Higher Oligomers Research Articles

Progress in Studies on the RNA World

The montmorillonite-catalyzed reactions of D, L-ImpA with D, L-ImpU generates RNA-like oligomers. The structures of the dimers to pentamers were investigated and homochiral products were identified in greater amounts than would be expected if theoretical amounts of each were formed. The homochirality increased from 64% to 97% as the chain length increased from dimers to pentamers. Investigation of the effect of pH, occupancy of the interlayer space and the influence of various cations in the reaction provided further insight into physical process in the mechanism of the catalysis. A detailed analysis of dimers was carried out in view of there being key intermediates towards formation of higher oligomers. The study was extended to the synthesis of non-standard dimers including those formed with deoxy-ribonucleotides.

Abstract 17176: Adiponectin, Metabolic Factors and CRP Changes with a Lifestyle Intervention for Weight Loss in Men and Women with Diabetes: Insights From the Look Ahead (Action for Health in Diabetes) Study

Adiponectin (ADN) has anti-inflammatory effects. In obese diabetic persons, ADN levels are low and CRP levels are high. The associations of changes in total (T) ADN and its high molecular-weight oligomer (HMW-ADN) with CRP change (Δ) in response to an intensive lifestyle intervention for weight loss (ILI) have not been examined within a clinical trial. Little is known about the effects of metabolic changes by sex on these associations in diabetes. Methods: Using regression analyses, we examined whether T-ADN and HMW-ADN changes with a 1-year ILI would be associated with CRP Δ in a subset of 1,397 Look AHEAD participants with biomarker data, when compared to usual care, after accounting for weight, HbA1c, HDL-C and fitness changes. Look AHEAD is a randomized trial examining whether ILI, when compared to usual care, will reduce CV events in obese type 2 diabetic persons. Results: ILI resulted in greater CRP reductions and increases in T-ADN and HMW-ADN, when compared to usual care (p≤ 0.0001 for all). CRP Δ with ILI was similar in men and women (≈ -40%). The increases in T-ADN and HMW-ADN were greater in men (23.6 and 13.5%) than in women (6.2% and 14.8%), respectively. Change in T-ADN and HMW-ADN in men and in T-ADN in women were significantly associated with CRP Δ, independently of weight, HDL-C, fitness and HbA1c changes (See Table). Fitness change in men and weight change in women, but not HbA1c or HDL-C changes, were associated with CRP Δ after adjusting for T-ADN and HMW-ADN change in separate models. Conclusion: In obese diabetic subjects, T-ADN and HMW-ADN are associated with changes in CRP with differential effects in male and female participants, with weight playing a greater role in women and fitness a greater role in men.

A novel dimer-tetramer transition captured by the crystal structure of the HIV-1 Nef.

HIV-1 Nef modulates disease progression through interactions with over 30 host proteins. Individual chains fold into membrane-interacting N-terminal and C-terminal core (Nefcore) domains respectively. Nef exists as small oligomers near membranes and associates into higher oligomers such as tetramers or hexadecamers in the cytoplasm. Earlier structures of the Nefcore in apo and complexed forms with the Fyn-kinase SH3 domain revealed dimeric association details and the role of the conserved PXXP recognition motif (residues 72–78) of Nef in SH3-domain interactions. The crystal structure of the tetrameric Nef reported here corresponds to the elusive cytoplasmic stage. Comparative analyses show that subunits of Nefcore dimers (open conformation) swing out with a relative displacement of ∼22 Å and rotation of ∼174° to form the ‘closed’ tetrameric structure. The changes to the association are around Asp125, a conserved residue important for viral replication and the important XR motif (residues 107–108). The tetramer associates through C4 symmetry instead of the 222 symmetry expected when two dimers associate together. This novel dimer-tetramer transition agrees with earlier solution studies including small angle X-ray scattering, analytical ultracentrifugation, dynamic laser light scattering and our glutaraldehyde cross-linking experiments. Comparisons with the Nefcore—Fyn-SH3 domain complexes reveal that the PXXP motif that interacts with the SH3-domain in the dimeric form is sterically occluded in the tetramer. However the 151–180 loop that is distal to the PXXP motif and contains several protein interaction motifs remains accessible. The results suggest how changes to the oligomeric state of Nef can help it distinguish between protein partners.

