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Introduction: Bayesian adaptive designs for clinical trials have gained popularity in the recent years due to the flexibility and efficiency that they offer. We consider the scenario where the outcome of interest comprises events with relatively low risk of occurrence and different case definitions resulting in varying control group risk assumptions. This is a scenario that occurs frequently for infectious diseases in global health research. Methods: We propose a Bayesian adaptive design that incorporates different case definitions of the outcome of interest that vary in stringency. A set of stopping rules are proposed where superiority and futility may be concluded with respect to different outcome definitions and therefore maintain a realistic probability of stopping in trials with low event rates. Through a simulation study, a variety of stopping rules and design configurations are compared. Results: The simulation results are provided in an interactive web application that allows the user to explore and compare the design operating characteristics for a variety of assumptions and design parameters with respect to different outcome definitions. The results for select simulation scenarios are provided in the article. Discussion: Bayesian adaptive designs offer the potential for maximizing the information learned from the data collected through clinical trials. The proposed design enables monitoring and utilizing multiple composite outcomes based on rare events to optimize the trial design operating characteristics.

To generate robust, demographically-adjusted regression-based norms for the Montreal Cognitive Assessment (MoCA) using a large sample of diverse older US adults. Baseline MoCA scores were examined for participants in the Systolic Blood Pressure Intervention Trial (SPRINT). A robust, cognitively-normal sample was drawn from individuals not subsequently adjudicated with cognitive impairment through 4 years of follow-up. Multivariable Beta-Binomial regression was used to model the association of demographic variables with MoCA performance and to create demographically-stratified normative tables. Participants' (N = 5,338) mean age was 66.9 ± 8.8 years, with 35.7% female, 63.1% White, 27.4% Black, 9.5% Hispanic, and 44.5% with a college or graduate education. A large proportion scored below published MoCA cutoffs: 61.4% scored below 26 and 29.2% scored below 23. A disproportionate number falling below these cutoffs were Black, Hispanic, did not graduate from college, or were ≥75 years of age. Multivariable modeling identified education, race/ethnicity, age, and sex as significant predictors of MoCA scores (p

In the SPRINT (Systolic Blood Pressure Intervention Trial), the number of strokes did not differ significantly by treatment group. However, stroke subtypes have heterogeneous causes that could respond differently to intensive blood pressure control. SPRINT participants (N=9361) were randomized to target systolic blood pressures of P =0.09). The interaction between baseline cardiovascular risk factor status and treatment arm on stroke risk did not reach significance ( P =0.05). Similar numbers of stroke subtypes occurred in the intensive BP control and standard control arms of SPRINT.

Background: In the SPRINT (Systolic Blood Pressure Intervention Trial), intensive BP treatment reduced acute decompensated heart failure (ADHF) events. Here, we report the effect on HF with preserved ejection fraction (HFpEF) and HF with reduced EF (HFrEF) and their subsequent outcomes. Methods: Incident ADHF was defined as hospitalization or emergency department visit, confirmed, and formally adjudicated by a blinded events committee using standardized protocols. HFpEF was defined as EF ≥45%, and HFrEF was EF Results: Among the 133 participants with incident ADHF who had EF assessment, 69 (52%) had HFpEF and 64 (48%) had HFrEF ( P value: 0.73). During average 3.3 years follow-up in those who developed incident ADHF, rates of subsequent all-cause and HF hospital readmission and mortality were high, but there were no significant differences between those who developed HFpEF versus HFrEF. Randomization to the intensive arm had no effect on subsequent mortality or readmissions after the initial ADHF event, irrespective of EF subtype. During follow-up among participants who developed HFpEF, although relatively modest number of events limited statistical power, age was an independent predictor of all-cause mortality, and Black race independently predicted all-cause and HF hospital readmission. Conclusions: In SPRINT, intensive BP reduction decreased both acute decompensated HFpEF and HFrEF events. After initial incident ADHF, rates of subsequent hospital admission and mortality were high and were similar for those who developed HFpEF or HFrEF. Randomization to the intensive arm did not alter the risks for subsequent all-cause, or HF events in either HFpEF or HFrEF. Among those who developed HFpEF, age and Black race were independent predictors of clinical outcomes. Registration: URL: https://www.clinicaltrials.gov ; Unique identifier: NCT01206062.

