2H-enrichment of cellulose and n-alkanes in heterotrophic plants.

Hydrogen (H) isotopes of plant organic compounds are rarely employed in ecological studies. If so, these values are interpreted as being indicative of the plant source and/or leaf water. Recent observations suggest, however, that variations in hydrogen isotope fractionation that occur during the biosynthesis of plant compounds (2H-εbio) imprint valuable metabolic information into the hydrogen isotope composition (δ2H values) of plant organic compounds. Here we show a consistent 2H-enrichment of compounds in heterotrophically growing plants across a series of autotrophic/heterotrophic plant pairs. We suggest that this is due to a higher recycling of compounds in the Calvin and tricarboxylic acid cycles in heterotrophic plants that is associated with a more complete exchange of C-bound H with the surrounding 2H-enriched foliar water. Interestingly, we found that 2H-enrichment in heterotrophic plants was larger for carbohydrates than for lipids, with an average 2H-enrichment of 76 ± 9‰ in α-cellulose and 23 ± 23‰ in n-alkanes. We propose that this systematically larger 2H-enrichment for carbohydrates than for lipids is either due to different level of 2H-fractionation associated with heterotrophically produced NADPH, or to the potential uptake of lipids by heterotrophic plants. With the work we present here, we contribute to a better mechanistic understanding of what the biochemical principles are that couple the carbohydrate dynamics of plants to their δ2H values and hope to foster as such the application of H isotopes in plant sciences.

Thyroglobulin fluctuations in patients with iodine-refractory differentiated thyroid carcinoma on lenvatinib treatment - initial experience.

Tyrosine kinase inhibitors (TKI) have shown clinical effectiveness in iodine-refractory differentiated thyroid cancer (DTC). The corresponding role of serum thyroglobulin (Tg) in iodine-refractory DTC has not been investigated yet. 9 patients (3 female, 61 ± 8y) with progressive iodine-refractory DTC starting on lenvatinib were considered. Tumor restaging was performed every 2-3 months including contrast-enhanced computed tomography (CT, RECIST 1.1). Serum Tg was measured and compared to imaging findings. After treatment initiation, serum Tg levels dropped in all patients with a median reduction of 86.2%. During long-term follow-up (median, 25.2 months), fluctuations in Tg could be observed in 8/9 subjects. According to RECIST, 6/9 subjects achieved a partial response or stable disease with the remaining 3/9 experiencing progressive disease (2/3 with Tg levels rising above baseline). All of the patients with disease progression presented with a preceding continuous rise in serum Tg, whereas tumor marker oscillations in the subjects with controlled disease were only intermittent. Initiation of lenvatinib in iodine-refractory DTC patients is associated with a significant reduction in serum Tg levels as a marker of treatment response. In the course of treatment, transient Tg oscillations are a frequent phenomenon that may not necessarily reflect morphologic tumor progression.

Diel variations in carbon isotopic composition and concentration of organic acids and their impact on plant dark respiration in different species.

Leaf respiration in the dark and its C isotopic composition (δ(13) CR ) contain information about internal metabolic processes and respiratory substrates. δ(13) CR is known to be less negative compared to potential respiratory substrates, in particular shortly after darkening during light enhanced dark respiration (LEDR). This phenomenon might be driven by respiration of accumulated (13) C-enriched organic acids, however, studies simultaneously measuring δ(13) CR during LEDR and potential respiratory substrates are rare. We determined δ(13) CR and respiration rates (R) during LEDR, as well as δ(13) C and concentrations of potential respiratory substrates using compound-specific isotope analyses. The measurements were conducted throughout the diel cycle in several plant species under different environmental conditions. δ(13) CR and R patterns during LEDR were strongly species-specific and showed an initial peak, which was followed by a progressive decrease in both values. The species-specific differences in δ(13) CR and R during LEDR may be partially explained by the isotopic composition of organic acids (e.g., oxalate, isocitrate, quinate, shikimate, malate), which were (13) C-enriched compared to other respiratory substrates (e.g., sugars and amino acids). However, the diel variations in both δ(13) C and concentrations of the organic acids were generally low. Thus, additional factors such as the heterogeneous isotope distribution in organic acids and the relative contribution of the organic acids to respiration are required to explain the strong (13) C enrichment in leaf dark-respired CO2 .

