The SMC-family Wadjet complex protects bacteria from plasmid transformation by recognition and cleavage of closed-circular DNA.

Self versus non-self discrimination is a key element of innate and adaptive immunity across life. In bacteria, CRISPR-Cas and restriction-modification systems recognize non-self nucleic acids through their sequence and their methylation state, respectively. Here, we show that the Wadjet defense system recognizes DNA topology to protect its host against plasmid transformation. By combining cryoelectron microscopy with cross-linking mass spectrometry, we show that Wadjet forms a complex similar to the bacterial condensin complex MukBEF, with a novel nuclease subunit similar to a type II DNA topoisomerase. Wadjet specifically cleaves closed-circular DNA in a reaction requiring ATP hydrolysis by the structural maintenance of chromosome (SMC) ATPase subunit JetC, suggesting that the complex could use DNA loop extrusion to sense its substrate's topology, then specifically activate the nuclease subunit JetD to cleave plasmid DNA. Overall, our data reveal how bacteria have co-opted a DNA maintenance machine to specifically recognize and destroy foreign DNAs through topology sensing.

DNA damage triggers Golgi dispersal via DNA-PK and GOLPH3.

The response to DNA damage, which regulates nuclear processes such as DNA repair, transcription, and cell cycle, has been studied thoroughly. However, the cytoplasmic response to DNA damage is poorly understood. Here, we demonstrate that DNA damage triggers dramatic reorganization of the Golgi, resulting in its dispersal throughout the cytoplasm. We further show that DNA-damage-induced Golgi dispersal requires GOLPH3/MYO18A/F-actin and the DNA damage protein kinase, DNA-PK. In response to DNA damage, DNA-PK phosphorylates GOLPH3, resulting in increased interaction with MYO18A, which applies a tensile force to the Golgi. Interference with the Golgi DNA damage response by depletion of DNA-PK, GOLPH3, or MYO18A reduces survival after DNA damage, whereas overexpression of GOLPH3, as is observed frequently in human cancers, confers resistance to killing by DNA-damaging agents. Identification of the DNA-damage-induced Golgi response reveals an unexpected pathway through DNA-PK, GOLPH3, and MYO18A that regulates cell survival following DNA damage.

An E1-E2 fusion protein primes antiviral immune signalling in bacteria.

In all organisms, innate immune pathways sense infection and rapidly activate potent immune responses while avoiding inappropriate activation (autoimmunity). In humans, the innate immune receptor cyclic GMP-AMP synthase (cGAS) detects viral infection to produce the nucleotide second messenger cyclic GMP-AMP (cGAMP), which initiates stimulator of interferon genes (STING)-dependent antiviral signalling1. Bacteria encode evolutionary predecessors of cGAS called cGAS/DncV-like nucleotidyltransferases2 (CD-NTases), which detect bacteriophage infection and produce diverse nucleotide second messengers3. How bacterial CD-NTase activation is controlled remains unknown. Here we show that CD-NTase-associated protein 2 (Cap2) primes bacterial CD-NTases for activation through a ubiquitin transferase-like mechanism. A cryo-electron microscopy structure of the Cap2-CD-NTase complex reveals Cap2 as an all-in-one ubiquitin transferase-like protein, with distinct domains resembling eukaryotic E1 and E2 proteins. The structure captures a reactive-intermediate state with the CD-NTase C terminus positioned in the Cap2 E1 active site and conjugated to AMP. Cap2 conjugates the CD-NTase C terminus to a target molecule that primes the CD-NTase for increased cGAMP production. We further demonstrate that a specific endopeptidase, Cap3, balances Cap2 activity by cleaving CD-NTase-target conjugates. Our data demonstrate that bacteria control immune signalling using an ancient, minimized ubiquitin transferase-like system and provide insight into the evolution of the E1 and E2 machinery across domains of life.

Defining the proximal interaction networks of Arf GTPases reveals a mechanism for the regulation of PLD1 and PI4KB.

The Arf GTPase family is involved in a wide range of cellular regulation including membrane trafficking and organelle-structure assembly. Here, we have generated a proximity interaction network for the Arf family using the miniTurboID approach combined with TMT-based quantitative mass spectrometry. Our interactome confirmed known interactions and identified many novel interactors that provide leads for defining Arf pathway cell biological functions. We explored the unexpected finding that phospholipase D1 (PLD1) preferentially interacts with two closely related but poorly studied Arf family GTPases, ARL11 and ARL14, showing that PLD1 is activated by ARL11/14 and may recruit these GTPases to membrane vesicles, and that PLD1 and ARL11 collaborate to promote macrophage phagocytosis. Moreover, ARL5A and ARL5B were found to interact with and recruit phosphatidylinositol 4-kinase beta (PI4KB) at trans-Golgi, thus promoting PI4KB's function in PI4P synthesis and protein secretion.

