Thermodynamically Stable o-Quinodimethane: Synthesis, Structure, and Reactivity.

Thermal isomerization of cyclobutaphenanthrene to o-quinodimethane was investigated. Sterically congested substituents or electron-donating substituents on the four-membered ring promoted the ring-opening, affording o-quinodimethane in a relatively stable form. Isolation of the newly prepared o-quinodimethane allowed its structural elucidation and investigation of its potential reactivities. Dual [4+2] cycloaddition of an aryne and o-quinodimethane afforded tetrabenzopentacene, demonstrating the synthetic application of the isolated compound.

A Scientific Proposal for Surgical Decision-Making in Occult Intertrochanteric Fractures Based on Finite Element Analysis.

Background In the treatment of femoral intertrochanteric fractures, there is still a lack of consensus on the optimal approach for isolated greater trochanteric fractures and insufficient intertrochanteric fractures. The limited number of patients and restricted access to accurate assessment of fracture extension using magnetic resonance imaging contribute to the unclear treatment strategy. This study aims to utilize finite element (FE) analysis to analyze stress values at the fracture line and investigate their influence on intertrochanteric fracture extension under different loading conditions. The hypothesis is that fracture extension occurs following certain conditions, supporting the need for surgery based on scientific evidence. Methodology Osseous data from a computed tomography (CT) scan was used to create a proximal femur FE model using FEA software. CT scan data were converted to Digital Imaging and Communications in Medicine format and used to generate the FE model. Trabecular bone and cortex were meshed into tetrahedral elements. The model consisted of 1,592,642 elements and 282,530 nodes. Two models were created, namely, healthy proximal femur (HF) and femoral insufficient intertrochanteric fracture (FIF). Material properties were assigned based on CT values and conversion equations. The distal end of the femur was constrained. Stress analysis using the dynamic explicit approach was performed. Von Mises stresses were calculated for the proximal femur. The number of elements exceeding yield stress was counted to predict fracture risk by focusing on fracture line spots. In this study, the distribution of von Mises stress was compared between the HF and the FIF models. Six loading combinations were considered, namely, two weight-bearing conditions (3 W loading simulating for walking and 1/3 W for touch-down standing) and three hip flexion angles (0°, 15°, and 23°). Results Under 3 W loading, no significant stress elevations were observed in the HF model at any flexion angles. However, the FIF model exhibited increased stress at the site of the posterior fracture line extension. This stress-induced element destruction was observed in both cortical and cancellous bone. For the 1/3 W loading condition, only minimal stress elevation was observed in both HF and FIF models. To assess the influence on fracture extension, the number of yielded elements was evaluated along the fracture line edges (greater trochanter and middle of the intertrochanteric ridge). Under 3 W loading, the HF model had only one yielded element, indicating minimal fracture risk. In contrast, the FIF model exhibited a notable presence of yield elements in various regions (total/greater trochanter/shaft) at different flexion angles: 0° (115/16/28), 15° (265/158/23), and 23° (446/233/34). Under the 1/3 W loading condition, neither the HF nor the FIF models showed any yielding elements, regardless of the direction of external force. Conclusions The results demonstrated elevated stress levels at the fracture line in the FIF model, particularly during walking, indicating a higher risk of fracture extension at the flex position. However, under reduced weight-bearing conditions, the stress at the fracture site remained within the yield stress range, suggesting a relatively low risk of fracture extension. These findings hold significant clinical implications for developing surgical protocols that consider patients' compliance with weight-bearing restrictions.

Cerebral histamine H1 receptor binding potential measured with PET under a test dose of olopatadine, an antihistamine, is reduced after repeated administration of olopatadine.

