Mechanisms of Autophagy Initiation.

Autophagy is the process of cellular self-eating by a double-membrane organelle, the autophagosome. A range of signaling processes converge on two protein complexes to initiate autophagy: the ULK1 (unc51-like autophagy activating kinase 1) protein kinase complex and the PI3KC3-C1 (class III phosphatidylinositol 3-kinase complex I) lipid kinase complex. Some 90% of the mass of these large protein complexes consists of noncatalytic domains and subunits, and the ULK1 complex has essential noncatalytic activities. Structural studies of these complexes have shed increasing light on the regulation of their catalytic and noncatalytic activities in autophagy initiation. The autophagosome is thought to nucleate from vesicles containing the integral membrane protein Atg9 (autophagy-related 9), COPII (coat protein complex II) vesicles, and possibly other sources. In the wake of reconstitution and super-resolution imaging studies, we are beginning to understand how the ULK1 and PI3KC3-C1 complexes might coordinate the nucleation and fusion of Atg9 and COPII vesicles at the start of autophagosome biogenesis.

Bidirectional Control of Autophagy by BECN1 BARA Domain Dynamics.

Membrane targeting of the BECN1-containing class III PI 3-kinase (PI3KC3) complexes is pivotal to the regulation of autophagy. The interaction of PI3KC3 complex II and its ubiquitously expressed inhibitor, Rubicon, was mapped to the first ß sheet of the BECN1 BARA domain and the UVRAG BARA2 domain by hydrogen-deuterium exchange and cryo-EM. These data suggest that the BARA ß sheet 1 unfolds to directly engage the membrane. This mechanism was confirmed using protein engineering, giant unilamellar vesicle assays, and molecular simulations. Using this mechanism, a BECN1 ß sheet-1 derived peptide activates both PI3KC3 complexes I and II, while HIV-1 Nef inhibits complex II. These data reveal how BECN1 switches on and off PI3KC3 binding to membranes. The observations explain how PI3KC3 inhibition by Rubicon, activation by autophagy-inducing BECN1 peptides, and inhibition by HIV-1 Nef are mediated by the switchable ability of the BECN1 BARA domain to partially unfold and insert into membranes.

Structural pathway for allosteric activation of the autophagic PI 3-kinase complex I.

Autophagy induction by starvation and stress involves the enzymatic activation of the class III phosphatidylinositol (PI) 3-kinase complex I (PI3KC3-C1). The inactive basal state of PI3KC3-C1 is maintained by inhibitory contacts between the VPS15 protein kinase and VPS34 lipid kinase domains that restrict the conformation of the VPS34 activation loop. Here, the proautophagic MIT domain-containing protein NRBF2 was used to map the structural changes leading to activation. Cryoelectron microscopy was used to visualize a 2-step PI3KC3-C1 activation pathway driven by NRFB2 MIT domain binding. Binding of a single NRBF2 MIT domain bends the helical solenoid of the VPS15 scaffold, displaces the protein kinase domain of VPS15, and releases the VPS34 kinase domain from the inhibited conformation. Binding of a second MIT stabilizes the VPS34 lipid kinase domain in an active conformation that has an unrestricted activation loop and is poised for access to membranes.

Does a history of periodontal disease affect implant survival?

