The Titrated Mannitol Improved Central [99mTc] Tc TRODAT-1 Uptake in an Animal Model-A Clinically Feasible Application.

[99mTc]Tc TRODAT-1 is a widely used single photon emission tomography (SPECT) radiopharmaceutical in Asian practice for early detection of central dopaminergic disorders. However, its imaging quality remains sub-optimal. To overcome this problem, mannitol, an osmotic agent was used to observe its effect on improving striatal [99mTc]Tc TRODAT-1 uptake in rat brain by titrated human dosages to investigate a clinically feasible way to improve human imaging quality. [99mTc]Tc TRODAT-1 synthesis and quality control were performed as described. Sprague-Dawley rats were used for this study. The animal in vivo nanoSPECT/CT and ex vivo autoradiography were employed to observe and verify the striatal [99mTc]Tc TRODAT-1 uptake in rat brains using clinically equivalent doses (i.e., 0, 1 and 2 mL groups, each n = 5) of mannitol (20% w/v, equivalent to 200 mg/mL) by an intravenous administration. Specific binding ratios (SBRs) were calculated to express the central striatal uptake in different experimental groups. In the NanoSPECT/CT imaging, the highest SBRs of striatal [99mTc]Tc TRODAT-1 were reached at 75-90 min post-injection. The averaged striatal SBRs were 0.85 ± 0.13 (2 mL normal saline, the control group), 0.94 ± 0.26 (1 mL mannitol group) and 1.36 ± 0.12 (2 mL mannitol group, p < 0.01 which were significantly different than the control as well as 1 mL mannitol groups (p < 0.05). The SBRs from ex vivo autoradiography also showed a comparable trend of the striatal [99mTc]Tc TRODAT-1 uptake in the 2 mL, 1 mL mannitol and the control groups (1.76 ± 0.52, 0.91 ± 0.29, and 0.21 ± 0.03, respectively, p < 0.05). No remarkable changes of vital signs were found in the mannitol groups and the controls. Pre-treated mannitol revealed a significant increase of the central striatal [99mTc]Tc TRODAT-1 uptake in a rat model which not only enabled us to perform pre-clinical studies of dopaminergic related disorders but also provided a potential way to further optimize image quality in clinical practice.

Prevalence and psychosocial risk factors associated with current cigarette smoking and hazardous alcohol drinking among adolescents in Taiwan.

BACKGROUND: Cigarette smoking and alcohol drinking are the most common types of substance use and misuse (SUM) among adolescents. This study aimed to estimate the prevalence and psychosocial factors associated with current cigarette smoking and hazardous alcohol drinking among adolescents in Taiwan. METHODS: Data were collected via self-administered questionnaires on computers from students at 14 senior high schools in Taipei, Taiwan. Hierarchical multiple regression strategies were used to determine the risk factors for SUM. RESULTS: A total of 5879 participants were recruited, the majority of whom were female (56.7%). The prevalence rates of current smoking and hazardous alcohol drinking were 3.84% and 7.38%, respectively. Risk factors associated with current smoking were similar to those for hazardous alcohol drinking, including male gender, low school ranking, and depression. In addition, current smoking was associated with increasing age, hazardous alcohol drinking, and fewer parents with whom they can talk, whereas hazardous alcohol drinking was associated with current smoking, not living with both biological parents, and more peers with whom they can talk. CONCLUSION: The potential coexistence of adolescent SUM and common psychosocial correlates demands an integrated approach. Health professionals should provide corresponding intervention programs and coordinate with parents and teachers to develop an anti-SUM environment, especially for males and high-risk schools. Preventive psychiatric services as an integral part of anti-SUM strategies for adolescents targeting to depression may be useful in reducing the risk.

Cyclodextrin- and dendrimer-conjugated graphene oxide as a nanocarrier for the delivery of selected chemotherapeutic and photosensitizing agents.

In this study, nanohybrid materials consisting of graphene oxide (GO), ߭cyclodextrin (CD) and poly(amido amine) dendrimer (DEN) were successfully prepared by covalent bonding. GO-CD and GO-CD-DEN were found to be potential nanocarriers for anticancer drugs including chemotherapeutics (doxorubicin (DOX), camptothecin (CPT)) and photosensitizer (protoporphyrin IX (PpIX)). GO-CD possessed 1.2 times higher DOX-loading capacity than GO due to inclusion of additional DOX to the CD. The drug loading on GO-CD-DEN increased in the order: DOX < PpIX < CPT. Enhanced cytotoxicity of DOX and CPT and also the photocytotoxicity of PpIX were observed when the drugs were loaded in GO-CD and GO-CD-DEN. Functionalization of GO with CD and CD-DEN increased the uptake in cancer cells, and both GO-CD and GO-CD-DEN nanohybrids remained in the cytoplasm and were not uptaken into the nucleus, as shown in the flow cytometry and high-content screening study.

