A network pharmacological study to unveil the mechanisms of xianlinggubao capsule in the treatment of osteoarthritis and osteoporosis.

Introduction: Xianlinggubao (XLGB) capsule is a traditional Chinese medicine, which is approved by the Chinese State Food and Drug Administration (CFDA) for osteoarthritis (OA) and osteoporosis (OP). However, as a capsule with complex ingredients, the molecular mechanisms supporting the therapeutic effects have not been explored. Material and methods: A network pharmacology-based approach was conducted to explore the complex interactome among the targets of the XLGB active compounds. Results: The herbs in the capsule contain 41 compounds with 246 high score targets, which cover four known OA targets (PTGS1, PTGS2, PTGER4 and TNF) and six known OP targets (AR, ESR1, PGR, PTGER2, TNFSF11 and VDR) of FDA-approved drugs or drugs undergoing clinical trials. The protein-protein interaction (PPI) network of the 246 targets had six key modules. Among the six modules, neuroactive ligand-receptor interaction, cAMP signaling pathway and calcium signaling pathway are the key pathways, which are all closely associated with the degeneration of joint cartilage and bone formation and resorption. Conclusions: Neuroactive ligand-receptor interaction, cAMP signaling pathway, and calcium signaling pathway might be the critical pathways upon which the capsule might act. The present study laid down a foundation to understand the molecular mechanisms of the XLGB capsule and also provided fundamental information for better improvement of the drug with the concept "less herbal materials for achieving equal treatment efficacy".

Miniaturized time-correlated single-photon counting module for time-of-flight non-line-of-sight imaging applications.

Single-photon time-of-flight (TOF) non-line-of-sight (NLOS) imaging enables the high-resolution reconstruction of objects outside the field of view. The compactness of TOF NLOS imaging systems, entailing the miniaturization of key components within such systems, is crucial for practical applications. Here, we present a miniaturized four-channel time-correlated single-photon counting module dedicated to TOF NLOS imaging applications. The module achieves excellent performance with a 10 ps bin size and 27.4 ps minimum root-mean-square time resolution. We present the results of the TOF NLOS imaging experiment using an InGaAs/InP single-photon detector and the time-correlated single-photon counting module and show that a 6.3 cm lateral resolution and 2.3 cm depth resolution can be achieved under the conditions of 5 m imaging distance and 1 ms pixel dwell time.

Architecture of the bacteriophage lambda tail.

Bacteriophage lambda has a double-stranded DNA genome and a long, flexible, non-contractile tail encoded by a contiguous block of 11 genes downstream of the head genes. The tail allows host recognition and delivery of viral DNA from the head shell to the cytoplasm of the infected cell. Here, we present a high-resolution structure of the tail complex of bacteriophage lambda determined by cryoelectron microscopy. Most component proteins of the lambda tail were determined at the atomic scale. The structure sheds light on the molecular organization of the extensively studied tail of bacteriophage lambda.

SARS-CoV-2 hijacks neutralizing dimeric IgA for nasal infection and injury in Syrian hamsters1.

Prevention of robust severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection in nasal turbinate (NT) requires in vivo evaluation of IgA neutralizing antibodies. Here, we report the efficacy of receptor binding domain (RBD)-specific monomeric B8-mIgA1 and B8-mIgA2, and dimeric B8-dIgA1, B8-dIgA2 and TH335-dIgA1 against intranasal SARS-CoV-2 challenge in Syrian hamsters. These antibodies exhibited comparable neutralization potency against authentic virus by competing with human angiotensin converting enzyme-2 (ACE2) receptor for RBD binding. While reducing viral loads in lungs significantly, prophylactic intranasal B8-dIgA unexpectedly led to high amount of infectious viruses and extended damage in NT compared to controls. Mechanistically, B8-dIgA failed to inhibit SARS-CoV-2 cell-to-cell transmission, but was hijacked by the virus through dendritic cell-mediated trans-infection of NT epithelia leading to robust nasal infection. Cryo-EM further revealed B8 as a class II antibody binding trimeric RBDs in 3-up or 2-up/1-down conformation. Neutralizing dIgA, therefore, may engage an unexpected mode of SARS-CoV-2 nasal infection and injury.

