Examining the Association of Social Isolation and Smoking in Older Adults.

Background: Tobacco use remains a leading cause of preventable death among older adults, but few studies have examined social isolation as a risk factor for smoking in US. older adults. Methods: Using National Health and Aging Trends Study (NHATS) data, we conducted multivariate analyses of smoking in a sample of 8136 adults ages 65 and older. Results: Social isolation and severe isolation were associated with higher odds of smoking (OR: 2.48 and 5.48, p = 0.002 and p < 0.001). Individuals with mild (OR: 1.46, p = 0.006), moderate (OR: 1.80, p = 0.001), or severe (OR: 3.05, p = 0.001) symptoms of depression/anxiety also had higher odds of smoking. Conclusions: Social isolation is a significant risk factor for smoking in US older adults. Further research is needed to support the development of interventions to reduce social isolation and smoking behavior in older adults.

Prevalence of Mental Health and Social Connection among Patients Seeking Tobacco Dependence Management: A Pilot Study.

INTRODUCTION: with regards to tobacco dependence management, there are certain barriers to successful smoking cessation for patients, such as untreated anxiety and depression. Complicating the impact of mental health morbidities on tobacco dependence may be the significant portion of patients whose mental health issues and limited social connections are undiagnosed and unaddressed. We hypothesize that patients with no prior mental health diagnoses who are treated for tobacco dependence have high rates of undiagnosed mental health morbidities. METHODS: patients were recruited from a tobacco treatment clinic in 2021. Every patient who came for an inaugural visit without a prior diagnosis of mental health disease was screened for depression, anxiety, social isolation and loneliness. Sociodemographic variables were collected. RESULTS: over a 12-month period, 114 patients were seen at the tobacco treatment clinic. Of these 114 patients, 77 (67.5%) did not have a prior diagnosis of a mental health disease. The mean age was 54.3 ± 11.2 years, 52 (67.5%) were females, and 64 (83.1%) were Black/African American. The mean age of starting smoking was 19.3 ± 5.2 years, and 43 (55.8%) had never attempted to quit smoking in the past. With regards to mental health screening, 32 (41.6%) patients had a score of 9 or greater on the Patient Health Questionnaire (PHQ) 9, 59 (76.6%) had a score of 7 or greater on the Generalized Anxiety Disorder (GAD) 7, 67 (87.0%) were identified with social isolation and 70 (90.1%) for loneliness on screening. CONCLUSION: there was a high prevalence of undiagnosed mental health morbidities and social disconnection in patients who were actively smoking and were struggling to achieve smoking cessation. While a larger scale study is necessary to reaffirm these results, screening for mental health morbidities and social disconnection may be warranted in order to provide effective tobacco dependence management.

Getting under the skin: Social isolation and biological markers in the National Health and Aging Trends Study.

BACKGROUND: Social isolation is a risk factor for morbidity and mortality comparable to well-established risk factors including smoking, hypertension, and a sedentary lifestyle. The specific biological mechanisms that connect social isolation to morbidity and mortality remain unclear. Interleukin-6 (IL-6) and C-reactive protein (CRP) are biological markers that are upregulated during inflammation and can have long-term negative consequences for the health of individuals as they age. METHODS: Utilizing Round 7 (2017) data from the National Health and Aging Trends Study (NHATS), we examine the relationship between social isolation and two biological markers: IL-6 and high-sensitivity CRP. This study included a nationally representative sample of 4648 Medicare beneficiaries 65 years and older who provided samples using dried blood spot (DBS) techniques. We defined social isolation utilizing a multi-domained typology that considers living arrangement, core discussion network, religious attendance, and social participation. IL-6 and CRP were obtained via DBS that were collected in Round 7 of the NHATS. We performed linear regression to examine the association between social isolation and biological markers IL-6 and CRP. RESULTS: After adjusting for age, gender, race/ethnicity, income, tobacco use, body mass index, and chronic conditions, we found that severe social isolation and social isolation were significantly associated with higher levels of IL-6 and CRP values among older adults. CONCLUSIONS: Social isolation is associated with higher levels of biological markers (IL-6 and CRP). Our findings inform the pathway between social isolation and morbidity and mortality among older adults. IL-6 or CRP could be a proximal outcome measures for future clinical and social interventions that seek to alter the trajectory of social isolation and its associated health outcomes.

