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Background Airway fibroblasts are major contributors to the histopathological feature of airway remodeling in asthma by their implication in the cell invasiveness and profibrogenic secretory phenotype observed in subepithelial fibrosis. 1,25 Dihydroxy vitamin D3 (1,25(OH)2D3) is an important therapeutic agent that blocks many features of airway remodeling induced by profibrogenic mediators, such as transforming growth factor beta 1 (TGF-β1) or T helper type 1 inflammatory cytokines. Objective We hypothesized that 1,25(OH)2D3 opposes the TGF-β1 or tumor necrosis factor alpha (TNF-α)-Interleukin 1 beta (IL-1β) stimulation on airway fibroblast profibrogenic secretory phenotype observed in severe asthmatic patients. Our aim was to investigate the anti-fibrogenic effect of 1,25(OH)2D3 in TGF-β1 or TNF-α-IL-1β-stimulated human bronchial fibroblast cells (HBFCs) from severe asthmatic compared with non-asthmatic subjects. Patients and methods All experiments were performed on primary HBFCs from asthmatic (DHBFCs, n=4) and non-asthmatic subjects (NHBFCs, n=4). mRNA expression and protein quantification of key fibrogenic markers were analyzed by RT-qPCR and ELISA, comparing HBFCs from asthmatic and non-asthmatic subjects. Vitamin D receptor (VDR) mRNA expression and its functionality in HBFCs were assessed by RT-qPCR. HBFCs proliferation was assessed by flow cytometry using BrdU-FITC/7AAD bivariate staining, while HBFCs apoptosis by Annexin V-FITC/7AAD. Results VDR is constitutively expressed in HBFCs and the addition of 1,25(OH)2D3 significantly increased mRNA expression of CYP24A1 (a direct VDRs' target gene) in both HBFCs groups. DHBFCs cultured in the presence of TGF-β1 or TNF-α-IL-1β showed increased mRNA expression and protein secretion of fibrogenic markers when compared to NHBFCs. Additionally, we observed decreased mRNA expression of FN 1, LUM, BGN, MMP2, COL5A1, TIMP1 and CC-chemokines (CCL2, CCL5, CCL11) in response to 1,25(OH)2D3 addition to the TGF-β1 or TNF-α-IL-1β-stimulated HBFCs. Cell culture media obtained from TGF-β1 or TNF-α-IL-1β-stimulated DHBFCs showed decreased protein secretion (fibronectin 1, lumican, MCP1, RANTES and eotaxin-1) in response to 1,25(OH)2D3 when compared to NHBFCs. 1,25(OH)2D3 inhibited proliferation in TGF-β1-stimulated HBFCs through G0/G1 cell cycle arrest and these effects were not correlated with the induction of apoptosis. Conclusion DHBFCs under TGF-β1 or TNF-α-IL-1β stimulation showed higher fibrogenic capacity when compared to NHBFCs. 1,25(OH)2D3 significantly blocked these effects and highlight 1,25(OH)2D3 as a possible therapeutic target for severe asthma.

Aim: To evaluate top-ranking genes identified through genome-wide association studies for an association with corticosteroid-related osteonecrosis in children with acute lymphoblastic leukemia (ALL) who received Dana–Farber Cancer Institute treatment protocols. Patients & methods: Lead SNPs from these studies, as well as other variants in the same genes, pooled from whole exome sequencing data, were analyzed for an association with osteonecrosis in childhood ALL patients from Quebec cohort. Top-ranking variants were verified in the replication patient group. Results: The analyses of variants in the ACP1-SH3YL1 locus derived from whole exome sequencing data showed an association of several correlated SNPs (rs11553746, rs2290911, rs7595075, rs2306060 and rs79716074). The rs79716074 defines *B haplotype of the APC1 gene, which is well known for its functional role. Conclusion: This study confirms implication of the ACP1 gene in the treatment-related osteonecrosis in childhood ALL and identifies novel, potentially causal variant of this complication.

