Cryo-EM Structure and Assembly of an Extracellular Contractile Injection System.

Contractile injection systems (CISs) are cell-puncturing nanodevices that share ancestry with contractile tail bacteriophages. Photorhabdus virulence cassette (PVC) represents one group of extracellular CISs that are present in both bacteria and archaea. Here, we report the cryo-EM structure of an intact PVC from P. asymbiotica. This over 10-MDa device resembles a simplified T4 phage tail, containing a hexagonal baseplate complex with six fibers and a capped 117-nanometer sheath-tube trunk. One distinct feature of the PVC is the presence of three variants for both tube and sheath proteins, indicating a functional specialization of them during evolution. The terminal hexameric cap docks onto the topmost layer of the inner tube and locks the outer sheath in pre-contraction state with six stretching arms. Our results on the PVC provide a framework for understanding the general mechanism of widespread CISs and pave the way for using them as delivery tools in biological or therapeutic applications.

Structure of a Pancreatic ATP-Sensitive Potassium Channel.

ATP-sensitive potassium channels (KATP) couple intracellular ATP levels with membrane excitability. These channels play crucial roles in many essential physiological processes and have been implicated extensively in a spectrum of metabolic diseases and disorders. To gain insight into the mechanism of KATP, we elucidated the structure of a hetero-octameric pancreatic KATP channel in complex with a non-competitive inhibitor glibenclamide by single-particle cryoelectron microscopy to 5.6-Å resolution. The structure shows that four SUR1 regulatory subunits locate peripherally and dock onto the central Kir6.2 channel tetramer through the SUR1 TMD0-L0 fragment. Glibenclamide-bound SUR1 uses TMD0-L0 fragment to stabilize Kir6.2 channel in a closed conformation. In another structural population, a putative co-purified phosphatidylinositol 4,5-bisphosphate (PIP2) molecule uncouples Kir6.2 from glibenclamide-bound SUR1. These structural observations suggest a molecular mechanism for KATP regulation by anti-diabetic sulfonylurea drugs, intracellular adenosine nucleotide concentrations, and PIP2 lipid.

Structure of the origin recognition complex bound to DNA replication origin.

The six-subunit origin recognition complex (ORC) binds to DNA to mark the site for the initiation of replication in eukaryotes. Here we report a 3 Å cryo-electron microscopy structure of the Saccharomyces cerevisiae ORC bound to a 72-base-pair origin DNA sequence that contains the ARS consensus sequence (ACS) and the B1 element. The ORC encircles DNA through extensive interactions with both phosphate backbone and bases, and bends DNA at the ACS and B1 sites. Specific recognition of thymine residues in the ACS is carried out by a conserved basic amino acid motif of Orc1 in the minor groove, and by a species-specific helical insertion motif of Orc4 in the major groove. Moreover, similar insertions into major and minor grooves are also embedded in the B1 site by basic patch motifs from Orc2 and Orc5, respectively, to contact bases and to bend DNA. This work pinpoints a conserved role of ORC in modulating DNA structure to facilitate origin selection and helicase loading in eukaryotes.

Mendelian Randomisation Analysis of Causal Association between Lifestyle, Health Factors, and Keratoconus.

