Therapeutic potential of saffron in brain disorders: From bench to bedside.

Saffron is a spice derived from the flower of Crocus sativus L., which has been used for centuries as a coloring and flavoring agent, as well as a source of medicinal compounds. Saffron contains various bioactive constituents, such as crocin, crocetin, safranal, picrocrocin, and kaempferol, that have shown potential benefits for human health. Among them, crocin is the most abundant and characteristic constituent of saffron, responsible for its bright red color and antioxidant properties. One of the most promising applications of saffron and its constituents is in the prevention and treatment of neurological disorders, such as depression, anxiety, Alzheimer's disease, Parkinson's disease, and other brain disorders. Saffron and its constituents have been reported to exert neuroprotective effects through various mechanisms, such as modulating neurotransmitters, enhancing neurogenesis, reducing neuroinflammation, regulating oxidative stress, activating the Nrf2 signaling pathway, and modulating epigenetic factors. Several clinical and preclinical studies have demonstrated the efficacy and safety of saffron and its constituents in improving cognitive function, mood, and other neurological outcomes. In this review, we summarize the current evidence on the therapeutic potential of saffron and its constituents in neurological disorders, from bench to bedside. We also discuss the challenges and future directions for the development of saffron-based therapies for brain health.

Improved Mitochondrial and Methylglyoxal-Related Metabolisms Support Hyperproliferation Induced by 50 Hz Magnetic Field in Neuroblastoma Cells.

Extremely low frequency magnetic fields (ELF-MF) are common environmental agents that are suspected to promote later stages of tumorigenesis, especially in brain-derived malignancies. Even though ELF magnetic fields have been previously linked to increased proliferation in neuroblastoma cells, no previous work has studied whether ELF-MF exposure may change key biomolecular features, such as anti-glycative defence and energy re-programming, both of which are currently considered as crucial factors involved in the phenotype and progression of many malignancies. Our study investigated whether the hyperproliferation that is induced in SH-SY5Y human neuroblastoma cells by a 50 Hz, 1 mT ELF magnetic field is supported by an improved defense towards methylglyoxal (MG), which is an endogenous cancer-static and glycating α-oxoaldehyde, and by rewiring of energy metabolism. Our findings show that not only the ELF magnetic field interfered with the biology of neuron-derived malignant cells, by de-differentiating further the cellular phenotype and by increasing the proliferative activity, but also triggered cytoprotective mechanisms through the enhancement of the defense against MG, along with a more efficient management of metabolic energy, presumably to support the rapid cell outgrowth. Intriguingly, we also revealed that the MF-induced bioeffects took place after an initial imbalance of the cellular homeostasis, which most likely created a transient unstable milieu. The biochemical pathways and molecular targets revealed in this research could be exploited for future approaches aimed at limiting or suppressing the deleterious effects of ELF magnetic fields. J. Cell. Physiol. 231: 2014-2025, 2016. © 2016 Wiley Periodicals, Inc.

Oxidative Stress and Neurodegeneration: Insights and Therapeutic Strategies for Parkinson's Disease.

Parkinson's disease (PD) is a progressive neurodegenerative condition marked by the gradual deterioration of dopaminergic neurons in the substantia nigra. Oxidative stress has been identified as a key player in the development of PD in recent studies. In the first part, we discuss the sources of oxidative stress in PD, including mitochondrial dysfunction, dopamine metabolism, and neuroinflammation. This paper delves into the possibility of mitigating oxidative stress as a potential treatment approach for PD. In addition, we examine the hurdles and potential of antioxidant therapy, including the challenge of delivering antioxidants to the brain and the requirement for biomarkers to track oxidative stress in PD patients. However, even if antioxidant therapy holds promise, further investigation is needed to determine its efficacy and safety in PD treatment.

Hydroalcoholic extract of Buxus sempervirens shows antiproliferative effect on melanoma, colorectal carcinoma and prostate cancer cells by affecting the autophagic flow.