Multiple roles of UV/KMnO4 in cyanobacteria containing water treatment: Cell inactivation & removal, and microcystin degradation.

Cyanobacterial blooms present great challenges to drinking water treatment and human health. The novel combination of potassium permanganate (KMnO4) and ultraviolet (UV) radiation is engaged as a promising advanced oxidation process in water purification. This study investigated the treatment of a typical cyanobacteria, Microcystis aeruginosa by UV/KMnO4. Cell inactivation was significantly improved by UV/KMnO4 treatment, compared to UV alone or KMnO4 alone, and cells were completely inactivated within 35 min by UV/KMnO4 in natural water. Moreover, effective degradation of associated microcystins was simultaneously achieved at UV fluence rate of 0.88 mW cm-2 and KMnO4 dosages of 3-5 mg L-1. The significant synergistic effect is possibly attributable to the highly oxidative species produced during UV photolysis of KMnO4. In addition, the cell removal efficiency via self-settling reached 87.9 % after UV/KMnO4 treatment, without additional coagulants. The fast in situ generated manganese dioxide was responsible for the enhancement of M. aeruginosa cell removal. This study firstly reports multiple roles of UV/KMnO4 process in cyanobacterial cell inactivation and removal, as well as simultaneous microcystin degradation under practical conditions.

Liensinine improves AngII-induced vascular remodeling via MAPK/TGF-ß1/Smad2/3 signaling.

ETHNOPHARMACOLOGICAL RELEVANCE: Liensinine(Lien, C37H42N2O6) is an alkaloid compound from plumula nelumbinis that demonstrates an antihypertensive effect. The protective effects of Lien on target organs during hypertension are still unclear. AIM OF THE STUDY: This study aimed to understand the mechanism of Lien during the treatment of hypertension, with emphasis on vascular protection. MATERIALS AND METHODS: Lien was extracted and isolated from plumula nelumbinis for further study. In vivo model of Ang II-induced hypertension, non-invasive sphygmomanometer was used to detect the blood pressure in and out of the context of Lien intervention. Ultrasound was used to detect the abdominal aorta pulse wave and media thickness of hypertensive mice, and RNA sequencing was used to detect the differential genes and pathways of blood vessels. The intersection of Lien and MAPK protein molecules was detected by molecular interconnecting technique. The pathological conditions of abdominal aorta vessels of mice were observed by HE staining. The expression of PCNA, α-SMA, Collagen Type Ⅰ and Collagen Type Ⅲ proteins were detected by IHC. The collagen expression in the abdominal aorta was detected by Sirius red staining. The MAPK/TGF-ß1/Smad2/3 signaling and the protein expression of PCNA and α-SMA was detected by Western blot. In vitro, MAPK/TGF-ß1/Smad2/3 signaling and the protein expression of PCNA and α-SMA were detected by Western blot, and the expression of α-SMA was detected by immunofluorescence; ELISA was used to detect the effect of ERK/MAPK inhibitor PD98059 on Ang Ⅱ-induced TGF-ß1secrete; and the detection TGF-ß1and α-SMA protein expression by Western blot; Western blot was used to detect the effect of ERK/MAPK stimulant12-O-tetradecanoyl phorbol-13-acetate (TPA) on the protein expression of TGF-ß1 and α-SMA. RESULTS: Lien displayed an antihypertensive effect on Ang Ⅱ-induced hypertension, reducing the pulse wave conduction velocity of the abdominal aorta and the thickness of the abdominal aorta vessel wall, ultimately improving the pathological state of blood vessels. RNA sequencing further indicated that the differential pathways expressed in the abdominal aorta of hypertensive mice were enriched in proliferation-related markers compared with the Control group. The profile of differentially expressed pathways was ultimately reversed by Lien. Particularly, MAPK protein demonstrated good binding with the Lien molecule. In vivo, Lien inhibited Ang Ⅱ-induced abdominal aorta wall thickening, reduced collagen deposition in the ventral aortic vessel, and prevented the occurrence of vascular remodeling by inhibiting MAPK/TGF-ß1/Smad2/3 signaling activation. In addition, Lien inhibited the activation of Ang II-induced MAPK and TGF-ß1/Smad2/3 signaling, attenuating the expression of PCNA and inhibiting the reduction of α-SMA, collectively playing a role in the inhibition of Ang Ⅱ-induced hypertensive vascular remodeling. PD98059 alone could inhibit Ang Ⅱ-induced elevation of TGF-ß1 and the decrease of α-SMA expression. Further, PD98059 combined with Lien had no discrepancy with the inhibitors alone. Simultaneously TPA alone could significantly increase the expression of TGF-ß1 and decrease the expression of α-SMA. Further, Lien could inhibit the effect of TPA. CONCLUSION: This study helped clarify the protective mechanism of Lien during hypertension, elucidating its role as an inhibitor of vascular remodeling and providing an experimental basis for the research and development of novel antihypertensive therapies.

