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A Fourier transform infrared microspectroscopic imaging investigation into an animal model exhibiting glioblastoma multiforme.

Glioblastoma multiforme (GBM) is a highly malignant human brain tumour for which no cure is available at present. Numerous clinical studies as well as animal experiments are under way with the goal being to understand tumour biology and develop potential therapeutic approaches. C6 cell glioma in the adult rat is a frequently used and well accepted animal model for the malignant human glial tumour. By combining standard analytical methods such as histology and immunohistochemistry with Fourier Transform Infrared (FTIR) microspectroscopic imaging and multivariate statistical approaches, we are developing a novel approach to tumour diagnosis which allows us to obtain information about the structure and composition of tumour tissues that could not be obtained easily with either method alone. We have used a "Stingray" FTIR imaging spectrometer to analyse and compare the compositions of coronal brain tissue sections of a tumour-bearing animal and those from a healthy animal. We have found that the tumour tissue has a characteristic chemical signature, which distinguishes it from tumour-free brain tissue. The physical-chemical differences, determined by image and spectral comparison are consistent with changes in total protein absorbance, phosphodiester absorbance and physical dispersive artefacts. The results indicate that FTIR imaging analysis could become a valuable analytic method in brain tumour research and possibly in the diagnosis of human brain tumours.

Animals↗

Infrared micro-spectroscopic studies of epithelial cells.

We report results from a study of human and canine mucosal cells, investigated by infrared micro-spectroscopy, and analyzed by methods of multivariate statistics. We demonstrate that the infrared spectra of individual cells are sensitive to the stage of maturation, and that a distinction between healthy and diseased cells will be possible. Since this report is written for an audience not familiar with infrared micro-spectroscopy, a short introduction into this field is presented along with a summary of principal component analysis.

Animals↗

Bill colour and correlates of male quality in blackbirds: an analysis using canonical ordination.

Carotenoid-dependent plumage displays are widely assumed to be honest indicators of individual health or quality, which are used as cues during mate choice and/or agonistic signalling. Despite the fact that red, yellow and orange pigmentation of bills is common, and also variable between individuals, comparatively little is known about bill colouration as a condition-dependent trait. Furthermore, many studies of avian colouration are confounded by the lack of objective colour quantification and the use of overly simplistic univariate techniques for analysis of the relationship between the condition-dependent trait and individual quality variables. In this study, we correlated male blackbird bill colour (a likely carotenoid-dependent sexually selected trait) with body/condition variables that reflect male quality. We measured bill colour using photometric techniques, thus ensuring objectivity. The data were analysed using the multivariate statistical techniques of canonical ordination. Analyses based on reflectance spectra of male blackbird bill samples and colour components (i.e. hue, chroma and brightness) derived from the reflectance spectra were very similar. Analysing the entire reflectance spectra of blackbird bill samples with Redundancy Analysis (RDA) allowed examination of individual wavelengths and their specific associations with the body/condition variables. However, hue, chroma and brightness values also provided useful information to explain colour variation, and the two approaches may be complimentary. We did not find any significant associations between male blackbird bill colour and percent incidence of ectoparasites or cloaca size. However, both the colour component and full spectral analyses showed that culmen length explained a significant amount of variation in male blackbird bill colour. Culmen length was positively associated with greater reflectance from the bill samples at longer wavelengths and a higher hue value (i.e. more orange-pigmented bills). Larger males may have larger territories or be better at defending territories during male-male interactions, ensuring access to carotenoid food sources. Future studies should elucidate the relationship between bill colour and behavioural measures such as aggressiveness, territory size, song rate and nest attendance.

Animals↗

Vascular depression: A distinct diagnostic subtype?

Vascular depression has been proposed as a unique and valid diagnostic subtype on the basis of studies of external (concurrent and predictive) validity. Validating a diagnostic entity on the basis of external validity is problematic, because it presupposes that the construct is well defined (i.e., the proposed features cluster together to define a distinct patient group). Because such evidence has not been obtained, we propose that the next critical step in evaluating this potential subtype is to establish internal (construct) validity and highlight taxometric analysis and latent class cluster analysis as illustrative multivariate statistical techniques that can be used in this effort. The psychometric approach advocated here (despite its inherent assumptions and limitations) might substantially improve on previous diagnostic efforts (e.g., expert consensus), and vascular depression might serve as a prototype for future psychiatric classification.

