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Carmofur, 1-hexylcarbamoyl-5-fluorouracil (HCFU) is an antineoplastic drug, which has been in clinics in Japan since 1981 for the treatment of colorectal cancer. Subsequently, it was also introduced in China, Korea, and Finland. Besides colorectal cancer, it has also shown antitumor activity in other cancers such as breast, head and neck, pancreatic, gastrointestinal, and solid brain tumors. A prodrug of 5-fluorouracil (5-FU), carmofur has shown better gastrointestinal stability and superior antiproliferative activity compared to its active counterpart 5-FU. Recently, carmofur has gained attention as an acid ceramidase inhibitor and as a potential lead compound against several noncancerous diseases such as coronavirus disease 2019, Krabbe disease, acute lung injury, Parkinson's disease, dementia, childhood ependymoma etc. Carmofur has also been reported to have antifungal, and antimicrobial properties. Nevertheless, no comprehensive review is available on this drug. Herein, we summarized the chemistry, pharmacokinetics, and pharmacology of carmofur based on the literature published between January 1976 and March 2022 as identified from PubMed and Google Scholar search engines.

Carmofur is an acid ceramidase inhibitor with superior efficacy in suppressing and killing fatally aggressive glioblastoma cell lines compared to the FDA-approved drug temozolomide. In addition to brain tumors, carmofur also gained attention as a potential lead inhibitor of the main protease (M

Xenorhabdus species are symbionts of entomopathogenic nematodes and pathogens of susceptible insects. Nematodes enter insect hosts and perforate the midgut to invade the haemocoel where Xenorhabdus bacteria are released transitioning to their pathogenic stage. During nematode invasion microbes from the insect gut translocate into the haemocoel. Different species of nematodes carrying specific strains of Xenorhabdus can also invade the same insect. Xenorhabdus species thereby compete for nutrients and space with both related strains and non-related gut microbes. While Xenorhabdus species produce diverse antimicrobial compounds in complex media, their functions in insect hosts are not well understood. We show that Xenorhabdus szentirmaii produced ngrA-dependent antibiotics that were active against both gut-derived microbes and Xenorhabdus nematophila whereas antibiotics of X. nematophila were not active against X. szentirmaii. X. nematophila growth was inhibited in co-cultures with wild-type X. szentirmaii in medium that mimics insect haemolymph. An antibiotic-deficient strain of X. szentirmaii was created by inactivating the ngrA gene that encodes the enzyme that attaches the 4' phosphopantetheinyl moiety to non-ribosomal peptide synthetases involved in antibiotic biosynthesis. X. nematophila growth was not inhibited in co-cultures with the ngrA strain. The growth of X. nematophila was suppressed in Manduca sexta co-injected with wild-type X. szentirmaii and X. nematophila. In contrast, growth of X. nematophila was not suppressed in M. sexta co-injected with the ngrA strain. Two unique compounds were detected by MALDI-TOF MS analysis in haemolymph infected with the wild-type but not with the ngrA strain. Finally, killing of M. sexta was delayed in insects infected with the ngrA strain. These findings indicate that in the insect host X. szentirmaii produces ngrA-dependent products involved in both interspecies competition and virulence.

Bexarotene has shown inhibition of lung and mammary gland tumorigenesis in preclinical models and in clinical trials. The main side effects of orally administered bexarotene are hypertriglyceridemia and hypercholesterolemia. We previously demonstrated that aerosolized bexarotene administered by nasal inhalation has potent chemopreventive activity in a lung adenoma preclinical model without causing hypertriglyceridemia. To facilitate its future clinical translation, we modified the formula of the aerosolized bexarotene with a clinically relevant solvent system. This optimized aerosolized bexarotene formulation was tested against lung squamous cell carcinoma mouse model and lung adenocarcinoma mouse model and showed significant chemopreventive effect. This new formula did not cause visible signs of toxicity and did not increase plasma triglycerides or cholesterol. This aerosolized bexarotene was evenly distributed to the mouse lung parenchyma, and it modulated the microenvironment in vivo by increasing the tumor-infiltrating T cell population. RNA sequencing of the lung cancer cell lines demonstrated that multiple pathways are altered by bexarotene. For the first time, these studies demonstrate a new, clinically relevant aerosolized bexarotene formulation that exhibits preventive efficacy against the major subtypes of lung cancer. This approach could be a major advancement in lung cancer prevention for high risk populations, including former and present smokers.

Glioblastoma is one of the most common and detrimental forms of solid brain tumor, with over 10,000 new cases reported every year in the United States. Despite aggressive multimodal treatment approaches, the overall survival period is reported to be less than 15 months after diagnosis. A widely used approach for the treatment of glioblastoma is surgical removal of the tumor, followed by radiotherapy and chemotherapy. While there are several drugs available that are approved by the Food and Drug Administration (FDA), significant efforts have been made in recent years to develop new chemotherapeutic agents for the treatment of glioblastoma. This review describes the molecular targets and pathogenesis as well as the current progress in chemotherapeutic development and other novel therapies in the clinical setting for the treatment of glioblastoma.

Pediatric glioblastoma (GBM) is a relatively rare brain tumor in children that has a dismal prognosis. Surgery followed by radiotherapy is the main treatment protocol used for older patients. The benefit of adjuvant chemotherapy is still limited due to a poor understanding of the underlying molecular and genetic changes that occur with irradiation of the tumor. In this study, we performed total RNA sequencing on an established stable radioresistant pediatric GBM cell line to identify mRNA expression changes following radiation. The expression of many genes was altered in the radioresistant pediatric GBM model. These genes have never before been reported to be associated with the development of radioresistant GBM. In addition to exhibiting an accelerated growth rate, radioresistant GBM cells also have overexpression of the DNA synthesis-rate-limiting enzyme ribonucleotide reductase, and pro-cathepsin B. These newly identified genes should be concertedly studied to better understand their role in pediatric GBM recurrence and progression after radiation. It was observed that the changes in multiple biological pathways protected GBM cells against radiation and transformed them to a more malignant form. These changes emphasize the importance of developing a treatment regimen that consists of a multiple-agent cocktail that acts on multiple implicated pathways to effectively target irradiated pediatric GBM. An alternative to radiation or a novel therapy that targets differentially expressed genes, such as metalloproteases, growth factors, and oncogenes and aim to minimize oncogenic changes following radiation is necessary to improve recurrent GBM survival.

The absence of major progress in the treatment of glioblastoma (GBM) is partly attributable to our poor understanding of both GBM tumor biology and the acquirement of treatment resistance in recurrent GBMs. Recurrent GBMs are characterized by their resistance to radiation. In this study, we used an established stable U87 radioresistant GBM model and total RNA sequencing to shed light on global mRNA expression changes following irradiation. We identified many genes, the expressions of which were altered in our radioresistant GBM model, that have never before been reported to be associated with the development of radioresistant GBM and should be concertedly further investigated to understand their roles in radioresistance. These genes were enriched in various biological processes such as inflammatory response, cell migration, positive regulation of epithelial to mesenchymal transition, angiogenesis, apoptosis, positive regulation of T-cell migration, positive regulation of macrophage chemotaxis, T-cell antigen processing and presentation, and microglial cell activation involved in immune response genes. These findings furnish crucial information for elucidating the molecular mechanisms associated with radioresistance in GBM. Therapeutically, with the global alterations of multiple biological pathways observed in irradiated GBM cells, an effective GBM therapy may require a cocktail carrying multiple agents targeting multiple implicated pathways in order to have a chance at making a substantial impact on improving the overall GBM survival.