Recognition of centromere-specific histone Cse4 by the inner kinetochore Okp1-Ame1 complex.

Successful mitosis depends on the timely establishment of correct chromosomal attachments to microtubules. The kinetochore, a modular multiprotein complex, mediates this connection by recognizing specialized chromatin containing a histone H3 variant called Cse4 in budding yeast and CENP-A in vertebrates. Structural features of the kinetochore that enable discrimination between Cse4/CENP-A and H3 have been identified in several species. How and when these contribute to centromere recognition and how they relate to the overall structure of the inner kinetochore are unsettled questions. More generally, this molecular recognition ensures that only one kinetochore is built on each chromatid and that this happens at the right place on the chromatin fiber. We have determined the crystal structure of a Cse4 peptide bound to the essential inner kinetochore Okp1-Ame1 heterodimer from budding yeast. The structure and related experiments show in detail an essential point of Cse4 contact and provide information about the arrangement of the inner kinetochore.

mTORC2 Regulates Amino Acid Metabolism in Cancer by Phosphorylation of the Cystine-Glutamate Antiporter xCT.

Mutations in cancer reprogram amino acid metabolism to drive tumor growth, but the molecular mechanisms are not well understood. Using an unbiased proteomic screen, we identified mTORC2 as a critical regulator of amino acid metabolism in cancer via phosphorylation of the cystine-glutamate antiporter xCT. mTORC2 phosphorylates serine 26 at the cytosolic N terminus of xCT, inhibiting its activity. Genetic inhibition of mTORC2, or pharmacologic inhibition of the mammalian target of rapamycin (mTOR) kinase, promotes glutamate secretion, cystine uptake, and incorporation into glutathione, linking growth factor receptor signaling with amino acid uptake and utilization. These results identify an unanticipated mechanism regulating amino acid metabolism in cancer, enabling tumor cells to adapt to changing environmental conditions.

Site-specific MCM sumoylation prevents genome rearrangements by controlling origin-bound MCM.

Timely completion of eukaryotic genome duplication requires coordinated DNA replication initiation at multiple origins. Replication begins with the loading of the Mini-Chromosome Maintenance (MCM) complex, proceeds by the activation of the Cdc45-MCM-GINS (CMG) helicase, and ends with CMG removal after chromosomes are fully replicated. Post-translational modifications on the MCM and associated factors ensure an orderly transit of these steps. Although the mechanisms of CMG activation and removal are partially understood, regulated MCM loading is not, leaving an incomplete understanding of how DNA replication begins. Here we describe a site-specific modification of Mcm3 by the Small Ubiquitin-like MOdifier (SUMO). Mutations that prevent this modification reduce the MCM loaded at replication origins and lower CMG levels, resulting in impaired cell growth, delayed chromosomal replication, and the accumulation of gross chromosomal rearrangements (GCRs). These findings demonstrate the existence of a SUMO-dependent regulation of origin-bound MCM and show that this pathway is needed to prevent genome rearrangements.

Recruitment of a SUMO isopeptidase to rDNA stabilizes silencing complexes by opposing SUMO targeted ubiquitin ligase activity.

Post-translational modification by SUMO (small ubiquitin-like modifier) plays important but still poorly understood regulatory roles in eukaryotic cells, including as a signal for ubiquitination by SUMO targeted ubiquitin ligases (STUbLs). Here, we delineate the molecular mechanisms for SUMO-dependent control of ribosomal DNA (rDNA) silencing through the opposing actions of a STUbL (Slx5:Slx8) and a SUMO isopeptidase (Ulp2). We identify a conserved region in the Ulp2 C terminus that mediates its specificity for rDNA-associated proteins and show that this region binds directly to the rDNA-associated protein Csm1. Two crystal structures show that Csm1 interacts with Ulp2 and one of its substrates, the rDNA silencing protein Tof2, through adjacent conserved interfaces in its C-terminal domain. Disrupting Csm1's interaction with either Ulp2 or Tof2 dramatically reduces rDNA silencing and causes a marked drop in Tof2 abundance, suggesting that Ulp2 promotes rDNA silencing by opposing STUbL-mediated degradation of silencing proteins. Tof2 abundance is rescued by deletion of the STUbL SLX5 or disruption of its SUMO-interacting motifs, confirming that Tof2 is targeted for degradation in a SUMO- and STUbL-dependent manner. Overall, our results demonstrate how the opposing actions of a localized SUMO isopeptidase and a STUbL regulate rDNA silencing by controlling the abundance of a key rDNA silencing protein, Tof2.

