Atrophy of the cholinergic regions advances from early to late mild cognitive impairment.

PURPOSE: We investigated the volumetric changes in the components of the cholinergic pathway for patients with early mild cognitive impairment (EMCI) and those with late mild cognitive impairment (LMCI). The effect of patients' apolipoprotein 4 (APOE-ε4) allele status on the structural changes were analyzed. METHODS: Structural magnetic resonance imaging data were collected. Patients' demographic information, plasma data, and validated global cognitive composite scores were included. Relevant features were extracted for constructing machine learning models to differentiate between EMCI (n = 312) and LMCI (n = 541) and predict patients' neurocognitive function. The data were analyzed primarily through one-way analysis of variance and two-way analysis of covariance. RESULTS: Considerable differences were observed in cholinergic structural changes between patients with EMCI and LMCI. Cholinergic atrophy was more prominent in the LMCI cohort than in the EMCI cohort (P < 0.05 family-wise error corrected). APOE-ε4 differentially affected cholinergic atrophy in the LMCI and EMCI cohorts. For LMCI cohort, APOE-ε4 carriers exhibited increased brain atrophy (left amygdala: P = 0.001; right amygdala: P = 0.006, and right Ch123, P = 0.032). EMCI and LCMI patients showed distinctive associations of gray matter volumes in cholinergic regions with executive (R2 = 0.063 and 0.030 for EMCI and LMCI, respectively) and language (R2 = 0.095 and 0.042 for EMCI and LMCI, respectively) function. CONCLUSIONS: Our data confirmed significant cholinergic atrophy differences between early and late stages of mild cognitive impairment. The impact of the APOE-ε4 allele on cholinergic atrophy varied between the LMCI and EMCI groups.

Macrophage circadian rhythms are differentially affected based on stimuli.

Macrophages are white blood cells that play disparate roles in homeostasis and immune responses. They can reprogram their phenotypes to pro-inflammatory (M1) or anti-inflammatory (M2) states in response to their environment. About 8-15% of the macrophage transcriptome has circadian oscillations, including genes closely related to their functioning. As circadian rhythms are associated with cellular phenotypes, we hypothesized that polarization of macrophages to opposing subtypes might differently affect their circadian rhythms. We tracked circadian rhythms in RAW 264.7 macrophages using luminescent reporters. Cells were stably transfected with Bmal1:luc and Per2:luc reporters, representing positive and negative components of the molecular clock. Strength of rhythmicity, periods and amplitudes of time series were assessed using multiple approaches. M1 polarization decreased amplitudes and rhythmicities of Bmal1:luc and Per2:luc, but did not significantly affect periods, while M2 polarization increased periods but caused no substantial alterations to amplitudes or rhythmicity. As macrophage phenotypes are also altered in the presence of cancer cells, we tested circadian effects of conditioned media from mouse breast cancer cells. Media from highly aggressive 4T1 cells caused loss of rhythmicity, while media from less aggressive EMT6 cells yielded no changes. As macrophages play roles in tumors, and oncogenic features are associated with circadian rhythms, we tested whether conditioned media from macrophages could alter circadian rhythms of cancer cells. Conditioned media from RAW 264.7 cells resulted in lower rhythmicities and periods, but higher amplitudes in human osteosarcoma, U2OS-Per2:luc cells. We show that phenotypic changes in macrophages result in altered circadian characteristics and suggest that there is an association between circadian rhythms and macrophage polarization state. Additionally, our data demonstrate that macrophages treated with breast cancer-conditioned media have circadian phenotypes similar to those of the M1 subtype, and cancer cells treated with macrophage-conditioned media have circadian alterations, providing insight to another level of cross-talk between macrophages and cancer.

The Effect of the APOE-ε4 Allele on the Cholinergic Circuitry for Subjects With Different Levels of Cognitive Impairment.

