Search

O-GlcNAc transferase (OGT) is an essential mammalian enzyme that binds thousands of different proteins, including substrates that it glycosylates and nonsubstrate interactors that regulate its biology. OGT also has one proteolytic substrate, the transcriptional coregulator host cell factor 1 (HCF-1), which it cleaves in a process initiated by glutamate side chain glycosylation at a series of central repeats. Although HCF-1 is OGT’s most prominent binding partner, its affinity for the enzyme has not been quantified. Here, we report a time-resolved Förster resonance energy transfer assay to measure ligand binding to OGT and show that an HCF-1-derived polypeptide (HCF3R) binds with picomolar affinity to the enzyme (KD≤ 85 pM). This high affinity is driven in large part by conserved asparagines in OGT’s tetratricopeptide repeat domain, which form bidentate contacts to the HCF-1 peptide backbone; replacing any one of these asparagines with alanine reduces binding by more than 5 orders of magnitude. Because the HCF-1 polypeptide binds so tightly to OGT, we tested its ability to inhibit enzymatic function. We found that HCF3R potently inhibits OGT both in vitro and in cells and used this finding to develop a genetically encoded, inducible OGT inhibitor that can be degraded with a small molecule, allowing for reversible and tunable inhibition of OGT.

The myeloid neoplasms encompass acute myeloid leukemia, myelodysplastic syndromes and myeloproliferative neoplasms. Most cases arise from the shared ancestor of clonal hematopoiesis (CH). Here we analyze data from 454,340 UK Biobank participants, of whom 1,808 developed a myeloid neoplasm 0–15 years after recruitment. We describe the differences in CH mutational landscapes and hematology/biochemistry test parameters among individuals that later develop myeloid neoplasms (pre-MN) versus controls, finding that disease-specific changes are detectable years before diagnosis. By analyzing differences between ‘pre-MN’ and controls, we develop and validate Cox regression models quantifying the risk of progression to each myeloid neoplasm subtype. We construct ‘MN-predict’, a web application that generates time-dependent predictions with the input of basic blood tests and genetic data. Our study demonstrates that many individuals that develop myeloid neoplasms can be identified years in advance and provides a framework for disease-specific prognostication that will be of substantial use to researchers and physicians.

The impermeable outer membrane of Pseudomonas aeruginosais bypassed by antibacterial proteins known as S-type pyocins. Because of their properties, pyocins are investigated as a potential new class of antimicrobials against Pseudomonasinfections. Their production and modification, however, remain challenging. To address this limitation, we employed automated fast-flow peptide synthesis for the rapid production of a pyocin S2 import domain. The N-terminal domain sequence (PyS2NTD) was synthesized in under 10 h and purified to yield milligram quantities of the desired product. To our knowledge, the 214 amino acid sequence of PyS2NTDis among the longest peptides produced from a “single-shot” synthesis, i.e., made in a single stepwise route without the use of ligation techniques. Biophysical characterization of the PyS2NTDwith circular dichroism was consistent with the literature reports. Fluorescently labeled PyS2NTDbinds to P. aeruginosaexpressing the cognate ferripyoverdine receptor and is taken up into the periplasm. This selective uptake was validated with confocal and super resolution microscopy, flow cytometry, and fluorescence recovery after photobleaching. These modified, synthetic S-type pyocin domains can be used to probe import mechanisms of P. aeruginosaand leveraged to develop selective antimicrobial agents that bypass the outer membrane.

Background: The purpose of this study was to evaluate the association between a novel psychometric 12-item questionnaire (U-qest) and other validated questionnaires to assess quality of life and work impairment in patients with non-infectious uveitis.Methods: Data were collected at baseline and 3 months postbaseline using U-qest and two other validated questionnaires: The National Eye Institute 25-Item Visual Function Questionnaire (VFQ-25) and the 12-Item Short-Form Health Survey (SF-12).Results: A total of 136 patients (52.2% female) aged 47.9 ± 14.8 years (mean ± SD) were enrolled in 14 uveitis referral centres. U-qest correlated moderately with VFQ-25 and SF-12 at baseline and at 3 months. Both U-qest and VFQ-25 scores improved as disease improved; however, U-qest also detected improvement in patients for whom VFQ-25 scores did not improve. Disease activity was shown to significantly affect activity impairment. Patients and physicians expressed positive perceptions regarding the use and benefit of this instrument. U-qest showed very good reliability in terms of internal consistency (Cronbach’s alpha = 0.91).Conclusions: U-qest can be considered a useful tool to assess the burden of uveitis on quality of life.

