Bax crystal structures reveal how BH3 domains activate Bax and nucleate its oligomerization to induce apoptosis.

In stressed cells, apoptosis ensues when Bcl-2 family members Bax or Bak oligomerize and permeabilize the mitochondrial outer membrane. Certain BH3-only relatives can directly activate them to mediate this pivotal, poorly understood step. To clarify the conformational changes that induce Bax oligomerization, we determined crystal structures of BaxΔC21 treated with detergents and BH3 peptides. The peptides bound the Bax canonical surface groove but, unlike their complexes with prosurvival relatives, dissociated Bax into two domains. The structures define the sequence signature of activator BH3 domains and reveal how they can activate Bax via its groove by favoring release of its BH3 domain. Furthermore, Bax helices α2-α5 alone adopted a symmetric homodimer structure, supporting the proposal that two Bax molecules insert their BH3 domain into each other's surface groove to nucleate oligomerization. A planar lipophilic surface on this homodimer may engage the membrane. Our results thus define critical Bax transitions toward apoptosis.

Two-Dimensional Polymers and Polymerizations.

Synthetic chemists have developed robust methods to synthesize discrete molecules, linear and branched polymers, and disordered cross-linked networks. However, two-dimensional polymers (2DPs) prepared from designed monomers have been long missing from these capabilities, both as objects of chemical synthesis and in nature. Recently, new polymerization strategies and characterization methods have enabled the unambiguous realization of covalently linked macromolecular sheets. Here we review 2DPs and 2D polymerization methods. Three predominant 2D polymerization strategies have emerged to date, which produce 2DPs either as monolayers or multilayer assemblies. We discuss the fundamental understanding and scope of each of these approaches, including: the bond-forming reactions used, the synthetic diversity of 2DPs prepared, their multilayer stacking behaviors, nanoscale and mesoscale structures, and macroscale morphologies. Additionally, we describe the analytical tools currently available to characterize 2DPs in their various isolated forms. Finally, we review emergent 2DP properties and the potential applications of planar macromolecules. Throughout, we highlight achievements in 2D polymerization and identify opportunities for continued study.

The Bak core dimer focuses triacylglycerides in the membrane.

Apoptosis, the intrinsic programmed cell death process, is mediated by the Bcl-2 family members Bak and Bax. Activation via formation of symmetric core dimers and oligomerization on the mitochondrial outer membrane (MOM) leads to permeabilization and cell death. Although this process is linked to the MOM, the role of the membrane in facilitating such pores is poorly understood. We recently described Bak core domain dimers, revealing lipid binding sites and an initial role of lipids in oligomerization. Here we describe simulations that identified localized clustering and interaction of triacylglycerides (TAGs) with a minimized Bak dimer construct. Coalescence of TAGs occurred beneath this Bak dimer, mitigating dimer-induced local membrane thinning and curvature in representative coarse-grain MOM and model membrane systems. Furthermore, the effects observed as a result of coarse-grain TAG cluster formation was concentration dependent, scaling from low physiological MOM concentrations to those found in other organelles. We find that increasing the TAG concentration in liposomes mimicking the MOM decreased the ability of activated Bak to permeabilize these liposomes. These results suggest that the presence of TAGs within a Bak-lipid membrane preserves membrane integrity and is associated with reduced membrane stress, suggesting a possible role of TAGs in Bak-mediated apoptosis.

Rapid removal of organic micropollutants from water by a porous ß-cyclodextrin polymer.

The global occurrence in water resources of organic micropollutants, such as pesticides and pharmaceuticals, has raised concerns about potential negative effects on aquatic ecosystems and human health. Activated carbons are the most widespread adsorbent materials used to remove organic pollutants from water but they have several deficiencies, including slow pollutant uptake (of the order of hours) and poor removal of many relatively hydrophilic micropollutants. Furthermore, regenerating spent activated carbon is energy intensive (requiring heating to 500-900 degrees Celsius) and does not fully restore performance. Insoluble polymers of ß-cyclodextrin, an inexpensive, sustainably produced macrocycle of glucose, are likewise of interest for removing micropollutants from water by means of adsorption. ß-cyclodextrin is known to encapsulate pollutants to form well-defined host-guest complexes, but until now cross-linked ß-cyclodextrin polymers have had low surface areas and poor removal performance compared to conventional activated carbons. Here we crosslink ß-cyclodextrin with rigid aromatic groups, providing a high-surface-area, mesoporous polymer of ß-cyclodextrin. It rapidly sequesters a variety of organic micropollutants with adsorption rate constants 15 to 200 times greater than those of activated carbons and non-porous ß-cyclodextrin adsorbent materials. In addition, the polymer can be regenerated several times using a mild washing procedure with no loss in performance. Finally, the polymer outperformed a leading activated carbon for the rapid removal of a complex mixture of organic micropollutants at environmentally relevant concentrations. These findings demonstrate the promise of porous cyclodextrin-based polymers for rapid, flow-through water treatment.