VTT-006, an anti-mitotic compound, binds to the Ndc80 complex and suppresses cancer cell growth in vitro.

Hec1 (Highly expressed in cancer 1) resides in the outer kinetochore where it works to facilitate proper kinetochore-microtubule interactions during mitosis. Hec1 is overexpressed in various cancers and its expression shows correlation with high tumour grade and poor patient prognosis. Chemical perturbation of Hec1 is anticipated to impair kinetochore-microtubule binding, activate the spindle assembly checkpoint (spindle checkpoint) and thereby suppress cell proliferation. In this study, we performed high-throughput screen to identify novel small molecules that target the Hec1 calponin homology domain (CHD), which is needed for normal microtubule attachments. 4 million compounds were first virtually fitted against the CHD, and the best hit molecules were evaluated in vitro. These approaches led to the identification of VTT-006, a 1,2-disubstituted-tetrahydro-beta-carboline derivative, which showed binding to recombinant Ndc80 complex and modulated Hec1 association with microtubules in vitro. VTT-006 treatment resulted in chromosome congression defects, reduced chromosome oscillations and induced loss of inter-kinetochore tension. Cells remained arrested in mitosis with an active spindle checkpoint for several hours before undergoing cell death. VTT-006 suppressed the growth of several cancer cell lines and enhanced the sensitivity of HeLa cells to Taxol. Our findings propose that VTT-006 is a potential anti-mitotic compound that disrupts M phase, impairs kinetochore-microtubule interactions, and activates the spindle checkpoint.

FRET detects lateral interaction between transmembrane domain of EGF receptor and ganglioside GM3 in lipid bilayers.

Ganglioside GM3 in the plasma membranes suppresses cell growth by preventing the autophosphorylation of the epidermal growth factor receptor (EGFR). Biological studies have suggested that GM3 interacts with the transmembrane segment of EGFR. Further biophysical experiments are particularly important for quantitative evaluation of the peptide-glycolipid interplay in bilayer membranes using a simple reconstituted system. To examine these interactions in this way, we synthesized the transmembrane segment of EGFR bearing a nitrobenzoxadiazole fluorophore (NBD-TM) at the N-terminus. The affinity between EGFR and GM3 was evaluated based on Förster resonance energy transfer (FRET) between NBD-TM and ATTO594-labeled GM3 in bilayers where their non-specific interaction due to lateral proximity was subtracted by using NBD-labeled phospholipid. This method for selectively detecting the specific lipid-peptide interactions in model lipid bilayers disclosed that the lateral interaction between GM3 and the transmembrane segment of EGFR plays a certain role in disturbing the formation of active EGFR dimers.

Aspirin/antiplatelet agent use improves disease-free survival and reduces the risk of distant metastases in Stage II and III triple-negative breast cancer patients.

PURPOSE: The objective is to define the therapeutic role of antiplatelet agents in a triple-negative breast cancer (TNBC) population. METHODS: We performed retrospective analysis using the UTSW TNBC registry containing data from 222 Stage II-III TNBC patients treated between 1998 and 2016. Univariate analysis and multivariable logistic regression models were constructed to identify factors associated with disease-free survival (DFS), distant metastases rate (DMR), and overall survival outcomes. Antiplatelet drug use was determined by review of electronic medical records. RESULTS: A total of 65 patients used antiplatelet (AP) agents, and 157 patients did not use AP agents. Median follow-up for AP and non-AP groups was 41.3 and 40.9 months, respectively. There was an improvement in the AP group compared with the control group in 5-year DFS (80.4% at 5 years compared with 62.3% in the control group, p = 0.04) and 5-year DMR (8.8 vs. 31.9%, p = 0.007). In multivariate analysis, AP use was found to be significantly associated with improvements in DFS and DMR. CONCLUSIONS: We illustrate that antiplatelet agent use improves DMR and DFS among a stage II and III TNBC population despite our short follow-up evaluation. Longer follow-up evaluation will be required to determine additional outcome advantage for antiplatelet agent use. Our findings support consideration of investigation of antiplatelet therapy as an adjunctive therapy for TNBC at high risk for disease recurrence.

Effect of Lung Surfactant Protein SP-C and SP-C-Promoted Membrane Fragmentation on Cholesterol Dynamics.

