Paradoxical activation of AMPK by glucose drives selective EP300 activity in colorectal cancer.

Coordination of gene expression with nutrient availability supports proliferation and homeostasis and is shaped by protein acetylation. Yet how physiological/pathological signals link acetylation to specific gene expression programs and whether such responses are cell-type-specific is unclear. AMP-activated protein kinase (AMPK) is a key energy sensor, activated by glucose limitation to resolve nutrient supply-demand imbalances, critical for diabetes and cancer. Unexpectedly, we show here that, in gastrointestinal cancer cells, glucose activates AMPK to selectively induce EP300, but not CREB-binding protein (CBP). Consequently, EP300 is redirected away from nuclear receptors that promote differentiation towards ß-catenin, a driver of proliferation and colorectal tumorigenesis. Importantly, blocking glycogen synthesis permits reactive oxygen species (ROS) accumulation and AMPK activation in response to glucose in previously nonresponsive cells. Notably, glycogen content and activity of the ROS/AMPK/EP300/ß-catenin axis are opposite in healthy versus tumor sections. Glycogen content reduction from healthy to tumor tissue may explain AMPK switching from tumor suppressor to activator during tumor evolution.

From obesity to diabetes and cancer: epidemiological links and role of therapies.

Increasing evidence suggests a complex relationship between obesity, diabetes and cancer. Here we review the evidence for the association between obesity and diabetes and a wide range of cancer types. In many cases the evidence for a positive association is strong, but for other cancer types a more complex picture emerges with some site-specific cancers associated with obesity but not to diabetes, and some associated with type I but not type II diabetes. The evidence therefore suggests the existence of cumulative common and differential mechanisms influencing the relationship between these diseases. Importantly, we highlight the influence of antidiabetics on cancer and antineoplastic agents on diabetes and in particular that antineoplastic targeting of insulin/IGF-1 signalling induces hyperglycaemia that often evolves to overt diabetes. Overall, a coincidence of diabetes and cancer worsens outcome and increases mortality. Future epidemiology should consider dose and time of exposure to both disease and treatment, and should classify cancers by their molecular signatures. Well-controlled studies on the development of diabetes upon cancer treatment are necessary and should identify the underlying mechanisms responsible for these reciprocal interactions. Given the global epidemic of diabetes, preventing both cancer occurrence in diabetics and the onset of diabetes in cancer patients will translate into a substantial socioeconomic benefit.

Androgenetic alopecia as an early marker of benign prostatic hyperplasia.

BACKGROUND: Androgenetic alopecia (AGA) and benign prostatic hyperplasia are both androgen-dependent entities that respond to the blocking of 5-alpha-reductase. OBJECTIVES: The objective of this study was to determine whether prostatic volumes and urinary flow changes were higher in patients with early-onset AGA than in healthy control subjects. METHODS: This was an observational case-control study of 87 men: 45 with early-onset AGA diagnosed in the dermatology department and 42 control subjects. End-point variables were prostatic volume, measured by transrectal ultrasound, and urinary flow, measured by urinary flowmetry. A hormone study was performed on all participants, and the International Prostate Symptom Score and International Index of Erectile Function score were determined. RESULTS: The groups did not significantly differ in mean age (cases, 52.7 years vs control subjects, 49.8 years; P = .12). Patients with AGA had significantly higher mean prostate volume (29.65 vs 20.24 mL, P < .0001), International Prostate Symptom Score (4.93 vs 1.23, P < .0001), and prostate-specific antigen value (1.53 vs 0.94 ng/mL, P < .0001) and significantly lower maximum urinary flow (14.5 vs 22.45 mL/s, P < .0001) versus control subjects. Binary logistic regression analysis showed a strong association between the presence of AGA and benign prostatic hyperplasia after adjusting for age, urinary volume, urination time, International Prostate Symptom Score, abdominal obesity, glucose levels, systolic blood pressure, insulin levels, fibrinogen, and C-reactive protein (odds ratio = 5.14, 95% confidence interval 1.23-47.36, P = .041). LIMITATIONS: The study of larger sample sizes would facilitate stratified analyses according to the Ebling type of androgenetic alopecia. CONCLUSION: There is a relationship between the presence of AGA and prostate growth-associated urinary symptoms, likely attributable to their pathophysiological similarity. This study suggests that early-onset AGA may be an early marker of urinary/prostatic symptomatology. Future studies may clarify whether treatment of patients with AGA may benefit the concomitant benign prostatic hypertrophy, which would be present at an earlier stage in its natural evolution.

