Association of alcohol use with olfactory function among older adults.

BACKGROUND/PURPOSE: Olfactory dysfunction (OD) has been recognized as an early biomarker for neurodegenerative diseases. Identifying behaviors that increase the risk of OD is crucial for early recognition of neurogenerative diseases. Alcohol consumption can potentially impact olfaction through its neurotoxic effects. This study aims to examine the relationship between alcohol consumption and OD, using data from the National Social Life, Health, and Aging Project (NSHAP). METHODS: This cross-sectional study was conducted on data for 2757 adults from Round 1 of NSHAP. OD was defined as correctly identifying 0-3 odors in the 5-item Sniffin' Sticks test while normal olfactory function was defined as correctly identifying 4-5 odors. Multivariable logistic regression was utilized to examine the association between alcohol consumption and OD, controlling for age, race, and comorbidities. Analyses were weighted to account for the sampling design. RESULTS: OD was present in 23.1 % of adults. The average age among those with OD was 71.2 ± 7.8 years, compared to 66.9 ± 7.2 years in those with normal olfaction. In terms of alcohol consumption, 31.1 % of adults with OD were light-to-moderate drinkers and 7.7 % were heavy drinkers, compared to 35.6 % light-to-moderate and 7.7 % heavy drinkers in the normal olfaction group. After adjusting for age, gender, race, and education, neither light-to-moderate drinking (aOR: 0.99; 95 % CI: 0.76-1.29) nor heavy drinking (aOR: 1.24; 95 % CI: 0.83-1.85) were significantly associated with OD. CONCLUSION: Alcohol consumption was not associated with OD after controlling for covariates. While this study provides insight into the relationship between alcohol consumption and OD, further research is needed due to conflicting results in previous studies.

Reducing Sample Size While Improving Equity in Vaccine Clinical Trials: A Machine Learning-Based Recruitment Methodology with Application to Improving Trials of Hepatitis C Virus Vaccines in People Who Inject Drugs.

Despite the availability of direct-acting antivirals that cure individuals infected with the hepatitis C virus (HCV), developing a vaccine is critically needed in achieving HCV elimination. HCV vaccine trials have been performed in populations with high incidence of new HCV infection such as people who inject drugs (PWID). Developing strategies of optimal recruitment of PWID for HCV vaccine trials could reduce sample size, follow-up costs and disparities in enrollment. We investigate trial recruitment informed by machine learning and evaluate a strategy for HCV vaccine trials termed PREDICTEE-Predictive Recruitment and Enrichment method balancing Demographics and Incidence for Clinical Trial Equity and Efficiency. PREDICTEE utilizes a survival analysis model applied to trial candidates, considering their demographic and injection characteristics to predict the candidate's probability of HCV infection during the trial. The decision to recruit considers both the candidate's predicted incidence and demographic characteristics such as age, sex, and race. We evaluated PREDICTEE using in silico methods, in which we first generated a synthetic candidate pool and their respective HCV infection events using HepCEP, a validated agent-based simulation model of HCV transmission among PWID in metropolitan Chicago. We then compared PREDICTEE to conventional recruitment of high-risk PWID who share drugs or injection equipment in terms of sample size and recruitment equity, with the latter measured by participation-to-prevalence ratio (PPR) across age, sex, and race. Comparing conventional recruitment to PREDICTEE found a reduction in sample size from 802 (95%: 642-1010) to 278 (95%: 264-294) with PREDICTEE, while also reducing screening requirements by 30%. Simultaneously, PPR increased from 0.475 (95%: 0.356-0.568) to 0.754 (95%: 0.685-0.834). Even when targeting a dissimilar maximally balanced population in which achieving recruitment equity would be more difficult, PREDICTEE is able to reduce sample size from 802 (95%: 642-1010) to 304 (95%: 288-322) while improving PPR to 0.807 (95%: 0.792-0.821). PREDICTEE presents a promising strategy for HCV clinical trial recruitment, achieving sample size reduction while improving recruitment equity.

Trends and Frequencies of Antibiotic Prescriptions for Acute Sinusitis Outpatient Visits in Adults.