To allow breathing and prevent alveolar collapse, lung surfactant (LS) develops a complex membranous system at the respiratory surface. LS is defined by a specific protein and lipid composition, including saturated and unsaturated phospholipid species and cholesterol. Surfactant protein C (SP-C) has been suggested to be an essential element for sustaining the presence of cholesterol in surfactant without functional impairment. In this work, we used a fluorescent sterol-partitioning assay to assess the effect of the surfactant proteins SP-B and SP-C on cholesterol distribution in membranes. Our results suggest that in the LS context, the combined action of SP-B and SP-C appears to facilitate cholesterol dynamics, whereas SP-C does not seem to establish a direct interaction with cholesterol that could increase the partition of free cholesterol into membranes. Interestingly, SP-C exhibits a membrane-fragmentation behavior, leading to the conversion of large unilamellar vesicles into highly curved vesicles ∼25 nm in diameter. Sterol partition was observed to be sensitive to the bending of bilayers, indicating that the effect of SP-C to mobilize cholesterol could be indirectly associated with SP-C-mediated membrane remodeling. Our results suggest a potential role for SP-C in generating small surfactant structures that may participate in cholesterol mobilization and pulmonary surfactant homeostasis at the alveolar interfaces.

Effects of cholesterol and saturated sphingolipids on acyl chain order in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine bilayers--a comparative study with phase-selective fluorophores.

Saturated sphingolipids have high acyl chain order. Our aim was to study how palmitoylated sphingomyelin (PSM), ceramide (PCer), glucosyl (GlcPCer)-, and galactosylceramide (GalPCer) were able to order the bulk acyl chains of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), in comparison with cholesterol. For this reason, we used lipid probes which had preferred phases that were either the disordered phase (1-oleoyl-2-propionyl[DPH-sn-glycero-3-phosphcholine (18:1-DPH-PC) or the ordered phase (trans parinaric acid (tPA). DPH was also used, although it has no clear phase preference. We measured steady-state anisotropy (all probes) and performed fluorescence lifetime analysis (tPA) as a function of composition and temperature. At concentrations where the saturated sphingolipids were not aggregated into ordered domains (and 23 °C), they did not increase POPC acyl chain order as determined from 18:1-DPH-PC anisotropy. As expected, cholesterol increased the POPC acyl chain order linearly as a function of concentration (0-28 mol %). Since PCer already forms ordered domains below 5 mol % (at 23 °C), we measured the acyl chain ordering effect of PCer at 50 °C (0-13 mol %) and observed that PCer ordered POPC acyl chains as efficiently as cholesterol. We conclude that the bulk acyl chain order of POPC was not markedly affected in bilayers where disordered and ordered domains coexist.

Sterol affinity for phospholipid bilayers is influenced by hydrophobic matching between lipids and transmembrane peptides.

Lipid self-organization is believed to be essential for shaping the lateral structure of membranes, but it is becoming increasingly clear that also membrane proteins can be involved in the maintenance of membrane architecture. Cholesterol is thought to be important for the lateral organization of eukaryotic cell membranes and has also been implicated to take part in the sorting of cellular transmembrane proteins. Hence, a good starting point for studying the influence of lipid-protein interactions on membrane trafficking is to find out how transmembrane proteins influence the lateral sorting of cholesterol in phospholipid bilayers. By measuring equilibrium partitioning of the fluorescent cholesterol analog cholestatrienol between large unilamellar vesicles and methyl-ß-cyclodextrin the effect of hydrophobic matching on the affinity of sterols for phospholipid bilayers was determined. Sterol partitioning was measured in 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) bilayers with and without WALP19, WALP23 or WALP27 peptides. The results showed that the affinity of the sterol for the bilayers was affected by hydrophobic matching. An increasing positive hydrophobic mismatch led to stronger sterol binding to the bilayers (except in extreme situations), and a large negative hydrophobic mismatch decreased the affinity of the sterol for the bilayer. In addition, peptide insertion into the phospholipid bilayers was observed to depend on hydrophobic matching. In conclusion, the results showed that hydrophobic matching can affect lipid-protein interactions in a way that may facilitate the formation of lateral domains in cell membranes. This could be of importance in membrane trafficking.

Probing the lipid-protein interface using model transmembrane peptides with a covalently linked acyl chain.

