Private Child Support: Current and Potential Impacts

This paper examines the effects of a number of methods for enhancing private child support collections: increasing the proportion of those children potentially eligible for child support who get child support awards; using a uniform standard for determining child support obligations; and collecting a greater percentage of current obligations. The paper also estimates the potential of all three methods used in combination to provide income to needy custodial families. The research demonstrates that the current private child support system falls far short of its potential to transfer income from noncustodial to custodial families. Although the use of a normative standard, improved collections, and extending child support to all those potentially eligible will greatly improve the economic circumstances of impoverished custodial families, private child support cannot be viewed as the sole answer for the economic plight of these families. Increased work opportunities and increased public support are also needed

Strategies for Proteome-Wide Quantification of Glycosylation Macro- and Micro-Heterogeneity

Protein glycosylation governs key physiological and pathological processes in human cells. Aberrant glycosylation is thus closely associated with disease progression. Mass spectrometry (MS)-based glycoproteomics has emerged as an indispensable tool for investigating glycosylation changes in biological samples with high sensitivity. Following rapid improvements in methodologies for reliable intact glycopeptide identification, site-specific quantification of glycopeptide macro- and micro-heterogeneity at the proteome scale has become an urgent need for exploring glycosylation regulations. Here, we summarize recent advances in N- and O-linked glycoproteomic quantification strategies and discuss their limitations. We further describe a strategy to propagate MS data for multilayered glycopeptide quantification, enabling a more comprehensive examination of global and site-specific glycosylation changes. Altogether, we show how quantitative glycoproteomics methods explore glycosylation regulation in human diseases and promote the discovery of biomarkers and therapeutic targets

The vascular bone marrow niche influences outcome in chronic myeloid leukemia via the E-selectin - SCL/TAL1-CD44 axis.

The endosteal bone marrow niche and vascular endothelial cells provide sanctuaries for leukemic cells. In murine chronic myeloid leukemia (CML) CD44 on leukemia cells and E-selectin on bone marrow endothelium are essential mediators for the engraftment of leukemic stem cells. We hypothesized that non-adhesion of CML-initiating cells to E-selectin on the bone marrow endothelium may lead to superior eradication of leukemic stem cells in CML after treatment with imatinib than imatinib alone. Indeed, here we show that treatment with the E-selectin inhibitor GMI-1271 in combination with imatinib prolongs survival of mice with CML via decreased contact time of leukemia cells with bone marrow endothelium. Non-adhesion of BCR-ABL1(+) cells leads to an increase of cell cycle progression and an increase of expression of the hematopoietic transcription factor and proto-oncogene Scl/Tal1 in leukemia-initiating cells. We implicate SCL/TAL1 as an indirect phosphorylation target of BCR-ABL1 and as a negative transcriptional regulator of CD44 expression. We show that increased SCL/TAL1 expression is associated with improved outcome in human CML. These data demonstrate the BCR-ABL1-specific, cell-intrinsic pathways leading to altered interactions with the vascular niche via the modulation of adhesion molecules - which could be exploited therapeutically in the future

A streamlined pipeline for multiplexed quantitative site-specific N-glycoproteomics

Regulation of protein N-glycosylation is essential in human cells. However, large-scale, accurate, and site-specific quantification of glycosylation is still technically challenging. We here introduce SugarQuant, an integrated mass spectrometry-based pipeline comprising protein aggregation capture (PAC)-based sample preparation, multi-notch MS3 acquisition (Glyco-SPS-MS3) and a data-processing tool (GlycoBinder) that enables confident identification and quantification of intact glycopeptides in complex biological samples. PAC significantly reduces sample-handling time without compromising sensitivity. Glyco-SPS-MS3 combines high-resolution MS2 and MS3 scans, resulting in enhanced reporter signals of isobaric mass tags, improved detection of N-glycopeptide fragments, and lowered interference in multiplexed quantification. GlycoBinder enables streamlined processing of Glyco-SPS-MS3 data, followed by a two-step database search, which increases the identification rates of glycopeptides by 22% compared with conventional strategies. We apply SugarQuant to identify and quantify more than 5,000 unique glycoforms in Burkitt’s lymphoma cells, and determine site-specific glycosylation changes that occurred upon inhibition of fucosylation at high confidence

Activation of CD44/PAK1/AKT signaling promotes resistance to FGFR1 inhibition in squamous-cell lung cancer

Lung cancer is the leading cause of cancer-related deaths worldwide. Fibroblast growth factor receptor 1 (FGFR1) gene amplification is one of the most prominent and potentially targetable genetic alterations in squamous-cell lung cancer (SQCLC). Highly selective tyrosine kinase inhibitors have been developed to target FGFR1; however, resistance mechanisms originally existing in patients or acquired during treatment have so far led to limited treatment efficiency in clinical trials. In this study we performed a wide-scale phosphoproteomic mass-spectrometry analysis to explore signaling pathways that lead to resistance toward FGFR1 inhibition in lung cancer cells that display (i) intrinsic, (ii) pharmacologically induced and (iii) mutationally induced resistance. Additionally, we correlated AKT activation to CD44 expression in 175 lung cancer patient samples. We identified a CD44/PAK1/AKT signaling axis as a commonly occurring resistance mechanism to FGFR1 inhibition in lung cancer. Co-inhibition of AKT/FGFR1, CD44/FGFR1 or PAK1/FGFR1 sensitized ‘intrinsically resistant’ and ‘induced-resistant’ lung-cancer cells synergetically to FGFR1 inhibition. Furthermore, strong CD44 expression was significantly correlated with AKT activation in SQCLC patients. Collectively, our phosphoproteomic analysis of lung-cancer cells resistant to FGFR1 inhibitor provides a large data library of resistance-associated phosphorylation patterns and leads to the proposal of a common resistance pathway comprising CD44, PAK1 and AKT activation. Examination of CD44/PAK1/AKT activation could help to predict response to FGFR1 inhibition. Moreover, combination between AKT and FGFR1 inhibitors may pave the way for an effective therapy of patients with treatment-resistant FGFR1-dependent lung cancer

Pretransplant assessment of human liver grafts by plasma lecithin: cholesterol acyltransferase (LCAT) activity in multiple organ donors.