Effect of non-steroidal anti-inflammatory drugs on the histology of basal cell carcinomas.

BACKGROUND: Aggressive histology is not rare in BCC. Large studies from referral centers report incidences of aggressive histology BCC ranging from 2.5- 44%. These aggressive BCC are characterized by subclinical extension, invasive behavior, local recurrence and challenging treatment. OBJECTIVES: To examine the association between non-steroidal anti-inflammatory drug (NSAID) use and the different histological subtypes of basal cell carcinoma (BCC). METHODS: The design was a nested case-control study. The two population-based cohorts were of patients with a primary BCC diagnosis during January and May 2010 (n=136) and NSAID use in the 15 years prior to baseline. All the lesions were excised and analyzed to determinate the histological subtype of BCC as aggressive or non-aggressive. Odds ratios (ORs) and 95% confidence intervals (CIs), using conditional logistic regression, were calculated with the SPSS software to estimate the association of aggressive histological subtypes of BCC and use of NSAID. We controlled the potential confounding factors. RESULTS: The rate of non-aggressive BCC associated with exposure to NSAID was increased (OD: 0.34; 95% CI: 0.14-0.84) after adjusting for covariants. LIMITATIONS: our sample is small. We collected data regarding use of NSAID over a wide time ranges, so that we are unable to propose when the potential benefits of NSAID on the histology of BCC would happen. CONCLUSION: According to our data, NSAID exposure is associated with a decreased risk of aggressive BCC.

Sex hormone-binding globulin and risk of hyperglycemia in patients with androgenetic alopecia.

BACKGROUND: Low circulating levels of sex hormone-binding globulin (SHBG) are a strong predictor of the risk of type 2 diabetes. Androgenetic alopecia (AGA) has been related to an increase in cardiovascular risk, but the mechanism of this association has not been elucidated. AGA can be associated with low levels of SHBG and insulin resistance, which could be related to hyperglycemia and type 2 diabetes. OBJECTIVE: The objective of this study was to evaluate SHBG and blood glucose levels in men and women with early-onset AGA and control subjects to determine whether low levels of SHBG are associated with hyperglycemia. METHODS: This case-control study included 240 patients consecutively admitted to the outpatient clinic (Dermatology Department of San Cecilio University Hospital, Granada, Spain), 120 with early-onset AGA (60 men and 60 women) and 120 control subjects (60 men and 60 women) with skin diseases other than alopecia. RESULTS: Of patients with AGA, 39.1% presented with hyperglycemia (>110 mg/dL) versus 12.5% of controls (P < 0.0001). AGA patients with hyperglycemia or diabetes presented lower significant levels of SHBG than alopecic patients without hyperglycemia or type 2 diabetes, respectively. Patients with AGA and hyperglycemia presented significantly lower levels of SHBG than controls with hyperglycemia (22.3 vs 39.4 nmol/L for AGA patients and controls, respectively, P = .004). No significant differences in SHBG levels were noticed between patients and controls without hyperglycemia. Binary logistic regression showed a strong association between lower SHBG levels and glucose levels greater than 110 mg/dL in patients with AGA even after additional adjustment for sex, abdominal obesity, and free testosterone (odds ratio = 3.35; 95% confidence interval = 1.9-5.7; P < .001). LIMITATIONS: The study of a wider sample of AGA patients would confirm these findings and would permit analysis of the pathogenic mechanisms underlying the increase in cardiovascular risk in patients with AGA. CONCLUSION: An association between early-onset AGA, hyperglycemia/diabetes, and low levels of SHBG was observed in the current study. Low levels of SHBG could be a marker of insulin resistance and hyperglycemia/diabetes in patients with AGA.

Metabolic and hormonal remodeling of colorectal cancer cell signalling by diabetes.