OBJECTIVE: The objective of this study was to analyze the trends and frequency in which recommended first-line therapy, amoxicillin with or without clavulanate, was prescribed for acute sinusitis based on current otolaryngology and other gold standard guidelines, as well as analyze differences in prescription behaviors of otolaryngologists compared with non-otolaryngologists for outpatient adult acute sinusitis visits. METHODS: Weighted patient data from the National Ambulatory Medical Care Survey were analyzed to calculate visit rates and trends of antibiotic prescriptions for adults diagnosed with acute sinusitis from 2007 to 2019. Visits with multiple prescribed antibiotics or concomitant diagnoses requiring antibiotics were excluded. Each visit was classified based on the type of antibiotic prescribed. RESULTS: Acute sinusitis was diagnosed in 0.63% of all outpatient visits from 2007 to 2019 (95% confidence interval: 0.56%-0.71%). Amoxicillin had the greatest increase in prescription frequency (13.4%), whereas macrolides had the largest decrease in prescription frequency (13.9%). Among adult acute sinusitis outpatient visits in which antibiotics were prescribed, recommended first-line antibiotic therapy of amoxicillin-clavulanate or amoxicillin alone was prescribed in 40.4% of visits. The most common antibiotic prescribed was amoxicillin-clavulanate at otolaryngologist visits (20.5%) and macrolides at non-otolaryngologist visits (26.0%). A greater proportion of otolaryngologist visits resulted in no antibiotics prescribed for acute sinusitis (36.8% vs. 22.5%, p < 0.001). CONCLUSION: Otolaryngologists engage in watchful waiting more than non-otolaryngologists. Broader dissemination of existing guidelines for acute sinusitis treatment to non-Otolaryngologist (ENT) primary care specialties that take care of acute sinusitis to improve antibiotic stewardship and appropriate antibiotic selection is needed. LEVEL OF EVIDENCE: 4 Laryngoscope, 134:2622-2625, 2024.

Diffuse-Type Pancreatic Ductal Adenocarcinoma Mimicking Autoimmune Pancreatitis.

Pancreatic ductal adenocarcinoma (PDAC) classically presents as a solitary mass on cross-sectional imaging. Diffuse-type PDAC is an unusual variant that accounts for 1%-5% of PDACs. Owing to its rarity, there are no established radiographic or endosonographic definitions. We report a unique case of diffuse-type PDAC presenting with imaging findings of 2 distinct masses in the pancreatic head and tail and with endoscopic ultrasound findings of diffuse gland enlargement mimicking autoimmune pancreatitis. The case illustrates the importance of sampling several areas of the pancreas when diffuse enlargement is present on endoscopic ultrasound and multiple masses are seen on cross-sectional imaging.

Depressive symptoms are associated with immunological failure among HIV-positive patients in Vietnam.

As the lives of people living with HIV (PLWH) become increasingly normalized, more focus is being given to the associated comorbidities of HIV, including those related to mental health such as depression. This study aims to evaluate the correlation between depressive symptoms and HIV outcomes in Vietnam through the measurement of CD4 cell count. A mixed design was utilized, in which both a longitudinal assessment of CD4 cell counts and a cross-sectional survey of depressive symptoms were conducted on 481 patients in the Bach Mai and Ha Dong HIV clinics (Hanoi, Vietnam). CD4 cell count data was extracted from the medical records of participants, and depressive symptoms were screened using the Patient Health Questionnaire (PHQ-9). The results illustrate that the presence of moderately severe to severe depressive symptoms is associated with lower CD4 cell counts, indicating poorer HIV outcomes resulting from comorbid depression. This correlation was especially noticeable in the PHQ-9 items for psychomotor agitation/retardation (p < 0.05) and suicidal ideation (p < 0.05). Future policy and treatment options for HIV in Vietnam should consider the presence of comorbid mental health conditions in order to provide more suitable and effective treatment in the goal of providing a higher quality of life for PLWH.

Cannabis Abuse and Perioperative Complications After Treatment of Intracranial Aneurysms: A Nationwide Analysis.

