Differential Internalization of Amphotericin B - Conjugated Nanoparticles in Human Cells and the Expression of Heat Shock Protein 70

Although a variety of nanoparticles (NPs) functionalized with amphotericin B, an antifungal agent widely used in the clinic, have been studied in the last years their cytotoxicity profile remains elusive. Here we show that human endothelial cells take up high amounts of silica nanoparticles (SNPs) conjugated with amphotericin B (AmB) (SNP-AmB) (65.4 12.4 pg of Si per cell) through macropinocytosis while human fibroblasts internalize relatively low amounts (2.3 0.4 pg of Si per cell) because of their low capacity for macropinocytosis. We further show that concentrations of SNP-AmB and SNP up to 400 mg/mL do not substantially affect fibroblasts. In contrast, endothelial cells are sensitive to low concentrations of NPs (above 10 mg/mL), in particular to SNP-AmB. This is because of their capacity to internalize high concentration of NPs and high sensitivity of their membrane to the effects of AmB. Low-moderate concentrations of SNP-AmB (up to 100 mg/mL) induce the production of reactive oxygen species (ROS), LDH release, high expression of pro-inflammatory cytokines and chemokines (IL-8, IL-6, G-CSF, CCL4, IL-1b and CSF2) and high expression of heat shock proteins (HSPs) at gene and protein levels. High concentrations of SNP-AmB (above 100 ug/mL) disturb membrane integrity and kill rapidly human cells(60% after 5 h). This effect is higher in SNP-AmB than in SNP.

A Novel Autosomal Recessive GJA1 Missense Mutation Linked to Craniometaphyseal Dysplasia

Craniometaphyseal dysplasia (CMD) is a rare sclerosing skeletal disorder with progressive hyperostosis of craniofacial bones. CMD can be inherited in an autosomal dominant (AD) trait or occur after de novo mutations in the pyrophosphate transporter ANKH. Although the autosomal recessive (AR)form of CMD had been mapped to 6q21-22 the mutation has been elusive. In this study, we performed whole-exome sequencing for one subject with AR CMD and identified a novel missense mutation (c.716G>A, p.Arg239Gln) in the C-terminus of the gap junction protein alpha-1 (GJA1) coding for connexin 43 (Cx43). We confirmed this mutation in 6 individuals from 3 additional families. The homozygous mutation cosegregated only with affected family members. Connexin 43 is a major component of gap junctions in osteoblasts, osteocytes, osteoclasts and chondrocytes. Gap junctions are responsible for the diffusion of low molecular weight molecules between cells. Mutations in Cx43 cause several dominant and recessive disorders involving developmental abnormalities of bone such as dominant and recessive oculodentodigital dysplasia (ODDD; MIM #164200, 257850) and isolated syndactyly type III (MIM #186100), the characteristic digital anomaly in ODDD. However, characteristic ocular and dental features of ODDD as well as syndactyly are absent in patients with the recessive Arg239Gln Cx43 mutation. Bone remodeling mechanisms disrupted by this novel Cx43 mutation remain to be elucidated.

Adult B-Cell Acute Lymphoblastic Leukemia Cells Display Decreased PTEN Activity and Constitutive Hyperactivation of PI3K/Akt Pathway Despite High PTEN Protein Levels

Adult B-cell acute lymphoblastic leukemia remains a major therapeutic challenge, requiring a better characterization of the molecular determinants underlying disease progression and resistance to treatment. Here, using a phospho-flow cytometry approach we show that adult diagnostic B-cell acute lymphoblastic leukemia specimens display PI3K/Akt pathway hyperactivation, irrespective of their BCR-ABL status and despite paradoxically high basal expression of PTEN, the major negative regulator of the pathway. Protein kinase CK2 is known to phosphorylate PTEN thereby driving PTEN protein stabilization and concomitant PTEN functional inactivation. In agreement, we found that adult B-cell acute lymphoblastic leukemia samples show significantly higher CK2 kinase activity and lower PTEN lipid phosphatase activity than healthy controls. Moreover, the clinical-grade CK2 inhibitor CX-4945 (Silmitasertib) reversed PTEN levels in leukemia cells to those observed in healthy controls, and promoted leukemia cell death without significantly affecting normal bone marrow cells. Our studies indicate that CK2-mediated PTEN posttranslational inactivation, associated with PI3K/Akt pathway hyperactivation, are a common event in adult B-cell acute lymphoblastic leukemia and suggest that CK2 inhibition may constitute a valid, novel therapeutic tool in this malignancy.