Background: Cholinergic deficiency has been suggested to associate with the abnormal accumulation of Aß and tau for patients with Alzheimer's disease (AD). However, no studies have investigated the effect of APOE-ε4 and group differences in modulating the cholinergic basal forebrain-amygdala network for subjects with different levels of cognitive impairment. We evaluated the effect of APOE-ε4 on the cholinergic structural association and the neurocognitive performance for subjects with different levels of cognitive impairment. Methods: We used the structural brain magnetic resonance imaging scans from the Alzheimer's Disease Neuroimaging Initiative dataset. The study included cognitively normal (CN, n = 167) subjects and subjects with significant memory concern (SMC, n = 96), early mild cognitive impairment (EMCI, n = 146), late cognitive impairment (LMCI, n = 138), and AD (n = 121). Subjects were further categorized according to the APOE-ε4 allele carrier status. The main effects of APOE-ε4 and group difference on the brain volumetric measurements were assessed. Regression analyses were conducted to evaluate the associations among cholinergic structural changes, APOE-ε4 status, and cognitive performance. Results: We found that APOE-ε4 carriers in the disease group showed higher brain atrophy than non-carriers in the cholinergic pathway, while there is no difference between carriers and non-carriers in the CN group. APOE-ε4 allele carriers in the disease groups also exhibited a stronger cholinergic structural correlation than non-carriers did, while there is no difference between the carriers and non-carriers in the CN subjects. Disease subjects exhibited a stronger structural correlation in the cholinergic pathway than CN subjects did. Moreover, APOE-ε4 allele carriers in the disease group exhibited a stronger correlation between the volumetric changes and cognitive performance than non-carriers did, while there is no difference between carriers and non-carriers in CN subjects. Disease subjects exhibited a stronger correlation between the volumetric changes and cognitive performance than CN subjects did. Conclusion: Our results confirmed the effect of APOE-ε4 on and group differences in the associations with the cholinergic structural changes that may reflect impaired brain function underlying neurocognitive degeneration in AD.

Circadian Alterations Increase with Progression in a Patient-Derived Cell Culture Model of Breast Cancer.

Circadian rhythm disruption can elicit the development of various diseases, including breast cancer. While studies have used cell lines to study correlations between altered circadian rhythms and cancer, these models have different genetic backgrounds and do not mirror the changes that occur with disease development. Isogenic cell models can recapitulate changes across cancer progression. Hence, in this study, a patient-derived breast cancer model, the 21T series, was used to evaluate changes to circadian oscillations of core clock protein transcription as cells progress from normal to malignant states. Three cell lines were used: H16N2 (normal breast epithelium), 21PT (atypical ductal hyperplasia), and 21MT-1 (invasive metastatic carcinoma). The cancerous cells are both HER2+. We assessed the transcriptional profiles of two core clock proteins, BMAL1 and PER2, which represent a positive and negative component of the molecular oscillator. In the normal H16N2 cells, both genes possessed rhythmic mRNA oscillations with close to standard periods and phases. However, in the cancerous cells, consistent changes were observed: both genes had periods that deviated farther from normal and did not have an anti-phase relationship. In the future, mechanistic studies should be undertaken to determine the oncogenic changes responsible for the circadian alterations found.

Evaluation of the Swallow-Tail Sign and Correlations of Neuromelanin Signal with Susceptibility and Relaxations.

The presence of a swallow-tail sign in the nigrosome-1 with hyperintense signal shown on the susceptibility-weighted imaging (SWI) has been shown to be sensitive in detecting the abnormal iron deposits in this area. A systematic evaluation in healthy subjects is required before this tool can be recommended in a widespread application. We evaluated a simple and practical SWI approach using a multiecho gradient-echo sequence with an improved contrast-to-noise ratio (CNR). We also evaluated the association of the neuromelanin imaging contrast behavior with the susceptibility and relaxation measurements. Twenty-five older and 23 young healthy adults were evaluated. The CNRs of the nigrosome-1 were compared along with method and group. Correlations of the nigrosome-1 neuromelanin signal in the neuromelanin-sensitive imaging with CNRs in the susceptibility, T1 and T2 mappings were examined. Two different coils were used to confirm the reproducibility. Compared with the single-echo, multiecho SWI can improve the CNR of the swallow-tail sign. We found significant correlations between neuromelanin signal and CNRs in the susceptibility and T2 mappings, and T1 value. The older subjects exhibited increased CNRs compared with the young adults. No significant difference was observed in the measurements between 20 and 64 channels. The multiecho technique allows the high-quality nigrosome-1 images in SWI and allows for a joint analysis of T2* and quantitative-susceptibility mapping at high spatial resolution. The correlations of neuromelanin-sensitive imaging with susceptibility and T2 imply that the iron content in the nigrosome-1 may have significant influences on the hyperintensity of neuromelanin in the magnetization transfer-related contrast.