Rationale: Cognitive impairment after critical illness is common in observational studies of older intensive care unit (ICU) survivors. The rate of screening for and diagnosis of cognitive impairment in ICU survivors in nonresearch settings is unknown. Objectives: To determine how often cognitive impairment was detected in older adults in the year after critical illness at an academic medical center as part of 1) the Medicare Annual Wellness Visit (AWV) and 2) routine clinical care. Methods: This study was a retrospective cohort study conducted at an urban academic medical center. The study included 696 patients aged 65 years and older admitted to the medical ICU between October 1, 2016, and October 1, 2018, and discharged alive. Patients were also required to have a health system–affiliated primary care provider. Patients were followed for 1 year. We defined cognitive impairment detected in the AWV as either an indicated diagnosis of cognitive impairment or dementia or patient, family, or provider indication of memory concerns during the AWV. We modeled the incidence of AWV completion and the detection of cognitive impairment using semiparametric additive models accounting for the competing risk of death. Results: Over 1 year of follow-up, the cumulative incidence of mortality was 23.0% (95% confidence interval [CI], 19.9–26.1%), with 24.7% (95% CI, 21.5–27.9%) completing the AWV. The cumulative incidence of cognitive impairment first detected through the AWV was 3.4% (95% CI, 1.8–5.0%) at 1 year, with a higher cumulative incidence for diagnoses of cognitive impairment or dementia first indicated via encounter diagnosis codes or the electronic health record problem list (5.9%; 95% CI, 3.9–7.9%). Conclusions: The results of our study suggest that the currently implemented AWV is unlikely to be an adequate mechanism for detecting cognitive impairment in a high-risk population such as those recovering from critical illness.

BACKGROUND: Frailty is associated with numerous post-operative adverse outcomes in older adults. Current pre-operative frailty screening tools require additional data collection or objective assessments, adding expense and limiting large-scale implementation. OBJECTIVE: To evaluate the association of an automated measure of frailty integrated within the Electronic Health Record (EHR) with post-operative outcomes for nonemergency surgeries. DESIGN: Retrospective cohort study. SETTING: Academic Medical Center. PARTICIPANTS: Patients 65 years or older that underwent nonemergency surgery with an inpatient stay 24 hours or more between October 8th, 2017 and June 1st, 2019. EXPOSURES: Frailty as measured by a 54-item electronic frailty index (eFI). OUTCOMES AND MEASUREMENTS: Inpatient length of stay, requirements for post-acute care, 30-day readmission, and 6-month all-cause mortality. RESULTS: Of 4,831 unique patients (2,281 females (47.3%); mean (SD) age, 73.2 (5.9) years), 4,143 (85.7%) had sufficient EHR data to calculate the eFI, with 15.1% categorized as frail (eFI > 0.21) and 50.9% pre-frail (0.10 < eFI ≤ 0.21). For all outcomes, there was a generally a gradation of risk with higher eFI scores. For example, adjusting for age, sex, race/ethnicity, and American Society of Anesthesiologists class, and accounting for variability by service line, patients identified as frail based on the eFI, compared to fit patients, had greater needs for post-acute care (odds ratio (OR) = 1.68; 95% confidence interval (CI) = 1.36–2.08), higher rates of 30-day readmission (hazard ratio (HR) = 2.46; 95%CI = 1.72–3.52) and higher all-cause mortality (HR = 2.86; 95%CI = 1.84–4.44) over 6 months’ follow-up. CONCLUSIONS: The eFI, an automated digital marker for frailty integrated within the EHR, can facilitate pre-operative frailty screening at scale.