// Ninh B. Doan 1, 2 , Hisham Alhajala 3 , Mona M. Al-Gizawiy 4 , Wade M. Mueller 2 , Scott D. Rand 4 , Jennifer M. Connelly 5 , Elizabeth J. Cochran 6 , Christopher R. Chitambar 3 , Paul Clark 7 , John Kuo 7 , Kathleen M. Schmainda 3, 8 and Shama P. Mirza 1, 9, 10 1 Biotechnology and Bioengineering Center, Medical College of Wisconsin, Milwaukee, Wisconsin, 53226, USA 2 Department of Neurosurgery, Medical College of Wisconsin, Milwaukee, Wisconsin, 53226, USA 3 Medicine, Hematology/Oncology, Medical College of Wisconsin, Milwaukee, Wisconsin, 53226, USA 4 Radiology, Medical College of Wisconsin, Milwaukee, Wisconsin, 53226, USA 5 Neurology, Medical College of Wisconsin, Milwaukee, Wisconsin, 53226, USA 6 Pathology, Medical College of Wisconsin, Milwaukee, Wisconsin, 53226, USA 7 Department of Neurological Surgery and Human Oncology, University of Wisconsin, Madison, Wisconsin, 53792, USA 8 Biophysics, Medical College of Wisconsin, Milwaukee, Wisconsin, 53226, USA 9 Obstetrics and Gynecology, Medical College of Wisconsin, Milwaukee, Wisconsin, 53226, USA 10 Department of Chemistry and Biochemistry, University of Wisconsin, Milwaukee, Wisconsin, 53211, USA Correspondence to: Shama P. Mirza, email: mirza@uwm.edu Keywords: glioblastoma, acid ceramidase, carmofur, glioblastoma cancer stem cells (GSCs), overall survival Received: December 28, 2016 Accepted: September 30, 2017 Published: November 07, 2017 ABSTRACT Glioblastoma remains the most common, malignant primary cancer of the central nervous system with a low life expectancy and an overall survival of less than 1.5 years. The treatment options are limited and there is no cure. Moreover, almost all patients develop recurrent tumors, which typically are more aggressive. Therapeutically resistant glioblastoma or glioblastoma stem-like cells (GSCs) are hypothesized to cause this inevitable recurrence. Identifying prognostic biomarkers of glioblastoma will potentially advance knowledge about glioblastoma tumorigenesis and enable discovery of more effective therapies. Proteomic analysis of more than 600 glioblastoma-specific proteins revealed, for the first time, that expression of acid ceramidase (ASAH1) is associated with poor glioblastoma survival. CD133+ GSCs express significantly higher ASAH1 compared to CD133- GSCs and serum-cultured glioblastoma cell lines, such as U87MG. These findings implicate ASAH1 as a plausible independent prognostic marker, providing a target for a therapy tailored toward GSCs. We further demonstrate that ASAH1 inhibition increases cellular ceramide level and induces apoptosis. Strikingly, U87MG cells, and three different patient-derived glioblastoma stem-like cancer cell lines were efficiently killed, through apoptosis, by three different known ASAH1 inhibitors with IC50’s ranging from 11–104 μM. In comparison, the standard glioblastoma chemotherapy agent, temozolomide, had minimal GSC-targeted effects at comparable or even higher concentrations (IC50 > 750 μM against GSCs). ASAH1 is identified as a de novo glioblastoma drug target, and ASAH1 inhibitors, such as carmofur, are shown to be highly effective and to specifically target glioblastoma GSCs. Carmofur is an ASAH1 inhibitor that crosses the blood-brain barrier, a major bottleneck in glioblastoma treatment. It has been approved in Japan since 1981 for colorectal cancer therapy. Therefore, it is poised for repurposing and translation to glioblastoma clinical trials.

Glioblastoma multiforme (GBM) is the most common primary, intracranial malignancy of the central nervous system. The standard treatment protocol, which involves surgical resection, and concurrent radiation with adjuvant temozolomide (TMZ), still imparts a grim prognosis. Ultimately, all GBMs exhibit recurrence or progression, developing resistance to standard treatment. This study demonstrates that GBMs acquire resistance to radiation via upregulation of acid ceramidase (ASAH1) and sphingosine-1-phosphate (Sph-1P). Moreover, inhibition of ASAH1 and Sph-1P, either with humanized monoclonal antibodies, small molecule drugs (i.e. carmofur), or a combination of both, led to suppression of GBM cell growth. These results suggest that ASAH1 and Sph-1P may be excellent targets for the treatment of new GBMs and recurrent GBMs, especially since the latter overexpresses ASAH1.

// Ninh B. Doan 1, 2 , Ha S. Nguyen 2 , Andrew Montoure 2 , Mona M. Al-Gizawiy 3 , Wade M. Mueller 2 , Shekar Kurpad 2 , Scott D. Rand 3 , Jennifer M. Connelly 4 , Christopher R. Chitambar 4 , Kathleen M. Schmainda 3, 6 , Shama P. Mirza 1, 7, 8 1 Biotechnology and Bioengineering Center, Medical College of Wisconsin, Milwaukee, Wisconsin, 53226, USA 2 Department of Neurosurgery, Medical College of Wisconsin, Milwaukee, Wisconsin, 53226, USA 3 Department of Radiology, Medical College of Wisconsin, Milwaukee, Wisconsin, 53226, USA 4 Department of Neurology, Medical College of Wisconsin, Milwaukee, Wisconsin, 53226, USA 5 Department of Medicine, Hematology/Oncology, Medical College of Wisconsin, Milwaukee, Wisconsin, 53226, USA 6 Department of Biophysics, Medical College of Wisconsin, Milwaukee, Wisconsin, 53226, USA 7 Obstetrics and Gynecology, Medical College of Wisconsin, Milwaukee, Wisconsin, 53226, USA 8 Department of Chemistry and Biochemistry, University of Wisconsin, Milwaukee, Wisconsin, 53211, USA Correspondence to: Ninh B. Doan, email: ndoan@mcw.edu Keywords: pediatric glioblastoma, acid ceramidase inhibitors, carmofur, glioblastoma, medulloblastoma Received: September 12, 2016 Accepted: February 15, 2017 Published: March 01, 2017 ABSTRACT Pediatric brain tumors are the most common solid tumors in children and are also a leading culprit of cancer-related fatalities in children. Pediatric brain tumors remain hard to treat. In this study, we demonstrated that medulloblastoma, pediatric glioblastoma, and atypical teratoid rhabdoid tumors express significant levels of acid ceramidase, where levels are highest in the radioresistant tumors, suggesting that acid ceramidase may confer radioresistance. More importantly, we also showed that acid ceramidase inhibitors are highly effective at targeting these pediatric brain tumors with low IC 50 values (4.6–50 μM). This data suggests acid ceramidase as a novel drug target for adjuvant pediatric brain tumor therapies. Of these acid ceramidase inhibitors, carmofur has seen clinical use in Japan since 1981 for colorectal cancers and is a promising drug to undergo further animal studies and subsequently a clinical trial as a treatment for pediatric patients with brain tumors.