TANGO1 facilitates cargo loading at endoplasmic reticulum exit sites.

A genome-wide screen revealed previously unidentified components required for transport and Golgi organization (TANGO). We now provide evidence that one of these proteins, TANGO1, is an integral membrane protein localized to endoplasmic reticulum (ER) exit sites, with a luminal SH3 domain and a cytoplasmic proline-rich domain (PRD). Knockdown of TANGO1 inhibits export of bulky collagen VII from the ER. The SH3 domain of TANGO1 binds to collagen VII; the PRD binds to the COPII coat subunits, Sec23/24. In this scenario, PRD binding to Sec23/24 subunits could stall COPII carrier biogenesis to permit the luminal domain of TANGO1 to guide SH3-bound cargo into a growing carrier. All cells except those of hematopoietic origin express TANGO1. We propose that TANGO1 exports other cargoes in cells that do not secrete collagen VII. However, TANGO1 does not enter the budding carrier, which represents a unique mechanism to load cargo into COPII carriers.

GOLPH3 bridges phosphatidylinositol-4- phosphate and actomyosin to stretch and shape the Golgi to promote budding.

Golgi membranes, from yeast to humans, are uniquely enriched in phosphatidylinositol-4-phosphate (PtdIns(4)P), although the role of this lipid remains poorly understood. Using a proteomic lipid-binding screen, we identify the Golgi protein GOLPH3 (also called GPP34, GMx33, MIDAS, or yeast Vps74p) as a PtdIns(4)P-binding protein that depends on PtdIns(4)P for its Golgi localization. We further show that GOLPH3 binds the unconventional myosin MYO18A, thus connecting the Golgi to F-actin. We demonstrate that this linkage is necessary for normal Golgi trafficking and morphology. The evidence suggests that GOLPH3 binds to PtdIns(4)P-rich trans-Golgi membranes and MYO18A conveying a tensile force required for efficient tubule and vesicle formation. Consequently, this tensile force stretches the Golgi into the extended ribbon observed by fluorescence microscopy and the familiar flattened form observed by electron microscopy.

A qualitative study on patients' and health care professionals' perspectives regarding care delivered during CIED operation.

BACKGROUND: Cardiac implantable electronic devices (CIEDs) has proven to be an invaluable tool in the practice of cardiology. Patients who have undergone CIED surgery with local anesthesia may result in fear, insecurity and suffering. Some studies have put efforts on ways to improve intraoperative experience of patients with local anesthesia, but researches concerning experiences of CIED patients during surgery is in its infancy. METHODS: Based on semi-structured and in-depth interviews, a qualitative design was conducted in a tertiary general hospital in China from May 2022 to July 2023.Purposeful sampling of 17 patients received CIED surgery and 20 medical staff were interviewed. Thematic analysis with an inductive approach was used to identify dominant themes. RESULTS: Four themes emerged from the data: (1) Safety and success is priority; (2) Humanistic Caring is a must yet be lacking; (3) Paradox of surgery information given; (4) Ways to improve surgery experiences in the operation. CONCLUSIONS: Intraoperative care is significant for CIED surgery. To improve care experience during surgery, healthcare professionals should pay attention to patients' safety and the factors that affecting humanistic caring in clinical practice. In addition, information support should consider information-seeking styles and personal needs. Besides, the four approaches presented in this study are effective to improve the intraoperative care experience.

Multi-phase FZA lensless imaging via diffusion model.

Lensless imaging shifts the burden of imaging from bulky and expensive hardware to computing, which enables new architectures for portable cameras. However, the twin image effect caused by the missing phase information in the light wave is a key factor limiting the quality of lensless imaging. Conventional single-phase encoding methods and independent reconstruction of separate channels pose challenges in removing twin images and preserving the color fidelity of the reconstructed image. In order to achieve high-quality lensless imaging, the multiphase lensless imaging via diffusion model (MLDM) is proposed. A multi-phase FZA encoder integrated on a single mask plate is used to expand the data channel of a single-shot image. The information association between the color image pixel channel and the encoded phase channel is established by extracting prior information of the data distribution based on multi-channel encoding. Finally, the reconstruction quality is improved through the use of the iterative reconstruction method. The results show that the proposed MLDM method effectively removes the influence of twin images and produces high-quality reconstructed images compared with traditional methods, and the results reconstructed using MLDM have higher structural similarity and peak signal-to-noise ratio.