Background Physical activity (PA) preserves mobility, but few practices screen older adults for mobility impairment or counsel on PA. Design "Promoting Active Aging" (PAA) was a mixed-methods randomized-controlled pilot, to test the feasibility and acceptability of a video-based PA counseling tool and implementation into practice of two mobility assessment tools. Setting Three primary care practices affiliated with Wake Forest Baptist Health. Participants Adults aged 65 years and older who presented for primary care follow-up and were willing and able to answer self-report questions and walk 4 meters (n = 59). Intervention Video-based PA counseling intervention versus control video, "Healthy Eating." Measurements Potential participants completed mobility assessments: self-report (Mobility Assessment Tool-short form (MAT-sf)) and performance based (4-meter walk test). We assessed PAA's implementation-feasibility, acceptability, and value-via interviews and surveys. Effectiveness was measured via participant attendance at a PA information session. Results Of 92 patients approached, 89 (96.7%) agreed to mobility assessment. Eighty-nine completed MAT-sf, and 97.8% (87/89) completed 4-meter walk test. Sixty-seven (75%) met eligibility criteria, and 59 (88%) consented to be randomized either to the PA counseling intervention (Video-PA) or to active control (Video-C). Most participants viewed the walk test positively (51/59; 86.4%). Staff reported that completion of patient surveys, MAT-sf, and videos required significant staff time and support (median = 26 minutes for all), resulting in low acceptability of MAT-sf and the videos. Attendance at a PA information session did not differ by randomization group (Video-PA = 11/29 (37.9%); Video-C = 12/30 (40%); 95% confidence interval for difference in proportion = -0.29 to 0.25). Conclusions Mobility assessment, particularly a 4-meter walk test, was feasible in primary care. Tablet-based assessment (MAT-sf) and video counseling tools, selected to reduce staff effort, instead required significant time to implement. Future work to promote PA should identify effective ways to facilitate adoption of PA in sedentary older adults that do not burden staff.

Advance care planning (ACP), especially among vulnerable older adults, remains underused in primary care. Additionally, many ACP initiatives fail to integrate directly into the electronic health record (EHR), resulting in infrequent and disorganized documentation.To determine whether a nurse navigator-led ACP pathway combined with a health care professional-facing EHR interface improves the occurrence of ACP discussions and their documentation within the EHR.This was a randomized effectiveness trial using the Zelen design, in which patients are randomized prior to informed consent, with only those randomized to the intervention subsequently approached to provide informed consent. Randomization began November 1, 2018, and follow-up concluded November 1, 2019. The study population included patients 65 years or older with multimorbidity combined with either cognitive or physical impairments, and/or frailty, assessed from 8 primary care practices in North Carolina.Participants were randomized to either a nurse navigator-led ACP pathway (n = 379) or usual care (n = 380).The primary outcome was documentation of a new ACP discussion within the EHR. Secondary outcomes included the usage of ACP billing codes, designation of a surrogate decision maker, and ACP legal form documentation. Exploratory outcomes included incident health care use.Among 759 randomized patients (mean age 77.7 years, 455 women [59.9%]), the nurse navigator-led ACP pathway resulted in a higher rate of ACP documentation (42.2% vs 3.7%, P .001) as compared with usual care. The ACP billing codes were used more frequently for patients randomized to the nurse navigator-led ACP pathway (25.3% vs 1.3%, P .001). Patients randomized to the nurse navigator-led ACP pathway more frequently designated a surrogate decision maker (64% vs 35%, P .001) and completed ACP legal forms (24.3% vs 10.0%, P .001). During follow-up, the incidence of emergency department visits and inpatient hospitalizations was similar between the randomized groups (hazard ratio, 1.17; 95% CI, 0.92-1.50).A nurse navigator-led ACP pathway integrated with a health care professional-facing EHR interface increased the frequency of ACP discussions and their documentation. Additional research will be required to evaluate whether increased EHR documentation leads to improvements in goal-concordant care.ClinicalTrials.gov Identifier: NCT03609658.