The existence of molecular links that facilitate colorectal cancer (CRC) development in the population with type 2 diabetes (T2D) is supported by substantial epidemiological evidence. This review summarizes how the systemic, metabolic and hormonal imbalances from T2D alter CRC cell metabolism, signalling and gene expression as well as their reciprocal meshing, with an overview of CRC molecular subtypes and animal models to study the diabetes-CRC cancer links. Metabolic and growth factor checkpoints ensure a physiological cell proliferation rate compatible with limited nutrient supply. Hyperinsulinaemia and hyperleptinaemia in prediabetes and excess circulating glucose and lipids in T2D overcome formidable barriers for tumour development. Increased nutrient availability favours metabolic reprogramming, alters signalling and generates mutations and epigenetic modifications through increased reactive oxygen species and oncometabolites. The reciprocal control between metabolism and hormone signalling is lost in diabetes. Excess adipose tissue at the origin of T2D unbalances adipokine (leptin/adiponectin) secretion ratios and function and disrupts the insulin/IGF axes. Leptin/adiponectin imbalances in T2D are believed to promote proliferation and invasion of CRC cancer cells and contribute to inflammation, an important component of CRC tumourigenesis. Disruption of the insulin/IGF axes in T2D targets systemic and CRC cell metabolic reprogramming, survival and proliferation. Future research to clarify the molecular diabetes-CRC links will help to prevent CRC and reduce its incidence in the diabetic population and must guide therapeutic decisions.

Remodelling of colorectal cancer cell signalling by microbiota and immunity in diabetes.

Obesity is the strongest known risk factor to develop type 2 diabetes (T2D) and both share a state of chronic, diffuse and low-grade inflammation, impaired immune responses and alterations in the composition and function of the microbiome. Notably, these hallmarks are shared with colorectal cancer (CRC), which is epidemiologically associated to obesity and T2D. Gut barrier damages in T2D destabilize the microbiome that metabolizes the diet and modulates the host immune response triggering inflammatory and proliferative pathways. In this review, we discuss the pathways altered by defects in the immune response and microbiota that may link T2D to CRC development. Stressed adipocytes, metabolic incongruity in blood and gut barrier failure with dysbiosis cooperate to establish imbalances between immune innate and adaptive cells and cytokines such as interleukin 6 (IL6) or TNFA that define low-grade diffuse inflammation in T2D. Inflammation drives tissue repair through proliferation and migration (critical mechanisms for tumourigenesis) and under physiological conditions feeds anti-inflammatory cytokine production to resolve the process. The disproportion in pro- vs anti-inflammatory cells and cytokines imposed by T2D will impact the tumour micro- and macro-environment, favouring tumour proliferation, angiogenesis and decreased immune responses. Complex bidirectional relationships between the metabolic environment of T2D, gut microbiota, and immune dysfunctions may favour tumour cell demands and will define the outcome. Animal models developed to study the relationships between T2D and CRC in the context of microbiota and immune system are discussed.

Androgenetic alopecia and cardiovascular risk factors in men and women: a comparative study.

BACKGROUND: Numerous studies in recent decades have associated male androgenetic alopecia (AGA) with the risk of cardiovascular disease. However, only 3 studies have addressed this association in female patients. Most studies considered the risk of myocardial infarction or mortality as a result of heart disease, without analyzing cardiovascular risk factors. OBJECTIVES: The objectives of this study were to analyze the presence of cardiovascular risk factors included in the Adult Treatment Panel-III criteria for metabolic syndrome, the prevalence of carotid atheromatosis, hormonal (aldosterone, insulin, testosterone, and sex hormone-binding globulin) factors, and acute phase reactant (C-reactive protein, fibrinogen, D-dimers, erythrocyte sedimentation rate) variables in male and female patients with AGA and in a control group, and to analyze differences among the groups. METHODS: This case-control study included 154 participants, 77 with early-onset AGA (40 male and 37 female) and 77 healthy control subjects (40 male and 37 female) from the dermatology department at a university hospital in Granada, Spain. RESULTS: Metabolic syndrome was diagnosed in 60% of male patients with AGA (odds ratio [OR] = 10.5, 95% confidence interval [CI] 3.3-32.5), 48.6% of female patients with AGA (OR = 10.73, 95% CI 2.7-41.2), 12.5% of male control subjects, and 8.1% of female control subjects (P < .0001). Atheromatous plaques were observed in 32.5% of male patients with AGA (OR = 5.93, 95% CI 1.5-22.9) versus 7.5% of male control subjects (P = .005) and 27% of female patients with AGA (OR = 4.19, 95% CI 1.05-16.7) versus 8.1% of female control subjects (P = .032). Aldosterone and insulin levels were significantly higher in the male and female patients with AGA versus their respective control subjects. Mean values of fibrinogen were significantly higher in male patients with AGA, whereas values of fibrogen, C-reactive protein, and D-dimers were significantly higher in female patients with AGA versus their respective control subjects. LIMITATIONS: The study of a wider sample of patients with AGA would confirm these findings and allow a detailed analysis of the above factors as a function of the degree of alopecia or between menopausal and premenopausal women. CONCLUSION: The determination of metabolic syndrome and ultrasound study of the carotid arteries may be useful screening methods to detect risk of developing cardiovascular disease in male and female patients with early-onset AGA and signal a potential opportunity for early preventive treatment.