Data sources Three electronic databases were searched (Medline, EMBASE and Cochrane Central) with date of publication between January 2003 and May 2018. Only articles written in English were included. Following electronic searches, the authors conducted manual searches of oral implant/periodontal journals from January 2012 to May 2018. In the event of disagreement on article selection, a further senior reviewer would make the final decision on its inclusion or exclusion following discussion.Study selection In total, 172 articles from the electronic search and ten from manual search were identified for initial screening. From the title and abstract, 18 articles were identified for full-text screening. Following this, 13 articles were included for quantitative synthesis and meta-analysis. The articles assessed the impact of history of periodontitis (HP) on implant survival, radiographic bone loss, pocket depth and bleeding on probing around the dental implant. All studies were either cohort or controlled studies. Seven of the 13 identified studies were prospective. Included studies fulfilled the following criteria: any human studies with supportive periodontal treatment (SPT) application, details of SPT provided in the studies for implant maintenance, compares the outcomes of implants from both patients with and without a HP and peri-implant conditions recorded.Data extraction and synthesis Data extraction followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidance. The Newcastle-Ottawa Scale was used to carry out quality assessment. Data was extracted to calculate risk ratio (RR) of implant survival, weighted mean difference (WMD) for radiographic bone loss, pocket depth, bleeding on probing and plaque index in patients with and without a HP.Results Implant survival rate was assessed as the primary outcome. Secondary outcomes also assessed were radiographic bone loss, pocket depth, bleeding on probing and plaque indices. In implants with rough surfaces, the HP group showed a reduced implant survival rate (RR: 0.96, 95% CI: 0.94-0.98, P <0.001) even under regular supportive post-implant treatment. They also showed more radiographic marginal bone loss (WMD: 0.34 mm, 95% CI: 0.2-0.48, P <0.001), pocket depth (WMD: 0.47 mm, 95% CI: 0.19-0.74, P <0.001) and bleeding on probing (WMD: 0.08 mm, 95% CI: 0.04-0.11, P <0.001) when compared to the non-HP group. In implants with a machined surface, again the HP group had more radiographic bone loss (WMD: 0.88 mm, 95% CI: 0.65-1.11, P <0.001) than the non-HP group. However, in implants with machined surfaces, there was no statistically significant difference in survival rate between HP and non-HP groups (RR: 0.98, 95% CI: 0.92-1.04, P = 0.895).Conclusion In implants with rough surfaces, a history of periodontal disease has a negative impact on survival rate, even with SPT.

How can local anaesthesia be improved in the management of irreversible pulpitis?

Background Pain management in endodontic treatment is often managed with local anaesthetic, occasionally supplemented with oral medication. Currently, there is little evidence to suggest the best combination of local anaesthetic and oral medication to provide optimal pain control in symptomatic irreversible pulpits. A network meta-analysis was carried out to identify the best agent/technique for pulpal anaesthesia in both the maxilla and mandible in irreversible pulpits.Methods Electronic searches of Medline, Cochrane Central and Google Scholar. Reference lists of suitable studies along with manual searches to identify further appropriate studies were also carried out. Sixty-one randomised controlled trials (RCTs) were identified, investigating different local anaesthetic agents, methods of administration, and the adjunct use of medications and alternative therapies. The Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines were used for data collection. Heterogeneity was assessed using chi-squared and I2 tests. Studies comparing the same interventions were pooled to define direct comparison estimates. A common comparator was used to compare indirect comparison estimates. Odds ratio and 95% confidence intervals were used to estimate effect.Results The results were presented on forest plots; 53 studies investigated irreversible pulpitis in the mandible, seven studies in the maxilla and one in both. In the mandible, inferior alveolar nerve block (IANB) with 2% lidocaine was the control. Direct comparison of outcomes found that the best interventions in the mandible were pre-medication with aceclofenac and paracetamol followed by IANB, or IANB with 2% lidocaine with buccal infiltration with 4% articaine, both compared to the control alone. Indirect comparison found pre-medication with ibuprofen and paracetamol before IANB to be the best intervention compared to the control. No significant differences were found between the interventions in the maxilla.Conclusion The quality of all the included evidence was very low and further studies need to be carried out, focusing on larger sample sizes and better quality of studies.

Icosahedral bacteriophage ΦX174 forms a tail for DNA transport during infection.