Prognostic Value of Serum Tumor Markers in Medullary Thyroid Cancer Patients Undergoing Vandetanib Treatment.

Tyrosine kinase inhibitors (TKIs) such as vandetanib have shown clinical effectiveness in advanced medullary thyroid cancer (MTC). During TKI treatment, fluctuations in the tumor markers carcinoembryonic antigen (CEA) and calcitonin (CTN) are frequently observed. Their role for treatment monitoring and the decision-making process has not been fully elucidated yet.Twenty-one patients (male, 16, female, 5; mean age, 49 ±â€Š13 years) with progressive MTC receiving vandetanib (300 mg orally per day) were considered. Tumor restaging was performed every 3 months including contrast-enhanced computed tomography (CT). Response was assessed according to recent criteria (Response Evaluation Criteria in Solid Tumors, RECIST 1.1). Additionally, CEA and CTN were measured at the day of CT imaging and alterations observed in tumor markers were compared to respective imaging findings (partial response, PR; stable disease, SD; progressive disease, PD).During long-term follow-up (510 ±â€Š350 days [range, 97-1140 days]), CTN and CEA levels initially dropped in 71.4% and 61.9% of the patients followed by fluctuations in serum marker levels. A rise in CTN ≥39.5% between 2 subsequent measurements (defined by ROC analysis) had a sensitivity of 70.6% and a specificity of 83.2% in predicting PD with an accuracy of 82.0% (area under the curve (AUC), 0.76). Oscillations in CEA levels were not predictive for PD.Whereas tumor marker fluctuations in MTC patients undergoing TKI treatment are a frequent phenomenon, a significant rise in CTN ≥40% turns out to as an early indicator of tumor progression.

The relationship between needle sugar carbon isotope ratios and tree rings of larch in Siberia.

Significant gaps still exist in our knowledge about post-photosynthetic leaf level and downstream metabolic processes and isotopic fractionations. This includes their impact on the isotopic climate signal stored in the carbon isotope composition (δ(13)C) of leaf assimilates and tree rings. For the first time, we compared the seasonal δ(13)C variability of leaf sucrose with intra-annual, high-resolution δ(13)C signature of tree rings from larch (Larix gmelinii Rupr.). The trees were growing at two sites in the continuous permafrost zone of Siberia with different growth conditions. Our results indicate very similar low-frequency intra-seasonal trends of the sucrose and tree ring δ(13)C records with little or no indication for the use of 'old' photosynthates formed during the previous year(s). The comparison of leaf sucrose δ(13)C values with that in other leaf sugars and in tree rings elucidates the cause for the reported (13)C-enrichment of sink organs compared with leaves. We observed that while the average δ(13)C of all needle sugars was 1.2‰ more negative than δ(13)C value of wood, the δ(13)C value of the transport sugar sucrose was on an average 1.0‰ more positive than that of wood. Our study shows a high potential of the combined use of compound-specific isotope analysis of sugars (leaf and phloem) with intra-annual tree ring δ(13)C measurements for deepening our understanding about the mechanisms controlling the isotope variability in tree rings under different environmental conditions.

SPECT- and PET-based patient-tailored treatment in neuroendocrine tumors: a comprehensive multidisciplinary team approach.

The overexpression of somatostatin receptors on the tumor cell surface of neuroendocrine tumors (NETs) detected by multimodal functional imaging modalities such as SPECT and PET tracers constitutes a therapeutic option using targeting radiolabeled compounds. We will introduce the theranostic concept in general, explain in more detail its development in NETs, and discuss available SPECT and PET tracers regarding their potential for diagnostic imaging, visualization of target expression, and treatment tailoring. Moreover, we will discuss the currently available peptide receptor radionuclide therapy principles and compare them to previously published studies. Finally, we will discuss which new concepts will most likely influence the theranostic treatment approach in NETs in the future.

Continuous flow 2H/1H and 18O/16O analysis of water samples with dual inlet precision.

