Yeast Ysl2p,homologous to Sec7 domain guanine nucleotide exchange factors,functions in endocytosis and maintenance of vacuole integrity and interacts with the Arf-Like small GTPase Arl1p

We previously described the isolation of ysl2-1 due to its genetic interaction with Delta ypt51/vps21, a mutant with a deletion of the coding sequence for the yeast Rab5 homolog, which regulates endocytic traffic between early and late endosomes. Here we report that Ysl2p is a novel 186.8-kDa peripheral membrane protein homologous to members of the Sec7 family. We provide multiple genetic and biochemical evidence for an interaction between Ysl12p and the Arf-like protein Arl1p, consistent with a potential function as an Arf guanine nucleotide exchange factor (GEF). The temperature-sensitive alleles ysl2-307 and ysl2-316 are specifically defective in ligand-induced degradation of Ste2p and alpha-factor and exhibit vacuole fragmentation directly upon a shift to 37 degrees C. In living cells, green fluorescent protein (GFP)-Ysl2p colocalizes with endocytic elements that accumulate FM4-64. The GFP-Ysl2p staining is sensitive to a mutation in VPS27 resulting in the formation of an aberrant class E compartment, but it is not affected by a sec7 mutation. Consistent with the idea that Ysl2p and Arl1p have closely related functions, Delta arl1 cells are defective in endocytic transport and in vacuolar protein sorting.     

Characterization of a Kunitz trypsin inhibitor with one disulfide bridge purified from Swartzia pickellii

Swartzia pickellii is a Leguminosae that belongs to the Caesalpinioideae sub-family the Swartzia pickellii Trypsin Inhibitor (SWTI), a serine proteinase inhibitor was isolated from its seeds. SWTI is a single polypeptide chain protein and it's structure has 174 amino acid residues, it homologous to other Kunitz plant inhibitors, however shows some major differences: it contains only one disulfide bridge, instead two which are usually found in plant Kunitz inhibitors, and the SWTI reactive site does not contain the usual Arg or Lys residues at the putative reactive site (position 65). A glycosylation site was detected at Asn38 with 1188 kDa carbohydrate portion. The primary structure micro heterogeneity was found combining the sequence determination and mass spectrometry. Three forms of SWTI were actually defined: two glycosylated forms a 20,204 kDa (Arg 165) and 20,185 kDa (His 165) and one deglycosylated form 19,016 kDa (Arg 165), all of them contain a Met residue at position 130.     

Elevated temperatures increase leaf senescence and root secondary metabolite concentrations in the understory herb Panax quinquefolius (Araliaceae)

The response of understory species to elevated temperatures is not well understood but is important because these plants are highly sensitive to their growth conditions. Three-year-old plants of Panax quinquefolius, an understory herb endemic to the eastern deciduous forests of North America, were grown in a greenhouse at 25/20°C (day/night) or 30/25°C for one growing season and analyzed each month. Plants grown at high temperatures had an early onset of leaf senescence and therefore accumulated less carbon. From May to July, P. quinquefolius grown at high temperatures had decreased photosynthesis (52%), stomatal conductance (60%), and root and total biomass (33% and 28%, respectively) compared to plants grown at low temperatures. As P. quinquefolius prepared to overwinter, plants grown at high temperatures had less root biomass (53%) than plants in low temperatures. The amount of storage-root ginsenosides was unaffected by temperature, and differences in storage root size may explain why plants grown at high temperatures had greater concentrations of storage root ginsenosides (49%) than plants grown at low temperatures. Panax quinquefolius is clearly sensitive to a 5°C increase in temperature, and therefore other understory species may be negatively impacted by future increases in global temperature.     

Fusarium head blight biological control with Lysobacter enzymogenes strain C3

Fusarium head blight (FHB), caused by Fusarium graminearum (= Gibberella zeae), is a destructive disease of wheat for which biological controls are needed. Lysobacter enzymogenes strain C3, a bacterial antagonist of fungal pathogens via lytic enzymes and induced resistance, was evaluated in this study for control of FHB. In greenhouse experiments, chitin broth cultures of C3 reduced FHB severity to <10% infected spikelets as compared to >80% severity in the controls in some experiments. C3 broth cultures heated to inactivate cells and lytic enzymes, but retaining the elicitor factor for induced resistance, also were effective in reducing FHB severity, suggesting induced resistance is one mechanism of action. C3 broth cultures also were effective when applied in highly diluted form and when applied 1 week prior to pathogen inoculation. When applied to 8 cultivars of hard red spring wheat in the greenhouse, C3 treatments reduced FHB in 5 cultivars but not in the others. These findings also are consistent with induced resistance. Protection offered by C3 treatments, however, was not systemic and required that C3 be applied uniformly to all susceptible florets. Field tests were conducted in South Dakota and Nebraska to evaluate the efficacy of C3 chitin broth cultures in spring and winter wheat, respectively. In experiments involving two hard red spring wheat cultivars, treatment with C3 reduced FHB severity in ‘Russ’ but not in ‘Ingot’. In three other field experiments comparing C3, the fungicide tebuconazole, and the combination of C3 and tebuconazole, treatments with the bacterial culture alone and the fungicide alone were inconsistent across experiments, each treatment being ineffective in controlling FHB in one experiment. The biocontrol agent–fungicide combination was more consistently effective, reducing FHB incidence or severity in all three experiments. Thus, the potential for using L. enzymogenes C3 as a biological control agent for FHB was demonstrated along with a number of factors that might affect control efficacy in the field.     