Keratoconus (KC), a leading cause of vision impairment, has an unclear aetiology. This study used Mendelian randomization (MR) to explore the causal links between various factors (smoking, asthma, Down syndrome, inflammatory bowel disease, atopic dermatitis, and serum 25-hydroxyvitamin D levels) and KC. A two-sample MR design, grounded in genome-wide association study (GWAS) summary statistics, was adopted using data from FinnGen, UK Biobank, and other GWAS-related articles. The inverse-variance weighted (IVW) method was employed, complemented by the Wald ratio method for factors with only one single-nucleotide polymorphism (SNP). Sensitivity and stability were assessed through Cochrane's Q test, the MR-Egger intercept test, MR-PRESSO outlier test, and the leave-one-out analysis. The IVW results for the ORA (Ocular Response Analyzer) biomechanical parameters indicated significant associations between tobacco smoking (CH: p < 0.001; CRF: p = 0.009) and inflammatory bowel disease (CH: p = 0.032; CRF: p = 0.001) and corneal biomechanics. The Wald ratio method showed tobacco smoking was associated with a lower risk of KC (p = 0.024). Conversely, asthma (p = 0.009), atopic dermatitis (p = 0.012), inflammatory bowel disease (p = 0.017), and serum 25-hydroxyvitamin D levels (p = 0.039) were associated with a higher risk of KC by IVW, and the same applied to Down syndrome (p = 0.004) using the Wald ratio. These results underscore the role of corneal biomechanics as potential mediators in KC risk, warranting further investigation using Corvis ST and Brillouin microscopy. The findings emphasise the importance of timely screening for specific populations in KC prevention and management.

CorNet: Autonomous feature learning in raw Corvis ST data for keratoconus diagnosis via residual CNN approach.

PURPOSE: To ascertain whether the integration of raw Corvis ST data with an end-to-end CNN can enhance the diagnosis of keratoconus (KC). METHOD: The Corvis ST is a non-contact device for in vivo measurement of corneal biomechanics. The CorNet was trained and validated on a dataset consisting of 1786 Corvis ST raw data from 1112 normal eyes and 674 KC eyes. Each raw data consists of the anterior and posterior corneal surface elevation during air-puff induced dynamic deformation. The architecture of CorNet utilizes four ResNet-inspired convolutional structures that employ 1 × 1 convolution in identity mapping. Gradient-weighted Class Activation Mapping (Grad-CAM) was adopted to visualize the attention allocation to diagnostic areas. Discriminative performance was assessed using metrics including the AUC of ROC curve, sensitivity, specificity, precision, accuracy, and F1 score. RESULTS: CorNet demonstrated outstanding performance in distinguishing KC from normal eyes, achieving an AUC of 0.971 (sensitivity: 92.49%, specificity: 91.54%) in the validation set, outperforming the best existing Corvis ST parameters, namely the Corvis Biomechanical Index (CBI) with an AUC of 0.947, and its updated version for Chinese populations (cCBI) with an AUC of 0.963. Though the ROC curve analysis showed no significant difference between CorNet and cCBI (p = 0.295), it indicated a notable difference between CorNet and CBI (p = 0.011). The Grad-CAM visualizations highlighted the significance of corneal deformation data during the loading phase rather than the unloading phase for KC diagnosis. CONCLUSION: This study proposed an end-to-end CNN approach utilizing raw biomechanical data by Corvis ST for KC detection, showing effectiveness comparable to or surpassing existing parameters provided by Corvis ST. The CorNet, autonomously learning comprehensive temporal and spatial features, demonstrated a promising performance for advancing KC diagnosis in ophthalmology.

Diagnosis of Forme Fruste Keratoconus Using Corvis ST Sequences with Digital Image Correlation and Machine Learning.