A comparative study of dyslipidaemia in men and woman with androgenic alopecia.

Several studies have analyzed the relationship between androgenetic alopecia and cardiovascular disease (mainly heart disease). However few studies have analyzed lipid values in men and women separately. This case-control study included 300 patients consecutively admitted to an outpatient clinic, 150 with early onset androgenetic alopecia (80 males and 70 females) and 150 controls (80 males and 70 females) with other skin diseases. Female patients with androgenic alopecia showed significant higher triglycerides values (123.8 vs 89.43 mg/dl, p = 0.006), total cholesterol values (196.1 vs 182.3 mg/dl, p = 0.014), LDL-C values (114.1 vs 98.8 mg/dl, p = 0.0006) and lower HDL-C values (56.8 vs 67.7 mg/dl, p <0.0001) versus controls respectively. Men with androgenic alopecia showed significant higher triglycerides values (159.7 vs 128.7 mg/dl, p = 0.04) total cholesterol values (198.3 vs 181.4 mg/dl, p = 0.006) and LDL-C values (124.3 vs 106.2, p = 0.0013) versus non-alopecic men. A higher prevalence of dyslipidemia in women and men with androgenic alopecia has been found. The elevated lipid values in these patients may contribute, alongside other mechanisms, to the development of cardiovascular disease in patient with androgenic alopecia.

Engineering hollow mesoporous silica nanoparticles to increase cytotoxicity.

Hollow mesoporous silica nanoparticles (HMSNs) consist of a network of cavities confined by mesoporous shells that have emerged as promising tools for drug delivery or diagnostic. The physicochemical properties of HMSNs are dictated by the synthesis conditions but which conditions affect which property and how it impacts on biological interactions is unclear. Here by changing the concentration of the structure-directing agent (SDA), the pH and the ratio between SDA and added salt (NaCl) we determine the effects in size, morphology, surface charge and density or degree of compaction (physicochemical properties) of HMSNs and define their impact on their biological interactions with human colon cancer or healthy cells at the level of cellular uptake and viability. Increased size or density/degree of compaction of HMSNs increases their cytotoxicity. Strikingly, high salt concentrations in the synthesis medium leads to a spiky-shell morphology that provokes nuclear fragmentation and irreversible cell damage turning HMSNs lethal and unveiling intrinsic therapeutic potential. This strategy may open new avenues to design HMSNs nanoarchitectures with intrinsic therapeutic properties without incorporation of external pharmaceutical ingredients.

Syringocystoadenoma papilliferum associated with apocrine hidrocystoma and verruca.

Syringocystoadenoma papilliferum is a benign adnexal tumor usually located in head and neck that occurs during childhood or adolescence. A case of a syringocystoadema papilliferum associated with apocrine hydrocystoma and verruca is presented. It is unusual to see the occurrence of three histopathologic types of tumors coexisting in one cutaneous lesion.

Basal cell carcinoma arising in outdoor workers versus indoor workers: a retrospective study.

Basal cell carcinoma (BCC) is the most prevalent malignancy in white individuals and continues to be a serious health problem. Individuals who have sustained exposure to UV radiation are at the highest risk for developing BCC. The aim of this study was to compare the features of BCC in outdoor workers (OWs) with a history of occupational exposure to UV radiation versus indoor workers (IWs). We found that OWs are more likely to develop nodular BCC with no increased risk for superficial BCC. The age of onset of BCC was older in OWs than in IWs. Truncal BCC was more common in IWs, which may suggest other etiological factors are involved in BCC such as genetic predisposition.