Transmembrane Ser/Thr kinases containing extracellular PASTA (penicillin-binding protein [PBP] and Ser/Thr-associated) domains are ubiquitous among Actinobacteria and Firmicutes species. Such PASTA kinases regulate critical bacterial processes, including antibiotic resistance, cell division, cell envelope homeostasis, and virulence, and are sometimes essential for viability. Previous studies of purified PASTA kinase fragments revealed they are capable of autophosphorylation in vitro, typically at multiple sites on the kinase domain. Autophosphorylation of a specific structural element of the kinase known as the activation loop is thought to enhance kinase activity in response to stimuli. However, the role of kinase phosphorylation at other sites is largely unknown. Moreover, the mechanisms by which PASTA kinases are deactivated once their stimulus has diminished are poorly understood. Enterococcus faecalis is a Gram-positive intestinal bacterium and a major antibiotic-resistant opportunistic pathogen. In E. faecalis, the PASTA kinase IreK drives intrinsic resistance to cell wall-active antimicrobials, and such antimicrobials trigger enhanced phosphorylation of IreK in vivo. Here we identify multiple sites of phosphorylation on IreK and evaluate their function in vivo and in vitro. While phosphorylation of the IreK activation loop is required for kinase activity, we found that phosphorylation at a site distinct from the activation loop reciprocally modulates IreK activity in vivo, leading to diminished activity (and diminished antimicrobial resistance). Moreover, this site is important for deactivation of IreK in vivo upon removal of an activating stimulus. Our results are consistent with a model in which phosphorylation of IreK at distinct sites reciprocally regulates IreK activity in vivo to promote adaptation to cell wall stresses. IMPORTANCE Transmembrane Ser/Thr kinases containing extracellular PASTA domains are ubiquitous among Actinobacteria and Firmicutes species and regulate critical processes, including antibiotic resistance, cell division, and cell envelope homeostasis. Previous studies of PASTA kinase fragments revealed autophosphorylation at multiple sites. However, the functional role of autophosphorylation and the relative impacts of phosphorylation at distinct sites are poorly understood. The PASTA kinase of Enterococcus faecalis, IreK, regulates intrinsic resistance to antimicrobials. Here we identify multiple sites of phosphorylation on IreK and show that modification of IreK at distinct sites reciprocally regulates IreK activity and antimicrobial resistance in vivo. Thus, these results provide new insights into the mechanisms by which PASTA kinases can regulate critical physiological processes in a wide variety of bacterial species.

Objective Endometrial cancer is the most common gynecologic cancer and yet much is still unknown about this disease. Our goal was to identify unique biomarkers of disease by performing a comprehensive proteomic analysis of early stage, low-grade endometrial cancer through analysis of serum collected from patients pre- and post-definitive surgery. Methods We used mass spectrometry (MS)-based proteomics to identify serum proteins from these patients. Serum samples from women undergoing hysterectomy with bilateral salpingo-oophorectomy for benign reasons served as control samples for the correlative studies. We then correlated our findings with The Cancer Genome Atlas (TCGA) database for additional confirmation. Results The Ingenuity Pathway Analysis of proteins that were differentially expressed in endometrial cancer showed increased cell survival and decreased organismal death, the most common hallmarks of cancer. We identified over expression of FAM83D (family with sequence similarity 83, member D) in the serum of patients with early stage low-grade endometrial cancer and verified the same in the endometrial cancer cell lines and patient tumors. We also confirmed our hypothesis that FAM83D may serve as a biomarker for endometrial cancer in a cohort of patients with endometrial cancer from The Cancer Genome Atlas (TCGA) project. Conclusion Comprehensive proteomic analysis is a feasible strategy for potential biomarker identification. Using this technique, FAM83D was identified as a candidate biomarker in early endometrial cancer in our patient samples and was not present in benign control samples. FAM83D has been associated with poor clinical outcomes in several human malignancies. Significance Our manuscript describes an alternative approach to comprehensive protein analysis in a model pre and post tumor removal for a sample of patients with early endometrial cancer. The model is innovative and the findings of over expression FAM83D in this population of early cancer may be useful in the study of a disease where there are few biomarkers or targetable therapies.

Background— Cardiac metabolism is critical for the functioning of the heart, and disturbance in this homeostasis is likely to influence cardiac disorders or cardiomyopathy. Our laboratory has previously shown that SNRK (sucrose nonfermenting related kinase) enzyme, which belongs to the AMPK (adenosine monophosphate–activated kinase) family, was essential for cardiac metabolism in mammals. Snrk global homozygous knockout (KO) mice die at postnatal day 0, and conditional deletion of Snrk in cardiomyocytes ( Snrk cmcKO) leads to cardiac failure and death by 8 to 10 months. Methods and Results— We performed additional cardiac functional studies using echocardiography and identified further cardiac functional deficits in Snrk cmcKO mice. Nuclear magnetic resonance-based metabolomics analysis identified key metabolic pathway deficits in SNRK knockdown cardiomyocytes in vitro. Specifically, metabolites involved in lipid metabolism and oxidative phosphorylation are altered, and perturbations in these pathways can result in cardiac function deficits and heart failure. A phosphopeptide-based proteomic screen identified ROCK (Rho-associated kinase) as a putative substrate for SNRK, and mass spec-based fragment analysis confirmed key amino acid residues on ROCK that are phosphorylated by SNRK. Western blot analysis on heart lysates from Snrk cmcKO adult mice and SNRK knockdown cardiomyocytes showed increased ROCK activity. In addition, in vivo inhibition of ROCK partially rescued the in vivo Snrk cmcKO cardiac function deficits. Conclusions— Collectively, our data suggest that SNRK in cardiomyocytes is responsible for maintaining cardiac metabolic homeostasis, which is mediated in part by ROCK, and alteration of this homeostasis influences cardiac function in the adult heart.

With increasing age, there is an observable loss of melanin in retinal pigment epithelial (RPE) cells. It is possible that degradation of the pigment contributes to the pathogenesis of retinal disease, as the cellular antioxidant material is depleted. Functionally, intact melanin maintains protective qualities, while oxidative degradation of melanin promotes reactive oxygen species (ROS) generation and formation of metabolic byproducts, such as melanolipofuscin. Understanding the structural and functional changes to RPE melanin with increasing age may contribute to a better understanding of disease progression and risk factors for conditions such as age-related macular degeneration (AMD). In this study, human donor RPE melanin is characterized using MALDI mass spectrometry to follow melanin degradation trends. In vitro models using ARPE-19 cells are used to assess photo-reactivity in repigmented cells. Significant protection against intracellular ROS produced by blue light is observed in calf melanin-pigmented cells versus unpigmented and black latex bead controls (P < 0.0001). UV-B exposure to aged human melanin-pigmented cells results in a significant increase in nitric oxide production versus control cells (P < 0.001). Peroxide-treated synthetic melanin is characterized to elucidate degradation products that may contribute to RPE cell damage.