UNLABELLED: Some antihistamine drugs that are used for rhinitis and pollinosis have a sedative effect as they enter the brain and block the H(1) receptor, potentially causing serious accidents. Receptor occupancy has been measured with PET under single-dose administration in humans to classify antihistamines as more sedating or as less sedating (or nonsedating). In this study, the effect of repeated administration of olopatadine, an antihistamine, on the cerebral H(1) receptor was measured with PET. METHODS: A total of 17 young men with rhinitis underwent dynamic brain PET with (11)C-doxepin at baseline, under an initial single dose of 5 mg of olopatadine (acute scan), and under another 5-mg dose after repeated administration of olopatadine at 10 mg/d for 4 wk (chronic scan). The H(1) receptor binding potential was estimated using Logan graphical analysis with cerebellum as reference region input. RESULTS: The acute scan showed a slight decrease in H(1) receptor binding potential across the cerebral cortex (by 15% in the frontal cortex), but the chronic scan showed a marked decrease (by 45% from the acute scan in the frontal cortex). Behavioral data before and after the PET scans did not reveal any sedative effect. CONCLUSION: The results may be interpreted as either intracerebral accumulation of olopatadine or H(1) receptor downregulation due to repeated administration. The study shows feasibility and potential value for PET in evaluating the pharmacologic effect of a drug not only after a single dose but also after repeated administration.

Relationship between beta4 hydrogen bond and beta6 hydrophobic interactions during aggregate, fiber or crystal formation in oversaturated solutions of hemoglobin A and S.

Oversaturated deoxy-alpha(2)beta(2)(T4V) aggregated instantly without a delay time, which is in contrast to the delay time before the generation of fibers of deoxy-HbS and deoxy-alpha(2)beta(2)(E6V,D73H). Solubility of deoxy-alpha(2)beta(2)(T4V) was approximately 10-fold lower than that of deoxy-HbS and was similar to oxy- and deoxy-alpha(2)beta(2)(E6V,T4V). These results indicate that beta4Val in HbA in the oxy and deoxy forms with or without beta6Val facilitates hydrophobic interaction of the A-helix with the EF helix of adjacent molecules without forming a beta4/beta73 hydrogen bond. Deoxy-HbA generated crystals following aggregation as does HbC-Harlem(alpha(2)beta(2)(E6V,D73N)), while alpha(2)beta(2)(T4V) and alpha(2)beta(2)(D73H) as well as HbS, alpha(2)beta(2)(E6V,D73H) and alpha(2)beta(2)(E6V,T4V) in the oxy and deoxy forms did not form crystals, indicating in addition to the strength of beta6 amino acid hydrophobicity that the synergism between the beta4Thr hydrogen bond and beta6 hydrophobic interaction free energies on the A-helix play a critical role in formation of fibers versus crystalline nuclei during phase transition.

Intramolecular benzoallene-alkyne cycloaddition initiated by site-selective SN2' reaction of epoxytetracene en route to π-extended pyracylene.

A hydrogen halide promoted cascade reaction of epoxytetracene to afford halo-benzoindenotetracene including a benzoallene intermediate was developed. The remaining two alkynyl groups in benzoindenotetracene were further reacted with norbornadiene or arylamine through transition metal-catalyzed cyclization to give π-extended pyracylene derivatives.

Development of multi-directional functional near-infrared spectroscopy system for human neuroimaging studies.

Multi-directional measurement using multi-directional light sources and multi-directional photodetectors drastically increases the amount of observation data without increasing the number of optical probes. In this study, we developed a novel multi-directional functional near-infrared spectroscopy (fNIRS) system for human neuroimaging studies. We tested our system by measuring the cortical hemodynamic changes of a single subject during a motor task and compared them with the same subject's functional magnetic resonance imaging (fMRI) data. We detected the direction-dependent fNIRS signals that originate from the cortical hemodynamic changes that are consistent with the fMRI data.

Role of the beta4Thr-beta73Asp hydrogen bond in HbS polymer and domain formation from multinucleate-containing clusters.

Fiber formation and domain formation from deoxy-HbS as well as from beta4 and beta73 HbS variants were investigated after temperature jump using DIC microscopy to gain a basic understanding of the determinants involved. Oversaturated deoxy-HbS generated numerous 14-stranded fibers and formed ovoid-shaped, multispherulitic domains. Domain number increased linearly as a function of time. Oversaturated deoxy-alpha2beta2(E6V,T4S) also generated time-dependent, ovoid-shaped spherulitic domains like HbS and alpha 2beta2(E6V,D73H) in the deoxy form. In contrast, alpha 2beta2(E6V,T4Y) and HbC-Harlem (alpha2beta2(E6V,D73N)) in the deoxy form generated time-dependent, ball-shaped domains containing many straight, crystalline-like fibers without evidence of branching. Some of these domains formed large needlelike crystals after overnight incubation. The inhibitory effect on polymer formation by beta4Tyr in HbS was stronger than that by beta4Ser but weaker than that by beta73Asn or beta73Leu. In contrast, both deoxy- and oxy-alpha2beta2(E6V,T4V) promoted formation of tiny, disordered amorphous aggregates without a delay time like oxy-HbS, which is in contrast to formation after a delay time of needlelike fibers for alpha 2beta2(E6V,D73L). Solubilities for both deoxy- and oxy-alpha 2beta2(E6V,T4V) were similar to that of deoxy-alpha 2beta2(E6V,D73H) but approximately 10-fold lower than that of deoxy-HbS. These results suggest that the strength of the hydrogen bond between beta4Thr and beta73Asp and the balance between the hydrogen bond and beta6Val hydrophobic interactions in deoxy-HbS polymers control formation of different types of fibers in a single domain or lead to formation of disordered, non-nucleated amorphous aggregates. These results also lead to a model in which multinucleation rather than a single-nucleation event occurs in a single cluster to generate numerous fibers growing from a single domain.

