Structural basis of peptide secretion for Quorum sensing by ComA.

Quorum sensing (QS) is a crucial regulatory mechanism controlling bacterial signalling and holds promise for novel therapies against antimicrobial resistance. In Gram-positive bacteria, such as Streptococcus pneumoniae, ComA is a conserved efflux pump responsible for the maturation and secretion of peptide signals, including the competence-stimulating peptide (CSP), yet its structure and function remain unclear. Here, we functionally characterize ComA as an ABC transporter with high ATP affinity and determined its cryo-EM structures in the presence or absence of CSP or nucleotides. Our findings reveal a network of strong electrostatic interactions unique to ComA at the intracellular gate, a putative binding pocket for two CSP molecules, and negatively charged residues facilitating CSP translocation. Mutations of these residues affect ComA's peptidase activity in-vitro and prevent CSP export in-vivo. We demonstrate that ATP-Mg2+ triggers the outward-facing conformation of ComA for CSP release, rather than ATP alone. Our study provides molecular insights into the QS signal peptide secretion, highlighting potential targets for QS-targeting drugs.

Practices for running a research-oriented shared cryo-EM facility.

The Harvard Cryo-Electron Microscopy Center for Structural Biology, which was formed as a consortium between Harvard Medical School, Boston Children's Hospital, Dana-Farber Cancer Institute, and Massachusetts General Hospital, serves both academic and commercial users in the greater Harvard community. The facility strives to optimize research productivity while training users to become expert electron microscopists. These two tasks may be at odds and require careful balance to keep research projects moving forward while still allowing trainees to develop independence and expertise. This article presents the model developed at Harvard Medical School for running a research-oriented cryo-EM facility. Being a research-oriented facility begins with training in cryo-sample preparation on a trainee's own sample, ideally producing grids that can be screened and optimized on the Talos Arctica via multiple established pipelines. The first option, staff assisted screening, requires no user experience and a staff member provides instant feedback about the suitability of the sample for cryo-EM investigation and discusses potential strategies for sample optimization. Another option, rapid access, allows users short sessions to screen samples and introductory training for basic microscope operation. Once a sample reaches the stage where data collection is warranted, new users are trained on setting up data collection for themselves on either the Talos Arctica or Titan Krios microscope until independence is established. By providing incremental training and screening pipelines, the bottleneck of sample preparation can be overcome in parallel with developing skills as an electron microscopist. This approach allows for the development of expertise without hindering breakthroughs in key research areas.

Early detection of COPD based on graph convolutional network and small and weakly labeled data.

Chronic obstructive pulmonary disease (COPD) is a common disease with high morbidity and mortality, where early detection benefits the population. However, the early diagnosis rate of COPD is low due to the absence or slight early symptoms. In this paper, a novel method based on graph convolution network (GCN) for early detection of COPD is proposed, which uses small and weakly labeled chest computed tomography image data from the publicly available Danish Lung Cancer Screening Trial database. The key idea is to construct a graph using regions of interest randomly selected from the segmented lung parenchyma and then input it into the GCN model for COPD detection. In this way, the model can not only extract the feature information of each region of interest but also the topological structure information between regions of interest, that is, graph structure information. The proposed GCN model achieves an acceptable performance with an accuracy of 0.77 and an area under a curve of 0.81, which is higher than the previous studies on the same dataset. GCN model also outperforms several state-of-the-art methods trained at the same time. As far as we know, it is also the first time using the GCN model on this dataset for COPD detection.

In situ Structure of Rotavirus VP1 RNA-Dependent RNA Polymerase.