Nobiletin affects circadian rhythms and oncogenic characteristics in a cell-dependent manner.

The natural product nobiletin is a small molecule, widely studied with regard to its therapeutic effects, including in cancer cell lines and tumors. Recently, nobiletin has also been shown to affect circadian rhythms via their enhancement, resulting in protection against metabolic syndrome. We hypothesized that nobiletin's anti-oncogenic effects, such as prevention of cell migration and formation of anchorage independent colonies, are correspondingly accompanied by modulation of circadian rhythms. Concurrently, we wished to determine whether the circadian and anti-oncogenic effects of nobiletin differed across cancer cell lines. In this study, we assessed nobiletin's circadian and therapeutic characteristics to ascertain whether these effects depend on cell line, which here also varied in terms of baseline circadian rhythmicity. Three cell culture models where nobiletin's effects on cell proliferation and migration have been studied previously were evaluated: U2OS (bone osteosarcoma), which possesses robust circadian rhythms; MCF7 (breast adenocarcinoma), which has weak circadian rhythms; and MDA-MB-231 (breast adenocarcinoma), which is arrhythmic. We found that circadian, migration, and proliferative effects following nobiletin treatment were subtle in the U2OS and MCF7 cells. On the other hand, changes were clear in MDA-MB-231s, where nobiletin rescued rhythmicity and substantially reduced oncogenic features, specifically two-dimensional cell motility and anchorage-independent growth. Based on these results and those previously described, we posit that the effects of nobiletin are indeed cell-type dependent, and that a positive correlation may exist between nobiletin's circadian and therapeutic effects.

Oncogenic and Circadian Effects of Small Molecules Directly and Indirectly Targeting the Core Circadian Clock.

Circadian rhythms are essential for controlling the cell cycle, cellular proliferation, and apoptosis, and hence are tightly linked to cell fate. Several recent studies have used small molecules to affect circadian oscillations; however, their concomitant cellular effects were not assessed, and they have not been compared under similar experimental conditions. In this work, we use five molecules, grouped into direct versus indirect effectors of the circadian clock, to modulate periods in a human osteosarcoma cell line (U2OS) and determine their influences on cellular behaviors, including motility and colony formation. Luciferase reporters, whose expression was driven via Bmal1- or Per2-promoters, were used to facilitate the visualization and quantitative analysis of circadian oscillations. We show that all molecules increase or decrease the circadian periods of Bmal1 and Per2 in a dose-dependent manner, but period length does not correlate with the extent of cell migration or proliferation. Nonetheless, molecules that affected circadian oscillations to a greater degree resulted in substantial influence on cellular behaviors (ie, motility and colony formation), which may also be attributable to noncircadian targets. Furthermore, we find that the ability and extent to which the molecules are able to affect oscillations is independent of whether they are direct or indirect modulators. Because of the numerous connections and feedback between the circadian clock and other pathways, it is important to consider the effects of both in assessing these and other compounds.

Chemical modulation of circadian rhythms and assessment of cellular behavior via indirubin and derivatives.