Buxus sempervirens (European Box, Buxaceae, boxwood) has been used in folk medicine to treat rheumatism, arthritis, fever, malaria and skin ulceration while, in recent years, interest has grown on possible employment of boxwood extracts in cancer therapy. We studied the effect of hydroalcoholic extract from dried leaves of Buxus sempervirens (BSHE) on four human cell lines (BMel melanoma cells, HCT116 colorectal carcinoma cells, PC3 prostate cancer cells, and HS27 skin fibroblasts) to ascertain its possible antineoplastic activity. This extract inhibited proliferation of all cell lines in different degree as shown, after 48 h-exposure and MTS assay, by the values of GR50 (normalized growth rate inhibition50) that were 72, 48, 38, and 32 µg/mL for HS27, HCT116, PC3 and BMel cells, respectively. At the above GR50 concentrations, 99% of all studied cells remained vital showing accumulation of acidic vesicles in the cytoplasm, mainly around nuclei, whereas a higher extract concentration (125 µg/mL) was cytotoxic causing, after 48 h-exposure, death of all BMel and HCT116 cells. Immunofluorescence showed microtubule-associated light chain three protein (LC3, a marker for autophagy) to be localized on the above acidic vesicles when cells were treated for 48 h with BSHE (GR50 concentrations). Western blot analysis revealed, in all treated cells, a significant increase (2.2-3.3 times at 24 h) of LC3II, i.e., the phosphatidylethanolamine conjugate of the cytoplasmic form LC3I that is recruited in autophagosome membranes during autophagy. Such increase was accompanied, in all cell lines treated for 24 h or 48 h with BSHE, by a significant increment (2.5-3.4 times at 24 h) of p62, an autophagic cargo protein undergoing degradation during the autophagic process. Therefore, BSHE appeared to promote autophagic flow with its following blockade and consequent accumulation of autophagosome or autolysosomes. The antiproliferative effects of BSHE also involved cell cycle regulators such as p21 (HS27, BMel and HCT116 cells) and cyclin B1 (HCT116, BMel and PC3 cells) whereas, among apoptosis markers, BSHE only decreased (30%-40% at 48 h) the expression of the antiapoptotic protein survivin. It was concluded that BSHE impairs autophagic flow with arrest of proliferation and death in both fibroblasts and cancer cells, being the latter much more sensitive to these effects.

MgO Nanoparticles Obtained from an Innovative and Sustainable Route and Their Applications in Cancer Therapy.

This paper aimed to evaluate the biological damages towards diseased cells caused by the use of MgO nanoparticles (NPs). The NPs are produced by a calcination process of a precursor, which is an aqueous suspension of nanostructured Mg(OH)2, in turn synthesized following our original, time-energy saving and scalable method able to guarantee short times, high yield of production (up to almost 10 kg/week of NPs), low environmental impact and low energy demand. The MgO NPs, in the form of dry powders, are organized as a network of intercrystallite channels, in turn constituted by monodispersed and roughly spherical NPs < 10 nm, preserving the original pseudo hexagonal-platelet morphology of the precursor. The produced MgO powders are diluted in a PBS solution to obtain different MgO suspension concentrations that are subsequently put in contact, for 3 days, with melanoma and healthy cells. The viable count, made at 24, 48 and 72 h from the beginning of the test, reveals a good cytotoxic activity of the NPs, already at low MgO concentrations. This is particularly marked after 72 h, showing a clear reduction in cellular proliferation in a MgO-concentration-dependent manner. Finally, the results obtained on human skin fibroblasts revealed that the use MgO NPs did not alter at all both the vitality and proliferation of healthy cells.

Particle Debris Generated from Passenger Tires Induces Morphological and Gene Expression Alterations in the Macrophages Cell Line RAW 264.7.

Air pollution in the urban environment is a topical subject. Aero-suspended particles can cause respiratory diseases in humans, ranging from inflammation to asthma and cancer. One of the components that is most prevalent in particulate matter (PM) in urban areas is the set of tire microparticles (1-20 µm) and nanoparticles (<1 µm) that are formed due to the friction of wheels with asphalt and are increased in slow-moving areas that involve a lot of braking actions. In this work, we studied the effect that microparticles generated from passenger tires (PTWP, passenger tire wear particles) have in vitro on murine macrophages cells RAW 264.7 at two concentrations of 25 and 100 µg/mL, for 24 and 48 h. In addition to the chemical characterization of the material and morphological characterization of the treated cells by transmission electron microscopy, gene expression analysis with RT-PCR and active protein analysis with Western blotting were performed. Growth curves were obtained, and the genotoxic effect was evaluated with a comet assay. The results indicate that initially, an induction of the apoptotic process is observable, but this is subsequently reversed by Bcl2. No genotoxic damage is present, but mild cellular abnormalities were observed in the treated cells.

Estimation of DNA Degradation in Archaeological Human Remains.

The evaluation of the integrity and quantity of DNA extracted from archaeological human remains is a fundamental step before using the latest generation sequencing techniques in the study of evolutionary processes. Ancient DNA is highly fragmented and chemically modified; therefore, the present study aims to identify indices that can allow the identification of potentially amplifiable and sequenceable DNA samples, reducing failures and research costs. Ancient DNA was extracted from five human bone remains from the archaeological site of Amiternum L'Aquila, Italy dating back to the 9th-12th century and was compared with standard DNA fragmented by sonication. Given the different degradation kinetics of mitochondrial DNA compared to nuclear DNA, the mitochondrially encoded 12s RNA and 18s ribosomal RNA genes were taken into consideration; fragments of various sizes were amplified in qPCR and the size distribution was thoroughly investigated. DNA damage degree was evaluated by calculating damage frequency (λ) and the ratio between the amount of the different fragments and that of the smallest fragment (Q). The results demonstrate that both indices were found to be suitable for identifying, among the samples tested, those less damaged and suitable for post-extraction analysis; mitochondrial DNA is more damaged than nuclear, in fact, amplicons up to 152 bp and 253 bp, respectively are obtained.

