MASH Native: a unified solution for native top-down proteomics data processing.

MOTIVATION: Native top-down proteomics (nTDP) integrates native mass spectrometry (nMS) with top-down proteomics (TDP) to provide comprehensive analysis of protein complexes together with proteoform identification and characterization. Despite significant advances in nMS and TDP software developments, a unified and user-friendly software package for analysis of nTDP data remains lacking. RESULTS: We have developed MASH Native to provide a unified solution for nTDP to process complex datasets with database searching capabilities in a user-friendly interface. MASH Native supports various data formats and incorporates multiple options for deconvolution, database searching, and spectral summing to provide a "one-stop shop" for characterizing both native protein complexes and proteoforms. AVAILABILITY AND IMPLEMENTATION: The MASH Native app, video tutorials, written tutorials, and additional documentation are freely available for download at https://labs.wisc.edu/gelab/MASH_Explorer/MASHSoftware.php. All data files shown in user tutorials are included with the MASH Native software in the download .zip file.

MASH Native: A Unified Solution for Native Top-Down Proteomics Data Processing.

Native top-down proteomics (nTDP) integrates native mass spectrometry (nMS) with top-down proteomics (TDP) to provide comprehensive analysis of protein complexes together with proteoform identification and characterization. Despite significant advances in nMS and TDP software developments, a unified and user-friendly software package for analysis of nTDP data remains lacking. Herein, we have developed MASH Native to provide a unified solution for nTDP to process complex datasets with database searching capabilities in a user-friendly interface. MASH Native supports various data formats and incorporates multiple options for deconvolution, database searching, and spectral summing to provide a one-stop shop for characterizing both native protein complexes and proteoforms. The MASH Native app, video tutorials, written tutorials and additional documentation are freely available for download at https://labs.wisc.edu/gelab/MASH_Explorer/MASHNativeSoftware.php . All data files shown in user tutorials are included with the MASH Native software in the download .zip file.

Absolute or relative stability in minimal invasive plate osteosynthesis of simple distal meta or diaphyseal tibia fractures?

INTRODUCTION: Minimal invasive plate osteosynthesis in simple distal meta or diaphyseal tibia fractures can be applied using absolute (lag screw and neutralisation plate; LSN) or relative stability (bridge plate; BP). The primary aim of the study was to compare time to radiological union and time to full weight bearing in the two groups (LSN vs. BP). Reduction was performed either percutaneously or by a minimally open approach (mini open). Secondary aim was to analyse the number of complications between both groups. METHODS: Retrospective single centre review of patients with a simple distal meta or diaphyseal tibia fracture operated with a Locking Compression Plate (LCP) between 2009 and 2015 in a Level one Trauma Centre. Postoperative radiographs were assessed in a standardised manner. Time to radiological fracture union and time to full weight bearing were observed. Callus index and postoperative complications were analysed. RESULTS: Fifty-seven patients with a minimum follow-up of 6 months were analysed. Forty-eight patients had a shaft (AO/OTA Type 42) and nine a distal tibia fracture (AO/OTA Type 43). Forty patients were treated with using the LSN concept and 17 patients with the BP concept. Median time to radiological fracture union was statistically significant shorter (p=0.04) in the LSN group with 19 weeks compared to 27 weeks in the BP-group. Time to full weight bearing was 10 weeks in both groups. A total (including implant removal) of 35 reoperations were performed in the LSN-group and 18 in the BP-group. Wound healing disorders (deep surgical site infections) were seen less the LSN group in 3/40 (7.5%) compared to the BP-group with 3/17 (17.6%). In the LSN group, there was no statistical difference in time to union or weight bearing between a percutaneous or mini open approach. CONCLUSION: Stable osteosynthesis of simple distal meta or diaphyseal tibia fractures leads to faster radiologic fracture healing without an increase in complications or number of revisions compared to bridge plating. If a percutaneous reduction is not feasible for the insertion of a lag screw, a mini-open approach does not lead to a delay in fracture healing.

MolProbity for the masses-of data.

