Modelling the Rhizosphere Priming Effect in Combination with Soil Food Webs to Quantify Interaction between Living Plant, Soil Biota and Soil Organic Matter.

A model of rhizosphere priming effect under impact of root exudate input into rhizosphere soil was developed as an important process of the plant-soil interaction. The model was based on the concept of nitrogen (N) mining, compensating for the N scarcity in exudates for microbial growth by accelerating SOM mineralisation. In the model, N deficiency for microbial growth is covered ("mined") by the increased SOM mineralisation depending on the C:N ratio of the soil and exudates. The new aspect in the model is a food web procedure, which calculates soil fauna feeding on microorganisms, the return of faunal by-products to SOM and mineral N production for root uptake. The model verification demonstrated similar magnitude of the priming effect in simulations as in the published experimental data. Model testing revealed high sensitivity of the simulation results to N content in exudates. Simulated CO2 emission from the priming can reach 10-40% of CO2 emission from the whole Ah horizon of boreal forest soil depending on root exudation rates. This modeling approach with including food web activity allows quantifying wider aspects of the priming effect functioning including ecologically important available N production.

Combined forest and soil management after a catastrophic event.

At the end of October 2018, a storm of unprecedented strength severely damaged the forests of the eastern sector of the Italian Alps. The affected forest area covers 42,500 ha. The president of one of the damaged regions asked for help from the University of Padua. After eight months of discussion, the authors of this article wrote a consensus text. The sometimes asper debate brought to light some crucial aspects: 1) even experienced specialists may have various opinions based on scientific knowledge that lead to conflicting proposals for action. For some of them there is evidence that to restore a destroyed natural environment it is more judicious to do nothing; 2) the soil corresponds to a living structure and every ecosystem's management should be based on it; 3) faced with a catastrophe, people and politicians find themselves unarmed, also because they rarely have the scientific background to understand natural processes. Yet politicians are the only persons who make the key decisions that drive the economy in play and therefore determine the near future of our planet. This article is an attempt to respond directly to a governor with a degree in animal production science, who formally and prudently asked a university department called "Land, Environment, Agriculture and Forestry" for help before taking decisions; 4) the authors also propose an artistic interpretation of facts (uncontrolled storm) and conclusions (listen to the soil). Briefly, the authors identify the soil as an indispensable source for the renewal of the destroyed forest, give indications on how to prepare a map of the soils of the damaged region, and suggest to anchor on this soil map a series of silvicultural and soil management actions that will promote the soil conservation and the faster recovery of the natural dynamic stability and resilience. ELECTRONIC SUPPLEMENTARY MATERIAL: Supplementary material is available for this article at 10.1007/s11629-019-5890-0 and is accessible for authorized users.

Uncovering a membrane-distal conformation of KRAS available to recruit RAF to the plasma membrane.

The small GTPase KRAS is localized at the plasma membrane where it functions as a molecular switch, coupling extracellular growth factor stimulation to intracellular signaling networks. In this process, KRAS recruits effectors, such as RAF kinase, to the plasma membrane where they are activated by a series of complex molecular steps. Defining the membrane-bound state of KRAS is fundamental to understanding the activation of RAF kinase and in evaluating novel therapeutic opportunities for the inhibition of oncogenic KRAS-mediated signaling. We combined multiple biophysical measurements and computational methodologies to generate a consensus model for authentically processed, membrane-anchored KRAS. In contrast to the two membrane-proximal conformations previously reported, we identify a third significantly populated state using a combination of neutron reflectivity, fast photochemical oxidation of proteins (FPOP), and NMR. In this highly populated state, which we refer to as "membrane-distal" and estimate to comprise ∼90% of the ensemble, the G-domain does not directly contact the membrane but is tethered via its C-terminal hypervariable region and carboxymethylated farnesyl moiety, as shown by FPOP. Subsequent interaction of the RAF1 RAS binding domain with KRAS does not significantly change G-domain configurations on the membrane but affects their relative populations. Overall, our results are consistent with a directional fly-casting mechanism for KRAS, in which the membrane-distal state of the G-domain can effectively recruit RAF kinase from the cytoplasm for activation at the membrane.

