Predictive Analyses of Biological Effects of Natural Products: From Plant Extracts to Biomolecular Laboratory and Computer Modeling

Year by year, the characterization of the biological activity of natural products is becoming more competitive and complex, with the involvement in this research area of experts belonging to different scientific fields, including chemistry, biochemistry, molecular biology, immunology and bioinformatics. These fields are becoming of great interest for several high-impact scientific journals, including eCAM. The available literature in general, and a survey of reviews and original articles recently published, establishes that natural products, including extracts from medicinal plants and essential oils, retain interesting therapeutic activities, including antitumor, antiviral, anti-inflammatory, pro-apoptotic and differentiating properties. In this commentary, we focus attention on interest in networks based on complementary activation and comparative evaluation of different experimental strategies applied to the discovery and characterization of bioactive natural products. A representative flow chart is shown in the paper

Predictive Analyses of Biological Effects of Natural Products: From Plant Extracts to Biomolecular Laboratory and Computer Modeling

Year by year, the characterization of the biological activity of natural products is becoming more competitive and complex, with the involvement in this research area of experts belonging to different scientific fields, including chemistry, biochemistry, molecular biology, immunology and bioinformatics. These fields are becoming of great interest for several high-impact scientific journals, including eCAM. The available literature in general, and a survey of reviews and original articles recently published, establishes that natural products, including extracts from medicinal plants and essential oils, retain interesting therapeutic activities, including antitumor, antiviral, anti-inflammatory, pro-apoptotic and differentiating properties. In this commentary, we focus attention on interest in networks based on complementary activation and comparative evaluation of different experimental strategies applied to the discovery and characterization of bioactive natural products. A representative flow chart is shown in the paper

Ground state naïve pluripotent stem cells and CRISPR/Cas9 gene correction for β-thalassemia

The β-thalassemias are a group of hereditary diseases caused by more than 300 mutations of the adult β-globin gene, leading to low or absent production of adult hemoglobin (HbA) (1-3). Together with sickle cell anemia (SCA), thalassemia syndromes are among the most impactful diseases in developing countries, in which the lack of genetic counselling and prenatal diagnosis have contributed to the maintenance of a very high frequency in the population. The management of β-thalassemia patients is mostly based on blood transfusion, chelation therapy and, alternatively, on bone marrow transplantation (2). Recently, novel therapeutic options have been explored, such as gene therapy (3) and fetal hemoglobin (HbF) induction (4). Despite the fact that these approaches are promising, they are at present still under deep experimental development and limited to a low number of clinical trials (2-4). With respect to gene therapy for β-thalassemia significant progresses are expected, also considering fundamental insights into globin switching and new technology developments which might have a strong impact on novel gene-therapy approaches (3). A robust information is however available regarding the management of β-thalassemia, i.e., that patients exhibiting high levels of endogenous HbF might exhibit a milder clinical status, as in the case of hereditary persistence of fetal hemoglobin (HPFH) (4). In this context, one of the most exciting strategies recently proposed for hereditary diseases, including β-thalassemia, is genome editing using a variety of strongly validated approaches. Among these strategies, the clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 nuclease system (5-7), in which a single guide RNA (sgRNA) directs the Cas9 nuclease for site-specific cleavage, is considered the most efficient

Combined approaches for increasing fetal hemoglobin (HbF) and de novo production of adult hemoglobin (HbA) in erythroid cells from β-thalassemia patients: treatment with HbF inducers and CRISPR-Cas9 based genome editing

Genome editing (GE) is one of the most efficient and useful molecular approaches to correct the effects of gene mutations in hereditary monogenetic diseases, including β-thalassemia. CRISPR-Cas9 gene editing has been proposed for effective correction of the β-thalassemia mutation, obtaining high-level “de novo” production of adult hemoglobin (HbA). In addition to the correction of the primary gene mutations causing β-thalassemia, several reports demonstrate that gene editing can be employed to increase fetal hemoglobin (HbF), obtaining important clinical benefits in treated β-thalassemia patients. This important objective can be achieved through CRISPR-Cas9 disruption of genes encoding transcriptional repressors of γ-globin gene expression (such as BCL11A, SOX6, KLF-1) or their binding sites in the HBG promoter, mimicking non-deletional and deletional HPFH mutations. These two approaches (β-globin gene correction and genome editing of the genes encoding repressors of γ-globin gene transcription) can be, at least in theory, combined. However, since multiplex CRISPR-Cas9 gene editing is associated with documented evidence concerning possible genotoxicity, this review is focused on the possibility to combine pharmacologically-mediated HbF induction protocols with the “de novo” production of HbA using CRISPR-Cas9 gene editing