A method for isotope ratio analysis of water samples is described comprising an on-line high-temperature reduction technique in a helium carrier gas. Using a gas-tight syringe, injection of 0.5 to 1 microL sample is made through a heated septum into a glassy carbon reactor at temperatures in excess of 1300 degrees C. More than 150 injections can be made per day and both isotope ratios of interest, delta2H and delta18O, can be measured with the same setup. The technique has the capability to transfer high-precision stable isotope ratio analysis of water samples from a specialized to a routine laboratory task compatible with other common techniques (automated injection for GC, LC, etc.). Experiments with an emphasis on the reactor design were made in two different laboratories using two different commercially available high-temperature elemental analyser (EA) systems. In the Jena TC/EA unit, sample-to-sample memory (single injection) has been reduced to approximately 1% and high precision of about 0.1 per thousand for delta18O and < 1 per thousand for delta2H has been achieved by a redesign of the glassy carbon reactor and by redirecting the gas flow of the commercial TC/EA unit. With the modified reactor, the contact of water vapour with surfaces other than glassy carbon is avoided completely. The carrier gas is introduced at the bottom of the reactor thereby flushing the outer tube compartment of the tube-in-tube assembly before entering the active heart of the reactor.With the Leipzig high-temperature reactor (HTP) similar precision was obtained with a minor modification (electropolishing) of the injector metal sleeve. With this system, the temperature dependence of the reaction has been studied between 1100 and 1450 degrees C. Complete yield and constant isotope ratio information has been observed only for temperatures above 1325 degrees C. For temperatures above 1300 degrees C the reactor produces an increasing amount of CO background from reaction of glass carbon with the ceramic tube. This limits the usable temperature to a maximum of 1450 degrees C. Relevant gas permeation through the Al2O3 walls has not been detected up to 1600 degrees C.

Prolonged median sensory latency as a predictor of future carpal tunnel syndrome.

The purpose of the study was to determine whether abnormal median sensory nerve conduction among asymptomatic workers was predictive of future symptoms suggestive of carpal tunnel syndrome (CTS). This was a prospective study involving 77 workers who were identified as asymptomatic cases with electrodiagnostic findings of median mononeuropathy compared to an age- and sex-matched control group. Follow-up was completed an average of 70 months later, and subjects who reported pain, numbness, tingling, or burning in the distribution of the median nerve, based upon a hand diagram, were classified as having CTS symptoms. The follow-up participation rate was 70%. Among subjects with abnormal median sensory latencies, 23% went on to develop symptoms consistent with CTS within the follow-up period, compared with 6% in the control group (P= .010). Age and hand repetition were also risk factors for CTS, but the majority of asymptomatic workers with a median mononeuropathy do not become symptomatic over an extended time.

Extraction of CO2 from air samples for isotopic analysis and limits to ultra high precision delta18O determination in CO2 gas.

The determination of delta18O values in CO2 at a precision level of +/-0.02 per thousand (delta-notation) has always been a challenging, if not impossible, analytical task. Here, we demonstrate that beyond the usually assumed major cause of uncertainty - water contamination - there are other, hitherto underestimated sources of contamination and processes which can alter the oxygen isotope composition of CO2. Active surfaces in the preparation line with which CO2 comes into contact, as well as traces of air in the sample, can alter the apparent delta18O value both temporarily and permanently. We investigated the effects of different surface materials including electropolished stainless steel, Duran glass, gold and quartz, the latter both untreated and silanized. CO2 frozen with liquid nitrogen showed a transient alteration of the 18O/16O ratio on all surfaces tested. The time to recover from the alteration as well as the size of the alteration varied with surface type. Quartz that had been ultrasonically cleaned for several hours with high purity water (0.05 microS) exhibited the smallest effect on the measured oxygen isotopic composition of CO2 before and after freezing. However, quartz proved to be mechanically unstable with time when subjected to repeated large temperature changes during operation. After several days of operation the gas released from the freezing step contained progressively increasing trace amounts of O2 probably originating from inclusions within the quartz, which precludes the use of quartz for cryogenically trapping CO2. Stainless steel or gold proved to be suitable materials after proper pre-treatment. To ensure a high trapping efficiency of CO2 from a flow of gas, a cold trap design was chosen comprising a thin wall 1/4" outer tube and a 1/8" inner tube, made respectively from electropolished stainless steel and gold. Due to a considerable 18O specific isotope effect during the release of CO2 from the cold surface, the thawing time had to be as long as 20 min for high precision delta18O measurements. The presence of traces of air in almost all CO2 gases that we analyzed was another major source of error. Nitrogen and oxygen in the ion source of our mass spectrometer (MAT 252, Finnigan MAT, Bremen, Germany) give rise to the production of NO2 at the hot tungsten filament. NO2+ is isobaric with C16O18O+ (m/z 46) and interferes with the delta18O measurement. Trace amounts of air are present in CO2 extracted cryogenically from air at -196 degrees C. This air, trapped at the cold surface, cannot be pumped away quantitatively. The amount of air present depends on the surface structure and, hence, the alteration of the measured delta18O value varies with the surface conditions. For automated high precision measurement of the isotopic composition of CO2 of air samples stored in glass flasks an extraction interface ('BGC-AirTrap') was developed which allows 18 analyses (including standards) per day to be made. For our reference CO2-in-air, stored in high pressure cylinders, the long term (>9 months) single sample precision was 0.012 per thousand for delta13C and 0.019 per thousand for delta18O.

