CCR5Δ32 Polymorphism Associated with a Slower Rate Disease Progression in a Cohort of RR-MS Sicilian Patients

Multiple sclerosis (MS) disease is carried through inflammatory and degenerative stages. Based on clinical feaures, it can be subdivided into three groups: relapsing-remitting MS, secondary progressive MS, and primary progressive MS. Multiple sclerosis has a multifactorial etiology with an interplay of genetic predisposition, environmental factors, and autoimmune inflammatory mechanism in which play a key role CC-chemokines and its receptors. In this paper, we studied the frequency of CCR5 gene Δ32 allele in a cohort of Sicilian RR-MS patients comparing with general Sicilian population. Also, we evaluate the association between this commonly polymorphism and disability development and age of disease onset in the same cohort. Our results show that presence of CCR5Δ32 is significantly associated with expanded disability status scale score (EDSS) but not with age of disease onset

Possible A2E Mutagenic Effects on RPE Mitochondrial DNA from Innovative RNA-Seq Bioinformatics Pipeline

Mitochondria are subject to continuous oxidative stress stimuli that, over time, can impair their genome and lead to several pathologies, like retinal degenerations. Our main purpose was the identification of mtDNA variants that might be induced by intense oxidative stress determined by N-retinylidene-N-retinylethanolamine (A2E), together with molecular pathways involving the genes carrying them, possibly linked to retinal degeneration. We performed a variant analysis comparison between transcriptome profiles of human retinal pigment epithelial (RPE) cells exposed to A2E and untreated ones, hypothesizing that it might act as a mutagenic compound towards mtDNA. To optimize analysis, we proposed an integrated approach that foresaw the complementary use of the most recent algorithms applied to mtDNA data, characterized by a mixed output coming from several tools and databases. An increased number of variants emerged following treatment. Variants mainly occurred within mtDNA coding sequences, corresponding with either the polypeptide-encoding genes or the RNA. Time-dependent impairments foresaw the involvement of all oxidative phosphorylation complexes, suggesting a serious damage to adenosine triphosphate (ATP) biosynthesis, that can result in cell death. The obtained results could be incorporated into clinical diagnostic settings, as they are hypothesized to modulate the phenotypic expression of mtDNA pathogenic variants, drastically improving the field of precision molecular medicine

Stargardt Phenotype Associated With Two ELOVL4 Promoter Variants and ELOVL4 Downregulation: New Possible Perspective to Etiopathogenesis?

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Two Novel KRIT1 and CCM2 Mutations in Patients Affected by Cerebral Cavernous Malformations: New Information on CCM2 Penetrance

Wide comprehension of genetic features of cerebral cavernous malformations (CCM) represents the starting point to better manage patients and risk rating in relatives. The causative mutations spectrum is constantly growing. KRIT1, CCM2, and PDCD10 are the three loci to date linked to familial CCM development, although germline mutations have also been detected in patients affected by sporadic forms. In this context, the main challenge is to draw up criteria to formulate genotype-phenotype correlations. Clearly, genetic factors determining incomplete penetrance of CCM need to be identified. Here, we report two novel intronic variants probably affecting splicing. Molecular screening of CCM genes was performed on DNA purified by peripheral blood. Coding exons and intron-exon boundaries were sequenced by the Sanger method. The first was detected in a sporadic patient and involves KRIT1. The second affects CCM2 and it is harbored by a woman with familial CCM. Interestingly, molecular analysis extended to both healthy and ill relatives allowed to estimate, for the first time, a penetrance for CCM2 lower than 100%, as to date reported. Moreover, heterogeneity of clinical manifestations among those affected carrying the same genotype further confirms involvement of modifier factors in CCM development