John B Christoforidis,1 Jillian Wang,2 Angela Jiang,2 James Willard,5 Cedric Pratt,2 Mahmoud Abdel-Rasoul,3 Sashwati Roy,4 Heather Powell51Department of Ophthalmology and Vision Science, College of Medicine, The University of Arizona, Tucson, AZ, USA; 2Department of Ophthalmology, College of Medicine, The Ohio State University, Columbus, OH, USA; 3Center for Biostatistics, The Ohio State University, Columbus, OH, USA; 4Center Surgery, The Ohio State University, Columbus, OH, USA; 5Department of Materials Science, College of Engineering, The Ohio State University, Columbus, OH, USAPurpose: To investigate the effect of intravitreal bevacizumab and ranibizumab on wound tension and by histopathology during cutaneous wound healing in a rabbit model and to compare this effect to placebo intravitreal saline controls 1 and 2 weeks following intravitreal injection.Methods: A total of 120 New Zealand white rabbits were randomly assigned to one of three treatment groups each consisting of 40 rabbits. Each group received intravitreal injections of bevacizumab, ranibizumab, or normal saline. Immediately afterwards, each rabbit underwent four 6 mm full-thickness dermatologic punch biopsies. Twenty rabbits from each agent group underwent wound harvesting on day 7 or day 14. The skin samples were stained for CD34 for vascular endothelial cells on day 7, and maximal wound tensile load was measured on days 7 and 14. Quantitative assessment of mean neovascularization (MNV) scores was obtained from 10 contiguous biopsy margin 400× fields of CD34-stained sections by two independent observers.Results: Wound tension reading means (N) with standard error and adjusted P-values on day 7 were: saline placebos, 7.46 ± 0.87; bevacizumab, 4.50 ± 0.88 (P = 0.041); and ranibizumab, 4.67 ± 0.84 (P = 0.025). On day 14 these were: saline placebos, 7.34 ± 0.55; bevacizumab, 6.05 ± 0.54 (P = 0.18); and ranibizumab 7.99 ± 0.54 (P = 0.40). MNV scores in CD34 stained sections were: saline controls, 18.31 ± 0.43; bevacizumab, 11.02 ± 0.45 (P < 0.0001); and ranibizumab, 13.55 ± 0.43 (P < 0.0001). The interobserver correlation coefficient was 0.928.Conclusion: At day 7, both anti–vascular endothelial growth factor (anti-VEGF) agents had significantly suppressed MNV scores and exerted a significant reduction of cutaneous wound tensile strength compared with saline controls. At day 14, neither agent produced a significant effect on tensile wound strength. Since angiogenesis is an integral component of the proliferative phase of wound healing, we encourage clinicians to be aware of their patients' recent surgical history during intravitreal anti-VEGF therapy and to consider refraining from their use during the perioperative period.Keywords: wound healing, tensile strength, bevacizumab, ranibizumab

The current standard of care for the prevention and treatment of scarring after burn injury is pressure garment therapy. Although this therapy has been used clinically for many years, controversy remains regarding its efficacy. The authors evaluated the efficacy of pressure garment therapy in a female red Duroc pig burn model in which wound depth could be tightly controlled.Full-thickness burn wounds were generated on female red Duroc pigs. At day 28 after burn, pressure garment therapy was applied to half the wounds (10 mmHg), with control wounds covered with garments that exerted no compression. Scar area, perfusion, hardness, and elasticity were quantified at days 0, 28, 42, 56, and 72 using computerized planimetry, laser Doppler, and torsional ballistometry. Scar morphology was assessed at days 28, 56, and 76 using histology, immunohistochemistry, and transmission electron microscopy.Pressure garment therapy significantly hindered scar contraction, with control scars contracting to 64.6 percent + 13.9 percent original area at day 72, whereas pressure garment therapy scars contracted to 82.7 percent + 17.9 percent original area. Pressure garments significantly reduced skin hardness and increased skin strength by 1.3 times. No difference in perfusion or blood vessel density was observed. The average collagen fiber diameter was greater in control burns than in pressure garment therapy.Pressure garment therapy was effective at reducing scar contraction and improving biomechanics compared with control scars. These results confirm the efficacy of pressure garments and highlight the need to further investigate the role of pressure magnitude and the time of therapy application to enhance efficacy for optimal biomechanics and patient mobility.

Tissue engineering scaffolds are commonly formed using proteins extracted from animal tissues, such as bovine hide. Risks associated with the use of these materials include hypersensitivity and pathogenic contamination. Human-derived proteins lower the risk of hypersensitivity, but possess the risk of disease transmission. Methods engineering recombinant human proteins using plant material provide an alternate source of these materials without the risk of disease transmission or concerns regarding variability. To investigate the utility of plant-derived human collagen (PDHC) in the development of engineered skin (ES), PDHC and bovine hide collagen were formed into tissue engineering scaffolds using electrospinning or freeze-drying. Both raw materials were easily formed into two common scaffold types, electrospun nonwoven scaffolds and lyophilized sponges, with similar architectures. The processing time, however, was significantly lower with PDHC. PDHC scaffolds supported primary human cell attachment and proliferation at an equivalent or higher level than the bovine material. Interleukin-1 beta production was significantly lower when activated THP-1 macrophages where exposed to PDHC electrospun scaffolds compared to bovine collagen. Both materials promoted proper maturation and differentiation of ES. These data suggest that PDHC may provide a novel source of raw material for tissue engineering with low risk of allergic response or disease transmission.

