Accumulation of potentially toxic elements in road deposited sediments in residential and light industrial neighborhoods of Singapore.

Road deposited sediments (RDS) are a valuable environmental medium for characterizing contaminant levels in urban areas; and their associated potentially toxic elements (PTEs) can directly impact both human and aquatic health. In this study, RDS were collected from 15 co-located industrial and residential roads throughout Singapore to determine the effect of land use on contaminant levels. A second pilot study was designed to quantify the efficiency of road sweeping in removing different RDS grain size fractions from industrial and residential roads. The fine fraction (<63 µm) of all RDSs was analyzed for over 40 elements. Eleven elements that reflect geogenic and anthropogenic sources were examined in detail (Al, Co, Cr, Cu, Fe, Ni, Pb, Sb, Sc, Si, and Zn). Industrial RDS had statistically higher concentrations of Co, Cr, Fe, and Ni than residential RDS. Potentially toxic elements Cu, Pb, Sb, and Zn were enriched >10-fold at all locations compared to upper continental crust values. Concentrations of Cu, Pb and Zn exceeded aquatic sediment probable effect concentration levels, suggesting they could generate a toxic response in bottom-dwelling aquatic organisms. Traffic was equally heavy at both industrial and residential sites, but large trucks and machinery comprised a larger proportion of the traffic in the industrial areas. Traffic was not significantly correlated with the PTE (i.e., Cu, Pb, Sb and Zn) concentrations. Plausible anthropogenic contaminant sources include vehicles (e.g., brake and tire wear, vehicle emissions) and several industrial activities including metal works, oil processing, and waste incineration. Street sweeping was effective in removal of large organic debris and inorganic RDS, but it was ineffective in removing the geochemically important fraction, i.e., <125 µm.

Mars Extant Life: What's Next? Conference Report.

On November 5-8, 2019, the "Mars Extant Life: What's Next?" conference was convened in Carlsbad, New Mexico. The conference gathered a community of actively publishing experts in disciplines related to habitability and astrobiology. Primary conclusions are as follows: A significant subset of conference attendees concluded that there is a realistic possibility that Mars hosts indigenous microbial life. A powerful theme that permeated the conference is that the key to the search for martian extant life lies in identifying and exploring refugia ("oases"), where conditions are either permanently or episodically significantly more hospitable than average. Based on our existing knowledge of Mars, conference participants highlighted four potential martian refugium (not listed in priority order): Caves, Deep Subsurface, Ices, and Salts. The conference group did not attempt to reach a consensus prioritization of these candidate environments, but instead felt that a defensible prioritization would require a future competitive process. Within the context of these candidate environments, we identified a variety of geological search strategies that could narrow the search space. Additionally, we summarized a number of measurement techniques that could be used to detect evidence of extant life (if present). Again, it was not within the scope of the conference to prioritize these measurement techniques-that is best left for the competitive process. We specifically note that the number and sensitivity of detection methods that could be implemented if samples were returned to Earth greatly exceed the methodologies that could be used at Mars. Finally, important lessons to guide extant life search processes can be derived both from experiments carried out in terrestrial laboratories and analog field sites and from theoretical modeling.

Exhaustive matching of the entire protein sequence database.

The entire protein sequence database has been exhaustively matched. Definitive mutation matrices and models for scoring gaps were obtained from the matching and used to organize the sequence database as sets of evolutionarily connected components. The methods developed are general and can be used to manage sequence data generated by major genome sequencing projects. The alignments made possible by the exhaustive matching are the starting point for successful de novo prediction of the folded structures of proteins, for reconstructing sequences of ancient proteins and metabolisms in ancient organisms, and for obtaining new perspectives in structural biochemistry.

Total synthesis and cloning of a gene coding for the ribonuclease S protein.

A gene for ribonuclease S protein, has been chemically synthesized and cloned. The gene is designed to have 25 specific restriction endonuclease sites spaced at short intervals, permitting its structure to be rapidly modified. This flexibility facilitates tests of hypotheses relating the primary structure of the enzyme to its physical and catalytic behavior.

The return of pancreatic ribonucleases.

A decade after losing favor as an 'uninteresting' digestive enzyme, pancreatic ribonuclease has been found to be homologous to a series of extracellular proteins that may influence tumor cell growth, neurological development and biological differentiation. One surprising outcome of these discoveries has been the confirmation of the hypothesis that extracellular 'communicator RNA' is a messenger important in cell growth and differentiation. The only question is: why wasn't this recognized earlier?

