologous recombination. RESULTS: The results showed that a recombinant virus without fluorescent labeling was obtained after multiple rounds of plaque screening. The recombinant virus successfully expressed the target proteins, and the empty capsid of FMDV could be observed by transmission electron microscope (TME), and the expression levels of foreign proteins (VP1 and VP3) detected by ELISA were like those detected in FMDV-infected cells. This study laid the foundation for the successful construction of a live vector vaccine against FMD and PPR. KEY POINTS: • A recombinant MVA expressing FMDVP12A3C and PRRV HF proteins • Both the FMDV and PRRV proteins inserted into the virus were expressed • The proteins expressed by the recombinant poxvirus were assembled into VLPs.

Maternal mRNA deadenylation and allocation via Rbm14 condensates facilitate vertebrate blastula development.

Early embryonic development depends on proper utilization and clearance of maternal transcriptomes. How these processes are spatiotemporally regulated remains unclear. Here we show that nuclear RNA-binding protein Rbm14 and maternal mRNAs co-phase separate into cytoplasmic condensates to facilitate vertebrate blastula-to-gastrula development. In zebrafish, Rbm14 condensates were highly abundant in blastomeres and markedly reduced after prominent activation of zygotic transcription. They concentrated at spindle poles by associating with centrosomal γ-tubulin puncta and displayed mainly asymmetric divisions with a global symmetry across embryonic midline in 8- and 16-cell embryos. Their formation was dose-dependently stimulated by m6 A, but repressed by m5 C modification of the maternal mRNA. Furthermore, deadenylase Parn co-phase separated with these condensates, and this was required for deadenylation of the mRNAs in early blastomeres. Depletion of Rbm14 impaired embryonic cell differentiations and full activations of the zygotic genome in both zebrafish and mouse and resulted in developmental arrest at the blastula stage. Our results suggest that cytoplasmic Rbm14 condensate formation regulates early embryogenesis by facilitating deadenylation, protection, and mitotic allocation of m6 A-modified maternal mRNAs, and by releasing the poly(A)-less transcripts upon regulated disassembly to allow their re-polyadenylation and translation or clearance.

Optimization of wavefront reconstruction accuracy for conjugate shift differential absolute testing.

The conjugate shift differential method, based on Fourier transforms, is critical for surface error testing of high-precision optical elements. However, this common approach is also prone to periodic spectrum loss. As such, this paper proposes conjugate double shift differential (CDSD) absolute testing, which can effectively compensate for spectrum loss and achieve accurate wavefront reconstructions. Spectrum loss in the single shift differential method is analyzed through a study of the Fourier reconstruction process. A calculation model for the proposed CDSD method is then established and constraint conditions for shift quantities are provided by analyzing double shear effects observed in transverse shear interference. Finally, the reconstruction accuracies of various spectrum compensation methods are compared. Results showed that spectrum loss became more evident with increasing shift amounts. However, the CDSD method produced the smallest measurement error compared with conventional direct zero filling and adjacent point averaging, suggesting our approach could effectively improve absolute shape measurement accuracy for planar optical elements.

Intertwined Wdr47-NTD dimer recognizes a basic-helical motif in Camsap proteins for proper central-pair microtubule formation.

Calmodulin-regulated spectrin-associated proteins (Camsaps) bind to the N-terminal domain of WD40-repeat 47 (Wdr47-NTD; featured with a LisH-CTLH motif) to properly generate axonemal central-pair microtubules (CP-MTs) for the planar beat pattern of mammalian motile multicilia. The underlying molecular mechanism, however, remains unclear. Here, we determine the structures of apo-Wdr47-NTD and Wdr47-NTD in complex with a characteristic Wdr47-binding region (WBR) from Camsap3. Wdr47-NTD forms an intertwined dimer with a special cross-over region (COR) in addition to the canonical LisH and globular α-helical core (GAC). The basic WBR peptide adopts an α-helical conformation and anchors to a tailored acidic pocket embedded in the COR. Mutations in this target-binding pocket disrupt the interaction between Wdr47-NTD and Camsap3. Impairing Wdr47-Camsap interactions markedly reduces rescue effects of Wdr47 on CP-MTs and ciliary beat of Wdr47-deficient ependymal cells. Thus, Wdr47-NTD functions by recognizing a specific basic helical motif in Camsap proteins via its non-canonical COR, a target-binding site in LisH-CTLH-containing domains.

Peste des Petits Ruminants Virus Upregulates STING to Activate ATF6-Mediated Autophagy.