A Multi-Scale Temporal Convolutional Network with Attention Mechanism for Force Level Classification during Motor Imagery of Unilateral Upper-Limb Movements.

In motor imagery (MI) brain-computer interface (BCI) research, some researchers have designed MI paradigms of force under a unilateral upper-limb static state. It is difficult to apply these paradigms to the dynamic force interaction process between the robot and the patient in a brain-controlled rehabilitation robot system, which needs to induce thinking states of the patient's demand for assistance. Therefore, in our research, according to the movement of wiping the table in human daily life, we designed a three-level-force MI paradigm under a unilateral upper-limb dynamic state. Based on the event-related de-synchronization (ERD) feature analysis of the electroencephalography (EEG) signals generated by the brain's force change motor imagination, we proposed a multi-scale temporal convolutional network with attention mechanism (MSTCN-AM) algorithm to recognize ERD features of MI-EEG signals. Aiming at the slight feature differences of single-trial MI-EEG signals among different levels of force, the MSTCN module was designed to extract fine-grained features of different dimensions in the time-frequency domain. The spatial convolution module was then used to learn the area differences of space domain features. Finally, the attention mechanism dynamically weighted the time-frequency-space domain features to improve the algorithm's sensitivity. The results showed that the accuracy of the algorithm was 86.4 ± 14.0% for the three-level-force MI-EEG data collected experimentally. Compared with the baseline algorithms (OVR-CSP+SVM (77.6 ± 14.5%), Deep ConvNet (75.3 ± 12.3%), Shallow ConvNet (77.6 ± 11.8%), EEGNet (82.3 ± 13.8%), and SCNN-BiLSTM (69.1 ± 16.8%)), our algorithm had higher classification accuracy with significant differences and better fitting performance.

Both decreased and increased SRPK1 levels promote cancer by interfering with PHLPP-mediated dephosphorylation of Akt.

Akt activation is a hallmark of human cancers. Here, we report a critical mechanism for regulation of Akt activity by the splicing kinase SRPK1, a downstream Akt target for transducing growth signals to regulate splicing. Surprisingly, we find that SRPK1 has a tumor suppressor function because ablation of SRPK1 in mouse embryonic fibroblasts induces cell transformation. We link the phenotype to constitutive Akt activation from genome-wide phosphoproteomics analysis and discover that downregulated SRPK1 impairs the recruitment of the Akt phosphatase PHLPP1 (pleckstrin homology (PH) domain leucine-rich repeat protein phosphatase) to Akt. Interestingly, SRPK1 overexpression is also tumorigenic because excess SRPK1 squelches PHLPP1. Thus, aberrant SRPK1 expression in either direction induces constitutive Akt activation, providing a mechanistic basis for previous observations that SRPK1 is downregulated in some cancer contexts and upregulated in others.

A Review of Circulating Tumor DNA in the Diagnosis and Monitoring of Esophageal Cancer.

Circulating tumor DNA (ctDNA) is a type of cell-free DNA released by tumor cells after necrosis and apoptosis, and it can be actively secreted by tumor cells. Since ctDNA is derived from various tumor sites, it can provide far more comprehensive genomic and epigenomic information than a single-site biopsy. Therefore, ctDNA can overcome tumor heterogeneity, which is the major limitation of a traditional tissue biopsy approach. Noninvasive ctDNA assays allow continuous real-time monitoring of the molecular status of cancers. Recently, ctDNA assays have been widely used in clinical practice, including cancer diagnosis, evaluation of therapeutic efficacy and prognosis, and monitoring of relapse and metastasis. Although ctDNA shows a high diagnostic performance in advanced esophageal cancer, it is far from satisfactory for early diagnosis of esophageal cancer. Monitoring the dynamic changes of ctDNA is beneficial for the evaluation of therapeutic efficacy and prediction of early recurrence in esophageal cancer. It is necessary to establish standards for individualized ctDNA detection in the evaluation of treatment response and surveillance of esophageal cancer and to develop clinical practice guideline for the systemic treatment of patients with "ctDNA recurrence." This review aims to provide an update on the role of ctDNA in the diagnosis and monitoring of esophageal cancer.

Recruitment of the Ulp2 protease to the inner kinetochore prevents its hyper-sumoylation to ensure accurate chromosome segregation.