Introduction: Ferlins are a family of proteins that play a variety of roles associated with plasma membrane dynamics in eukaryotes, including plasma membrane repair, regulation of membrane expression of receptors, and endocytosis. Dysferlin was recently reported to regulate platelet endothelial cellular adhesion molecule-1 (PECAM-1) in endothelial cells (1). We have shown that ablation of myoferlin (MYOF) in breast tumor cells attenuates invasion in vitro (in review, J Cell Sci). Moreover, loss of MYOF was found to be associated with reduced phosphorylation of a number of receptor tyrosine kinases (in press, PNAS), and others have reported on the role of MYOF in tyrosine kinase receptors expression at the plasma membrane (2). These results prompted us to hypothesize that MYOF may be involved in cell-matrix adhesion. Methods & Results: We used lentiviral-mediated RNA interference (RNAi) gene silencing to generate stable MYOF-deficient MDA-MB-231 cells and confirmed MYOF reduction with immunoblotting and qRT-PCR. RNAi control cells were generated in tandem with a non-human gene targeting construct. Relative adhesion and spreading of the wild-type, control, and MYOF-deficient epithelial cells were evaluated using a real-time electrical impedance measurement system (xCELLigence, Roche Applied Science). Results show a significant increase in the adhesion and spreading of the MYOF-deficient cells in comparision to wild-type and control cells. A trypsin assay developed using the same electrical impedance system demonstrated greater adhesion strength in the MYOF-deficient cells. This was measured by the duration of time necessary to reduce the pre-trypsin-treatment impedance by one-half. These results were further validated using a conventional parallel plate device, in which MYOF-deficient cells were found to be more adherent than controls when subjected to a range of shear stresses (1-10 dynes/cm2). PCR array screening of extracellular matrix related genes (SABiosciences) in MYOF-deficient and RNAi control MDA-MB-231 cells indicated that intercellular adhesion molecule 1 (ICAM-1/CD54) and vitronectin mRNA levels are increased following MYOF-diminution, and may potentially contribute to the mechanism behind the altered adhesion in MYOF-deficient cells. Conclusion: Myoferlin is important for the regulation of breast cancer epithelial cell adhesion. Since dysregulation of adhesion is often associated with cancer invasion and metastasis, myoferlin may play a role in these processes in breast carcinoma. (1) Sharma A, Yu C, Leung C, Trane A, Lau M, Utokaparch S, et al. A new role for the muscle repair protein dysferlin in endothelial cell adhesion and angiogenesis. Arterioscler Thromb Vasc Biol 2010 Nov;30 (11):2196-204. (2) Yu C, Sharma A, Trane A, Utokaparch S, Leung C, Bernatchez P. Myoferlin gene silencing decreases Tie-2 expression in vitro and angiogenesis in vivo. Vascul Pharmacol 2011 May 6. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5180. doi:1538-7445.AM2012-5180

Personalized cancer therapies drive the need for devices that rapidly and accurately segregate cancer cells from solid tumors. One potential sorting strategy is to segregate populations of cells based on their relative strength of adhesion. To investigate the effect of surface hydrophilicity and cell phenotype on adhesion, primary human breast skin fibroblasts and keratinocytes and MCF-7 breast cancer cells were seeded onto air and CF(4) plasma-treated nanofibers followed by exposure to three shear stresses (200, 275 and 350 dynes per cm(2)) 1 hour after inoculation. No difference in strength of adhesion was measured in either fibroblasts or keratinocytes on either plasma treated-surface: all exhibited60% of the initial cell count after a 5 minute exposure to 350 dynes per cm(2) of shear stress. In contrast, a significant difference between relative strength of adhesion on air versus CF(4) plasma-treated surfaces was observed for MCF-7 cells: 26% and 6.6% of cells remained on the air and CF(4) plasma-treated surfaces, respectively. The ability to sort this cancer cell line from two non-cancerous primary human cells was evaluated by inoculating a mixture of all three cell types simultaneously onto CF(4) treated nanofibers followed by 1 hour of culture and exposure to 350 dynes per cm(2) shear stress. The majority of MCF-7 cells were removed (0.7% remained) while a majority of fibroblasts and keratinocytes remained adhered (74 and 57%). Post-sorted MCF-7 viability and morphology remained unchanged, preserving the possibility of post-separation and analysis. These data suggest that the plasma treatment of electrospun scaffolds provides a tool useful in sorting cancer cells from a mixed cell population based on adhesion strength.