Structural specializations of the sperm tail.

Sperm motility is crucial to reproductive success in sexually reproducing organisms. Impaired sperm movement causes male infertility, which is increasing globally. Sperm are powered by a microtubule-based molecular machine-the axoneme-but it is unclear how axonemal microtubules are ornamented to support motility in diverse fertilization environments. Here, we present high-resolution structures of native axonemal doublet microtubules (DMTs) from sea urchin and bovine sperm, representing external and internal fertilizers. We identify >60 proteins decorating sperm DMTs; at least 15 are sperm associated and 16 are linked to infertility. By comparing DMTs across species and cell types, we define core microtubule inner proteins (MIPs) and analyze evolution of the tektin bundle. We identify conserved axonemal microtubule-associated proteins (MAPs) with unique tubulin-binding modes. Additionally, we identify a testis-specific serine/threonine kinase that links DMTs to outer dense fibers in mammalian sperm. Our study provides structural foundations for understanding sperm evolution, motility, and dysfunction at a molecular level.

Causality of gut microbiome and hypertension: A bidirectional mendelian randomization study.

Background & Aims: The pathogenesis of hypertension involves a diverse range of genetic, environmental, hemodynamic, and more causative factors. Recent evidence points to an association between the gut microbiome and hypertension. Given that the microbiota is in part determined by host genetics, we used the two-sample Mendelian randomization (MR) analysis to address the bidirectional causal link between gut microbiota and hypertension. Methods: We selected genetic variants (P < 1 × 10-5) for gut microbiota (n = 18,340) from the MiBioGen study. Genetic association estimates for hypertension were extracted from genome-wide association study (GWAS) summary statistics on 54,358 cases and 408,652 controls. Seven complementary MR methods were implemented, including the inverse-variance weighted (IVW) method, followed by sensitivity analyses to verify the robustness of the results. Reverse-direction MR analyses were further conducted to probe if there was a reverse causative relationship. Bidirectional MR analysis then examines a modulation of gut microbiota composition by hypertension. Results: At the genus level, our MR estimates from gut microbiome to hypertension showed that there were 5 protective factors Allisonella, Parabacteroide, Phascolarctobacterium, Senegalimassilia, and unknowngenus (id.1000000073), while 6 genera Clostridiuminnocuum, Eubacteriumcoprostanoligenes, Eubacteriumfissicatena, Anaerostipes, LachnospiraceaeFCS020, and unknowngenus (id.2041) are risk factors. The Alcaligenaceae and ClostridialesvadinBB60 were detrimental and beneficial at the family level, respectively. In contrast, the MR results of hypertension-gut flora showed hypertensive states can lead to an increased abundance of Eubacteriumxylanophilum, Eisenbergiella, and Lachnospiraceae and a lower abundance of Alistipes, Bilophila, Butyricimonas, and Phascolarctobacterium. Conclusion: Altered gut microbiota is a causal factor in the development of hypertension, and hypertension causes imbalances in the intestinal flora. Substantial research is still needed to find the key gut flora and explore the specific mechanisms of their effects so that new biomarkers can be found for blood pressure control.

Cryoelectron microscopy structures of a human neutralizing antibody bound to MERS-CoV spike glycoprotein.