A cancer phenotype is driven by several proteins and targeting a cluster of functionally interdependent molecules should be more effective for therapeutic intervention. This is specifically important for Ras-dependent cancer, as mutated (MT) Ras is non-druggable and targeting its interaction with effectors may be essential for therapeutic intervention. Here, we report that a protein-complex activated by the Ras effector p38γ MAPK is a novel therapeutic target for K-Ras-dependent colon cancer. Unbiased proteomic screening and immune-precipitation analyses identified p38γ interaction with heat shock protein 90 (Hsp90) and K-Ras in K-Ras MT, but not wild-type (WT), colon cancer cells, indicating a role of this complex in Ras-dependent growth. Further experiments showed that this complex requires p38γ and Hsp90 activity to maintain MT, but not WT, K-Ras protein expression. Additional studies demonstrated that this complex is activated by p38γ-induced Hsp90 phosphorylation at S595, which is important for MT K-Ras stability and for K-Ras dependent growth. Of most important, pharmacologically inhibition of Hsp90 or p38γ activity disrupts the complex, decreases K-Ras expression, and selectively inhibits the growth of K-Ras MT colon cancer in vitro and in vivo. These results demonstrated that the p38γ-activated ternary complex is a novel therapeutic target for K-Ras-dependent colon cancer.

Glioblastoma remains the most common, malignant primary cancer of the central nervous system with a low life expectancy and an overall survival of less than 1.5 years. The treatment options are limited and there is no cure. Moreover, almost all patients develop recurrent tumors, which typically are more aggressive. Therapeutically resistant glioblastoma or glioblastoma stem-like cells (GSCs) are hypothesized to cause this inevitable recurrence. Identifying prognostic biomarkers of glioblastoma will potentially advance knowledge about glioblastoma tumorigenesis and enable discovery of more effective therapies. Proteomic analysis of more than 600 glioblastoma-specific proteins revealed, for the first time, that expression of acid ceramidase (ASAH1) is associated with poor glioblastoma survival. CD133+ GSCs express significantly higher ASAH1 compared to CD133- GSCs and serum-cultured glioblastoma cell lines, such as U87MG. These findings implicate ASAH1 as a plausible independent prognostic marker, providing a target for a therapy tailored toward GSCs. We further demonstrate that ASAH1 inhibition increases cellular ceramide level and induces apoptosis. Strikingly, U87MG cells, and three different patient-derived glioblastoma stem-like cancer cell lines were efficiently killed, through apoptosis, by three different known ASAH1 inhibitors with IC50's ranging from 11–104 μM. In comparison, the standard glioblastoma chemotherapy agent, temozolomide, had minimal GSC-targeted effects at comparable or even higher concentrations (IC50 > 750 μM against GSCs). ASAH1 is identified as a de novo glioblastoma drug target, and ASAH1 inhibitors, such as carmofur, are shown to be highly effective and to specifically target glioblastoma GSCs. Carmofur is an ASAH1 inhibitor that crosses the blood-brain barrier, a major bottleneck in glioblastoma treatment. It has been approved in Japan since 1981 for colorectal cancer therapy. Therefore, it is poised for repurposing and translation to glioblastoma clinical trials.

BACKGROUND: Chemo- and radioresistance are characteristic features of post-treatment high-grade glioma (HGG). The cell populations responsible for this treatment-resistant phenotype are glioma stem cells (GSCs). These GSCs have a high requirement for iron which is essential for tumor cell viability. Intriguingly, the transferrin receptors that play a crucial role in iron uptake by both adult and pediatric astrocytomas exhibit a high affinity for gallium maltolate (GaM), a novel iron mimetic. Given the added commonality between adult and pediatric glioma stem cell signatures, especially post-treatment, we believe targeting treatment-resistant cell populations via their inherent iron metabolism is a viable approach to combat treatment-resistant HGG in adults and children. Here, we demonstrate the profound effects of GaM in a novel in vivo model of recurrent glioblastoma. METHODS: Irradiated human GBM cells (adult or pediatric) were stereotactically implanted into the right striatum of male athymic rats. Following confirmation of in vivo tumor growth by MRI at 9.4T, animals received GaM (50 mg/kg/day) in an oral preparation for voluntary ingestion. Tumor growth was monitored weekly by MRI, and lesion volume and associated advanced MRI parameter maps were determined using enhancing tumor ROIs. RESULTS: In a first set of animals, the mean weekly tumor growth rates of enhancing lesions were 65.8% and 156% in GaM-treated and control rats, respectively (p=0.002). Median disease-specific survival was 51 days in GaM-treated animals and 28 days in controls (p=0.004). Complete response was observed in 20% of the animals, with complete resolution of the disease confirmed histologically. A partial response was observed in 40% of the animals. Follow-up data is being collected in a second set of animals for verification and updated results will be discussed. CONCLUSION: We present compelling evidence that iron-targeted therapy using the novel iron mimetic GaM is highly effective in treatment-resistant HGG.

Human cytomegalovirus (HCMV) is a common agent of congenital infection and causes severe disease in immunocompromised patients. Current approved therapies focus on inhibiting viral DNA replication. The HCMV kinase pUL97 contributes to multiple stages of viral infection including DNA replication, controlling the cell cycle, and virion maturation. Our studies demonstrate that pUL97 also functions by influencing immediate early (IE) gene expression during the initial stages of infection. Inhibition of kinase activity using the antiviral compound maribavir or deletion of the UL97 gene resulted in decreased expression of viral immediate early genes during infection. Expression of pUL97 was sufficient to transactivate IE1 gene expression from the viral genome, which was dependent on viral kinase activity. We observed that pUL97 associates with histone deacetylase 1 (HDAC1). HDAC1 is a transcriptional corepressor that acts to silence expression of viral genes. We observed that inhibition or deletion of pUL97 kinase resulted in increased HDAC1 and decreased histone H3 lysine 9 acetylation associating with the viral major immediate early (MIE) promoter. IE expression during pUL97 inhibition or deletion was rescued following inhibition of deacetylase activity. HDAC1 associates with chromatin by protein-protein interactions. Expression of active but not inactive pUL97 kinase decreased HDAC1 interaction with the transcriptional repressor protein DAXX. Finally, using mass spectrometry, we found that HDAC1 is uniquely phosphorylated upon expression of pUL97. Our results support the conclusion that HCMV pUL97 kinase regulates viral immediate early gene expression by phosphorylation-mediated disruption of HDAC1 binding to the MIE promoter.