Hydroxymethanesulfonate from Volcanic Sulfur Dioxide: A "Mineral" Reservoir for Formaldehyde and Other Simple Carbohydrates in Prebiotic Chemistry.

While formaldehyde (HCHO) was likely generated in Earth's prebiotic atmosphere by ultraviolet light, electrical discharge, and/or volcano-created lightning, HCHO could not have accumulated in substantial amounts in prebiotic environments, including those needed for prebiotic processes that generate nucleosidic carbohydrates. HCHO at high concentrations in alkaline solutions self-reacts in the Cannizzaro reaction to give methanol and formate, neither having prebiotic value. Here, we explore the possibility that volcanic sulfur dioxide (SO2) might have generated a reservoir for Hadean HCHO by a reversible reaction with HCHO to give hydroxymethanesulfonate (HMS). We show that salts of HMS are stable as solids at 90°C and do not react with themselves in solution, even at high (>8 M) concentrations. This makes them effective stores of HCHO, since the reverse reaction slowly delivers HCHO back into an environment where it can participate in prebiotically useful reactions. Specifically, we show that in alkaline borate solutions, HCHO derived from HMS allows formation of borate-stabilized carbohydrates as effectively as free HCHO, without losing material to Cannizzaro products. Further, we show that SO2 can perform similar roles for glycolaldehyde and glyceraldehyde, two intrinsically unstable carbohydrates that are needed by various models as precursors for RNA building blocks. Zircons from the Hadean show that the Hadean mantle likely provided volcanic SO2 at rates at least as great as the rates of atmospheric HCHO generation, making the formation of Hadean HMS essentially unavoidable. Thus, hydroxymethylsulfonate adducts of formaldehyde, glycolaldehyde, and glyceraldehyde, including the less soluble barium, strontium, and calcium salts, are likely candidates for prebiotically useful organic minerals on early Earth.

Evolution, language and analogy in functional genomics.

Almost a century ago, Wittgenstein pointed out that theory in science is intricately connected to language. This connection is not a frequent topic in the genomics literature. But a case can be made that functional genomics is today hindered by the paradoxes that Wittgenstein identified. If this is true, until these paradoxes are recognized and addressed, functional genomics will continue to be limited in its ability to extrapolate information from genomic sequences.

Regression of oral leukoplakia with alpha-tocopherol: a community clinical oncology program chemoprevention study.

BACKGROUND: Oral leukoplakia is an important model for developing chemoprevention approaches for lesions in the upper aerodigestive tract. These lesions most often result from exposure to carcinogens such as tobacco and alcohol and may precede development of invasive cancer. The potent antioxidant alpha-tocopherol (vitamin E) has prevented the development of cancers of the oral cavities in animal models. PURPOSE: The objectives of this study were to evaluate the toxicity and efficacy of alpha-tocopherol in patients with oral leukoplakia and to assess the feasibility of performing chemoprevention trials through the network of the Community Clinical Oncology Program (CCOP). METHODS: A single-arm phase II study using the nontoxic agent alpha-tocopherol to treat oral premalignant leukoplakia was performed at seven institutions affiliated with the CCOP through The University of Texas M. D. Anderson Cancer Center. Patients with symptomatic leukoplakia or dysplasia were treated orally with alpha-tocopherol (400 IU) twice daily for 24 weeks. Follow-up was performed at 6, 12, and 24 weeks after the start of treatment to assess toxicity and response, and serum alpha-tocopherol levels were determined at baseline and at 6 and 24 weeks. RESULTS: Of the 43 patients who have completed 24 weeks of treatment, 20 (46%) had clinical responses and nine (21%) had histologic responses. Mean serum alpha-tocopherol levels were 16.1 micrograms/mL at baseline and increased to 34.29 micrograms/mL after 24 weeks of treatment. Patient-recorded drug calendars, as well as serum drug levels, indicated excellent patient compliance; an average of 95% of the prescribed pills were taken. Treatment was extremely well tolerated; no grade 3 or 4 toxic effects were reported. CONCLUSIONS: Administration of alpha-tocopherol resulted in both clinical and histologic responses in premalignant leukoplakia lesions. The study also demonstrated that chemoprevention trials can be performed through the CCOP. The major problems were that a high percentage of patients were not assessable for response, some patients withdrew because expenses were not reimbursable, and there was limited participation within the CCOP network. These problems may reflect difficulties inherent in the implementation of multi-institutional chemoprevention trials. IMPLICATIONS: The efficacy of alpha-tocopherol alone and in combination with other chemopreventive agents for carcinogenesis in the upper aerodigestive tract should be explored in future trials.