A series of neopentyl carboxylic acids bearing functionality amenable to click chemistry were prepared and then appended to high-generation bis-MPA dendrons via fluoride-promoted esterification. The nucleophilic stability of neopentyl and non-neopentyl dendrons in acidic to basic phosphate buffers was compared by monitoring degradation via quantitative 1H nuclear magnetic resonance (NMR). The neopentyl periphery dendrons were found to be highly resistant to hydrolysis under all experimental conditions. The neopentyl groups also did not impede click functionalization onto the dendrons.

Glycans are ubiquitous and play important biological roles, yet chemical methods for probing their structure and function within cells remain limited. Strategies for studying other biomacromolecules, such as proteins, often exploit chemoselective reactions for covalent modification, capture, or imaging. Unlike amino acids that constitute proteins, glycan building blocks lack distinguishing reactivity because they are composed primarily of polyol isomers. Moreover, encoding glycan variants through genetic manipulation is complex. Therefore, we formulated a new, generalizable strategy for chemoselective glycan modification that directly takes advantage of cellular glycosyltransferases. Many of these enzymes are selective for the products they generate yet promiscuous in their donor preferences. Thus, we designed reagents with bioorthogonal handles that function as glycosyltransferase substrate surrogates. We validated the feasibility of this approach by synthesizing and testing probes of d-arabinofuranose (d-Araf), a monosaccharide found in bacteria and an essential component of the cell wall that protects mycobacteria, including Mycobacterium tuberculosis. The result is the first probe capable of selectively labeling arabinofuranose-containing glycans. Our studies serve as a platform for developing new chemoselective labeling agents for other privileged monosaccharides. This probe revealed an asymmetric distribution of d-Arafresidues during mycobacterial cell growth and could be used to detect mycobacteria in THP1-derived macrophages.

Altered transcription is a cardinal feature of acute myeloid leukemia (AML); however, exactly how mutations synergize to remodel the epigenetic landscape and rewire three-dimensional DNA topology is unknown. Here, we apply an integrated genomic approach to a murine allelic series that models the two most common mutations in AML: Flt3-ITDand Npm1c. We then deconvolute the contribution of each mutation to alterations of the epigenetic landscape and genome organization, and infer how mutations synergize in the induction of AML. Our studies demonstrate that Flt3-ITDsignals to chromatin to alter the epigenetic environment and synergizes with mutations in Npm1cto alter gene expression and drive leukemia induction. These analyses also allow the identification of long-range cis-regulatory circuits, including a previously unknown superenhancer of Hoxalocus, as well as larger and more detailed gene-regulatory networks, driven by transcription factors including PU.1 and IRF8, whose importance we demonstrate through perturbation of network members.

Small molecules can self-assemble in aqueous solution into a wide range of nanoentity types and sizes (dimers, n-mers, micelles, colloids, etc.), each having their own unique properties. This has important consequences in the context of drug discovery including issues related to nonspecific binding, off-target effects, and false positives and negatives. Here, we demonstrate the use of the spin–spin relaxation Carr–Purcell–Meiboom–Gill NMR experiment, which is sensitive to molecular tumbling rates and can expose larger aggregate species that have slower rotational correlations. The strategy easily distinguishes lone-tumbling molecules versus nanoentities of various sizes. The technique is highly sensitive to chemical exchange between single-molecule and aggregate states and can therefore be used as a reporter when direct measurement of aggregates is not possible by NMR. Interestingly, we found differences in solution behavior for compounds within structurally related series, demonstrating structure–nanoentity relationships. This practical experiment is a valuable tool to support drug discovery efforts.