MolProbity is a powerful software program for validating structures of proteins and nucleic acids. Although MolProbity includes scripts for batch analysis of structures, because these scripts analyze structures one at a time, they are not well suited for the validation of a large dataset of structures. We have created a version of MolProbity (MolProbity-HTC) that circumvents these limitations and takes advantage of a high-throughput computing cluster by using the HTCondor software. MolProbity-HTC enables the longitudinal analysis of large sets of structures, such as those deposited in the PDB or generated through theoretical computation-tasks that would have been extremely time-consuming using previous versions of MolProbity. We have used MolProbity-HTC to validate the entire PDB, and have developed a new visual chart for the BioMagResBank website that enables users to easily ascertain the quality of each model in an NMR ensemble and to compare the quality of those models to the rest of the PDB.

Post-ischaemic silencing of p66Shc reduces ischaemia/reperfusion brain injury and its expression correlates to clinical outcome in stroke.

AIM: Constitutive genetic deletion of the adaptor protein p66(Shc) was shown to protect from ischaemia/reperfusion injury. Here, we aimed at understanding the molecular mechanisms underlying this effect in stroke and studied p66(Shc) gene regulation in human ischaemic stroke. METHODS AND RESULTS: Ischaemia/reperfusion brain injury was induced by performing a transient middle cerebral artery occlusion surgery on wild-type mice. After the ischaemic episode and upon reperfusion, small interfering RNA targeting p66(Shc) was injected intravenously. We observed that post-ischaemic p66(Shc) knockdown preserved blood-brain barrier integrity that resulted in improved stroke outcome, as identified by smaller lesion volumes, decreased neurological deficits, and increased survival. Experiments on primary human brain microvascular endothelial cells demonstrated that silencing of the adaptor protein p66(Shc) preserves claudin-5 protein levels during hypoxia/reoxygenation by reducing nicotinamide adenine dinucleotide phosphate oxidase activity and reactive oxygen species production. Further, we found that in peripheral blood monocytes of acute ischaemic stroke patients p66(Shc) gene expression is transiently increased and that this increase correlates with short-term neurological outcome. CONCLUSION: Post-ischaemic silencing of p66(Shc) upon reperfusion improves stroke outcome in mice while the expression of p66(Shc) gene correlates with short-term outcome in patients with ischaemic stroke.

Prospective study on complications following a lower body lift after massive weight loss.

BACKGROUND: Lower body lift procedures are in high demand following the increase of massive weight loss patients. As surgical complication rates in this patient group are generally high, patients need to be prepared for risk factors and complications in lower body lift surgery. The aim of this study was to identify the complications and possible risk factors of a lower body lift as concrete data for this procedure are limited. METHODS: A prospective study on 50 consecutive patients who underwent a lower body lift procedure was performed. Measures included co-morbidities and complications. Risk factors assessed included patient age, gender, highest lifetime body mass index (BMI) (BMI max), current BMI, excess weight loss (EWL), type of weight loss and nicotine consumption. RESULTS: There were 50 patients (44 females, six males) with a mean age of 41±10.8 years and a mean EWL of 86.4±15.6%. Mean BMI max was 49.5±10.5 kg m(-2), current BMI was 27.8±4.0 kg m(-2). A total of 35 (70%) patients developed at least one complication. Five patients (10%) suffered a major complication that necessitated surgical revision. Wound dehiscence occurred in 30 patients (60%), followed by seroma in 17 patients (34%). A surgical complication was directly related to BMI max (p=0.02) and age of the patient at the time of surgery (p=0.03). CONCLUSIONS: The overall complication rate following a lower body lift was 70%, which is comparable with that known for high-risk patient groups. The most important risk factors are BMI max and age of the patient (Clinical trial registration number (ISRCTN): NCT01551862).

Non-canonical HIF-2α function drives autonomous breast cancer cell growth via an AREG-EGFR/ErbB4 autocrine loop.