Epidemiological bases and molecular mechanisms linking obesity, diabetes, and cancer. / Bases epidemiológicas y mecanismos moleculares implicados en las asociaciones de obesidad y diabetes con cáncer.

The association between diabetes and cancer was hypothesized almost one century ago. Today, a vast number of epidemiological studies support that obese and diabetic populations are more likely to experience tissue-specific cancers, but the underlying molecular mechanisms remain unknown. Obesity, diabetes, and cancer share many hormonal, immune, and metabolic changes that may account for the relationship between diabetes and cancer. In addition, antidiabetic treatments may have an impact on the occurrence and course of some cancers. Moreover, some anticancer treatments may induce diabetes. These observations aroused a great controversy because of the ethical implications and the associated commercial interests. We report an epidemiological update from a mechanistic perspective that suggests the existence of many common and differential individual mechanisms linking obesity and type 1 and 2 diabetes mellitus to certain cancers. The challenge today is to identify the molecular links responsible for this association. Classification of cancers by their molecular signatures may facilitate future mechanistic and epidemiological studies.

Basal cell carcinoma: analysis of factors associated with incomplete excision at a referral hospital in southern Spain.

Basal cell carcinoma (BCC) is the most prevalent malignancy, with excision as the best therapeutic approach. Incomplete excision of nonmelanoma skin cancer is a clinical indicator of the surgical technique performed. This retrospective study of 292 patients with BCC assessed the rate of incomplete excision in a tertiary referral hospital in southern Spain and its relationship with tumor location as well as histologic and surgical features.

Dual specificity phosphatase 1 expression inversely correlates with NF-κB activity and expression in prostate cancer and promotes apoptosis through a p38 MAPK dependent mechanism.

Dual specificity phosphatase 1 (DUSP1) and the transcription factor NF-κB are implicated in prostate cancer since their expression levels are altered along this disease, although there are no evidences up to date demonstrating a crosstalk between them. In this report, we show for the first time that DUSP1 over-expression in DU145 cells promotes apoptosis and decreases NF-κB activity by blocking p65/NF-κB nuclear translocation. Moreover, although DUSP1 impairs TNF-α-induced p38 MAPK and JNK activation, only the specific inhibition of p38 MAPK exerts the same effects than DUSP1 over-expression on both apoptosis and NF-κB activity. Consistently, DUSP1 promotes apoptosis and decreases NF-κB activity in cells in which p38 MAPK is induced by TNF-α treatment. These results demonstrate that p38 MAPK is specifically involved in DUSP1-mediated effects on both apoptosis and NF-κB activity. Interestingly, we show an inverse correlation between DUSP1 expression and activation of both p65/NF-κB and p38 MAPK in human prostate tissue specimens. Thus, most of apparently normal glands, benign prostatic hyperplasia and low-grade prostatic intraepithelial neoplasia samples show high DUSP1 expression and low levels of both nuclear p65/NF-κB and activated p38 MAPK. By contrast, DUSP1 expression levels are low or even absent in high-grade prostatic intraepithelial neoplasia and prostatic adenocarcinoma samples, whereas nuclear p65/NF-κB and activated p38 MAPK are highly expressed in the same samples. Overall, our results provide evidence for a role of DUSP1 in the apoptosis of prostate cancer cells, through a mechanism involving the inhibition of p38 MAPK and NF-κB. Furthermore, our findings suggest that the ratio between DUSP1 and p65/NF-κB expression levels, rather than the individual expression of both molecules, is a better marker for diagnostic purposes in prostate cancer.

Factors related to delay in the diagnosis of basal cell carcinoma.

BACKGROUND: There is often a delay between the clinical emergence of a basal cell carcinoma (BCC) and the point in time at which the patient presents for definitive diagnosis and treatment. Previously published studies on delays regarding skin cancer have focused on melanoma rather than BCC. We conducted a study aimed at identifying factors associated with the detection of BCC and reasons for the delay in diagnosis. METHOD: A monocentric study was performed. Patients with a primary BCC diagnosed in 2010 were included in the study. They were asked about factors concerning BCC awareness and detection, tumor characteristics, previous history of nonmelanoma cutaneous cancer, family history of nonmelanoma cutaneous cancer, and the presence of comorbidities. Data were analyzed using SPSS software. RESULTS: The mean diagnostic delay for BCC in our hospital setting was estimated at 19.79 ± 14.71 months. Delayed diagnosis was significantly associated with patients over 65 years, those without a previous history of BCC, those without a family history of BCC, those with BCC located elsewhere than the head or neck, and those with lesions not associated with itching or bleeding. CONCLUSION: This study revealed considerable delay in the diagnosis of BCC. The main reason for delay in the diagnosis seems to be the initial decision of the patient to seek medical advice. These data suggest a need for greater information for the general public on the symptoms and signs that should prompt suspicion of a BCC.