Despite the ubiquity and importance of glycans in biology, methods to probe their structures in cells are limited. Mammalian glycans can be modulated using metabolic incorporation, a process in which non-natural sugars are taken up by cells, converted to nucleotide–sugar intermediates, and incorporated into glycans via biosynthetic pathways. These studies have revealed that glycan intermediates can be shunted through multiple pathways, and this complexity can be heightened in bacteria, as they can catabolize diverse glycans. We sought to develop a strategy that probes structures recalcitrant to metabolic incorporation and that complements approaches focused on nucleotide sugars. We reasoned that lipid-linked glycans, which are intermediates directly used in glycan biosynthesis, would offer an alternative. We generated synthetic arabinofuranosyl phospholipids to test this strategy in Corynebacterium glutamicumand Mycobacterium smegmatis, organisms that serve as models of Mycobacterium tuberculosis. Using a C. glutamicummutant that lacks arabinan, we identified synthetic glycosyl donors whose addition restores cell wall arabinan, demonstrating that non-natural glycolipids can serve as biosynthetic intermediates and function in chemical complementation. The addition of an isotopically labeled glycan substrate facilitated cell wall characterization by NMR. Structural analysis revealed that all five known arabinofuranosyl transferases could process the exogenous lipid-linked sugar donor, allowing for the full recovery of the cell envelope. The lipid-based probe could also rescue wild-type cells treated with an inhibitor of cell wall biosynthesis. Our data indicate that surrogates of natural lipid-linked glycans can intervene in the cell’s traditional workflow, indicating that biosynthetic incorporation is a powerful strategy for probing glycan structure and function.

Epigenetic regulators, such as EZH2, are frequently mutated in cancer, and loss-of-function EZH2 mutations are common in myeloid malignancies. We have examined the importance of cellular context for Ezh2 loss during the evolution of acute myeloid leukemia (AML), where we observed stage-specific and diametrically opposite functions for Ezh2 at the early and late stages of disease. During disease maintenance, WT Ezh2 exerts an oncogenic function that may be therapeutically targeted. In contrast, Ezh2 acts as a tumor suppressor during AML induction. Transcriptional analysis explains this apparent paradox, demonstrating that loss of Ezh2 derepresses different expression programs during disease induction and maintenance. During disease induction, Ezh2 loss derepresses a subset of bivalent promoters that resolve toward gene activation, inducing a feto-oncogenic program that includes genes such as Plag1, whose overexpression phenocopies Ezh2 loss to accelerate AML induction in mouse models. Our data highlight the importance of cellular context and disease phase for the function of Ezh2 and its potential therapeutic implications.

Systolic blood pressure of more than 185 mm Hg is a contraindication to thrombolytic treatment with intravenous alteplase in patients with acute ischaemic stroke, but the target systolic blood pressure for optimal outcome is uncertain. We assessed intensive blood pressure lowering compared with guideline-recommended blood pressure lowering in patients treated with alteplase for acute ischaemic stroke.

Durum wheat (Triticum turgidumL. subsp. durum(Desf.) Husn) is a major food source in Mediterranean countries since it is utilized for the production of pasta, leavened and unleavened breads, couscous, and other traditional foods. The technological and nutritional properties of durum wheat semolina depend mainly on the type of gluten proteins and on their amount, which is a genotype- and environment-dependent trait. Gluten proteins are also responsible for celiac disease (CD), an autoimmune enteropathy with a prevalence of about 0.7–2% in the human population. At this purpose, two Italian durum wheat cultivars, Saragolla and Cappelli, currently used for monovarietal pasta, were chosen to compare (i) the reserve and embryo proteome, (ii) the free and bound phenolics, antioxidant activity, and amino acid composition, and (iii) the content of immunogenic peptides produced after a simulated gastrointestinal digestion. The results obtained from 2 years of field cultivation on average showed a higher amount of gluten proteins, amino acids, and immunogenic peptides in Cappelli. Saragolla showed a higher abundance in bound phenolics, antioxidant enzymes, and stress response proteins in line with its higher antioxidant activity. However, the impact of the year of cultivation, largely depending on varying rainfall regimes through the wheat growth cycle, was significant for most of the parameters investigated. Differences in technological and nutritional characteristics observed between the two cultivars are discussed in relation to the influence of genetic and environmental factors.

Introduction:

Introduction:

Only two trials have compared acceptance and commitment therapy (ACT) and traditional cognitive behavior therapy (tCBT) in the treatment of social anxiety disorder (SAD), with both finding no significant differences. These trials did not examine effects on observer-rated behavioral outcomes and did not explicitly quantify the dose of exposure therapy within each treatment. In a replication trial, one hundred and two individuals with SAD (per DSM-IV criteria) were randomized to 12 sessions of ACT (n = 49) or tCBT (n = 53) controlling for exposure dose and assessing behavioral outcomes. Assessments were completed at pre- and post-treatment using clinician-rated and self-report measures of social anxiety, quality of life, and overall functioning. Observer-rated behavioral measures of social performance were completed for a subsample of participants. Results indicated that participants across conditions received equivalent doses of exposure. Those who received tCBT evidenced greater improvements in self-reported social anxiety symptoms and overall functioning, which contrasts with prior studies finding no differences between tCBT and ACT in the treatment of social anxiety. Medium effect sizes, while not statistically significant, indicate that ACT participants may have had greater improvements in observer-rated social behavior than tCBT participants. The discrepancy between these symptom and behavioral outcomes, building upon prior literature, calls for more research to assess the differences between tCBT and ACT treatments in behavioral domains.