Dynamic finite element analysis of implants for femoral neck fractures simulating walking.

BACKGROUND: To examine postoperative complications for osteosynthesizing femoral neck fractures (Pauwels III), biomechanical analysis should be conducted under dynamic conditions simulating for walking, not static conditions. Among the two main aims of this study, one is to pioneer the technique of dynamic finite element (FE) analysis, and the other is to compare stress distribution between two implants during walking. MATERIALS AND METHODS: First, we performed an inverse dynamic analysis with optimization method using a musculoskeletal model to calculate the inter-segmental and muscular forces during walking. Second, three FE models were prepared: (I) intact hip joint, (II) fractures treated with two Hansson pins (HP), and (III) fractures with Dual SC Screws (DSCS) maintaining an angular stability. The direction and magnitude of the loadings varied continuously. Stress distribution during the walking was evaluated by using a dynamic explicit method. We examined the time-dependent von Mises stresses at two representative spots: medial cortex at the femoral neck fracture site and lateral pin (presumed) insertion holes. RESULTS: In general, stress values are always changing during walking cycle. Regarding medial femoral neck cortex at the fracture line, intact model showed almost consistent value. Both HP model and DSCS model amounted the highest around 30 MPa. At lateral holes, highest values were 18.8, 104.0, and 63.1 MPa of intact, HP, and DSCS models, respectively. CONCLUSION: Thus, our analysis simulating the real walking will be useful in evaluating time-varying stress distribution to assess postoperative complication. CLINICAL RELEVANCE: DSCS is expected to be paramount for treatment of unstable femoral neck fractures.

Loss of alpha-hemoglobin-stabilizing protein impairs erythropoiesis and exacerbates beta-thalassemia.

Hemoglobin (Hb) A production during red blood cell development is coordinated to minimize the deleterious effects of free alpha- and beta-Hb subunits, which are unstable and cytotoxic. The alpha-Hb-stabilizing protein (AHSP) is an erythroid protein that specifically binds alpha-Hb and prevents its precipitation in vitro, which suggests that it may function to limit free alpha-Hb toxicities in vivo. We investigated this possibility through gene ablation and biochemical studies. AHSP(-/-) erythrocytes contained hemoglobin precipitates and were short-lived. In hematopoietic tissues, erythroid precursors were elevated in number but exhibited increased apoptosis. Consistent with unstable alpha-Hb, AHSP(-/-) erythrocytes contained increased ROS and evidence of oxidative damage. Moreover, purified recombinant AHSP inhibited ROS production by alpha-Hb in solution. Finally, loss of AHSP worsened the phenotype of beta-thalassemia, a common inherited anemia characterized by excess free alpha-Hb. Together, the data support a model in which AHSP binds alpha-Hb transiently to stabilize its conformation and render it biochemically inert prior to Hb A assembly. This function is essential for normal erythropoiesis and, to a greater extent, in beta-thalassemia. Our findings raise the possibility that altered AHSP expression levels could modulate the severity of beta-thalassemia in humans.

The Hb A variant (beta73 Asp-->Leu) disrupts Hb S polymerization by a novel mechanism.