Rotaviruses, like other non-enveloped, double-strand RNA viruses, package an RNA-dependent RNA polymerase (RdRp) with each duplex of their segmented genomes. Rotavirus cell entry results in loss of an outer protein layer and delivery into the cytosol of an intact, inner capsid particle (the "double-layer particle," or DLP). The RdRp, designated VP1, is active inside the DLP; each VP1 achieves many rounds of mRNA transcription from its associated genome segment. Previous work has shown that one VP1 molecule lies close to each 5-fold axis of the icosahedrally symmetric DLP, just beneath the inner surface of its protein shell, embedded in tightly packed RNA. We have determined a high-resolution structure for the rotavirus VP1 RdRp in situ, by local reconstruction of density around individual 5-fold positions. We have analyzed intact virions ("triple-layer particles"), non-transcribing DLPs and transcribing DLPs. Outer layer dissociation enables the DLP to synthesize RNA, in vitro as well as in vivo, but appears not to induce any detectable structural change in the RdRp. Addition of NTPs, Mg2+, and S-adenosylmethionine, which allows active transcription, results in conformational rearrangements, in both VP1 and the DLP capsid shell protein, that allow a transcript to exit the polymerase and the particle. The position of VP1 (among the five symmetrically related alternatives) at one vertex does not correlate with its position at other vertices. This stochastic distribution of site occupancies limits long-range order in the 11-segment, double-strand RNA genome.

Radioprotective activity of glutathione on cognitive ability in X-ray radiated tumor-bearing mice.

OBJECTIVES: The use of X-ray for therapeutics always raises the problem of radiation hazards to living beings. In this research, we explored the radioprotective activity of glutathione (GSH) on cognitive ability of X-ray radiated tumor-bearing mice. METHODS: Forty C57BL/6 mice were chosen to establish the GL261 glioma model and randomly divided into four groups: Model group, X-ray group, Pre-GSH group and Pos-GSH group. Morris water maze test was used to test cognitive ability. Moreover, histopathological observation of hippocampus was observed by hematoxylin and eosin (HE) staining. The protein expression of choline acetyl transferase (ChAT) was measured by western blot, simultaneously the contents of acetylcholinesterase (Ach), superoxide dismutase (SOD), methane dicarboxylic aldehyde (MDA),TNF-α and IL-6 were detected by the respective kit. RESULTS: There was a significant difference in X-ray group of the escape latency from the Model group (P<0.05). Besides, HE staining revealed that nucleus in hippocampus cells were pyknotic, glial cells were hyperplastic and the nerve cells were swelling in X-ray group. In X-ray group the expression of ChAT and Ache were decreased versus Model group. Finally, the cognitive ability in Pre-GSH and Pos-GSH group was enhanced than X-ray group, in which the cognitive ability of Pos-GSH group was higher than the Pre-GSH group. DISCUSSION: X-ray impaired the brain tissues and cognitive ability of tumor-bearing mice. The damages of brain tissues were alleviated by Pre-GSH and Pos-GSH protection and the efficacy of Pos-GSH protection was superior to Pre-GSH protection. Abbreviation Ach: Acetylcholinesterase; GSH: Glutathione; HE: Hematoxylin and eosin; MDA: methane dicarboxylic aldehyde; SOD: Superoxide dismutase; TV: Tumor volume; TW: Tumor weight.

Cryo-EM Structure of Caspase-8 Tandem DED Filament Reveals Assembly and Regulation Mechanisms of the Death-Inducing Signaling Complex.

Caspase-8 activation can be triggered by death receptor-mediated formation of the death-inducing signaling complex (DISC) and by the inflammasome adaptor ASC. Caspase-8 assembles with FADD at the DISC and with ASC at the inflammasome through its tandem death effector domain (tDED), which is regulated by the tDED-containing cellular inhibitor cFLIP and the viral inhibitor MC159. Here we present the caspase-8 tDED filament structure determined by cryoelectron microscopy. Extensive assembly interfaces not predicted by the previously proposed linear DED chain model were uncovered, and were further confirmed by structure-based mutagenesis in filament formation in vitro and Fas-induced apoptosis and ASC-mediated caspase-8 recruitment in cells. Structurally, the two DEDs in caspase-8 use quasi-equivalent contacts to enable assembly. Using the tDED filament structure as a template, structural analyses reveal the interaction surfaces between FADD and caspase-8 and the distinct mechanisms of regulation by cFLIP and MC159 through comingling and capping, respectively.

A mechanism of viral immune evasion revealed by cryo-EM analysis of the TAP transporter.