FLT3 internal tandem duplication (FLT3ITD) mutations are common in acute myeloid leukemia (AML) associated with poor patient prognosis. Although new-generation FLT3 tyrosine kinase inhibitors (TKI) have shown promising results, the outcome of FLT3ITD AML patients remains poor and demands the identification of novel, specific, and validated therapeutic targets for this highly aggressive AML subtype. Utilizing an unbiased genome-wide clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 screen, we identify GLS, the first enzyme in glutamine metabolism, as synthetically lethal with FLT3-TKI treatment. Using complementary metabolomic and gene-expression analysis, we demonstrate that glutamine metabolism, through its ability to support both mitochondrial function and cellular redox metabolism, becomes a metabolic dependency of FLT3ITD AML, specifically unmasked by FLT3-TKI treatment. We extend these findings to AML subtypes driven by other tyrosine kinase (TK) activating mutations and validate the role of GLS as a clinically actionable therapeutic target in both primary AML and in vivo models. Our work highlights the role of metabolic adaptations as a resistance mechanism to several TKI and suggests glutaminolysis as a therapeutically targetable vulnerability when combined with specific TKI in FLT3ITD and other TK activating mutation–driven leukemias.

Mindfulness-informed cognitive behavioral interventions for obesity are promising. However, results on the efficacy of such treatments are inconsistent which in part may be due to their substantially different methods of practice. This study is the first direct comparison of two theoretically distinct mindfulness-based weight loss approaches: increasing awareness of homeostatic/innate physiological cues versus hedonic/externally-driven cues for eating.

Supplemental Digital Content is Available in the Text.

Mycobacterium tuberculosis(Mtb) is one of history’s most successful human pathogens. By subverting typical immune responses, Mtbcan persist within a host until conditions become favorable for growth and proliferation. Virulence factors that enable mycobacteria to modulate host immune systems include a suite of mannose-containing glycolipids: phosphatidylinositol mannosides, lipomannan, and lipoarabinomannan (LAM). Despite their importance, tools for their covalent capture, modification, and imaging are limited. Here, we describe a chemical biology strategy to detect and visualize these glycans. Our approach, biosynthetic incorporation, is to synthesize a lipid-glycan precursor that can be incorporated at a late-stage step in glycolipid biosynthesis. We previously demonstrated selective mycobacterial arabinan modification by biosynthetic incorporation using an exogenous donor. This report reveals that biosynthetic labeling is general and selective: it allows for cell surface mannose-containing glycolipid modification without nonspecific labeling of mannosylated glycoproteins. Specifically, we employed azido-(Z,Z)-farnesyl phosphoryl-β-d-mannose probes and took advantage of the strain-promoted azide–alkyne cycloaddition to label and directly visualize the localization and dynamics of mycobacterial mannose-containing glycolipids. Our studies highlight the generality and utility of biosynthetic incorporation as the probe structure directs the selective labeling of distinct glycans. The disclosed agents allowed for direct tracking of the target immunomodulatory glycolipid dynamics in cellulo. We anticipate that these probes will facilitate investigating the diverse biological roles of these glycans.

The recent notable increase in refugees' flows, with refugee children and adolescents relocating worldwide in recent years, posed severe challenges to the different national healthcare systems. Social groups such as refugees fleeing from their countries because of persecution, wars and violence are considered at high risk of developing mental health-related problems. Despite international and national policies legally regulating the reception process and protecting health-related rights, including the mental well-being of refugee migrants, there is a theoretical and applied need for evidence-based instruments and procedures to support mental health within this population. Recent evidence refers to the Refugee Health Screener-15 (RHS-15) as a reliable and valid instrument for the early detection of trauma-related mental health problems. In this scenario, this study aimed to test the RHS screening process within a multidisciplinary first intervention reception context for unaccompanied refugee minors.

High ambient temperatures are associated with many health effects, including premature mortality. The combination of global warming due to climate change and the expansion of the global built environment mean that the intensification of urban heat islands (UHIs) is expected, accompanied by adverse effects on population health. Urban green infrastructure can reduce local temperatures. We aimed to estimate the mortality burden that could be attributed to UHIs and the mortality burden that would be prevented by increasing urban tree coverage in 93 European cities.