18O pattern and biosynthesis of natural plant products.

Oxygen atoms in plant products originate from CO(2), H(2)O and O(2), precursors with quite different delta18O values. Furthermore their incorporation by different reactions implies isotope effects. On this base the resulting non-statistical 18O distributions in natural compounds are discussed. The delta18O value of cellulose is correlated to that of the leaf water, and the observed 18O enrichment (approximately +27 per thousand) is generally attributed to an equilibrium isotope effect between carbonyl groups and water. However, as soluble and heterotrophically synthesised carbohydrates show other correlations, a non-statistical 18O distribution - originating from individual biosynthetic reactions - is postulated for carbohydrates. Similarly, the delta18O values of organic acids, carbonyl compounds, alcohols and esters indicate water-correlated, but individual 18O abundances (e.g. O from acyl groups approximately +19% above water), depending upon origin and biosyntheses. Alcoholic groups introduced by monooxygenase reactions, e.g. in sterols and phenols, show delta18O values near +5 per thousand, in agreement with an assumed isotope fractionation factor of approximately 1.02 on the reaction with atmospheric oxygen (delta18O=+23.5 per thousand). Correspondingly, a "thermodynamically ordered isotope distribution" is only observed for oxygen in some functional groups correlated to an origin from CO(2) and H(2)O, not from O(2). The individual isotopic increments of functional groups permit the prediction of global delta18O values of natural compounds on the basis of their biosynthesis.

Referencing strategies and techniques in stable isotope ratio analysis.

Stable isotope ratios are reported in the literature in terms of a deviation from an international standard (delta-values). The referencing procedures, however, differ from instrument to instrument and are not consistent between measurement facilities. This paper reviews an attempt to unify the strategy for referencing isotopic measurements. In particular, emphasis is given to the importance of identical treatment of sample and reference material ('IT principle'), which should guide all isotope ratio determinations and evaluations. The implementation of the principle in our laboratory, the monitoring of our measurement quality, the status of the international scales and reference materials and necessary correction procedures are discussed.

Unipedal stance testing in the assessment of peripheral neuropathy.

OBJECTIVE: To define further the relation between unipedal stance testing and peripheral neuropathy. DESIGN: Prospective cohort. SETTING: Electroneuromyography laboratory of a Veterans Affairs medical center and a university hospital. PATIENTS: Ninety-two patients referred for lower extremity electrodiagnostic studies. MAIN OUTCOME MEASURES: A standardized history and physical examination designed to detect peripheral neuropathy, 3 trials of unipedal stance, and electrodiagnostic studies. RESULTS: Peripheral neuropathy was identified by electrodiagnostic testing in 32%. These subjects had a significantly shorter (p <.001) unipedal stance time (15.7s, longest of 3 trials) than the patients without peripheral neuropathy (37.1s). Abnormal unipedal stance time (<45s) identified peripheral neuropathy with a sensitivity of 83% and a specificity of 71%, whereas a normal unipedal stance time had a negative predictive value of 90%. Abnormal unipedal stance time was associated with an increased risk of having peripheral neuropathy on univariate analysis (odds ratio = 8.8, 95% confidence interval = 2.5--31), and was the only significant predictor of peripheral neuropathy in the regression model. Aspects of the neurologic examination did not add to the regression model compared with abnormal unipedal stance time. CONCLUSIONS: Unipedal stance testing is useful in the clinical setting both to identify and to exclude the presence of peripheral neuropathy.

Prevalence of carpal tunnel syndrome and median mononeuropathy among dentists.