Prognostic value of MIB-1 proliferation index in solitary fibrous tumors of the pleura implemented in a new score – a multicenter study

Background

Although the majority of solitary fibrous tumors of the pleura (SFTP) follow a benign course, 10–25% of patients suffer from recurrence or metastatic disease. Several scoring models have been proposed to predict the outcome. However, none of these included immunohistochemical (IHC) markers as possible prognosticators.

Methods

In this multicenter study, we collected clinical data and formalin-fixed and paraffin-embedded (FFPE) tissue blocks of patients with histologically proven SFTP which had been surgically resected between 2000 und 2015. After systematic and extensive IHC staining on tissue microarrays, the results were analyzed and compared to histomorphological and clinical data for their possible prognostic value.

Results

In total, 78 patients (mean age 61?±?11 years) were included. Of these, 9 patients (11%) had an adverse outcome including SFTP recurrence (n?=?6) or SFTP-related death (n?=?3). Mean overall survival was 172?±?13 months. 1 and 10-year event-free survival rates were 99% and 93%. In the multivariable analysis only MIB-1 proliferation index (Ki-67) ≥10% (HR 12.3, CI 1.1–139.5, p?=?0.043), ≥4 mitoses per 10 high power fields (HR 36.5, CI 1.2–1103.7, p?=?0.039) and tumor size larger than 10 cm (HR 81.8, CI 1.7–4016.8, p?=?0.027) were independently associated with adverse outcome.

Conclusion

A high proliferation rate by MIB-1 IHC was associated with impaired outcome. Upon this, we established a new score using mitosis, necrosis, size of the tumor and MIB-1, which performed better than the traditional scores in our data set. This prognostic score could help to better evaluate outcome of SFTP, but requires external validation.

     

Cellular senescence induced by cathepsin X downregulation

Cellular senescence represents a powerful tumor suppressor mechanism to prevent proliferation and invasion of malignant cells. Since tumor cells as well as primary fibroblasts lacking the lysosomal cysteine-type carboxypeptidase cathepsin X exhibit a reduced invasive capacity, we hypothesized that the underlying reason may be the induction of cellular senescence. To investigate the cellular and molecular mechanisms leading to diminished migration/invasion of cathepsin X-deficient cells, we have analyzed murine embryonic fibroblasts (MEF) derived from cathepsin X-deficient mice and neonatal human dermal fibroblasts (NHDF) transfected with siRNAs targeting cathepsin X. Remarkably, both cell types exhibited a flattened and enlarged cell body, a characteristic phenotype of senescent cells. Additional evidence for accelerated senescence was obtained by detection of the common senescence marker β-galactosidase. Further examination revealed increased expression levels of senescence-associated genes such as p16, p21, p53, and caveolin in these cells along with a reduced proliferation rate. The accelerated cellular senescence induced by cathepsin X deficiency was rescued by simultaneous expression of exogenous cathepsin X. Finally, cell cycle analysis confirmed a marked reduction of the synthesis rate and prolongation of the S-phase, while susceptibility to apoptosis of cathepsin X-deficient cells remained unchanged. In conclusion, cathepsin X deficiency leads to accelerated cellular senescence and consequently to diminished cellular proliferation and migration/invasion implying a potential role of cathepsin X in bypassing cellular senescence.     

EBV-gp350 confers B-cell tropism to tailored exosomes and is a neo-antigen in normal and malignant B cells--a new option for the treatment of B-CLL

gp350, the major envelope protein of Epstein-Barr-Virus, confers B-cell tropism to the virus by interacting with the B lineage marker CD21. Here we utilize gp350 to generate tailored exosomes with an identical tropism. These exosomes can be used for the targeted co-transfer of functional proteins to normal and malignant human B cells. We demonstrate here the co-transfer of functional CD154 protein on tailored gp350+ exosomes to malignant B blasts from patients with B chronic lymphocytic leukemia (B-CLL), rendering B blasts immunogenic to tumor-reactive autologous T cells. Intriguingly, engulfment of gp350+ exosomes by B-CLL cells and presentation of gp350-derived peptides also re-stimulated EBV-specific T cells and redirected the strong antiviral cellular immune response in patients to leukemic B cells. In essence, we show that gp350 alone confers B-cell tropism to exosomes and that these exosomes can be further engineered to simultaneously trigger virus- and tumor-specific immune responses. The simultaneous exploitation of gp350 as a tropism molecule for tailored exosomes and as a neo-antigen in malignant B cells provides a novel attractive strategy for immunotherapy of B-CLL and other B-cell malignancies.