Christian Scerri

Haploinsufficiency for the erythroid transcription factor KLF1 causes hereditary persistence of fetal hemoglobin

Hereditary persistence of fetal hemoglobin (HPFH) is characterized by persistent high levels of fetal hemoglobin (HbF) in adults. Several contributory factors, both genetic and environmental, have been identified but others remain elusive. HPFH was found in 10 of 27 members from a Maltese family. We used a genome-wide SNP scan followed by linkage analysis to identify a candidate region on chromosome 19p13.12–13. Sequencing revealed a nonsense mutation in the KLF1 gene, p.K288X, which ablated the DNA-binding domain of this key erythroid transcriptional regulator. Only family members with HPFH were heterozygous carriers of this mutation. Expression profiling on primary erythroid progenitors showed that KLF1 target genes were downregulated in samples from individuals with HPFH. Functional assays suggested that, in addition to its established role in regulating adult globin expression, KLF1 is a key activator of the BCL11A gene, which encodes a suppressor of HbF expression. These observations provide a rationale for the effects of KLF1 haploinsufficiency on HbF levels.

Deregulation of the protein phosphatase 2A, PP2A in cancer: complexity and therapeutic options.

The complexity of the phosphatase, PP2A, is being unravelled and current research is increasingly providing information on the association of deregulated PP2A function with cancer initiation and progression. It has been reported that decreased activity of PP2A is a recurrent observation in many types of cancer, including colorectal and breast cancer (Baldacchino et al. EPMA J. 5:3, 2014; Cristobal et al. Mol Cancer Ther. 13:938–947, 2014). Since deregulation of PP2A and its regulatory subunits is a common event in cancer, PP2A is a potential target for therapy (Baldacchino et al. EPMA J. 5:3, 2014). In this review, the structural components of the PP2A complex are described, giving an in depth overview of the diversity of regulatory subunits. Regulation of the active PP2A trimeric complex, through phosphorylation and methylation, can be targeted using known compounds, to reactivate the complex. The endogenous inhibitors of the PP2A complex are highly deregulated in cancer, representing cases that are eligible to PP2A-activating drugs. Pharmacological opportunities to target low PP2A activity are available and preclinical data support the efficacy of these drugs, but clinical trials are lacking. We highlight the importance of PP2A deregulation in cancer and the current trends in targeting the phosphatase.

The β+ IVS, I‐NT no. 6 (T→ C) thalassaemia in heterozygotes with an associated Hb Valletta or Hb S heterozygosity in homozygotes from Malta

Summary. In vitro DNA amplification and dot blot analysis with synthetic allele specific oligonucleotides (ASO) identified the β IVS, I‐6 (T→C) thalassaemia in 78% of 32 chromosomes from 16 β‐thalassaemia homozygotes in Malta. The preponderance of a single thalassaemia mutation in one population is unusual. The β+ IVS, I‐6C thalassaemia mutation was also found in three carriers who had an associated β globin heterozygosity, i.e. Hb Valletta (or α2β287PRO) or Hb S (or α2β26VAL). The proportion of Hb A in these cases (av. = 29.7%) provided objective documentation of the relatively mild effect of this mutation on in vivo globin gene expression. However, the expression of homozygous disease was more severe in developing children compared to adults. The β+ IVS, I‐6C mutation complicates population testing because heterozygotes can have Hb A2 levels below those classically associated with β thalassaemia.

Suppressive role exerted by microRNA-29b-1-5p in triple negative breast cancer through SPIN1 regulation

MiR-29 family dysregulation occurs in various cancers including breast cancers. We investigated miR-29b-1 functional role in human triple negative breast cancer (TNBC) the most aggressive breast cancer subtype. We found that miR-29b-1-5p was downregulated in human TNBC tissues and cell lines. To assess whether miR-29b-1-5p correlated with TNBC regenerative potential, we evaluated cancer stem cell enrichment in our TNBC cell lines, and found that only MDA-MB-231 and BT-20 produced primary, secondary and tertiary mammospheres, which were progressively enriched in OCT4, NANOG and SOX2 stemness genes. MiR-29b-1-5p expression inversely correlated with mammosphere stemness potential, and miR-29b-1 ectopic overexpression decreased TNBC cell growth, self-renewal, migration, invasiveness and paclitaxel resistance repressing WNT/βcatenin and AKT signaling pathways and stemness regulators. We identified SPINDLIN1 (SPIN1) among predicted miR-29b-1-5p targets. Consistently, SPIN1 was overexpressed in most TNBC tissues and cell lines and negatively correlated with miR-29b-1-5p. Target site inhibition showed that SPIN1 seems to be directly controlled by miR-29b-1-5p. Silencing SPIN1 mirrored the effects triggered by miR-29b-1 overexpression, whereas SPIN1 rescue by SPIN1miScript protector, determined the reversal of the molecular effects produced by the mimic-miR-29b-1-5p. Overall, we show that miR-29b-1 deregulation impacts on multiple oncogenic features of TNBC cells and their renewal potential, acting, at least partly, through SPIN1, and suggest that both these factors should be evaluated as new possible therapeutic targets against TNBC.

