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Background and purpose: Reduced cerebral perfusion has been observed in multiple sclerosis (MS) and may contribute to tissue loss both acutely and chronically. Here, we test the hypothesis that hypoperfusion occurs in MS and relates to the presence of irreversible tissue damage.Methods: In 91 patients with relapsing MS and 26 healthy controls (HC), gray matter (GM) cerebral blood flow (CBF) was assessed using pulsed arterial spin labeling. GM volume, T1 hypointense and T2 hyperintense lesion volumes (T1LV and T2LV, respectively), and the proportion of T2-hyperintense lesion volume that appears hypointense on T1-weighted magnetic resonance imaging (T1LV/T2LV) were quantified. GM CBF and GM volume were evaluated globally, as well as regionally, using an atlas-based approach.Results: Global GM CBF was lower in patients (56.9 ± 12.3 mL/100 g/min) than in HC (67.7 ± 10.0 mL/100 g/min; p < 0.001), a difference that was widespread across brain regions. Although total GM volume was comparable between groups, significant reductions were observed in a subset of subcortical structures. GM CBF negatively correlated with T1LV (r = -0.43, p = 0.0002) and T1LV/T2LV (r = -0.37, p = 0.0004), but not with T2LV.Conclusions: GM hypoperfusion occurs in MS and is associated with irreversible white matter damage, thus suggesting that cerebral hypoperfusion may actively contribute and possibly precede neurodegeneration by hampering tissue repair abilities in MS.

В течение последних нескольких десятилетий западные исследователи считали, что Россия в 1990 г. рухнула вместе со своей экономической и политической системой. В связи с этим ей отводилась роль «восходящей» азиатской державы. Однако основные направления внешней политики России остаются верны ее традиционным интересам и связям с Европой. Сближение с Азией в целом, и в особенности с Китаем, — это лишь ответ на ухудшение отношений с Евросоюзом. Укрепление отношений со странами Средиземноморья является доказательством того, что Россия не откажется от Европы. Сегодня, как и вчера, без участия России невозможно решить ни одну проблему, затрагивающую европейские страны. Over the last few decades western observers have believed that Russia, in 1990, collapsed along with its economic and political system. Therefore, it was relegated to the role of aspiring Asian power. However, the basic lines of Russian foreign policy remain true to its traditional links and interests with Europe. The rapprochement with Asia in general and notably with China is only a response to the worsening of relations with the European Union. The strengthening of relations with Mediterranean area countries is proof that Russia will not give up on Europe. Today as yesterday, no problem involving European countries can really be solved without the participation of Russia.

""Neural stem cells (NSCs) are the self-renewing, multipotent cells that generate the main phenotypes of the nervous system. NSCs hold the promise for cell-based therapies in human neurodevelopmental and neurodegenerative diseases such as Alzheimer’s and Parkinson’s dis- eases. Unfortunately, several con- cerns with NSC cultures currently may limit their therapeutic promise. One major problem is the inability to precisely control stem cell behavior in culture, such as proliferation and specification into different cell types. NSCs are grown in serum-free media supplemented with several hormones and cytokines. It has been established that the adult mouse forebrain con- tains NSCs that can be cultivated in vitro when EGF or FGF-2 or their combination is provided. In particu- lar, FGF-2 was shown to promote the growth rate of NSCs in vitro thereby maintaining their multilineage dif- ferentiation potential. However, FGF-2’s susceptibility to enzymatic degradation may limit its clinical applications. We evaluated whether a device containing heparan sulfate (HS), which is a co-factor in growth factor-mediated cell proliferation and differentiation, could potentiate and prolong the delivery of fibroblast growth factor-2 (FGF-2) and thus improve in vitro NSC cultivation. Our research showed that cultivation of NSCs in media with a controlled release of FGF-2 increased their pro-liferation rate, reduced the apoptosis and the senescence. In these experi- mental conditions NSCs preserve their stemness properties for a longer period of time compared with con- trols.""