Prokaryotic viruses have evolved various mechanisms to transport their genomes across bacterial cell walls. Many bacteriophages use a tail to perform this function, whereas tail-less phages rely on host organelles. However, the tail-less, icosahedral, single-stranded DNA ΦX174-like coliphages do not fall into these well-defined infection processes. For these phages, DNA delivery requires a DNA pilot protein. Here we show that the ΦX174 pilot protein H oligomerizes to form a tube whose function is most probably to deliver the DNA genome across the host's periplasmic space to the cytoplasm. The 2.4 Å resolution crystal structure of the in vitro assembled H protein's central domain consists of a 170 Å-long α-helical barrel. The tube is constructed of ten α-helices with their amino termini arrayed in a right-handed super-helical coiled-coil and their carboxy termini arrayed in a left-handed super-helical coiled-coil. Genetic and biochemical studies demonstrate that the tube is essential for infectivity but does not affect in vivo virus assembly. Cryo-electron tomograms show that tubes span the periplasmic space and are present while the genome is being delivered into the host cell's cytoplasm. Both ends of the H protein contain transmembrane domains, which anchor the assembled tubes into the inner and outer cell membranes. The central channel of the H-protein tube is lined with amide and guanidinium side chains. This may be a general property of viral DNA conduits and is likely to be critical for efficient genome translocation into the host.

A comparison of techniques for the explantation of osseointegrated dental implants.

Data sources A search of electronic databases (Embase and PubMed) was carried out along with manual and grey searches of published and unpublished journals. Publication year was from first available until 23 August 2018.Study selection Titles and abstracts from the original search were reviewed by two authors. Studies were chosen for full-text analysis and data extraction after inter-reviewer agreement. Disagreement was resolved by discussion and Cohen's kappa was used to measure inter-reviewer agreement. An initial search gave 2,197 articles and, following screening, 18 publications were included in the study. Five articles were case series and ten were case reports describing one to nine cases. Three publications reported on comparatively large sample sizes, one prospectively and two retrospectively. None of the studies had control groups or blinding. The QUADAS-2 tool was used for quality assessment. Studies were deemed to have high, low or unclear levels of bias by two examiners. All were considered high risk of bias. Publications included fulfilled the following criteria: English language, human studies, endosseous osseointegrated dental implants, explantation technique described and reason for explantation clearly reported.Data extraction and synthesis Data extraction followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guideline process. Studies chosen for analysis were examined and the following data parameters were included: study design, number of patients, number of implants removed, implant system, reason for explantation, explantation technique and its success or failure, complications, flap access, socket grafting and immediate implant placement.Results The following five techniques for explantation of dental implants were identified: reverse torque, trephines, piezosurgery, burs and laser-assisted explantation. Reverse torque was the most commonly described technique (284 implants) with 87.7% success. Burs were used to remove 49 implants with 100% success, while trephines were used for explantation of 35 implants with 94% success. Piezosurgery and Er, Cr: YSGG laser removed 11 implants and one implant, respectively, with 100% success. One study reported perforation of the maxillary sinus floor following the use of a trephine technique, while another reported the fracture of three implants using reverse torque. The quality of the studies and lack of available data prevented further analysis. Results were presented in a narrative format.Conclusion The authors recommend reverse torque as the first choice for explantation. Despite its inferior success rate, it is the most conservative technique in terms of bone removal and flap access, meaning there is a greater opportunity for immediate implant placement.

Do viruses play a role in peri-implantitis?

Data sources A search of electronic databases (EMBASE, MEDLINE, Cochrane Oral Group Trials Register and the Cochrane Central Register of Controlled Trials) along with a manual search of various Science Citation Indexed journals.Study selection Four cross-sectional studies and one case-control study were included where percentage levels of Herpes Simplex Virus Type 1 (HSV1), Epstein-Barr Virus (EBV) and Cytomegalovirus (CMV) were sampled for in both peri-implantitis affected and healthy implant sites, with the latter used as the control. Studies were excluded that investigated any other infective agent, had fewer than ten participants, was performed in vitro or involved subjects with only periodontal disease.Data extraction and synthesis Data extraction followed the Preferred Reporting Items for Systematic Review and Meta-analysis (PRISMA) guideline process. Two examiners used the Newcastle Ottawa Scale to determine overall study quality while the key information was extracted and tabulated for comparison. The data was analysed using Chi-squared test and I2 test for heterogenicity with a random effects or fixed affect models applied as appropriate. Risk difference of outcomes was displayed via a forest plot with 95% confidence intervals. Funnel plots were generated to evaluate publication bias.Results All four cross-sectional studies searched for EBV, while three also looked for CMV. The case-control study included investigated for HSV1 presence only. EBV presence in peri-implantitis sites was found to be statistically significant in three of the four studies despite obvious heterogeneity. CMV presence at peri-implantitis sites was statistically significant in all relevant studies, but the data displayed notable heterogeneity so as to render it insignificant. HSV1 exhibited similar percentage frequency in both healthy and diseased implant sites.Conclusions Virus prevalence was found to be increased in patients with peri-implantitis when compared to healthy sites but this assertion must be treated with caution as the data supporting it is weak due to the limited number of studies involved and the significant inherent heterogeneity they displayed.