Assembly and Separation of Semiconductor Quantum Dot Dimers and Trimers

Repeated precipitation of colloidal semiconductor quantum dots (QD) from a good solvent by adding a poor solvent leads to an increasing number of QD oligomers after redispersion in the good solvent. By using density gradient ultracentrifugation we have been able to separate QD monomer, dimer, and trimer fractions from higher oligomers in such solutions. In the corresponding fractions QD dimers and trimers have been enriched up to 90% and 64%, respectively. Besides directly coupled oligomers, QD dimers and trimers were also assembled by linkage with a rigid terrylene diimide dye (TDI) and separated again by ultracentrifugation. High-resolution transmission electron micrographs show that the interparticle distances are clearly larger than those for directly coupled dots proving that the QDs indeed are cross-linked by the dye. Moreover, energy transfer from the QDs to the TDI "bridge" has been observed. Individual oligomers (directly coupled or dye-linked) can be readily deposited on a substrate and studied simultaneously by scanning force and optical microscopy. Our simple and effective scheme is applicable to a wide range of ligand stabilized colloidal nanoparticles and opens the way to a detailed study of electronic coupling in, e.g., QD molecules.

Bax dimerizes via a symmetric BH3:groove interface during apoptosis

During apoptotic cell death, Bax and Bak change conformation and homo-oligomerize to permeabilize mitochondria. We recently reported that Bak homodimerizes via an interaction between the BH3 domain and hydrophobic surface groove, that this BH3:groove interaction is symmetric, and that symmetric dimers can be linked via the α6-helices to form the high order oligomers thought responsible for pore formation. We now show that Bax also dimerizes via a BH3:groove interaction after apoptotic signaling in cells and in mitochondrial fractions. BH3:groove dimers of Bax were symmetric as dimers but not higher order oligomers could be linked by cysteine residues placed in both the BH3 and groove. The BH3:groove interaction was evident in the majority of mitochondrial Bax after apoptotic signaling, and correlated strongly with cytochrome c release, supporting its central role in Bax function. A second interface between the Bax α6-helices was implicated by cysteine linkage studies, and could link dimers to higher order oligomers. We also found that a population of Bax:Bak heterodimers generated during apoptosis formed via a BH3:groove interaction, further demonstrating that Bax and Bak oligomerize via similar mechanisms. These findings highlight the importance of BH3:groove interactions in apoptosis regulation by the Bcl-2 protein family.

Tuning of diglycerol yield and isomer distribution in oligomerization of glycerol supported by DFT-calculations

Diglycerol (DG) is a useful compound for food, cosmetic and chemical applications being available from cheap glycerol of biodiesel manufacture. Homogeneous catalyzed conversions led to a distribution of the three isomers (58(αα′) : 35(αβ) : 7(ββ′); glycerol conversion (X) = 55%, DG yield (Y-DG) = 40%; 7 h). This can be shifted in favor of αβ-DG by using basic zeolites (e.g. CsX, 24(αα′) : 52(αβ) : 24(ββ′); X = 70%, Y-DG ca. 45%; 8 h). Otherwise, acidic Nafion® catalyst revealed 69(αα′) : 27(αβ) : 4(ββ′) distribution (X = 93%, Y-DG ca. 68%; 24 h) using a falling film reactor under reduced pressure. ► Glycerol can be converted to diglycerols by basic as well as acidic catalysts. ► Higher oligomers are formed in consecutive reactions. ► Basic zeolites dramatically change composition of the diglycerol isomer pool. ► Acidic ionomers used at reduced reaction pressure seem to be applicable catalysts. ► Falling film reactor set-up prevents consecutive reactions of diglycerols.

Detection of Amyloid-β Protein Precursor Homo-Interactions Using Beta-Galactosidase Enzyme Fragment Complementation

Amyloidogenic processing of the amyloid-β protein precursor (AβPP) produces amyloid-β peptides (Aβ), the major constituent of amyloid plaques in the brains of Alzheimer's disease (AD) patients. Experimental evidence suggests that increased dimerization of AβPP increases Aβ while decreased dimerization of AβPP decreases Aβ production. If true, developing tools for detecting AβPP-AβPP interactions to understand AβPP processing leading to Aβ production would be important. Here, we developed the method of β-galactosidase (β-gal) enzyme fragment complementation as a means to detect AβPP-AβPP interactions. Inactive β-gal fragments are independently tagged to the C-terminal ends of monomeric AβPPs, and will come together to form a functional enzyme upon AβPP-AβPP interactions. Successful detection of β-gal activity has been used to qualitatively visualize and quantify the amount of AβPP dimers or higher oligomers. This method can be used to enhance our understanding of the biological processes dependent upon AβPP-AβPP interactions.