OBJECTIVE: In the present retrospective cohort analysis, we examined the differences in baseline characteristics and peri- and postoperative outcomes stratified by 3 groups: cannabis abuse or dependence versus none, surgical versus endovascular treatment, and unruptured and ruptured intracranial aneurysms. METHODS: A study population of 26,868 patients was defined using the 2009-2016 National Inpatient Sample database. The baseline characteristics were compared between the cannabis and no-cannabis groups, and the traits that differed significantly were factored into the multivariate analysis using 1:1 propensity score matching. The matched groups were analyzed to compare the cannabis and no-cannabis cohorts for the following endpoints: mortality, length of stay, discharge disposition, total hospital charges, and several peri- and postoperative outcomes. RESULTS: In the surgically and endovascularly treated groups for unruptured intracranial aneurysms, those in the cannabis group were more likely to be male and younger and to smoke tobacco than were those in the no-cannabis group. After matching, no significant endpoint differences were noted. Similarly, in the surgically and endovascularly treated ruptured aneurysm groups, those in the cannabis group were more likely to be male and younger and to smoke tobacco. After matching, the cannabis group within the endovascular treatment group had had a longer length of stay and were more likely to have developed any hydrocephalus, obstructive hydrocephalus, sepsis, and acute kidney injury. Those in the cannabis group who had undergone surgery were more likely to have developed any hydrocephalus, specifically, communicating hydrocephalus. CONCLUSIONS: The cannabis group with ruptured intracranial aneurysms was more likely to experience certain adverse outcomes after surgical or endovascular treatment compared with the no-cannabis group. However, such was not the case for cannabis abusers treated for unruptured aneurysms.

Distribution of Paycheck Protection Program to otolaryngology practices during the COVID-19 pandemic.

BACKGROUND: The Coronavirus disease of 2019 (COVID-19) has impacted physician practices in many ways with some ENT clinics reporting around a 50% drop in completed scheduled ENT visits during the first wave of the pandemic compared to 2019. AIMS: This study surveyed first round PPP loan disbursement to otolaryngology practices in the United States in response to COVID-19. METHODS: A cross-sectional study was conducted using publicly available data published on PPP by the SBA. Otolaryngology clinics receiving loans greater than $0.15M were filtered using the following terms: "otolaryngology", "otolaryngologist","sinus", "head and neck", "throat", "ENT", and "facial plastic". 481 ENT clinics that received loans greater than $0.15 M from the Paycheck Protection Program (PPP) were identified. Loan amount, business type, geographicregion, owner race, owner gender, and the number of jobs per business were recorded for each clinic. Chi-square analysis was performed to determine significance (P < 0.05) of each characteristic. RESULTS: Loan distribution was significantly different based on jobs reported (P < .001) and business type (P < .001). 100% of loans ranging from $0.15 M to $0.35 M went to micro and small practices whereas 33% of medium-sized practices received loans greater than $1 M. Higher proportions of Subchapter corporations (60.00%) received smaller loans of $0.15 to $0.35 M than Limited Liability Companies (39.13%) and Corporations (51.69%) which generally employ more people. DISCUSSION: Loan distribution was significantly different between businesses based on jobs reported (P < 0.001), with micro/small practices recieving smaller loans than their medium counterparts. All large businesses recived loans in in excess of $2 M. This suggests proportional distribution of loans in accordance with jobs reported. CONCLUSION: This study suggests PPP funding was objectively distributed to ENT clinics based on staff size. LEVEL OF EVIDENCE: Level 4.

Suppression of Nestin reveals a critical role for p38-EGFR pathway in neural progenitor cell proliferation.

