Nutrition education tools used in phenylketonuria: clinician, parent and patient perspectives from three international surveys.

BACKGROUND: Three international surveys were developed aiming to identify the current nutrition educational tools used in the management of phenylketonuria (PKU) and the perceived effectiveness of these tools by clinicians, parents and patients. METHODS: The first two surveys were distributed through the Metabolic Dietitians ListServe (pno-metabl@listserv.cc.emory.edu), and the third survey was distributed by international clinics and the National PKU Alliance website (www.npkua.org). A total of 888 responses (S1, n = 88; S2, n = 81; S3, n = 719) were collected from all three surveys. The surveys represent participants from 17 countries, in Europe; North America (USA and Canada); Mexico; Argentina; Turkey; Australia; and Africa (Tunisia). RESULTS: A consistent decline in 'parents as role models' as an educational tool was observed starting at age 10 years. Patients responded they feel their families are the most effective form of education, whereas handouts were selected as the least effective educational tool by patients. Parents responded they feel the most effective educational tool is one-on-one counselling. Patients and parents show a desirable trend in wanting to attend group clinic, even in centres where this type of educational tool is not offered. CONCLUSIONS: There was a discrepancy between clinicians and patient views regarding the perceived effectiveness of the nutrition education tools. Future research is needed surrounding the impact nutrition education may have on improved dietary compliance in patients with PKU.

Carpal tunnel syndrome and sleep, a systematic review and meta-analysis.

BACKGROUND: The most common symptom and reason patients seek treatment for carpal tunnel syndrome is lack of sleep. Our purpose was to determine how much sleep-related symptoms of carpal tunnel syndrome improve after carpal tunnel release using validated patient-reported outcome measures (PROMs) and objective sleep data as primary measures of interest. METHODS: A PRISMA-guided literature search was conducted using Ovid MEDLINE, PubMed, Cochrane, and ClinicalTrials.gov. Only interventional clinical trials that examined primary outcome measures of interest were included. Patient-reported outcome measures underwent meta-analysis to determine how much scores improved following carpal tunnel release. RESULTS: The Pittsburgh Sleep Quality Index improved significantly after carpal tunnel release, by 4.43 points and 6.02 points at 1-3 and 6-12 months postoperatively, respectively, and continued to improve up to 2 years. Improvement on the Insomnia Severity Index after carpal tunnel release was also significant, with improvement up to 1 year postoperatively, by 8.54 points and 9.05 points at 1-3 and 6-12 months, respectively. Insomnia Severity Index scores improved significantly after splinting as well. CONCLUSIONS: The present meta-analysis determined to what extent patients can expect their sleep to improve after operative and non-operative intervention, as measured by various patient-reported outcome measures that assess sleep. The Pittsburgh Sleep Quality Index and Insomnia Severity Index correlated very well between studies and across hundreds of patients with carpal tunnel syndrome. Data are lacking to define the minimal clinically important difference and assess whether patients achieve a minimal clinically important difference for sleep questionnaires; more information on this topic is needed. LEVEL OF EVIDENCE: III.

Dietary practices in pyridoxine non-responsive homocystinuria: a European survey.

BACKGROUND: Within Europe, the management of pyridoxine (B6) non-responsive homocystinuria (HCU) may vary but there is limited knowledge about treatment practice. AIM: A comparison of dietetic management practices of patients with B6 non-responsive HCU in European centres. METHODS: A cross-sectional audit by questionnaire was completed by 29 inherited metabolic disorder (IMD) centres: (14 UK, 5 Germany, 3 Netherlands, 2 Switzerland, 2 Portugal, 1 France, 1 Norway, 1 Belgium). RESULTS: 181 patients (73% >16 years of age) with HCU were identified. The majority (66%; n=119) were on dietary treatment (1-10 years, 90%; 11-16 years, 82%; and >16 years, 58%) with or without betaine and 34% (n=62) were on betaine alone. The median natural protein intake (g/day) on diet only was, by age: 1-10 years, 12 g; 11-16 years, 11 g; and >16 years, 45 g. With diet and betaine, median natural protein intake (g/day) by age was: 1-10 years, 13 g; 11-16 years, 20 g; and >16 years, 38 g. Fifty-two percent (n=15) of centres allocated natural protein by calculating methionine rather than a protein exchange system. A methionine-free l-amino acid supplement was prescribed for 86% of diet treated patients. Fifty-two percent of centres recommended cystine supplements for low plasma concentrations. Target treatment concentrations for homocystine/homocysteine (free/total) and frequency of biochemical monitoring varied. CONCLUSION: In B6 non-responsive HCU the prescription of dietary restriction by IMD centres declined with age, potentially associated with poor adherence in older patients. Inconsistencies in biochemical monitoring and treatment indicate the need for international consensus guidelines.