Neutralizing monoclonal antibodies (mAbs) against highly pathogenic coronaviruses represent promising candidates for clinical intervention. Here, we isolated a potent neutralizing monoclonal antibody, MERS-S41, from a yeast displayed scFv library using the S protein as a bait. To uncover the neutralization mechanism, we determined structures of MERS-S41 Fab in complex with the trimeric spike glycoprotein by cryoelectron microscopy (cryo-EM). We observed four distinct classes of the complex structure, which showed that the MERS-S41 Fab bound to the "up" receptor binding domain (RBD) with full saturation and also bound to an accessible partially lifted "down" RBD, providing a structural basis for understanding how mAbs bind to trimeric spike glycoproteins. Structure analysis of the epitope and cell surface staining assays demonstrated that virus entry is blocked predominantly by direct competition with the host receptor, dipeptidyl peptidase-4 (DPP4).

Accuracy and stability evaluation of different blood sampling methods in blood gas analysis in emergency patients: A retrospective study.

BACKGROUND: To evaluate the accuracy and stability of arterial blood gas (ABG) results by comparison with venous measurements from routine blood tests, and to compare the accuracy and performance of two sampling syringes, pre-heparinized syringe (PHS) and disposable arterial blood syringe (DABS), in ABG analysis. METHODS: We retrospectively analyzed the practical use of PHS and DABS in collecting ABG samples, involving 500 and 400 patients, respectively. For each patient, in addition to the ABG sample, a venous blood sample was also collected using a venous blood collection tube (VBCT) and used for routine blood tests. Accordingly, patients were referred to as the PHS + VBCT group and DABS + VBCT group. The correlation between arterial and venous values of each blood parameter in each group was evaluated using the interclass correlation coefficient (ICC). Bland-Altman was performed to evaluate the agreement between arterial and venous values and compare the performance of PHS and DABS in ABG sample collection. RESULTS: In the PHS + VBCT group, arterial K+ , Na+ , hemoglobin (Hb), and hematocrit (HCT) were 0.32 mmol/L, 2.90 mmol/L, 2.21 g/L, and 1.27% significantly lower their corresponding venous values while arterial Cl- was 7.60 mmol/L significantly higher than venous Cl- . In the DABS + VBCT group, arterial K+ and Na+ were 0.20 mmol/L and 1.19 mmol/L significantly lower while Cl- and HCT in arterial blood were 5.34 mmol/L and 0.66% significantly higher than their corresponding venous values. In both groups, arterial K+ , Na+ , Hb, and HCT values were highly consistent with their corresponding venous values, with all ICCs greater than 0.70, especially Hb and HCT. Bland-Altman analysis demonstrated that arterial K+ and Na+ were more consistent with venous counterparts in the DABS + VBCT group, with a narrower 95% limits of agreement than the PHS + VBCT group (K+ , -0.7-0.3 mmol/L vs. -1.1 to 0.5 mmol/L; Na+ , -5.8 to 3.4 mmol/L vs. -8.2 to 2.4 mmol/L). CONCLUSION: Arterial blood gas analysis of K+ , Na+ , Hb, and HCT using PHS or DABS for blood sampling is accurate and stable, especially DABS, which can provide clinicians with fast and reliable blood gas results.

Protective role of Achyranthes bidentata polysaccharides against chondrocyte extracellular matrix degeneration through lncRNA GAS5 in osteoarthritis.