Dynamics and architecture of the NRBF2-containing phosphatidylinositol 3-kinase complex I of autophagy.

The class III phosphatidylinositol 3-kinase complex I (PI3KC3-C1) is central to autophagy initiation. We previously reported the V-shaped architecture of the four-subunit version of PI3KC3-C1 consisting of VPS (vacuolar protein sorting) 34, VPS15, BECN1 (Beclin 1), and ATG (autophagy-related) 14. Here we show that a putative fifth subunit, nuclear receptor binding factor 2 (NRBF2), is a tightly bound component of the complex that profoundly affects its activity and architecture. NRBF2 enhances the lipid kinase activity of the catalytic subunit, VPS34, by roughly 10-fold. We used hydrogen-deuterium exchange coupled to mass spectrometry and negative-stain electron microscopy to map NRBF2 to the base of the V-shaped complex. NRBF2 interacts primarily with the N termini of ATG14 and BECN1. We show that NRBF2 is a homodimer and drives the dimerization of the larger PI3KC3-C1 complex, with implications for the higher-order organization of the preautophagosomal structure.

Increasing intracellular trehalose is sufficient to confer desiccation tolerance to Saccharomyces cerevisiae.

Diverse organisms capable of surviving desiccation, termed anhydrobiotes, include species from bacteria, yeast, plants, and invertebrates. However, most organisms are sensitive to desiccation, likely due to an assortment of different stresses such as protein misfolding and aggregation, hyperosmotic stress, membrane fracturing, and changes in cell volume and shape leading to an overcrowded cytoplasm and metabolic arrest. The exact stress(es) that cause lethality in desiccation-sensitive organisms and how the lethal stresses are mitigated in desiccation-tolerant organisms remain poorly understood. The presence of trehalose in anhydrobiotes has been strongly correlated with desiccation tolerance. In the yeast Saccharomyces cerevisiae, trehalose is essential for survival after long-term desiccation. Here, we establish that the elevation of intracellular trehalose in dividing yeast by its import from the media converts yeast from extreme desiccation sensitivity to a high level of desiccation tolerance. This trehalose-induced tolerance is independent of utilization of trehalose as an energy source, de novo synthesis of other stress effectors, or the metabolic effects of trehalose biosynthetic intermediates, indicating that a chemical property of trehalose is directly responsible for desiccation tolerance. Finally, we demonstrate that elevated intracellular maltose can also make dividing yeast tolerant to short-term desiccation, indicating that other disaccharides have stress effector activity. However, trehalose is much more effective than maltose at conferring tolerance to long-term desiccation. The effectiveness and sufficiency of trehalose as an antagonizer of desiccation-induced damage in yeast emphasizes its potential to confer desiccation tolerance to otherwise sensitive organisms.

Mutations in the N terminus of the oX174 DNA pilot protein H confer defects in both assembly and host cell attachment.