Mithramycin encapsulated in polymeric micelles by microfluidic technology as novel therapeutic protocol for beta-thalassemia

This report shows that the DNA-binding drug, mithramycin, can be efficiently encapsulated in polymeric micelles (PM-MTH), based on Pluronic® block copolymers, by a new microfluidic approach. The effect of different production parameters has been investigated for their effect on PM-MTH characteristics. The compared analysis of PM-MTH produced by microfluidic and conventional bulk mixing procedures revealed that microfluidics provides a useful platform for the production of PM-MTH with improved controllability, reproducibility, smaller size, and polydispersity. Finally, an investigation of the effects of PM-MTH, produced by microfluidic and conventional bulk mixing procedures, on the erythroid differentiation of both human erythroleukemia and human erythroid precursor cells is reported. It is demonstrated that PM-MTH exhibited a slightly lower toxicity and more pronounced differentiative activity when compared to the free drug. In addition, PM-MTH were able to upregulate preferentially ?-globin messenger ribonucleic acid production and to increase fetal hemoglobin (HbF) accumulation, the percentage of HbF-containing cells, and their HbF content without stimulating ?-globin gene expression, which is responsible for the clinical symptoms of ß-thalassemia. These results represent an important first step toward a potential clinical application, since an increase in HbF could alleviate the symptoms underlying ß-thalassemia and sickle cell anemia. In conclusion, this report suggests that PM-MTH produced by microfluidic approach warrants further evaluation as a potential therapeutic protocol for ß-thalassemia.<br/

Upstream stimulatory factors are involved in the P1 promoter directed transcription of the AbetaH-J-J locus

<p>Abstract</p> <p>Background</p> <p>Alternative splicing of the locus AβH-J-J generates functionally distinct proteins: the enzyme aspartyl (asparaginyl) β-hydroxylase (AAH), truncated homologs of AAH with a role in calcium homeostasis humbug and junctate and a structural protein of the sarcoplasmic reticulum membranes junctin. AAH and humbug are over expressed in a broad range of malignant neoplasms. We have previously reported that this locus contains two promoters, P1 and P2. While AAH and humbug are expressed in most tissues under the regulation of the P1 promoter, AAH, junctin and junctate are predominantly expressed in excitable tissues under the control of the P2 promoter. We previously demonstrated that Sp transcription factors positively regulate the P1 promoter.</p> <p>Results</p> <p>In the present study, we extended the functional characterization of the P1 promoter of the AβH-J-J locus. We demonstrated by quantitative Real-time RT-PCR that mRNAs from the P1 promoter are actively transcribed in all the human cell lines analysed. To investigate the transcription mechanism we transiently transfected HeLa cells with sequentially deleted reporter constructs containing different regions of the -661/+81 P1 nucleotide sequence. Our results showed that (i) this promoter fragment is a powerful activator of the reporter gene in HeLa cell line, (ii) the region spanning 512 bp upstream of the transcription start site exhibits maximal level of transcriptional activity, (iii) progressive deletions from -512 gradually reduce reporter expression.</p> <p>The region responsible for maximal transcription contains an E-box site; we characterized the molecular interactions between USF1/2 with this E-box element by electrophoretic mobility shift assay and supershift analysis. In addition, our USF1 and USF2 chromatin immunoprecipitation results demonstrate that these transcription factors bind the P1 promoter <it>in vivo</it>.</p> <p>A functional role of USF1/USF2 in upregulating P1-directed transcription was demonstrated by analysis of the effects of (i) <it>in vitro </it>mutagenesis of the P1/E-box binding site, (ii) RNA interference targeting USF1 transcripts.</p> <p>Conclusion</p> <p>Our results suggest that USF factors positively regulate the core of P1 promoter, and, together with our previously data, we can conclude that both Sp and USF DNA interaction and transcription activity are involved in the P1 promoter dependent expression of AAH and humbug.</p