Randomized phase III intergroup trial of isotretinoin to prevent second primary tumors in stage I non-small-cell lung cancer.

BACKGROUND: Promising data have suggested that retinoid chemoprevention may help to control second primary tumors (SPTs), recurrence, and mortality of stage I non-small-cell lung cancer (NSCLC) patients. METHODS: We carried out a National Cancer Institute (NCI) Intergroup phase III trial (NCI #I91-0001) with 1166 patients with pathologic stage I NSCLC (6 weeks to 3 years from definitive resection and no prior radiotherapy or chemotherapy). Patients were randomly assigned to receive a placebo or the retinoid isotretinoin (30 mg/day) for 3 years in a double-blind fashion. Patients were stratified at randomization by tumor stage, histology, and smoking status. The primary endpoint (time to SPT) and the secondary endpoints (times to recurrence and death) were analyzed by log-rank test and the Cox proportional hazards model. All statistical tests were two-sided. RESULTS: After a median follow-up of 3.5 years, there were no statistically significant differences between the placebo and isotretinoin arms with respect to the time to SPTs, recurrences, or mortality. The unadjusted hazard ratio (HR) of isotretinoin versus placebo was 1.08 (95% confidence interval [CI] = 0.78 to 1.49) for SPTs, 0.99 (95% CI = 0.76 to 1.29) for recurrence, and 1.07 (95% CI = 0.84 to 1.35) for mortality. Multivariate analyses showed that the rate of SPTs was not affected by any stratification factor. Rate of recurrence was affected by tumor stage (HR for T(2) versus T(1) = 1.77 [95% CI = 1.35 to 2.31]) and a treatment-by-smoking interaction (HR for treatment-by-current-versus-never-smoking status = 3.11 [95% CI = 1.00 to 9.71]). Mortality was affected by tumor stage (HR for T(2) versus T(1) = 1.39 [95% CI = 1.10 to 1.77]), histology (HR for squamous versus nonsquamous = 1.31 [95% CI = 1.03 to 1.68]), and a treatment-by-smoking interaction (HR for treatment-by-current-versus-never-smoking = 4.39 [95% CI = 1.11 to 17.29]). Mucocutaneous toxicity (P<.001) and noncompliance (40% versus 25% at 3 years) were higher in the isotretinoin arm than in the placebo arm. CONCLUSIONS: Isotretinoin treatment did not improve the overall rates of SPTs, recurrences, or mortality in stage I NSCLC. Secondary multivariate and subset analyses suggested that isotretinoin was harmful in current smokers and beneficial in never smokers.

Process-based reactive transport modeling of a permeable reactive barrier for the treatment of mine drainage.

Reactive transport modeling of a permeable reactive barrier for the treatment of mine drainage was used to integrate a comprehensive data set including pore water chemistry and solid phase data from several sampling events over a >3-year time period. The simulations consider the reduction of sulfate by the organic carbon-based treatment material and the removal of sulfate and iron by precipitation of reduced mineral phases including iron monosulfides and siderite. Additional parameters constraining the model include dissolved H2S, alkalinity and pH, as well as a suite of solid phase S-fractions identified by extractions. Influences of spatial heterogeneity necessitated the use of a 2-dimensional modeling approach. Simulating observed seasonal fluctuations and long-term changes in barrier reactivity required the use of temperature dependent rate coefficients and a multimodal Monod-type rate expression accounting for the variable reactivity of different organic carbon fractions. Simulated dissolved concentrations of SO4, Fe, H2S, alkalinity and pH, as well as solid phase accumulations of reduced sulfur phases generally compare well to observed trends over 23 months. Spatial variations, seasonal fluctuations and the time-dependent decline in reactivity were also captured. The modeling results generally confirm, and further strengthen, the existing conceptual model for the site. Overall sulfate reduction and S-accumulation rates are constrained with confidence within a factor of 1.5.

Chemoprevention strategies for lung and upper aerodigestive tract cancer.

