Optogenetic control of cancer cell survival in ChR2-transfected HeLa cells.

Optogenetics is a molecular biological technique involving transfection of cells with photosensitive proteins and the subsequent study of their biological effects. The aim of this study was to evaluate the effect of blue light on the survival of HeLa cells, transfected with channelrhodopsin-2 (ChR2). HeLa wild-type cells were transfected with a plasmid that contained the gene for ChR2. Transfection and channel function were evaluated by real-time polymerase chain reaction (RT-PCR), fluorescence imaging using green fluorescent protein (GFP) and flow cytometry for intracellular calcium changes using a Fura Red probe. We developed a platform for optogenetic stimulation for use within the cell culture incubator. Different stimulation procedures using blue light (467 nm) were applied for up to 24 h. Cell survival was determined by flow cytometry using propidium iodide and rhodamine probes. Change in cell survival showed a statistically significant (p < 0.05) inverse association with the frequency and time of application of the light stimulus. This change seemed to be associated with the ChR2 cis-trans-isomerization cycle. Cell death was associated with high concentrations of calcium in the cytoplasm and stimulation intervals less than the period of isomerization. It is possible to transfect HeLa cells with ChR2 and control their survival under blue light stimulation. We suggest that this practice should be considered in the future development of optogenetic systems in biological or biomedical research.

Molecular weight and guluronic/mannuronic ratio of alginate produced by Azotobacter vinelandii at two bioreactor scales under diazotrophic conditions.

Alginates can be used to elaborate hydrogels, and their properties depend on the molecular weight (MW) and the guluronic (G) and mannuronic (M) composition. In this study, the MW and G/M ratio were evaluated in cultures of Azotobacter vinelandii to 3 and 30 L scales at different oxygen transfer rates (OTRs) under diazotrophic conditions. An increase in the maximum OTR (OTRmax) improved the alginate production, reaching 3.3 ± 0.2 g L-1. In the cultures conducted to an OTR of 10.4 mmol L-1 h-1 (500 rpm), the G/M increased during the cell growth phase and decreased during the stationary phase; whereas, in the cultures at 19.2 mmol L-1 h-1 was constant throughout the cultivation. A higher alginate MW (520 ± 43 kDa) and G/M ratio (0.86 ± 0.01) were obtained in the cultures conducted at 10.4 mmol L-1 h-1. The OTR as a criterion to scale up alginate production allowed to replicate the concentration and the alginate production rate; however, it was not possible reproduce the MW and G/M ratio. Under a similar specific oxygen uptake rate (qO2) (approximately 65 mmol g-1 h-1) the alginate MW was similar (approximately 365 kDa) in both scales. The evidences revealed that the qO2 can be a parameter adequate to produce alginate MW similar in two bioreactor scales. Overall, the results have shown that the alginate composition could be affected by cellular respiration, and from a technological perspective the evidences contribute to the design process based on oxygen consumption to produce alginates defined.

Poly(3-hydroxybutyrate) accumulation by Azotobacter vinelandii under different oxygen transfer strategies.

Azotobacter vinelandii OP is a bacterium that produces poly(3-hydroxybutyrate) (PHB). PHB production in a stirred bioreactor, at different oxygen transfer strategies, was evaluated. By applying different oxygen contents in the inlet gas, the oxygen transfer rate (OTR) was changed under a constant agitation rate. Batch cultures were performed without dissolved oxygen tension (DOT) control (using 9% and 21% oxygen in the inlet gas) and under DOT control (4%) using gas blending. The cultures that developed without DOT control were limited by oxygen. As result of varying the oxygen content in the inlet gas, a lower OTR (4.6 mmol L-1 h-1) and specific oxygen uptake rate (11.6 mmol g-1 h-1) were obtained using 9% oxygen in the inlet gas. The use of 9% oxygen in the inlet gas was the most suitable for improving the intracellular PHB content (56 ± 6 w w-1). For the first time, PHB accumulation in A. vinelandii OP cultures, developed with different OTRs, was compared under homogeneous mixing conditions, demonstrating that bacterial respiration affects PHB synthesis. These results can be used to design new oxygen transfer strategies to produce PHB under productive conditions.

Evidence for the facultative intracellular behaviour of the fish pathogen Vibrio ordalii.