Hydrodynamic Noise of Pulsating Jets through Bileaflet Mechanical Mitral Valve.

Experimental research results of hydrodynamic noise of pulsating flow through a bileaflet mechanical mitral valve are presented. The pulsating flow of pure water corresponds to the diastolic mode of the cardiac rhythm heart. The valve was located between the model of the left atrium and the model of the left ventricle of the heart. A coordinate device, on which a block of miniature sensors of absolute pressure and pressure fluctuations was installed, was located inside the model of the left ventricle. It is found that the hydrodynamic noise of the pulsating side jet of the semiclosed valve is higher than for the open valve. The pressure fluctuation levels gradually decrease with the removal from the mitral valve. It is established that at the second harmonic of the pulsating flow frequency, the spectral levels of the hydrodynamic noise of the semiclosed bileaflet mechanical mitral valve are almost 5 times higher than the open valve. With the removal from the mitral valve, spectral levels of hydrodynamic noise are decreased, especially strongly at the frequency of the pulsating water flow and its higher harmonics.

Prediction of nocturnal hypoglycemia by an aggregation of previously known prediction approaches: proof of concept for clinical application.

BACKGROUND AND OBJECTIVE: Nocturnal hypoglycemia (NH) is common in patients with insulin-treated diabetes. Despite the risk associated with NH, there are only a few methods aiming at the prediction of such events based on intermittent blood glucose monitoring data and none has been validated for clinical use. Here we propose a method of combining several predictors into a new one that will perform at the level of the best involved one, or even outperform all individual candidates. METHODS: The idea of the method is to use a recently developed strategy for aggregating ranking algorithms. The method has been calibrated and tested on data extracted from clinical trials, performed in the European FP7-funded project DIAdvisor. Then we have tested the proposed approach on other datasets to show the portability of the method. This feature of the method allows its simple implementation in the form of a diabetic smartphone app. RESULTS: On the considered datasets the proposed approach exhibits good performance in terms of sensitivity, specificity and predictive values. Moreover, the resulting predictor automatically performs at the level of the best involved method or even outperforms it. CONCLUSION: We propose a strategy for a combination of NH predictors that leads to a method exhibiting a reliable performance and the potential for everyday use by any patient who performs self-monitoring of blood glucose.

Reduced Shedding of Surface Mesothelin Improves Efficacy of Mesothelin-Targeting Recombinant Immunotoxins.

Mesothelin (MSLN) is a differentiation antigen that is highly expressed in many epithelial cancers. MSLN is an important therapeutic target due to its high expression in cancers and limited expression in normal human tissues. Although it has been assumed that shed antigen is a barrier to immunotoxin action, a modeling study predicted that shed MSLN may enhance the action of MSLN-targeting recombinant immunotoxins such as SS1P and similar therapeutics by facilitating their redistribution within tumors. We aimed to determine whether shed MSLN enhances or reduces the antitumor effect of MSLN-targeting immunotoxins SS1P and RG7787. We engineered a cell line, A431/G9 (TACE mutant) that expresses a mutant form of MSLN in which the TNF-converting enzyme protease site is replaced with GGGS. We compared the response of the TACE-mutant cells with immunotoxins SS1P and RG7787 with that of the parental A431/H9 cell line. We show that TACE-mutant cells shed 80% less MSLN than A431/H9 cells, that TACE-mutant cells show a 2- to 3-fold increase in MSLN-targeted immunotoxin uptake, and that they are about 5-fold more sensitive to SS1P killing in cell culture. Tumors with reduced shedding respond significantly better to treatment with SS1P and RG7787. Our data show that MSLN shedding is an impediment to the antitumor activity of SS1P and RG7787. Approaches that decrease MSLN shedding could enhance the efficacy of immunotoxins and immunoconjugates targeting MSLN-expressing tumors. Mol Cancer Ther; 15(7); 1648-55. ©2016 AACR.

Evolutionary approach to violating group anonymity using third-party data.