Using nomogram of the Barcelona Clinic Liver Cancer system for treatment selection in patients with stage C hepatocellular carcinoma.

BACKGROUND: The nomogram of the Barcelona Clinic Liver Cancer (BCLC) for hepatocellular carcinoma (HCC) has been used for outcome prediction. Patients with BCLC stage C HCC often undergo anti-cancer therapy against current treatment guidelines in real world practice. We aimed to use the nomogram to provide guidance on treatment selection for BCLC stage C patients. METHODS: A total of 1317 patients with stage C HCC were retrospectively analyzed and divided into four groups by nomogram points. One-to-one matched pairs between patients receiving different treatments were generated by the propensity score with matching model within these groups. Survival analysis was performed by Kaplan-Meier method with log-rank test. RESULTS: Patients with higher nomogram points were more often treated with targeted or supportive therapies (p <  0.001). Patients receiving targeted or supportive therapies had a decreased survival compared to patients undergoing aggressive treatments (surgical resection, ablation, transarterial chemo-embolization or transplantation) across all four groups (p <  0.001). After matching for baseline differences in the propensity model, patients receiving different treatments had comparable age, gender, etiology of liver disease, tumor burden, severity of cirrhosis and performance status. Survival analyses were re-performed and disclosed that patients with nomogram points < 15 had better overall outcome after aggressive treatments (p <  0.05). For patients with nomogram points > 15, there was no significant difference in survival between patients receiving two different treatment strategies. CONCLUSIONS: The nomogram of BCLC system is a feasible tool to help stage C HCC patients to select primary anti-cancer treatment in pursuance of better overall survival.

Impact of tumor burden on prognostic prediction for patients with terminal stage hepatocellular carcinoma: A nomogram study.

BACKGROUND: The recently proposed nomogram of Barcelona Clinic Liver Cancer (BCLC) lacks predictive accuracy for patients with stage D hepatocellular carcinoma (HCC). Tumor burden is crucial in prognostic prediction but is not included in the criteria of stage D HCC. This study aims to develop a nomogram with tumor burden as the core element for BCLC stage D patients. METHODS: A total of 386 patients were randomly grouped into derivation and validation sets (1:1 ratio). The multivariate Cox proportional hazards model was used to select factors with significant prognostic effect and generate the nomogram. Concordance indices and calibration plots were used to evaluate the performance of nomogram. RESULTS: Overall survival of study patients was significantly associated with tumor burden as well as hepatitis B, serum α-fetoprotein level, cirrhosis and performance status in multivariate Cox regression (all p<0.05). Beta-coefficients of these variables in derivation set were used to generate the nomogram. Each patient was assigned with a total nomogram point that predicted individualized 6-month and 1-year survival. The derivation and validation sets had a c-index of 0.759 (95% confidence interval [CI]: 0.552-0.923) and 0.741 (95% CI: 0.529-0.913), respectively. The calibration plots were close to the 45-degree line for 6-month and 1-year survival prediction for all quarters of patients in both derivation and validation sets. CONCLUSION: Tumor burden is significantly associated with the outcome for patients with stage D HCC. The tumor burden-incorporated nomogram may serve as a feasible and easy-to-use tool in predicting survival on an individual level.

Prognostic role of noninvasive liver reserve markers in patients with hepatocellular carcinoma undergoing transarterial chemoembolization.