Intact cultured tomato cells eluted with dilute salt solutions yielded two soluble precursors (P1 and P2) to the bound extensin network. HF-deglycosylation of P1 and P2 (with methanol as scavenger) followed by tryptic degradation and HPLC on Hamilton PRP-1, gave unique tryptic peptide maps. The P1 map consisted predominantly of the deca- and hexadecapeptides, H5 and H20: (P1-H5) Ser-Hyp-Hyp-Hyp-Hyp-Thr-Hyp-Val-Tyr-Lys, and (H20) Ser-Hyp-Hyp-Hyp-Hyp-Val-Lys-Pro-Tyr-His-Pro-Thr-Hyp-Val-Tyr-Lys; the P2 map consisted almost entirely of the di- and octapeptides, H3 and H4: (P2-H3) Tyr-Lys; (H4) Ser-Hyp-Hyp-Hyp-Hyp-Val-Tyr-Lys, and small amounts of a closely related decapeptide containing intramolecularly-linked isodityrosine: (H11) Ser-Hyp-Hyp-Hyp-Hyp-Val- 1 2 IDT-Lys- 1 2 IDT-Lys. Both P1 and P2 are therefore highly periodic structures: P1 consists to a considerable extent of repeated H5 and H20 peptide blocks, while P2 may consist entirely of a single repeating decapeptide Ser-Hyp-Hyp-Hyp-Hyp-Val-Tyr-Lys-Tyr-Lys with occasional isoleucine for valine substitutions, and varying only in the extent of intramolecular IDT formation, which could stiffen the molecular rod. In P1, single tyrosine residues occur within the hexapeptide Val-Lys-Pro-Tyr-His-Pro region ofH20. This nonglycosylated region is sterically unhindered. Assuming a polyproline II conformation, all the basic residues of H20 lie in the same plane (available for pectic binding), while the hexapeptide tyrosine residue lies out of that plane and is therefore a prime candidate for the postulated inter molecular isodityrosine crosslink. Our sequences also reveal fundamental tetrapeptide and tripeptide periodicities of tomato extensin precursors: the contiguous decamers and hexadecamers consist of hydroxyproline tetrapeptides separated by a relatively few tripeptide sequences, (notably Tyr-Lys-Ser, Val-Tyr-Lys, Thr-Hyp-Val, Val-Lys-Pro, and Tyr-His-Pro). Furthermore, the presence of the pentapeptide Ser-Hyp-Hyp-Hyp-Hyp in all the repeat peptides shows that extensin, like many synthetic block copolymers, consists of relatively rigid domains (glycosylated Ser-Hyp-Hyp-Hyp-Hyp) separated by intervening (non-glycosylated) flexible spacers. Thus extensin is well-designed for its suggested role in mechanically coupling the cellulosic load-bearing polymers of the primary cell wall through formation of a network of defined porosity.

Rapidly growing tomato (Lycopersicon esculentum) cell suspension cultures contain transiently high levels of cell surface, salt-elutable, monomeric precursors to the covalently cross-linked extensin network of the primary cell wall. Thus, we purified a highly soluble monomeric extensin substrate from rapidly growing cells, and devised a soluble in vitro cross-linking assay based on Superose-6 fast protein liquid chromatography separation, which resolved extensin monomers from the newly formed oligomers within 25 minutes. Salt elution of slowly growing (early stationary phase) cells yielded little or no extensin monomers but did give a highly active enzymic preparation that specifically cross-linked extensin monomers in the presence of hydrogen peroxide, judging from: (a) a decrease in the extensin monomer peak on fast protein liquid chromatography gel filtration, (b) appearance of oligomeric peaks, and (c) direct electron microscopical observation of the cross-linked oligomers. The cross-linking reaction had a broad pH optimum between 5.5 and 6.5. An approach to substrate saturation of the enzyme required extensin monomer concentrations of 20 to 40 milligrams per milliliter. Preincubation with catalase completely inhibited the cross-linking reaction, which was highly dependent on hydrogen peroxide and optimal at 15 to 50 micromolar. We therefore identified the cross-linking activity as extensin peroxidase.