Decreased concentration of adiponectin together with a selective reduction of its high molecular weight oligomers is involved in metabolic complications of myotonic dystrophy type 1

The hormone adiponectin exerts beneficial pleiotropic effects on biological and metabolic processes. Although a well-recognized insulin sensitizer, its characteristic has yet to be clearly defined. Myotonic dystrophy type 1 (DM1) is a rare genetic disorder that features muscle wasting and metabolic comorbidity, and patients have an increased risk of developing type 2 diabetes. We analyzed circulating levels of adiponectin and its oligomers to determine whether their expression correlates with metabolic alterations in DM1 patients. We measured the anthropometric and biochemical features and three insulin resistance (IR) indices (homeostasis model assessment, quantitative insulin sensitivity check index, and McAuley) of 21 DM1 patients and of 82 age-, sex-, and weight-matched controls. In the blood samples of patients and controls, adiponectin levels were measured by ELISA, and its oligomers were characterized by using western blotting and gel filtration. The adiponectin gene was molecularly analyzed in patients. DM1 patients had significantly higher body mass index, waist circumference, triglycerides (TGs), glucose, tumor necrosis factor α, and IR; conversely, they had significantly lower concentrations of total serum adiponectin with a selective, pronounced decrease of its high molecular weight (HMW) oligomers. There was a strong negative correlation between adiponectin and TGs in DM1 patients. Our results endorse the hypothesis that decreased expression of adiponectin together with a selective reduction of its HMW oligomers contributes to the worsening of IR and its metabolic complications in DM1 patients. These findings suggest that adiponectin and HMW oligomers may serve as biomarkers and are promising therapeutic agents for IR and its consequences in DM1.

Structure of solvent-free grafted nanoparticles: Molecular dynamics and density-functional theory

The structure of solvent-free oligomer-grafted nanoparticles has been investigated using molecular dynamics simulations and density-functional theory. At low temperatures and moderate to high oligomer lengths, the qualitative features of the core particle pair probability, structure factor, and the oligomer brush configuration obtained from the simulations can be explained by a density-functional theory that incorporates the configurational entropy of the space-filling oligomers. In particular, the structure factor at small wave numbers attains a value much smaller than the corresponding hard-sphere suspension, the first peak of the pair distribution function is enhanced due to entropic attractions among the particles, and the oligomer brush expands with decreasing particle volume fraction to fill the interstitial space. At higher temperatures, the simulations reveal effects that differ from the theory and are likely caused by steric repulsions of the expanded corona chains.

A single copy of SecYEG is sufficient for preprotein translocation

The heterotrimeric SecYEG complex comprises a protein-conducting channel in the bacterial cytoplasmic membrane. SecYEG functions together with the motor protein SecA in preprotein translocation. Here, we have addressed the functional oligomeric state of SecYEG when actively engaged in preprotein translocation. We reconstituted functional SecYEG complexes labelled with fluorescent markers into giant unilamellar vesicles at a natively low density. Förster's resonance energy transfer and fluorescence (cross-) correlation spectroscopy with single-molecule sensitivity allowed for independent observations of the SecYEG and preprotein dynamics, as well as complex formation. In the presence of ATP and SecA up to 80% of the SecYEG complexes were loaded with a preprotein translocation intermediate. Neither the interaction with SecA nor preprotein translocation resulted in the formation of SecYEG oligomers, whereas such oligomers can be detected when enforced by crosslinking. These data imply that the SecYEG monomer is sufficient to form a functional translocon in the lipid membrane.