BACKGROUND: Various noninvasive liver reserve markers were proposed to indicate the severity of liver damage. However, the role and feasibility of these markers to predict the prognosis of patients with hepatocellular carcinoma (HCC) are unknown. We aimed to identify the prognostic role of the 8 currently used hepatic reserve markers in patients with HCC undergoing transarterial chemoembolization (TACE). METHODS: Between 2002 and 2013, a total of 881 patients with HCC undergoing TACE were prospectively identified and retrospectively analyzed. The baseline characteristics, tumor status and noninvasive markers were collected. Homogeneity and corrected Akaike information criteria (AICc) were compared between these markers. The Cox proportional hazards model was used to identify independent predictors of survival. RESULTS: Significant differences in survival distribution were found for albumin-bilirubin (ALBI) grade, Child-Turcotte-Pugh (CTP) class, Lok index, fibrosis index based on 4 factors (FIB-4), Göteborg University cirrhosis index (GUCI), cirrhosis discriminant index (CDI) and model for end-stage liver disease (MELD) score (all p values <0.05). Among these markers, the ALBI grade showed the highest homogeneity and lowest AICc value, indicating a better prognostic performance. Cox multivariate analysis confirmed that ALBI grade 2, ascites, serum alkaline phosphatase and α-fetoprotein level, tumor diameter, vascular invasion and performance status were significant independent prognostic predictors. The distribution of the ALBI score well correlated with baseline CTP and MLED scores. CONCLUSIONS: Our data suggest that among the currently used liver reserve markers, ALBI grade may serve as an objective and feasible surrogate to predict the prognosis of HCC patients undergoing TACE.

Active Treatments Prolong the Survival in Patients With Hepatocellular Carcinoma and Performance Status 3 or 4: A Propensity Score Analysis.

GOALS AND BACKGROUNDS: Best supportive care is suggested as the standard treatment for hepatocellular carcinoma (HCC) patients with performance status (PS) 3-4 by the Barcelona Clinic Liver Cancer (BCLC) system. To investigate the rationale of treatment allocation. STUDY: A total of 2660 HCC patients were reviewed. One-to-one matched pairs between PS 3 and 4 patients receiving supportive care and anti-HCC treatments were generated by using the propensity score with matching model. The survival analysis was performed with the Kaplan-Meier method and log-rank test. The hazard ratio was calculated with the Cox proportional hazards model. RESULTS: Among 328 patients with PS 3-4, 38% of patients received active anti-HCC treatments against the BCLC system. Compared with patients undergoing supportive care, patients receiving anti-HCC treatments more often had milder cirrhosis, smaller tumor burden, and lower serum α-fetoprotein levels (all P<0.05). Patients undergoing supportive care had significantly decreased survival (P<0.0001). With propensity scores, 101 pairs of similar HCC patients with PS 3-4 were selected from different treatment groups. They were comparable in age, sex, etiologies of liver disease, severity of cirrhosis, tumor burden, and prevalence of diabetes mellitus (all P>0.05) at baseline. In the matching model, patients with PS 3-4 undergoing supportive care had significantly shortened survival with an adjusted hazard ratio of 4.711 (confidence interval: 3.041-7.297, P<0.0001). CONCLUSIONS: Over one-third of patients with PS 3-4 receive active anti-HCC treatments against the BCLC allocation algorithm in this study. Active anticancer therapies rather than best supportive care should be performed if there is no apparent contraindication.

Self-aggregation of amphiphilic [60]fullerenyl focal point functionalized PAMAM dendrons into pseudodendrimers: DNA binding involving dendriplex formation.

In this research, we successfully performed a "click" synthesis of amphiphilic poly(amido amine) dendron-bearing fullerenyl conjugate (C60 G1 ) using a copper(I)-catalyzed azide-alkyne cycloaddition reaction. The strong hydrophobicity of the C60 moiety induces self-assembly of C60 G1 into core-shell-like "pseudodendrimers" with a uniform size distribution and positively charged peripherals. The pseudodendrimers were well-characterized by atomic force microscopy (AFM), transmission electron microscopy, and dynamic light scattering. On the basis of electrostatic interactions, the polycationic C60 G1 assembly can serve as a nonviral gene vector. An ethidium bromide displacement assay and agarose gel electrophoresis both indicated that C60 G1 assembly forms stable complexes with the cyclic reporter gene (pEGFP-C1) at low nitrogen-to-phosphorous (N/P) ratios. AFM analysis revealed a dynamic complex-formation process, and confirmed the synthesis of C60 G1 /pEGFP-C1 hybrids with a particle dimensions less than 200 nm. Fluorescence microscopy and flow cytometry revealed that 51% of HeLa and 43% of MCF-7 cells are positive to the YOYO-1-labeled hybrids at an N/P ratio of 2, being comparable to TurboFect-mediated delivery.

Impact of renal insufficiency on patients with hepatocellular carcinoma undergoing radiofrequency ablation.