Background Heart failure with ejection fraction (HFpEF) is a syndrome resulting from several co-morbidities in which specific mediators are unknown. The platelet proteome responds to disease processes. We hypothesize that the platelet proteome will change composition in patients with HFpEF and may uncover mediators of the syndrome. Methods and results Proteomic changes were assessed in platelets from hospitalized subjects with symptoms of HFpEF (n = 9), the same subjects several weeks later without symptoms (n = 7) and control subjects (n = 8). Mass spectrometry identified 6102 proteins with five scans with peptide probabilities of ≥0.85. Of the 6102 proteins, 165 were present only in symptomatic subjects, 78 were only found in outpatient subjects and 157 proteins were unique to the control group. The S100A8 protein was identified consistently in HFpEF samples when compared with controls. We validated the fining that plasma S100A8 levels are increased in subjects with HFpEF (654 ± 391) compared to controls (352 ± 204) in an external cohort (p = 0.002). Recombinant S100A8 had direct effects on the electrophysiological and calcium handling profile in human induced pluripotent stem cell-derived cardiomyocytes. Conclusions Platelets may harbor proteins associated with HFpEF. S100A8 is present in the platelets of subjects with HFpEF and increased in the plasma of the same subjects. We further established a bedside-to-bench translational system that can be utilized as a secondary screen to ascertain whether the biomarkers may be an associated finding or causal to the disease process. S100A8 has been linked with other cardiovascular disease such as atherosclerosis and risk for myocardial infarction, stroke, or death. This is the first report on association of S100A8 with HFpEF. Electronic supplementary material The online version of this article (doi:10.1186/s12967-016-0774-3) contains supplementary material, which is available to authorized users.

Phosphatase plays a crucial role in determining cellular fate by inactivating its substrate kinase, but it is not known whether a kinase can vice versa phosphorylate its phosphatase to execute this function. Protein-tyrosine phosphatase H1 (PTPH1) is a specific phosphatase of p38γ mitogen-activated protein kinase (MAPK) through PDZ binding, and here, we show that p38γ is also a PTPH1 kinase through which it executes its oncogenic activity and regulates stress response. PTPH1 was identified as a substrate of p38γ by unbiased proteomic analysis, and its resultant phosphorylation at Ser-459 occurs in vitro and in vivo through their complex formation. Genetic and pharmacological analyses showed further that Ser-459 phosphorylation is directly regulated by Ras signaling and is important for Ras, p38γ, and PTPH1 oncogenic activity. Moreover, experiments with physiological stimuli revealed a novel stress pathway from p38γ to PTPH1/Ser-459 phosphorylation in regulating cell growth and cell death by a mechanism dependent on cellular environments but independent of canonical MAPK activities. These results thus reveal a new mechanism by which a MAPK regulates Ras oncogenesis and stress response through directly phosphorylating its phosphatase.

Unilateral ureteropelvic junction obstruction (UPJO) is the most common prenatally detected disease leading to hydronephrosis. The obstructive anatomic lesion leads to varying degrees of hydronephrosis, ranging from no apparent effect on renal function to atrophy. Furthermore, the natural course of hydronephrosis varies from spontaneous resolution to progressive deterioration and may take upwards of 3 years for a kidney to declare itself. The objectives of this article are to update our knowledge regarding the evaluation and management of UPJO in depth and to discuss the emerging value of urinary proteome analysis to the clinical arena.

Phosphorylation has been the most studied of all the post-translational modifications of proteins. Mass spectrometry has emerged as a powerful tool for phosphomapping on proteins/peptides. Collision-induced dissociation (CID) of phosphopeptides leads to the loss of phosphoric or metaphosphoric acid as a neutral molecule, giving an intense neutral loss product ion in the mass spectrum. Dissociation of the neutral loss product ion identifies peptide sequence. This method of data-dependent constant neutral loss (DDNL) scanning analysis has been commonly used for mapping phosphopeptides. However, preferential losses of groups other than phosphate are frequently observed during CID of phosphopeptides. Ions that result from such losses are not identified during DDNL analysis due to predetermined scanning for phosphate loss. In this study, we describe an alternative approach for improved identification of phosphopeptides by sequential abundant ion fragmentation analysis (SAIFA). In this approach, there is no predetermined neutral loss molecule, thereby undergoing sequential fragmentation of abundant peak, irrespective of the moiety lost during CID. In addition to improved phosphomapping, the method increases the sequence coverage of the proteins identified, thereby increasing the confidence of protein identification. To the best of our knowledge, this is the first report to use SAIFA analysis for phosphopeptide identification.

BACKGROUND: Recurrent glioblastoma (rGBM) is a distinctly different disease condition than de novo GBM, characterized by chemo- and radioresistance. Consequently, treatment options remain limited. Given our recently published promising results using the novel iron mimetic, gallium maltolate (GaM), in a rat xenograft of de novo GBM, we set out to investigate the effects of oral GaM in an in vivo model of rGBM. METHODS: Irradiated adult human GBM cells were stereotactically implanted into the right striatum of 12 male athymic rats. Following confirmation of in vivo tumor growth by advanced MRI at 9.4T on day 14 post-implantation, animals received GaM (50 mg/kg/day, n=5) in an oral preparation for voluntary ingestion. Two animals received daily oral GaM continuously throughout the study period, while three underwent a 2-week on, 1-week off treatment cycle. Tumor growth was monitored weekly by MRI, and lesion volume and associated parameter maps were determined using enhancing tumor ROIs. RESULTS: Mean compliance with voluntary ingestion of GaM was 97% (49 mg/kg/day). Complete longitudinal MRI data was available for 5 GaM rats and 5 controls. The mean weekly tumor growth rates of enhancing lesions were 78% and 156% in GaM-treated and control animals, respectively (p=0.006). Median disease-specific survival was 28 days in controls and 49 days in GaM-treated animals, with 3/5 treated animals still alive (p=0.004). Preliminary histological findings in GaM-treated tumor tissue indicate treatment effect (swollen cells, degeneration and/or granulation of cytoplasm) and apoptotic cells throughout the lesions, compared to pseudopalisading necrosis and standard tumor cell size in control tissues. No adverse physiological effects due to treatment were noted. CONCLUSION: We presented compelling evidence that oral GaM significantly impairs tumor growth and extends disease-specific survival in a xenograft model of rGBM. We propose that GaM shows great promise as a new interventional strategy for GBM therapy.

The implication of the various lipoprotein classes in the development of atherosclerotic cardiovascular disease has served to focus a great deal of attention on these particles over the past half-century. Using knowledge gained by the sequencing of the human genome, recent research efforts have been directed toward the elucidation of the proteomes of several lipoprotein subclasses. One of the challenges of such proteomic experimentation is the ability to initially isolate plasma lipoproteins subsequent to their analysis by mass spectrometry. Although several methods for the isolation of plasma lipoproteins are available, the most commonly utilized techniques require large sample volumes and may cause destruction and dissociation of lipoprotein particle-associated proteins. Fast protein liquid chromatography (FPLC) is a nondenaturing technique that has been validated for the isolation of plasma lipoproteins from relatively small sample volumes. In this study, we present the use of FPLC in conjunction with nano-HPLC-ESI-tandem mass spectrometry as a new integrated methodology suitable for the proteomic analysis of human lipoprotein fractions. Results from our analysis show that only 200 μl of human plasma suffices for the isolation of whole high density lipoprotein (HDL) and the identification of the majority of all known HDL-associated proteins using mass spectrometry of the resulting fractions.