Nitration of Tyrosine 10 Critically Enhances Amyloid β Aggregation and Plaque Formation

Part of the inflammatory response in Alzheimer's disease (AD) is the upregulation of the inducible nitric oxide synthase (NOS2) resulting in increased NO production. NO contributes to cell signaling by inducing posttranslational protein modifications. Under pathological conditions there is a shift from the signal transducing actions to the formation of protein tyrosine nitration by secondary products like peroxynitrite and nitrogen dioxide. We identified amyloid β (Aβ) as an NO target, which is nitrated at tyrosine 10 (3NTyr(10)-Aβ). Nitration of Aβ accelerated its aggregation and was detected in the core of Aβ plaques of APP/PS1 mice and AD brains. NOS2 deficiency or oral treatment with the NOS2 inhibitor L-NIL strongly decreased 3NTyr(10)-Aβ, overall Aβ deposition and cognitive dysfunction in APP/PS1 mice. Further, injection of 3NTyr(10)-Aβ into the brain of young APP/PS1 mice induced β-amyloidosis. This suggests a disease modifying role for NOS2 in AD and therefore represents a potential therapeutic target.

Mechanism of actin filament nucleation by Vibrio VopL and implications for tandem W domain nucleation

Pathogen proteins targeting the actin cytoskeleton often serve as model systems to understand their more complex eukaryotic analogs. We show that the strong actin filament nucleation activity of Vibrio VopL depends on its three W domains and dimerization through a unique VopL C-terminal domain (VCD). The VCD displays a novel all-helical fold and interacts with the pointed end of the actin nucleus, contributing to the nucleation activity directly and through duplication of the W domain repeat. VopL promotes rapid cycles of filament nucleation and detachment, but generally has no effect on elongation. Profilin inhibits VopL-induced nucleation by competing for actin binding to the W domains. Combined, the results suggest that VopL stabilizes a hexameric double-stranded pointed end nucleus. Analysis of hybrid constructs of VopL and the eukaryotic nucleator Spire suggest that Spire may also function as a dimer in cells.

A Series of Energy‐Transfer Copolymers Derived from Fluorene and 4,7‐Dithienylbenzotriazole for High Efficiency Yellow, Orange, and White Light‐Emitting Diodes

AbstractEight random and alternating copolymers PF‐DTBTA derived from 2,7‐fluorene and 4,7‐dithienylbenzotriazole (DTBTA) were synthesized. Thin solid films of the energy‐transfer copolymers possess high absolute photoluminescence (PL) quantum yields (ΦPL) between 60−72%. Inserting PVK layer between anode and emissive layer could show higher electroluminescence (EL) performances due to PVK‐enhanced hole injection. Random copolymers PF‐DTBTA1−15, with DTBTA molar contents from 1% to 15%, displayed yellow EL spectra with high external quantum efficiency (EQEmax) up to 5.78%. PF‐DTBTA50, the alternating copolymer, showed an orange EL with EQEmax of 3.3%. The good ΦPL and EQEmax of the PF‐DTBTA50 with very high DTBTA content indicate that DTBTA is a high efficiency chromophore with very low concentration quenching effects in the solid state PL and EL processes. PF‐DTBTA0.03−0.1 could emit white EL due to partial energy transfer from fluorene segments to DTBTA units. Moreover, white EL devices, with forward‐viewing maximum luminous efficiency up to 11 cd/A and stable white EL spectra (CIE coordinates of (0.33, 0.43)) in high current range from 5 mA to 60 mA, could be realized from the non‐doped polymer with simple binary structure. Our results suggest that DTBTA has big potential to construct high performanced EL polymers or oligomers.

Laboratory Investigation on the Role of Organics in Atmospheric Nanoparticle Growth

The uptake of organic vapors by 4-20 nm H(2)SO(4) particles has been investigated to assess the role of organics in atmospheric nanoparticle growth. Sulfuric acid nanoparticles are generated from homogeneous binary nucleation of H(2)SO(4) and H(2)O vapors in a laminar flow chamber. The growth factor of H(2)SO(4) nanoparticles exposed to methyglyoxal, ethanol, 1-butanol, 1-heptanol, and 1-decanol is measured using a nanotandem differential mobility analyzer (nano-TDMA). The measured growth factor is close to unity when nanoparticles are exposed to methylglyoxal, ethanol, 1-butanol, 1-heptanol, and 1-decanol, indicating no apparent growth within the experimental uncertainty. In addition, spectroscopic evolution of functional groups in H(2)SO(4) particles of ∼40 nm diameter size, deposited on ZnSe crystal and subsequently exposed to glyoxal and 2,4-hexadienal, is studied using attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FT-IR). The ATR-FT-IR measurements present the first spectroscopic signatures of high molecular weight aldol and oligomer products and show that polymerization and oligomerization reactions are partially reversible. The implications of the present results to nanoparticle growth in the atmosphere are discussed.