Dietary management of urea cycle disorders: European practice.

BACKGROUND: There is no published data comparing dietary management of urea cycle disorders (UCD) in different countries. METHODS: Cross-sectional data from 41 European Inherited Metabolic Disorder (IMD) centres (17 UK, 6 France, 5 Germany, 4 Belgium, 4 Portugal, 2 Netherlands, 1 Denmark, 1 Italy, 1 Sweden) was collected by questionnaire describing management of patients with UCD on prescribed protein restricted diets. RESULTS: Data for 464 patients: N-acetylglutamate synthase (NAGS) deficiency, n=10; carbamoyl phosphate synthetase (CPS1) deficiency, n=29; ornithine transcarbamoylase (OTC) deficiency, n=214; citrullinaemia, n=108; argininosuccinic aciduria (ASA), n=80; arginase deficiency, n=23 was reported. The majority of patients (70%; n=327) were aged 0-16y and 30% (n=137) >16y. Prescribed median protein intake/kg body weight decreased with age with little variation between disorders. The UK tended to give more total protein than other European countries particularly in infancy. Supplements of essential amino acids (EAA) were prescribed for 38% [n=174] of the patients overall, but were given more commonly in arginase deficiency (74%), CPS (48%) and citrullinaemia (46%). Patients in Germany (64%), Portugal (67%) and Sweden (100%) were the most frequent users of EAA. Only 18% [n=84] of patients were prescribed tube feeds, most commonly for CPS (41%); and 21% [n=97] were prescribed oral energy supplements. CONCLUSIONS: Dietary treatment for UCD varies significantly between different conditions, and between and within European IMD centres. Further studies examining the outcome of treatment compared with the type of dietary therapy and nutritional support received are required.

Assessing the agreement between the pneumatic and the flow-centrifuge method for estimating xylem safety in temperate diffuse-porous tree species.

The increasing frequency of global change-type droughts has created a need for fast, accurate and widely applicable techniques for estimating xylem embolism resistance to improve forecasts of future forest changes. We used data from 12 diffuse-porous temperate tree species covering a wide range of xylem safety to compare the pneumatic and flow-centrifuge method, two rapid methods used for constructing xylem vulnerability curves. We evaluated the agreement between parameters estimated with both methods and the sensitivity of pneumatic measurements to the duration of air discharge (AD) measurements. There was close agreement between xylem water potentials at 50% air discharged (PAD), estimated with the Pneumatron, and 50% loss of hydraulic conductivity (PLC), estimated with the flow-centrifuge method (mean signed deviation: 0.12 MPa, Pearson correlation: 0.96 after 15 s of gas extraction). However, the relationship between the estimated slopes was more variable, resulting in lower agreement in the xylem water potential at 12% and 88% PAD/PLC. The agreement between the two methods was not affected by species-specific vessel length distributions. All pneumatic parameters were sensitive to AD time. Overall agreement was highest at relatively short AD times, with an optimum at 16 s. Our results highlight the value of the Pneumatron as an easy and reliable tool to estimate 50% embolism thresholds for a wide range of diffuse-porous temperate angiosperms. Further, our study provides a set of useful metrics for methodological comparisons of vulnerability curves in terms of systematic and random deviations, as well as overall agreement.

Powder plasma spheroidization treatment and the characterization of microstructure and mechanical properties of SS 316L powder and L-PBF builds.

A plasma spheroidization treatment was applied to stock stainless steel 316L powder for additive manufacturing. The normal and treated powders were compared both in the powder state as well as in the resulting laser powder bed fusion (L-PBF) builds. The plasma spheroidization process slightly increased treated powder aspect ratio and sphericity and shifted the size distribution to larger diameters relative to the normal powder. The normal powder was austenitic in nature whereas the plasma spheroidization process introduced a small fraction (∼3.5 vol %) of ferrite in the treated powder. Ferrite in the powder was not retained in the printed samples and was not shown to negatively affect the build quality. Porosity areal fraction was generally smaller in the treated powder builds. The normal powder builds had a 6% higher yield strength than treated, however the scatter was significantly larger in the 45° and horizontal orientations compared to the treated powder builds.

Root water uptake depth determines the hydraulic vulnerability of temperate European tree species during the extreme 2018 drought.