Achyranthes bidentata polysaccharides (ABPS) is an active ingredient of the flowering plant Achyranthes bidentata that has been previously reported to be effective for the treatment of osteoarthritis (OA). However, the underlying molecular mechanism remain to be fully clarified. Emerging studies have shown that the long non-coding RNA (lncRNA) growth arrest-specific transcript 5 (GAS5) is involved in the pathogenesis of OA. Therefore, the present study aimed to investigate the potential mechanism of ABPS by focusing on its effects on the regulation of chondrocyte extracellular matrix (ECM) homeostasis, with particular emphasis on lncRNA GAS5. In the present study, the modified Hulth method was used to construct OA rats, which were gavaged with 400 mg/kg ABPS for 8 weeks. Histopathological changes in cartilage and subchondral bone were evaluated by hematoxylin-eosin staining and Safranin O-fast green staining. In in vitro experiments, IL-1ß-treated chondrocytes were infected with Lenti-lncRNA GAS5. Fluorescence in situ hybridization assay was performed to measure the expression of the lncRNA GAS5 in chondrocytes. Moreover, the relative expression level of lncRNA GAS5 in cartilage tissue and chondrocytes was detected using reverse transcription-quantitative PCR. Western blot analysis was used to detect protein expression levels of MMP-9, MMP-13, TIMP-1, TIMP-3 and type II collagen in cartilage tissue and chondrocytes. The results indicated that ABPS delayed the degradation of the ECM by chondrocytes in addition to reducing lncRNA GAS5 expression both in vivo and in vitro. Furthermore, silencing of lncRNA GAS5 expression in IL-1ß-treated chondrocytes downregulated the protein expression of MMP-9 and MMP-13 whilst upregulating the expression of tissue inhibitor matrix metalloproteinase (TIMP)-1, TIMP-3 and type II collagen. To conclude, the present study provides evidence that ABPS can inhibit the expression of lncRNA GAS5 in chondrocytes to regulate the homeostasis of ECM, which in turn may delay the occurrence of cartilage degeneration during OA.

Structural Basis of the Immunity Mechanisms of Pediocin-like Bacteriocins.

Pediocin-like bacteriocins, also designated class IIa bacteriocins, are ribosomally synthesized antimicrobial peptides targeting species closely related to the producers. They act on the cytoplasmic membrane of Gram-positive cells by dissipating the transmembrane electrical potential through pore formation with the mannose phosphotransferase system (man-PTS) as the target/receptor. Bacteriocin-producing strains also synthesize a cognate immunity protein that protects them against their own bacteriocins. Herein, we report the cryo-electron microscopy structure of the bacteriocin-receptor-immunity ternary complex from Lactobacillus sakei. The complex structure reveals that pediocin-like bacteriocins bind to the same position on the Core domain of man-PTS, while the C-terminal helical tails of bacteriocins delimit the opening range of the Core domain away from the Vmotif domain to facilitate transmembrane pore formation. Upon attack of bacteriocins from the extracellular side, man-PTS exposes its cytosolic side for recognition of the N-terminal four-helix bundle of the immunity protein. The C-terminal loop of the immunity protein then inserts into the pore and blocks leakage induced by bacteriocins. Elucidation of the toxicity and immunity mechanisms of pediocin-like bacteriocins could support the design of novel bacteriocins against antibiotic-resistant pathogenic bacteria. IMPORTANCE Pediocin-like bacteriocins, ribosomally synthesized antimicrobial peptides, are generally co-expressed with cognate immunity proteins to protect the bacteriocin-producing strain from its own bacteriocin. Bacteriocins are considered potential alternatives to conventional antibiotics in the context of the bacterial resistance crisis, but the immunity mechanism is unclear. This study uncovered the mechanisms of action and immunity of class IIa bacteriocins.

Jiangu granule ameliorated OVX rats bone loss by modulating gut microbiota-SCFAs-Treg/Th17 axis.

BACKGROUND: Postmenopausal osteoporosis (PMOP) is a common disease that has decreased bone strength as its main symptom after menopause. Effective treatment for PMOP remains lacking, but traditional Chinese medicine has some advantages in delaying bone loss. Jiangu granule is a traditional Chinese medicine prescription commonly used to treat PMOP. Previous studies have demonstrated its efficacy, but the mechanism of action remains uncharacterized. PURPOSE: This study aims to observe and discuss the mechanism of Jiangu granule to ameliorate bone loss in OVX rats by regulating the gut microbiota (GM)-short-chain fatty acids (SCFAs)- Treg/Th17 axis. METHODS: Female SD rats were divided into the sham operation (S), Jiangu granule (J), and model group (M). Bilateral ovaries were surgically removed from the rats in the J and M groups. After 6 and 12 weeks, rats were sacrificed, and femur, tibia, vertebrae, serum, spleen, colon, and feces samples were collected. We detected the strength of bones, gut microbiota structure, and SCFAs in feces, the Treg and Th17 cell levels in the spleen, and cytokine levels in the serum. RESULT: Jiangu granule restored the abundance of gut microbiota, increased the content of SCFAs, reduced the permeability of colon epithelium, increased the proportion of Treg cells in the spleen, changed the osteoimmunomodulation-related cytokines, effectively prevented bone loss, and enhanced bone strength. CONCLUSION: Jiangu granule can effectively improve bone loss in OVX rats, possibly by regulating the "GM-SCFAs-Treg/Th17″ axis.