Osteonecrosis (ON) is corticosteroid-related complication, reported in children with acute lymphoblastic leukemia (ALL). We have previously found that polymorphisms in BCL2L11 gene coding for pro-apoptotic Bim protein influence reduction of overall survival (OS) in a corticosteroid (CS) dose-dependent manner in childhood ALL patients. The same set of SNPs was here investigated for an association with CS-related ON assessed retrospectively in 304 children with ALL from Quebec (QcALL cohort) who received Dana-Farber Cancer Institute (DFCI) ALL treatment protocols. Two-year cumulative incidence of symptomatic ON was 10.6%. Two BCL2L11 polymorphisms, the 891T>G (rs2241843) in all QcALL patients and 29201C>T (rs724710) in high-risk group were significantly associated with ON, P = 0.009 and P = 0.003, respectively. The association remained significant in multivariate model (HR891TT = 2.4, 95% CI 1.2–4.8, P = 0.01 and HR29201CC = 5.7, 95% CI 1.6–20.9, P = 0.008). Both polymorphisms influenced viability of dexamethasone treated lymphoblastoid cell lines (P ≤ 0.03). The 891T>G influenced Bim gamma isoform levels (0.03) and its association with ON was also confirmed in replication DFCI cohort (N = 168, P = 0.03). QcALL children had a high incidence of ON during therapy, which was highly associated with BCL2L11 polymorphisms.

Neutropenia and infection are major dose-limiting side effects of chemotherapy. The risk of initial infection and subsequent complications are directly related to the depth and duration of neutropenia. Recent genome-wide association studies identified variants in DARC and CXCL2 genes, and in ORMDL3-GSDMA-CSF3 locus on chromosome 17q21 that influence white blood cell and neutrophil counts in healthy individuals. To investigate whether polymorphisms in these loci in conjunction with chemotherapy may modulate risk of treatment complications, we analyzed 21 SNPs across these genes for an association with chemotherapy-related neutropenia and infection in 286 Caucasian children with acute lymphoblastic leukemia. After correction for multiple testing, DARC polymorphism rs3027012 in 5'-UTR was associated with higher risk of low absolute phagocyte count (APC

Background Asthma is a heterogenous disease characterized by chronic inflammation and airway remodeling. An increase in the severity of airway remodeling is associated with a more severe form of asthma. There is increasing interest in the epithelial to mesenchymal transition process and mechanisms involved in the differentiation and repair of the airway epithelium, especially as they apply to severe asthma. Growing evidence suggests that Epithelial-Mesenchymal transition (EMT) could contribute to airway remodeling and fibrosis in asthma. Severe asthmatic patients with remodeled airways have a neutrophil driven inflammation. Neutrophils are an important source of TGF-β1, which plays a role in recruitment and activation of inflammatory cells, extracellular matrix (ECM) production and fibrosis development, and is a potent inducer of EMT. Objective As there is little data examining the contribution of neutrophils and/or their mediators to the induction of EMT in airway epithelial cells, the objective of this study was to better understand the potential role of neutrophils in severe asthma in regards to EMT. Methods We used an in vitro system to investigate the neutrophil-epithelial cell interaction. We obtained peripheral blood neutrophils from severe asthmatic patients and control subjects and examined for their ability to induce EMT in primary airway epithelial cells. Results Our data indicate that neutrophils from severe asthmatic patients induce changes in morphology and EMT marker expression in bronchial epithelial cells consistent with the EMT process when co-cultured. TGF-β1 levels in the culture medium of severe asthmatic patients were increased compared to that from co-cultures of non-asthmatic neutrophils and epithelial cells. Conclusions and clinical relevance As an inducer of EMT and an important source of TGF-β1, neutrophils may play a significant role in the development of airway remodeling and fibrosis in severe asthmatic airways.