-We map DDX41 germline variation in 454,792 volunteers and define the risk of MDS/AML development associated with different variant types.-DDX41-mutant MDS/AML evolves differently to sporadic disease, but individuals at high risk often have somatic DDX41 mutations or a high MCV.

Sleep deprivation and time of day have been shown to play a critical role in decreasing ability to sustain attention, such as when driving long distances. However, a gap in the literature exists regarding external factors, such as workload. One way to examine workload is via modulating time on task. This study investigated the combined effect of sleep deprivation, time of day, and time on task as a workload factor on driving performance. Twenty-one participants (18–34 years, 10 females) underwent 62 h of sleep deprivation within a controlled laboratory environment. Participants received an 8-h baseline and 9.5-h recovery sleep. Every 8 h, participants completed a Psychomotor Vigilance Task (PVT), Karolinska Sleepiness Scale (KSS), 30-min monotonous driving task and NASA-Task Load Index (TLX). Driving variables examined were lane deviation, number of crashes, speed deviation and time outside the safe zone. Workload was measured by comparing two 15-min loops of the driving track. A mixed model ANOVA revealed significant main effects of day and time of day on all driving performance measures (p< .001). There was a significant main effect of workload on lane deviation (p< .05), indicating that a longer time on task resulted in greater lane deviation. A significant main effect of day (p< .001) but not time of day for the NASA-TLX, PVT and KSS was found. Time on task has a significant further impact on driving performance and should be considered alongside sleep deprivation and time of day when implementing strategies for long-distance driving.

The role of circulating factors in regulating colonic stem cells (CoSCs) and colonic epithelial homeostasis is unclear. Individuals with long-standing type 1 diabetes (T1D) frequently have intestinal symptoms, termed diabetic enteropathy (DE), though its etiology is unknown. Here, we report that T1D patients with DE exhibit abnormalities in their intestinal mucosa and CoSCs, which fail to generate in vitro mini-guts. Proteomic profiling of T1D+DE patient serum revealed altered levels of insulin-like growth factor 1 (IGF-I) and its binding protein 3 (IGFBP3). IGFBP3 prevented in vitro growth of patient-derived organoids via binding its receptor TMEM219, in an IGF-I-independent manner, and disrupted in vivo CoSC function in a preclinical DE model. Restoration of normoglycemia in patients with long-standing T1D via kidney-pancreas transplantation or in diabetic mice by treatment with an ecto-TMEM219 recombinant protein normalized circulating IGF-I/IGFBP3 levels and reestablished CoSC homeostasis. These findings demonstrate that peripheral IGF-I/IGFBP3 controls CoSCs and their dysfunction in DE.

In this study, we aimed to evaluate the effects of exenatide (EXE) treatment on exocrine pancreas of nonhuman primates. To this end, 52 baboons (Papio hamadryas) underwent partial pancreatectomy, followed by continuous infusion of EXE or saline (SAL) for 14 weeks. Histological analysis, immunohistochemistry, Computer Assisted Stereology Toolbox morphometry, and immunofluorescence staining were performed at baseline and after treatment. The EXE treatment did not induce pancreatitis, parenchymal or periductal inflammatory cell accumulation, ductal hyperplasia, or dysplastic lesions/pancreatic intraepithelial neoplasia. At study end, Ki-67–positive (proliferating) acinar cell number did not change, compared with baseline, in either group. Ki-67–positive ductal cells increased after EXE treatment (P = 0.04). However, the change in Ki-67–positive ductal cell number did not differ significantly between the EXE and SAL groups (P = 0.13). M-30–positive (apoptotic) acinar and ductal cell number did not change after SAL or EXE treatment. No changes in ductal density and volume were observed after EXE or SAL. Interestingly, by triple-immunofluorescence staining, we detected c-kit (a marker of cell transdifferentiation) positive ductal cells co-expressing insulin in ducts only in the EXE group at study end, suggesting that EXE may promote the differentiation of ductal cells toward a β-cell phenotype. In conclusion, 14 weeks of EXE treatment did not exert any negative effect on exocrine pancreas, by inducing either pancreatic inflammation or hyperplasia/dysplasia in nonhuman primates.

Issues are presented concerning the investments made in California by a variety of high-tech industries and by foreign companies. The economic benefits gained by the state are discussed.