The aim of this study was to gain insight into how interactions between proteins and lipids in membranes are sensed at the protein-lipid interface. As a probe to analyze this interface, we used deuterium-labeled acyl chains that were covalently linked to a model transmembrane peptide. First, a perdeuterated palmitoyl chain was coupled to the Trp-flanked peptide WALP23 (Ac-CGWW(LA)(8)LWWA-NH(2)), and the deuterium NMR spectrum was analyzed in di-C18:1-phosphatidylcholine (PC) bilayers. We found that the chain order of this peptide-linked chain is rather similar to that of a noncovalently coupled perdeuterated palmitoyl chain, except that it exhibits a slightly lower order. Similar results were obtained when site-specific deuterium labels were used and when the palmitoyl chain was attached to the more-hydrophobic model peptide WLP23 (Ac-CGWWL(17)WWA-NH(2)) or to the Lys-flanked peptide KALP23 (Ac-CGKK(LA)(8)LKKA-NH(2)). The experiments showed that the order of both the peptide-linked chains and the noncovalently coupled palmitoyl chains in the phospholipid bilayer increases in the order KALP23 < WALP23 < WLP23. Furthermore, changes in the bulk lipid bilayer thickness caused by varying the lipid composition from di-C14:1-PC to di-C18:1-PC or by including cholesterol were sensed rather similarly by the covalently coupled chain and the noncovalently coupled palmitoyl chains. The results indicate that the properties of lipids adjacent to transmembrane peptides mostly reflect the properties of the surrounding lipid bilayer, and hence that (at least for the single-span model peptides used in this study) annular lipids do not play a highly specific role in protein-lipid interactions.

Construction of chimeric dual-chain avidin by tandem fusion of the related avidins.

BACKGROUND: Avidin is a chicken egg-white protein with high affinity to vitamin H, also known as D-biotin. Many applications in life science research are based on this strong interaction. Avidin is a homotetrameric protein, which promotes its modification to symmetrical entities. Dual-chain avidin, a genetically engineered avidin form, has two circularly permuted chicken avidin monomers that are tandem-fused into one polypeptide chain. This form of avidin enables independent modification of the two domains, including the two biotin-binding pockets; however, decreased yields in protein production, compared to wt avidin, and complicated genetic manipulation of two highly similar DNA sequences in the tandem gene have limited the use of dual-chain avidin in biotechnological applications. PRINCIPAL FINDINGS: To overcome challenges associated with the original dual-chain avidin, we developed chimeric dual-chain avidin, which is a tandem fusion of avidin and avidin-related protein 4 (AVR4), another member of the chicken avidin gene family. We observed an increase in protein production and better thermal stability, compared with the original dual-chain avidin. Additionally, PCR amplification of the hybrid gene was more efficient, thus enabling more convenient and straightforward modification of the dual-chain avidin. When studied closer, the generated chimeric dual-chain avidin showed biphasic biotin dissociation. SIGNIFICANCE: The improved dual-chain avidin introduced here increases its potential for future applications. This molecule offers a valuable base for developing bi-functional avidin tools for bioseparation, carrier proteins, and nanoscale adapters. Additionally, this strategy could be helpful when generating hetero-oligomers from other oligomeric proteins with high structural similarity.

Transmembrane peptides influence the affinity of sterols for phospholipid bilayers.

Cholesterol is distributed unevenly between different cellular membrane compartments, and the cholesterol content increases from the inner bilayers toward the plasma membrane. It has been suggested that this cholesterol gradient is important in the sorting of transmembrane proteins. Cholesterol has also been to shown play an important role in lateral organization of eukaryotic cell membranes. In this study the aim was to determine how transmembrane proteins influence the lateral distribution of cholesterol in phospholipid bilayers. Insight into this can be obtained by studying how cholesterol interacts with bilayer membranes of different composition in the presence of designed peptides that mimic the transmembrane helices of proteins. For this purpose we developed an assay in which the partitioning of the fluorescent cholesterol analog CTL between LUVs and mbetaCD can be measured. Comparison of how cholesterol and CTL partitioning between mbetaCD and phospholipid bilayers with different composition suggests that CTL sensed changes in bilayer composition similarly as cholesterol. Therefore, the results obtained with CTL can be used to understand cholesterol distribution in lipid bilayers. The effect of WALP23 on CTL partitioning between DMPC bilayers and mbetaCD was measured. From the results it was clear that WALP23 increased both the order in the bilayers (as seen from CTL and DPH anisotropy) and the affinity of the sterol for the bilayer in a concentration dependent way. Although WALP23 also increased the order in DLPC and POPC bilayers the effects on CTL partitioning was much smaller with these lipids. This indicates that proteins have the largest effect on sterol interactions with phospholipids that have longer and saturated acyl chains. KALP23 did not significantly affect the acyl chain order in the phospholipid bilayers, and inclusion of KALP23 into DMPC bilayers slightly decreased CTL partitioning into the bilayer. This shows that transmembrane proteins can both decrease and increase the affinity of sterols for the lipid bilayers surrounding proteins. This is likely to affect the sterol distribution within the bilayer and thereby the lateral organization in biomembranes.