BACKGROUND AND AIM: Renal insufficiency (RI) is commonly seen in patients with hepatocellular carcinoma (HCC). We aimed to investigate the impact of RI on the long-term survival of HCC patients undergoing radiofrequency ablation (RFA) and to determine the optimal staging strategy for these patients. METHODS: RI was defined as an estimated glomerular filtration rate < 60 mL/min/1.73 m(2) . A total of 123 and 344 patients with and without RI undergoing RFA, respectively, were enrolled. A one-to-one propensity score matching analysis with preset caliper width was performed. The prognostic ability of four currently used staging systems was compared by the Akaike information criterion (AIC). RESULTS: HCC patients with RI undergoing RFA were older (P < 0.001) and had significantly different baseline characteristics. Of all patients, RI was significantly associated with a decreased long-term survival (P = 0.03). After matching in the propensity model, the baseline characteristics were similar between patients with (n = 92) and without (n = 92) RI. In the propensity model, RI was not significantly associated with a shortened survival (P = 0.273). In the Cox multivariate analysis, Child-Turcotte-Pugh class B or C was identified as the only independent predictor of poor prognosis. Among patients with RI undergoing RFA, the Taipei Integrated Scoring (TIS) system provided the highest homogeneity and lowest AIC value among the currently used staging systems. CONCLUSIONS: The long-term survival of HCC patients undergoing RFA is not affected by RI. The TIS staging system may provide a better prognostic prediction for HCC patients with RI undergoing RFA.

Aggressive therapeutic strategies improve the survival of hepatocellular carcinoma patients with performance status 1 or 2: a propensity score analysis.

BACKGROUND: Targeted therapy or chemotherapy is suggested as standard treatment for hepatocellular carcinoma (HCC) patients with performance status (PS) 1-2 according to the Barcelona Clinic Liver Cancer (BCLC) system. The underlying rationales have not been fully studied. METHODS: This study enrolled 2,620 HCC patients. One-to-one matched pairs between HCC patients receiving aggressive anti-HCC treatments (resection, transplantation, ablation, and transarterial chemoembolization) and those receiving targeted therapy or chemotherapy or best supportive care were generated by using the propensity score with a matching model. Survival analysis was performed with the Kaplan-Meier method and the log-rank test. Mortality risk was calculated with the Cox proportional hazards model. RESULTS: Of 793 patients with PS 1-2, 64 % received aggressive anti-HCC treatments against the suggestion of the BCLC system. The patients receiving aggressive anti-HCC treatments had significantly milder cirrhosis, a smaller tumor burden, and better long-term survival than the patients undergoing targeted therapy or chemotherapy or best supportive care (all p < 0.05). With the use of propensity scores, 166 pairs of matched HCC patients with PS 1-2 were selected from different treatment groups. After matching, patients were comparable in age, gender, severity of cirrhosis, tumor burden, and prevalence of diabetes mellitus (all p > 0.05) at baseline. In the propensity score model, patients with PS 1-2 undergoing aggressive anti-HCC treatments had significantly better long-term survival (p < 0.0001). The adjusted hazard ratio of the choice for targeted therapy or chemotherapy or best supportive care to the choice for aggressive anti-HCC treatments was 2.028 (p < 0.0001). CONCLUSIONS: According to the findings, HCC patients with PS 1-2 should consider aggressive anticancer treatments if no contraindication is noted. Adjustment of the BCLC treatment allocation is needed to enhance its prognostic accuracy.

18F-fluorodeoxyglucose positron emission tomography for the initial evaluation and monitoring of therapeutic response in bilateral adrenal histoplasmosis.

(18)F-fluorodeoxyglucose positron emission tomography (FDG-PET) of a 74-year-old man showed high FDG uptake in bilateral adrenal histoplasmosis. Four months after the administration of an appropriate antifungal treatment, we observed a significant decrease in FDG uptake into the lesions, with a 40% reduction in activity (maximal standardized uptake value). This imaging result indicated partial resolution of the disease and was consistent with clinical outcome. Our study results suggest that FDG-PET is a useful modality for initial whole-body evaluation, selection of an appropriate antifungal treatment regimen, and monitoring of therapeutic response in bilateral adrenal histoplasmosis.

Risk factors for falling in psychiatric inpatients: a prospective, matched case-control study.