Cellular immunity against viral infection and tumour cells depends on antigen presentation by major histocompatibility complex class I (MHC I) molecules. Intracellular antigenic peptides are transported into the endoplasmic reticulum by the transporter associated with antigen processing (TAP) and then loaded onto the nascent MHC I molecules, which are exported to the cell surface and present peptides to the immune system. Cytotoxic T lymphocytes recognize non-self peptides and program the infected or malignant cells for apoptosis. Defects in TAP account for immunodeficiency and tumour development. To escape immune surveillance, some viruses have evolved strategies either to downregulate TAP expression or directly inhibit TAP activity. So far, neither the architecture of TAP nor the mechanism of viral inhibition has been elucidated at the structural level. Here we describe the cryo-electron microscopy structure of human TAP in complex with its inhibitor ICP47, a small protein produced by the herpes simplex virus I. Here we show that the 12 transmembrane helices and 2 cytosolic nucleotide-binding domains of the transporter adopt an inward-facing conformation with the two nucleotide-binding domains separated. The viral inhibitor ICP47 forms a long helical hairpin, which plugs the translocation pathway of TAP from the cytoplasmic side. Association of ICP47 precludes substrate binding and prevents nucleotide-binding domain closure necessary for ATP hydrolysis. This work illustrates a striking example of immune evasion by persistent viruses. By blocking viral antigens from entering the endoplasmic reticulum, herpes simplex virus is hidden from cytotoxic T lymphocytes, which may contribute to establishing a lifelong infection in the host.

Structure of the L Protein of Vesicular Stomatitis Virus from Electron Cryomicroscopy.

The large (L) proteins of non-segmented, negative-strand RNA viruses, a group that includes Ebola and rabies viruses, catalyze RNA-dependent RNA polymerization with viral ribonucleoprotein as template, a non-canonical sequence of capping and methylation reactions, and polyadenylation of viral messages. We have determined by electron cryomicroscopy the structure of the vesicular stomatitis virus (VSV) L protein. The density map, at a resolution of 3.8 Å, has led to an atomic model for nearly all of the 2109-residue polypeptide chain, which comprises three enzymatic domains (RNA-dependent RNA polymerase [RdRp], polyribonucleotidyl transferase [PRNTase], and methyltransferase) and two structural domains. The RdRp resembles the corresponding enzymatic regions of dsRNA virus polymerases and influenza virus polymerase. A loop from the PRNTase (capping) domain projects into the catalytic site of the RdRp, where it appears to have the role of a priming loop and to couple product elongation to large-scale conformational changes in L.

Age-related activation of MKK/p38/NF-κB signaling pathway in lung: from mouse to human.

We and others previously reported that the pro-inflammatory cytokine tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß) and IL-6 significantly accumulate with age in mouse lung. This is accompanied by elevated phosphorylation of p38. Here, we further investigate whether aging affects activation of p38 signaling and the inflammatory reaction after exposure to lipopolysaccharide (LPS) in the lungs of mice in vivo and humans ex vivo. The data showed that activation of p38 peaked at 0.5h and then rapidly declined in young (2-month-old) mouse lung, after intranasal inhalation challenge with LPS. In contract, activation of p38 peaked at 24h and was sustained longer in aged (20-month-old) mice. As well as altered p38, activations of its upstream activator MKK and downstream substrate NF-κB were also changed in the lungs of aged mice, which corresponded with the absence in the early phase but delayed increases in concentrations of TNF-α, IL-1ß and IL-6. Consistent with the above observations in mice, similar patterns of p38 signaling also occurred in human lungs. Compared with younger lungs from adult-middle aged subjects, the activation of p38, MKK and NF-κB, as well as the production of pro-inflammatory cytokines were significantly increased in the lungs of older subjects ex vivo. Exposure of human lung cells to LPS induced rapid activation of p38, MKK and NF-κB in these cells from adult-middle aged subjects, but not older subjects, with increases in the production of the pro-inflammatory cytokines. The LPS-induced rapid activation in the lung cells from adult-middle aged subjects occurred as early as 0.25h after exposure, and then declined. Compared with adult-middle aged subjects, the LPS exposure did not induce marked changes in the early phase, either in the activation of p38, MKK and NF-κB, or in the production of TNF-α, IL-1ß or IL-6 in the lung cells from older subjects. In contrast, these changes occurred relatively late, peaked at 16h and were sustained longer in the lungs of older subjects. These data support the hypothesis that the sustained activation of the p38 signaling pathway at baseline and the absence in the early phase but delayed of p38 signaling pathway response to LPS in the elderly may play important roles in increased susceptibility of aged lungs to inflammatory injury.