We took advantage of the European 2018 drought and assessed the mechanisms causing differences in drought vulnerability among mature individuals of nine co-occurring tree species at the Swiss Canopy Crane II site in Switzerland. Throughout the drought we monitored leaf water status and determined native embolism formation in the canopy of the trees as indicators of drought vulnerability. We also determined hydraulic vulnerability thresholds (Ψ12 -, Ψ50 - and Ψ88 -values), corresponding hydraulic safety margins (HSMs) and carbohydrate reserves for all species as well as total average leaf area per tree, and used stable isotopes to assess differences in root water uptake depth among the nine species as variables predicting differences in drought vulnerability among species. Marked differences in drought vulnerability were observed among the nine tree species. Six species maintained their water potentials above hydraulic thresholds, while three species, Fagus sylvatica, Carpinus betulus and Picea abies, were pushed beyond their hydraulic thresholds and showed loss of hydraulic conductivity in their canopies at the end of the drought. Embolism resistance thresholds and associated HSMs did not explain why the co-existing species differed in their drought vulnerability, neither did their degree of isohydry, nor their regulation of carbohydrate reserves. Instead, differences in structural-morphological traits, in particular root water uptake depth, were associated with the risk of reaching hydraulic vulnerability thresholds and embolism formation among the nine species. Our study shows that structural-morphological traits, such as root water uptake depth, determine how quickly different species approach hydraulic vulnerability thresholds during a drought event and can thus explain species differences in drought vulnerability among mature field-grown trees.

Leaf trait modification in European beech trees in response to climatic and edaphic drought.

Leaf morphological and physiological traits control the carbon and water relations of mature trees and are determinants of drought tolerance, but it is not well understood how they are modified in response to water deficits. We analysed five sun-canopy leaf traits (mean leaf size (LS), specific leaf area (SLA), Huber value (HV), water potential at turgor loss point (Ψtlp ) and foliar carbon isotope signature (δ13 C)) in European beech (Fagus sylvatica L.) across three precipitation gradients sampled in moist (2010), dry (2019) and very dry (2018) summers, and tested their response to short-term water deficits (climatic water balance (CWB) preceding sample collection) and long-term water availability (mean annual precipitation (MAP), plant-available soil water capacity (AWC) and neighbourhood competition). Across the 34 sites, LS varied seven-fold (3.9-27.0 cm2 ), SLA four-fold (77.1-306.9 cm²·g-1 ) and HV six-fold (1.0-6.65 cm2 ·m-2 ). In the 2018 dataset, LS showed a negative and HV a positive relationship to MAP, which contradicts relations found in multi-species samples. Average Ψtlp ranged from -1.90 to -2.62 MPa and decreased across the sites with decreasing CWB in the month prior to measurement, as well as with decreasing MAP and AWC in 2019. Studied leaf traits varied considerably between years, suggesting that mast fruiting and the severe 2018 drought caused the formation of smaller leaves. We conclude that sun-canopy leaf traits of European beech exhibit considerable plasticity in response to climatic and edaphic aridity, and that osmotic adjustment may be an important element in the drought response strategy of this anisohydric tree species.

Estimating the likelihood of sustained virological response in chronic hepatitis C therapy.

The likelihood of a sustained virological response (SVR) is the most important factor for physicians and patients in the decision to initiate and continue therapy for chronic hepatitis C (CHC) infection. This study identified predictive factors for SVR with peginterferon plus ribavirin (RBV) in patients with CHC treated under 'real-life' conditions. The study cohort consisted of patients from a large, retrospective German multicentre, observational study who had been treated with peginterferon alfa-2a plus RBV or peginterferon alfa-2b plus RBV between the years 2000 and 2007. To ensure comparability regarding peginterferon therapies, patients were analysed in pairs matched by several baseline variables. Univariate and multivariate logistic regression analyses were used to determine the effect of nonmatched baseline variables and treatment modality on SVR. Among 2378 patients (1189 matched pairs), SVR rates were 57.9% overall, 46.5% in HCV genotype 1/4-infected patients and 77.3% in genotype 2/3-infected patients. In multivariate logistic regression analysis, positive predictors of SVR were HCV genotype 2 infection, HCV genotype 3 infection, low baseline viral load and treatment with peginterferon alfa-2a. Negative predictors of SVR were higher age (≥40 years), elevated baseline gamma-glutamyl transpeptidase (GGT) and low baseline platelet count (<150,000/µL). Among patients treated with peginterferon plus RBV in routine clinical practice, genotype, baseline viral load, age, GGT level and platelet levels all predict the likelihood of treatment success. In patients matched by baseline characteristics, treatment with peginterferon alfa-2a may be a positive predictor of SVR when compared to peginterferon alfa-2b.