Iron acquisition at the outer membrane (OM) of Gram-negative bacteria is powered by the proton motive force (PMF) of the cytoplasmic membrane (CM), harnessed by the CM-embedded complex of ExbB, ExbD, and TonB. Its stoichiometry, ensemble structural features, and mechanism of action are unknown. By panning combinatorial phage libraries, periplasmic regions of dimerization between ExbD and TonB were predicted. Using overexpression of full-length His 6 -tagged exbB-exbD and S-tagged tonB , we purified detergent-solubilized complexes of ExbB-ExbD-TonB from Escherichia coli . Protein-detergent complexes of ∼230 kDa with a hydrodynamic radius of ∼6.0 nm were similar to previously purified ExbB 4 -ExbD 2 complexes. Significantly, they differed in electronegativity by native agarose gel electrophoresis. The stoichiometry was determined to be ExbB 4 -ExbD 1 -TonB 1 . Single-particle electron microscopy agrees with this stoichiometry. Two-dimensional averaging supported the phage display predictions, showing two forms of ExbD-TonB periplasmic heterodimerization: extensive and distal. Three-dimensional (3D) particle classification showed three representative conformations of ExbB 4 -ExbD 1 -TonB 1 . Based on our structural data, we propose a model in which ExbD shuttles a proton across the CM via an ExbB interprotein rearrangement. Proton translocation would be coupled to ExbD-mediated collapse of extended TonB in complex with ligand-loaded receptors in the OM, followed by repositioning of TonB through extensive dimerization with ExbD. Here we present the first report for purification of the ExbB-ExbD-TonB complex, molar ratios within the complex (4:1:1), and structural biology that provides insights into 3D organization. IMPORTANCE Receptors in the OM of Gram-negative bacteria allow entry of iron-bound siderophores that are necessary for pathogenicity. Numerous iron-acquisition strategies rely upon a ubiquitous and unique protein for energization: TonB. Complexed with ExbB and ExbD, the Ton system links the PMF to OM transport. Blocking iron uptake by targeting a vital nanomachine holds promise in therapeutics. Despite much research, the stoichiometry, structural arrangement, and molecular mechanism of the CM-embedded ExbB-ExbD-TonB complex remain unreported. Here we demonstrate in vitro evidence of ExbB 4 -ExbD 1 -TonB 1 complexes. Using 3D EM, we reconstructed the complex in three conformational states that show variable ExbD-TonB heterodimerization. Our structural observations form the basis of a model for TonB-mediated iron acquisition.

Mutations in human LMBRD1 and ABCD4 prevent lysosomal export of vitamin B(12) to the cytoplasm, impairing the vitamin B(12)-dependent enzymes methionine synthase and methylmalonyl-CoA mutase. The gene products of LMBRD1 and ABCD4 are implicated in vitamin B(12) transport at the lysosomal membrane and are proposed to act in complex. To address the mechanism for lysosomal vitamin B(12) transport, we report the novel recombinant production of LMBD1 and ABCD4 for detailed biophysical analyses. Using blue native PAGE, chemical crosslinking, and size exclusion chromatography coupled to multi-angle light scattering (SEC-MALS), we show that both detergent-solubilized LMBD1 and detergent-solubilized ABCD4 form homodimers. To examine the functional binding properties of these proteins, label-free surface plasmon resonance (SPR) provides direct in vitro evidence that: (i) LMBD1 and ABCD4 interact with low nanomolar affinity; and (ii) the cytoplasmic vitamin B(12)-processing protein MMACHC also interacts with LMBD1 and ABCD4 with low nanomolar affinity. Accordingly, we propose a model whereby membrane-bound LMBD1 and ABCD4 facilitate the vectorial delivery of lysosomal vitamin B(12) to cytoplasmic MMACHC, thus preventing cofactor dilution to the cytoplasmic milieu and protecting against inactivating side reactions.

A healthy binocular vision offers perceptual skills absolutely necessary for a normal life: depth perception and stereoscopic vision. For this to happen, it is important to conduct an examination from the youngest ages. Any deprivation of visual stimulus in this critical period of development can lead to strabismus, amblyopia or abnormal retinal correspondence which compromises the binocular vision. As a result, visual therapy can be defined as a series of exercises that engage the eyeballs and the brain. The subject recovers or develops normal binocular vision skills through progressive therapeutic procedures in which are used a wide variety of tools and devices. This paper presents two of uncommon optometric techniques which can be applied at preschool children.