PURPOSE: This study aimed to employ the incremental digital image correlation (DIC) method to obtain displacement and strain field data of the cornea from Corvis ST (CVS) sequences and access the performance of embedding these biomechanical data with machine learning models to distinguish forme fruste keratoconus (FFKC) from normal corneas. METHODS: 100 subjects were categorized into normal (N = 50) and FFKC (N = 50) groups. Image sequences depicting the horizontal cross-section of the human cornea under air puff were captured using the Corvis ST tonometer. The high-speed evolution of full-field corneal displacement, strain, velocity, and strain rate was reconstructed utilizing the incremental DIC approach. Maximum (max-) and average (ave-) values of full-field displacement V, shear strain γxy, velocity VR, and shear strain rate γxyR were determined over time, generating eight evolution curves denoting max-V, max-γxy, max-VR, max-γxyR, ave-V, ave-γxy, ave-VR, and ave-γxyR, respectively. These evolution data were inputted into two machine learning (ML) models, specifically Naïve Bayes (NB) and Random Forest (RF) models, which were subsequently employed to construct a voting classifier. The performance of the models in diagnosing FFKC from normal corneas was compared to existing CVS parameters. RESULTS: The Normal group and the FFKC group each included 50 eyes. The FFKC group did not differ from healthy controls for age (p = 0.26) and gender (p = 0.36) at baseline, but they had significantly lower bIOP (p < 0.001) and thinner central cornea thickness (CCT) (p < 0.001). The results demonstrated that the proposed voting ensemble model yielded the highest performance with an AUC of 1.00, followed by the RF model with an AUC of 0.99. Radius and A2 Time emerged as the best-performing CVS parameters with AUC values of 0.948 and 0.938, respectively. Nonetheless, no existing Corvis ST parameters outperformed the ML models. A progressive enhancement in performance of the ML models was observed with incremental time points during the corneal deformation. CONCLUSION: This study represents the first instance where displacement and strain data following incremental DIC analysis of Corvis ST images were integrated with machine learning models to effectively differentiate FFKC corneas from normal ones, achieving superior accuracy compared to existing CVS parameters. Considering biomechanical responses of the inner cornea and their temporal pattern changes may significantly improve the early detection of keratoconus.

Cholesterol reduction by immunization with a PCSK9 mimic.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a plasma protein that controls cholesterol homeostasis. Here, we design a human PCSK9 mimic, named HIT01, with no consecutive 9-residue stretch in common with any human protein as a potential heart attack vaccine. Murine immunizations with HIT01 reduce low-density lipoprotein (LDL) and cholesterol levels by 40% and 30%, respectively. Immunization of cynomolgus macaques with HIT01-K21Q-R218E, a cleavage-resistant variant, elicits high-titer PCSK9-directed antibody responses and significantly reduces serum levels of cholesterol 2 weeks after each immunization. However, HIT01-K21Q-R218E immunizations also increase serum PCSK9 levels by up to 5-fold, likely due to PCSK9-binding antibodies altering the half-life of PCSK9. While vaccination with a PCSK9 mimic can induce antibodies that block interactions of PCSK9 with the LDL receptor, PCSK9-binding antibodies appear to alter homeostatic levels of PCSK9, thereby confounding its vaccine impact. Our results nevertheless suggest a mechanism for increasing the half-life of soluble regulatory factors by vaccination.

In-vivo high-speed biomechanical imaging of the cornea using Corvis ST and digital image correlation.

In-vivo corneal biomechanical characterization has gained significant clinical relevance in ophthalmology, especially in the early diagnosis of eye disorders and diseases (e.g. keratoconus). In clinical medicine, the air-puff-based tonometers such as Ocular Response Analyzer (ORA) and Corvis ST have been used in the in-vivo biomechanical testing. In the test, the high-speed dynamic deformation of the cornea under air-puff excitation is analyzed to identify the abnormities in the morphological and biomechanical properties of the cornea. While most existing measurements reflect the overall corneal biomechanical properties, in-vivo high-speed strain and strain rate fields at the tissue level have not been assessed. In this study, 20 subjects were classified into two different groups: the normal (NORM, N = 10) group and the keratoconus (KC, N = 10) group. Image sequences of the horizontal cross-section of the human cornea under air puff were captured by the Corvis ST tonometer. The macroscale mechanical response of the cornea was determined through image analysis. The high-speed evolution of full-field corneal displacement, strain, velocity, and strain rate was reconstructed using the incremental digital image correlation (DIC) approach. Differences in the parameters between the NORM and KC groups were statistically analyzed and compared. Statistical results indicated that compared with the NORM group, the KC corneas absorbed more energy (KC: 8.98 ± 2.76 mN mm; NORM: 4.79 ± 0.62 mN mm; p-value <0.001) with smaller tangent stiffness (KC: 22.49 ± 2.62 mN/mm; NORM: 24.52 ± 3.20 mN/mm; p-value = 0.15) and larger maximum deflection (KC: 0.99 ± 0.07 mN/mm; NORM: 0.92 ± 0.06 mN/mm; p-value <0.05) on the macro scale. Further, we also observed that The maximum displacement (KC: 1.17 ± 0.06 mm; NORM: 1.06 ± 0.07 mm; p-value <0.005), velocity (KC: 236 ± 29 mm/s; NORM: 203 ± 17 mm/s; p-value <0.01), shear strain (KC: 24.43 ± 2.59%; NORM: 20.26 ± 1.54%; p-value <0.001), and shear strain rate (KC: 69.74 ± 11.99 s-1; NORM: 54.84 ± 3.03 s-1; p-value <0.005) in the KC group significantly increased at the tissue level. This is the first time that the incremental DIC method was applied to the in-vivo high-speed corneal deformation measurement in combination with the Corvis ST tonometer. Through the image registration using incremental DIC analysis, spatiotemporal dynamic strain/strain rate maps of the cornea can be estimated at the tissue level. This is constructive for the clinical recognition and diagnosis of keratoconus at a more underlying level.