Introduction: Clinical applica- tions of RNA-based therapeutics such as small interfering RNAs (siR- NAs) have been limited mainly due to low intracellular delivery efficien- cy in vitro and in vivo. To enhance gene delivery effect, various cationic complexes have been developed for delivering plasmid DNA, antisense, or siRNA into cells. However, the use of cationic vectors for clinical appli- cations has been severely limited by their high toxicity, low serum stabil- ity, nonspecific immune-stimulating effects, and poor biodegradability. In order to overcome these hurdles in gene therapy and improve gene delivery efficiency, we developed a copolymer composed of acetylated PEI (AcPEI) and poly(D,L-lactide- co-glycolide) (PLGA). The biode- gradable PLGA-AcPEI nanoparticles were tested by loading the siRNA of mutant huntingtin in fibroblasts from control and heterozygotic HD patients [1]. Materials and methods: The PLGA-based nanoparticles pre- sented in this study were synthesized by emulsion evaporation method, characterized by dynamic light scat- tering, and used as vector carrier for siRNA transfection in fibroblast cells. Quantitative PCR reactions were performed with primers to am- plify mutant huntingtin or β-actin mRNA using the Quantitect SYBR Green PCR kit. Data were analyzed using the 2-ΔΔCT method [S3] and β-actin mRNA for normalization. Results: PLGA-AcPEI nanoparticles exhibited a higher transfection ef- ficiency and lower cytotoxicity in fibroblasts from control and hetero- zygotic HD patients compared to the PEI/siRNA complex. Real-time PCR experiments showed decreased expression levels of mutant Htt and almost normal expression level of wild-type Htt. We achieved improved gene selectivity and allele selectivity of mutant HTT allele silencing. Con- clusions: Targeted reduction of mu- tant Huntingtin mRNA is considered an ideal strategy for treating HD. Our results demonstrate that PLGA- AcPEI nanoparticles are promising non-viral vectors for gene delivery in neurodegerative disease. Reference [1] Fiszer A, Mykowska A, Krzyzosiak WJ. Inhibition of mutant huntingtin expres- sion by RNA duplex targeting expanded CAG repeats. Nucleic Acids Res. 2011; 39: 5578-85.

Accumulation of unwanted/misfolded proteins in aggregates has been observed in airways of patients with cystic fibrosis (CF), a life-threatening genetic disorder caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR). Here we show how the defective CFTR results in defective autophagy and decreases the clearance of aggresomes. Defective CFTR-induced upregulation of reactive oxygen species (ROS) and tissue transglutaminase (TG2) drive the crosslinking of beclin 1, leading to sequestration of phosphatidylinositol-3-kinase (PI(3)K) complex III and accumulation of p62, which regulates aggresome formation. Both CFTR knockdown and the overexpression of green fluorescent protein (GFP)-tagged-CFTR(F508del) induce beclin 1 downregulation and defective autophagy in non-CF airway epithelia through the ROS-TG2 pathway. Restoration of beclin 1 and autophagy by either beclin 1 overexpression, cystamine or antioxidants rescues the localization of the beclin 1 interactome to the endoplasmic reticulum and reverts the CF airway phenotype in vitro, in vivo in Scnn1b-transgenic and Cftr(F508del) homozygous mice, and in human CF nasal biopsies. Restoring beclin 1 or knocking down p62 rescued the trafficking of CFTR(F508del) to the cell surface. These data link the CFTR defect to autophagy deficiency, leading to the accumulation of protein aggregates and to lung inflammation.

Tissue engineering represents a possible approach to replace the lost or defective muscle. The purpose of this study was to assess a new scaffold design for muscle tissue engineering, by comparing the growth of C2C12 mouse cells on different polymers. Polymeric substrata were prepared with a soft lithography technique (replica molding) from three different masters (a milled aluminium plate, a laser-processed alumina sheet, and a microgrooved modeling paste mould obtained from a home-made fibers array). Each master imprinted with an elastomeric material (Elastosil RT601 Wacker), was in turn used as a mould to prepare the microgrooved polymeric substrata by solvent-casting. Polymer selected was a medical grade biodegradable poly(L-lactic acid)/trimethylencarbonate (PLLA/TMC 68:32, Boerhinger Ingelheim). Four different groups based on fiber spacing (30–35, 50–55, 70–75, and 90–95 lm) were evaluated. We compared 3- week growth of C2C12 cells cultured on scaffolds alone, or supplementing the scaffold with two key cytokines involved in muscle regeneration, bFGF(30 ± 120 pM) and HGF(70 ± 280 pM). Both methods facilitated cell attachment, growth, and viability. The cells lined the inner and outer surfaces of the scaffold, filling the pores, as demonstrated by scanning electron microscopy and histology. BrdUrd incorporation confirmed high levels of cellular proliferation. Yet, the combination of both cytokines showed a strong synergistic stimulation of C2C12 myoblast proliferation and chemotactic activity. After 21 days in the HGF-treated samples large areas of well-formed and aligned myotube fascicles were present which appeared to be more frequent than in the scaffold-alone samples and to show higher expression of differentiation-associated proteins. Our results show that the combination of PLLA/TMC polymer as a vehicle for myoblasts and exogenous HGF as a proliferative stimulator is a good candidate for the generation of skeletal muscle in vitro.