Cerebrovascular Disorders↗

Laboratory variables and stratification of metastatic colorectal cancer patients: recommendations for therapeutic trials and for clinical practice guidelines.

OBJECTIVE: To identify, through a systematic review of the literature, the laboratory variables that, in addition to performance status and to the degree of tumor invasion, would allow a more accurate stratification of metastatic colorectal cancer patients who participate in chemotherapy trials, with or without radiotherapy. SECONDARY AIM: To compare the results of our systematic review with the recommendations made in current clinical practice guidelines, and with the results of related systematic reviews. METHODS: Update of two recently published systematic reviews, without metaanalysis, following the recommendations of the International Federation of Clinical Chemistry and Laboratory Medicine, and taking into account the Consolidated Standards of Reporting Trials statement. RESULTS: Of 877 publications retrieved, reasonable exclusion and inclusion criteria allow us to include 15 studies in our systematic review, thus confirming the low quality of clinical research in laboratory medicine. Four variables were most often found "significant" in multivariate statistical analysis: pretherapeutic levels of laboratory tests (13/15, 87%), degree of tumor invasion (9/13, 69%), treatment, or response to treatment (6/9, 67%), and performance status (8/13, 62%). The laboratory variable whose measurements are quite often recommended in the 10 clinical practice guidelines or in the four related systematic reviews that we retrieved are carcinoembryonic antigen (CEA), and liver function tests to a lesser extent. CONCLUSIONS: Available evidence supports the recommendation that in all metastatic colorectal cancer patients who participate in therapeutic trials, the following pretreatment laboratory variables should be systematically measured: blood cell counts, and haemoglobin, plasma prothrombin time, serum alkaline phosphatase (ALP), lactate dehydrogenase, transaminases, albumin, bilirubin, and CEA. If other tests were to be added, gamma glutamyl transferase, and erythrocyte sedimentation rate might perhaps be proposed. Further studies would be necessary to support the addition to this list, of other tests [e.g., cancer antigen (CA) 19-9]. Rather than using laboratory variables according to arbitrary thresholds, it seems recommendable to use them as continuous variables, and if possible, in terms of kinetics. Many clinical practice guidelines do not use levels of evidence in order to grade the strength of their recommendations, but rather seem to be based on experts opinions which are not always in agreement with the results of systematic reviews.

Antineoplastic Agents↗

Study of the topographic distribution of ets-1 protein expression in invasive breast carcinomas in relation to tumor phenotype.

BACKGROUND: Ets-1 is a transcription factor, implicated in the regulation of expression of various genes'. The aim of the present study was to investigate the expression of ets-1 protein in invasive breast carcinomas and its correlation with classic clinicopathological parameters, patients' survival and various biological markers. METHODS: Immunohistochemistry was performed in paraffin-embedded tissue specimens from 149 invasive breast carcinomas to detect the proteins ets-1, p53, topoisomerase IIalpha, matrix metalloproteinase-7 (MMP-7) and urokinase-type plasminogen activator receptor (uPAR). Results were subjected to univariate and multivariate statistic analysis. RESULTS: Ets-1 protein was detected in the 77.9% of the cases in the cytoplasm, in the 46.3% in the nucleus of the malignant cells, and in stromal fibroblasts as well. Cytoplasmic ets-1 was inversely correlated with nuclear and histologic grade of the tumor (p=0.004 and 0.033, respectively) and topoisomerase IIotaalpha (p=0.057), while nuclear ets-1 showed a positive association with p53 (p=0.002). Stromal ets-1 revealed a negative correlation with estrogen receptors (ER) (p=0.003) and a positive one with stromal uPAR and MMP-7 as well (p=0.048 and 0.066, respectively). The univariate statistic analysis showed nuclear ets-1 to be related to a shortened overall survival of the postmenopausal patients (p=0.032). CONCLUSIONS: Ets-1 seems to be related to a different tumor phenotype according to its topographic distribution, with nuclear localization being associated with decreased apoptotic potential of the malignant cells through its relation to the mutant p53 protein, cytoplasmic being related to a favorable tumor phenotype and stromal ets-1 being related to tumor invasion.

Adult↗

Characterization of human breast epithelial cells by confocal Raman microspectroscopy.