Circadian rhythms are critical regulators of many physiological and behavioral functions. The use and abilities of small molecules to affect oscillations have recently received significant attention. These manipulations can be reversible and tunable, and have been used to study various biological mechanisms and molecular properties. Here, we outline procedures for assessment of cellular circadian changes following treatment with small molecules, using luminescent reporters. We describe reporter generation, luminometry experiments, and data analysis. Protocols for studies of accompanying effects on cells, including motility, viability, and anchorage-independent proliferation assays are also presented. As examples, we use indirubin-3'-oxime and two derivatives, 5-iodo-indirubin-3'-oxime and 5-sulfonic acid-indirubin-3'-oxime. In this case study, we analyze effects of these compounds on Bmal1 and Per2 (positive and negative core circadian elements) oscillations and provide step-by-step protocols for data analysis, including removal of trends from raw data, period estimations, and statistical analysis. The reader is provided with detailed protocols, and guidance regarding selection of and alternative approaches.

Design and Synthesis of Benzimidazole-Chalcone Derivatives as Potential Anticancer Agents.

Numerous reports have shown that conjugated benzimidazole derivatives possess various kinds of biological activities, including anticancer properties. In this report, we designed and synthesized 24 new molecules comprising a benzimidazole ring, arene, and alkyl chain-bearing cyclic moieties. The results showed that the N-substituted benzimidazole derivatives bearing an alkyl chain and a nitrogen-containing 5- or 6-membered ring enhanced the cytotoxic effects on human breast adenocarcinoma (MCF-7) and human ovarian carcinoma (OVCAR-3) cell lines. Among the 24 synthesized compounds, (2E)-1-(1-(3-morpholinopropyl)-1H-benzimidazol-2 -yl)-3-phenyl-2-propen-1-one) (23a) reduced the proliferation of MCF-7 and OVCAR-3 cell lines demonstrating superior outcomes to those of cisplatin.

Circadian oscillations persist in low malignancy breast cancer cells.

Epidemiological studies have shown that humans with altered circadian rhythms have higher cancer incidence, with breast cancer being one of the most cited examples. To uncover how circadian disruptions may be correlated with breast cancer and its development, prior studies have assessed the expression of BMAL1 and PER2 core clock genes via RT-qPCR and western blot analyses. These and our own low-resolution data show that BMAL1 and PER2 expression are suppressed and arrhythmic. We hypothesized that oscillations persist in breast cancer cells, but due to limitations of protocols utilized, cannot be observed. This is especially true where dynamic changes may be subtle. In the present work, we generated luciferase reporter cell lines representing high- and low-grade breast cancers to assess circadian rhythms. We tracked signals for BMAL1 and PER2 to determine whether and to what extent oscillations exist and provide initial correlations of circadian rhythm alterations with breast cancer aggression. In contrast to previous studies, where no oscillations were apparent in any breast cancer cell line, our luminometry data reveal that circadian oscillations of BMAL1 and PER2 in fact exist in the low-grade, luminal A MCF7 cells but are not present in high-grade, basal MDA-MB-231 cells. To our knowledge, this is the first evidence of core circadian clock oscillations in breast cancer cells. This work also suggests that circadian rhythms are further disrupted in more aggressive/high tumor grades of breast cancer, and that use of real time luminometry to study additional representatives of breast and other cancer subtypes is merited.

Analyses of BMAL1 and PER2 Oscillations in a Model of Breast Cancer Progression Reveal Changes With Malignancy.

From an epidemiological standpoint, disruptions to circadian rhythms have been shown to contribute to the development of various disease pathologies, including breast cancer. However, it is unclear how altered circadian rhythms are related to malignant transformations at the molecular level. In this article, a series of isogenic breast cancer cells representing disease progression was used to investigate the expression patterns of core circadian clock proteins BMAL1 and PER2. Our model is indicative of 4 stages of breast cancer and includes the following cells: MCF10A (non-malignant), MCF10AT.Cl2 (pre-malignant), MCF10Ca1h (well-differentiated, malignant), and MCF10Ca1a (poorly differentiated, malignant). While studies of circadian rhythms in cancer typically use low-resolution reverse transcription polymerase chain reaction assays, we also employed luciferase reporters BMAL1:Luc and PER2:Luc in real-time luminometry experiments. We found that across all 4 cancer stages, PER2 showed relatively stable oscillations compared with BMAL1. Period estimation using both wavelet-based and damped-sine-fitting methods showed that the periods are distributed over a wide circadian range and there is no clear progression in mean period as cancer severity progresses. Additionally, we used the K-nearest neighbors algorithm to classify the recordings according to cancer line, and found that cancer stages were largely differentiated from one another. Taken together, our data support that there are circadian discrepancies between normal and malignant cells, but it is difficult and insufficient to singularly use period evaluations to differentiate them. Future studies should employ other progressive disease models to determine whether these findings are representative across cancer types or are specific to this series.