Polymerization of a 1:1 mixture of hemoglobin S (Hb S) and the artificial mutant HbAbeta73Leu produces a dramatic morphological change in the polymer domains in 1.0 M phosphate buffer that are a characteristic feature of polymer formation. Instead of feathery domains with quasi 2-fold symmetry that characterize polymerization of Hb S and all previously known mixtures such as Hb A/S and Hb F/S mixtures, these domains are compact structures of quasi-spherical symmetry. Solubility of Hb S/Abeta73Leu mixtures was similar to that of Hb S/F mixtures. Kinetics of polymerization indicated that homogeneous nucleation rates of Hb S/Abeta73Leu mixtures were the same as those of Hb S/F mixtures, while exponential polymer growth (B) of Hb S/Abeta73Leu mixtures were about three times slower than those of Hb S/F mixtures. Differential interference contrast (DIC) image analysis also showed that fibers in the mixture appear to elongate between three and five times more slowly than in equivalent Hb S/F mixtures by direct measurements of exponential growth of mass of polymer in a domain. We propose that these results of Hb S/Abeta73Leu mixtures arise from a non-productive binding of the hybrid species of this mixture to the end of the growing polymer. This "cap" prohibits growth of polymers, but by nature is temporary, so that the net effect is a lowered growth rate of polymers. Such a cap is consistent with known features of the structure of the Hb S polymer. Domains would be more spherulitic because slower growth provides more opportunity for fiber bending to spread domains from their initial 2-fold symmetry. Moreover, since monomer depletion proceeds more slowly in this mixture, more homogeneous nucleation events occur, and the resulting gel has a far more granular character than normally seen in mixtures of non-polymerizing hemoglobins with Hb S. This mixture is likely to be less stiff than polymerized mixtures of other hybrids such as Hb S with HbF, potentially providing a novel approach to therapy.

Star-Shaped Polycyclic Aromatic Ketones via 3-Fold Cycloadditions of Isobenzofuran Trimer Equivalent.

Three-directional annulations of isobenzofuran trimer equivalent are developed. Importantly, the successive cycloadditions could be controlled under suitable conditions, selectively affording the dual or triple cycloadduct, which leads to the alternative preparation of the symmetrical and unsymmetrical star-shaped polycyclic aromatic ketones. 1H NMR analysis of the star-shaped aromatic ketone indicated the π-π interactions through the aggregation in solution.

Whole-body biodistribution and the influence of body activity on brain kinetic analysis of the 11C-PiB PET scan.

Dynamic 11C-PiB PET imaging with kinetic analysis has been performed for accurate quantification of amyloid binding in patients with Alzheimer's disease (AD). In this study, we measured the whole-body biodistribution of 11C-PiB in nine subjects. We then evaluated the effect of body activity on quantitative accuracy of brain 11C-PiB three-dimensional (3D) dynamic PET. Based on clinical biodistribution data, we conducted phantom experiments to estimate the effect of body activity on quantification of the brain 3D dynamic 11C-PiB PET data and the error introduced by body activity using six different PET camera models. One of the PET cameras was used to acquire 11C-PiB brain 3D dynamic PET data on a patient with AD. We calculated the distribution volume ratio (DVR) in two kinetic methods using both the original human time-activity-curve (TAC) data and the TAC corrected for the error caused by body activity. In the early phase, both healthy subjects and patients with AD showed a biodistribution of 11C-PiB that reflected regional blood flow. In the simulated early phase of the phantom experiments, activity outside the field of view led to a maximum 6.0% overestimation of brain activity in the vertex region. Conversely, the effect of body activity on the DVR estimate was small (≤1.2%), probably because the tested kinetic methods did not rely heavily on early phase data. These results indicate that the effect of body activity on brain 11C-PiB PET quantification is generally small and that it depends on the method of kinetic analysis, the region of interest, and the PET camera model used.

Diminished abductor muscular strength in patients with valgus-impacted femoral neck fractures treated by internal fixation: Clinical study and biomechanical considerations.