Membrane-bound and membrane-associated proteins are difficult to analyze by mass spectrometry, since the association with lipids impedes the isolation and solubilization of the proteins in buffers suitable for mass spectrometry and the efficient generation of positively charged peptide ions by electrospray ionization. Current methods mostly utilize detergents for the isolation of proteins from membranes. In this study, we present an improved detergent-free method for the isolation and mass spectrometric identification of membrane-bound and membrane-associated proteins. We delipidate proteins from the membrane bilayer by chloroform extraction to overcome dissolution and ionization problems during analysis. Comparison of our results to results obtained by direct tryptic digestion of insoluble membrane pellets identifies an increased number of membrane proteins, and a higher quality of the resulting mass spectral data.

BACKGROUND: The Cancer Genome Atlas (TCGA) project previously identified genetic changes in the DNA sequence and copy number, DNA methylation, and gene expression of glioblastoma multiforme (GBM) tumors, leading to an enthused effort to compile and characterize changes in the genome, transcriptome, and proteome of human GBM tumors, so that aberrantly functioning molecular pathways and tumor subtypes could be identified. METHODS: Agilent GeneSpring and Mass Profiler Professional (MPP) software was used to perform integrated multi-omics analysis of mRNA expression, miRNA expression, protein expression, and copy number aberration associated with GBM. Additionally, analysis was performed on an independent discovery proteomics study of GBM and control (brain fragments from epilepsy surgery) patients measured using a label-free proteomics approach. RESULTS: Correlation analysis performed between mRNA and miRNA datasets from TCGA identified potential candidate miRNAs regulating the known Proneural signature genes OLIG2, NKX2-2, and SOX11. It also highlighted the molecular events leading to suppression of p53 in Proneural tumor signaling. Sample-sample correlation and PCA analysis on patient specimens resulted in clear separation between control and tumor samples. In addition, the tumor samples were segregated into two to three subgroups, suggesting that different TCGA subtypes may be present. Indeed, combined analysis of mRNA expression data from TCGA study and proteomics data from the label-free study identified a core subset of 54 GBM subtype signatures, clearly differentiating the known subtypes in the larger TCGA cohort. CONCLUSION: Our observation suggests that GBM tumor heterogeneity may be best characterized by both mRNA and protein abundance of the signature genes, opening a possibility for follow-up studies with potential diagnostic and clinical significance.

Proteolytic digestion of a complicated protein mixture from an organelle or whole-cell lysate is usually carried out in a dilute solution of a denaturing buffer, such as 1-2 M urea. Urea must be subsequently removed by C18 beads before downstream analysis such as HPLC/MS/MS or complete methylation followed by IMAC isolation of phosphopeptides. Here we describe a procedure for digesting a complicated protein mixture in the absence of denaturants. Proteins in the mixture are precipitated with trichloroacetic acid/acetone for denaturation and salt removal and resuspended in NH4HCO3 buffer. After trypsinolysis, the resulting peptides are not contaminated by urea or other nonvolatile salts and can be dried in a SpeedVac to remove NH4HCO3. When this protocol was applied to an extract of A431 cells, 96.8% of the tryptic peptides were completely digested (i.e., had no missed cleavage sites), in contrast to 87.3% of those produced by digestion in urea buffer. We successfully applied this digestion method to analysis of the phosphoproteome of adiposomes from HeLa cells, identifying 33 phosphorylation sites in 28 different proteins. Our digestion method avoids the need to remove urea before HPLC/MS/MS analysis or methylation and IMAC, increasing throughput while reducing sample loss and contamination from sample handling. We believe that this method should be valuable for proteomics studies.

A series of meso-cycloalkyl calix(4)pyrroles (I) and meso-dialkyl calix(4)pyrroles (II) has been studied under electron ionization (EI) mass spectral conditions. All the calix(4)pyrroles showed prominent molecular ions. The cleavage of the CC bond linked at position 2 of the pyrrole ring (β-cleavage) is the foremost and dominant fragmentation process. The β-cleavage process, either through ring opening or directly, results in the loss of an alkyl radical from the molecular ion. The collision-induced dissociation (CID) spectra of I showed specific sequential expulsion of pyrrole and/or cycloalkyl rings from the molecular ion with or without hydrogen migrations, revealing more information on the structure of individual compounds than was available from the EI spectra. The isomeric cycloalkyl calix(4)pyrroles showed distinguishable CID spectra, indicating structure specificity in initial ring opening whereas, in the case of II, the EI mass spectrum contains all the structure-indicative fragment ions. The CID spectra of II resulted in a dominant [M–R]+ ion, with other characteristic ions being less abundant. Copyright © 2004 John Wiley & Sons, Ltd.

The kinetics of oxidation of 16 meta-, ortho-, and para-substituted anilines with nine oxo(salen)chromium(V) ions have been studied by spectrophotometric, ESIMS, and EPR techniques. During the course of the reaction, two new peaks with lambda(max) at 470 and 730 nm appear in the absorption spectrum, and these peaks are due to the formation of emeraldine forms of oligomers of aniline supported by the ESIMS peaks with m/z values 274 and 365 (for the trimer and tetramer of aniline). The rate of the reaction is highly sensitive to the change of substituents in the aryl moiety of aniline and in the salen ligand of chromium(V) complexes. Application of the Hammett equation to analyze kinetic data yields a rho value of -3.8 for the substituent variation in aniline and +2.2 for the substituent variation in the salen ligand of the metal complex. On the basis of the spectral, kinetic, and product analysis studies, a mechanism involving an electron transfer from the nitrogen of aniline to the metal complex in the rate controlling step has been proposed. The Marcus equation has been successfully applied to this system, and the calculated values are compliant with the measured values.

The kinetic method for measuring proton affinities (PA) and gas-phase basicities (GB) was applied to the methyl esters of simple amino acids. The experiments show that the GB and PA values for deuterium labeled glycine methyl ester are indeed greater than that of the corresponding unlabelled glycine methyl ester. The PA of l -Ala-OCD3 is also slightly greater than that of the unlabeled alanine methyl ester. The secondary isotope effects originate, as shown by density functional theory, in differences in zero-point energies and thermal-energy corrections between H and D-bearing molecules.

Some dialkylaminoethanols, precursors of chemical warfare agents such as V-agents and nitrogen mustards, were analyzed by electron impact (EI) and electrospray ionization (ESI) mass spectrometry. The fragmentation pathways in EI and ESI-MS/MS methods are rationalized. The collision-induced dissociation (CID) spectra of [M+H]+ ions of aminoethanols in ESI mode are clearly distinguishable from one another, including those of isomeric normal and branched chain dialkylaminoethanols. Structures can be proposed based on the general fragmentation pathways of these molecules. Copyright © 2003 John Wiley & Sons, Ltd.