Characterization of Photorhabdus Virulence Cassette as a causative agent in the emerging pathogen Photorhabdus asymbiotica.

The extracellular contractile injection systems (eCISs) are encoded in the genomes of a large number of bacteria and archaea. We have previously characterized the overall structure of Photorhabdus Virulence Cassette (PVC), a typical member of the eCIS family. PVC resembles the contractile tail of bacteriophages and exerts its action by the contraction of outer sheath and injection of inner tube plus central spike. Nevertheless, the biological function of PVC effectors and the mechanism of effector translocation are still lacking. By combining cryo-electron microscopy and functional experiments, here we show that the PVC effectors Pdp1 (a new family of widespread dNTP pyrophosphatase effector in eCIS) and Pnf (a deamidase effector) are loaded inside the inner tube lumen in a "Peas in the Pod" mode. Moreover, we observe that Pdp1 and Pnf can be directly injected into J774A.1 murine macrophage and kill the target cells by disrupting the dNTP pools and actin cytoskeleton formation, respectively. Our results provide direct evidence of how PVC cargoes are loaded and delivered directly into mammalian macrophages.

N-terminal signal peptides facilitate the engineering of PVC complex as a potent protein delivery system.

Extracellular contractile injection systems (eCISs) are widespread bacterial nanomachines that resemble T4 phage tail. As a typical eCIS, Photorhabdus virulence cassette (PVC) was proposed to inject toxins into eukaryotic cells by puncturing the cell membrane from outside. This makes it an ideal tool for protein delivery in biomedical research. However, how to manipulate this nanocomplex as a molecular syringe is still undetermined. Here, we identify that one group of N-terminal signal peptide (SP) sequences are crucial for the effector loading into the inner tube of PVC complex. By application of genetic operation, cryo-electron microscopy, in vitro translocation assays, and animal experiments, we show that, under the guidance of the SP, numerous prokaryotic and eukaryotic proteins can be loaded into PVC to exert their functions across cell membranes. We therefore might customize PVC as a potent protein delivery nanosyringe for biotherapy by selecting cargo proteins in a broad spectrum, regardless of their species, sizes, and charges.

Structural Insight into the MCM double hexamer activation by Dbf4-Cdc7 kinase.