Therapy for Cystic Fibrosis Caused by Nonsense Mutations

Nonsense mutations cover about 10% of cystic fibrosis (CF) patients and generate premature termination codons (PTCs) leading to premature translational termination and causing the synthesis of truncated non-functional or partially functional CFTR (cystic fibrosis transmembrane conductance regulator) protein. The read-through approach is the suppression of translation terminations at PTCs and it has been developed as a therapeutic strategy to restore full-length protein using aminoglycoside antibiotics or PTC124. Phenotypic consequences of PTCs can be exacerbated by the nonsense-mediated mRNA decay (NMD) pathway, which detects and degrades mRNA containing PTC. Therefore, modulation of NMD is also of interest as a potential target for suppression therapy. Not all PTCs are susceptible to the read-through treatment alone, especially where the nonsense mutations are combined with other CFTR mutations. For example, many CF patients present the highly frequent F508del CF mutation, causing an alteration of the cell membrane positioning of the CFTR channel. Pharmacological correctors that rescue the trafficking of F508del CFTR may overcome this defect. A combined administration of correctors/potentiators, read-through molecules, and/or NMD inhibitors, depending on the genotype of the CF patients, could be the basis for the design of a personalized therapeutic approach

Induction by TNF- α

We have developed a microencapsulation procedure for the entrapment and manipulation of IB3-1 cystic fibrosis cells. The applied method is based on generation of monodisperse droplets by a vibrational nozzle. Different experimental parameters were analyzed, including frequency and amplitude of vibration, polymer pumping rate and distance between the nozzle and the gelling bath. We have found that the microencapsulation procedure does not alter the viability of the encapsulated IB3-1 cells. The encapsulated IB3-1 cells were characterized in term of secretomic profile, analyzing the culture medium by Bio-Plex strategy. The experiments demonstrated that most of the analyzed proteins, were secreted both by the free and encapsulated cells, even if in a different extent. In order to determine the biotechnological applications of this procedure, we determined whether encapsulated IB3-1 cells could be induced to pro-inflammatory responses, after treatment with TNF-α. In this experimental set-up, encapsulated and free IB3-1 cells were treated with TNF-α, thereafter the culture media from both cell populations were collected. As expected, TNF-α induced a sharp increase in the secretion of interleukins, chemokines and growth factors. Of great interest was the evidence that induction of interleukin-6 and interleukin-8 occurs also by encapsulated IB3-1 cells

Applications to cancer research of "lab-on-a-chip" devices based on dielectrophoresis (DEP).

The recent development of advanced analytical and bioseparation methodologies based on microarrays and biosensors is one of the strategic objectives of the so-called post-genomic. In this field, the development of microfabricated devices could bring new opportunities in several application fields, such as predictive oncology, diagnostics and anti-tumor drug research. The so called "Laboratory-on-a-chip technology", involving miniaturisation of analytical procedures, is expected to enable highly complex laboratory testing to move from the central laboratory into non-laboratory settings. The main advantages of Lab-on-a-chip devices are integration of multiple steps of different analytical procedures, large variety of applications, sub-microliter consumption of reagents and samples, and portability. One of the requirement for new generation Lab-on-a-chip devices is the possibility to be independent from additional preparative/analytical instruments. Ideally, Lab-on-a-chip devices should be able to perform with high efficiency and reproducibility both actuating and sensing procedures. In this review, we discuss applications of dielectrophoretic(DEP)-based Lab-on-a-chip devices to cancer research. The theory of dielectrophoresis as well as the description of several devices, based on spiral-shaped, parallel and arrayed electrodes are here presented. In addition, in this review we describe manipulation of cancer cells using advanced DEP-based Lab-on-a-chip devices in the absence of fluid flow and with the integration of both actuating and sensing procedures

A Rational Approach to Drug Repositioning in β-thalassemia: Induction of Fetal Hemoglobin by Established Drugs

Drug repositioning and the relevance of orphan drug designation for β-thalassemia is reviewed. Drug repositioning and similar terms ('drug repurposing', 'drug reprofiling', 'drug redirecting', 'drug rescue', 'drug re-tasking' and/or 'drug rediscovery') have gained great attention, especially in the field or rare diseases (RDs), and represent relevant novel drug development strategies to be considered together with the 'off-label' use of pharmaceutical products under clinical trial regimen. The most significant advantage of drug repositioning over traditional drug development is that the repositioned drug has already passed a significant number of short- and long-term toxicity tests, as well as it has already undergone pharmacokinetic and pharmacodynamic (PK/PD) studies. The established safety of repositioned drugs is known to significantly reduce the probability of project failure. Furthermore, development of repurposed drugs can shorten much of the time needed to bring a drug to market. Finally, patent filing of repurposed drugs is expected to catch the attention of pharmaceutical industries interested in the development of therapeutic protocols for RDs. Repurposed molecules that could be proposed as potential drugs for β-thalassemia, will be reported, with some of the most solid examples, including sirolimus (rapamycin) that recently has been tested in a pilot clinical trial