Vibrio ordalii is an extracellular, Gram-negative bacterium that produces vibriosis in salmonids. While pathogenesis is not fully understood, this bacterium has numerous likely genes for adhesion, colonization, invasion factors and, as recently suggested, intracellular behaviour. Therefore, this study aimed to clarify possible intracellular behaviour for V. ordalii Vo-LM-18 and ATCC 33509T in the fish-cell lines SHK-1 and CHSE-214. Confocal microscopy revealed Vo-LM-18 and ATCC 33509T inside cytoplasm in both fish-cell lines at 4 hr post-inoculation (hpi). At 8 and 16 hpi, the proportion of fish cells invaded by both strains increased. Moreover, intracellular V. ordalii were observed after 8 hpi inside mouse embryonic fibroblasts (MEF), demonstrating that entry was not due to a cellular phagocytosis process. Flow cytometry confirmed immunocytochemistry results, with both V. ordalii evidencing statistically significant differences in the number of infected cells between 8 and 16 hpi. Interestingly, V. ordalii infection did not significantly damage fish cells, as determined by LDH liberation. Viable counts at 8 hpi detected, on average for both lines, 176 ± 47 CFU/ml of culturable intracellular Vo-LM-18 and ATCC 33509T cells. These in vitro findings support the facultative intracellular behaviour of V. ordalii and may be of importance for understanding pathogenicity and survival in aquatic environments.

Default Mode Network, Meditation, and Age-Associated Brain Changes: What Can We Learn from the Impact of Mental Training on Well-Being as a Psychotherapeutic Approach?

Aging is a physiological process accompanied by cognitive decline, principally in memory and executive functions. Alterations in the connectivity of the default mode network (DMN) have been found to participate in cognitive decline, as well as in several neurocognitive disorders. The DMN has antisynchronic activity with attentional networks (task-positive networks (TPN)), which are critical to executive function and memory. Findings pointing to the regulation of the DMN via activation of TPN suggest that it can be used as a strategy for neuroprotection. Meditation is a noninvasive and nonpharmacological technique proven to increase meta-awareness, a cognitive ability which involves the control of both networks. In this review, we discuss the possibility of facilitating healthy aging through the regulation of networks through meditation. We propose that by practicing specific types of meditation, cognitive decline could be slowed, promoting a healthy lifestyle, which may enhance the quality of life for the elderly.

Proteostasis and Mitochondrial Role on Psychiatric and Neurodegenerative Disorders: Current Perspectives.

Proteostasis involves processes that are fundamental for neural viability. Thus, protein misfolding and the formation of toxic aggregates at neural level, secondary to dysregulation of the conservative mechanisms of proteostasis, are associated with several neuropsychiatric conditions. It has been observed that impaired mitochondrial function due to a dysregulated proteostasis control system, that is, ubiquitin-proteasome system and chaperones, could also have effects on neurodegenerative disorders. We aimed to critically analyze the available findings regarding the neurobiological implications of proteostasis on the development of neurodegenerative and psychiatric diseases, considering the mitochondrial role. Proteostasis alterations in the prefrontal cortex implicate proteome instability and accumulation of misfolded proteins. Altered mitochondrial dynamics, especially in proteostasis processes, could impede the normal compensatory mechanisms against cell damage. Thereby, altered mitochondrial functions on regulatory modulation of dendritic development, neuroinflammation, and respiratory function may underlie the development of some psychiatric conditions, such as schizophrenia, being influenced by a genetic background. It is expected that with the increasing evidence about proteostasis in neuropsychiatric disorders, new therapeutic alternatives will emerge.

Walking Speed, Risk Factors, and Cardiovascular Events in Older Adults-Systematic Review.

Fonseca Alves, DJ, Bartholomeu-Neto, J, Júnior, ER, Ribeiro Zarricueta, BS, Nóbrega, OT, and Córdova, C. Walking speed, risk factors, and cardiovascular events in older adults-systematic review. J Strength Cond Res 31(11): 3235-3244, 2017-It is important that new clinical measures can identify risk factors and predict cardiovascular events. Although the walking speed (WS) test is a potential candidate, consolidating data from multiple studies is required to determine comparative references. We examined the associations of WS measures with markers of cardiovascular risk and with cardiovascular events in noninstitutionalized subjects older than 60 years. A systematic review of observational studies was conducted using MEDLINE and SCOPUS from inception of the databases to December 2014, aiming at studies that evaluated WS as the primary outcome (usual or maximal pace) within a distance ≤20 m associated with cardiovascular health. All 15 included studies (29,845 subjects) reported significant associations of WS with different cardiovascular risk factors (coronary artery calcification, C-reactive protein, hypertension, diabetes, and intima-media thickness) and occurrence of cardiovascular events (peripheral artery disease, stroke, and mortality). Approximately 80% of the studies used a distance ≤6 m and WS at usual pace. There was high heterogeneity in the risk thresholds established by different studies. Our results suggest usefulness of the WS test as a tool for cardiovascular risk stratification in older adults. However, the variation in speed thresholds and diversity of protocols among studies suggest caution when generalizing results to different older adult populations. Because the WS test is a simple, cheap, and safe tool to administer, we make suggestions for its standardization in future studies.