Tissue engineering represents a possible approach to replace the lost or defective muscle. The purpose of this study was to assess a new scaffold design for muscle tissue engineering, by comparing the growth of C2C12 mouse cells on different polymers. Polymeric substrata were prepared with a soft lithography technique (replica molding) from three different masters (a milled aluminium plate, a laser-processed alumina sheet, and a microgrooved modeling paste mould obtained from a home-made fibers array). Each master imprinted with an elastomeric material (Elastosilw RT601 Wacker), was in turn used as a mould to prepare the microgrooved polymeric substrata by solvent-casting. Polymer selected was a medical grade biodegradable poly(L-lactic acid) /trimethylencarbonate (PLLA/TMC 68:32, Boerhinger Ingelheim). Four different groups based on fiber spacing (30–35, 50–55, 70–75, and 90–95 mm) were evaluated. We compared 3-week growth of C2C12 cells cultured on scaffolds alone, or supplementing the scaffold with two key cytokines involved in muscle regeneration, bFGF(30G120 pM) and HGF(70G280 pM). Both methods facilitated cell attachment, growth, and viability. The cells lined the inner and outer surfaces of the scaffold, filling the pores, as demonstrated by scanning electron microscopy and histology. BrdUrd incorporation confirmed high levels of cellular proliferation. Yet, the combination of both cytokines showed a strong synergistic stimulation of C2C12 myoblast proliferation and chemotactic activity. After 21 days in the HGF-treated samples large areas of well-formed and aligned myotube fascicles were present which appeared to be more frequent than in the scaffold-alone samples and to show higher expression of differentiation-associated proteins. Our results show that the combination of PLLA/TMC polymer as a vehicle for myoblasts and exogenous HGF as a proliferative stimulator is a good candidate for the generation of skeletal muscle in vitro.

The autosomal dominant, giant-platelet disorders, May-Hegglin anomaly (MHA; MIM 155100), Fechtner syndrome (FTNS; MIM 153640) and Sebastian syndrome (SBS), share the triad of thrombocytopenia, large platelets and characteristic leukocyte inclusions ('Döhle-like' bodies). MHA and SBS can be differentiated by subtle ultrastructural leukocyte inclusion features, whereas FTNS is distinguished by the additional Alport-like clinical features of sensorineural deafness, cataracts and nephritis. The similarities between these platelet disorders and our recent refinement of the MHA (ref. 6) and FTNS (ref. 7) disease loci to an overlapping region of 480 kb on chromosome 22 suggested that all three disorders are allelic. Among the identified candidate genes is the gene encoding nonmuscle myosin heavy chain 9 (MYH9; refs 8-10), which is expressed in platelets and upregulated during granulocyte differentiation. We identified six MYH9 mutations (one nonsense and five missense) in seven unrelated probands from MHA, SBS and FTNS families. On the basis of molecular modelling, the two mutations affecting the myosin head were predicted to impose electrostatic and conformational changes, whereas the truncating mutation deleted the unique carboxy-terminal tailpiece. The remaining missense mutations, all affecting highly conserved coiled-coil domain positions, imparted destabilizing electrostatic and polar changes. Thus, our results suggest that mutations in MYH9 result in three megakaryocyte/platelet/leukocyte syndromes and are important in the pathogenesis of sensorineural deafness, cataracts and nephritis.

