The Arabidopsis thaliana F-Box Protein FBL17 Is Essential for Progression through the Second Mitosis during Pollen Development

In fungi and metazoans, the SCF-type Ubiquitin protein ligases (E3s) play a critical role in cell cycle regulation by degrading negative regulators, such as cell cycle-dependent kinase inhibitors (CKIs) at the G1-to-S-phase checkpoint. Here we report that FBL17, an Arabidopsis thaliana F-box protein, is involved in cell cycle regulation during male gametogenesis. FBL17 expression is strongly enhanced in plants co-expressing E2Fa and DPa, transcription factors that promote S-phase entry. FBL17 loss-of-function mutants fail to undergo pollen mitosis II, which generates the two sperm cells in mature A. thaliana pollen. Nonetheless, the single sperm cell-like cell in fbl17 mutants is functional but will exclusively fertilize the egg cell of the female gametophyte, giving rise to an embryo that will later abort, most likely due to the lack of functional endosperm. Seed abortion can, however, be overcome by mutations in FIE, a component of the Polycomb group complex, overall resembling loss-of-function mutations in the A. thaliana cyclin-dependent kinase CDKA;1. Finally we identified ASK11, as an SKP1-like partner protein of FBL17 and discuss a possible mechanism how SCFFBL17 may regulate cell division during male gametogenesis

Pyrrolidine dithiocarbamate administered during ex-vivo lung perfusion promotes rehabilitation of injured donor rat lungs obtained after prolonged warm ischemia.

Damaged lung grafts obtained after circulatory death (DCD lungs) and warm ischemia may be at high risk of reperfusion injury after transplantation. Such lungs could be pharmacologically reconditioned using ex-vivo lung perfusion (EVLP). Since acute inflammation related to the activation of nuclear factor kappaB (NF-κB) is instrumental in lung reperfusion injury, we hypothesized that DCD lungs might be treated during EVLP by pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF-κB. Rat lungs exposed to 1h warm ischemia and 2 h cold ischemia were subjected to EVLP during 4h, in absence (CTRL group, N = 6) or in presence of PDTC (2.5g/L, PDTC group, N = 6). Static pulmonary compliance (SPC), peak airway pressure (PAWP), pulmonary vascular resistance (PVR), and oxygenation capacity were determined during EVLP. After EVLP, we measured the weight gain of the heart-lung block (edema), and the concentration of LDH (cell damage), proteins (permeability edema) and of the cytokines IL-6, TNF-α and CINC-1 in bronchoalveolar lavage (BAL), and we evaluated NF-κB activation by the degree of phosphorylation and degradation of its inhibitor IκBα in lung tissue. In CTRL, we found significant NF-κB activation, lung edema, and a massive release of LDH, proteins and cytokines. SPC significantly decreased, PAWP and PVR increased, while oxygenation tended to decrease. Treatment with PDTC during EVLP inhibited NF-κB activation, did not influence LDH release, but markedly reduced lung edema and protein concentration in BAL, suppressed TNFα and IL-6 release, and abrogated the changes in SPC, PAWP and PVR, with unchanged oxygenation. In conclusion, suppression of innate immune activation during EVLP using the NF-κB inhibitor PDTC promotes significant improvement of damaged rat DCD lungs. Future studies will determine if such rehabilitated lungs are suitable for in vivo transplantation

Chemical genetics approach to restoring p27Kip1 reveals novel compounds with antiproliferative activity in prostate cancer cells

<p>Abstract</p> <p>Background</p> <p>The cyclin-dependent kinase (CDK) inhibitor p27^Kip1is downregulated in a majority of human cancers due to ectopic proteolysis by the ubiquitin-proteasome pathway. The expression of p27 is subject to multiple mechanisms of control involving several transcription factors, kinase pathways and at least three different ubiquitin ligases (SCF^SKP2, KPC, Pirh2), which regulate p27 transcription, translation, protein stability and subcellular localization. Using a chemical genetics approach, we have asked whether this control network can be modulated by small molecules such that p27 protein expression is restored in cancer cells.</p> <p>Results</p> <p>We developed a cell-based assay for measuring the levels of endogenous nuclear p27 in a high throughput screening format employing LNCaP prostate cancer cells engineered to overexpress SKP2. The assay platform was optimized to Z' factors of 0.48 - 0.6 and piloted by screening a total of 7368 chemical compounds. During the course of this work, we discovered two small molecules of previously unknown biological activity, SMIP001 and SMIP004, which increase the nuclear level of p27 at low micromolar concentrations. SMIPs (small molecule inhibitors of p27 depletion) also upregulate p21^Cip1, inhibit cellular CDK2 activity, induce G1 delay, inhibit colony formation in soft agar and exhibit preferential cytotoxicity in LNCaP cells relative to normal human fibroblasts. Unlike SMIP001, SMIP004 was found to downregulate SKP2 and to stabilize p27, although neither SMIP is a proteasome inhibitor. Whereas the screening endpoint - nuclear p27 - was robustly modulated by the compounds, SMIP-mediated cell cycle arrest and apoptosis were not strictly dependent on p27 and p21 - a finding that is explained by parallel inhibitory effects of SMIPs on positive cell cycle regulators, including cyclins E and A, and CDK4.</p> <p>Conclusions</p> <p>Our data provide proof-of-principle that the screening platform we developed, using endogenous nuclear p27 as an endpoint, presents an effective means of identifying bioactive molecules with cancer selective antiproliferative activity. This approach, when applied to larger and more diverse sets of compounds with refined drug-like properties, bears the potential of revealing both unknown cellular pathways globally impinging on p27 and novel leads for chemotherapeutics targeting a prominent molecular defect of human cancers.</p