Functional capacity and obesity reflect the cognitive performance of older adults living in long-term care facilities.

BACKGROUND: With the increase in life expectancy, the elderly Brazilian population has grown considerably. However, longevity is usually accompanied by problems such as the loss of functional capacity, cognitive decline, and deterioration in anthropometric parameters, particularly among those living in long-term care facilities. We analyzed the relationship among cognitive performance, anthropometry, and functional capacity in institutionalized elderly individuals. METHODS: In a sample of 38 older adults (20 men, 18 women) aged 73.4 ± 8.1 years, data regarding anthropometric and functional measurements as well as the Mini-Mental State Examination were collected. Functional tests were based on a battery adapted from the Functional Fitness Test. The independent t-test and χ2 test were applied. P < 0.05 was set as the significance threshold. RESULTS: The rate of cognitive impairment among obese individuals (78.6%) differed from that among eutrophic subjects (45.8%), but there was no difference between sexes. There was a significant positive correlation between handgrip strength and Mini-Mental State Examination score, and the scores of the timed up-and-go test and the sitting-rising test were negatively correlated with Mini-Mental State Examination score. CONCLUSION: Cognitive impairment in the elderly living in long-term care facilities was more pronounced and frequent in obese individuals. This study also demonstrated that better functional capacity reflects better cognitive performance.

Habitual dietary intake and mediators of the inflammaging process in Brazilian older women.

AIM: This study aimed to assess whether the habitual intake of macronutrients by older women associates with circulating levels of important inflammaging mediators by means of a cross-sectional design with 229 Brazilian elderly women. METHODS: Laboratory tests determined serum IL1α, IL1ß, IL6, IL8, IL10, IL12 and TNFα by specific immunoassays. Food records of three alternate days were decomposed into usual intake of carbohydrates, proteins and lipids (and fractions), as well as total energy value (TEV) per patient. Moreover, the study has identified and controlled results for metabolic conditions known to influence the inflammatory profile: hypercholesterolemia, hypertension and diabetes. RESULTS AND DISCUSSION: Pearson's correlation test revealed that log10IL8 expressed a positive association with levels of saturated fatty acid (FA) (r = 0.173; p = 0.009) and total cholesterol intake (r = 0.223; p = 0.001). Similar analysis of the other mediators revealed no association with dietary intake. CONCLUSION: Higher intakes of total cholesterol and saturated FA seem to correlate with increased serum IL8 levels, being a possible mechanism by which this pro-atherogenic intake pattern may increase the risk of age-related chronic diseases with important inflammatory contribution.

Serum Immune Mediators Independently Associate with Atherosclerosis in the Left (But Not Right) Carotid Territory of Older Individuals.

BACKGROUND AND AIM: Disturbance in the carotid arteries strongly predicts cerebrovascular events and correlates with a systemic inflammatory milieu. We investigated the relationship of a profile of 10 circulating inflammatory mediators with measures of carotid intima-media thickness (cIMT) in elderly subjects, taking traditional risk factors into account. METHODS: Clinical inspection for present and past chronic conditions and events, as well as biochemical and anthropometric measurements, was performed for patients in ambulatory setting. Scores of cIMT were obtained bilaterally in the distal common carotid artery wall. Serum concentrations of cytokines were assessed by bead-based, multiplexed flow cytometry immunoassays. RESULTS: Correlation analysis between log-transformed cytokines levels implicated the mediators interleukin-1ß (IL1ß), IL6, IL8, IL10, and tumor necrosis factor-α (TNFα) (P ≤ .005) with scores of the left cIMT. Stepwise multivariate regression showed that TNFα, IL1ß, and IL6 levels accounted for most of the variance in the cIMT scores. Comparison of cytokine levels across increasing tertiles of the left cIMT reproduced the positive association with TNFα and IL1ß levels. CONCLUSION: Five out of ten immune mediators independently correlated with cIMT of older subjects in a territory-sensitive manner. This possible contribution of immune mediators to an atherosclerotic process probably relates to the inflammaging process.