The kinetochore is the central molecular machine that drives chromosome segregation in all eukaryotes. Genetic studies have suggested that protein sumoylation plays a role in regulating the inner kinetochore; however, the mechanism remains elusive. Here, we show that Saccharomyces cerevisiae Ulp2, an evolutionarily conserved SUMO specific protease, contains a previously uncharacterized kinetochore-targeting motif that recruits Ulp2 to the kinetochore via the Ctf3CENP-I-Mcm16CENP-H-Mcm22CENP-K complex (CMM). Once recruited, Ulp2 selectively targets multiple subunits of the kinetochore, specifically the Constitutive Centromere-Associated Network (CCAN), via its SUMO-interacting motif (SIM). Mutations that impair the kinetochore recruitment of Ulp2 or its binding to SUMO result in an elevated rate of chromosome loss, while mutations that affect both result in a synergistic increase of chromosome loss rate, hyper-sensitivity to DNA replication stress, along with a dramatic accumulation of hyper-sumoylated CCAN. Notably, sumoylation of CCAN occurs at the kinetochore and is perturbed by DNA replication stress. These results indicate that Ulp2 utilizes its dual substrate recognition to prevent hyper-sumoylation of CCAN, ensuring accurate chromosome segregation during cell division.

Single photon counting compressive imaging using a generative model optimized via sampling and transfer learning.

Single photon counting compressive imaging, a combination of single-pixel-imaging and single-photon-counting technology, is provided with low cost and ultra-high sensitivity. However, it requires a long imaging time when applying traditional compressed sensing (CS) reconstruction algorithms. A deep-learning-based compressed reconstruction network refrains iterative computation while achieving efficient reconstruction. This paper proposes a compressed reconstruction network (OGTM) based on a generative model, adding sampling sub-network to achieve joint-optimization of sampling and generation for better reconstruction. To avoid the slow convergence caused by alternating training, initial weights of the sampling and generation sub-network are transferred from an autoencoder. The results indicate that the convergence speed and imaging quality are significantly improved. The OGTM validated on a single-photon compressive imaging system performs imaging experiments on specific and generalized targets. For specific targets, the results demonstrate that OGTM can quickly generate images from few measurements, and its reconstruction is better than the existing compressed sensing recovery algorithms, compensating defects of the generative models in compressed sensing.

Investigation of the extended focusing capability of the spherical aberration to enlarge the field of view in light-sheet fluorescence microscopy.

In light-sheet fluorescence microscopy (LSFM), using Gaussian beams for light-sheet generation results in a trade-off between the thickness and the field of view (FOV). Here we present a theoretical analysis of using spherical aberration to enlarge the FOV while keeping the light-sheet thickness small. Such spherical aberration can arise when focusing beams through an interface between materials of mismatched refractive indices. The depth-of-focus extension of the Gaussian beam is achieved when using air objectives to focus light into the samples dipped in the immersion medium with a higher refractive index. By scanning this elongated beam, a thin light sheet with a wide FOV can be used for LSFM imaging. Meanwhile, the accompanied sidelobes with the spherical aberrated light sheet, which are mainly distributed in the rear part of the light sheet, are also discussed. Simulation results show that an extended FOV of 64.4µm is possible for an objective lens of NA=0.3, which is about 5 times that of the unaberrated case. For such an extended FOV, a comparatively thin thickness of 1.38µm as well as the first sidelobe about 11.1% of the peak intensity in the center are also demonstrated.

SUMO E3 ligase Mms21 prevents spontaneous DNA damage induced genome rearrangements.

Mms21, a subunit of the Smc5/6 complex, possesses an E3 ligase activity for the Small Ubiquitin-like MOdifier (SUMO). Here we show that the mms21-CH mutation, which inactivates Mms21 ligase activity, causes increased accumulation of gross chromosomal rearrangements (GCRs) selected in the dGCR assay. These dGCRs are formed by non-allelic homologous recombination between divergent DNA sequences mediated by Rad52-, Rrm3- and Pol32-dependent break-induced replication. Combining mms21-CH with sgs1Δ caused a synergistic increase in GCRs rates, indicating the distinct roles of Mms21 and Sgs1 in suppressing GCRs. The mms21-CH mutation also caused increased rates of accumulating uGCRs mediated by breakpoints in unique sequences as revealed by whole genome sequencing. Consistent with the accumulation of endogenous DNA lesions, mms21-CH mutants accumulate increased levels of spontaneous Rad52 and Ddc2 foci and had a hyper-activated DNA damage checkpoint. Together, these findings support that Mms21 prevents the accumulation of spontaneous DNA lesions that cause diverse GCRs.