Falling is one the most common types of inpatient adverse events. Most fall-related research was conducted retrospectively and focused on elderly population in general hospital settings. This study aimed to timely identify all potential risk factors associated with falls and fall-related injury in a psychiatric inpatient setting. We recruited 145 fall events and 145 sex- and room-matched psychiatric control inpatients without fall in a 1002-bed psychiatric teaching hospital in northern Taiwan. In addition to medical records, the study variables included patient characteristics, circumstances and medications, which were collected from the patients and/or their families within 24 h of receiving reports right after obtaining written informed consent. A psychiatrist and three head nurses conducted a comprehensive assessment of risk factors immediately after falls occurred. A conditional logistic regression model revealed four variables significantly associated with an increased risk of falling: the clinical global impression-severity (adjusted odds ratio (aOR) = 2.19; 95% confidence interval, CI = 1.13-4.24), the parkinsonism scores of the extrapyramidal syndrome rating scale (aOR = 1.14; 95% CI = 1.08-1.21), equivalent dosage of benzodiazepines use (aOR = 1.15; 95% CI = 1.03-1.30), and medication changes within 24 h (aOR = 10.3; 95% CI = 1.37-76.8). Acute settings (aORs = 2.06, 95% CI = 1.01-4.18), a fall history in the past six months and a lack of history of medical problems (aORs = 3.04; 95% CI = 1.46-6.33) were associated with fall-related injury (aOR = 2.70; 95% CI = 1.29-5.69). Our study identified the severity of psychotic symptoms, extrapyramidal symptoms, medications usage and other several specific measures for prevention of falls in psychiatric inpatient settings.

Different involvement of ventral and dorsal norepinephrine pathways on norepinephrine reuptake inhibitor-induced locomotion and antidepressant-like effects in rats.

Two experiments were performed to investigate the involvements of ventral and dorsal norepinephrine bundle (VNB and DNB) in a selective norepinephrine reuptake inhibitor (NRI) reboxetine (RBX)-induced locomotion and antidepressant-like effects. Rats in the Experiment 1 received a local brain injection of 6-hydroxydopamine (6-OHDA) or an intraperitoneal injection of N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4) to lesion their VNB or DNB, respectively. Acute effects of RBX (10 mg/kg, i.p.) on the locomotor activity and the forced swim test (FST) were measured 14 days after the lesion. In Experiment 2, activities of the norepinephrine transporter (NET) and the protein kinase C (PKC) in cortex, hippocampus, and locus coeruleus (LC) were detected by western blot after 14 days chronic RBX treatment. Results showed that the antidepressant-like effect of RBX was blocked in VNB lesion but augmented in DNB lesion, and was not relevant to the RBX-induced hypoactivity. Fourteen-days RBX treatment altered the activities of NET and PKC in LC but not hippocampus or cortex. The possible involvement of VNB and DNB in the RBX-induced antidepressant-like effect is discussed.

Uterine cervical melanoma presenting with rapid progression detected by PET/CT.

Malignant melanoma of the uterine cervix is a rare extracutaneous melanoma which develops aggressively and is associated with a bleak prognosis. To our knowledge, no prior published reports have discussed the role of 18F-FDG positron emission tomography/computed tomography (PET/CT) in managing this disease. Our case study involved a 66-year-old woman with a malignant melanoma of the uterine cervix. The patient received PET/CT that identified metastases and lesions which had not been detected from her MRI. Serial PET/CT elucidated that the disease was initially limited to the pelvis, but then metastasized to the abdominal para-aortic lymph nodes, followed by extensive metastases to the brain, lungs, breast, supraclavicular, neck, and other abdominal lymph nodes, as observed at 6-month follow-up. PET/CT was used to complement conventional anatomic imaging modalities, and provided a novel modality for whole body screening. Visualization of the metabolic activity of indeterminate lesions may help in staging, re-staging, treatment planning, and prognostic prediction for patients with this rare disease.

Intrinsically fluorescent PAMAM dendrimer as gene carrier and nanoprobe for nucleic acids delivery: bioimaging and transfection study.