BACKGROUND: The authors undertook a study to determine the prevalence in dentists of abnormal sensory nerve conduction and/or symptoms of carpal tunnel syndrome, or CTS, the most common nerve entrapment syndrome. METHODS: In a cross-sectional study, dentists (n = 1,079) were screened during the American Dental Association's Annual Health Screening Program in 1997 and 1998 by means of standard electrodiagnostic measures in the dominant hand and a self-reported symptom questionnaire. The authors diagnosed a median mononeuropathy from a 0.5- or 0.8-millisecond, or ms, prolongation of the median sensory-evoked peak latency compared to the ulnar latency. They diagnosed CTS if the subject also had accompanying symptoms of numbness, tingling or pain. RESULTS: Thirteen percent of screened dentists were diagnosed with a median mononeuropathy (using a 0.5-ms prolongation as the criterion), but only 32 percent of these had symptoms consistent with CTS (4.8 percent overall). When the 0.8-ms prolongation was used as the electrodiagnostic criterion, only 2.9 percent (overall) were diagnosed with CTS. People with diabetes, rheumatoid arthritis and obesity were more likely to have a median mononeuropathy. CONCLUSIONS: The prevalence of symptoms consistent with CTS in the dominant hand among dentists was higher than the prevalence in the general population. However, when electrodiagnostic confirmation is added, the prevalence of CTS was nearly the same as that among the general population. CLINICAL IMPLICATIONS: Early recognition of CTS can lead to more effective management. Education regarding ergonomic risk factors can be an effective preventive measure.

Test-retest reliability of the Upper Extremity Questionnaire among keyboard operators.

BACKGROUND: Questionnaires are often used in research among workers although few have been tested in the working population. The Upper Extremity Questionnaire is a self-administered questionnaire designed for epidemiological studies and tested among workers. This study assessed reliability of the instrument. METHODS: A two-part assessment was conducted among 138 keyboard operators as part of a large medical survey. Test-retest reliability was analyzed using the kappa statistic, paired t-test, and intraclass correlation coefficient (ICC). Logistic regression models were used to test the effect of demographic and work-related factors on reliability. RESULTS: The average respondent was a white woman, age 35 years, with some college education, in permanent employment with tenure of 1.4 years. Overall, reports of symptoms were stable from Round 1 to 2. Most kappa values for symptom reports were between 0.60 and 0.89. Kappa values for right and left hand diagrams were 0.57 and 0.28, respectively. Among psychosocial items, Perceived Stress and Job Dissatisfaction Scales were most reliable (ICC = 0.88); co-worker support was least reliable (ICC = 0.44). CONCLUSION: Reliability of items on the Upper Extremity Questionnaire were generally good to excellent. Reports of symptom severity and interference with work were less stable. Demographic and work-related factors were not statistically significant in modeling the variation in reliability. Repeated use of the questionnaire with similar results suggests findings are applicable to a larger working population.

Unipedal stance testing as an indicator of fall risk among older outpatients.

OBJECTIVE: To test the hypothesis that a decreased unipedal stance time (UST) is associated with a history of falling among older persons. DESIGN: Fifty-three subjects underwent a standardized history and physical examination and three trials of timed unipedal stance. SETTING: The electroneuromyography laboratories of tertiary care Veterans Administration and university hospitals. SUBJECTS: Ambulatory outpatients 50 years and older referred for electrodiagnostic studies. OUTCOME MEASURES: UST and fall histories during the previous year. RESULTS: Twenty subjects (38%) reported falling. Compared with the subjects who had not fallen, those who fell had a significantly shorter UST (9.6 [SD 11.6] vs 31.3 [SD 16.3] seconds, using the longest of the three trials, p < .00001). An abnormal UST (<30sec) was associated with an increased risk of having fallen on univariate analysis and in a regression model (odds ratio 108; 95% confidence interval 3.8, >100; p < .007). The sensitivity of an abnormal UST in the regression model was 91% and the specificity 75%. When UST was considered age was not a predictor of a history of falls. CONCLUSIONS: UST of <30sec in an older ambulatory outpatient population is associated with a history of falling, while a UST of > or = 30sec is associated with a low risk of falling.

Reliability of physical examination of the upper extremity among keyboard operators.