Notas sobre a Carta de Veneza

This paper presents a critical reading of the Venice Charter, an Icomos key document, fruit of a conference held in 1964. The Charter is often quoted in Brazil but is not always properly understood. The conservation and restoration charters - especially those produced by international institutions - are documents that have an indicatory or, at the most, prescriptive character. They constitute the deontological foundation of many professionals involved in preservation, but they are not recipes for immediate use. In order to elaborate a well-founded reading of the document, its ideas must be understood in connection to the theoretical postulates of the time they were engendered and to the developments of the field. Thus this paper will examine these subjects, commenting and enlightening the Charter's articles and pointing out the origins of specific ideas. It also discusses how the Charter relates to previous documents and their theoretical foundations. This approach, based in a critical analysis, is necessary in order to reach a fuller interpretation of the Charter's indications so that they can be used in the present

ANNURCA APPLE POLYPHENOL EXTRACT DECREASES THE IN VITRO MIGRATORY AND INVASIVE POTENTIAL OF TRIPLE NEGATIVE BREAST CANCER CELLS

Breast cancer is the most common invasive cancer among women in the world. Over the past two decades, although the treatment for breast cancer has substantially improved, its metastasis is still a major cause of mortality and poor prognosis. Cancer cell migration is essential for the early steps of metastasis, during which cancer cells move through the primary tumor and reach the blood vessels. Thus, suppression of cancer cell migration and invasion represents an important therapeutic target and the development of new therapeutic agents to prevent cancer cell migration and invasion is highly desirable. Triple-negative breast cancer (TNBC) is a highly invasive subgroup of breast carcinomas that lacks expression of estrogen receptor, progesterone receptor, and human epidermal growth factor receptor-2. Proven active targeted therapy is presently unavailable for patients with TNBC and currently, chemotherapy represents the standard of care for patients with early advanced TNBC. Dietary polyphenols are among the naturally-occurring substances that have shown promising anti-cancer properties and low toxicity in comparison to standard chemotherapeutic agents. Previous works from our group led us to select polyphenol extract from Annurca apple (APE) as a potential candidate for drug development against breast cancer. We have indeed reported that APE displays a potent prooxidant cytotoxic effect in MCF-7 human breast carcinoma cells and, more recently, we demonstrated that APE is able to selectively kill MDA-MB-231 TNBC cells through ROS generation, sustained JNK activation and cell growth and survival inhibition while exerting a protective antioxidant effect in normal cells. The wound healing assay is widely used to investigate cell migrationin vitro and video-time lapse microscopy allows to monitor in real time the migration process. Several experimental factors, such as uneven cell density among different samples can affect the reproducibility and reliability of this assay. In this study the dose-dependent inhibitory effect exerted by APE on migration of MDAMB- 231 TNBC cells has been highlighted by means of a novel methodological approach to quantify wound healing process that is based on the analysis of the wound closure as a diffusion reaction process, and the potential underlying molecular mechanisms have been explored

Annurca apple polyphenol extract promotes mesenchymal-to-epithelial transition and inhibits migration in triple-negative breast cancer cells through ROS/JNK signaling

Aberrant activation of epithelial-to-mesenchymal transition has been shown to correlate with triple-negative breast cancer (TNBC) progression and metastasis. Thus, the induction of the reverse process might offer promising opportunities to restrain TNBC metastatic spreading and related mortality. Recently, the Annurca apple polyphenol extract (APE) has been highlighted as a multi-faceted agent that selectively kills TNBC cells by ROS generation and sustained JNK activation. Here, by qualitatively and quantitatively monitoring the real-time movements of live cells we provided the first evidence that APE inhibited the migration of MDA-MB-231 and MDA-MB-468 TNBC cells and downregulated metalloproteinase-2 and metalloproteinase-9. In MDA-MB-231 cells APE decreased SMAD-2/3 and p-SMAD-2/3 levels, increased E-cadherin/N-cadherin protein ratio, induced the switch from N-cadherin to E-cadherin expression and greatly reduced vimentin levels. Confocal and scanning electron microscopy imaging of APE-treated MDA-MB-231 cells evidenced a significant cytoskeletal vimentin and filamentous actin reorganization and revealed considerable changes in cell morphology highlighting an evident transition from the mesenchymal to epithelial phenotype with decreased migratory features. Notably, all these events were reverted by N-acetyl-L-cysteine and JNK inhibitor SP600125 furnishing evidence that APE exerted its effects through the activation of ROS/JNK signaling. The overall data highlighted APE as a potential preventing agent for TNBC metastasis