Absorptive clearance of DTPA as an aerosol-based biomarker in the cystic fibrosis airway.

Biomarkers providing in vivo quantification of the basic elements of cystic fibrosis (CF) lung disease are needed. A study was performed to determine whether the absorption of a small radiolabelled hydrophilic molecule (Indium-111 (In-)DTPA) would be increased in CF airways. DTPA clearance has been used previously to assess epithelial permeability and may also be useful for quantifying liquid absorption. The absorptive clearance rate of DTPA was quantified in 10 CF and 11 control subjects using a novel aerosol technique. Subjects inhaled an aerosol containing nonabsorbable technetium-99m sulfur colloid (Tc-SC) particles and In-DTPA. Tc-SC clearance from the lung is exclusively mucociliary, while In-DTPA is cleared by both absorption and mucociliary clearance. The difference between the In-DTPA and Tc-SC clearance rates estimates In-DTPA absorption. Tc-SC (mucociliary) clearance was similar in central and peripheral zones in CF and non-CF lungs. Total In-DTPA clearance was increased in both zones in CF lungs. The absorptive component of In-DTPA clearance was increased in the airway-dominated central lung zones in CF (42% x h( -1) versus 32% x h(-1), p = 0.03). The absorption of In-DTPA is increased in the CF airway. Further study is needed to understand the relative roles of fluid absorption, inflammation and other mechanisms potentially affecting epithelial permeability and DTPA absorption.

Genetics of age-related macular degeneration.

PURPOSE OF REVIEW: Age-related macular degeneration (AMD) was until recently viewed as a part of the normal aging process; however, we are increasingly aware that genetic factors play a much greater role than previously suspected. This review will provide an up-to-date snapshot of the genetics of AMD to help guide our thoughts about its causes and the risk for family members. RECENT FINDINGS: Epidemiological research and basic bench research have identified pathways of oxidative stress, lipid metabolism and inflammation as playing causative roles in the pathogenesis of AMD. Emerging research is focusing on the biology of the retinal pigment epithelium as secreting pro and anti-inflammatory mediators in the eye. Antivascular endothelial growth factor therapy has dramatically improved the prognosis for neovascular or wet AMD patients. Nutritional supplementation with antioxidants and omega-3 fatty acids has provided treatment options for patients with atrophic or dry AMD. We should expect that some of the response to therapy might be genetically determined. SUMMARY: First-degree relatives of patients with AMD tend to have a higher risk of AMD. Recognizing an inherent genetic risk of AMD in these patients will improve their management and potentially help prevent blindness.

Influence of trifluoroethanol on membrane interfacial anchoring interactions of transmembrane alpha-helical peptides.

Interfacial anchoring interactions between aromatic amino acid residues and the lipid-water interface are believed to be important determinants for membrane protein structure and function. Thus, it is possible that molecules that partition into the lipid-water interface can influence membrane protein activity simply by interfering with these anchoring interactions. Here we tested this hypothesis by investigating the effects of 2,2,2-trifluoroethanol (TFE) on the interaction of a Trp-flanked synthetic transmembrane peptide (acetyl-GW(2)(LA)(8)LW(2)A-NH(2)) with model membranes of dimyristoylphosphatidylcholine. Two striking observations were made. First, using (2)H nuclear magnetic resonance on acyl chain deuterated lipids, we found that addition of 4 or 8 vol % of TFE completely abolishes the ability of the peptide to order and stretch the lipid acyl chains in these relatively thin bilayers. Second, we observed that addition of 8 vol % TFE reduces the tilt angle of the peptide from 5.3 degrees to 2.5 degrees, as measured by (2)H NMR on Ala-d(4) labeled peptides. The "straightening" of the peptide was accompanied by an increased exposure of Trp to the aqueous phase, as shown by Trp-fluorescence quenching experiments using acrylamide. The observation of a reduced tilt angle was surprising because we also found that TFE partioning results in a significant thinning of the membrane, which would increase the extent of hydrophobic mismatch. In contrast to the Trp-flanked peptide, no effect of TFE was observed on the interaction of a Lys-flanked analog (acetyl-GK(2)(LA)(8)LK(2)A-NH(2)) with the lipid bilayer. These results emphasize the importance of interfacial anchoring interactions for membrane organization and provide new insights into how molecules such as TFE that can act as anesthetics may affect the behavior of membrane proteins that are enriched in aromatic amino acids at the lipid-water interface.