Lack of association between apolipoprotein E genotypes and cognitive performance in the non-demented elderly.

AIM: The ε4 alelle of the apolipoprotein E gene is known to be a key genetic risk factor for Alzheimer's disease and possibly for other neurological disorders. Some evidence in the literature indicates that the ε4 allele interferes with human cognition independently of chronological age and diagnosis of Alzheimer's disease. The present study investigated the correlation of allelic variants of apolipoprotein E with the cognitive performance of elderly individuals without apparent cognitive impairment. METHODS: This was a cross-sectional analysis that included 213 non-demented elderly individuals (age ≥60 years) from the Brazilian Federal District. The analysis assessed the subjects for cognitive domains including short- and long-term episodic memory, processing speed, and attention and executive functions. Sociodemographic and other clinical characteristics were gathered and analyzed as covariates. RESULTS: Being sufficiently powered, the present study did not identify differential performance across apolipoprotein E genotypes. There was no influence of age, gender, marital status, schooling, depressive symptoms or use of central nervous system depressants when the analyses were controlled for such factors. CONCLUSIONS: Our findings suggest that the ε4 allele does not contribute to detectable cognitive decline within the context of non-dementia.

Osteopontin in bone mineral density of very old Brazilians.

Recent evidence suggests that changes in plasma levels of osteopontin (OPN) may be a promising marker for early diagnosis of bone disorders. We hypothesized that a frequent OPN gene polymorphism may be useful for identifying very old individuals with alterations in plasma OPN levels and consequently at risk of abnormal bone density scores. Men and women (80 years or older) living in the Brazilian Federal District underwent assessments with dual energy X-ray absorptiometry for bone mineral density (BMD) of the femoral neck, femoral head and lumbosacral (L1 to S5) regions. Clinical inspection was performed to assess other physical traits and to exclude co-morbidities (cardiovascular, autoimmunity, infections or neoplastic diseases). Serum concentrations of OPN were determined with an enzyme-linked immunosorbent assay, whereas the A7385G polymorphism (rs1126772) was determined by direct sequencing of a polymerase chain reaction product. Among the two hundred and ten subjects enrolled, no differential scores for bone mineral density could be observed across genotypes, but a greater content of circulating OPN was found among carriers of the A allele (P ≤ 0.05) even after adjustments. Serum OPN levels were negatively correlated with femoral neck density (P = 0.050 for BMD; P = 0.032 for T scores) but not with scores of the other regions investigated. Analyses with the sample dichotomized to age and body mass revealed that this inverse relationship was noticeable only among those aged within the highest and weighing within the lowest intervals. Our findings indicate elevated circulating osteopontin levels in patients with decreased bone mineral density, consistent with a modest contribution of an OPN allelic variation to its expression. Assessing the clinical relevance of our findings demands forthcoming studies.

Cytokine gene polymorphisms and Alzheimer's disease in Brazil.

BACKGROUND: Single-nucleotide polymorphisms in genes encoding immunological mediators can affect the biological activity of these molecules by regulating transcription, translation, or secretion, modulating the genetic risk of inflammatory damage in Alzheimer's disease (AD). Nonetheless, the Brazilian contingent is highly admixed, and few association trials performed herein with AD patients have considered genetic ancestry estimates as co-variables when investigating markers for this complex trait. METHODS: We analyzed polymorphisms in 10 inflammatory genes and compared the genotype distribution across outpatients with late-onset AD and noncognitively impaired subjects from Midwest Brazil under a strict criterion, and controlling for ancestry heritage and ApoE genotype. RESULTS: Our findings show an almost 40% lower chance of AD (p = 0.004) among homozygotes of the IL10 -1082A allele (rs1800896). Dichotomization to ApoE and mean ancestry levels did not affect protection, except among those with greater European or minor African heritage. CONCLUSION: The IL10 locus seems to affect the onset of AD in a context sensitive to the genetic ancestry of Brazilian older adults.

Exercise intensity modulates nitric oxide and blood pressure responses in hypertensive older women.