The expression of intermediate filament Nestin is necessary for the neural progenitor cells (NPCs) to maintain stemness, but the underlying cellular and molecular mechanism remains unclear. In this study, we demonstrated that Nestin is required for the self-renew of NPCs through activating MAPK and EGFR pathways. Knockdown of Nestin by shRNA inhibited cell cycle progression and proliferation in mouse NPCs. Moreover, suppression of Nestin reduced expression of the epidermal growth factor receptor (EGFR) in NPCs and inhibited the mitogenic effects of EGF on these cells. Treatment of NPCs with p38-MAPK inhibitor PD169316 reversed cell cycle arrest caused by the knockdown of Nestin. Our findings indicate that Nestin promotes NPC proliferation via p38-MAPK and EGFR pathways, and reveals the necessity of these pathways in NPCs self-renewal.

Curettage and cryosurgery for low-grade cartilage tumors is associated with low recurrence and high function.

BACKGROUND: Chondrosarcomas of bone traditionally have been treated by wide or radical excision, procedures that may result in considerable lifelong disability. Grade 1 chondrosarcomas have little or no metastatic potential and are often difficult to distinguish from painful benign enchondromas. Curettage with adjuvant cryosurgery has been proposed as an alternative therapy for Grade 1 chondrosarcomas given the generally better function after the procedure. However, because it is an intralesional procedure, curettage and cryosurgery may be associated with higher rates of recurrence. QUESTIONS/PURPOSES: We asked whether Grade 1 chondrosarcomas and enchondromas of uncertain malignant potential treated by curettage and cryosurgery are associated with low recurrence rates and high functional scores. PATIENTS AND METHODS: We retrospectively reviewed the records of 46 patients with Grade 1 chondrosarcomas and enchondromas of uncertain malignant potential treated by curettage and cryosurgery. Forty-one patients had tumors of the long bones. Patients were followed a minimum of 18 months (average, 47.2. months; range, 18-134 months) for evidence of recurrence and for assessment of Musculoskeletal Tumor Society (MSTS) functional score. RESULTS: Two of the 46 patients had recurrences in the original tumor site (4.3% recurrence rate), which subsequently were removed by wide excision, and both patients were confirmed to be disease-free 36 and 30 months, respectively, after the second surgery. The mean MSTS score was 27.2 of 30 points (median, 29 points). CONCLUSIONS: Our observations show curettage with cryosurgery is associated with low recurrence of Grade 1 chondrosarcoma and high functional scores. Curettage with cryosurgery is a reasonable alternative to wide or radical excision as the treatment for Grade 1 chondrosarcomas, and allows for more radical surgery in the event of local recurrence. LEVEL OF EVIDENCE: Level IV, therapeutic study. See the Guidelines for Authors for a complete description of levels of evidence.

Polymethylmethacrylate particle exposure causes changes in p38 MAPK and TGF-beta signaling in differentiating MC3T3-E1 cells.

Periprosthetic osteolysis of joint replacements caused by wear debris is a significant complication of joint replacements. Polymethylmethacrylate (PMMA) particles have been shown to inhibit osteogenic differentiation, but the molecular mechanism has not been previously determined. In this study, we exposed differentiating MC3T3-E1 preostoblast cells to PMMA particles and determined the changes that occurred with respect to p38 mitogen-activated protein kinase (MAPK) activity and the transforming growth factor (TGF)-beta1 and bone morphogenetic protein (BMP) signaling pathways. In the absence of particles, MC3T3-E1 cells demonstrate activation of p38 MAPK on day 8 of differentiation; however, when treated with PMMA particles, differentiating MC3T3-E1 cells demonstrate the suppression of p38 activity on day 8 and show activation of p38 on days 1 and 4. On day 4 of particle exposure, the differentiating MC3T3-E1 cells show significant downregulation of TGF-beta1 expression, which is involved in osteoblast differentiation, and a significant upregulation of the expression of BMP3 and Sclerostin (SOST), which are negative regulators of osteoblast differentiation. By day 8 of particle exposure, the changes in TGF-beta1, BMP3, and SOST expression are opposite of those seen on day 4. This study has demonstrated the distinct changes in the molecular profile of MC3T3-E1 cells during particle-induced inhibition of osteoblast differentiation. (c) 2010 Wiley Periodicals, Inc. J Biomed Mater Res, 2010.

OP-1 (BMP-7) stimulates osteoprogenitor cell differentiation in the presence of polymethylmethacrylate particles.