BACKGROUND: The past decade has seen an explosion of interest in utilizing Raman spectroscopy in cancer diagnosis, due to its capability to probe changes in the biochemical composition of tissue that accompany disease progression. However, most of the existing methods used multivariate statistical analysis/chemometrics to differentiate normal and diseased tissues, which did not identify the compositional and chemical changes associated with the tumorigenic transition explicitly; also the sub-cellular level spatial resolution achievable through Confocal Raman microscopy was not fully utilized. METHODS: Confocal Raman microspectroscopy was used to characterize normal and transformed human breast epithelial cell lines. Key molecular components (DNA, RNA, and proteins) were extracted from cell nuclei and their Raman spectra were measured and used as a basis set to fit the spectra of cell nuclei. Contributions of each component and their relative contents were evaluated based on the fitting coefficients. RESULTS: Spectrum-fitting revealed that DNA duplication activities in tumorigenic cell nuclei are significantly higher than in normal cells. The fitting coefficients could serve as good spectral markers for disease state identification. CONCLUSIONS: A spectroscopic approach that yields compositional information of cell nuclei could be a powerful tool for rapid cell characterization and assessment of cellular activities at the sub-cellular level.

Breast↗

Pharmaceuticals in the river Elbe and its tributaries.

Medicinal drugs were found to be ubiquitous in the river Elbe, its tributary the river Saale and in other tributaries at their points of entry into the Elbe. The distribution of concentration peaks along the investigated river stretches provides an indication that they are mainly due to the emission of treated waste water from municipal sewage treatment works. This leads to the conclusion that medicinal substances can be regarded as faecal indicators for water pollution caused by human activity. The main substances found in the Elbe in 1998 were diclofenac, ibuprofen and carbamazepine as well as various antibiotics and lipid regulators in the concentration range of <20-140 ng/l. The more thorough investigations carried out in 1999 and 2000 show that in addition to the drugs (phenazone, isopropyl-phenazone and paracetamol) metabolite concentrations contributed significantly to the total concentration of pharmaceuticals in the Elbe. The metamizole metabolites N-acetyl-4-aminoantipyrine (AAA) and N-formyl-4-aminoantipyrine (FAA) were found in concentrations from <20 to 939 ng/l. A multivariate statistical analysis revealed a high correlation in respect of the distribution of persistent substances. The metoprolol distribution throughout the Saale demonstrated that the tributaries cause either an increase (Weisse Elster, Unstrut, Ilm) or a reduction (Wipper, Bode) in the concentration, depending on the respective load of waste water. Wide scale sampling in Saxony during 2002 showed the ubiquitous occurrence of carbamazepine in surface waters. The ecotoxicological effects of this contamination cannot be assessed at present. This is due to the fact that no legal framework in respect of these medicinal drugs for human consumption has been established and therefore little research and no risk assessment has been carried out. Therefore it is urgently necessary to include at least the quantitatively most significant substances in the new assessment concept of the EC White Paper.

Cluster Analysis↗

The hydrochemistry of surface waters in andesitic volcanic area, Norikura volcano, central Japan.

Many hydrochemical studies have been reported on surface waters and a major contributor of the water chemistry is considered to be rock dissolution. However, the extent of rock dissolution effect on surface water chemistry has still not been fully understood. The purpose of this research is to clarify the effect to give better understanding on the nature of surface water chemistry. Natural surface water samples were collected from a typical andesitic volcanic area, and major chemicals were investigated in terms of water-rock interaction. The analytical results were subjected to multivariate statistical techniques to understand the geographical distribution and to extract geochemical potential factors affecting the chemical concentration of waters. High correlation between the major chemicals and the altitude of sampling points was found among non-geothermal waters. The mole ratios of summation Sigmacations/Si were significantly high in meteoric waters (2.5), low in the surface waters from the summit area of the volcano (0.4), and the median in the surface waters at the mountainside region (1-2) and in geothermal waters (1-2). The basement rocks around the observation areas showed a relatively low ratio (0.4). On the basis of the water chemical compositions, these chemical variation were quantitatively interpreted by the incongruent dissolution reactions for primary minerals in the basement rocks followed by the formation of secondary minerals. The variations of Na+/(Ca2+ + Na+) ratio took positive correlation with [Si] for the non-geothermal waters, and high linear correlation was observed between log(a(Ca2+)/a2(H+)) and log(a(Na+)/a(H+)). These relations were interpreted by an ion-exchange reaction. The factor analysis (FA) gave two major factors to describe the chemical behaviors mentioned above. The contribution proportionate for each factor was calculated as; 65% was the incongruent rock dissolution, and 16% was the ion-exchange reaction.