Altered Circadian Rhythms and Breast Cancer: From the Human to the Molecular Level.

Circadian clocks are fundamental, time-tracking systems that allow organisms to adapt to the appropriate time of day and drive many physiological and cellular processes. Altered circadian rhythms can result from night-shift work, chronic jet lag, exposure to bright lights at night, or other conditioning, and have been shown to lead to increased likelihood of cancer, metabolic and cardiovascular diseases, and immune dysregulation. In cases of cancer, worse patient prognoses and drug resistance during treatment have also been observed. Breast, colon, prostate, lung, and ovarian cancers and hepatocellular carcinoma have all been linked in one way or another with altered circadian rhythms. Critical elements at the molecular level of the circadian system have been associated with cancer, but there have been fairly few studies in this regard. In this mini-review, we specifically focus on the role of altered circadian rhythms in breast cancer, providing an overview of studies performed at the epidemiological level through assessments made in animal and cellular models of the disease. We also address the disparities present among studies that take into account the rhythmicity of core clock and other proteins, and those which do not, and offer insights to the use of small molecules for studying the connections between circadian rhythms and cancer. This article will provide the reader with a concise, but thorough account of the research landscape as it pertains to altered circadian rhythms and breast cancer.

Synthesis and Evaluation of Aminothiazole-Paeonol Derivatives as Potential Anticancer Agents.

In this study, novel aminothiazole-paeonol derivatives were synthesized and characterized using ¹H-NMR, (13)C-NMR, IR, mass spectroscopy, and high performance liquid chromatography. All the new synthesized compounds were evaluated according to their anticancer effect on seven cancer cell lines. The experimental results indicated that these compounds possess high anticancer potential regarding human gastric adenocarcinoma (AGS cells) and human colorectal adenocarcinoma (HT-29 cells). Among these compounds, N-[4-(2-hydroxy-4-methoxyphenyl)thiazol-2-yl]-4-methoxybenzenesulfonamide (13c) had the most potent inhibitory activity, with IC50 values of 4.0 µM to AGS, 4.4 µM to HT-29 cells and 5.8 µM to HeLa cells. The 4-fluoro-N-[4-(2-hydroxy-4-methoxyphenyl)thiazol-2-yl]benzenesulfonamide (13d) was the second potent compound, showing IC50 values of 7.2, 11.2 and 13.8 µM to AGS , HT-29 and HeLa cells, respectively. These compounds are superior to 5-fluorouracil (5-FU) for relatively higher potency against AGS and HT-29 human cancer cell lines along with lower cytotoxicity to fibroblasts. Novel aminothiazole-paeonol derivatives in this work might be a series of promising lead compounds to develop anticancer agents for treating gastrointestinal adenocarcinoma.

Design, synthesis, and bioevaluation of paeonol derivatives as potential anti-HBV agents.

Hepatitis B virus (HBV) is a causative reagent that frequently causes progressive liver diseases, leading to the development of acute, chronic hepatitis, cirrhosis, and eventually hepatocellular carcinoma (HCC). Despite several antiviral drugs including interferon-α and nucleotide derivatives are approved for clinical treatment for HBV, critical issues remain unresolved, e.g., low-to-moderate efficacy, adverse side effects, and resistant strains. In this study, novel Paeonol-phenylsulfonyl derivatives were synthesized and their antiviral effect against HBV was evaluated. The experimental results indicated that these compounds process significant antiviral potential, including the inhibition of viral antigen expression and secretion, and the suppression of HBV viral DNA replication. Among compounds synthesized in this research, compound 2-acetyl-5-methoxyphenyl 4-methoxybenzenesulfonate (7f) had the most potent inhibitory activity with IC50 value of 0.36 µM, and high selectivity index, SI (TC50/IC50) 47.75; which exhibited an apparent inhibition effect on viral gene expression and viral propagation in cell culture model. So, we believe our compounds could serve as reservoir for antiviral drug development.