Mutational Analysis of Portuguese Families with Multiple Endocrine Neoplasia Type 1 Reveals Large Germline Deletions

OBJECTIVE: To determine the spectrum of MEN1 mutations in Portuguese kindreds, and identify mutation-carriers. PATIENTS, DESIGN AND RESULTS: Six unrelated MEN1 families were studied for MEN1 gene mutations by single-strand conformational polymorphism (SSCP) and DNA sequence analysis of the coding region and exon-intron boundaries of the MEN1 gene. These methods identified 4 different heterozygous mutations in four families: two mutations are novel (mt 1539 delG and mt 655 ims 11 bp) and two have been previously observed (mt 735 del 46p and mt 1656 del C) all resulting in a premature stop codon. In the remaining two families, in whom no mutations or abnormal MEN1 transcripts were detected, segregation studies of the 5' intragenic marker D11S4946 and codon 418 polymorphism in exon 9 revealed two large germline deletions of the MEN1 gene. Southern blot and tumour loss of heterozygosity analysis confirmed and refined the limits of these deletions, which spanned the MEN1 gene at least from: exon 7 to the 3' untranslated region, in one family, and the 5' polymorphic site D11S4946 to exon 9 (obliterating the initiation codon), in the other family. Twenty-six mutant-gene carriers were identified, 6 of which were asymptomatic. CONCLUSIONS: These results emphasize the importance of the detection of MEN1 germline deletions in patients who do not have mutations of the coding region. Important clues indicating the presence of such deletions may be obtained by segregation studies using the intragenic polymorphisms D11S4946 and at codon 418. The detection of these mutations will help in the genetic counselling of clinical management of the MEN1 families in Portugal.

Urinary Tract Effects of HPSE2 Mutations

Urofacial syndrome (UFS) is an autosomal recessive congenital disease featuring grimacing and incomplete bladder emptying. Mutations of HPSE2, encoding heparanase 2, a heparanase 1 inhibitor, occur in UFS, but knowledge about the HPSE2 mutation spectrum is limited. Here, seven UFS kindreds with HPSE2 mutations are presented, including one with deleted asparagine 254, suggesting a role for this amino acid, which is conserved in vertebrate orthologs. HPSE2 mutations were absent in 23 non-neurogenic neurogenic bladder probands and, of 439 families with nonsyndromic vesicoureteric reflux, only one carried a putative pathogenic HPSE2 variant. Homozygous Hpse2 mutant mouse bladders contained urine more often than did wild-type organs, phenocopying human UFS. Pelvic ganglia neural cell bodies contained heparanase 1, heparanase 2, and leucine-rich repeats and immunoglobulin-like domains-2 (LRIG2), which is mutated in certain UFS families. In conclusion, heparanase 2 is an autonomic neural protein implicated in bladder emptying, but HPSE2 variants are uncommon in urinary diseases resembling UFS.

Thoracic Duct Decompression for Protein-Losing Enteropathy in Failing Fontan Circulation

An infrequent but devastating late complication of Fontan circulation is protein-losing enteropathy (PLE), which results from unbalanced lymphatic homeostasis. Surgical decompression of the thoracic duct by redirecting its drainage to the pulmonary venous atrium has been introduced recently as a possible treatment. This report describes a single-institution experience with this innovative procedure in 2 patients with failing Fontan circulation with PLE refractory to optimized medical therapy.