In the era of Big Data, it is almost impossible to completely restrict access to primary non-aggregated statistical data. However, risk of violating privacy of individual respondents and groups of respondents by analyzing primary data has not been reduced. There is a need in developing subtler methods of data protection to come to grips with these challenges. In some cases, individual and group privacy can be easily violated, because the primary data contain attributes that uniquely identify individuals and groups thereof. Removing such attributes from the dataset is a crude solution and does not guarantee complete privacy. In the field of providing individual data anonymity, this problem has been widely recognized, and various methods have been proposed to solve it. In the current work, we demonstrate that it is possible to violate group anonymity as well, even if those attributes that uniquely identify the group are removed. As it turns out, it is possible to use third-party data to build a fuzzy model of a group. Typically, such a model comes in a form of a set of fuzzy rules, which can be used to determine membership grades of respondents in the group with a level of certainty sufficient to violate group anonymity. In the work, we introduce an evolutionary computing based method to build such a model. We also discuss a memetic approach to protecting the data from group anonymity violation in this case.

Farnesylated and methylated KRAS4b: high yield production of protein suitable for biophysical studies of prenylated protein-lipid interactions.

Prenylated proteins play key roles in several human diseases including cancer, atherosclerosis and Alzheimer's disease. KRAS4b, which is frequently mutated in pancreatic, colon and lung cancers, is processed by farnesylation, proteolytic cleavage and carboxymethylation at the C-terminus. Plasma membrane localization of KRAS4b requires this processing as does KRAS4b-dependent RAF kinase activation. Previous attempts to produce modified KRAS have relied on protein engineering approaches or in vitro farnesylation of bacterially expressed KRAS protein. The proteins produced by these methods do not accurately replicate the mature KRAS protein found in mammalian cells and the protein yield is typically low. We describe a protocol that yields 5-10 mg/L highly purified, farnesylated, and methylated KRAS4b from insect cells. Farnesylated and methylated KRAS4b is fully active in hydrolyzing GTP, binds RAF-RBD on lipid Nanodiscs and interacts with the known farnesyl-binding protein PDEδ.

Increased secretory leukocyte protease inhibitor (SLPI) production by highly metastatic mouse breast cancer cells.

The precise molecular mechanisms enabling cancer cells to metastasize from the primary tumor to different tissue locations are still largely unknown. Secretion of some proteins by metastatic cells could facilitate metastasis formation. The comparison of secreted proteins from cancer cells with different metastatic capabilities in vivo might provide insight into proteins involved in the metastatic process. Comparison of the secreted proteins from the mouse breast cancer cell line 4T1 and its highly metastatic 4T1.2 clone revealed a prominent differentially secreted protein which was identified as SLPI (secretory leukocyte protease inhibitor). Western blotting indicated higher levels of the protein in both conditioned media and whole cell lysates of 4T1.2 cells. Additionally higher levels of SLPI were also observed in 4T1.2 breast tumors in vivo following immunohistochemical staining. A comparison of SLPI mRNA levels by gene profiling using microarrays and RT-PCR did not detect major differences in SLPI gene expression between the 4T1 and 4T1.2 cells indicating that SLPI secretion is regulated at the protein level. Our results demonstrate that secretion of SLPI is drastically increased in highly metastatic cells, suggesting a possible role for SLPI in enhancing the metastatic behavior of breast cancer cell line 4T1.

Methylation of the DPH1 promoter causes immunotoxin resistance in acute lymphoblastic leukemia cell line KOPN-8.

Moxetumomab pasudotox (HA22) is an immunotoxin with an anti-CD22 Fv fused to a portion of Pseudomonas exotoxin A that kills CD22 expressing ALL cells. HA22 produced significant responses in some cases of ALL. To understand how to increase response rate, we isolated HA22-resistant KOPN-8 cells and found that HA22 cannot inactivate elongation factor-2 (EF2) due to low levels of DPH1 RNA and protein. Resistance was associated with methylation of the CpG island in the DPH1 promoter. 5-Azacytidine prevented resistance and methylation of the CpG residues and merits evaluation to determine if it can increase the efficacy of HA22 in ALL.

Characterization and favorable in vivo properties of heterodimeric soluble IL-15·IL-15Rα cytokine compared to IL-15 monomer.