Simultaneous visualization of the extracellular and cytoplasmic domains of the epidermal growth factor receptor.

To our knowledge, no structural study to date has characterized, in an intact receptor, the coupling of conformational change in extracellular domains through a single-pass transmembrane domain to conformational change in cytoplasmic domains. Here we examine such coupling, and its unexpected complexity, using nearly full-length epidermal growth factor receptor (EGFR) and negative-stain EM. The liganded, dimeric EGFR ectodomain can couple both to putatively active, asymmetrically associated kinase dimers and to putatively inactive, symmetrically associated kinase dimers and monomers. Inhibitors that stabilize the active or inactive conformation of the kinase active site, as well as mutations in the kinase dimer interface and a juxtamembrane phosphorylation site, shift the equilibrium among the three kinase association states. This coupling of one conformation of an activated receptor ectodomain to multiple kinase-domain arrangements reveals previously unanticipated complexity in transmembrane signaling and facilitates regulation of receptor function in the juxtamembrane and cytoplasmic environments.

Age-induced augmentation of p38 MAPK phosphorylation in mouse lung.

The p38 mitogen-activated protein kinase (p38 MAPK) pathway is a key regulator of pro-inflammatory cytokine biosynthesis, which may contribute to the chronic low-grade inflammation observed with aging. We hypothesize that aging up-regulates the activation of p38 MAPK as well as the pro-inflammatory cytokines tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß) and interleukin-6 (IL-6) in mouse lung, and is accompanied by disturbances in oxidant-antioxidant status. In addition, the elevated protein levels of phosphorylated active form of p38 MAPK (phospho-p38 MAPK) with age are tissue-specific. To test this hypothesis, protein levels of phospho-p38 MAPK were determined using Western blot analysis in isolated lung, brain, heart, spleen, kidney and muscle of young (2-month-old) and aged (20-month-old) male C57BL/6J mice. Results show that phospho-p38 MAPK protein levels, not total-p38 MAPK, increased significantly (p<0.01, n=8) in lung and brain of 20-month-old mice. The activation of p38 MAPK in other tissues was not altered with age. Immunostaining showed that epithelial cells and alveolar macrophages in lung parenchyma were the major cellular sources of phospho-p38 MAPK immunity. As measured by enzyme-linked immunosorbent assay (ELISA), TNF-α, IL-1ß and IL-6 in lung homogenates were elevated significantly with age, but there were no differences with age in serum levels except for IL-6. In addition, IL-1ß and IL-6 were increased notably while TNF-α was not different with age in bronchoalveolar lavage fluid (BALF). Furthermore, the oxidant-antioxidant status was evaluated by measuring pro-oxidant malondialdehyde (MDA) levels and the activity of reactive oxygen species scavenging enzymes (i.e. superoxide dismutase (SOD) and glutathione (GSH)) in lung homogenates. The results showed that SOD and GSH decreased with age, while MDA did not change. In conclusion, our data demonstrate that p38 MAPK is activated during lung aging with a corresponding increase in pro-inflammatory cytokines and decrease in antioxidant capacity.

Rotavirus architecture at subnanometer resolution.