A Deep Insight into the Sialotranscriptome of the Gulf Coast Tick, Amblyomma maculatum

Background: Saliva of blood sucking arthropods contains compounds that antagonize their hosts ’ hemostasis, which include platelet aggregation, vasoconstriction and blood clotting; saliva of these organisms also has anti-inflammatory and immunomodullatory properties. Perhaps because hosts mount an active immune response against these compounds, the diversity of these compounds is large even among related blood sucking species. Because of these properties, saliva helps blood feeding as well as help the establishment of pathogens that can be transmitted during blood feeding. Methodology/Principal Findings: We have obtained 1,626,969 reads by pyrosequencing a salivary gland cDNA library from adult females Amblyomma maculatum ticks at different times of feeding. Assembly of this data produced 72,441 sequences larger than 149 nucleotides from which 15,914 coding sequences were extracted. Of these, 5,353 had.75 % coverage to their best match in the non-redundant database from the National Center for Biotechnology information, allowing for the deposition of 4,850 sequences to GenBank. The annotated data sets are available as hyperlinked spreadsheets. Putative secreted proteins were classified in 133 families, most of which have no known function. Conclusions/Significance: This data set of proteins constitutes a mining platform for novel pharmacologically activ

Stage- and Gender-Specific Proteomic Analysis of Brugia malayi Excretory-Secretory Products

To succeed in infection, parasites must have ways to reach the host, penetrate its tissues and escape its defense systems. As they are not necessarily fatal, most helminth parasites remain viable within their host for many years, exerting a strong influence over the host immune function. Many of these functions are performed by products that are released from the parasite. We exploited the remarkable sensitivity of modern proteomics tools together with the availability of a sequenced genome to identify and compare the proteins released in vitro by adult males, adult females and the microfilariae of the filarial nematode Brugia malayi. This parasite is one of the etiological agents of lymphatic filariasis, a disease that poses continuing and significant threats to human health. The different forms of the parasite inhabit different compartments in the mammalian host. We found that the set of proteins released by each form is unique; they must reflect particular developmental processes and different strategies for evasion of host responses. The identification of these proteins will allow us to illuminate the biology of secretory processes in this organism and to establish a path for developing an understanding of how these parasite proteins function in immune evasion events

A Natural Combination Extract of Viscum album L. Containing Both Triterpene Acids and Lectins Is Highly Effective against AML In Vivo

Aqueous Viscum album L. extracts are widely used in complementary cancer medicine. Hydrophobic triterpene acids also possess anti-cancer properties, but due to their low solubility they do not occur in significant amounts in aqueous extracts. Using cyclodextrins we solubilised mistletoe triterpenes (mainly oleanolic acid) and investigated the effect of a mistletoe whole plant extract on human acute myeloid leukaemia cells in vitro, ex vivo and in vivo. Single Viscum album L. extracts containing only solubilised triterpene acids (TT) or lectins (viscum) inhibited cell proliferation and induced apoptosis in a dose-dependent manner in vitro and ex vivo. The combination of viscum and TT extracts (viscumTT) enhanced the induction of apoptosis synergistically. The experiments demonstrated that all three extracts are able to induce apoptosis via caspase-8 and -9 dependent pathways with down-regulation of members of the inhibitor of apoptosis and Bcl-2 families of proteins. Finally, the acute myeloid leukaemia mouse model experiment confirmed the therapeutic effectiveness of viscumTT-treatment resulting in significant tumour weight reduction, comparable to the effect in cytarabine-treated mice. These results suggest that the combination viscumTT may have a potential therapeutic value for the treatment AML

Demographic, clinical and antibody characteristics of patients with digital ulcers in systemic sclerosis: data from the DUO Registry