Interleukin-15 (IL-15), a 114-amino acid cytokine related to IL-2, regulates immune homeostasis and the fate of many lymphocyte subsets. We reported that, in the blood of mice and humans, IL-15 is present as a heterodimer associated with soluble IL-15 receptor α (sIL-15Rα). Here, we show efficient production of this noncovalently linked but stable heterodimer in clonal human HEK293 cells and release of the processed IL-15·sIL-15Rα heterodimer in the medium. Purification of the IL-15 and sIL-15Rα polypeptides allowed identification of the proteolytic cleavage site of IL-15Rα and characterization of multiple glycosylation sites. Administration of the IL-15·sIL-15Rα heterodimer reconstituted from purified subunits resulted in sustained plasma IL-15 levels and in robust expansion of NK and T cells in mice, demonstrating pharmacokinetics and in vivo bioactivity superior to single chain IL-15. These identified properties of heterodimeric IL-15 provide a strong rationale for the evaluation of this molecule for clinical applications.

A modified form of diphthamide causes immunotoxin resistance in a lymphoma cell line with a deletion of the WDR85 gene.

HA22 is a recombinant immunotoxin that kills CD22-expressing cells by ADP-ribosylating and inactivating elongation factor-2 (EF2). HA22 is composed of an Fv that binds to CD22 fused to a portion of Pseudomonas exotoxin A. HA22 is very active in drug-resistant hairy cell leukemia but is less active in children with acute lymphoblastic leukemia. To understand why some patients do not respond to HA22, we isolated an HA22-resistant lymphoma cell line and showed that resistance was due to the inability of HA22 to ADP-ribosylate and inactivate EF2. We analyzed the diphthamide synthesis genes and found that the WDR85 gene was deleted. We show that WDR85 knockdown conferred HA22 resistance to sensitive cells and that sensitivity was restored by introduction of a WDR85 cDNA into resistant cells. Analysis of EF2 in the mutant cells revealed a novel form of diphthamide with an additional methyl group that prevented ADP-ribosylation and inactivation of EF2. The abnormal methylation appeared to be catalyzed by DPH5. Inactivation of the WDR85 gene could be a mechanism of immunotoxin resistance in patients undergoing immunotoxin therapy.

Immunotoxin resistance via reversible methylation of the DPH4 promoter is a unique survival strategy.

HA22 is a recombinant immunotoxin composed of an anti-CD22 Fv fused to a portion of Pseudomonas exotoxin A. HA22 produced a high rate of complete remissions in drug-resistant hairy cell leukemia and has a lower response rate in pediatric acute lymphoblastic leukemia (ALL). To understand why patients with ALL have poorer responses, we isolated an ALL cell line that is resistant to killing by HA22. The resistance is unstable; without HA22 the cells revert to HA22 sensitivity in 4 mo. We showed that in the resistant cell line, HA22 is unable to ADP ribosylate and inactivate elongation factor-2 (EF2), owing to a low level of DPH4 mRNA and protein, which prevents diphthamide biosynthesis and renders EF2 refractory to HA22. Analysis of the promoter region of the DPH4 gene shows that the CpG island was hypomethylated in the HA22-sensitive cells, heavily methylated in the resistant cells, and reverted to low methylation in the revertant cells. Our data show that immunotoxin resistance is associated with reversible CpG island methylation and silencing of DPH4 gene transcription. Incubation of sensitive cells with the methylation inhibitor 5-azacytidine prevented the emergence of resistant cells, suggesting that this agent in combination with HA22 may be useful in the treatment of some cases of ALL.

Matrix-assisted laser desorption/ionization mass spectrometry reveals decreased calcylcin expression in small cell lung cancer.

To date, most of the proteomic analyses on lung cancer tissue samples have been performed using surgical specimens, which are obtained after a diagnosis is made. To determine if a proteomic signature obtained from bronchoscopic biopsy samples could be found to assist with diagnosis, 50 lung cancer bronchoscopic biopsy samples and 13 adjacent normal lung tissue samples were analyzed using histology-directed, matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS). Lung tissue samples were cryosectioned, and sinapinic acid was robotically deposited on areas of each tissue section enriched in epithelial cells, either tumor or normal. Mass spectra were acquired using a MALDI-time of flight instrument. Small cell lung cancers (SCLCs) demonstrated clearly different protein profiles from normal lung tissue and from non-small cell lung cancers (NSCLCs). Calcyclin (m/z= 10,094.7) was identified to be underexpressed in small cell lung cancers, as compared with non-small cell lung cancers and normal lung tissue. An immunohistochemistry study using 152 NSCLCs and 21 SCLCs confirmed significantly reduced calcyclin stain in SCLCs. Thus, protein profiles obtained from bronchoscopic biopsy samples via MALDI MS distinguish cancerous epithelium from normal lung tissue and between NSCLCs and SCLCs.