BACKGROUND AND OBJECTIVE: Fanconi anemia (FA) is an autosomal recessive disease characterized by pancytopenia, congenital malformation and high predisposition to developing malignancies. The phenotypical heterogeneity is associated with genetic heterogeneity: at least 8 complementation groups (FA-A to FA-H) have been identified, each group presumably corresponding to a separate disease gene (FAA to FAH). The FAA and FAC genes, which account for 75-80% of the patients, have been cloned. Their protein products have no significant homology to any known protein, or to each other, and may therefore represent elements of a new pathway. This review describes some of the recent contributions to the understanding of the molecular basis of FA. EVIDENCE AND INFORMATION SOURCES: The authors of the present review have been working in the field of FA for several years. In the present review they have critically examined articles published in journals covered by the Science Citation Index and Medline. STATE OF ART AND PERSPECTIVE(S): A variety of biochemical and cellular approaches have been used to determine the molecular defect of FA. Evidence for a possible role of the defective proteins in cell cycle regulation, apoptosis, or DNA stability have been reported. Recently, it has been demonstrated that FAA and FAC proteins bind each other and form a complex found in similar abundance in both cytoplasm and nucleus, suggesting a possible function in nucleus activities. Knowledge of the mutation spectrum occurring in the FA genes may contribute significantly to pathogenesis studies in FA and help to design mutation screening strategies. Further functional studies and the cloning of other FA genes will provide insights into the biological basis of FA and information for developing specific therapies for the disease.

Item does not contain fulltext Here, we report the aquaporin 2 (AQP2) mutational analysis of a patient with nephrogenic diabetes insipidus heterozygote due to two novel missense mutations. Direct sequencing of DNA in the male patient revealed that he was compound heterozygote for two mutations in the AQP2 gene: a thymine-to-adenine transversion at position 450 (c.450T>A) in exon 2 and a guanine-to-thymine at nucleotide position 643 (c.643G>T) in exon 4. The double heterozygous 450T>A and 643G>T transversion causes the amino acid substitution D150E and G215C. Direct sequencing of exons 2 and 4 of the AQP2 gene from each of the parents revealed that the c.450T>A mutation was inherited from the father while the c.643G>T mutation was inherited from the mother. Analysis of AQP2 excretion demonstrated that no AQP2 was detectable in the urine of the proband, whereas normal AQP2 levels were measured in both parents. When expressed in renal cells, both proteins were retarded in the endoplasmic reticulum and no redistribution was observed after forskolin stimulation. Of note, homology modeling revealed that the two mutations involve two highly conserved residues providing important clues about the role of the wt residues in AQP2 stability and function.

Non-small cell lung cancer (NSCLC) is frequently complicated by central nervous system (CNS) metastases affecting patients’ life expectancy and quality. At the present clinical trials including neurosurgery, radiotherapy (RT) and systemic treatments alone or in combination have provided controversial results. CNS involvement is even more frequent in NSCLC patients with EGFR activating mutations or ALK rearrangement suggesting a role of target therapy in the upfront treatment in place of loco-regionals treatments (i.e. RT and/or surgery). So far clinical research has not explored the potential role of accurate brain imaging (i.e. MRI instead of the routine total-body contrast CT and/or PET/CT staging) to identify patients that could benefit of local therapies. Moreover, for patients who require concomitant RT there are no clear guidelines on the timing of intervention with respect to innovative precision medicine approaches with Tyrosine Kinase Inhibitors, ALK-inhibitors and/or immuno-oncological therapies. On this basis the present review describes the therapeutic strategies integrating medical and radiation oncology in patients with metastatic NSCLC (mNSCLC) adenocarcinoma with CNS involvement and EGFR activating mutations or ALK rearrangement.

Systemic sclerosis (SSc) is a heterogeneous, chronic connective tissue disease, characterized by skin fibrosis as well as vascular and visceral lesions. It can involve the lungs, heart, kidneys, gastrointestinal tract, and bones. The orofacial manifestations of SSc can cause functional, aesthetic, and social distress, resulting in significant psychological implications for the patients. In recent decades, fat grafting improved the aesthetic outcomes in terms of volume deficiency, contour asymmetry, and skin elasticity of the face thanks to the regenerative action of the stem cells contained within it. We describe five cases of a patient with SSc treated with fat grafting used to correct volume loss and facial elasticity of the lips and perioral region on the middle and lower third of the face. All the patients received regular postoperative checks at weeks 1 and 2. A multiple choice questionnaire was administered to assess the degree of tolerability of the procedure. The reliability of the questionnaire was evaluated by calculating the Cronbach alpha using the MedCalc Statistical Software version 20.113. The aim of our study is to describe three different types of fat grafting used to correct volume loss and restore facial elasticity of the lips and perioral region on the middle and lower third of the face.