Peste des petits ruminants virus (PPRV) infection leads to autophagy, and the molecular mechanisms behind this phenomenon are unclear. Here, we demonstrate that PPRV infection results in morphological changes of the endoplasmic reticulum (ER) and activation of activating transcription factor 6 (ATF6) of the ER stress unfolded protein response (UPR). Knockdown of ATF6 blocked the autophagy process, suggesting ATF6 is necessary for PPRV-mediated autophagy induction. Further study showed that PPRV infection upregulates expression of the ER-anchored adaptor protein stimulator of interferon genes (STING), which is well-known for its pivotal roles in restricting DNA viruses. Knockdown of STING suppressed ATF6 activation and autophagy induction, implying that STING functions upstream of ATF6 to induce autophagy. Moreover, the STING-mediated autophagy response originated from the cellular pattern recognition receptor melanoma differentiation-associated gene 5 (MDA5). The absence of MDA5 abolished the upregulation of STING and the activation of autophagy. The deficiency of autophagy-related genes (ATG) repressed the autophagy process and PPRV replication, while it had no effect on MDA5 or STING expression. Overall, our work revealed that MDA5 works upstream of STING to activate ATF6 to induce autophagy. IMPORTANCEPPRV infection induces cellular autophagy; however, the intracellular responses and signaling mechanisms that occur upon PPRV infection are obscure, and whether innate immune responses are linked with autophagy to regulate viral replication is largely unknown. Here, we uncovered that the innate immune sensor MDA5 initiated the signaling cascade by upregulating STING, which is best known for its role in anti-DNA virus infection by inducing interferon expression. We first provide evidence that STING regulates PPRV replication by activating the ATF6 pathway of unfolded protein responses (UPRs) to induce autophagy. Our results revealed that in addition to mediating responses to foreign DNA, STING can cross talk with MDA5 to regulate the cellular stress response and autophagy induced by RNA viruses; thus, STING works as an adaptor protein for cellular stress responses and innate immune responses. Modulation of STING represents a promising approach to control both DNA and RNA viruses.

Purification and Immunostaining of Mouse Ependymal Ciliary Shafts.

Cilia and flagella are microtubule-based hair-like organelles protruding from the surface of most eukaryotic cells, and play essential roles in cell locomotion, left-right asymmetry, embryo development, and tissue homeostasis. With isolated cilia and flagella, great progress has been made in understanding the composition, structure, and function of cilia. However, the current cilia/flagella isolation methods are deficient in the integrity or productivity of purified cilia when applied to mammalian motile cilia. Here, we describe a new protocol that isolates cilia shafts from mouse ependymal cells, by horizontal shear force and mild detergent. This method enables the production of virtually integral cilia with high yields and less cell body contamination. It is suitable for immunostaining, puromycin labeling assay, and proximity ligation assay of mammalian motile cilia. Graphical abstract.

TUBright: A Peptide Probe for Imaging Microtubules.

In vitro assays using reconstituted microtubules have provided molecular insights into the principles of microtubule dynamics and the roles of microtubule-associated proteins. Emerging questions that further uncover the complexity in microtubule dynamics, especially those on tubulin isotypes and post-translational modifications, raise new technical challenges on how to visualize microtubules composed of tubulin purified from limited sources, primarily due to the low efficiency of the conventional tubulin labeling protocol. Here, we develop a peptide probe, termed TUBright, that labels in vitro reconstituted microtubules. TUBright, when coupled with different fluorescent dyes, provides flexible labeling of microtubules with a high signal-to-noise ratio. TUBright does not interfere with the dynamic behaviors of microtubules and microtubule-associated proteins. Therefore, TUBright is a useful tool for imaging microtubules, making it feasible to use tubulin from limited sources for answering many open questions on microtubule dynamics.

Identification of TAZ as the essential molecular switch in orchestrating SCLC phenotypic transition and metastasis.

Small-cell lung cancer (SCLC) is a recalcitrant cancer characterized by high metastasis. However, the exact cell type contributing to metastasis remains elusive. Using a Rb1 L/L /Trp53 L/L mouse model, we identify the NCAMhiCD44lo/- subpopulation as the SCLC metastasizing cell (SMC), which is progressively transitioned from the non-metastasizing NCAMloCD44hi cell (non-SMC). Integrative chromatin accessibility and gene expression profiling studies reveal the important role of the SWI/SNF complex, and knockout of its central component, Brg1, significantly inhibits such phenotypic transition and metastasis. Mechanistically, TAZ is silenced by the SWI/SNF complex during SCLC malignant progression, and its knockdown promotes SMC transition and metastasis. Importantly, ectopic TAZ expression reversely drives SMC-to-non-SMC transition and alleviates metastasis. Single-cell RNA-sequencing analyses identify SMC as the dominant subpopulation in human SCLC metastasis, and immunostaining data show a positive correlation between TAZ and patient prognosis. These data uncover high SCLC plasticity and identify TAZ as the key molecular switch in orchestrating SCLC phenotypic transition and metastasis.

Self-construction of actin networks through phase separation-induced abLIM1 condensates.

The abLIM1 is a nonerythroid actin-binding protein critical for stable plasma membrane-cortex interactions under mechanical tension. Its depletion by RNA interference results in sparse, poorly interconnected cortical actin networks and severe blebbing of migrating cells. Its isoforms, abLIM-L, abLIM-M, and abLIM-S, contain, respectively four, three, and no LIM domains, followed by a C terminus entirely homologous to erythroid cortex protein dematin. How abLIM1 functions, however, remains unclear. Here we show that abLIM1 is a liquid-liquid phase separation (LLPS)-dependent self-organizer of actin networks. Phase-separated condensates of abLIM-S-mimicking ΔLIM or the major isoform abLIM-M nucleated, flew along, and cross-linked together actin filaments (F-actin) to produce unique aster-like radial arrays and interconnected webs of F-actin bundles. Interestingly, ΔLIM condensates facilitated actin nucleation and network formation even in the absence of Mg2+. Our results suggest that abLIM1 functions as an LLPS-dependent actin nucleator and cross-linker and provide insights into how LLPS-induced condensates could self-construct intracellular architectures of high connectivity and plasticity.