Les pratiques pharmaceutiques en milieu hospitalier ont évolué différemment entre la France et le Québec. Certes, cette évolution s’inscrit dans un cadre plus large, alors que les systèmes de santé français et québécois ont subi d’importantes transformations au fil des ans pour faire face aux défis liés, entre autres, aux réalités économiques et démographiques.

Acinar cell carcinoma (ACC) of the pancreas is a very rare tumor that has various morphologic features, which may give rise to diagnostic difficulties. Because of its rarity, many clinicopathologic characteristics remain to be further elucidated, and prognostic factors are yet to be well established. With the aim of better characterizing this carcinoma and searching for prognostic indicators, we collected 62 ACCs and investigated the following parameters: site, size, local infiltration, node and distant metastases, architectural pattern, nuclear atypia, presence of necrosis, lymphovascular and perineural invasion, proliferation, BCL10, trypsin, carboxyl ester lipase, amylase, lipase, PDX1, cytokeratin 19 (CK19), CK7, p53, and -catenin expression. Twelve cases showing >30 of endocrine cells were reclassified as mixed acinar-neuroendocrine carcinomas, whereas 1 tumor was reclassified as a mixed ductal-acinar carcinoma and was excluded from the statistical prognostic evaluations. BCL10 and trypsin were the most reliable immunohistochemical markers, whereas amylase and lipase were not. Surgery was statistically correlated with a better prognosis (P=0.0008). Among resected tumors there was no difference in survival between ACCs and mixed acinar-neuroendocrine carcinomas, and factors that significantly correlated with poor prognosis were size >6.5 cm (P=0.004), lymph node (P=0.0039) and distant (P=0.008) metastases, and UICC stage (P=0.009). Stage was the only independent prognostic factor at multivariable analysis, and the best prognostic discrimination was observed on grouping together stages I and II and grouping together stages III and IV, suggesting a simplification of the UICC staging for such cancers. In addition, vascular and perineural invasion and CK19 and p53 expression showed a trend for poor prognosis, not reaching statistical significance.

Colorectal poorly differentiated neuroendocrine carcinomas (NECs) and mixed adenoneuroendocrine carcinomas (MANECs) are well-recognized entities generally known to be associated with biological aggressiveness and poor patient survival. However, a few published papers have highlighted the existence of a subgroup of tumors with a better survival than expected; however, to date, there are no established parameters that usefully identify this category. In the present study we have investigated the morphologic features, the CpG methylator phenotype (CIMP), microsatellite instability (MSI), and the immunohistochemical profile, including the expression of transcription factors (TTF1, ASH1, CDX2, and PAX5), stem cell markers (CD117 and CD34), and cytokeratins 7 and 20, in a series of 39 carcinomas (27 NECs and 12 MANECs) to better characterize such neoplasms and to search for prognostic indicators. No different patient survival was observed between NECs and MANECs. Neoplasms showed a heterogenous spectrum of morphologic and immunohistochemical features; however, only large-cell subtype, significant peritumoral lymphoid reaction, CD117 immunoreactivity, vascular invasion, and MSICIMP status were significantly correlated with prognosis on univariable analysis. Furthermore, vascular invasion and CD117 immunoreactivity were independent prognostic markers on multivariable analysis. In addition to these prognostic features, neoplasms showed different expression of transcription factors, stem cell markers, and cytokeratins that should be considered for diagnostic purposes and, especially, for discriminating among possible differential diagnoses.

In paroxysmal nocturnal hemoglobinuria (PNH) hemolytic anemia is due mainly to deficiency of the complement regulator CD59 on the surface of red blood cells (RBCs). Eculizumab, an antibody that targets complement fraction 5 (C5), has proven highly effective in abolishing complement-mediated intravascular hemolysis in PNH; however, the hematologic benefit varies considerably among patients. In the aim to understand the basis for this variable response, we have investigated by flow cytometry the binding of complement fraction 3 (C3) on RBCs from PNH patients before and during eculizumab treatment. There was no evidence of C3 on RBCs of untreated PNH patients; by contrast, in all patients on eculizumab (n = 41) a substantial fraction of RBCs had C3 bound on their surface, and this was entirely restricted to RBCs with the PNH phenotype (CD59−). The proportion of C3+RBCs correlated significantly with the reticulocyte count and with the hematologic response to eculizumab. In 3 patients in whom 51Cr labeling of RBCs was carried out while on eculizumab, we have demonstrated reduced RBC half-life in vivo, with excess 51Cr uptake in spleen and in liver. Binding of C3 by PNH RBCs may constitute an additional disease mechanism in PNH, strongly enhanced by eculizumab treatment and producing a variable degree of extravascular hemolysis.