The Dbf4-dependent kinase Cdc7 (DDK) regulates DNA replication initiation by phosphorylation of the MCM double hexamer (MCM-DH) to promote helicase activation. Here, we determine a series of cryo electron microscopy (cryo-EM) structures of yeast DDK bound to the MCM-DH. These structures, occupied by one or two DDKs, differ primarily in the conformations of the kinase core. The interactions of DDK with the MCM-DH are mediated exclusively by subunit Dbf4 straddling across the hexamer interface on the three N-terminal domains (NTDs) of subunits Mcm2, Mcm6, and Mcm4. This arrangement brings Cdc7 close to its only essential substrate, the N-terminal serine/threonine-rich domain (NSD) of Mcm4. Dbf4 further displaces the NSD from its binding site on Mcm4-NTD, facilitating an immediate targeting of this motif by Cdc7. Moreover, the active center of Cdc7 is occupied by a unique Dbf4 inhibitory loop, which is disengaged when the kinase core assumes wobbling conformations. This study elucidates the versatility of Dbf4 in regulating the ordered multisite phosphorylation of the MCM-DH by Cdc7 kinase during helicase activation.

Structural insights into TSC complex assembly and GAP activity on Rheb.

Tuberous sclerosis complex (TSC) integrates upstream stimuli and regulates cell growth by controlling the activity of mTORC1. TSC complex functions as a GTPase-activating protein (GAP) towards small GTPase Rheb and inhibits Rheb-mediated activation of mTORC1. Mutations in TSC genes cause tuberous sclerosis. In this study, the near-atomic resolution structure of human TSC complex reveals an arch-shaped architecture, with a 2:2:1 stoichiometry of TSC1, TSC2, and TBC1D7. This asymmetric complex consists of two interweaved TSC1 coiled-coil and one TBC1D7 that spans over the tail-to-tail TSC2 dimer. The two TSC2 GAP domains are symmetrically cradled within the core module formed by TSC2 dimerization domain and central coiled-coil of TSC1. Structural and biochemical analyses reveal TSC2 GAP-Rheb complimentary interactions and suggest a catalytic mechanism, by which an asparagine thumb (N1643) stabilizes γ-phosphate of GTP and accelerate GTP hydrolysis of Rheb. Our study reveals mechanisms of TSC complex assembly and GAP activity.

Structural insight into the assembly and conformational activation of human origin recognition complex.

The function of the origin recognition complex (ORC) in DNA replication is highly conserved in recognizing and marking the initiation sites. The detailed molecular mechanisms by which human ORC is reconfigured into a state competent for origin association remain largely unknown. Here, we present structural characterizations of human ORC1-5 and ORC2-5 assemblies. ORC2-5 exhibits a tightly autoinhibited conformation with the winged-helix domain of ORC2 completely blocking the central DNA-binding channel. The binding of ORC1 partially relieves the autoinhibitory effect of ORC2-5 through remodeling ORC2-WHD, which makes ORC2-WHD away from the central channel creating a still autoinhibited but more dynamic structure. In particular, the AAA+ domain of ORC1 is highly flexible to sample a variety of conformations from inactive to potentially active states. These results provide insights into the detailed mechanisms regulating the autoinhibition of human ORC and its subsequent activation for DNA binding.

Autophagy functions as an antiviral mechanism against geminiviruses in plants.

Autophagy is an evolutionarily conserved process that recycles damaged or unwanted cellular components, and has been linked to plant immunity. However, how autophagy contributes to plant immunity is unknown. Here we reported that the plant autophagic machinery targets the virulence factor ßC1 of Cotton leaf curl Multan virus (CLCuMuV) for degradation through its interaction with the key autophagy protein ATG8. A V32A mutation in ßC1 abolished its interaction with NbATG8f, and virus carrying ßC1V32A showed increased symptoms and viral DNA accumulation in plants. Furthermore, silencing of autophagy-related genes ATG5 and ATG7 reduced plant resistance to the DNA viruses CLCuMuV, Tomato yellow leaf curl virus, and Tomato yellow leaf curl China virus, whereas activating autophagy by silencing GAPC genes enhanced plant resistance to viral infection. Thus, autophagy represents a novel anti-pathogenic mechanism that plays an important role in antiviral immunity in plants.