Altitude↗

Assessment of heavy metal pollution in surface soils of urban parks in Beijing, China.

Assessing the concentration of potentially harmful heavy metals in the soil of urban parks is imperative in order to evaluate the potential risks to residents and tourists. To date, little research on soil pollution in China's urban parks has been conducted. To identify the concentrations and sources of heavy metals, and to assess the soil environmental quality, samples were collected from 30 urban parks located in the city of Beijing. Subsequently, the concentrations of Cu, Ni, Pb and Zn in the samples were analyzed. The investigation revealed that the accumulations of Cu and Pb were readily apparent in the soils. The integrated pollution index (IPI) of these four metals ranged from 0.97 to 9.21, with the highest IPI in the densely populated historic center district (HCD). Using multivariate statistic approaches (principal components analysis and hierarchical cluster analysis), two factors controlling the heavy metal variability were obtained, which accounted for nearly 80% of the total variance. Nickel and Zn levels were controlled by parent material in the soils, whereas Cu, Pb and, in part, Zn were accounted for mainly by anthropogenic activities. The findings presented here indicate that the location and the age of the park are important factors in determining the extent of heavy metal, particularly Cu and Pb, pollution. In addition, the accumulation of Zn did not appear to reach pollution levels, and no obvious pollution by Ni was observed in the soils of the parks in Beijing.

China↗

Blood concentrations of polycyclic musks in healthy young adults.

Knowledge on the concentration of polycyclic musk fragrance compounds in human blood is sparse. This study examined the concentrations of six polycyclic musks in blood samples from healthy volunteers. Blood was taken from hundred healthy students of the Medical University of Vienna. The lipophilic fraction was extracted and after purification analyzed by GC-MS. Study participants also completed a questionnaire on the use of cosmetics, about nutrition and other life-style aspects. Two compounds -- galaxolide and tonalide -- were identified in higher percentages of the blood plasma samples. Maximum plasma levels over 100 ng/l were also only found for galaxolide (4100 ng/l) and tonalide (800 ng/l). Women showed significantly higher levels than men. In a statistical multivariate approach only use of body lotion and age were predictive of positive galaxolide concentrations. For tonalide no significant predictor could be found. The findings mirror the replacement of nitro musk fragrances by polycyclic musks, mainly galaxolide. The high concentrations of galaxolide in human blood raise concern since few toxicological data are available.

Adult↗

Influence of a municipal solid waste incinerator on ambient air and soil PCDD/Fs levels.

To examine the influence of polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans (PCDD/Fs) emissions from a municipal solid waste incinerator (MSWI) on the environment, we measured the levels of PCDD/Fs in ambient air and soil samples collected near a MSWI in Bucheon, Korea. The PCDD/Fs concentrations in the ambient air samples ranged from 0.22 to 1.16 pg I-TEQm(-3) (13.39-75.16 pg m(-3)), with an average of 0.66 pg I-TEQ m(-3) (35.62 pg m(-3)). The soil samples contained between 1.25 and 74.98 pg I-TEQ g(-1) (38.15-3,303.33 pg g(-1)), with an average of 19.06 pg I-TEQ g(-1) (1,077.11 pg g(-1)). These levels were higher than those previously reported by other investigators in a number of surveys. The furan homologues predominated in the air samples and some soil samples, and the soil PCDD/Fs levels decreased with increasing distance from the MSWI. Comparison of the homologue patterns and a multivariate statistical analysis showed that PCDD/Fs emission from the MSWI directly affected the pattern of PCDD/Fs in air, while the PCDD/Fs patterns in soil differed according to the location relative to the MSWI, roads, and construction sites. These results collectively indicate that the MSWI was the major PCDD/Fs emission source in this area, but that unidentified combustion sources and vehicles might influence the environment to some extent.

Air Pollutants↗

Identification of isomeric 4-nonylphenol structures by gas chromatography-tandem mass spectrometry combined with cluster analysis.