Tumor progression is intrinsically tied to the clonal selection of tumor cells with acquired phenotypes allowing to cope with a hostile microenvironment. Hypoxia-inducible factors (HIFs) master the transcriptional response to local tissue hypoxia, a hallmark of solid tumors. Here, we report significantly longer patient survival in breast cancer with high levels of HIF-2α. Amphiregulin (AREG) and WNT1-inducible signaling pathway protein-2 (WISP2) expression was strongly HIF-2α-dependent and their promoters were particularly responsive to HIF-2α. The endogenous AREG promoter recruited HIF-2α in the absence of a classical HIF-DNA interaction motif, revealing a novel mechanism of gene regulation. Loss of AREG expression in HIF-2α-depleted cells was accompanied by reduced activation of epidermal growth factor (EGF) receptor family members. Apparently opposing results from patient and in vitro data point to an HIF-2α-dependent auto-stimulatory tumor phenotype that, while promoting EGF signaling in cellular models, increased the survival of diagnosed and treated human patients. Our findings suggest a model where HIF-2α-mediated autocrine growth signaling in breast cancer sustains a state of cellular self-sufficiency, thereby masking unfavorable microenvironmental growth conditions, limiting adverse selection and improving therapy efficacy. Importantly, HIF-2α/AREG/WISP2-expressing tumors were associated with luminal tumor differentiation, indicative of a better response to classical treatments. Shifting the HIF-1/2α balance toward an HIF-2-dominated phenotype could thus offer a novel approach in breast cancer therapy.

Prolyl-4-hydroxylase PHD2- and hypoxia-inducible factor 2-dependent regulation of amphiregulin contributes to breast tumorigenesis.

Hypoxia-elicited adaptations of tumor cells are essential for tumor growth and cancer progression. Although ample evidence exists for a positive correlation between hypoxia-inducible factors (HIFs) and tumor formation, metastasis and bad prognosis, the function of the HIF-α protein stability regulating prolyl-4-hydroxylase domain enzyme PHD2 in carcinogenesis is less well understood. In this study, we demonstrate that downregulation of PHD2 leads to increased tumor growth in a hormone-dependent mammary carcinoma mouse model. Tissue microarray analysis of PHD2 protein expression in 281 clinical samples of human breast cancer showed significantly shorter survival times of patients with low-level PHD2 tumors over a period of 10 years. An angiogenesis-related antibody array identified, amongst others, amphiregulin to be increased in the absence of PHD2 and normalized after PHD2 reconstitution. Cultivation of endothelial cells in conditioned media derived from PHD2-downregulated cells resulted in enhanced tube formation that was blocked by the addition of neutralizing anti-amphiregulin antibodies. Functionally, amphiregulin was regulated on the transcriptional level specifically by HIF-2 but not HIF-1. Our data suggest that PHD2/HIF-2/amphiregulin signaling has a critical role in the regulation of breast tumor progression and propose PHD2 as a potential tumor suppressor in breast cancer.

Onconeuronal cerebellar degeneration-related antigen, Cdr2, is strongly expressed in papillary renal cell carcinoma and leads to attenuated hypoxic response.

The onconeuronal cerebellar degeneration-related antigen Cdr2 is associated with paraneoplastic syndromes. Neoplastic expression of Cdr2 in ovary and breast tumors triggers an autoimmune response that suppresses tumor growth by developing tumor immunity, but culminates in cerebellar degeneration when Cdr2-specific immune cells recognize neuronal Cdr2. We identified Cdr2 as a novel interactor of the hypoxia-inducible factor (HIF) prolyl-4-hydroxylase PHD1 and provide evidence that Cdr2 might represent a novel important tumor antigen in renal cancer. Strong Cdr2 protein expression was observed in 54.2% of papillary renal cell carcinoma (pRCC) compared with 7.8% of clear-cell RCC and no staining was observed in chromophobe RCC or oncocytoma. High Cdr2 protein levels correlated with attenuated HIF target gene expression in these solid tumors, and Cdr2 overexpression in tumor cell lines reduced HIF-dependent transcriptional regulation. This effect was because of both attenuation of hypoxic protein accumulation and suppression of the transactivation activity of HIF-1alpha. pRCC is known for its tendency to avascularity, usually associated with a lower pathological stage and higher survival rates. We provide evidence that Cdr2 protein strongly accumulates in pRCC, attenuates the HIF response to tumor hypoxia and may become of diagnostic importance as novel renal tumor marker.