Community-based rapid HIV testing in homeless and marginally housed adults in San Francisco.

BACKGROUND: Standard two-step HIV testing is limited by poor return-for-results rates and misses high-risk individuals who do not access conventional testing facilities. METHODS: We describe a community-based rapid HIV testing programme in which homeless and marginally housed adults recruited from shelters, free meal programmes and single room occupancy hotels in San Francisco received OraQuick Rapid HIV-1 Antibody testing (OraSure Technologies, Bethlehem, PA, USA). RESULTS: Over 8 months, 1614 adults were invited to participate and 1213 (75.2%) underwent testing. HIV seroprevalence was 15.4% (187 of 1213 individuals) overall and 3.5% (37 of 1063) amongst high-risk individuals reporting no previous testing, a prior negative test, or previous testing without result disclosure. All 1213 participants received their results. Of 30 newly diagnosed persons who received confirmatory results, 26 (86.7%) reported at least one contact with a primary healthcare provider in the 6 months following diagnosis. CONCLUSIONS: We conclude that community-based rapid testing is feasible, acceptable and effective based on the numbers of high-risk persons tested over a short period, the participation rate, the prevalence of new infection, the rate of result disclosure, and the proportion linked to care.

Peptides in lipid bilayers: the power of simple models.

Interactions between proteins and lipids lie at the heart of virtually all membrane processes, but on a molecular level they are still poorly understood. Nowadays, simple model systems comprising designed transmembrane peptides in synthetic lipid bilayers are increasingly being recognized as powerful tools to uncover basic principles of protein-lipid interactions. Such model systems enable detailed analysis of how the properties of lipids influence the structure and dynamics of transmembrane helices, how these helices are anchored at the lipid-water interface, and how the length and composition of transmembrane segments influence the organization and dynamics of membrane lipids. In addition, well-characterized model systems have proven useful to refine computational approaches and to develop new techniques for studies of protein-lipid interactions.

A new case of oculoectodermal syndrome.

An 11-month-old infant girl presented with right-sided features of aplasia cutis congenita of the scalp, unilateral epibulbar dermoids, eccentric pupil, coloboma of the right upper eyelid, and depigmentation of the fundus surrounding the right optic nerve. These findings were similar to the oculoectodermal syndrome reported by other clinicians and researchers.

Dual-affinity avidin molecules.

A recently reported dual-chain avidin was modified further to contain two distinct, independent types of ligand-binding sites within a single polypeptide chain. Chicken avidin is normally a tetrameric glycoprotein that binds water-soluble d-biotin with extreme affinity (K(d) approximately 10(-15) M). Avidin is utilized in various applications and techniques in the life sciences and in the nanosciences. In a recent study, we described a novel avidin monomer-fusion chimera that joins two circularly permuted monomers into a single polypeptide chain. Two of these dual-chain avidins were observed to associate spontaneously to form a dimer equivalent to the wt tetramer. In the present study, we successfully used this scaffold to generate avidins in which the neighboring biotin-binding sites of dual-chain avidin exhibit two different affinities for biotin. In these novel avidins, one of the two binding sites in each polypeptide chain, the pseudodimer, is genetically modified to have lower binding affinity for biotin, whereas the remaining binding site still exhibits the high-affinity characteristic of the wt protein. The pseudotetramer (i.e., a dimer of dual-chain avidins) has two high and two lower affinity biotin-binding sites. The usefulness of these novel proteins was demonstrated by immobilizing dual-affinity avidin with its high-affinity sites. The sites with lower affinity were then used for affinity purification of a biotinylated enzyme. These "dual-affinity" avidin molecules open up wholly new possibilities in avidin-biotin technology, where they may have uses as novel bioseparation tools, carrier proteins, or nanoscale adapters.