In spite of the improved outcome of orthotopic liver transplantation (OLTx), primary graft nonfunction remains one of the life-threatening problems following OLTx. The purpose of this study was to evaluate plasma lecithin: cholesterol acyltransferase (LCAT) activity in multiple organ donors as a predictor of liver allograft viability prior to OLTx. Thirty-nine donors were studied during a 5-month period between April and August 1988. Allograft hepatectomy was performed using a rapid technique or its minor modification with hilar dissections, and the allografts were stored cold (4 degrees C) in University of Wisconsin (UW) solution. Early post-transplant allograft function was classified as good, fair, or poor, according to the highest SGOT, SGPT, and prothrombin time within 5 days following OLTx. Procurement records were reviewed to identify donor data, which included conventional liver function tests, duration of hospital stay, history of cardiac arrest, and graft ischemic time. Blood samples from the donors were drawn immediately prior to aortic crossclamp, and from these plasma LCAT activity was determined. Plasma LCAT activity of all donors was significantly lower than that of healthy controls (12.4 +/- 8.0 vs 39.2 +/- 13.3 micrograms/ml per hour, P less than 0.01). LCAT activity (16.4 +/- 8.3 micrograms/ml per hour) in donors of grafts with good function was significantly higher than that in those with fair (8.6 +/- 4.5 micrograms/ml per hour, P less than 0.01) or poor (7.3 +/- 2.4 micrograms/ml per hour, P less than 0.01) function.(ABSTRACT TRUNCATED AT 250 WORDS

Targeting BTK for the treatment of FLT3-ITD mutated acute myeloid leukemia

Approximately 20% of patients with acute myeloid leukaemia (AML) have a mutation in FMS-like-tyrosine-kinase-3 (FLT3). FLT3 is a trans-membrane receptor with a tyrosine kinase domain which, when activated, initiates a cascade of phosphorylated proteins including the SRC family of kinases. Recently our group and others have shown that pharmacologic inhibition and genetic knockdown of Bruton's tyrosine kinase (BTK) blocks AML blast proliferation, leukaemic cell adhesion to bone marrow stromal cells as well as migration of AML blasts. The anti-proliferative effects of BTK inhibition in human AML are mediated via inhibition of downstream NF-κB pro-survival signalling however the upstream drivers of BTK activation in human AML have yet to be fully characterised. Here we place the FLT3-ITD upstream of BTK in AML and show that the BTK inhibitor ibrutinib inhibits the survival and proliferation of FLT3-ITD primary AML blasts and AML cell lines. Furthermore ibrutinib inhibits the activation of downstream kinases including MAPK, AKT and STAT5. In addition we show that BTK RNAi inhibits proliferation of FLT3-ITD AML cells. Finally we report that ibrutinib reverses the cyto-protective role of BMSC on FLT3-ITD AML survival. These results argue for the evaluation of ibrutinib in patients with FLT3-ITD mutated AML

Quantitative proteomics identifies biomarkers to distinguish pulmonary from head and neck squamous cell carcinomas by immunohistochemistry

The differentiation between a pulmonary metastasis and a newly developed squamous cell carcinoma of the lung in patients with prior head and neck squamous cell carcinoma (HNSCC) is difficult due to a lack of biomarkers but is crucially important for the prognosis and therapy of the affected patient. By using high-resolution mass spectrometry in combination with stable isotope labelling by amino acids in cell culture, we identified 379 proteins that are differentially expressed in squamous cell carcinomas of the lung and the head and neck. Of those, CAV1, CAV2, LGALS1, LGALS7, CK19, and UGDH were tested by mmunohistochemistry on 194 tissue samples (98 lung and 96 HNSCCs). The combination of CAV1 and LGALS7 was able to distinguish the origin of the squamous cell carcinoma with high accuracy (area under the curve 0.876). This biomarker panel was tested on a cohort of 12 clinically classified lung tumours of unknown origin after HNSCC. Nine of those tumours were immunohistochemically classifiable

Sequential phosphorylation of SLP-76 at tyrosine 173 is required for activation of T and mast cells.

Cooperatively assembled signalling complexes, nucleated by adaptor proteins, integrate information from surface receptors to determine cellular outcomes. In T and mast cells, antigen receptor signalling is nucleated by three adaptors: SLP-76, Gads and LAT. Three well-characterized SLP-76 tyrosine phosphorylation sites recruit key components, including a Tec-family tyrosine kinase, Itk. We identified a fourth, evolutionarily conserved SLP-76 phosphorylation site, Y173, which was phosphorylated upon T-cell receptor stimulation in primary murine and Jurkat T cells. Y173 was required for antigen receptor-induced phosphorylation of phospholipase C-γ1 (PLC-γ1) in both T and mast cells, and for consequent downstream events, including activation of the IL-2 promoter in T cells, and degranulation and IL-6 production in mast cells. In intact cells, Y173 phosphorylation depended on three, ZAP-70-targeted tyrosines at the N-terminus of SLP-76 that recruit and activate Itk, a kinase that selectively phosphorylated Y173 in vitro. These data suggest a sequential mechanism whereby ZAP-70-dependent priming of SLP-76 at three N-terminal sites triggers reciprocal regulatory interactions between Itk and SLP-76, which are ultimately required to couple active Itk to its substrate, PLC-γ1