Divalent metal transporter 1 (DMT1) mediates apical iron uptake in duodenal enterocytes and iron transfer from the transferrin receptor endosomal cycle into the cytosol in erythroid cells. Both mk mice and Belgrade rats, which carry an identical DMT1 mutation, exhibit severe microcytic anemia at birth and defective intestinal iron use and erythroid iron use. We report the hematologic phenotype of a child, compound heterozygote for 2 DMT1 mutations, who was affected by severe anemia since birth and showed hepatic iron overload. The novel mutations were a 3-bp deletion in intron 4 (c.310-3_5del CTT) resulting in a splicing abnormality and a C>T transition at nucleotide 1246(p. R416C). A striking reduction of DMT1 protein in peripheral blood mononuclear cells was demonstrated by Western blot analysis. The proband required blood transfusions until erythropoietin treatment allowed transfusion independence when hemoglobin levels between 75 and 95 g/L (7.5 and 9.5 g/dL) were achieved. Hematologic data of this patient at birth and in the first years of life strengthen the essential role of DMT1 in erythropoiesis. The early onset of iron overload indicates that, as in animal models, DMT1 is dispensable for liver iron uptake, whereas its deficiency in the gut is likely bypassed by the up-regulation of other pathways of iron use.

In this commentary, using existing clinical trial data and FDA approvals we propose that there is currently a critical need for an appropriate balancing between the financial impact of new cancer drugs and their actual benefit for patients. By adopting “pleural mesothelioma” as our clinical model we summarize the most relevant pertinent and available literature on this topic, and use an analysis of the reliability of the trials submitted for registration and/or recently published as a case in point to raise concerns with respect to appropriate trial design, biomarker based stratification and to highlight the ongoing need for balancing the benefit/cost ratio for both patients and healthcare providers.

Chestnut seeds are used for fresh consumption and for the industrial preparation of derivatives, such as chestnut flour. During industrial processing, large amounts of by-products are generally produced, such as leaves, flowers, shells and burs. In the present study, chestnut shells were extracted by boiling water in order to obtain polyphenol-rich extracts. Moreover, for the removal or non-phenolic compounds, a separation by preparative reverse phase chromatography in ten fractions was carried out. The richest fractions in terms of phenolic content were characterized by means of untargeted high-resolution mass spectrometric analysis together with a dedicated and customized data processing workflow. A total of 243 flavonoids, phenolic acids, proanthocyanidins and ellagitannins were tentatively identified in the five richest fractions. Due its high phenolic content (450.03 &micro, g GAE per mg of fraction), one tumor cell line (DU 145) and one normal prostate epithelial cell line (PNT2) were exposed to increasing concentration of fraction 3 dry extract for 24, 48 and 72 h. Moreover, for DU 145 cell lines, increase of apoptotic cells and perturbation of cell cycle was demonstrated for the same extract. Those outcomes suggest that chestnut industrial by-products could be potentially employed as a source of bioresources.

In this study, chestnut shells (CS) were used in order to obtain bioactive compounds through different extraction procedures. The aqueous extracts were chemically characterized. The highest extraction yield and total phenolic content was obtained by conventional liquid extraction (CLE). Gallic and protocatechuic acids were the main simple phenols in the extract, with 86.97 and 11.20 mg/g chestnut shells dry extract (CSDE), respectively. Six tumor cell lines (DU 145, PC-3, LNCaP, MDA-MB-231, MCF-7, and HepG2) and one normal prostate epithelial cell line (PNT2) were exposed to increasing concentration of CSDE (1–100 µg/mL) for 24 h, and cell viability was evaluated using 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide MTT assay. A reduced rate in cell viability was observed in DU 145, PC-3, LNCaP, and MCF-7 cells, while viability of the other assessed cells was not affected, except for PNT2 cells at a concentration of 100 μg/mL. Furthermore, CSDE—at concentrations of 55.5 and 100 µg/mL—lead to a significant increase of apoptotic cells in DU 145 cells of 28.2% and 61%, respectively. In conclusion, these outcomes suggested that CS might be used for the extraction of several polyphenols that may represent good candidates for alternative therapies or in combination with current chemotherapeutics.