Evaluation of the increase in permeability of the blood-brain barrier during tumor progression after pulsed focused ultrasound.

PURPOSE: The purpose of this study was to evaluate the permeability of the blood-brain barrier after sonication by pulsed high-intensity focused ultrasound and to determine if such an approach increases the tumor:ipsilateral brain permeability ratio. MATERIALS AND METHODS: F98 glioma-bearing Fischer 344 rats were injected intravenously with Evans blue with or without blood-tumor barrier disruption induced by transcranial pulsed high-intensity focused ultrasound. Sonication was applied at a frequency of 1 MHz with a 5% duty cycle and a repetition frequency of 1 Hz. The permeability of the blood-brain barrier was assessed by the extravasation of Evans blue. Contrast-enhanced magnetic resonance images were used to monitor the gadolinium deposition path associated with transcranial pulsed high-intensity focused ultrasound, and the influencing size and location was also investigated. In addition, whole brain histological analysis was performed. The results were compared by two-tailed unpaired t-test. RESULTS: The accumulation of Evans blue in brains and the tumor:ipsilateral brain permeability ratio of Evans blue were significantly increased after pulsed high-intensity focused ultrasound exposure. Evans blue injection followed by sonication showed an increase in the tumor:ipsilateral brain ratio of the target tumors (9.14:1) of about 2.23-fold compared with the control tumors (x4.09) on day 6 after tumor implantation. Magnetic resonance images showed that pulsed high-intensity focused ultrasound locally enhances the permeability of the blood-tumor barrier in the glioma-bearing rats. CONCLUSION: This method could allow enhanced synergistic effects with respect to other brain tumor treatment regimens.

Micro-SPECT/CT-based pharmacokinetic analysis of 99mTc-diethylenetriaminepentaacetic acid in rats with blood-brain barrier disruption induced by focused ultrasound.

UNLABELLED: This study evaluated the pharmacokinetics of (99m)Tc-diethylenetriamine pentaacetate acid ((99m)Tc-DTPA) after intravenous administration in healthy and F98 glioma-bearing F344 rats in the presence of blood-brain barrier disruption (BBB-D) induced by focused ultrasound (FUS). The pharmacokinetics of the healthy and tumor-containing brains after BBB-D were compared to identify the optimal time period for combined treatment. METHODS: Healthy and F98 glioma-bearing rats were injected intravenously with Evans blue (EB) and (99m)Tc-DTPA; these treatments took place with or without BBB-D induced by transcranial FUS of 1 hemisphere of the brain. The permeability of the BBB was quantified by EB extravasation. Twelve rats were scanned for 2 h to estimate uptake of (99m)Tc radioactivity with respect to time for the pharmacokinetic analysis. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining was performed to examine tissue damage. RESULTS: The accumulations of EB and (99m)Tc-DTPA in normal brains or brains with a tumor were significantly elevated after the intravenous injection when BBB-D was induced. The disruption-to-nondisruption ratio of the brains and the tumor-to-ipsilateral brain ratio of the tumors in terms of radioactivity reached a peak at 45 and 60 min, respectively. EB injection followed by sonication showed that there was an increase of about 2-fold in the tumor-to-ipsilateral brain EB ratio of the target tumors (7.36), compared with the control tumors (3.73). TUNEL staining showed no significant differences between the sonicated tumors and control tumors. CONCLUSION: This study demonstrates that (99m)Tc-DTPA micro-SPECT/CT can be used for the pharmacokinetic analysis of BBB-D induced by FUS. This method should be able to provide important information that will help with establishing an optimal treatment protocol for drug administration after FUS-induced BBB-D in clinical brain disease therapy.