The øX174 DNA pilot protein H forms an oligomeric DNA-translocating tube during penetration. However, monomers are incorporated into 12 pentameric assembly intermediates, which become the capsid's icosahedral vertices. The protein's N terminus, a predicted transmembrane helix, is not represented in the crystal structure. To investigate its functions, a series of absolute and conditional lethal mutations were generated. The absolute lethal proteins, a deletion and a triple substitution, were efficiently incorporated into virus-like particles lacking infectivity. The conditional lethal mutants, bearing cold-sensitive (cs) and temperature-sensitive (ts) point mutations, were more amenable to further analyses. Viable particles containing the mutant protein can be generated at the permissive temperature and subsequently analyzed at the restrictive temperature. The characterized cs defect directly affected host cell attachment. In contrast, ts defects were manifested during morphogenesis. Particles synthesized at permissive temperature were indistinguishable from wild-type particles in their ability to recognize host cells and deliver DNA. One mutation conferred an atypical ts synthesis phenotype. Although the mutant protein was efficiently incorporated into virus-like particles at elevated temperature, the progeny appeared to be kinetically trapped in a temperature-independent, uninfectious state. Thus, substitutions in the N terminus can lead to H protein misincorporation, albeit at wild-type levels, and subsequently affect particle function. All mutants exhibited recessive phenotypes, i.e., rescued by the presence of the wild-type H protein. Thus, mixed H protein oligomers are functional during DNA delivery. Recessive and dominant phenotypes may temporally approximate H protein functions, occurring before or after oligomerization has gone to completion.

An immunohistochemical analysis of SERT in the blood-brain barrier of the male rat brain.

5-Hydroxytryptamine (5-HT) was originally discovered as a vasoconstrictor. 5-HT lowers blood pressure when administered peripherally to both normotensive and hypertensive male rats. Because the serotonin transporter (SERT) can function bidirectionally, we must consider whether 5-HT can be transported from the bloodstream to the central nervous system (CNS) in facilitating the fall in blood pressure. The blood-brain barrier (BBB) is a highly selective barrier that restricts movement of substances from the bloodstream to the CNS and vice versa, but the rat BBB has not been investigated in terms of SERT expression. This requires us to determine whether the BBB of the rat, the species in which we first observed a fall in blood pressure to infused 5-HT, expresses SERT. We hypothesized that SERT is present in the BBB of the male rat. To test this hypothesis, over 500 blood vessels were sampled from coronal slices of six male rat brains. Immunofluorescence of these coronal slices was used to determine whether SERT and RecA-1 (an endothelial cell marker) colocalized to the BBB. Blood vessels were considered to be capillaries if they were between 1.5 and 23 µm (intraluminal diameter). SERT was identified in the largest pial vessels of the BBB (mean ± SEM = 228.70 ± 18.71 µm, N = 9) and the smallest capillaries (mean ± SEM = 2.75 ± 0.12 µm, N = 369). SERT was not identified in the endothelium of blood vessels ranging from 20 to 135 µm (N = 45). The expression of SERT in the rat BBB means that 5-HT entry into the CNS must be considered a potential mechanism when investigating 5-HT-induced fall in blood pressure.

Analysis of bone structure in PEROMYSCUS: Effects of burrowing behavior.

We compare the effects of burrowing behavior on appendicular bone structure in two Peromyscus (deer mouse) species. P. polionotus creates complex burrows in their territories, while P. eremicus is a non-burrowing nesting mouse. We examined museum specimens' bones of wild-caught mice of the two species and lab-reared P. polionotus not given the opportunity to burrow. Bones were scanned using micro-computed tomography, and cortical and trabecular bone structural properties were quantified. Wild P. polionotus mice had a larger moment of area in the ulnar and tibial cortical bone compared with their lab-reared counterparts, suggesting developmental adaptation to bending resistance. Wild P. polionotus had a larger normalized second moment of area and cross-sectional area in the tibia compared with P. eremicus. Tibial trabecular analysis showed lower trabecular thickness and spacing in wild P. polionotus than in P. eremicus and femoral analysis showed wild P. polionotus had lower thickness than P. eremicus and lower spacing than lab-reared P. polionotus, suggesting adaptation to high loads from digging. Results lay the groundwork for future exploration of the ontogenetic and evolutionary basis of mechanoadaptation in Peromyscus.

Bringing Structure to Cell Biology with Cryo-Electron Tomography.

Recent advances in cryo-electron microscopy have marked only the beginning of the potential of this technique. To bring structure into cell biology, the modality of cryo-electron tomography has fast developed into a bona fide in situ structural biology technique where structures are determined in their native environment, the cell. Nearly every step of the cryo-focused ion beam-assisted electron tomography (cryo-FIB-ET) workflow has been improved upon in the past decade, since the first windows were carved into cells, unveiling macromolecular networks in near-native conditions. By bridging structural and cell biology, cryo-FIB-ET is advancing our understanding of structure-function relationships in their native environment and becoming a tool for discovering new biology.