The field cancerization hypothesis suggests that carcinogen exposure affects the entire epithelial lining of the lungs and upper aerodigestive tract. The concept that common exposures place the entire epithelium at risk for the development of invasive cancer is supported both by the occurrence of premalignant lesions such as leukoplakia and squamous metaplasia, and by the development of multiple primary tumors within the field. Chemoprevention trials in lung and upper aerodigestive tract cancer have included studies to reverse premalignant lesions and to prevent second primary tumors. Promising results have been reported in both settings using the retinoid 13-cis-retinoid acid. Several clinical trials are in progress which attempt both to reduce cancer incidence and to determine the mechanisms and biological markers of successful chemoprevention.

Retinoid chemoprevention studies in upper aerodigestive tract and lung carcinogenesis.

Chemoprevention is a clinical strategy to block or reverse carcinogenesis before the development of invasive cancer. Studies of chemoprevention in the lungs and upper aerodigestive tract have relied on the field carcinogenesis hypothesis, which predicts that diffuse epithelial injury will result from exposure of that epithelium to carcinogens. This hypothesis is supported by the frequent occurrence of multiple primary tumors within the exposed field. In addition, the understanding of carcinogenesis as a multistep process supports the use of interventions in damaged epithelium before the development of clinically invasive cancer. Current efforts are focused on applying to chemoprevention studies the increasing knowledge of the molecular events in carcinogenesis. In our program, clinical trials in lung and head and neck chemoprevention have focused on individuals with evidence of field carcinogenesis, i.e., a history of previous epithelial cancer or the presence of premalignant lesions. These trials include studies to develop clinically applicable intermediate markers of carcinogenesis and large Phase III trials to evaluate the efficacy of the retinoid isotretinoin in preventing second primary tumors following head and neck or lung cancers.

Uracil-ftorafur: an oral fluoropyrimidine active in colorectal cancer.

PURPOSE AND DESIGN: This review describes the early clinical development of uracil-ftorafur (UFT), an oral fluoropyrimidine, designed in 1978 by adding uracil to ftorafur. The review focuses on the treatment of colorectal cancer and summarizes the Japanese experience and the phase I and II trials performed in the United States and Europe. RESULTS: Clinical trials of UFT published in the Western world have included 581 patients with colorectal cancer. UFT has been administered in these trials as a single agent or biomodulated by leucovorin (LV). UFT was administered daily in split doses for periods that ranged from 14 to 28 days. The activity of oral UFT in large-bowel cancer when administered with oral LV (approximately 50 mg/dose) has resulted in objective response rates of approximately 40%. Response rates of approximately 25% (range, 17% to 39%) were reported when UFT was administered as a single agent or with lower doses of LV. The highest dose-intensities of UFT are achieved with 28-day schedules of administration. The maximum-tolerated dose (MTD) of UFT with this schedule, when administered concomitantly with oral LV 150 mg daily, is 300 mg/m2 daily. The dose-limiting toxicity (DLT) of UFT has generally been diarrhea. Other commonly described toxicities include nausea and vomiting, fatigue, and stomatitis. Myelosuppression occurs infrequently. Typically, hand-foot syndrome and neurologic toxicity are lacking. CONCLUSION: UFT is a fluoropyrimidine active in colorectal cancer. The oral route of administration and improved safety profile represent important advantages over both conventional and infusional fluorouracil (5-FU) regimens.

Cancer chemoprevention.

PURPOSE: To review the most important recent advances in clinical trials and biologic studies within the growing field of chemoprevention. METHODS: The most critical methods issue concerns the definitive end point of phase III trials, which is now cancer incidence. This end point usually needs thousands of subjects monitored for 5 to 10 or more years to determine efficacy. Biologic markers of potential intermediate end points are under intensive study and may one day replace cancer incidence. Validated intermediate end point biomarkers could greatly reduce phase III trial populations, durations, and costs. RESULTS: Randomized clinical trials over the last 5 years have produced significant activity in reversing oral, skin, colon, and cervical premalignancy; in preventing primary skin and stomach cancer; and in preventing second primary tumors associated with head and neck and lung cancer. These clinical advances have been paralleled at the basic science level by elegant molecular studies of premalignant carcinogenesis and of chemopreventive agents' mechanisms of action. One major laboratory advance is the discovery of nuclear retinoic acid receptors and strong evidence of their roles both in carcinogenic progression and in its response to retinoids. CONCLUSION: Chemoprevention has matured greatly in recent years with the significant reversal or suppression of premalignancy by chemopreventive agents in several sites. The future of chemoprevention will be determined largely by several ongoing phase III trials, including trials of retinoids, beta-carotene, and alpha-tocopherol in the aerodigestive tract, of tamoxifen and fenretinide in the breast, and of finasteride in the prostate.