This study successfully evaluated gene delivery and transfection toward rat C6 glioma cell lines mediated by intrinsic blue fluorescent poly(amido amine) (PAMAM) dendrimer. We used three antisense oligonucleotides, (AS-ODN) p75, NGF1, and NGF2 for knocking down specific protein expressions. The three oligonucleotides were electrostatically associated with the photoluminescent amino-terminated PAMAM dendrimer to yield fluorescent complexes at various nitrogen-to-phosphorus (N/P) ratios. Compared with pristine PAMAM dendrimer and hyperbranched polyethylenimine (PEI), the fluorescent PAMAM dendrimer revealed lower in vitro cytotoxicity toward C6 cells, allowing us to transfect the cells with the AS-ODN complexes under a higher N/P ratio. Due to the intrinsic fluorescence, cellular uptake behavior could be directly analyzed by fluorescence microscopy and flow cytometry, without additional fluorescence labeling. As expected, the result clearly suggested that the uptake efficiency increased as the N/P value increased. Furthermore, the quantified data obtained from flow cytometry indicated relatively higher uptake efficiency for the p75 complex, which is mainly due to different association patterns between the fluorescent dendrimer and AS-ODNs. At N/P = 20, atomic force microscopic analysis confirmed that the p75 complex formed well-condensed, spherical particles with dimensions less than 200 nm, but that NGF2 AS-ODN associated poorly with the dendrimer. Finally, Western blot analysis indicated that these complexes were capable of knocking down the specific protein expression to a certain level, being comparable to the hyperbranched PEI-mediated gene transfection. Our preliminary results clearly indicated that intrinsic fluorescent PAMAM dendrimers show promise as gene vehicles that can achieve delivery, transfection, and bioimaging at the same time.

Early identification of poor responders to transarterial chemoembolization for hepatocellular carcinoma.

PURPOSE: Transarterial chemoembolization (TACE) is used to treat unresectable hepatocellular carcinoma (HCC). However, HCC patients may have an even shorter survival after TACE. This study aimed to identify poor responders to TACE at an early stage. PATIENTS AND METHODS: A total of 624 and 122 patients with HCC undergoing TACE and best supportive care (BSC), respectively, were analyzed. Poor responders were defined as patients who died after TACE or had viable tumor(s), but not eligible for further treatment at 3 months of treatment. RESULTS: A total of 102 (16%) patients were identified as poor responders. Poor responders had a significantly decreased long-term survival than other patients receiving TACE and a tendency of higher risk of mortality than patients receiving BSC (p < 0.001 and p = 0.054, respectively). The comparison of 24-month survival showed significantly worse outcome in poor responders than patients receiving BSC (p = 0.04). Serum α-fetoprotein (AFP) level >40 ng/mL (p = 0.024) and albumin level 3.8 g/dL (p = 0.016), Child-Turcotte-Pugh (CTP) class B (p = 0.011), performance status 1 (p < 0.001), total tumor volume (TTV) >65 cm(3) (p = 0.001), and vascular invasion (p = 0.005) were independent risk factors predicting poor response at 3 months in the multivariate logistic regression analysis. Among the four HCC staging systems, the Barcelona Clinic Liver Cancer (BCLC) classification showed the highest predictive accuracy for the identification of poor responders. CONCLUSIONS: Poor responders have an increased risk of mortality due to rapid disease progression after TACE. Advanced BCLC stages may better predict a poor response to TACE.

Developmental expression of genes involved in neural estrogen biosynthesis and signaling in the brain of the orange-spotted grouper Epinephelus coioides during gonadal sex differentiation.

In the brain, the synthesis of neurosteroids and receptor activation during gonadal sex differentiation in teleosts are poorly understood. In the present study, the protogynous orange-spotted grouper (Epinephelus coioides) was selected as a model fish, and we hypothesized that de novo synthesis of neural estrogen may play a role in the female grouper brain during gonadal sex differentiation. We investigated the temporal expression of the genes StAR, cyp19a1b and pcna and the sex steroid nuclear receptors for estrogen (ERα, ERß1 and ERß2), androgen (AR) and the plasma membrane-associated estrogen receptor (GPR30) in the brain during early developmental ages from 90 days after hatching (dah) to 180dah after gonadal sex differentiation. Our results revealed that mRNA for ERs and GPR30 but not AR was significantly increased at 110dah (a time close to gonadal sex differentiation) in the forebrain and midbrain and for cyp19a1b at 110dah in the forebrain. Brain aromatase activity and estradiol (E2) levels, but not testosterone (T), were increased in the forebrain at 110 and 120dah, respectively. Furthermore, exogenous E2 stimulated cyp19a1b transcripts in the forebrain and hypothalamus and immunoreactive (ir)Cyp19a1b (aromatase enzyme) in the forebrain. irCyp19a1b localized in the glial cells of the forebrain regions. Therefore, we identified a peak of functional aromatase activity and estrogen signaling in the early grouper brain during gonadal sex differentiation. Moreover, pcna transcripts (a marker for cell proliferation activity) were higher in the early brain at 110-150dah. Thus, a peak time of development in the brain is suggested to occur during gonadal sex differentiation in the grouper.