State-of-the-art review of the clinical research on menopause and hormone replacement therapy association with Parkinson's disease: What meta-analysis studies cannot tell us.

The menopause is a midlife endocrinological process that greatly affects women's central nervous system functions. Over the last 2 decades numerous clinical studies have addressed the influence of ovarian hormone decline on neurological disorders like Parkinson's disease and Alzheimer's disease. However, the findings in support of a role for age at menopause, type of menopause and hormone replacement therapy on Parkinson's disease onset and its core features show inconsistencies due to the heterogeneity in the study design. Here, we provide a unified overview of the clinical literature on the influence of menopause and ovarian hormones on Parkinson's disease. We highlight the possible sources of conflicting evidence and gather considerations for future observational clinical studies that aim to explore the neurological impact of menopause-related features in Parkinson's disease.

Combining CRISPR-Cas9 and brain imaging to study the link from genes to molecules to networks.

Receptors, transporters, and ion channels are important targets for therapy development in neurological diseases, but their mechanistic role in pathogenesis is often poorly understood. Gene editing and in vivo imaging approaches will help to identify the molecular and functional role of these targets and the consequence of their regional dysfunction on the whole-brain level. We combine CRISPR-Cas9 gene editing with in vivo positron emission tomography (PET) and functional MRI (fMRI) to investigate the direct link between genes, molecules, and the brain connectome. The extensive knowledge of the Slc18a2 gene encoding the vesicular monoamine transporter (VMAT2), involved in the storage and release of dopamine, makes it an excellent target for studying the gene network relationships while structurally preserving neuronal integrity and function. We edited the Slc18a2 in the substantia nigra pars compacta of adult rats and used in vivo molecular imaging besides behavioral, histological, and biochemical assessments to characterize the CRISPR-Cas9-mediated VMAT2 knockdown. Simultaneous PET/fMRI was performed to investigate molecular and functional brain alterations. We found that stage-specific adaptations of brain functional connectivity follow the selective impairment of presynaptic dopamine storage and release. Our study reveals that recruiting different brain networks is an early response to the dopaminergic dysfunction preceding neuronal cell loss. Our combinatorial approach is a tool to investigate the impact of specific genes on brain molecular and functional dynamics, which will help to develop tailored therapies for normalizing brain function.

Universal Gene Correction Approaches for ß-hemoglobinopathies Using CRISPR-Cas9 and Adeno-Associated Virus Serotype 6 Donor Templates.

Mutations in the human ß-globin gene are the cause of ß-hemoglobinopathies, one of the most common inherited single-gene blood disorders in the world. Novel therapeutic approaches are based on lentiviral vectors (LVs) or CRISPR-Cas9-mediated gene disruption to express adult hemoglobin (HbA), or to reactivate the completely functional fetal hemoglobin, respectively. Nonetheless, LVs present a risk of insertional mutagenesis, while gene-disrupting transcription factors (BCL11A, KLF1) involved in the fetal-to-adult hemoglobin switch might generate dysregulation of other cellular processes. Therefore, universal gene addition/correction approaches combining CRISPR-Cas9 and homology directed repair (HDR) by delivering a DNA repair template through adeno-associated virus could mitigate the limitations of both lentiviral gene transfer and gene disruption strategies, ensuring targeted integration and controlled transgene expression. In this study, we attained high rates of gene addition (up to 12%) and gene correction (up to 38%) in hematopoietic stem and progenitor cells from healthy donors without any cell sorting/enrichment or the application of HDR enhancers. Furthermore, these approaches were tested in heterozygous (ß0/ß+) and homozygous (ß0/ß0, ß+/ß+) ß-thalassemia patients, achieving a significant increase in HbA and demonstrating the universal therapeutic potential of this study for the treatment of ß-hemoglobinopathies.

Quantitative Rodent Brain Receptor Imaging.

Positron emission tomography (PET) is a non-invasive imaging technology employed to describe metabolic, physiological, and biochemical processes in vivo. These include receptor availability, metabolic changes, neurotransmitter release, and alterations of gene expression in the brain. Since the introduction of dedicated small-animal PET systems along with the development of many novel PET imaging probes, the number of PET studies using rats and mice in basic biomedical research tremendously increased over the last decade. This article reviews challenges and advances of quantitative rodent brain imaging to make the readers aware of its physical limitations, as well as to inspire them for its potential applications in preclinical research. In the first section, we briefly discuss the limitations of small-animal PET systems in terms of spatial resolution and sensitivity and point to possible improvements in detector development. In addition, different acquisition and post-processing methods used in rodent PET studies are summarized. We further discuss factors influencing the test-retest variability in small-animal PET studies, e.g., different receptor quantification methodologies which have been mainly translated from human to rodent receptor studies to determine the binding potential and changes of receptor availability and radioligand affinity. We further review different kinetic modeling approaches to obtain quantitative binding data in rodents and PET studies focusing on the quantification of endogenous neurotransmitter release using pharmacological interventions. While several studies have focused on the dopamine system due to the availability of several PET tracers which are sensitive to dopamine release, other neurotransmitter systems have become more and more into focus and are described in this review, as well. We further provide an overview of latest genome engineering technologies, including the CRISPR/Cas9 and DREADD systems that may advance our understanding of brain disorders and function and how imaging has been successfully applied to animal models of human brain disorders. Finally, we review the strengths and opportunities of simultaneous PET/magnetic resonance imaging systems to study drug-receptor interactions and challenges for the translation of PET results from bench to bedside.