Cardiovascular regulation in mice lacking alpha2-adrenergic receptor subtypes b and c.

alpha2-Adrenergic receptors (alpha2ARs) are essential components of the neural circuitry regulating cardiovascular function. The role of specific alpha2AR subtypes (alpha2a, alpha2b, and alpha2c) was characterized with hemodynamic measurements obtained from strains of genetically engineered mice deficient in either alpha2b or alpha2c receptors. Stimulation of alpha2b receptors in vascular smooth muscle produced hypertension and counteracted the clinically beneficial hypotensive effect of stimulating alpha2a receptors in the central nervous system. There were no hemodynamic effects produced by disruption of the alpha2c subtype. These results provide evidence for the clinical efficacy of more subtype-selective alpha2AR drugs.

Unifying Theories of Psychedelic Drug Effects.

How do psychedelic drugs produce their characteristic range of acute effects in perception, emotion, cognition, and sense of self? How do these effects relate to the clinical efficacy of psychedelic-assisted therapies? Efforts to understand psychedelic phenomena date back more than a century in Western science. In this article I review theories of psychedelic drug effects and highlight key concepts which have endured over the last 125 years of psychedelic science. First, I describe the subjective phenomenology of acute psychedelic effects using the best available data. Next, I review late 19th-century and early 20th-century theories-model psychoses theory, filtration theory, and psychoanalytic theory-and highlight their shared features. I then briefly review recent findings on the neuropharmacology and neurophysiology of psychedelic drugs in humans. Finally, I describe recent theories of psychedelic drug effects which leverage 21st-century cognitive neuroscience frameworks-entropic brain theory, integrated information theory, and predictive processing-and point out key shared features that link back to earlier theories. I identify an abstract principle which cuts across many theories past and present: psychedelic drugs perturb universal brain processes that normally serve to constrain neural systems central to perception, emotion, cognition, and sense of self. I conclude that making an explicit effort to investigate the principles and mechanisms of psychedelic drug effects is a uniquely powerful way to iteratively develop and test unifying theories of brain function.

Real-world use, effectiveness and safety of anti-viral treatment in chronic hepatitis C genotype 3 infection.

BACKGROUND: Treatment of chronic hepatitis C genotype 3 (GT3) is more challenging compared with other genotypes. Since 2014, several new treatment regimens have been approved but sometimes based on limited data. AIM: To validate the use, effectiveness and safety of anti-viral treatment in chronic hepatitis C genotype 3 infection under real-word conditions. METHODS: The German Hepatitis C-Registry is a large national non-interventional real-world study for patients with chronic hepatitis C. A total of 1322 GT3 patients were enrolled (211 untreated and 1111 treated patients). RESULTS: Between February 2014 and September 2015, five different treatment strategies have been used (PegIFN+RBV, PegIFN+RBV+SOF, SOF+RBV, DCV+SOF±RBV, LDV/SOF±RBV). Treatment uptake and use of treatment concepts changed markedly and rapidly during the study influenced by new approvals, guideline recommendations, and label updates. PegIFN-based therapies constantly declined while DCV-based therapies increased with one interruption after the approval of LDV/SOF, which was frequently used until new guidelines recommended not using this combination for GT3. Per-protocol SVR ranged from 80.9% in the PegIFN+RBV group to 96.1% in PegIFN+RBV+SOF treated patients. Treatment-experienced patients with cirrhosis showed a suboptimal SVR of 68% for SOF+RBV but a high SVR of 90-95% for DCV+SOF±RBV. The safety analysis showed more adverse events and a stronger decline of haemoglobin for RBV containing regimens. CONCLUSIONS: Real-world data can validate the effectiveness and safety for treatment regimens that had previously been approved with limited data, in particular for specific subgroups of patients. The present study demonstrates how rapid new scientific data, new treatment guidelines, new drug approvals and label changes are implemented into routine clinical practice today.

Brief report: Failure of an electronic medical record tool to improve pain assessment documentation.