Insights into the inhibition of type I-F CRISPR-Cas system by a multifunctional anti-CRISPR protein AcrIF24.

CRISPR-Cas systems are prokaryotic adaptive immune systems and phages use anti-CRISPR proteins (Acrs) to counteract these systems. Here, we report the structures of AcrIF24 and its complex with the crRNA-guided surveillance (Csy) complex. The HTH motif of AcrIF24 can bind the Acr promoter region and repress its transcription, suggesting its role as an Aca gene in self-regulation. AcrIF24 forms a homodimer and further induces dimerization of the Csy complex. Apart from blocking the hybridization of target DNA to the crRNA, AcrIF24 also induces the binding of non-sequence-specific dsDNA to the Csy complex, similar to AcrIF9, although this binding seems to play a minor role in AcrIF24 inhibitory capacity. Further structural and biochemical studies of the Csy-AcrIF24-dsDNA complexes and of AcrIF24 mutants reveal that the HTH motif of AcrIF24 and the PAM recognition loop of the Csy complex are structural elements essential for this non-specific dsDNA binding. Moreover, AcrIF24 and AcrIF9 display distinct characteristics in inducing non-specific DNA binding. Together, our findings highlight a multifunctional Acr and suggest potential wide distribution of Acr-induced non-specific DNA binding.

The effect of heart rate on blood pressure measurement in patients with atrial fibrillation: a cross-sectional study.

This study explored the effect of heart rate (HR) on the stability and accuracy of blood pressure (BP) measurement and the optimal HR range for the most accurate blood pressure measurement in atrial fibrillation (AF) patients. A total of 583 patients (383 and 200 with AF and sinus rhythm (SR), respectively) were included in this study. The noninvasive blood pressure (NIBP), invasive blood pressure (IBP), and HR were repeatedly measured ten times at 30-second intervals for every patient. Both the AF and SR groups were then subdivided into five groups depending on the HR (i.e., < 60, 60-80, 80-100, 100-120, and ≥120 bpm). The difference between the IBP and NIBP (i.e., △SBP) and the coefficient of variation (CV) were calculated, and the stability and accuracy of NIBP measurements were analyzed. CV and △SBP were significantly higher in the AF group. In the AF group, the CV of NIBP was highest when the HR was ≥ 100 bpm; and △SBP was significantly lower in the HR groups with 60-80 and 80-100 bpm (< 60 bpm, △SBP 11.62 ± 2.64 mmHg; 60-80 bpm, △SBP 7.10 ± 1.92 mmHg; 80-100 bpm, △SBP 7.10 ± 2.95 mmHg; 100-120 bpm, △SBP 10.52 ± 2.72 mmHg; ≥120 bpm, △SBP 14.15 ± 3.61 mmHg, P < 0.05). The stability and accuracy of the NIBP in the SR groups were not affected by the HR. In AF patients, the NIBP stability was low when the HR was high, and the NIBP was often underestimated when the HR was high or low. Sixty to 100 bpm is the best HR range for measuring blood pressure in AF patients.

Structural basis of von Willebrand factor multimerization and tubular storage.

The von Willebrand factor (VWF) propeptide (domains D1D2) is essential for the assembly of VWF multimers and its tubular storage in Weibel-Palade bodies. However, detailed molecular mechanism underlying this propeptide dependence is unclear. Here, we prepared Weibel-Palade body-like tubules using the N-terminal fragment of VWF and solved the cryo-electron microscopy structures of the tubule at atomic resolution. Detailed structural and biochemical analysis indicate that the propeptide forms a homodimer at acidic pH through the D2:D2 binding interface and then recruits 2 D'D3 domains, forming an intertwined D1D2D'D3 homodimer in essence. Stacking of these homodimers by the intermolecular D1:D2 interfaces brings 2 D3 domains face-to-face and facilitates their disulfide linkages and multimerization of VWF. Sequential stacking of these homodimers leads to a right-hand helical tubule for VWF storage. The clinically identified VWF mutations in the propeptide disrupted different steps of the assembling process, leading to diminished VWF multimers in von Willebrand diseases (VWD). Overall, these results indicate that the propeptide serves as a pH-sensing template for VWF multimerization and tubular storage. This sheds light on delivering normal propeptide as a template to rectify the defects in multimerization of VWD mutants.