BACKGROUND: Physical examination is a traditional outcome measure in epidemiological research. Its value as a reliable measure depends, in part, on the prevalence of positive findings. The purpose of this paper is to determine the empirical reliability of physical examination and anthropometry in a field study of upper extremity disorders among keyboard operators. METHODS: Two experienced examiners independently performed common provocative tests and procedures in physical examinations of the neck and upper extremity among 160 keyboard operators. Two additional examiners conducted anthropometric surveys among 137 workers. Inter-examiner reliability was assessed with observed agreement, kappa statistics, and intra-class correlations (ICC). RESULTS: Observed agreement was between 96% and 100% for neck and upper extremity signs, muscle stretch reflexes, and muscle strength, however, with the exception of provocative tests, reliability statistics were unstable. Among the provocative tests, Phalen and Tinel tests had modest agreement after adjusting for chance (kappa range: 0.20-0.43). The carpal compression test had the best reliability (kappa=0.60 and kappa=0.67, left and right side, respectively). The ICCs for anthropometry ranged from 0.36-0.91. CONCLUSIONS: Results from the study showed that statistically, except for the carpal compression test, physical examination contributed minimal reliable information. This was attributed mainly to the low prevalence of positive findings, and generally mild nature of upper extremity disorders in this population. The results are the best estimate of what would be found in a field study with experienced examiners. While it may reduce bias, separating physical examination from medical history may contribute to the poor reliability of findings. With a shift toward reliable measures, resources can be allocated to more effective tools, like questionnaires, in epidemiological research of upper extremity disorders among keyboard operators.

Reliability of nerve conduction studies among active workers.

Nerve conduction studies play an important role in clinical practice and research. Given their widespread use, reliability of tests merits careful attention. We assessed interexaminer and intraexaminer reliability of median and ulnar sensory nerve measures of amplitude, onset latency, and peak latency. In a two-phase cross-sectional study, two examiners tested 158 workers. Reliability was assessed with intraclass correlations (ICC) and kappa statistics. Median nerve measures were more reliable (ICC range, 0.76 to 0.92) than ulnar measures (ICC range, 0.22 to 0.85). Ulnar-onset latencies had the worst reliability. The median-ulnar peak latency difference was a particularly stable measure (ICC range, 0.79 to 0.92). The median-ulnar peak latency difference had high interexaminer reliability (kappa range, 0.71 to 0.79) for normal tests defined by cut points of 0.8 ms and 0.5 ms. Intraexaminer reliability was higher with the 0.8-ms cut point (kappa = 0.90 and kappa = 0.85 for examiners 1 and 2, respectively). Rather than absolute cut points to describe normality, a more rational interpretation of results can be made with ordered categories or continuous measures.

Cross-sectional study of the relationship between repetitive work and the prevalence of upper limb musculoskeletal disorders.

BACKGROUND: This study examined the relationship of repetitive work and other physical stressors to prevalence of upper limb discomfort, tendinitis, and carpal tunnel syndrome. METHODS: Three hundred fifty-two workers from three companies participated. Job exposure levels for repetition and other physical stressors were quantified using an observational rating technique. Ergonomic exposures were rated on a 10-point scale, where 0 corresponded to no stress and 10 corresponded to maximum stress. Job selection was based on repetition (three categories: high, medium, and low) to ensure a wide range of exposures. Physical evaluations on all participating workers were performed by medical professionals and included a self-administered questionnaire, physical exam, and limited electrodiagnostic testing. RESULTS: Repetitiveness of work was found to be significantly associated with prevalence of reported discomfort in the wrist, hand, or fingers (odds ratio (OR) = 1.17 per unit of repetition; OR = 2.45 for high vs. low repetition), tendinitis in the distal upper extremity (OR = 1.23 per unit of repetition; OR = 3.23 for high vs. low repetition), and symptoms consistent with carpal tunnel syndrome (OR = 1.16 per unit of repetition; OR = 2.32 for high vs. low repetition). An association was also found between repetitiveness of work and carpal tunnel syndrome, indicated by the combination of positive electrodiagnostic results and symptoms consistent with carpal tunnel syndrome (OR = 1. 22 per unit of repetition; OR = 3.11 for high vs. low repetition). CONCLUSIONS: These findings indicate that repetitive work is related to upper limb discomfort, tendinitis, and carpal tunnel syndrome in workers. Further research with a wider range of exposures is needed to evaluate the effects of other physical stresses alone and in combination.