Polymethylmethacrylate (PMMA) particles have been shown to inhibit the differentiation, proliferation, and mineralization of osteoprogenitor cells in vitro. In this study, we investigated the effects of OP-1 (BMP-7) on the osteogenesis of MC3T3-E1 osteoprogenitor cells exposed to PMMA particles in vitro. MC3T3-E1 cells challenged with PMMA particles on the 1st day of differentiation in osteogenic culture showed a significant dose-dependent decrease in mineralization and alkaline phosphatase expression over a 20-day culture period. Exposure of these cells to OP-1 (200 ng/mL) during days 1-4, 1-20, and 4-20 in the presence of PMMA particles resulted in significant increases in mineralization and alkaline phosphatase expression at all particle doses. Addition of OP-1 to MC3T3-E1 cultures challenged with PMMA particles on the 4th day of differentiation in osteogenic media also resulted in significant increases in mineralization and alkaline phosphatase expression. This study has shown that OP-1 stimulates osteogenesis in MC3T3-E1 osteoprogenitor cells that have been inhibited by PMMA particles. Local administration of OP-1 to the site of osteolysis may be a potential adjunctive therapy to reverse the bone destruction due to wear particles.

Polymethylmethacrylate particles impair osteoprogenitor viability and expression of osteogenic transcription factors Runx2, osterix, and Dlx5.

Polymethylmethacrylate (PMMA) particles have been shown to inhibit the differentiation of osteoprogenitor cells, but the mechanism of this inhibitory effect has not been investigated. We hypothesize that the inhibitory effects of PMMA particles involve impairment of osteoprogenitor viability and direct inhibition of transcription factors that regulate osteogenesis. We challenged MC3T3-E1 osteoprogenitors with PMMA particles and examined the effects of these materials on osteoprogenitor viability and expression of transcription factors Runx2, osterix, Dlx5, and Msx2. MC3T3-E1 cells treated with PMMA particles over a 72-h period showed a significant reduction in cell viability and proliferation as indicated by a dose- and time-dependent increase in supernatant levels of lactate dehydrogenase, an intracellular enzyme released from dead cells, a dose-dependent decrease in cell number and BrdU uptake, and the presence of large numbers of positively labeled Annexin V-stained cells. The absence of apoptotic cells on TUNEL assay indicated that cell death occurred by necrosis, not apoptosis. MC3T3-E1 cells challenged with PMMA particles during the first 6 days of differentiation in osteogenic medium showed a significant dose-dependent decrease in the RNA expression of Runx2, osterix, and Dlx5 on all days of measurement, while the RNA expression of Msx2, an antagonist of Dlx5-induced osteogenesis, remained relatively unaffected. These results indicate that PMMA particles impair osteoprogenitor viability and inhibit the expression of transcription factors that promote osteoprogenitor differentiation.

Ultrahigh molecular weight polyethylene wear debris inhibits osteoprogenitor proliferation and differentiation in vitro.

Polyethylene wear debris induces progressive osteolysis by increasing bone degradation and suppressing bone formation. Polyethylene particles inhibit the function of mature osteoblasts, but whether polyethylene particles also interfere with the proliferation and differentiation of osteoprogenitor cells is unknown. In this study, we investigated the effects of ultrahigh molecular weight polyethylene (UHMWPE) particles on the osteogenic activity of primary murine bone marrow osteoprogenitors and MC3T3-E1 preosteoblastic cells in vitro. Submicron-sized UHMWPE particles generated from wear simulator tests were isolated from serum-containing solution by density gradient centrifugation. The particles were coated onto the surface of culture wells at concentrations of 0.038, 0.075, 0.150, 0.300, and 0.600% v/v in a layer of type I collagen matrix. Primary murine bone marrow cells and MC3T3-E1 preosteoblasts were seeded onto the particle-collagen matrix and induced to differentiate in osteogenic medium for 20 days. Exposure of both cell populations to UHMWPE particles resulted in a dose-dependent decrease in mineralization, proliferation, alkaline phosphatase activity, and osteocalcin production when compared with control cells cultured on collagen matrix without particles. Complete suppression of osteogenesis was observed at particle concentrations > or =0.150% v/v. This study demonstrated that UHMWPE particles inhibit the osteogenic activity of osteoprogenitor cells, which may result in reduced periprosthetic bone regeneration and repair.