The endocrine-disrupting effect of 4-nonylphenols (NP) formed from industrial detergents such as nonylphenol polyethoxylates is well known today. The technical mixture contains a great variety of 4-iso-nonylphenol isomers having different endocrine-disrupting activities. Currently used gas chromatography-mass spectrometry (GC-MS) protocols allow the detection of about 20 peaks, mostly co-eluting isomers. In the present study, Product Ion mass spectrometry obtained by ion trap technology enhanced the selectivity in NP detection resulting in improved gas chromatographic resolution as well as structure assignment of the isomers. The structure proposals of 4-nonylphenol isomers given were derived from GC-MS-MS data processed by multivariate statistics. The cluster analysis allowed the classification of NP due to common structural features that were reflected in the mass spectra. The fragmentation pathways of three reference NP isomers, 4-(1-ethyl-1,4-dimethylpentyl)phenol (NP1), 4-(1,1,5-trimethylhexyl)phenol (NP2) and 4-(1-ethyl-1,3-dimethylpentyl)phenol (NP3), were investigated in more detail. They served as model compounds to aid the interpretation of spectra from unknown NP isomers. Structures of two groups of isomers, characterized by alpha-ethyl as well as alpha-propyl substituents, could be proposed.

Cluster Analysis↗

Effects of multipurpose contact lens solutions on the protein composition of the tear film.

OBJECTIVES: To analyze the influence of multipurpose contact lens cleaning solutions on tear proteins. Changes in tear film protein profiles of contact lens wearers who used several marketed brands of multipurpose contact lens care solutions, were assessed by ProteinChip analysis. METHODS: Three studies were conducted. Study I was a comparison of Complete and OptiFree multipurpose solutions. Study II was a study with Complete Moisture Plus solution, Study II was a comparison of Renu and Solocare contact lens solutions. Wearers of soft contact lenses were assigned to use the contact lens care solutions for 4 weeks. Non-contact lens wearing patients were used as controls. Tear samples of each participant were analyzed with the ProteinChip (SELDI-TOF) system. Multivariate statistical analysis and artificial neural networks were used to determine the tear protein profiles of each study group. RESULTS: Before starting the use of the solutions, the tear protein composition in all contact lens wearers deviated from the tear composition of the non-contact lens wearing controls. After 4 weeks of using the different care regimens, the tear protein composition of the patients using Complete or Complete Moisture Plus solutions tended to move toward that of the non-contact lens wearing controls. The tear protein composition of patients using the OptiFree, Renu or Solocare solutions did not undergo a measureable change in the protein level. CONCLUSIONS: The ProteinChip system can analyze protein profiles for large-scale applications as in clinical studies. Two multipurpose solutions, Complete and Complete Moisture Plus, demonstrated a beneficial effect on the tear proteins in contact lens wearers.

Contact Lens Solutions↗

Typical and atypical carcinoid tumours: analysis of the experience of the Spanish Multi-centric Study of Neuroendocrine Tumours of the Lung.

BACKGROUND: This study examines the experience of the Spanish Multi-centric Study of Neuroendocrine Tumours of the Lung with patients treated surgically for typical and atypical carcinoid tumours. METHODS: From 1980 to 2002, 661 patients were treated surgically for 569 typical carcinoid tumours and 92 atypical carcinoid tumours. Three hundred and four cases were studied retrospectively from 1980 to 1997 (261 typical carcinoid and 43 atypical carcinoid tumours); the other 357 new cases (308 typical carcinoid and 49 atypical carcinoid tumours) were collected prospectively from 1998 to 2002. Tumours were classified according the 1999 classification from the WHO and the International Association for the Study of Lung Cancer (IASLC). Several variables were reviewed in all patients. Univariate and multivariate statistical analyses were performed in order to determine whether clinical characteristics were associated with significant differences in survival. RESULTS: In the total of the patients, 5-year survival for different tumours was as follows: typical carcinoid: overall survival 97%; with nodal involvement 100%; atypical carcinoid: overall 78%; with nodal involvement 60%. A significant difference in survival was found between patients in the retrospective and prospective groups with atypical carcinoid and nodal involvement. The comparative analysis of several factors in typical and atypical carcinoid tumours showed a significant difference for mean age, tumour size, nodal involvement and distant metastases. CONCLUSION: Nodal involvement and histological sub-type appear as the most important factors influencing the prognosis. Adequate lung resection and systematic radical mediastinal lymphadenectomy should always be performed. Sleeve resection could be performed in central typical and atypical carcinoid tumours, avoiding pneumonectomy.