Characterization and locus assignment of two α-globin variants present in the maltese population: Hb St. Luke's [α95(G2)Pro→Arg] and Hb Setif [α94(G1)Asp→Tyr]

Two types of α-globin variants were found in 0.2% of a large number of newborn from Malta. The two hemoglobins were identified from tryptic maps on a Vydac C18 column and by α-globin gene sequencing as Hb St. Lukes (isoelectric point = 7.18 ± 0.017) and Hb Setif (isoelectric point = 7.26 ± 0.010). Hb St. Lukes [α95(G2)Pr→Arg] was found to result from a C→G mutation at the second position of codon 95 on an cd-globin gene, and Hb Setif [α94(Gl) Asp→Tyr] resulted from a G→T mutation at the first position of codon 94 on an α2-globin gene. Quantification of Hb St. Lukes (11.1 ± 1.12%) and Hb Setif (14.7 ± 2.22%) in peripheral blood hemolysates indicated that, in the absence of either an α- or a β-thalassemia allele, the protein products of the α1- and α2-gIobin genes were nearly equal in quantity.

Variants within protectin (CD59) and CD44 genes linked to an inherited haplotype in a family with coeliac disease

Coeliac disease (CD) is an autoimmune disorder characterised by inflammation, villous atrophy and hyperplasia of the small intestinal mucosa that affects genetically susceptible individuals. A genome-wide scan was performed in 17 family members with high incidence of CD. Highest nonparametric linkage (NPL) and logarithm of odds (LOD) scores were of 6.21 (P = 0.0107) and 2.57, respectively, to a region on chromosome 11p13-12. Following fine mapping, NPL and LOD scores did not change, but the linkage interval on chromosome 11 was narrowed to a region that is approximately 50.94 cM from pTer. Two inherited haplotypes on chromosomes 11p13-12 and 9q21 were observed in all affected members but not in the majority of clinically normal individuals. Sequencing of genes at region 11p13-12 showed a number of sequence variants, two of which were linked with the inherited haplotype. One of these variants in the CD59 gene was found at a very low frequency in the population and could possibly affect pre-messenger RNA splicing. This study is of particular importance for the identification of novel genes that might be responsible for CD other than human leukocyte antigen.

Unusual roles of caspase-8 in triple-negative breast cancer cell line MDA-MB-231

Triple-negative breast cancer (TNBC) is a clinically aggressive form of breast cancer that is unresponsive to endocrine agents or trastuzumab. TNBC accounts for ~10-20% of all breast cancer cases and represents the form with the poorest prognosis. Patients with TNBC are at higher risk of early recurrence, mainly in the lungs, brain and soft tissue, therefore, there is an urgent need for new therapies. The present study was carried out in MDA-MB-231 cells, where we assessed the role of caspase-8 (casp-8), a critical effector of death receptors, also involved in non‑apoptotic functions. Analysis of casp-8 mRNA and protein levels indicated that they were up-regulated with respect to the normal human mammalian epithelial cells. We demonstrated that silencing of casp-8 by small interfering-RNA, strongly decreased MDA-MB-231 cell growth by delaying G0/G1- to S-phase transition and increasing p21, p27 and hypo-phosphorylated/active form of pRb levels. Surprisingly, casp-8-knockdown, also potently increased both the migratory and metastatic capacity of MDA-MB‑231 cells, as shown by both wound healing and Matrigel assay, and by the expression of a number of related-genes and/or proteins such as VEGFA, C-MYC, CTNNB1, HMGA2, CXCR4, KLF4, VERSICAN V1 and MMP2. Among these, KLF4, a transcriptional factor with a dual role (activator and repressor), seemed to play critical roles. We suggest that in MDA-MB‑231 cells, the endogenous expression of casp-8 might keep the cells perpetually cycling through downregulation of KLF4, the subsequent lowering of p21 and p27, and the inactivation by hyperphosphorylation of pRb. Simultaneously, by lowering the expression of some migratory and invasive genes, casp-8 might restrain the metastatic ability of the cells. Overall, our findings showed that, in MDA-MB-231 cells, casp-8 might play some unusual roles which should be better explored, in order to understand whether it might be identified as a molecular therapeutic target.