Ion currents through Kir potassium channels are gated by anionic lipids.

Ion currents through potassium channels are gated. Constriction of the ion conduction pathway at the inner helix bundle, the textbook gate of Kir potassium channels, has been shown to be an ineffective permeation control, creating a rift in our understanding of how these channels are gated. Here we present evidence that anionic lipids act as interactive response elements sufficient to gate potassium conduction. We demonstrate the limiting barrier to K+ permeation lies within the ion conduction pathway and show that this gate is operated by the fatty acyl tails of lipids that infiltrate the conduction pathway via fenestrations in the walls of the pore. Acyl tails occupying a surface groove extending from the cytosolic interface to the conduction pathway provide a potential means of relaying cellular signals, mediated by anionic lipid head groups bound at the canonical lipid binding site, to the internal gate.

First-in-Human Phase I Clinical Trial of Pharmacologic Ascorbate Combined with Radiation and Temozolomide for Newly Diagnosed Glioblastoma.

PURPOSE: Standard treatment for glioblastoma (GBM) includes surgery, radiation therapy (RT), and temozolomide (TMZ), yielding a median overall survival (OS) of approximately 14 months. Preclinical models suggest that pharmacologic ascorbate (P-AscH-) enhances RT/TMZ antitumor effect in GBM. We evaluated the safety of adding P-AscH- to standard RT/TMZ therapy. PATIENTS AND METHODS: This first-in-human trial was divided into an RT phase (concurrent RT/TMZ/P-AscH-) and an adjuvant (ADJ) phase (post RT/TMZ/P-AscH- phase). Eight P-AscH- dose cohorts were evaluated in the RT phase until targeted plasma ascorbate levels were achieved (≥20 mmol/L). In the ADJ phase, P-AscH- doses were escalated in each subject at each cycle until plasma concentrations were ≥20 mmol/L. P-AscH- was infused 3 times weekly during the RT phase and 2 times weekly during the ADJ phase continuing for six cycles or until disease progression. Adverse events were quantified by CTCAE (v4.03). RESULTS: Eleven subjects were evaluable. No dose-limiting toxicities occurred. Observed toxicities were consistent with historical controls. Adverse events related to study drug were dry mouth and chills. Targeted ascorbate plasma levels of 20 mmol/L were achieved in the 87.5 g cohort; diminishing returns were realized in higher dose cohorts. Median progression-free survival (PFS) was 9.4 months and median OS was 18 months. In subjects with undetectable MGMT promoter methylation (n = 8), median PFS was 10 months and median OS was 23 months. CONCLUSIONS: P-AscH-/RT/TMZ is safe with promising clinical outcomes warranting further investigation.

Insight into the crystallization of amorphous imine-linked polymer networks to 2D covalent organic frameworks.

We explore the crystallization of a high surface area imine-linked two-dimensional covalent organic framework (2D COF). The growth process reveals rapid initial formation of an amorphous network that subsequently crystallizes into the layered 2D network. The metastable amorphous polymer may be isolated and resubjected to growth conditions to form the COF. These experiments provide the first mechanistic insight into the mechanism of imine-linked 2D COF formation, which is distinct from that of boronate-ester linked COFs.

Case report: an unorthodox orthodontic retainer.

This case report describes the management of a patient who presented with an interdental eyelet-style retainer placed around maxillary central incisors ten years ago. At that time, the patient underwent orthodontic treatment to close a large maxillary midline diastema. The maxillary central incisors were then crowned to close the small residual midline diastema remaining after the orthodontic treatment. The wire retainer was apparently placed to prevent the diastema from reopening. The patient sought treatment ten years later when pain from necrotic pulps in these central incisors became unbearable.