Cytotoxic activity of immunotoxin SS1P is modulated by TACE-dependent mesothelin shedding.

Mesothelin is a cell-surface tumor-associated antigen expressed in several human cancers. The limited expression of mesothelin on normal tissues and its high expression in many cancers make it an attractive candidate for targeted therapies using monoclonal antibodies, immunoconjugates, and immunotoxins. Mesothelin is actively shed from the cell surface and is present in the serum of patients with malignant mesothelioma, which could negatively affect the response to these therapies. We have found that mesothelin sheddase activity is mediated by a TNF-α converting enzyme (TACE), a member of the matrix metalloproteinase/a disintegrin and metalloprotease family. We showed that EGF and TIMP-3 act through TACE as endogenous regulators of mesothelin shedding. We also found that reducing shedding significantly improved the in vitro cytotoxicity of immunotoxin SS1P, which targets mesothelin and is currently in clinical trials for the treatment of patients with mesothelioma and lung cancer. Our findings provide a mechanistic understanding of mesothelin shedding and could help improve mesothelin-based targeted therapies.

Novel peptides based on HIV-1 gp120 sequence with homology to chemokines inhibit HIV infection in cell culture.

The sequential interaction of the envelope glycoprotein of the human immunodeficiency virus type 1 (HIV-1) with CD4 and certain chemokine coreceptors initiates host cell entry of the virus. The appropriate chemokines have been shown to inhibit viral replication by blocking interaction of the gp120 envelope protein with the coreceptors. We considered the possibility that this interaction involves a motif of the gp120 that may be structurally homologous to the chemokines. In the amino acid sequences of most chemokines there is a Trp residue located at the beginning of the C-terminal α-helix, which is separated by six residues from the fourth Cys residue. The gp120 of all HIV-1 isolates have a similar motif, which includes the C-terminal part of a variable loop 3 (V3) and N-terminal part of a conserved region 3 (C3). Two synthetic peptides, derived from the relevant gp120 sequence inhibited HIV-1 replication in macrophages and T lymphocytes in sequence-dependent manner. The peptides also prevented binding of anti-CXCR4 antibodies to CXCR4, and inhibited the intracellular Ca(2+) influx in response to CXCL12/SDF-1α. Thus these peptides can be used to dissect gp120 interactions with chemokine receptors and could serve as leads for the design of new inhibitors of HIV-1.

Gastric cancer-specific protein profile identified using endoscopic biopsy samples via MALDI mass spectrometry.

To date, proteomic analyses on gastrointestinal cancer tissue samples have been performed using surgical specimens only, which are obtained after a diagnosis is made. To determine if a proteomic signature obtained from endoscopic biopsy samples could be found to assist with diagnosis, frozen endoscopic biopsy samples collected from 63 gastric cancer patients and 43 healthy volunteers were analyzed using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. A statistical classification model was developed to distinguish tumor from normal tissues using half the samples and validated with the other half. A protein profile was discovered consisting of 73 signals that could classify 32 cancer and 22 normal samples in the validation set with high predictive values (positive and negative predictive values for cancer, 96.8% and 91.3%; sensitivity, 93.8%; specificity, 95.5%). Signals overexpressed in tumors were identified as alpha-defensin-1, alpha-defensin-2, calgranulin A, and calgranulin B. A protein profile was also found to distinguish pathologic stage Ia (pT1N0M0) samples (n = 10) from more advanced stage (Ib or higher) tumors (n = 48). Thus, protein profiles obtained from endoscopic biopsy samples may be useful in assisting with the diagnosis of gastric cancer and, possibly, in identifying early stage disease.

HER2- and EGFR-specific affiprobes: novel recombinant optical probes for cell imaging.