BACKGROUND AND AIMS: Whether intensity or other characteristics of physical activity can better promote the release of nitric oxide (NO) and reduction of blood pressure in hypertensive older-adults is still unknown. In this study, the post-exercise blood pressure (BP) response and NO release after different intensities of aerobic exercise in elderly women were analyzed. METHODS: Blood pressure response and NO were analyzed in 23 elderly mildly hypertensive women. Participants underwent (1) high-intensity incremental exercise (IT); (2) moderate-intensity 20 min exercise at 90% of the anaerobic threshold (AT), and (3) control (CONT) session. BP was measured before and after interventions; volunteers remained seated for 1 h. NO estimates were made through NO2- analyses. RESULTS: After CONT session, both diastolic BP and mean arterial pressure (MAP) were significantly higher than during pre-exercise resting. Post-exercise hypotension (PEH) was observed after exercise at IT and 90% of AT. Although exercise in both sessions lowered SBP and MAP compared with CONT, exercise at the highest intensity (IT) was more effective on lowering systolic BP after exercise. In comparison with pre-exercise resting, NO2- increased significantly only after IT, but both exercise sessions caused NO2- to increase compared with CONT. CONCLUSION: Exercise intensity and NO release may exert a role in eliciting PEH in mildly hypertensive elderly women.

HER2 classification in breast cancer cells: A new explainable machine learning application for immunohistochemistry.

The immunohistochemical (IHC) evaluation of epidermal growth factor 2 (HER2) for the diagnosis of breast cancer is still qualitative with a high degree of inter-observer variability, and thus requires the incorporation of complementary techniques such as fluorescent in situ hybridization (FISH) to resolve the diagnosis. Implementing automatic algorithms to classify IHC biomarkers is crucial for typifying the tumor and deciding on therapy for each patient with better performance. The present study aims to demonstrate that, using an explainable Machine Learning (ML) model for the classification of HER2 photomicrographs, it is possible to determine criteria to improve the value of IHC analysis. We trained a logistic regression-based supervised ML model with 393 IHC microscopy images from 131 patients, to discriminate between upregulated and normal expression of the HER2 protein. Pathologists' diagnoses (IHC only) vs. the final diagnosis complemented with FISH (IHC + FISH) were used as training outputs. Basic performance metrics and receiver operating characteristic curve analysis were used together with an explainability algorithm based on Shapley Additive exPlanations (SHAP) values to understand training differences. The model could discriminate amplified IHC from normal expression with better performance when the training output was the IHC + FISH final diagnosis (IHC vs. IHC + FISH: area under the curve, 0.94 vs. 0.81). This may be explained by the increased analytical impact of the membrane distribution criteria over the global intensity of the signal, according to SHAP value interpretation. The classification model improved its performance when the training input was the final diagnosis, downplaying the weighting of the intensity of the IHC signal, suggesting that to improve pathological diagnosis before FISH consultation, it is necessary to emphasize subcellular patterns of staining.

Synthesis of fluorescent chalcones, photophysical properties, quantitative structure-activity relationship and their biological application.

The development of fluorescent pigments is an area of interest in several research fields due to their high sensitivity. In the current study-eight known and three new N,N-dimethylamino-chalcones (12a-k) were synthesized with good yields using the Claisen-Schmidt reaction. For each molecular system, the photophysical properties, including the maximum absorption wavelength (λAbsorption), molar absorption coefficient (ε), maximum excitation wavelength (λExcitation), maximum emission wavelength (λEmission), Stokes Shift (Δλ), fluorescence quantum yield (Φfl), fluorescence lifetime (τfl), radiative and non-radiative rate constants (kR and kNR, respectively) were evaluated. Variations in each of these properties were analyzed depending on the substituents present on each compound. To relate the chemical structures of the synthesized compounds to their photophysical properties, Hansch analysis (2D-QSPR) was applied. As a result of Hansch analysis, we found different photophysical properties related to molecular orbitals and the energy of their derivatives (Highest Occupied Molecular Orbital-HOMO, Lowest Unoccupied Molecular Orbital-LUMO, Difference between LUMO-HOMO-ΔLH, Chemical potential-µ, Hardness-η, Softness-S, and electrophilic global index-ω) as well as to the atomic charges on atoms C5, Cα, Cß, and CO. The application of this type of analysis has made it possible to understand and subsequently design new molecules with defined photophysical properties. Finally, the compounds were use as fluorescent pigment to get living cell imaging on breast cancer cells, obtaining the compound 12a as promissory alternative.

Amerindian genetic ancestry protects against Alzheimer's disease.