BACKGROUND: Valgus-impacted femoral neck fractures treated with internal fixation occasionally result in unsatisfactory postoperative locomotive function, partially due to muscle shortening and a decrease in the moment arm. This study quantifies the degree of diminished abduction strength both clinically and biomechanically. METHODS: Fifteen patients were enrolled in this study. Twelve patients with fracture healed in valgus-impacted position were further evaluated. Muscular strength around hip was examined, and values between the nonoperated and operated side were compared and analyzed. For the biomechanical study, two three-dimensional models were prepared: model I (control model without displacement) and model II (simulated malunion of a 15° valgus-impacted fracture). Two sets of hip flexion angles in each of the models were simulated with flexion angles of 0° and 23°. RESULTS: Mean and standard deviation values for muscle strength from the nonoperative/operative side among the valgus group are as follows: flexion strength was 9.2 ± 4.0/9.2 ± 3.2, extension strength was 5.8 ± 2.8/6.1 ± 3.2, abduction strength at 0° was 9.1 ± 3.7/7.4 ± 3.6, abduction strength at 10° was 6.7 ± 3.0/5.5 ± 2.2, and knee extension strength was 15.3 ± 6.2/15.1 ± 6.0 (kgf). When comparing values between the nonoperative and operative sides, statistical significance was only observed in abduction strength ( p < 0.01). The biomechanical models prove that valgus impaction decreases the moment arm by approximately 10% at both flexion angle. CONCLUSIONS: A significant decrease in abductor strength at 0° and 10° was observed in the valgus-healed group. This may be related to a decrease in the moment arm. Further research should be done to define the acceptable limit of deformity for the satisfactory postoperative functioning.

Molecular crowding limits the role of fetal hemoglobin in therapy for sickle cell disease.

The dominant assumption central to most treatments for sickle cell anemia has been that replacement of sickle hemoglobin (HbS) by fetal hemoglobin (HbF) would have major clinical benefit. Using laser photolysis, we have measured polymerization kinetics including rates of homogeneous and heterogeneous nucleation on mixtures of 20% and 30% HbF with HbS. We find that the present model for polymerization, including molecular crowding, can accurately predict the rates of such mixtures, by using the single assumption that no significant amount of HbF enters the polymer. The effects of replacing HbS by HbF on the rates of polymer formation are found to be significantly lower than previous measurements appeared to indicate because the impact of the replacement is also highly dependent on the total hemoglobin concentration. This is because the molecular crowding of non-polymerizing HbF offsets substantially the effects of decreasing the concentration of HbS concentration, an effect that increases with concentration. Most strikingly, the demonstrated benefit of hydroxyurea therapy in slowing the kinetics of intracellular polymerization cannot be primarily due to enhanced HbF, but must have some other origin, which could itself represent a promising therapeutic approach.

Biomechanical Study Using the Finite Element Method of Internal Fixation in Pauwels Type III Vertical Femoral Neck Fractures.

BACKGROUND: Several factors are known to influence osseous union of femoral neck fractures. Numerous clinical studies have reported different results, hence with different recommendations regarding treatment of Pauwels III fractures: femoral neck fractures with a more vertically oriented fracture line. The current study aimed to analyze biomechanically whether this fracture poses a higher risk of nonunion. OBJECTIVES: To analyze the influence of one designated factor, authors believe that a computerized fracture model, using a finite element Finite Element Method (FEM), may be essential to negate the influence of other factors. The current study aimed to investigate a single factor, i.e. orientation of the fracture line toward a horizontal line, represented by Pauwels classification. It was hypothesized that a model with a vertically oriented fracture line maintaining parity of all other related factors has a higher stress at the fracture site, which would delay fracture healing. This result can be applicable to other types of pinning. PATIENTS AND METHODS: The finite element models were constructed from computed tomography data of the femur. Three fracture models, treated with pinning, were constructed based on Pauwels classification: Type I, 30° between the fracture line and a horizontal line; Type II, 50°; and Type III, 70°. All other factors were matched between the models. The Von Mises stress and principal stress distribution were examined along with the fracture line in each model. RESULTS: The peak Von Mises stresses at the medial femoral neck of the fracture site were 35, 50 and 130 MPa in Pauwels type I, II, and III fractures, respectively. Additionally, the peak Von Mises stresses along with the fracture site at the lateral femoral neck were 140, 16, and 8 MPa in Pauwels type I, II, and III fractures, respectively. The principal stress on the medial femoral neck in Pauwels type III fracture was identified as a traction stress, whereas the principal stress on the lateral femoral neck in Pauwels type I fracture was a compression stress. CONCLUSIONS: The most relevant finding was that hook pinning in Pauwels type III fracture may result in delayed union or nonunion due to significantly increased stress of a traction force at the fracture site that works to displace the fracture. However, in a Pauwels type I fracture, increased compression stress contributes to stabilize it. Surgeons are recommended not to treat Pauwels type III femoral neck fractures by pinning.