OBJECTIVE: To comply with pain management standards, Bellevue Hospital in New York City implemented a mandatory computerized pain assessment screen (PAS) in its electronic medical record (EMR) system for every outpatient encounter. We assessed provider acceptance of the instrument and examined whether the intervention led to increased documentation of pain-related diagnoses or inquiries. DESIGN: Cross-sectional survey; a pre- and posthistorically controlled observational study. SUBJECTS AND MEASUREMENTS: The utility of the computerized tool to medicine housestaff and attendings was assessed by an anonymous survey. We conducted an electronic chart review comparing all adult primary care patient encounters over a 2-day period 6 months prior to implementation of the PAS and on 2 days 6 months after its implementation. RESULTS: Forty-seven percent of survey respondents felt that the computerized assessment tool was "somewhat difficult" or "very difficult" to use. The majority of respondents (79%) felt the tool did not change their pain assessment practice. Of 265 preintervention patients and 364 postintervention patients seen in the clinic, 42% and 37% had pain-related diagnoses, respectively (P=.29). Pain inquiry by the physician was noted for 49% of preintervention patients and 44% of the postintervention patients (P=.26). In 55% of postintervention encounters, there was discordance between the pain documentation using the PAS tool and the free text section of the medical note. CONCLUSION: A mandatory computerized pain assessment tool did not lead to an increase in pain-related diagnoses and may have hindered the documentation of pain assessment because of the perceived burden of using the application.

Use of a validated quality of life questionnaire to assess sexual function following laparoscopic radical prostatectomy.

Wide variations exist in the methods for evaluating potency following radical prostatectomy. We describe our technique of laparoscopic radical prostatectomy (LRP), present our methods for assessing the return of potency following LRP, and discuss the relevant literature. Sexual function was assessed pre- and postoperatively using the Expanded Prostate Cancer Index Composite questionnaire (EPIC). Sexual function subscale scores (SFSS) were reported as a percentage of preoperative baseline sexual function. The EPIC was also used for single-question assessment of successful intercourse. We also reviewed the literature on prospective health-related quality of life results following LRP and open radical retropubic prostatectomy. Only patients reporting preoperative intercourse were analyzed. Of these, 72 and 35% undergoing bilateral and unilateral nerve sparing (NS) reported postoperative intercourse at 12 months (P=0.01). Mean SFSS at 12 months was 61 and 57% of baseline after bilateral and unilateral NS, respectively (P=0.71). Following NS procedures, 74% of patients < or =58 years of age and 41% of patients >58 years of age reported successful intercourse at 12 months (P=0.015). Mean SFSS was 64 and 52% of baseline function (P=0.249) at 12 months for patients < or =58 and >58 years of age, respectively. In patients <58 years of age who underwent bilateral NS surgery, 82% reported intercourse at 12 months. In conclusion, return of sexual function following NS LRP in our experience is comparable to reports from centers of excellence in open prostatectomy. Standardizing data collection using validated quality of life instruments can provide both surgeon and patient with a realistic forecast of relative return to normal sexual function following prostatectomy.

The Predictive Processing Paradigm Has Roots in Kant.

Predictive processing (PP) is a paradigm in computational and cognitive neuroscience that has recently attracted significant attention across domains, including psychology, robotics, artificial intelligence and philosophy. It is often regarded as a fresh and possibly revolutionary paradigm shift, yet a handful of authors have remarked that aspects of PP seem reminiscent of the work of 18th century philosopher Immanuel Kant. To date there have not been any substantive discussions of how exactly PP links back to Kant. In this article, I argue that several core aspects of PP were anticipated by Kant (1996/1787) in his works on perception and cognition. Themes from Kant active in PP include: (1) the emphasis on "top-down" generation of percepts; (2) the role of "hyperpriors"; (3) the general function of "generative models"; (4) the process of "analysis-by-synthesis"; and (5) the crucial role of imagination in perception. In addition to these, I also point out that PP echoes Kant's general project in that it aims to explain how minds track causal structure in the world using only sensory data, and that it uses a reverse-engineer or "top-down" method of analysis. I then locate a possible source of Kant's influence on PP by tracing the paradigm back to Hermann von Helmholtz, who saw himself as providing a scientific implementation of Kant's conclusions. I conclude by arguing that PP should not be regarded as a new paradigm, but is more appropriately understood as the latest incarnation of an approach to perception and cognition initiated by Kant and refined by Helmholtz.

Histone phosphorylation in phorbol ester stimulated and beta-adrenergically stimulated mouse epidermis in vivo and characterization of an epidermal protein phosphorylation system.