Adolescent↗

The association of progesterone, infant formula use and pacifier use with the return of menstruation in breastfeeding women: a prospective cohort study.

OBJECTIVES: This study sought to explore hormonal and postnatal factors, which are associated with the return of menstruation in breastfeeding mothers. DESIGN: A prospective cohort study in Bristol, UK. Ninety-one breastfeeding mothers of mixed parity were recruited at community-based antenatal clinics. Eighty-five mothers completed all stages and informed us about the return of menstruation. Data were collected from interviews and questionnaires antenatally, immediately postnatally, and 1 week, 4 weeks and 6 months later. Blood samples were taken at all time points except at 6 months. Associations between hormone levels, postnatal factors and the return of menstruation were explored using univariate and multivariate statistical survival analysis. RESULTS: Sixty-five (76%) mothers were still breastfeeding at 6 months and 46 (54%) of these were fully breastfeeding (not giving formula milk); 21 (32%) mothers started menstruating in the first 6 months whilst they were still breastfeeding and 14 were fully breastfeeding at 6 months. Survival analysis modelling showed that formula use (RR=4.27; 95% CI=1.89, 9.67), pacifier (dummy) use (RR=3.26; 95% CI=1.35, 7.84) and lower postnatal progesterone levels (P=0.006) each made a significant contribution to the chance of earlier resumption of menstruation. Prolactin and oestradiol levels showed no associations with the return of menstruation. CONCLUSIONS: Higher postnatal progesterone levels are associated with delayed menstruation; while the use of pacifiers and infant formula milk are associated with an earlier return to menstruation.

Adult↗

Do psychological factors increase the risk for back pain in the general population in both a cross-sectional and prospective analysis?

This study aimed to conduct a comprehensive evaluation of background, individual and workplace psychological risk factors to investigated their relationship with spinal pain. Because there is some doubt as to whether the results of cross-sectional findings hold in longitudinal studies, a prospective study was superimposed upon a cross-sectional design of the effects of psychological variables on back pain and function to determine, whether similar results are obtained. Participants were workers randomly selected from the general population, where 372 had not experienced pain during the past year, and 209 had experienced considerable pain problems. A cross-sectional comparison of these groups using multivariate statistics indicated that the most potent risk factors were psychological distress (odds ratio=13.2) and poor function (odds ratio=6.4). Much smaller levels of risk were found for perceived workload, gender and foreign birth. Those participants with no pain were followed for one year to determine development of a spinal pain problem. Although few participants developed a significant pain problem, the prospective analyses showed that psychological distress (odds ratio=2.2), catastrophizing (odds ratio=3.0), and workload (odds ratio=2.3) produced the highest odds ratios. Taken together these results underscore the need for a multidimensional view of the development of pain disability. Moreover, individual psychological factors such as distress and catastrophizing as well as work place factors like work load were found to be highly related to the development of back pain in a sample of workers from the general population. The cross-sectional and prospective results were similar in character and demonstrate that cross-sectional studies may provide valuable information. Because psychological variables were relevant very early on, these factors may be important targets for pain prevention programs.

Activities of Daily Living↗

A comparative assessment of heavy metal accumulation in soft parts and byssus of mussels from subarctic, temperate, subtropical and tropical marine environments.

Existing data on metal concentrations in mussels from subarctic, temperate, subtropical and tropical waters were analyzed using multivariate statistics in order to assess regional variations in metal contamination. Potential errors were reduced by only analyzing data from surveys that employed the same protocols, analytical methodologies and analysts. Factor analysis demonstrated that mussels inhabiting extremely contaminated areas (e.g. from Japanese and Swedish metallurgy sources) could be separated from mussels from other contaminated areas, and that metals such as Cd, Pb, Cu and Zn could be used to identify heavily contaminated samples while Co, Fe, Cr and Ni concentrations were good markers for exposure to inputs from different industrial sources. Furthermore byssus, like soft tissue, selectively and sensitively reflects variations of certain metal concentrations in ambient waters and thus serves as a reliable biomonitor for these contaminants in a variety of coastal and estuarine areas.

Animals↗