A qPCR-duplex assay for sex determination in ancient DNA.

Molecular biology techniques are increasingly being used in sex identification of skeletal remains when traditional anthropometric analyzes are not successful in identifying sex of remains that are incomplete, fragmented and /or of immature individuals. In the present work, we investigated the possibility of determining sex by using the qPCR-duplex method for both ancient and modern DNA samples. This method involves the co-amplification of two genes in a single reaction system and the subsequent analysis of the fusion curves; the gene sequences used for the construction of suitable primers are those of steroid sulfatase (STS) and testis specific protein Y-linked 1 (TSPY) genes which turned out to be two sensitive markers as they have a detection limit of 60 pg and 20 pg respectively on modern DNA. The validity of the method was verified on modern DNA in which gender was identified in all the samples with 100% accuracy; thus, allowing for the same results as the classic method with amelogenin, but in a faster and more immediate way, as it allows for sex determination solely by analyzing the denaturation curves without having to perform an electrophoretic run. The proposed molecular technique proves to be sensitive and precise even on degraded DNA, in fact on 9 archaeological finds dating from the VII-XII century in which sex had been identified through anthropometric analysis, it confirmed the sex of 8 out of 9 finds correctly.

Transcriptional analysis of tyrosinase gene expression during Bufo bufo development.

Tyrosinase (EC.1.14.18.1.) is a widespread enzyme, in the phylogenetic scale, that produces melanin, from bacteria to man, by using as substrates monophenols, o-diphenols and molecular oxygen. In this work we have confirmed and demonstrated that during Bufo bufo development tyrosinase activity and gene expression first occur at developmental stages 17-18 (tail bud-muscular response) as detected by a spectrophotometric assay and qRT-PCR. As expected, also during B. bufo development tyrosinase gene is expressed after the late gastrula (stage 12), differently from Rana pipiens development when tyrosinase mRNA appears at the neural plate stage and enzyme activity at stage 20 (gill circulation). We have cloned and sequenced the B. bufo tyrosinase cDNA in order to prepare B. bufo tyrosinase cDNA specific primers (forward and reverse). Tyrosinase mRNA cloning has been performed by using degenerate primers prepared according to the anuran tyrosinase gene sequence coding for the copper binding sites. The expressions of tyrosinase gene and enzymatic activity during B. bufo development support that until the developmental stage 17, embryo melanin is of maternal origin and at this stage can start embryo melanin synthesis. A correlation exists between tyrosinase expression and O2 consumption during B. bufo development.

Analysis of ancient mtDNA from the medieval archeological site of Amiternum (L'Aquila), central Italy.

Study of ancient DNA makes it possible to analyze genetic relationships between individuals and populations of past and present. In this paper we have analyzed remains of human bones, dating back to the 8th-10th century AD, from the burials found in the Cathedral of Santa Maria in Civitate, archaeological site of Amiternum, L'Aquila, Italy. As a genetic marker, the hypervariable region 1 of mitochondrial DNA (HVR1) was selected. To obtain reliable sequences from the hypervariable region 1 of mtDNA (HVR1) were performed: multiple extractions, template quantification and cloning of PCR products. The sequences obtained were compared with Anderson's sequence for the identification of polymorphisms (SNP) and haplogroups. The data obtained were analyzed with various software and phylogenetic methods. For the comparison between populations, ancient and modern sequences found in databases and literature have been used. This work provides preliminary information on the correlation between the population of Amiternum, the migrant populations transited and/or established in the territory of Amiternum such as Byzantines, Longobards (Lombards), which dominated the Italian peninsula between 568 and 774 AD, and the current populations of Italy. The study of haplogroups, the analysis of genetic variability and phylogenesis studies on the sequences considered show a genetic closeness between the individuals of Amiternum, the current population of central-northern Italy and the Germanic tribe of Longobards, however, also highlights genetic traits of Byzantines in some samples of Amiternum. Using the analysis of amelogenin gene fragments, we successfully determined the sex of the bone remains on all samples.

The genomic tool-kit of the truffle Tuber melanosporum programmed cell death.