Rotavirus, a nonturreted member of the Reoviridae, is the causative agent of severe infantile diarrhea. The double-stranded RNA genome encodes six structural proteins that make up the triple-layer particle. X-ray crystallography has elucidated the structure of one of these capsid proteins, VP6, and two domains from VP4, the spike protein. Complementing this work, electron cryomicroscopy (cryoEM) has provided relatively low-resolution structures for the triple-layer capsid in several biochemical states. However, a complete, high-resolution structural model of rotavirus remains unresolved. Combining new structural analysis techniques with the subnanometer-resolution cryoEM structure of rotavirus, we now provide a more detailed structural model for the major capsid proteins and their interactions within the triple-layer particle. Through a series of intersubunit interactions, the spike protein (VP4) adopts a dimeric appearance above the capsid surface, while forming a trimeric base anchored inside one of the three types of aqueous channels between VP7 and VP6 capsid layers. While the trimeric base suggests the presence of three VP4 molecules in one spike, only hints of the third molecule are observed above the capsid surface. Beyond their interactions with VP4, the interactions between VP6 and VP7 subunits could also be readily identified. In the innermost T=1 layer composed of VP2, visualization of the secondary structure elements allowed us to identify the polypeptide fold for VP2 and examine the complex network of interactions between this layer and the T=13 VP6 layer. This integrated structural approach has resulted in a relatively high-resolution structural model for the complete, infectious structure of rotavirus, as well as revealing the subtle nuances required for maintaining interactions in such a large macromolecular assembly.

Structure of the influenza virus A H5N1 nucleoprotein: implications for RNA binding, oligomerization, and vaccine design.

The threat of a pandemic outbreak of influenza virus A H5N1 has become a major concern worldwide. The nucleoprotein (NP) of the virus binds the RNA genome and acts as a key adaptor between the virus and the host cell. It, therefore, plays an important structural and functional role and represents an attractive drug target. Here, we report the 3.3-A crystal structure of H5N1 NP, which is composed of a head domain, a body domain, and a tail loop. Our structure resolves the important linker segments (residues 397-401, 429-437) that connect the tail loop with the remainder of the molecule and a flexible, basic loop (residues 73-91) located in an arginine-rich groove surrounding Arg150. Using surface plasmon resonance, we found the basic loop and arginine-rich groove, but mostly a protruding element containing Arg174 and Arg175, to be important in RNA binding by NP. We also used our crystal structure to build a ring-shaped assembly of nine NP subunits to model the miniribonucleoprotein particle previously visualized by electron microscopy. Our study of H5N1 NP provides insight into the oligomerization interface and the RNA-binding groove, which are attractive drug targets, and it identifies the epitopes that might be used for universal vaccine development.

Transglutaminase induces protofibril-like amyloid beta-protein assemblies that are protease-resistant and inhibit long-term potentiation.

An increasing body of evidence suggests that soluble assemblies of amyloid beta-protein (Abeta) play an important role in the initiation of Alzheimer disease (AD). In vitro studies have found that synthetic Abeta can form soluble aggregates through self-assembly, but this process requires Abeta concentrations 100- to 1000-fold greater than physiological levels. Tissue transglutaminase (TGase) has been implicated in neurodegeneration and can cross-link Abeta. Here we show that TGase induces rapid aggregation of Abeta within 0.5-30 min, which was not observed with chemical cross-linkers. Both Abeta40 and Abeta42 are good substrates for TGase but show different aggregation patterns. Guinea pig and human TGase induced similar Abeta aggregation patterns, and oligomerization was observed with Abeta40 concentrations as low as 50 nm. The formed Abeta40 species range from 5 to 6 nm spheres to curvilinear structures of the same width, but up to 100 nm in length, that resemble the previously described self-assembled Abeta protofibrils. TGase-induced Abeta40 assemblies are resistant to a 1-h incubation with either neprilysin or insulin degrading enzyme, whereas the monomer is rapidly degraded by both proteases. In support of these species being pathological, TGase-induced Abeta40 assemblies (100 nm) inhibited long term potentiation recorded in the CA1 region of mouse hippocampus slices. Our data suggest that TGase can contribute to AD by initiating Abeta oligomerization and aggregation at physiological levels, by reducing the clearance of Abeta due to the generation of protease-resistant Abeta species, and by forming Abeta assemblies that inhibit processes involved in memory and learning. Our data suggest that TGase might constitute a specific therapeutic target for slowing or blocking the progression of AD.