Polymethylmethacrylate particles inhibit osteoblastic differentiation of MC3T3-E1 osteoprogenitor cells.

Orthopedic wear debris has been implicated as a significant inhibitory factor of osteoblast differentiation. Polymethylmethacrylate (PMMA) particles have been previously shown to inhibit the differentiation of osteoprogenitors in heterogeneous murine marrow stromal cell cultures, but the effect of PMMA particles on pure osteoprogenitor populations remains unknown. In this study, we challenged murine MC3T3-E1 osteoprogenitor cells with PMMA particles during their initial differentiation in osteogenic medium. MC3T3-E1 cultures challenged with PMMA particles showed a gradual dose-dependent decrease in mineralization, cell number, and alkaline phosphatase activity at low particle doses (0.038-0.150% v/v) and complete reduction of these outcome parameters at high particle doses (> or =0.300% v/v). MC3T3-E1 cultures challenged with a high particle dose (0.300% v/v) showed no rise in these outcome parameters over time, whereas cultures challenged with a low particle dose (0.075% v/v) showed a normal or reduced rate of increase compared to controls. Osteocalcin production was not significantly affected by particles at all doses tested. MC3T3-E1 cells grown in conditioned medium from particle-treated MC3T3-E1 cultures showed a significant reduction in mineralization only. These results indicate that direct exposure of MC3T3-E1 osteoprogenitors to PMMA particles results in suppression of osteogenic proliferation and differentiation.

Kinetics of polymethylmethacrylate particle-induced inhibition of osteoprogenitor differentiation and proliferation.

Periprosthetic bone loss induced by implant wear debris may be a combined effect of osteolysis and reduced bone formation resulting from particle-induced suppression of osteoprogenitor differentiation. This study investigated the time-dependent effects of polymethylmethacrylate (PMMA) particles on the osteogenic capability of bone marrow osteoprogenitor cells during the early phase of differentiation. Murine bone marrow cells were challenged with PMMA particles (0.30% v/v) on the first day of growth in osteogenic medium. Particles were removed from culture after 1, 3, and 5 days, respectively, after which cell growth in osteogenic medium was continued until the 15th day. Bone marrow osteoprogenitor cells exposed to particles during the first 5 days of differentiation showed complete, irreversible inhibition of proliferation, alkaline phosphatase expression, and mineralization. Osteoprogenitors exposed to particles for more than 5 days showed the same degree of inhibition, while those exposed to particles for less than 5 days showed a diminished inhibitory response. Conditioned medium from particle-treated cells did not suppress osteogenic development, demonstrating that suppression of osteogenesis was not due to secreted inhibitory factors. This study has shown that the early phase of osteoprogenitor differentiation is a crucial time period during which exposure to PMMA particles causes irreversible inhibition of osteogenesis.

Effects of orthopaedic wear particles on osteoprogenitor cells.

Wear particles from total joint arthroplasties are constantly being generated throughout the lifetime of an implant. Since mesenchymal stem cells and osteoprogenitors from the bone marrow are the precursors of osteoblasts, the reaction of these cells to orthopaedic wear particles is critical to both initial osseointegration of implants and ongoing regeneration of the periprosthetic bed. Particles less than 5 microm can undergo phagocytosis by mature osteoblasts, with potential adverse effects on cellular viability, proliferation and function. The specific effects are dependent on particle composition and dose. Metal and polymer particles in non-toxic doses stimulate pro-inflammatory factor release more than ceramic particles of a similar size. The released factors inhibit markers of bone formation and are capable of stimulating osteoclast-mediated bone resorption. Mesenchymal stem cells and osteoprogenitors are also profoundly affected by wear particles. Titanium and polymethylmethacrylate particles inhibit bone cell viability and proliferation, and downregulate markers of bone formation in a dose- and time-dependent manner. Future studies should delineate the molecular mechanisms by which particles adversely affect mesenchymal stems cells and the bone cell lineage and provide strategies to modulate these effects.