A survey of the truffle Tuber melanosporum genome has shown the presence of 67 programmed cell death (PCD)-related genes. The 67 genes are all expressed during fruit body (FB) development of T. melanosporum development; their expression has been detected by DNA microarrays and qPCR. A set of 14 PCD-related genes have been chosen, those with the highest identities to the homologs of other species, for a deeper investigation. That PCD occurs during T. melanosporum development has been demonstrated by the TUNEL reaction and transmission electron microscopy. The findings of this work, in addition to the discovery of PCD-related genes in the T. melanosporum genome and their expression during the differentiation and development of the FB, would suggest that one of the PCD subroutines, maybe autophagy, is involved in the FB ripening, i.e., sporogenesis.

Leptin contributes to long-term stabilization of HIF-1α in cancer cells subjected to oxygen limiting conditions.

Leptin, a cytokine produced by the adipose tissue in response to food intake, is a key player in the regulation of energy balance and body weight control. Physiological action of leptin in modulating the metabolic adaptation of different peripheral tissues supports the hypothesis that it could also exert a direct effect on cancer cells. In vitro, treatment with leptin up-regulated HIF-1α and stimulated adhesion and invasion of prostate cancer cells cultured in hypoxia. Leptin action was effective in both low and high glycolytic cancer cell lines, and determined the up-regulation of lactate exporter MCT4 and its associated protein CD147. HIF-1α stabilization was oligomycin-independent and was associated with an important modulation of mitochondrial homeostasis. In fact, leptin treatment produced mitochondrial biogenesis, stabilization of mitochondrial membrane potential and increased uncoupled respiration through the up-regulation of UCP2. Furthermore, leptin counteracted the downmodulation of SIRT1 induced by hypoxia, and persistent high levels of SIRT1 were directly involved in HIF-1α stabilization. Leptin can sustain cancer progression in hypoxic environment and when mitochondrial respiration is impaired. Leptin signaling axis, including the new proposed intermediate SIRT1, could represent a new diagnostic and therapeutic target in prostate cancer.

Validation of reference genes for quantitative real-time PCR in Périgord black truffle (Tuber melanosporum) developmental stages.

The symbiotic fungus Tuber melanosporum Vittad. (Périgord black truffle) belongs to the Ascomycota and forms mutualistic symbiosis with tree and shrub roots. This truffle has a high value in a global market and is cultivated in many countries of both hemispheres. The publication of the T. melanosporum genome has given researchers unique opportunities to learn more about the biology of the fungus. Real-time quantitative PCR (qRT-PCR) is a definitive technique for quantitating differences in transcriptional gene expression levels between samples. To facilitate gene expression studies and obtain more accurate qRT-PCR data, normalization relative to stable housekeeping genes is required. These housekeeping genes must show stable expression under given experimental conditions for the qRT-PCR results to be accurate. Unfortunately, there are no studies on the stability of housekeeping genes used in T. melanosporum development. In this study, we present a morphological and microscopical classification of the developmental stages of T. melanosporum fruit body, and investigate the expression levels of 12 candidate reference genes (18S rRNA; 5.8S rRNA; Elongation factor 1-alpha; Elongation factor 1-beta; α-tubulin; 60S ribosomal protein L29; ß-tubulin; 40S ribosomal protein S1; 40S ribosomal protein S3; Glucose-6-phosphate dehydrogenase; ß-actin; Ubiquitin-conjugating enzyme). To evaluate the suitability of these genes as endogenous controls, five software-based approaches and one web-based comprehensive tool (RefFinder) were used to analyze and rank the tested genes. We demonstrate here that the 18S rRNA gene shows the most stable expression during T. melanosporum development and that a set of three genes, 18S rRNA, Elongation factor 1-alpha and 40S ribosomal protein S3, is the most suitable to normalize qRT-PCR data from all the analyzed developmental stages; conversely, 18S rRNA, Glucose-6-phosphate dehydrogenase and Elongation factor 1-alpha are the most suitable genes for fruiting body developmental stages.

Truffle thio-flavours reversibly inhibit truffle tyrosinase.

Tyrosinase is an enzyme having two copper atoms at the reactive site occurring in prokaryotic and eukaryotic organisms. In animals tyrosinase is responsible for pigmentation, in plants for protection of injured tissues or, as in fungi, to harden cell walls. Some of us have previously shown that tyrosinase is involved in truffle development and differentiation. Here we present the purification, the molecular properties and the reversible inhibition of Tuber melanosporum tyrosinase by dimethyl-sulfide and bis[methylthio]methane, the main flavour compounds of black and whitish truffles. The MW(r) is 39000. L-3,4-dihydroxyphenylalanine and L-tyrosine stain corresponding bands as expected for a true tyrosinase. Phenylthiourea, diethyldithiocarbamate and mimosine inhibit L-tyrosine and L-3,4-dihydroxyphenylalanine oxidation.