Silica/quercetin sol-gel hybrids as antioxidant dental implant materials.

The development of biomaterials with intrinsic antioxidant properties could represent a valuable strategy for preventing the onset of peri-implant diseases. In this context, quercetin, a naturally occurring flavonoid, has been entrapped at different weight percentages in a silica-based inorganic material by a sol-gel route. The establishment of hydrogen bond interactions between the flavonol and the solid matrix was ascertained by Fourier transform infrared spectroscopy. This technique also evidenced changes in the stretching frequencies of the quercetin dienonic moiety, suggesting that the formation of a secondary product occurs. Scanning electron microscopy was applied to detect the morphology of the synthesized materials. Their bioactivity was shown by the formation of a hydroxyapatite layer on sample surface soaked in a fluid that simulates the composition of human blood plasma. When the potential release of flavonol was determined by liquid chromatography coupled with ultraviolet and electrospray ionization tandem mass spectrometry techniques, the eluates displayed a retention time that was 0.5 min less than quercetin. Collision-activated dissociation mass spectrometry and untraviolet-visible spectroscopy were in accordance with the release of a quercetin derivative. The antiradical properties of the investigated systems were evaluated by DPPH and ABTS methods, whereas the 2,7-dichlorofluorescein diacetate assay highlighted their ability to inhibit the H2O2-induced intracellular production of reactive oxygen species in NIH-3T3 mouse fibroblast cells. Data obtained, along with data gathered from the MTT cytotoxicity test, revealed that the materials that entrapped the highest amount of quercetin showed notable antioxidant effectiveness.

LC-MS/MS profiling of a mastic leaf phenol enriched extract and its effects on H2O2 and Aß(25-35) oxidative injury in SK-B-NE(C)-2 cells.

The development of polyphenol neuroprotective nutraceuticals useful for functional foods could be a valuable strategy for counteracting oxidative stress relative diseases as Alzheimer's Disease (AD). Oxidative stress is one of the AD earliest event and seems to play a central role in Aß generation, neuroinflammation, and neuronal apoptosis. In order to counteract AD neurodegeneration, the inhibition of the vicious cycle of Aß generation and oxidation is an attractive therapeutic strategy, and antiamyloidogenic and antioxidant plant drugs could represent an alternative and valid approach. In this context, an alcoholic extract (Pl-M) from deterpenated Pistacia lentiscus L. leaves was investigated for its phenol composition through LC-ESI-MS/MS analysis. Besides the identified metabolites, ten compounds were reported for the first time as constituents of Pistacia lentiscus leaves. Through DPPH, ABTS, and ORAC methods, the antioxidant potential of the extract was initially investigated. In order to evaluate the preparation of a safe and no toxic extract, MTT, SRB, and LDH assays toward SH-5YSY, and SK-N-BE(2)-C human neuronal cell lines, as well as on C6 mouse glial cell line, were performed. Evaluating the protective effects from oxidant injury in SK-N-BE(2)-C cells cotreated with the plant complex and H2O2, or Aß(25-35) fragment, it was observed that Pl-M extract exerted a significant cytoprotective response in both the oxidized cell systems. In particular, Pl-M extract was able to reduce by nearly 50% the Aß(25-35) induced toxicity at 25.0 µg/mL dose level, whereas it counteracted almost completely the cytotoxic action at 100.0 µg/mL. Data obtained allow us to hypothesize the use of Pistacia lentiscus leaves, a broadly available and renewable source, as an alternative strategy for the enrichment of food matrices with polyphenol bioactives. The present study put the basis for bioavailability and preclinical studies, able to define Pl-M extract safety and efficacy.

Influence of seasonal variation on Thymus longicaulis C. Presl chemical composition and its antioxidant and anti-inflammatory properties.