Comparison of restorative dentistry referrals to a district general hospital and to a teaching hospital using a pilot Index of Restorative Dental Treatment Need.

The purpose of this study was to identify differences between referral patterns and the clinical conditions of patients referred to consultant-led Restorative Dentistry clinics in a district general hospital and a London dental teaching hospital. The Index of Restorative Dental Treatment Need was used to classify patients into those patients whose treatment was appropriate for any dental graduate in general practice, for an experienced dentist in general practice, and for a dentist with specialist training. Demographic and clinical details were recorded for one hundred patients at each hospital. The results of the study showed that the proposed treatment of 21% of the patients seen at the district general hospital and 16% of the patients seen at the teaching hospital was appropriate for specialist care. The results support the expansion of specialist restorative dentistry services in district general hospitals.

The removal of fractured post fragments in general dental practice using ultrasonic vibration.

This paper describes an evidence-based clinical procedure that is suitable for the removal of fractured metal post fragments using ultrasonic vibration in general dental practice, particularly when non-resin-based cements have been used. Fragments of posts cemented with resin-based cements are likely to be extremely difficult to remove. The use of non-resin-based cements is recommended for luting posts, as fragment or total post removal is much easier with these materials.

A family of leukemia inhibitory factor-binding peptides that can act as antagonists when conjugated to poly(ethylene glycol).

A panel of six naïve 14-residue random peptide libraries displayed polyvalently on M13 phage was pooled and sorted against human leukemia inhibitory factor (LIF). After four rounds of selection, a single large family of peptides with the consensus sequence XCXXXXG(A/S)(D/E)(W/F)WXCF was found to bind specifically to LIF. Peptides within this family did not bind related members of the interleukin-6 family of cytokines, nor to murine LIF that has 80% sequence identity with human LIF. A representative peptide from this family was synthesized and found to bind to LIF with an affinity of approximately 300 nM. The phage-displayed form of this peptide was able to compete with the LIF receptor alpha chain (LIFR) for binding to LIF; however, the free synthetic peptide was unable to inhibit LIF-LIFR binding or inhibit LIF bioactivity in vitro. Using a panel of human/murine chimeric LIF molecules, the peptide-binding site on LIF was mapped to a groove located between the B and the C helices of the LIF structure, which is distinct from the surfaces involved in binding to receptor. To mimic the effect of the phage particle and convert the free peptide into an antagonist of LIFR binding, a 40 kDa poly(ethylene glycol) (PEG) moiety was conjugated to the synthetic LIF-binding peptide. This PEG-peptide conjugate was found to be both an antagonist of LIF-LIFR binding and of LIF signaling in engineered Ba/F3 cells expressing LIFR and the gp130 coreceptor.

Properties of GDP-mannose pyrophosphorylase, a critical enzyme and drug target in Leishmania mexicana.

Leishmania parasites synthesize a range of mannose-containing glycoconjugates thought to be essential for virulence in the mammalian host and sandfly vector. A prerequisite for the synthesis of these molecules is the availability of the activated mannose donor, GDP-Man, the product of the catalysis of mannose-1-phosphate and GTP by GDP-mannose pyrophosphorylase (GDP-MP). In contrast to the lethal phenotype in fungi, the deletion of the gene in Leishmania mexicana did not affect parasite viability but led to a total loss of virulence, making GDP-MP an ideal target for anti-Leishmania drug development. We show by immunofluorescence and subcellular fractionation that GDP-MP is a cytoplasmic protein, and we describe a colorimetric activity assay suitable for the high throughput screening of small molecule inhibitors. We expressed recombinant GDP-MP as a fusion with maltose-binding protein and separated the enzyme from maltose-binding protein by thrombin cleavage, ion-exchange, and size exclusion chromatography. Size exclusion chromatography and analytical ultracentrifugation studies demonstrate that GDP-MP self-associates to form an enzymatically active and stable hexamer. However, sedimentation studies show that the GDP-MP hexamer dissociates to trimers and monomers in a time-dependent manner, at low protein concentrations, at low ionic strength, and at alkaline pH. Circular dichroism spectroscopy reveals that GDP-MP is comprised of mixed alpha/beta structure, similar to its closest related homologue, N-acetyl-glucoseamine-1-phosphate uridyltransferase (Glmu) from Streptococcus pneumoniae. Our studies provide insight into the structure of a novel target for the development of anti-Leishmania drugs.