Differential Impact of Caveolae and Caveolin-1 Scaffolds on The Membrane Raft Proteome

Caveolae, a class of cholesterol-rich lipid rafts, are smooth invaginations of the plasma membrane whose formation in nonmuscle cells requires caveolin-1 (Cav1). The recent demonstration that Cav1-associated cavin proteins, in particular PTRF/cavin-1, are also required for caveolae formation supports a functional role for Cav1 independently of caveolae. In tumor cells deficient for Golgi β-1,6N-acetylglucosaminyltransferase V (Mgat5), reduced Cav1 expression is associated not with caveolae but with oligomerized Cav1 domains, or scaffolds, that functionally regulate receptor signaling and raft-dependent endocytosis. Using subdiffraction-limit microscopy, we show that Cav1 scaffolds are homogenous subdiffraction-limit sized structures whose size distribution differs from that of Cav1 in caveolae expressing cells. These cell lines displaying differing Cav1/caveolae phenotypes are effective tools for probing the structure and composition of caveolae. Using stable isotope labeling by amino acids in cell culture, we are able to quantitatively distinguish the composition of caveolae from the background of detergent-resistant membrane proteins and show that the presence of caveolae enriches the protein composition of detergent-resistant membrane, including the recruitment of multiple heterotrimeric G-protein subunits. These data were further supported by analysis of immuno-isolated Cav1 domains and of methyl-β-cyclodextrin-disrupted detergent-resistant membrane. Our data show that loss of caveolae results in a dramatic change to the membrane raft proteome and that this change is independent of Cav1 expression. The proteomics data, in combination with subdiffraction-limit microscopy, indicates that noncaveolar Cav1 domains, or scaffolds are structurally and functionally distinct from caveolae and differentially impact on the molecular composition of lipid rafts.

ATP and Mg2+ Promote the Reversible Oligomerization and Aggregation of Chloroplast 2-Cys Peroxiredoxin

2-Cys peroxiredoxins (2-Cys Prxs) are ubiquitous peroxidases with important roles in cellular antioxidant defense and hydrogen peroxide-mediated signaling. Post-translational modifications of conserved cysteines cause the transition from low to high molecular weight oligomers, triggering the functional change from peroxidase to molecular chaperone. However, it remains unclear how non-covalent interactions of 2-Cys Prx with metabolites modulate the quaternary structure. Here, we disclose that ATP and Mg(2+) (ATP/Mg) promote the self-polymerization of chloroplast 2-Cys Prx (polypeptide 23.5 kDa) into soluble higher order assemblies (>2 MDa) that proceed to insoluble aggregates beyond 5 mM ATP. Remarkably, the withdrawal of ATP or Mg(2+) brings soluble oligomers and insoluble aggregates back to the native conformation without compromising the associated functions. As confirmed by transmission electron microscopy, ATP/Mg drive the toroid-like decamers (diameter 13 nm) to the formation of large sphere-like particles (diameter ∼30 nm). Circular dichroism studies on ATP-labeled 2-Cys Prx reveal that ATP/Mg enhance the proportion of β-sheets with the concurrent decrease in the content of α-helices. In line with this observation, the formation of insoluble aggregates is strongly prevented by 2,2,2-trifluoroethanol, a cosolvent employed to induce α-helical conformations. We further find that the response of self-polymerization to ATP/Mg departs abruptly from that of the associated peroxidase and chaperone activities when two highly conserved residues, Arg(129) and Arg(152), are mutated. Collectively, our data uncover that non-covalent interactions of ATP/Mg with 2-Cys Prx modulate dynamically the quaternary structure, thereby coupling the non-redox chemistry of cell energy with redox transformations at cysteine residues.