BACKGROUND: Pediatric high grade glioma (pHGG) is a devastating cancer of the central nervous system. The prognosis of this most malignant form of brain tumor remains poor, largely due to the inherent resistance of pHGG to therapy, and inevitably leads to recurrence. Unfortunately, because recurrent glioma, or glioma remaining after conventional therapy, frequently has very different molecular characteristics from the original tumor in addition to a vastly different biological behavior, recurrent pHGG has proven exceedingly difficult to treat, with very few treatment options providing clinically meaningful outcomes. As part of the NCI Pediatric Preclinical Testing Program, several pediatric brain cancer cell lines, including the ones used in this study, were developed and characterized. However, to our knowledge, none have been utilized in development of a rat xenograft model of recurrent pHGG. METHODS: Pediatric glioblastoma cells (SJ-GBM2) were obtained from the Children’s Oncology Group Cell Culture and Xenograft Repository. Irradiated SJGBM2-10gy cells were generated by exposure to a total radiation dose of 10 Gy. Following irradiation, the resultant SJGBM2-10gy cells were tested for cell viability and allowed to reach confluence prior to stereotactical implantation into the right striatum of male athymic rats. RESULTS: Less than 1% of total cells survived radiation. Both irradiated and parental cell lines formed tumors in 100% of the animals following intracranial implantation. Irradiated SJGBM2-10gy cells exhibited a more aggressive tumor growth than the parental SJ-GBM2 cell line, resulting in mortality on average 14 days earlier. Histopathological analysis is underway and more advanced imaging studies are planned. CONCLUSION: To our knowledge this is the first report of an intracranial rat xenograft model mimicking recurrent pHGG using irradiated cell lines. It represents a robust and expedient in vivo model for studying recurrent pHGG biology.

// Anna Alexanian 1 , Bradley Miller 1 , Marla Chesnik 2 , Shama Mirza 2 and Andrey Sorokin 1 1 Division of Nephrology and Kidney Disease Center, Department of Medicine, Medical College of Wisconsin, Milwaukee, Wisconsin, USA, 2 Department of Biochemistry, Biotechnology and Bioengineering Center, Medical College of Wisconsin, Milwaukee, Wisconsin, USA Correspondence: Andrey Sorokin, email: // Keywords : COX2 regulation/DU145 cells/prostaglandins/phosphorylation/prostate cancer/Src family kinases Received : March 7, 2014 Accepted : May 16, 2014 Published : May 18, 2014 Abstract While increased COX2 expression and prostaglandin levels are elevated in human cancers, the mechanisms of COX2 regulation at the post-translational level are unknown. Initial observation that COX2 forms adduct with non-receptor tyrosine kinase FYN, prompted us to study FYN-mediated post-translational regulation of COX2. We found that FYN increased COX2 activity in prostate cancer cells DU145, independent of changes in COX2 or COX1 protein expression levels. We report that FYN phosphorylates human COX2 on Tyr 446, and while corresponding phospho-mimetic COX2 mutation promotes COX2 activity, the phosphorylation blocking mutation prevents FYN-mediated increase in COX2 activity.

Cancer is a complex disease; glioblastoma (GBM) is no exception. Short survival, poor prognosis, and very limited treatment options make it imperative to unravel the disease pathophysiology. The critically important identification of proteins that mediate various cellular events during disease is made possible with advancements in mass spectrometry (MS)-based proteomics. The objective of our study is to identify and characterize proteins that are differentially expressed in GBM to better understand their interactions and functions that lead to the disease condition. Further identification of upstream regulators will provide new potential therapeutic targets. We analyzed GBM tumors by SDS-PAGE fractionation with internal DNA markers followed by liquid chromatography-tandem mass spectrometry (MS). Brain tissue specimens obtained for clinical purposes during epilepsy surgeries were used as controls, and the quantification of MS data was performed by label-free spectral counting. The differentially expressed proteins were further characterized by Ingenuity Pathway Analysis (IPA) to identify protein interactions, functions, and upstream regulators. Our study identified several important proteins that are involved in GBM progression. The IPA revealed glioma activation with z score 2.236 during unbiased core analysis. Upstream regulators STAT3 and SP1 were activated and CTNNα was inhibited. We verified overexpression of several proteins by immunoblot to complement the MS data. This work represents an important step towards the identification of GBM biomarkers, which could open avenues to identify therapeutic targets for better treatment of GBM patients. The workflow developed represents a powerful and efficient method to identify biomarkers in GBM.

A preliminary investigation of the use of 5-ethyl-2-mercaptothiazole as matrix in matrix-assisted laser desorption/ionization (MALDI) of a broad spectrum of analytes is reported. The analytes studied are substance P, insulin, beta-cyclodextrin, triacylglycerols of coconut oil and polypropylene glycol 2000 (PPG 2000). In the positive ion mass spectra of the matrix/analyte combinations, the formation of [M + H]+ and [M + cation]+ species were observed and compared with those obtained by using well-established matrices such as alpha-cyano-4-hydroxycinnamic acid, genticic acid, sinapinic acid and dithranol. In addition, the usefulness of this new matrix for MALDI in negative ion mode is also described using substance P and beta-cyclodextrin as examples.

The electron impact (EI) mass spectra of isomeric S- and O-aryl (2-naphthyl, R-Ph-= aryl, where R = H, NO 2 , COOCH 3 , COCH 3 and OCH 3 ) dimethylthiocarbamates have been studied in detail. The EI spectra of O-aryl dimethylthiocarbamates include some of the fragment ions that are characteristic of the corresponding S-aryl isomer, which could be explained by a Newman-Kwart rearrangement in the ion source of the mass spectrometer, after ionization. The EI mass spectra of S-aryl dimethylthiocarbamates showed specific fragment ions resulting from cleavage of the molecular ion involving the bond between sulfur and the carbonyl group of the carbamate moiety (α-cleavage). The EI mass spectra of O-aryl dimethylthiocarbamates, however, showed the fragmentation of the molecular ion by α-cleavage involving the bonds on both sides of oxygen atom. Further, the EI mass spectra of O-aryl dimethylthiocarbamates containing an electron-withdrawing substituent (-R) on the aryl group showed a characteristic fragment ion corresponding to [M - R] + .