The PAS-domain kinase PASKIN: a new sensor in energy homeostasis.

The PAS domain kinase PASKIN, also termed PAS kinase or PASK, is an evolutionarily conserved potential sensor kinase related to the heme-based oxygen sensors of nitrogen-fixing bacteria. In yeast, the two PASKIN homologs link energy flux and protein synthesis following specific stress conditions. In mammals, PASKIN may regulate glycogen synthesis and protein translation. Paskin knock-out mice do not show any phenotype under standard animal husbandry conditions. Interestingly, these mice seem to be protected from the symptoms of the metabolic syndrome when fed a high-fat diet. Energy turnover might be increased in specific PASKIN-deficient cell types under distinct environmental conditions. According to the current model, binding of a putative ligand to the PAS domain disinhibits the kinase domain and activates PASKIN auto- and target phosphorylation. Future research needs to be conducted to elucidate the nature of the putative ligand and the molecular mechanisms of downstream signalling by PASKIN.

Anti-Trypanosoma cruzi effects of cyclosporin A derivatives: possible role of a P-glycoprotein and parasite cyclophilins.

Cyclophilins are target molecules for cyclosporin A (CsA), an immunosuppressive antimicrobial drug. We have previously reported the in vitro anti-Trypanosoma cruzi activity of H-7-94 and F-7-62 non-immunosuppressive CsA analogues. In this work, we continue the study of the parasiticidal effect of H-7-94 and F-7-62 CsA analogues in vitro and in vivo and we analyse 3 new CsA derivatives: MeIle-4-CsA (NIM 811), MeVal-4-CsA (MeVal-4) and D-MeAla-3-EtVal-4-CsA, (EtVal-4). The most efficient anti-T. cruzi effect was observed with H-7-94, F-7-62 and MeVal-4 CsA analogues evidenced as inhibition of epimastigote proliferation, trypomastigote penetration, intracellular amastigote development and in vivo T. cruzi infection. This trypanocidal activity could be due to inhibition of the peptidyl prolyl cis-trans isomerase activity on the T. cruzi recombinant cyclophilins tested. Furthermore, CsA and F-7-62 derivative inhibited the efflux of rhodamine 123 from T. cruzi epimastigotes, suggesting an interference with a P-glycoprotein activity. Moreover, H-7-94 and F-7-62 CsA analogues were not toxic as shown by cell viability and by aminopyrine-N-demethylase activity on mammalian cells. Our results show that H-7-94, F-7-62 and MeVal-4 CsA analogues expressed the highest inhibiting effects on T. cruzi, being promissory parasiticidal drugs worthy of further studies.

BioMagResBank.

The BioMagResBank (BMRB: www.bmrb.wisc.edu) is a repository for experimental and derived data gathered from nuclear magnetic resonance (NMR) spectroscopic studies of biological molecules. BMRB is a partner in the Worldwide Protein Data Bank (wwPDB). The BMRB archive consists of four main data depositories: (i) quantitative NMR spectral parameters for proteins, peptides, nucleic acids, carbohydrates and ligands or cofactors (assigned chemical shifts, coupling constants and peak lists) and derived data (relaxation parameters, residual dipolar couplings, hydrogen exchange rates, pK(a) values, etc.), (ii) databases for NMR restraints processed from original author depositions available from the Protein Data Bank, (iii) time-domain (raw) spectral data from NMR experiments used to assign spectral resonances and determine the structures of biological macromolecules and (iv) a database of one- and two-dimensional (1)H and (13)C one- and two-dimensional NMR spectra for over 250 metabolites. The BMRB website provides free access to all of these data. BMRB has tools for querying the archive and retrieving information and an ftp site (ftp.bmrb.wisc.edu) where data in the archive can be downloaded in bulk. Two BMRB mirror sites exist: one at the PDBj, Protein Research Institute, Osaka University, Osaka, Japan (bmrb.protein.osaka-u.ac.jp) and the other at CERM, University of Florence, Florence, Italy (bmrb.postgenomicnmr.net/). The site at Osaka also accepts and processes data depositions.