The human epidermal growth factor receptors, EGFR and HER2, are members of the EGFR family of cell-surface receptors/tyrosine kinases. EGFR- and HER2-positive cancers represent a more aggressive disease with greater likelihood of recurrence, poorer prognosis, and decreased survival rate, compared to EGFR- or HER2-negative cancers. The details of HER2 proto-oncogenic functions are not deeply understood, partially because of a restricted availability of tools for EGFR and HER2 detection (A. Sorkin and L. K. Goh, Exp. Cell Res. 2009, 315, 683-696). We have created photostable and relatively simple-to-produce imaging probes for in vitro staining of EGFR and HER2. These new reagents, called affiprobes, consist of a targeting moiety, a HER2- or EGFR-specific Affibody molecule, and a fluorescent moiety, mCherry (red) or EGFP (green). Our flow cytometry and confocal microscopy experiments demonstrated high specificity and signal/background ratio of affiprobes. Affiprobes are able to stain both live cells and frozen tumor xenograph sections. This type of optical probe can easily be extended for targeting other cell-surface antigens/ receptors.

Affitoxin--a novel recombinant, HER2-specific, anticancer agent for targeted therapy of HER2-positive tumors.

Expression of the human epidermal growth factor receptor 2 (HER2) is amplified in 25% to 30% of breast cancers and has been associated with an unfavorable prognosis. Here we report the construction, purification, and characterization of Affitoxin-a novel class of HER2-specific cytotoxic molecules combining HER2-specific Affibody molecule as a targeting moiety and PE38KDEL, which is a truncated version of Pseudomonas exotoxin A, as a cell killing agent. It is highly soluble and does not require additional refolding, oxidation, or reduction steps during its purification. Using surface plasmon resonance technology and competitive binding assays, we have shown that Affitoxin binds specifically to HER2 with nanomolar affinity. We have also observed a high correlation between HER2 expression and retention of Affitoxin bound to the cell surface. Affitoxin binding and internalization is followed by Pseudomonas exotoxin A activity domain-mediated ADP-ribosylation of translation elongation factor 2 and, consequently, inhibition of protein synthesis as shown by protein expression analysis of HER2-positive cells treated with Affitoxin. Measured IC50 value for HER2-negative cells MDA-MB468 (65+/-2.63 pM) was more than 20 times higher than the value for low HER2 level-expressing MCF7 cells (2.56+/-0.1 pM), and almost 3 orders of magnitude higher for its HER2-overexpressing derivative MCF7/HER2 (62.7+/-5.9 fM). These studies suggest that Affitoxin is an attractive PE38-based candidate for treatment of HER2-positive tumors.

TREM-like transcript-1 protects against inflammation-associated hemorrhage by facilitating platelet aggregation in mice and humans.

Triggering receptor expressed on myeloid cells-like (TREM-like) transcript-1 (TLT-1), a type 1 single Ig domain orphan receptor specific to platelet and megakaryocyte alpha-granules, relocates to the platelet surface upon platelet stimulation. We found here that patients diagnosed with sepsis, in contrast to healthy individuals, had substantial levels of soluble TLT-1 (sTLT-1) in their plasma that correlated with the presence of disseminated intravascular coagulation. sTLT-1 bound to fibrinogen and augmented platelet aggregation in vitro. Furthermore, the cytoplasmic domain of TLT-1 could also bind ezrin/radixin/moesin family proteins, suggesting its ability to link fibrinogen to the platelet cytoskeleton. Accordingly, platelets of Treml1-/- mice failed to aggregate efficiently, extending tail-bleeding times. Lipopolysaccharide-treated Treml1-/- mice developed higher plasma levels of TNF and D-dimers than wild-type mice and were more likely to succumb during challenge. Finally, Treml1-/- mice were predisposed to hemorrhage associated with localized inflammatory lesions. Taken together, our findings suggest that TLT-1 plays a protective role during inflammation by dampening the inflammatory response and facilitating platelet aggregation at sites of vascular injury. Therefore, therapeutic modulation of TLT-1-mediated effects may provide clinical benefit to patients with hypercoagulatory conditions, including those associated with inflammation.