The electron impact (EI) mass spectrum of allyl phenyl ether (1) includes an ion at m/z 106 that is formed mainly by the loss of CO from the molecular ion, as supported by high resolution and MS/MS data. The formation of the [M - CO](+) ion from 1 can be explained in terms of the Claisen rearrangement of 1 after ionization in the ion source of the mass spectrometer. Similarly, allyl phenyl sulfide (2) and allyl phenyl selenide (3) showed characteristic ions corresponding to [M - CH(3)](+), [M - XH](+) (X = S or Se) and [M - C(2)H(4)](+.), and the formation of these ions are explained via Claisen rearrangement of 2 and 3 in the ion source of the mass spectrometer resulting in a mixture of rearrangement products. The formation of molecular ions of 2-allyl thiophenol and 2-allyl selenophenol as intermediates, that cannot be isolated as the neutrals from the solution phase Claisen rearrangement of 2 and 3, respectively, is clearly indicated in the gas phase. The mass spectra of the rearrangement products obtained from the solution phase reaction were also consistent with the proposal of formation of these products in the ion source of the mass spectrometer. The formation of characteristic fragment ions attributed to the Claisen rearrangement products are also evident in the collision induced dissociation spectra of the corresponding molecular ions. Copyright 2000 John Wiley & Sons, Ltd.

Cardiovascular proteomics is the branch of proteomics specifically applied to heart and vasculature. The field of proteomics has recently gained greater importance based on its potential to unravel the complex physiological and biochemical mechanisms and pathways in organisms and cells at the molecular level. The goal is to identify proteins that generate/mediate disease process, thereby, opening avenues to new diagnostics and therapeutic strategies. Proteins are much closer to the actual disease process, in most cases, than parent genes. They are the ultimate regulators of cell functions, and the vast majorities serve as drug targets. Differential protein expression analysis, by measuring the upregulated or downregulated proteins in disease state as compared with the normal healthy condition, is vital to understand the disease mechanism. In conjunction with various separation techniques, mass spectrometry (MS)-based methods evolved as the best techniques for the comprehensive characterization of proteins.1,2 After the initial protein identification, a more challenging approach in proteomics is the quantification of the proteins identified.3 Various methods of quantification by MS have been developed recently, for both relative and absolute measurements (Figure 1), and the technology is still expanding to find better and more efficient quantification approaches. This, in turn, is facilitated by the advancements in the MS instrumentation, including powerful analyzers and fragmentation technology. Most of the quantification methods are based on measurements at peptide level and fall under the bottom-up proteomics approach. In this review article, we will outline various MS-based approaches available for measuring the variation in protein abundances and how these techniques are playing a key role in cardiovascular research. Figure 1. An overview of mass spectrometry-based quantitative proteomics: Mass spectrometry (MS)-based quantification can be achieved either by relative or absolute quantification (AQUA). Both relative and AQUA can be further classified into labeled and label-free quantification, each of …

Unilateral ureteropelvic junction obstruction (UPJO) is the most common prenatally detected disease leading to hydronephrosis. The obstructive anatomic lesion leads to varying degrees of hydronephrosis, ranging from no apparent effect on renal function to atrophy. Furthermore, the natural course of hydronephrosis varies from spontaneous resolution to progressive deterioration and may take upwards of 3 years for a kidney to declare itself. The objectives of this article are to update our knowledge regarding the evaluation and management of UPJO in depth and to discuss the emerging value of urinary proteome analysis to the clinical arena.

derwent redo RALP. Eight patients had rightand five had left-sided repairs. Mean age at first pyeloplasty was 4.8 years (range 10 days– 18.0 years). Initially, patients underwent open repair (12) or RALP (1). Mean age at redo RALP was 11.0 years (range 1.2–19.4 years) and mean interval between surgeries was 74.5 months. Indications for repeat repair were presence of clinical symptoms and/or radiographic evidence of poor/worsening renal function. Patients underwent transperitoneal RALP via the Anderson-Hynes (10), Y-V plasty (1) or pyeloureterostomy (2) techniques. Mean operative time was 202 minutes (range 132–285) from incision to port closure. Mean blood loss was 28 mL. No patient required conversion to an open procedure. Double J stents were placed in all patients preor intraoperatively. Mean length of stay was 2.7 days (range 1.2–4.4 days). Complications immediately post-op included ileus (1) and ileus and pneumonia (1). Three patients developed culture-proven urinary tract infection within one month of surgery. Mean length of follow-up was 15.2 months (range 1.2–41.4 months). Ureteral stents were removed on average 32 days postoperatively. In 6 of 7 patients with pain complaints, symptoms completely resolved. Of the 12 with follow-up, most recent renal imaging demonstrated improved (9), unchanged (2), or worsening hydronephrosis (1). Four children underwent MAG3 scans which showed either improved function or a normal t1/2. Following redo RALP, one patient required subsequent temporary stent placement and another underwent stent placement twice with eventual nephrectomy. CONCLUSIONS: RALP following previous pyeloplasty is technically feasible with acceptable operative times, lengths of stay and complication rates. In the short-term, 83% have not required further operative intervention, and excellent results were achieved in 9 of 12 children with post-operative follow-up in terms of symptom resolution and/or improved imaging results.

Maspin, a 42-kDa non classical serine protease inhibitor (serpin) is expressed by epithelial cells of various tissues including the cornea. The protein localizes to the nucleus and cytosol, and is present in the extracellular space. While extracellular maspin regulates corneal stromal fibroblast adhesion and inhibits angiogenesis during wound healing in the cornea, the molecular mechanism of its extracellular functions is unclear. We hypothesized that identifying post-translational modifications of maspin, such as phosphorylation, may help decipher its mode of action. The focus of this study was on the identification of phosphorylation sites on extracellular maspin, since the extracellular form of the molecule is implicated in several functions. Multi-stage fragmentation mass spectrometry was used to identify sites of phosphorylation on extracellular corneal epithelial cell maspin. A total of eight serine and threonine phosphorylation sites (Thr50, Ser97, Thr118, Thr157, Ser240, Ser298, Thr310, Ser316) were identified on the extracellular forms of the molecule. Phosphorylation of tyrosine residues on extracellular maspin was not detected on extracellular maspin from corneal epithelial cell, in contrast to breast epithelial cells. This study provides the basis for further investigation into the functional role of phosphorylation of corneal epithelial maspin.

The regulation of gene transcription is fundamental to the existence of complex multicellular organisms such as humans. Although it is widely recognized that much of gene regulation is controlled by gene-specific protein-DNA interactions, there presently exists little in the way of tools to identify proteins that interact with the genome at locations of interest. We have developed a novel strategy to address this problem, which we refer to as GENECAPP, for Global ExoNuclease-based Enrichment of Chromatin-Associated Proteins for Proteomics. In this approach, formaldehyde cross-linking is employed to covalently link DNA to its associated proteins; subsequent fragmentation of the DNA, followed by exonuclease digestion, produces a single-stranded region of the DNA that enables sequence-specific hybridization capture of the protein-DNA complex on a solid support. Mass spectrometric (MS) analysis of the captured proteins is then used for their identification and/or quantification. We show here the development and optimization of GENECAPP for an in vitro model system, comprised of the murine insulin-like growth factor-binding protein 1 (IGFBP1) promoter region and FoxO1, a member of the forkhead rhabdomyosarcoma (FoxO) subfamily of transcription factors, which binds specifically to the IGFBP1 promoter. This novel strategy provides a powerful tool for studies of protein-DNA and protein-protein interactions.