Induction of activating transcription factor 3 by anoxia is independent of p53 and the hypoxic HIF signalling pathway.

Solid tumors often have an inadequate blood supply, which results in large regions that are subjected to hypoxic or anoxic stress. Hypoxia-inducible factor-1 (HIF-1) is a transcription factor that regulates much of the transcriptional response of cells to hypoxia. Activating transcription factor 3 (ATF3) is another transcription factor that responds to a variety of stresses and is often upregulated in cancer. We investigated the regulation of ATF3 by oxygen deprivation. ATF3 induction occurred most robustly under anoxia, is common, and it is not dependent on presence of HIF-1 or p53, but is sensitive to the inhibition of c-Jun NH2-terminal kinase activation and the antioxidant N-acetylcystein. ATF3 could also be induced by desferrioxamine but not by the mitochondrial poison cyanide or the nonspecific 2-oxoglutarate dioxygenase inhibitor dimethyloxalylglycine. We also show that anoxic ATF3 mRNA is more stable than normoxic mRNA providing a mechanism for this induction. Thus, this study demonstrates that the regulation of ATF3 under anoxia is independent of 2-oxoglutarate dioxygenase, HIF-1 and p53, presumably involving multiple regulatory pathways.

DNA copy number gains in head and neck squamous cell carcinoma.

Gene amplification, a common mechanism for oncogene activation in cancer, has been used as a tag for the identification of novel oncogenes. DNA amplification is frequently observed in head and neck squamous cell carcinoma (HNSCC) and potential oncogenes have already been reported. We applied restriction landmark genome scanning (RLGS) to study gene amplifications and low-level copy number changes in HNSCC in order to locate previously uncharacterized regions with copy number gains in primary tumor samples. A total of 63 enhanced RLGS fragments, indicative of DNA copy number changes, including gains of single alleles, were scored. Enhanced sequences were identified from 33 different chromosomal regions including those previously reported (e.g. 3q26.3 and 11q13.3) as well as novel regions (e.g. 3q29, 8q13.1, 8q22.3, 9q32, 10q24.32, 14q32.32, 17q25.1 and 20q13.33). Furthermore, our data suggest that amplicons 11q13.3 and 3q26.3-q29 may be divided into possibly two and three independent amplicons, respectively, an observation supported by published microarray expression data.

Conversion of cyclosporine A prodrugs in human tears vs rabbits tears.

The aim of this study was to evaluate the rate and mechanism of conversion of two water-soluble prodrugs of cyclosporine A (CsA) intended for topical delivery to the eye. The new molecules were designed according to the double prodrug concept: a solubilizing moiety was grafted onto CsA via an ester function, which could be hydrolysed via a two-step process (enzymatic and chemical). Prodrug solutions were prepared extemporaneously in an isotonic and neutral aqueous medium compatible with ophthalmic use. The rates of conversion into the parent molecule were determined by incubating the prodrugs in fresh rabbit or human tears or in a phosphate buffer solution (PBS) at pH 7.4. Both prodrugs were converted into CsA within the first minute in the presence of rabbit tears with rate constants of k=5.9x10(-3)min(-1) and k=3.8x10(-3)min(-1), respectively, for UNIL088 and UNIL089, whereas chemical conversion in PBS was negligible (k=0.5x10(-3)min(-1) for both molecules). Incubation of UNIL088 in human tears showed a significantly high conversion rate. It is concluded that the developed double prodrugs underwent a bioconversion in physiological media and thus represent promising candidates for topical delivery of CsA to the eye.

[Oxygen homeostasis in tissues and cells: how hypoxia regulates gene expression]. / Sauerstoffhomöostase in Gewebe und Zelle: Wie Sauerstoffmangel die Genexpression reguliert.