ULK complex organization in autophagy by a C-shaped FIP200 N-terminal domain dimer.

The autophagy-initiating human ULK complex consists of the kinase ULK1/2, FIP200, ATG13, and ATG101. Hydrogen-deuterium exchange mass spectrometry was used to map their mutual interactions. The N-terminal 640 residues (NTD) of FIP200 interact with the C-terminal IDR of ATG13. Mutations in these regions abolish their interaction. Negative stain EM and multiangle light scattering showed that FIP200 is a dimer, while a single molecule each of the other subunits is present. The FIP200NTD is flexible in the absence of ATG13, but in its presence adopts the shape of the letter C ∼20 nm across. The ULK1 EAT domain interacts loosely with the NTD dimer, while the ATG13:ATG101 HORMA dimer does not contact the NTD. Cryo-EM of the NTD dimer revealed a structural similarity to the scaffold domain of TBK1, suggesting an evolutionary similarity between the autophagy-initiating TBK1 kinase and the ULK1 kinase complex.

The BARA necessities of PtdIns 3-kinase activation in autophagy.

Macroautophagy/autophagy is an evolutionarily conserved degradation system with fundamental biological functions. The activation of the class III phosphatidylinositol 3-kinase (PtdIns3K) complexes and the subsequent production of phosphatidylinositol 3-phosphate (PtdIns3P) are pivotal to autophagy. Using a combination of structural biology, biochemistry, and biophysics, we revealed how the non-catalytic subunit BECN1 serves as a membrane-binding switch in the regulation of PtdIns3K complexes and autophagy.

Structural mechanism of a Rag GTPase activation checkpoint by the lysosomal folliculin complex.

The tumor suppressor folliculin (FLCN) enables nutrient-dependent activation of the mechanistic target of rapamycin complex 1 (mTORC1) protein kinase via its guanosine triphosphatase (GTPase) activating protein (GAP) activity toward the GTPase RagC. Concomitant with mTORC1 inactivation by starvation, FLCN relocalizes from the cytosol to lysosomes. To determine the lysosomal function of FLCN, we reconstituted the human lysosomal FLCN complex (LFC) containing FLCN, its partner FLCN-interacting protein 2 (FNIP2), and the RagAGDP:RagCGTP GTPases as they exist in the starved state with their lysosomal anchor Ragulator complex and determined its cryo-electron microscopy structure to 3.6 angstroms. The RagC-GAP activity of FLCN was inhibited within the LFC, owing to displacement of a catalytically required arginine in FLCN from the RagC nucleotide. Disassembly of the LFC and release of the RagC-GAP activity of FLCN enabled mTORC1-dependent regulation of the master regulator of lysosomal biogenesis, transcription factor E3, implicating the LFC as a checkpoint in mTORC1 signaling.

Architecture and dynamics of the autophagic phosphatidylinositol 3-kinase complex.

The class III phosphatidylinositol 3-kinase complex I (PI3KC3-C1) that functions in early autophagy consists of the lipid kinase VPS34, the scaffolding protein VPS15, the tumor suppressor BECN1, and the autophagy-specific subunit ATG14. The structure of the ATG14-containing PI3KC3-C1 was determined by single-particle EM, revealing a V-shaped architecture. All of the ordered domains of VPS34, VPS15, and BECN1 were mapped by MBP tagging. The dynamics of the complex were defined using hydrogen-deuterium exchange, revealing a novel 20-residue ordered region C-terminal to the VPS34 C2 domain. VPS15 organizes the complex and serves as a bridge between VPS34 and the ATG14:BECN1 subcomplex. Dynamic transitions occur in which the lipid kinase domain is ejected from the complex and VPS15 pivots at the base of the V. The N-terminus of BECN1, the target for signaling inputs, resides near the pivot point. These observations provide a framework for understanding the allosteric regulation of lipid kinase activity.