Phase I study of preoperative oral uracil and tegafur plus leucovorin and radiation therapy in rectal cancer.

PURPOSE: Preoperative combined-modality therapy for rectal cancer may allow for sphincter preservation, while decreasing recurrence rates and improving the overall prognosis. Oral chemotherapy with uracil and tegafur (UFT) plus leucovorin (LV) may reduce costs and complications associated with protracted infusions of fluorouracil. Our goal was to evaluate the safety of UFT plus LV combined with preoperative radiation and determine the maximum-tolerated dose (MTD) and dose-limiting toxicity (DLT) of UFT plus LV in this setting. PATIENTS AND METHODS: Patients with tumor-node-metastasis stage II or III rectal cancer received escalating doses of UFT (starting at 250mg/m(2)/d, with 50-mg/m(2)/d increments between consecutive cohorts) and fixed doses of LV (90 mg/d). The UFT and LV combination was given 5 days per week concurrently with a 5-week course of preoperative radiation totaling 45 Gy (1.8 Gy/fraction). Surgery was performed 4 to 6 weeks after radiation and was followed by four 35-day cycles of fixed doses of UFT and LV (28 days of therapy each cycle). RESULTS: Fifteen patients were treated, and 13 received the full preoperative chemotherapy. All planned radiation was delivered successfully. The MTD of UFT with radiation was 350 mg/m(2)/d with 90 mg/d of LV. Diarrhea was the DLT. Sphincter-preserving surgery was performed in 12 of 14 patients. One patient had progressive disease before surgery. Pathologic evaluation of 14 resected specimens showed a complete response in three cases. CONCLUSION: Preoperative chemoradiation with oral UFT plus LV is feasible and well tolerated and should be further investigated.

Phase II study of docetaxel for advanced or metastatic platinum-refractory non-small-cell lung cancer.

PURPOSE: We conducted a phase II study to determine the response to and toxicity of docetaxel (Taxotere; Rhône-Poulenc Rorer Pharmaceuticals, Inc, Collegeville, PA) in patients with advanced non-small-cell lung cancer refractory to prior platinum-containing chemotherapy (PCC) regimens. PATIENTS AND METHODS: Forty-four patients with stage IIIb or IV platinum-refractory non-small-cell lung cancer were treated with 100 mg/m2 of docetaxel intravenously over 1 hour every 3 weeks. The responses of 42 of 44 patients were assessable. Most patients had a Zubrod performance status of 1; the predominant histologic type was adenocarcinoma (61%), and 91% of patients had stage IV disease. RESULTS: Nine of 42 assessable patients (21%) achieved a partial response to treatment. The median response duration (from response to progression) was 17 weeks, and the projected median survival duration of all patients is 42 weeks (51 weeks for adenocarcinoma and 22 weeks for nonadenocarcinoma). Grade 3/4 neutropenia occurred in 85% of patients and was associated with fever that required intravenous antibiotics in 16% of patients (3% of cycles). Other acute side effects included easily treated hypersensitivity reactions and dermatitis. Cumulative side effects included fluid retention and neuropathy. CONCLUSION: Docetaxel administered at 100 mg/m2 intravenously every 3 weeks has notable activity against platinum-refractory non-small-cell lung cancer, with a 21% major response rate. Primary side effects were neutropenia, hypersensitivity, and fluid retention.

Phase II study of docetaxel for recurrent or metastatic non-small-cell lung cancer.