Alcohol intake and risk of aggressive histological basal cell carcinoma: a case-control study.

BACKGROUND: Aggressive basal cell carcinomas (BCC) are not rare. These subtypes of skin cancer are characterized by an infiltrative behavior and rapid progression. Often, management may be difficult. Recent evidence suggests that minimal UV exposure in combination with other behavioral and/or environmental factors may lead to higher incidence of BCC and, therefore, more risk of aggressive subtypes of this malignancy. Alcohol is a very commonly consumed beverage in Western societies, especially in association with outdoors activities. OBJECTIVE: To investigate a possible relationship between alcohol intake and aggressive histological variants of BCC. MATERIALS AND METHOD: We designed a prospective study. Patients who underwent surgery for BCC in our hospital were interviewed to collect data regarding alcohol intake. The specimens were reviewed by a pathologist and classified into aggressive and non-aggressive subtypes. Statistical analysis was performed using SPSS software. RESULTS: 136 patients were included. Of participants with aggressive BCCs, 10 (26.3%) were abstainers, 4 (10.4%) had light consumption, 18 (47.5%) moderate consumption and 6 (15.8%) heavy consumption, while among participants with non-aggressive BCCs, 57 (58.2%) were abstainers, 29 (29.5%) had light consumption, 10 (10.2%) moderate consumption and 2 (2.1%) heavy consumption. In the multivariate analysis we found a positive significant association between alcohol consumption and the presence of aggressive BBCs. CONCLUSIONS: According to our results, alcohol intake may be linked with a higher incidence of aggressive subtypes of BCC.

Bases epidemiológicas y mecanismos moleculares implicados en las asociaciones de obesidad y diabetes con cáncer / Epidemiological bases and molecular mechanisms linking obesity, diabetes, and cancer

Hace casi un siglo que se hipotetizó la asociación entre la diabetes y el cáncer. Hoy, numerosos estudios epidemiológicos sostienen que las poblaciones con obesidad y/o diabetes poseen una mayor predisposición a padecer cáncer en órganos específicos. Los mecanismos moleculares subyacentes se desconocen. Las alteraciones metabólicas, hormonales e inmunológicas que comparten la obesidad, la diabetes y el cáncer pueden contribuir a justificar la relación existente. Por otra parte, la influencia de los tratamientos antidiabéticos en la aparición/evolución de algunos cánceres y la inducción de la diabetes por los tratamientos antineoplásicos han despertado una gran controversia debido a las implicaciones éticas y los intereses comerciales asociados. Esta actualización de los datos epidemiológicos presenta un enfoque mecanístico que sugiere la existencia de múltiples mecanismos comunes y diferenciales que asocian la obesidad y la diabetes tipo1 y tipo2 a ciertos cánceres. Identificar los mecanismos responsables de la asociación diabetes-cáncer es un reto de la investigación actual; la clasificación de los cáncer por sus firmas moleculares podría facilitar futuros estudios mecanísticos y epidemiológicos (AU)

The association between diabetes and cancer was hypothesized almost one century ago. Today, a vast number of epidemiological studies support that obese and diabetic populations are more likely to experience tissue-specific cancers, but the underlying molecular mechanisms remain unknown. Obesity, diabetes, and cancer share many hormonal, immune, and metabolic changes that may account for the relationship between diabetes and cancer. In addition, antidiabetic treatments may have an impact on the occurrence and course of some cancers. Moreover, some anticancer treatments may induce diabetes. These observations aroused a great controversy because of the ethical implications and the associated commercial interests. We report an epidemiological update from a mechanistic perspective that suggests the existence of many common and differential individual mechanisms linking obesity and type 1 and 2 diabetes mellitus to certain cancers. The challenge today is to identify the molecular links responsible for this association. Classification of cancers by their molecular signatures may facilitate future mechanistic and epidemiological studies (AU)