Polymethylmethacrylate particles inhibit osteoblastic differentiation of bone marrow osteoprogenitor cells.

Aseptic implant loosening of total joint replacements often results from particle-mediated bone loss, which may be a combined effect of osteolysis and suppressed bone formation. Bone regeneration in the prosthetic bed depends on the activity of osteoblasts and their differentiation from osteoprogenitors in the bone marrow. This study investigated the effects of polymethylmethacrylate (PMMA) particles on the ability of bone marrow osteoprogenitors to differentiate into osteoblasts in vitro. Murine bone marrow cells challenged with PMMA particles on the first day of differentiation in osteogenic medium showed a dose-dependent decrease in osteoprogenitor proliferation, alkaline phosphatase expression, and mineralization. Undifferentiated bone marrow cells pretreated with PMMA particles in nonosteogenic medium for 5 days also showed a dose-dependent loss in osteogenic potential, which was sustained throughout subsequent growth in particle-free, osteogenic medium. Bone marrow cells challenged with PMMA particles after the fifth day of differentiation in osteogenic medium showed significant reductions in cellular proliferation, but not alkaline phosphatase expression and mineralization, indicating that bone marrow cells were most sensitive to particle treatment during the first 5 days of differentiation. This study demonstrated that PMMA particles inhibit osteoblastic differentiation of bone marrow osteoprogenitor cells, which may contribute to periprosthetic bone loss and implant failure.

Conformational states of cytochrome P450cam revealed by trapping of synthetic molecular wires.

Members of the ubiquitous cytochrome P450 family catalyze a vast range of biologically significant reactions in mammals, plants, fungi, and bacteria. Some P450s display a remarkable promiscuity in substrate recognition, while others are very specific with respect to substrate binding or regio and stereo-selective catalysis. Recent results have suggested that conformational flexibility in the substrate access channel of many P450s may play an important role in controlling these effects. Here, we report the X-ray crystal structures at 1.8A and 1.5A of cytochrome P450cam complexed with two synthetic molecular wires, D-4-Ad and D-8-Ad, consisting of a dansyl fluorophore linked to an adamantyl substrate analog via an alpha,omega-diaminoalkane chain of varying length. Both wires bind with the adamantyl moiety in similar positions at the camphor-binding site. However, each wire induces a distinct conformational response in the protein that differs from the camphor-bound structure. The changes involve significant movements of the F, G, and I helices, allowing the substrate access channel to adapt to the variable length of the probe. Wire-induced opening of the substrate channel also alters the I helix bulge and Thr252 at the active site with binding of water that has been proposed to assist in peroxy bond cleavage. The structures suggest that the coupling of substrate-induced conformational changes to active-site residues may be different in P450cam and recently described mammalian P450 structures. The wire-induced changes may be representative of the conformational intermediates that must exist transiently during substrate entry and product egress, providing a view of how substrates enter the deeply buried active site. They also support observed examples of conformational plasticity that are believed be responsible for the promiscuity of drug metabolizing P450s. Observation of such large changes in P450cam suggests that substrate channel plasticity is a general property inherent to all P450 structures.

Fluorescent probes for cytochrome p450 structural characterization and inhibitor screening.

We have synthesized two luminescent probes (D-4-Ad and D-8-Ad) that target cytochrome P450cam. D-4-Ad luminescence is quenched by Förster energy transfer upon binding (Kd = 0.83 muM) but is restored when the probe is displaced from the active site by camphor. In contrast, D-8-Ad (Kd approximately 0.02 muM) is not displaced from the enzyme, even in the presence of a large excess of camphor. The 2.2 A resolution crystal structure of the D-8-Ad:P450cam complex reveals extensive hydrophobic contacts between the probe and the enzyme, which result from the conformational flexibility of the B', F, and G helices. Probes with properties similar to those of D-4-Ad potentially could be useful for screening P450 inhibitors.