Every cell of the body is capable of measuring the environmental oxygen concentration. When oxygen supply falls below oxygen demand of the cell (hypoxia), a series of genes will be activated whose products help the cell, the tissue as well as the whole organism in adapting to these altered oxygen concentrations. The discovery of hypoxia-inducible transcription factors (HIFs) allowed the molecular understanding of these processes.

Cyclosporin A prodrugs: design, synthesis and biophysical properties.

The cyclic undecapeptide cyclosporin A (CsA) has a remarkable spectrum of diverse biological activities, including anti-inflammatory, antifungal, antiparasitic as well as immunosuppressive activities. However, the low water solubility of this drug is a serious problem causing undesirable pharmacological properties such as erratic oral absorption. In order to overcome this problem, the design and synthesis of water-soluble prodrugs of CsA are described. Using the OH-MeBmt-1-group as attachment site, we investigate dipeptide systems exhibiting differential tendencies for intramolecular cyclization [diketopiperazine (DKP) formation] for tailoring the chemoreversible release of the parent CsA. In modulating the chemical and structural features of the dipeptide esters (N-alkylation, side chains, C-terminal Pro), we find conversion rates at physiological conditions ranging from minutes to several days. Together with their thermodynamic stability in the solid state and strongly enhanced solubility in water, these chemoreversible CsA prodrugs represent versatile candidates for therapeutical use.

Oxygen-regulated expression of TGF-beta 3, a growth factor involved in trophoblast differentiation.

The transforming growth factor-beta 3 (TGF-beta 3) is involved in oxygen-dependent differentiation processes during placental development and pregnancy disorders. However, the importance of oxygen partial pressure for the regulation of TGF-beta 3 expression is presently unclear. We and others presented preliminary evidence that the hypoxia-inducible factor-1 (HIF-1) confers TGF-beta 3 transcription but it was unknown whether this occurred directly or indirectly. To analyze how HIF-1 regulates TGF-beta 3 gene transcription, we cloned and sequenced the mouse TGF-beta 3 promoter region. Multiple putative HIF-1 binding sites (HBSs) were identified, many of which co-localized with two G+C rich CpG islands 5' to the TGF-beta 3 transcription start site. A 6.8 kb fragment of the TGF-beta 3 promoter induced reporter gene expression under hypoxic conditions or when treated with an iron chelator known to stabilize and activate the HIF-1 alpha subunit. Deletion of a 2.4 kb fragment upstream of the distal CpG island abolished inducibility of reporter gene expression. Two HBSs (HBS1 and HBS6) that bound the HIF-1 protein could be identified within this 2.4 kb fragment. These results suggest that TGF-beta 3 gene expression is directly regulated by HIF-1.

Hypoxia affects expression of circadian genes PER1 and CLOCK in mouse brain.

The key elements of circadian clockwork and oxygen homeostasis are the PAS protein family members PER and CLOCK and hypoxia-inducible factor 1alpha (HIF-1alpha). The PAS domain serves as an interface for protein-protein interactions. We asked whether a cross-talk exists between the PAS components of hypoxic and circadian pathways. We found several isoforms of PER1 protein that exhibit tissue-specific size differences. In the mouse brain, a predominantly nuclear 48 kDa isoform that followed a daily rhythm was observed. The 48 kDa form was found in the nuclear fractions derived from mouse liver, Swiss3T3 fibroblasts, and N2A neuroblastoma cells. In mouse kidney and human 293 kidney cells, a 55 kDa PER1 form was detected. CLOCK was observed as a predicted 100 kDa protein in rat-1 cells and in all analyzed mouse tissues including brain, liver, kidney, and spleen. In contrast to PER1, CLOCK protein expression was not rhythmic. Exposure to hypoxia led to increased PER1 and CLOCK protein levels in mice. Based on coimmunoprecipitation experiments that showed protein-protein interaction between PER1 and the alpha subunit of HIF-1, we suggest that these hypoxic effects may be modulated by HIF-1alpha.-Chilov, D., Hofer, T., Bauer, C., Wenger, R. H., Gassmann, M. Hypoxia affects expression of circadian genes PER1 and CLOCK in mouse brain.