OBJECTIVES: The Digital Ulcers Outcome (DUO) Registry was designed to describe the clinical and antibody characteristics, disease course and outcomes of patients with digital ulcers associated with systemic sclerosis (SSc). METHODS: The DUO Registry is a European, prospective, multicentre, observational, registry of SSc patients with ongoing digital ulcer disease, irrespective of treatment regimen. Data collected included demographics, SSc duration, SSc subset, internal organ manifestations, autoantibodies, previous and ongoing interventions and complications related to digital ulcers. RESULTS: Up to 19 November 2010 a total of 2439 patients had enrolled into the registry. Most were classified as either limited cutaneous SSc (lcSSc; 52.2%) or diffuse cutaneous SSc (dcSSc; 36.9%). Digital ulcers developed earlier in patients with dcSSc compared with lcSSc. Almost all patients (95.7%) tested positive for antinuclear antibodies, 45.2% for anti-scleroderma-70 and 43.6% for anticentromere antibodies (ACA). The first digital ulcer in the anti-scleroderma-70-positive patient cohort occurred approximately 5 years earlier than the ACA-positive patient group. CONCLUSIONS: This study provides data from a large cohort of SSc patients with a history of digital ulcers. The early occurrence and high frequency of digital ulcer complications are especially seen in patients with dcSSc and/or anti-scleroderma-70 antibodies

The effects of methionine acquisition and synthesis on Streptococcus pneumoniae growth and virulence

Extent: 14 p.Bacterial pathogens need to acquire nutrients from the host, but for many nutrients their importance during infection remain poorly understood. We have investigated the importance of methionine acquisition and synthesis for Streptococcus pneumoniae growth and virulence using strains with gene deletions affecting a putative methionine ABC transporter lipoprotein (Sp_0149, metQ) and/or methionine biosynthesis enzymes (Sp_0585 - Sp_0586, metE and metF). Immunoblot analysis confirmed MetQ was a lipoprotein and present in all S. pneumoniae strains investigated. However, vaccination with MetQ did not prevent fatal S. pneumoniae infection in mice despite stimulating a strong specific IgG response. Tryptophan fluorescence spectroscopy and isothermal titration calorimetry demonstrated that MetQ has both a high affinity and specificity for L-methionine with a KD of ~ 25 nM, and a DmetQ strain had reduced uptake of C14-methionine. Growth of the ΔmetQ/ΔmetEF strain was greatly impaired in chemically defined medium containing low concentrations of methionine and in blood but was partially restored by addition of high concentrations of exogenous methionine. Mixed infection models showed no attenuation of the ΔmetQ, ΔmetEF and ΔmetQ/DmetEF strains in their ability to colonise the mouse nasopharnyx. In a mouse model of systemic infection although significant infection was established in all mice, there were reduced spleen bacterial CFU after infection with the ΔmetQ/ΔmetEF strain compared to the wild-type strain. These data demonstrate that Sp_0149 encodes a high affinity methionine ABC transporter lipoprotein and that Sp_0585 – Sp_0586 are likely to be required for methionine synthesis. Although Sp_0149 and Sp_0585-Sp_0586 make a contribution towards full virulence, neither was essential for S. pneumoniae survival during infection.Shilpa Basavanna, Suneeta Chimalapati, Abbas Maqbool, Bruna Rubbo, Jose Yuste, Robert J. Wilson, Arthur Hosie, Abiodun D. Ogunniyi, James C. Paton, Gavin Thomas and Jeremy S. Brow

Practical value of anti-xa activity in the evaluation of extracorporeal circuit anticoagulation during haemodialysis:Results of a cross-sectional single-centre study

\u3cp\u3eBackground/Aims: Anticoagulation of the extracorporeal circuit is essential for adequate haemodialysis (HD). Low molecular weight heparins (LMWHs) are safe and sufficient towards achieving this goal. In the Netherlands, dosage is based on bodyweight and adjusted based on clinical events. LMWH levels during dialysis can be quantified through measurement of the anti-Xa activity and a target range of 0.5-1.0 IU/mL has been proposed. We aimed to evaluate the practical value of the anti-Xa activity to guide LMWH dosage in HD patients. Additionally, the value of the activated partial thromboplastin time (APTT) was investigated. Methods: All prevalent adult HD patients of our dialysis clinic were included. APTT and anti-Xa activity were measured before, during and after 2 dialysis sessions. Clinical and dialysis characteristics, including LMWH dosage, were derived from digital patient charts. Results: Our final study cohort consisted of 83 patients. LMWH dosage during dialysis was appropriate for bodyweight in 61% of cases, of which 50% reached an anti-Xa activity within the putative target range of 0.5-1.0 IU/mL. Forty-six percent of patients had an anti-Xa activity &gt;1.0 IU/mL. Anti-Xa levels during and after dialysis were significantly correlated (r = 0.803, p &lt; 0.01). No thrombotic or haemorrhagic complications were observed in this study. Correlation of APTT with anti-Xa activity was poor. Conclusion: Anti-Xa activity measurements during dialysis can identify patients in whom LMWH dosage should be lowered in a subsequent dialysis session. Whether such an intervention leads to a decrease in haemorrhagic complications needs to be evaluated in prospective studies.\u3c/p\u3