Granulocyte–colony stimulating factor plus plerixafor in patients with β-thalassemia major results in the effective mobilization of primitive CD34+ cells with specific gene expression profile

Successful gene therapy for β-thalassemia requires optimal numbers of autologous gene-transduced hematopoietic stem and progenitor cells (HSPCs) with high repopulating capacity. Previous studies suggested superior mobilization in these patients by the combination of granulocyte–colony stimulating factor (G-CSF) plus plerixafor over single agents. We mobilized four adult patients using G-CSF+plerixafor to assess the intra-individual variation of the circulating CD34+ cells number and subtypes preand post-plerixafor administration. The procedure was well-tolerated and the target cell dose of ≥8×106 CD34+ cells/kg was achieved in three of them with one apheresis procedure. The addition of plerixafor unanimously increased the number of circulating CD34+ cells, and the frequency of the most primitive CD34+ subtypes: CD34+/38- and CD34+/133+/38- as well as the in vitro clonogenic potency. Microarray analyses of CD34+ cells purified from the leukapheresis of one patient mobilized twice, with G-CSF and with G-CSF+plerixafor, highlighted in G-CSF+plerixafor-mobilized CD34+ cells, higher levels of expression genes involved in HSPC motility, homing, and cell cycles. In conclusion, G-CSF+plerixafor in β-thalassemia patients mobilizes optimal numbers of HSPCs with characteristics that suggest high capacity of engraftment after transplantation.   β地中海贫血的成功基因治疗需要最佳数量具有较高再生能力的自体基因转导的造血干细胞和祖细胞（HSPC）。之前的研究表明，与单药相比，通过组合粒细胞集落刺激因子（G-CSF）加普乐沙福在这些患者中有出色的动员作用。我们使用G-CSF+普乐沙福对四例成年患者进行了动员，以评估服用普乐沙福之前和之后的循环CD34+细胞数量和亚型的个体内差异。这种方式的耐受性好，其中的三例患者仅通过一次分离技术即获得≥8×106 CD34+细胞/kg的细胞采集目标。加用普乐沙福毫无例外地增加了循环CD34+细胞的数量和最原始CD34+亚型（CD34+/38-和CD34+/133+/38+）的频率以及体外克隆效力。一例血细胞分离术中纯化的CD34+细胞微阵列分析（患者使用G-CSF和G-CSF+普乐沙福动员两次）强调，在G-CSF+普乐沙福动员的CD34+细胞中，有更高水平的表达基因牵涉到HSPC运动性、归巢和细胞周期。总之，G-CSF+普乐沙福在β地中海贫血病患者中可以动员最优数量的HSPC，具有移植后的移植成活率高的特征

Update on Novel CCM Gene Mutations in Patients with Cerebral Cavernous Malforzmations

Cerebral cavernous malformations (CCMs) are lesions affecting brain microvessels. The pathogenesis is not clearly understood. Conventional classification criterion is based on genetics, and thus, familial and sporadic forms can be distinguished; however, classification of sporadic cases with multiple lesions still remains uncertain. To date, three CCM causative genes have been identified: CCM1/KRIT1, CCM2/MGC4607 and CCM3/PDCD10. In our previous mutation screening, performed in a cohort of 95 Italian patients, with both sporadic and familial cases, we identified several mutations in CCM genes. This study represents further molecular screening in a cohort of 19 Italian patients enrolled by us in the few last years and classified into familial, sporadic and sporadic with multiple lesions cases. Direct sequencing and multiplex ligation-dependent probe amplification (MLPA) analysis were performed to detect point mutations and large genomic rearrangements, respectively. Effects of detected mutations and single-nucleotide polymorphisms (SNPs) were evaluated by an in silico approach and by western blot analysis. A novel nonsense mutation in CCM1 and a novel missense mutation in CCM2 were detected; moreover, several CCM2 gene polymorphisms in sporadic CCM patients were reported. We believe that these data enrich the mutation spectrum of CCM genes, which is useful for genetic counselling to identify both familial and sporadic CCM cases, as early as possible