Under certain physiological conditions a change in the phosphorylation of histones in mouse epidermis in vivo was observed. Thus a single local application of the tumor-promoting mitogen 12-O-tetradecanoylphorbol-13-acetate caused a long-lasting increase of histone H1 phosphorylation which paralleled stimulated cell proliferation. Injection of the antimitotic beta-adrenergic agonist isoproterenol led to a temporary decrease in the rate of phosphorylation of H1, H2A and H2B immediately after cyclic AMP accumulation. A complete protein phosphorylation system could be demonstrated in mouse epidermis homogenates. The following enzyme activities were partially purified and characterized: a cyclic AMP-dependent histone kinase; a 'casein kinase' and an 'unspecific' protein kinase; a histone-specific protein phosphatase; and two 'unspecific' phosphoprotein phosphatases. In addition, a stimulatory effect of cyclic GMP on histone phosphorylation was observed. The enzymes were found to be predominantly localized in the 105000 X g supernatant, but a small proportion of protein kinase and phosphatase activity could be regularly demonstrated in cell nuclei.

Primary structure of the mouse beta 1-adrenergic receptor gene.

The mouse beta 1-adrenergic receptor was isolated from a genomic library and cloned into pBluescript SK-. Characterization of the clone revealed an open reading frame which encodes a predicted protein of 466 amino acids. The mouse beta 1 receptor is 92.7% identical to the human sequence, 98.5% identical to the rat sequence, and contains a consensus site for N-linked glycosylation at Asn-15 and a cAMP-dependent protein kinase phosphorylation site at Ser-301.

Protein-based medical adhesives.

There are many naturally occurring adhesive proteins which have potential for application in medicine and dentistry. Cloning and expression of their genes enables the modes of action of these proteins to be better understood and increases their availability for practical applications. This article concentrates on the adhesive protein from the blue mussel Mytilus edulis but also describes medical adhesives based on fibrin isolated from human blood.

Dispase, a neutral protease from Bacillus polymyxa, is a powerful fibronectinase and type IV collagenase.

Dispase, a neutral protease isolated from culture filtrates of Bacillus polymyxa, has proven to be a rapid, effective, but gentle agent for separating intact epidermis from the dermis and intact epithelial sheets in culture from the substratum. In both cases it effects separation by cleaving the basement membrane zone region while preserving the viability of the epithelial cells. Because it is not known what or where in the basement membrane zone Dispase cleaves, we set up studies to define its substrate specificity. Using purified basement membrane components and sodium dodecyl sulfate-polyacrylamide gel electrophoresis we show that Dispase cleaves fibronectin and type IV collagen, but not laminin, type V collagen, serum albumin, or transferrin. The action of Dispase on collagen appears to be selective for type IV collagen in that several stable degradation products are formed, whereas the enzyme degrades type I collagen only minimally. In newborn human skin, as seen by electron microscopy, Dispase removes the lamina densa, rich in type IV collagen, but preserves the anchoring fibrils (structures known to contain type VII collagen) and the epidermal cells. Because its action is so selective, it suggests that Dispase can serve as a powerful tool for dissecting epithelial-mesenchymal interactions.

Epithelial growth by rat vibrissae follicles in vitro requires mesenchymal contact via native extracellular matrix.

An in vitro assay utilizing the rat vibrissa anagen follicle as a model for studying the epithelial-mesenchymal interactions (EMI) in hair growth is described. Through selective disruption of the epithelial-mesenchymal interface, we investigate whether the specialized extracellular matrix (ECM) of the dermal papilla and basement membrane zone (BMZ) serves a crucial function in hair follicle EMI. Epithelial bulbs incubated intact within their follicular sheaths incorporate thymidine primarily into cells of the hair matrix and outer root sheath, as shown by autoradiography. However, after removal of its mesenchymal associations (dermal papilla and extrabulbar connective tissue), the epithelial bulb showed no incorporation. Neither externally added collagen (type I or IV) nor the basement membrane components in Matrigel could substitute for the growth supporting influence of native surrounding stroma. Mechanical separation of the bulb from the dermal papilla in the basement membrane zone inhibited thymidine incorporation by the epithelium even though mesenchyme was still in close proximity. Enzymatic digestion of the dermal papilla ECM and the basal lamina by Dispase, a fibronectinase and type IV collagenase, also inhibited bulb growth without evidence of cytotoxicity. These experiments suggest that direct epithelial to mesenchymal contact is required for the support of follicular epithelial growth in vitro and that specific ECM components, possibly fibronectin and/or type IV collagen, rather than diffusable factors alone, play a crucial role in the mechanism of hair follicle EMI. The in vitro system described here provides an alternative to developmental EMI models and may serve as a valuable tool for studying EMI in the adult mammalian organism.