Human-Computer Interaction Patterns within the Mobile Nutrition Landscape: A Review of Literature

Advances in mobile computing offer the potential to deliver better solutions in the growing health problem of obesity. Poor nutrition and a lack of exercise have led to a situation where obesity is rapidly on the rise. As a result, mobile health interventions in the area of nutrition have been a great asset into changing the lives of many through the use of their devices. Smartphones and other mobile devices can be transformed into effective nutrition tracking tools that consumers can easily make use of to help improve their lives and wellbeing. The academic community has conducted a vast amount of research in the area of mobile nutrition however a number of challenges still exist in the area of human-computer interaction (HCI). HCI has been found to be a key contributing factor when developing systems that constantly and directly interact with their respective users. Effective HCI is required to bridge the communication barrier between a user and the device, thus ensuring a seam-less approach. This research paper highlights the efforts done by academics in the area of mobile HCI within the nutrition landscape. Every study will be discussed in detailed by highlighted the key benefits together with a number of challenges that still exist. As a result, this review of literature will identify key lacunae that still exist in the field of mobile HCI within the nutrition landscape.

Parthenolide prevents resistance of MDA-MB231 cells to doxorubicin and mitoxantrone: the role of Nrf2.

Triple-negative breast cancer is a group of aggressive cancers with poor prognosis owing to chemoresistance, recurrence and metastasis. New strategies are required that could reduce chemoresistance and increases the effectiveness of chemotherapy. The results presented in this paper, showing that parthenolide (PN) prevents drug resistance in MDA-MB231 cells, represent a contribution to one of these possible strategies. MDA-MB231 cells, the most studied line of TNBC cells, were submitted to selection treatment with mitoxantrone (Mitox) and doxorubicin (DOX). The presence of resistant cells was confirmed through the measurement of the resistance index. Cells submitted to this treatment exhibited a remarkable increment of NF-E2-related factor 2 (Nrf2) level, which was accompanied by upregulation of catalase, MnSOD, HSP70, Bcl-2 and P-glycoprotein. Moreover, as a consequence of overexpression of Nrf2 and correlated proteins, drug-treated cells exhibited a much lower ability than parental cells to generate ROS in response to a suitable stimulation. The addition of PN (2.0 μM) to Mitox and DOX, over the total selection time, prevented both the induction of resistance and the overexpression of Nrf2 and correlated proteins, whereas the cells showed a good ability to generate ROS in response to adequate stimulation. To demonstrate that Nrf2 exerted a crucial role in the induction of resistance, the cells were transiently transfected with a specific small interfering RNA for Nrf2. Similarly to the effects induced by PN, downregulation of Nrf2 was accompanied by reductions in the levels of catalase, MnSOD, HSP70 and Bcl-2, prevention of chemoresistance and increased ability to generate ROS under stimulation. In conclusion, our results show that PN inhibited the development of the resistance toward Mitox and DOX, and suggest that these effects were correlated with the prevention of the overexpression of Nrf2 and its target proteins, which occurred in the cells submitted to drug treatment.

Hb Valletta [β87(F3)Thr→Pro] and Hb Marseille/Long Island [β2(NA2)His→Pro; (–1)Met-(+1)Val-(+2)Pro-Leu], in a Unique Compound Heterozygote with a Normal Hemoglobin Phenotype

This study refers to the quantitative hemoglobin (Hb) phenotype of a 19-year-old female with Hb Valletta [β87(F3)Thr→Pro] in association with Hb Marseille/Long Island [β2(NA2)His→Pro; (–1)Met-(+1)Val-(+2)Pro-Leu] and a normal Hb electrophoretogram. The data serve to alert investigators to the possibility that relatives with apparently normal Hb phenotypes may be transmitting mutant alleles and suggest methods for identification.