An intraoperative photograph taken with a smartphone improves accurate documentation of complex ocular surface and eyelid biopsies for enhanced communication with pathologists and future surgical planning.

Epstein-Barr virus (EBV) is present in over 90% of the world's population. This infection is considered benign, even though in limited cases EBV is associated with infectious and neoplastic conditions. Over the past decade, the EBV association with breast cancer has been constantly debated. Adding to this clinical and biological uncertainty, different techniques gave contradictory results for the presence of EBV in breast carcinoma specimens. In this study, minor groove binding (MGB)-TaqMan real time PCR was used to detect the presence of EBV DNA in both peripheral blood and tumor samples of selected patients. Peripheral blood and breast carcinoma specimens from 24 patients were collected. DNA was extracted and then amplified by MGB-TaqMan real time PCR. Of 24 breast tumor specimens, 11 (46%) were positive for EBV DNA. Of these 11 breast tumor specimens, 7 (64%) were also positive for EBV DNA in the peripheral blood, while 4 (36%) were positive for EBV DNA in the tumor, but negative in the blood. EBV was found at extremely low levels, with a mean of 0.00004 EBV genomes per cell (range 0.00014 to 0.00001 EBV genomes per cell). Furthermore, our finding of the presence of EBV in the tumor specimens coupled to the absence of detection of EBV genomic DNA in the peripheral blood is consistent with the epithelial nature of the virus. Because of the low levels of viral DNA in tumor tissue, further studies are needed to assess the biological input of EBV in breast cancer.

ALL AUTHORS: Phillip C.C. Liu, Xiangdong Liu, Yanlong Li, Maryanne Covington, Richard Wynn, Reid Huber, Milton Hillman, Gengjie Yang, Dawn Ellis, Cindy Marando, Kamna Katiyar, Jodi Bradley, Kenneth Abremski, Mark Stow, Mark Rupar, Jincong Zhuo, Yun-Long Li, Qiyan Lin, David Burns, Meizhong Xu, Colin Zhang, Ding-Quan Qian, Chunhong He, Vaqar Sharief, Lingkai Weng, Costas Agrios, Eric Shi, Brian Metcalf, Robert Newton, Steven Friedman, Wenqing Yaol, Peggy Scherlel, Gregory Hollis, Timothy C. BurnOverexpression and activating mutations of ErbB family members have been implicated in the development and progression of a variety of tumor types. Cleavage of the HER2 receptor by an as yet unidentified ectodomain sheddase has been shown to liberate the HER2 extracellular domain (ECD) leaving a fragment with constitutive kinase activity that can provide ligand-independent growth and survival signals to the cell. This process is clinically relevant since HER2 ECD serum levels in metastatic breast cancer patients are associated with a poorer prognosis. Thus, inhibition of the HER2 sheddase may provide a novel therapeutic approach for breast cancer. We describe the use of transcriptional profiling, pharmacological and in vitro approaches to identify the major source of HER2 sheddase activity. Real-time PCR was used to identify those ADAM family members which were expressed in HER2 shedding cell lines. siRNAs that selectively inhibited ADAM10 expression reduced HER2 shedding. In addition, we profiled over 1000 small molecules for in vitro inhibition of a panel of ADAM and MMP proteins; a positive correlation was observed only between ADAM10 inhibition and reduction of HER2 ECD shedding in a cell based assay. Finally, in vitro studies demonstrate that in combination with low doses of Herceptin, selective ADAM10 inhibitors decrease proliferation in HER2 overexpressing cell lines while inhibitors, that do not inhibit ADAM10, have no impact. These results are consistent with ADAM10 being a major determinant of HER2 shedding, the inhibition of which, may provide a novel therapeutic approach for treating a variety of cancers with active HER2 signaling.