BACKGROUND: Alzheimer's disease (AD) is the most common form of dementia worldwide, and bears remarkable evidence for a differential prevalence among continental populations. In this scenario, estimating ancestry proportions in recently admixed populations is a strategy that can help increasing knowledge about the genetic structure of this complex trait. AIM/METHODS: Our purpose was to assess mean ancestry estimates for the three main parental contributors to the Brazilian contingent (European, African and Amerindian) using a panel of 12 ancestry informative markers. Outpatients with the late-onset form of AD (n = 120) were compared for ancestry levels with non-cognitively impaired subjects (n = 412) in the Midwest Brazil, controlling for classic clinical, social and anthropometric risk factors. RESULTS: Our findings show a 3-fold greater genetic Amerindian content among control subjects compared to AD patients (p < 0.001). CONCLUSION: Our results suggest that the allelic architecture of Native Americans can confer protection against the onset of the disease.

Usual dietary intake and cardiovascular risk factors in older Brazilian women.

AIM: To evaluate habitual macronutrient intake and its association with common cardiovascular risk factors in Brazilian elderly women. METHODS: Analytical cross-sectional study with 293 subjects. Carbohydrate, protein and lipid intakes were determined based on a non-consecutive three-day dietary record. The following conditions were evaluated: dyslipidemia, systemic arterial hypertension, and type 2 diabetes. RESULTS: Anthropometric, clinical and biochemical data revealed an elevated prevalence of classic cardiovascular risk factors in the sample. Higher energy intake from omega-3 fatty acid was associated with elevated levels of high-density lipoprotein cholesterol (p<0.05), whereas a diet pattern with a relatively lower energy content from monounsaturated fatty acids was associated with the presence of type 2 diabetes (p<0.05). CONCLUSIONS: Results corroborate experimental reports and contribute by suggesting that the usual diet, independently of supplementation, may be valuable in promoting health and preventing chronic diseases of aging.

Methods of body mass reduction by combat sport athletes.

The aim of this study was to investigate the methods adopted to reduce body mass (BM) in competitive athletes from the grappling (judo, jujitsu) and striking (karate and tae kwon do) combat sports in the state of Minas Gerais, Brazil. An exploratory methodology was employed through descriptive research, using a standardized questionnaire with objective questions self-administered to 580 athletes (25.0 ± 3.7 yr, 74.5 ± 9.7 kg, and 16.4% ± 5.1% body fat). Regardless of the sport, 60% of the athletes reported using a method of rapid weight loss (RWL) through increased energy expenditure. Strikers tend to begin reducing BM during adolescence. Furthermore, 50% of the sample used saunas and plastic clothing, and only 26.1% received advice from a nutritionist. The authors conclude that a high percentage of athletes uses RWL methods. In addition, a high percentage of athletes uses unapproved or prohibited methods such as diuretics, saunas, and plastic clothing. The age at which combat sport athletes reduce BM for the first time is also worrying, especially among strikers.

Estrogen, Cognitive Performance, and Functional Imaging Studies: What Are We Missing About Neuroprotection?

Menopause transition can be interpreted as a vulnerable state characterized by estrogen deficiency with detrimental systemic effects as the low-grade chronic inflammation that appears with aging and partly explains age-related disorders as cancer, diabetes mellitus and increased risk of cognitive impairment. Over the course of a lifetime, estrogen produces several beneficial effects in healthy neurological tissues as well as cardioprotective effects, and anti-inflammatory effects. However, clinical evidence on the efficacy of hormone treatment in menopausal women has failed to confirm the benefit reported in observational studies. Unambiguously, enhanced verbal memory is the most robust finding from longitudinal and cross-sectional studies, what merits consideration for future studies aiming to determine estrogen neuroprotective efficacy. Estrogen related brain activity and functional connectivity remain, however, unexplored. In this context, the resting state paradigm may provide valuable information about reproductive aging and hormonal treatment effects, and their relationship with brain imaging of functional connectivity may be key to understand and anticipate estrogen cognitive protective effects. To go in-depth into the molecular and cellular mechanisms underlying rapid-to-long lasting protective effects of estrogen, we will provide a comprehensive review of cognitive tasks used in animal studies to evaluate the effect of hormone treatment on cognitive performance and discuss about the tasks best suited to the demonstration of clinically significant differences in cognitive performance to be applied in human studies. Eventually, we will focus on studies evaluating the DMN activity and responsiveness to pharmacological stimulation in humans.