Thymus longicaulis C. Presl. (Lamiaceae) is a small aromatic perennial herb typical of the Illyric-Mediterranean flora, traditionally used as remedy for cold, flu, cough, nephritis and abdominal pain. In order to carry out a thorough chemical and biological screening of the plant and to explore phenophases influence on its polyphenol content, samples of the plant were collected at different phases during its life cycle (July/October 2012 and January/April 2013). Each sample, previously extracted using a hydroalcoholic solution, was phytochemically analyzed for its metabolic constitution applying LC-DAD-ESI-MS/MS techniques. Although identified metabolites were differently concentrated at the various collection times, T. longicaulis leaf extracts were mainly constituted by low molecular weight phenols, and flavonoids. Rosmarinic acid was found as the main metabolite in Oct12 sample. Chemopreventive efficacy of the investigated extracts, by means of their anti-inflammatory, cytotoxic and antioxidant activities, was assessed. To this purpose, each extract underwent an extensive screening towards five human cell lines: CCRF-CEM (leukemia); U251 (glioblastoma); MDA-MB-231 (breast cancer); HCT-116 (colon cancer) and MRC-5 (lung fibroblasts) through XTT [2,3bis(2-metoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H tetrazolium hydroxide] test. The ability of the extracts to counteract cyclooxygenase-2 (COX-2) expression was also evaluated by COX-2 expression assay in human THP-1 monocyte-derived macrophages. COX-2 inhibition could represent a valuable anticancer strategy as it is associated with carcinogenesis and over-expressed in a variety of human malignancies. Oct12 extract, which was particularly rich in rosmarinic acid and methylapigenin, exhibited a strong antioxidant and anti-inflammatory effectiveness.

Neuroprotective potential of Laurus nobilis antioxidant polyphenol-enriched leaf extracts.

Oxidative stress has been proposed to be an important factor in the pathogenesis of Alzheimer's disease (AD), playing a central role in amyloid ß-protein (Aß) generation and neuronal apoptosis. Oxidative damage directly correlates with the presence of Aß deposits. Aß and oxidative stress jointly induce neuronal death, Aß deposits, gliosis, and memory impairment in AD. In order to counteract AD neurodegeneration, the inhibition of the vicious cycle of Aß generation and oxidation is an attractive therapeutic strategy, and antiamyloidogenic and antioxidant herbal drugs could represent an alternative and valid approach. In this context, an alcoholic extract from Laurus nobilis leaves (LnM) and seven fractions obtained therefrom were of interest. All extracts prepared through extractive and chromatographic techniques were phytochemically studied by chromatographic techniques including gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-tandem mass spectrometry (LC-MS(n)). The potential antioxidant efficacy of the obtained fractions was screened by DPPH(•) and ABTS(•+) assays, as well as specific assay media characterized from the presence of highly reactive ROS and RNS species (ROO(•), OH(•), O2(•-), and NO). In order to evaluate the preparation of safe and nontoxic extracts, MTT, SRB, and LDH assays toward SH-5YSY and SK-N-BE(2)-C human neuronal cell lines, as well as on C6 mouse glial cell line, were performed. The apoptosis-inducing properties by spectroscopic evaluation of the extracts' ability to activate caspase-3 and by a DNA fragmentation assay were also investigated. Data thus obtained allowed us to state the absence of toxic effects induced by phenolic-rich fractions (LnM, LnM-1, LnM-1a, LnM-1b, and LnM-2c), which at the same time exerted significant cytoprotective and antioxidant responses in hydrogen peroxide and Aß(25-35)-fragment-oxidized cell systems. The potential antiamyloidogenic efficacy of Laurus nobilis leaf polar extracts in the Aß(25-35) fragment oxidized cell systems was further analyzed by Congo red staining.

Apolar Laurus nobilis leaf extracts induce cytotoxicity and apoptosis towards three nervous system cell lines.

In the course of a bioactivity screening of Mediterranean plants, the assessment of neuroprotective properties of Laurus nobilis L. was of interest. Dried leaves were extracted by sonication using CHCl3 as solvent. The CHCl3 parental extract (CHCl3-pe) was fractionated to yield CHCl3 (LnC-1), EtOAc (LnC-2), MeOH (LnC-3) fractions. Each fraction underwent an extensive screening towards human neuroblastoma (SK-N-BE(2)-C, and SH-SY5Y) and rat glioma (C6) cell lines. MTT and SRB cytotoxicity tests were performed. The effect on the plasma membrane integrity was evaluated by assessment of LDH release. The caspase-3 activation enzyme and DNA fragmentation were also evaluated. The oxidant/antioxidant ability of all the extracts were evaluated using different methods. Furthermore, a metabolite profiling of the investigated extracts was carried out by GC-EI-MS. CHCl3-pe contained terpenes, allylphenols, and α-tocopherol. Dehydrocostus lactone was the main constituent. As result of the fractionation technique, the LnC-1 extract was mainly composed of α-tocopherol, whereas the LnC-2 fraction was enriched in guaiane and eudesmane terpenes. The most cytotoxic LnC-2 fraction induced apoptosis; it was ineffective in preventing in vitro free radicals production. Overall, the experimental results support a possible role of LnC-2 preparation as a chemopreventive agent for neuronal cells or other cells of the CNS.