The HER-2 receptor tyrosine kinase is an important regulator of cell proliferation and survival, and it is a clinically validated target of therapeutic intervention for HER-2 positive breast cancer patients. Its extracellular domain (ECD) is frequently cleaved by protease(s) in HER-2 overexpressing breast cancer patients, rendering the remaining membrane-bound portion (p95) a constitutively activated kinase. The presence of both serum ECD and cellular p95 protein has been linked to poor clinical outcome as well as reduced effectiveness of some therapeutic treatments. We have identified a series of potent, selective small molecule inhibitors of ADAM proteases, exemplified here by INCB003619, and demonstrate that these inhibitors effectively block HER-2 cleavage in HER-2 overexpressing human breast cancer cell lines. Intriguingly, when used in combination, INCB003619 dramatically enhances the antiproliferative activity of suboptimal doses of the anti-HER-2 antibody, trastuzumab, in HER-2 overexpressing/shedding breast cancer cell lines, accompanied by reduced ERK and AKT phosphorylation. Furthermore, INCB003619, in combination with trastuzumab, augments the pro-apoptotic and antiproliferative effects of the chemotherapeutic agent paclitaxel. Consistent with these in vitro data, INCB003619 reduces serum ECD levels and enhances the antitumor effect of trastuzumab in a xenograft tumor model derived from the HER-2 overexpressing BT-474 breast cancer cell line. Collectively, these findings suggest that blocking HER-2 cleavage with selective ADAM inhibitors may represent a novel therapeutic approach for treating HER-2 overexpressing breast cancer patients.

Liver transplantation (LT) was achieved for factor V Leiden-induced thrombophilia in a neonate with hepatic veno-occlusive disease. Initial LT was performed with a liver segment removed from a child with primary oxalosis. Four months later, a second, definitive LT was performed. The child remains well without recurrent thrombosis.

Three enzyme systems (2 amylase-based and 1 protease-based) were tested in shelf-stable bread to determine effectiveness in preserving texture during storage for eight weeks. Each enzyme was tested in formulations without glycerol or with 6% glycerol. Bread samples were analyzed to determine physical properties (crumb density, crust-to-crumb ratio, rate of moisture distribution from crumb to crust), mechanical properties (modulus, and a parameter [C1] describing resistance to high levels of deformation obtained by fitting stress-strain data to a three-parameter function), and thermal properties (thermal stability and enthalpy of transitions) as a function of storage time. Mechanical properties were further analyzed to predict asymptotic firmness. Bread firmness after storage as evaluated in terms of modulus and C1 were lower in all enzyme-added systems, the effect of protease being the most significant. Enzymes had less effect on glycerol-containing systems with no apparent trend. The breads had complex thermal behavior and exhibited multiple transitions. Both amylase preparations in the presence of glycerol reduced the amount of starch recrystallization.

Focuses on the affect of canine lymphoma on middle-aged dogs. Importance of early diagnosis; Observation of canine lymphoma with generalized lymphadenopathy during petting and grooming of pets; Use of different modalities to treat canine lymphoma.

A critical prerequisite to the promotion of public health in local communities is gaining political support for local public health agencies and their activities. County governments are particularly important sources of support for public health agencies at the county level. This article deals with the issue of the priority given to public health by top county government officials. We determine that public health is generally a low priority issue for county governmental leaders. The low priority given to public health is, in part, linked to top county officials’ lack of recognition of important public health problems and low levels of community group advocacy for public health issues. The problems that face public health in the political arena are related to the fact that many public health activities are not highly visible as political issues. An understanding of the relationship between public health and more politically salient issues can help health officials to raise awareness among county governmental leaders in regard to the importance of supporting public health activities. The data used in this study were collected from a survey questionnaire, which was mailed to 1,430 top county officials in 596 U.S. counties. The survey results were supplemented by observations made during site visits to 12 counties selected from four states.

This research examines how sixteen state commissions on local governments concep tualized and defined urban problems. The research indicates that the commissions had a reform-oriented definition which stressed governmental fragmentation and multi plicity. It was found that the commissions took their cues on defining urban problems from organizations such as the Advisory Commission on Intergovernmental Relations and the Committee for Economic Development. The note concludes with observations on how urban research is related to urban policy formation.

Proteins have been directed into either the top or bottom phase of a polyethylene glycol/dextran aqueous two-phase system by applying an electric field perpendicular to the phase interface. Protein migration across the interface was manipulated by varying polarity, pH, electrophoresis time, field strength, and phase volume ratio. Mixtures of hemoglobin and albumin were separated by operating between isoelectric points and directing oppositely charged proteins into separate phases. Applying 50 V/cm for 2 hours to 58 mL of an equal phase volume two-phase system containing 0.2 g/L of each protein at pH 6 resulted in a bottom phase containing 99% of the hemoglobin and a top phase containing 95% of the albumin. This represents a significant improvement over the separation obtained either by partitioning in the same two-phase system with no applied field or by electrophoresis under the same conditions in homogeneous buffer. The two-phase system divides the electrophoresis device into two distinct regions, providing stability against convective mixing and facilitating product isolation.