PURPOSE: We conducted a phase II study to determine the response and toxicity of docetaxel (Taxotere; Rhône-Poulenc Rorer Pharmaceuticals, Inc, Collegeville, PA) in chemotherapy-naive patients with advanced non-small-cell lung cancer. PATIENTS AND METHODS: We treated 41 chemotherapy-naive patients who had stage IIIb or IV non-small-cell lung cancer with 100 mg/m2 of docetaxel intravenously over 1 hour every 3 weeks. Responses were assessed after every one to two treatment courses. Responses of 39 of 41 patients were assessable. The patient's median age was 63 years; 90% of patients had a Zubrod performance status of 0 or 1. The predominant histology was adenocarcinoma (54%), and 90% of patients had stage IV disease. RESULTS: Thirteen patients (33%) achieved a partial response to treatment, and the median response duration was 14 weeks. Grade 3 or 4 neutropenia occurred in 97% of patient; this was usually of brief duration and was associated with serious infection in 17% of patients. Other acute toxic effects included easily treated hypersensitivity reactions (36% of patients) and dermatitis (74%). We also observed fluid retention (with peripheral edema or pleural effusion or both) in 54% of patients. This was a cumulative side effect that generally occurred late in treatment. CONCLUSION: Docetaxel administered at 100 mg/m2 intravenously every 3 weeks has significant activity against non-small-cell lung cancer, with a 33% major response rate. Primary toxicities were neutropenia, hypersensitivity, and fluid retention.

Randomized placebo-controlled trial of isotretinoin in chemoprevention of bronchial squamous metaplasia.

PURPOSE: Retinoids have proven chemopreventive efficacy in both preclinical and clinical studies. This trial was designed to confirm the finding of an earlier uncontrolled trial that the synthetic retinoid etretinate had major activity in reversing squamous metaplasia found in the bronchial epithelium of chronic smokers. PATIENTS AND METHODS: We prospectively evaluated 152 smokers with bronchoscopy and obtained biopsies from six sites. Subjects with dysplasia and/or a metaplasia index of greater than 15% were randomly assigned to receive either 1 mg/kg isotretinoin or placebo daily for 6 months. Of 86 subjects randomized (41 isotretinoin, 45 placebo), 69 were reevaluated at the completion of treatment. RESULTS: In the group as a whole, the metaplasia index decreased over time from a mean +/- SE of 35.8% +/- 2.7% at baseline to 28.1% +/- 3.3% at the completion of treatment (P = .01) by repeated measures analysis of variance [ANOVA]); a reduction in the metaplasia index (> 8%) was noted in both isotretinoin and placebo groups (19 of 35 [54.3%] and 20 of 34 [58.8%], respectively). Complete reversal of squamous metaplasia was noted in nine subjects from each group. However, the magnitudes of the mean metaplasia index changes did not differ significantly in the two treatment groups. In both groups, smoking cessation resulted in significant declines in the extent of squamous metaplasia, whereas no significant change in metaplasia index was found among those who continued to smoke. CONCLUSION: Squamous metaplasia was frequently observed in bronchial biopsy samples from chronic smokers. From this study, we conclude that isotretinoin has no effect on squamous metaplasia, a potential intermediate end point of bronchial carcinogenesis. Although determining the exact role of isotretinoin in lung cancer prevention requires further study, the finding that there was a significant decrease in squamous metaplasia in the placebo group emphasizes the critical importance of a placebo-controlled study design in chemoprevention trials using intermediate end points.

Predicted secondary structure for the Src homology 3 domain.

A de novo secondary structure prediction has been prepared for Src homology domain 3, in advance of any crystallographic information concerning any member of this interesting protein family. The prediction can be compared with a crystal structure that will be published in Nature on October 29, 1992. The prediction is based on analysis of a multiple alignment of homologous proteins. The patterns of variation and conservation of amino acids across the alignment allow the determination of surface and internal positions, which then allow the assignment of secondary structure. The prediction is quite different both in method and, in this case, result from predictions based on propensities (e.g. Garnier-Osgurthorpe-Robson) of particular amino acids to appear in particular types of secondary structure.

Bona fide prediction of aspects of protein conformation. Assigning interior and surface residues from patterns of variation and conservation in homologous protein sequences.

Heuristics have been developed for analyzing patterns of conservation and variation within a set of aligned homologous protein sequences for the purpose of assigning amino acids whose side-chains lie on the surface and inside the folded structure of a protein. These were used in several recent bona fide predictions of the secondary structure of proteins from sequence data, made and published before crystallographic information became available. Heuristics based on concurrent hydrophilic variation identify positions that lie on the surface. Heuristics based on concurrent hydrophobic conservation and variation identify positions lying in the interior. These heuristics are described here in detail and their performance evaluated when applied to seven protein families with known three-dimensional structures. The performance of individual heuristics is shown to depend on the nature of the multiple alignment within the protein family, and a strategy is presented for obtaining surface and interior assignments useful for predicting secondary structure.