Synthesis of sexithiophene-bridged cage compound: a new class of three-dimensionally expanded oligothiophenes.

A bicyclo-type cage-shaped compound consisting of three sexithiophenes was successfully synthesized and characterized by NMR, HRMS, and X-ray crystallographic analysis. The strained cage architecture was reflected in the blue-shifted absorption spectrum relative to its linear analogue. Intramolecular interaction between three-dimensionally fixed sexithiophenes was suggested by electrochemical analysis.

Assembly of recently translated full-length and C-terminal truncated human gamma-globin chains with a pool of alpha-globin chains to form Hb F in a cell-free system.

Assembly of alpha-globin with translated, full-length and C-terminal truncated human gamma-globin to form Hb F was assessed in a cell-free transcription/translation system. Polysome profiles showed two amino acid C-terminal-truncated gamma-chains retained on polysomes can assemble with unlabeled holo alpha-chains only after puromycin-induced chain release. Two amino acid C-terminal truncated gamma-chains encoded from vectors containing a stop codon at the translation termination site were released from polysomes and assembled with alpha-chains in the absence of puromycin addition, while removal of 11 or more amino acids from the gamma-chain carboxy-terminus inhibited assembly with alpha-chains. These results suggest that amino acids in the HC- and H-helix gamma-chain regions including amino acids 135-144 at the C-terminus in the translated gamma-chains play a key role in assembly with alpha-chains, and that assembly occurs soon after exit of translated gamma-chains from the ribosome tunnel and release from polysomes thereby preventing stable gamma(2) homo-dimer formation.

Inhibition of hemoglobin S polymerization in vitro by a novel 15-mer EF-helix beta73 histidine-containing peptide.

Our mutational studies on Hb S showed that the Hb S beta73His variant (beta6Val and beta73His) promoted polymerization, while Hb S beta73Leu (beta6Val and beta73Leu) inhibited polymerization. On the basis of these results, we speculated that EF-helix peptides containing beta73His interact with beta4Thr in Hb S and compete with Hb S, resulting in inhibition of Hb S polymerization. We, therefore, studied inhibitory effects of 15-, 11-, 7-, and 3-mer EF-helix peptides containing beta73His on Hb S polymerization. The delay time prior to Hb S polymerization increased only in the presence of the 15-mer His peptide; the higher the amount, the longer the delay time. DIC image analysis also showed that the fiber elongation rate for Hb S polymers decreased with increasing concentration of the 15-mer His peptide. In contrast, the same 15-mer peptide containing beta73Leu instead of His and peptides shorter than 11 amino acids containing beta73His including His alone showed little effect on the kinetics of polymerization and elongation of polymers. Analysis by protein-chip arrays showed that only the 15-mer beta73His peptide interacted with Hb S. CD spectra of the 15-mer beta73His peptide did not show a specific helical structure; however, computer docking analysis suggested a lower energy for interaction of Hb S with the 15-mer beta73His peptide compared to peptides containing other amino acids at this position. These results suggest that the 15-mer beta73His peptide interacts with Hb S via the beta4Thr in the betaS-globin chain in Hb S. This interaction may influence hydrogen bond interaction between beta73Asp and beta4Thr in Hb S polymers and interfere in hydrophobic interactions of beta6Val, leading to inhibition of Hb S polymerization.

Hemoglobin equilibrium analysis by the multiangle laser light-scattering method.

Dimer-tetramer and monomer-dimer-tetramer equilibria of tetrameric hemoglobins and their single chains in the CO form, respectively, were evaluated using the microbatch multiangle light-scattering (MALS) analysis system. The molecular weights of human Hb A and Hb F in the CO form were dependent on concentration. The dissociation constants to dimers of Hb A and Hb F were 2.58 x 10(-6) and 0.66 x 10(-6), respectively. Equilibration of single globin chains, including alpha, beta, and gamma chains, was also evaluated by the same method. The dissociation constants of alpha-chain dimers to monomers, of beta-chain tetramers to monomers, and of gamma-chain tetramers to dimers were 14 x 10(-6), 25 x 10(-17), and 6.86 x 10(-6) M, respectively. These results indicate that the MALS analysis system can not only determine molecular weight but also characterize protein-protein interactions of multi-subunit proteins.