Structural basis of bacteriophage lambda capsid maturation.

Bacteriophage lambda is an excellent model system for studying capsid assembly of double-stranded DNA (dsDNA) bacteriophages, some dsDNA archaeal viruses, and herpesviruses. HK97 fold coat proteins initially assemble into a precursor capsid (procapsid) and subsequent genome packaging triggers morphological expansion of the shell. An auxiliary protein is required to stabilize the expanded capsid structure. To investigate the capsid maturation mechanism, we determined the cryo-electron microscopy structures of the bacteriophage lambda procapsid and mature capsid at 3.88 Å and 3.76 Å resolution, respectively. Besides primarily rigid body movements of common features of the major capsid protein gpE, large-scale structural rearrangements of other domains occur simultaneously. Assembly of intercapsomers within the procapsid is facilitated by layer-stacking effects at 3-fold vertices. Upon conformational expansion of the capsid shell, the missing top layer is fulfilled by cementing the gpD protein against the internal pressure of DNA packaging. Our structures illuminate the assembly mechanisms of dsDNA viruses.

Mechanistic insights into the inhibition of the CRISPR-Cas surveillance complex by anti-CRISPR protein AcrIF13.

Clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins provide prokaryotes with nucleic acid-based adaptive immunity against infections of mobile genetic elements, including phages. To counteract this immune process, phages have evolved various anti-CRISPR (Acr) proteins which deactivate CRISPR-Cas-based immunity. However, the mechanisms of many of these Acr-mediated inhibitions are not clear. Here, we report the crystal structure of AcrIF13 and explore its inhibition mechanism. The structure of AcrIF13 is unique and displays a negatively charged surface. Additionally, biochemical studies identified that AcrIF13 interacts with the type I-F CRISPR-Cas surveillance complex (Csy complex) to block target DNA recognition and that the Cas5f-8f tail and Cas7.6f subunit of the Csy complex are specific binding targets of AcrIF13. Further mutational studies demonstrated that several negatively charged residues of AcrIF13 and positively charged residues of Cas8f and Cas7f of the Csy complex are involved in AcrIF13-Csy binding. Together, our findings provide mechanistic insights into the inhibition mechanism of AcrIF13 and further suggest the prevalence of the function of Acr proteins as DNA mimics.

Crystal Structure of the Core Module of the Yeast Paf1 Complex.

The highly conserved multifunctional polymerase-associated factor 1 (Paf1) complex (PAF1C), which consists of five core subunits: Ctr9, Paf1, Leo1, Cdc73, and Rtf1, acts as a diverse hub that regulates all stages of RNA polymerase II-mediated transcription and various other cellular functions. However, the underlying mechanisms remain unclear. Here, we report the crystal structure of the core module derived from a quaternary Ctr9/Paf1/Cdc73/Rtf1 complex of S. cerevisiae PAF1C, which reveals interfaces between the tetratricopeptide repeat module in Ctr9 and Cdc73 or Rtf1, and find that the Ctr9/Paf1 subcomplex is the key scaffold for PAF1C assembly. Our study demonstrates that Cdc73 binds Ctr9/Paf1 subcomplex with a very similar conformation within thermophilic fungi or human PAF1C, and that the binding of Cdc73 to PAF1C is important for yeast growth. Importantly, our structure reveals for the first time that the extreme C-terminus of Rtf1 adopts an "L"-shaped structure, which interacts with Ctr9 specifically. In addition, disruption of the binding of either Cdc73 or Rtf1 to PAF1C greatly affects the normal level of histone H2B K123 monoubiquitination in vivo. Collectively, our results provide a structural insight into the architecture of the quaternary Ctr9/Paf1/Cdc73/Rtf1 complex and PAF1C functional regulation.