Sequence-Controlled Oligomers Fold into Nanosolenoids and Impart Unusual Optical Properties

Controlled syntheses give unique block oligomers with alternating flexible ethylene glycol and rigid perylenetetracarboxylic diimide (PDI) units. The number of rigid units vary from n=1 to 10. PDI units were stitched together by using efficient phosphoramidite chemistry. The resulting oligomers undergo folding in most solvents, including chloroform. In their ground state, these folded oligomers were characterized by using Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS), NMR spectroscopy, and electronic absorption spectroscopy. FTICR-MS revealed the exact masses of these sequence-controlled oligomers, which confirmed the chemical composition and validated the synthetic strategy. The NMR neighboring ring-current effect (NRE) indicates the formation of cofacial π stacks; the stacked aromatic rings have nearly coaxial alignment akin to a nanosoleniod. Nanosolenoidal shielding in π stacks causes all aromatic protons to shift upfield, whereas NOE in a cyclic hetero-chromophoric dimer supports a rotated, cofacial π-stacking orientation separated by about 3.5 Å. Electron-phonon coupling is much stronger than excitonic coupling in these self-folded PDI oligomers; thus, Franck-Condon factors dictate the observed spectral features in visible spectra. The absorbance spectrum exhibits weak hypochromism due to π stacking with increasing stacking units n. Finally, ab initio calculations support the experimental observations, indicating 3.5 Å cofacial spacing in which one molecule is rotated 30° from the eclipsed orientation and higher oligomers can adopt, without a compensating energy penalty, either the right/left-handed helices or the 1,3-eclipsed structures. Both theory and experiments validate the nano-π-solenoids and their novel photophysical properties.

Arp you ready for actin in the nucleus?

Actin and actin‐related proteins (Arps) are critical components of chromatin remodelling and modifying complexes, where they are commonly found in pairs. Yet, little is known about their role in these complexes. In this issue of The EMBO Journal , Fenn et al (2011) report the first structural investigation of two nuclear Arps, the INO80 complex subunits Arp4 and Arp8. Importantly, their results also shed light on the critical role that the nucleotide state of actin may have on its interactions with the Arps.

Recombinant HPV16 E7 assembled into particles induces an immune response and specific tumour protection administered without adjuvant in an animal model

BackgroundThe HPV16 E7 protein is both a tumour-specific and a tumour-rejection antigen, the ideal target for developing therapeutic vaccines for the treatment of HPV16-associated cancer and its precursor lesions. E7, which plays a key role in virus-associated carcinogenesis, contains 98 amino acids and has two finger-type structures which bind a Zn++ ion. The ability of an Escherichia coli-produced E7-preparation, assembled into particles, to induce protective immunity against a HPV16-related tumour in the TC-1-C57BL/6 mouse tumour model, was evaluated.MethodsE7 was expressed in E. coli, purified via a one-step denaturing protocol and prepared as a soluble suspension state after dialysis in native buffer. The presence in the E7 preparation of particulate forms was analysed by non-reducing SDS-PAGE and negative staining electron microscopy (EM). The Zn++ ion content was analysed by mass-spectrometry. Ten μg of protein per mouse was administered to groups of animals, once, twice or three times without adjuvant. The E7-specific humoral response was monitored in mice sera using an E7-based ELISA while the cell-mediated immune response was analysed in mice splenocytes with lymphoproliferation and IFN-γ ELISPOT assays. The E7 immunized mice were challenged with TC-1 tumour cells and the tumour growth monitored for two months.ResultsIn western blot analysis E7 appears in multimers and high molecular mass oligomers. The EM micrographs show the protein dispersed as aggregates of different shape and size. The protein appears clustered in micro-, nano-aggregates, and structured particles. Mice immunised with this protein preparation show a significant E7-specific humoral and cell-mediated immune response of mixed Th1/Th2 type. The mice are fully protected from the tumour growth after vaccination with three E7-doses of 10 μg without any added adjuvant.ConclusionsThis report shows that a particulate form of HPV16 E7 is able to induce, without adjuvant, an E7-specific tumour protection in C57BL/6 mice. The protective immunity is sustained by both humoral and cell-mediated immune responses. The E. coli-derived HPV16 E7 assembled in vitro into micro- and nanoparticles represents not only a good substrate for antigen-presenting cell uptake and processing, but also a cost-effective means for the production of a new generation of HPV subunit vaccines.

ChemInform Abstract: Solid‐State [2 + 2] Photodimerization and Photopolymerization of α,ω‐Diarylpolyene Monomers: Effective Utilization of Noncovalent Intermolecular Interactions in Crystals

AbstractReview: 171 refs.