Root Suberin Forms an Extracellular Barrier That Affects Water Relations and Mineral Nutrition in Arabidopsis

Though central to our understanding of how roots perform their vital function of scavenging water and solutes from the soil, no direct genetic evidence currently exists to support the foundational model that suberin acts to form a chemical barrier limiting the extracellular, or apoplastic, transport of water and solutes in plant roots. Using the newly characterized enhanced suberin1 (esb1) mutant, we established a connection in Arabidopsis thaliana between suberin in the root and both water movement through the plant and solute accumulation in the shoot. Esb1 mutants, characterized by increased root suberin, were found to have reduced day time transpiration rates and increased water-use efficiency during their vegetative growth period. Furthermore, these changes in suberin and water transport were associated with decreases in the accumulation of Ca, Mn, and Zn and increases in the accumulation of Na, S, K, As, Se, and Mo in the shoot. Here, we present direct genetic evidence establishing that suberin in the roots plays a critical role in controlling both water and mineral ion uptake and transport to the leaves. The changes observed in the elemental accumulation in leaves are also interpreted as evidence that a significant component of the radial root transport of Ca, Mn, and Zn occurs in the apoplast

Proteomic identification of putative biomarkers of neo-adjuvant chemotherapy resistance in luminal (ER+) breast cancer

Background: Neoadjuvant chemotherapy is a standard treatment for locally advanced breast cancer however chemoresistance can be a major obstacle in ER+ cancers. Using comparative proteomic approaches (antibody microarray/AbMA and 2D-PAGE with MALDI-TOF/TOF MS) to investigate a pilot series of breast cancer samples our research group recently identified 14-3-3 theta/tau, tBID and BcL-XL as putative biomarkers of response to neoadjuvant chemotherapy (Hodgkinson et al J Prot 2012, 75:1276-1283 and 75:2745-2752). Here we aimed to analyse further samples using the AbMA approach and to re-analyse the combined data. Methods: Samples from chemoresistant and chemosensitive breast cancers were selected following anthracycline-taxane chemotherapy and 4 experiments were performed using ductal ER+ tumours. Differential protein expression was compared between chemoresistant and chemosensitive samples using the Panorama XPRESS Profiler725 AbMA kit. The combined data from 9 AbMA assays and 3 2D-PAGE/MS experiments was then analysed using Ingenuity Pathway Analysis (IPA; Ingenuity Systems). A pilot series of archival samples was used for clinical validation of putative predictive biomarkers. Results: 89 differentially expressed proteins (DEPs) were seen in the 4 further AbMA experiments. In the combined dataset (12 experiments from 2 proteomic platforms), 8 DEPs were seen in at least 3 experiments. These were 14-3-3 theta, 14-3-3 epsilon, 14-3-3 gamma, Bcl-xl, Bid, Phosphokinase B, Vimentin and FAK. 121 DEPs from the combined data were analysed using IPA; 13 DEPs were mapped onto the PI3K/AKT pathway. Clinical validation in a pilot series of archival samples revealed AkT-1 Ser473 and FAKY397 alongside the previously identified and validated 14-3-3 theta/tau, and tBID to be significantly associated with chemotherapy resistance. Conclusion: We have now identified at least 8 proteins which could play a role in breast chemoresistance. We propose a potential role for AkT-1, FAK, 14-3-3 theta/tau and tBID as predictive biomarkers of neoadjuvant chemotherapy resistance in breast cancer. Further validation in a larger sample series is now required

Proteomic identification of putative biomarkers of neo-adjuvant chemotherapy resistance in luminal (ER+) breast cancer

Background:Neoadjuvant chemotherapy is a standard treatment for locally advanced breast cancer however chemoresistance can be a major obstacle in ER+ cancers. Using comparative proteomic approaches (antibody microarray/AbMA and 2D-PAGE with MALDI-TOF/TOF MS) to investigate a pilot series of breast cancer samples our research group recently identified 14-3-3 theta/tau, tBID and BcL-XL as putative biomarkers of response to neoadjuvant chemotherapy (Hodgkinson et al J Prot 2012, 75:1276-1283 and 75:2745-2752). Here we aimed to analyse further samples using the AbMA approach and to re-analyse the combined data.Methods:Samples from chemoresistant and chemosensitive breast cancers were selected following anthracycline-taxane chemotherapy and 4 experiments were performed using ductal ER+ tumours. Differential protein expression was compared between chemoresistant and chemosensitive samples using the Panorama XPRESS Profiler725 AbMA kit. The combined data from 9 AbMA assays and 3 2D-PAGE/MS experiments was then analysed using Ingenuity Pathway Analysis (IPA; Ingenuity Systems). A pilot series of archival samples was used for clinical validation of putative predictive biomarkers.Results:89 differentially expressed proteins (DEPs) were seen in the 4 further AbMA experiments. In the combined dataset (12 experiments from 2 proteomic platforms), 8 DEPs were seen in at least 3 experiments. These were 14-3-3 theta, 14-3-3 epsilon, 14-3-3 gamma, Bcl-xl, Bid, Phosphokinase B, Vimentin and FAK. 121 DEPs from the combined data were analysed using IPA; 13 DEPs were mapped onto the PI3K/AKT pathway. Clinical validation in a pilot series of archival samples revealed AkT-1 Ser473 and FAKY397 alongside the previously identified and validated 14-3-3 theta/tau, and tBID to be significantly associated with chemotherapy resistance.Conclusion: We have now identified at least 8 proteins which could play a role in breast chemoresistance. We propose a potential role for AkT-1, FAK, 14-3-3 theta/tau and tBID as predictive biomarkers of neoadjuvant chemotherapy resistance in breast cancer. Further validation in a larger sample series is now required

Root Suberin Forms an Extracellular Barrier That Affects Water Relations and Mineral Nutrition in Arabidopsis

Though central to our understanding of how roots perform their vital function of scavenging water and solutes from the soil, no direct genetic evidence currently exists to support the foundational model that suberin acts to form a chemical barrier limiting the extracellular, or apoplastic, transport of water and solutes in plant roots. Using the newly characterized enhanced suberin1 (esb1) mutant, we established a connection in Arabidopsis thaliana between suberin in the root and both water movement through the plant and solute accumulation in the shoot. Esb1 mutants, characterized by increased root suberin, were found to have reduced day time transpiration rates and increased water-use efficiency during their vegetative growth period. Furthermore, these changes in suberin and water transport were associated with decreases in the accumulation of Ca, Mn, and Zn and increases in the accumulation of Na, S, K, As, Se, and Mo in the shoot. Here, we present direct genetic evidence establishing that suberin in the roots plays a critical role in controlling both water and mineral ion uptake and transport to the leaves. The changes observed in the elemental accumulation in leaves are also interpreted as evidence that a significant component of the radial root transport of Ca, Mn, and Zn occurs in the apoplast.</p

A minimalistic co-culture platform for alpha-synuclein spreading in human dopaminergic neurons

Parkinson’s disease (PD) is the second major neurodegenerative disease and the most common movement disorder. Due to age being a critical risk factor, the rapid ageing of the world population further increases the prevalence of PD. So far no treatment is available and therapies mainly focus on motor symptoms by pharmacologically substituting striatal dopamine, caused by the loss of dopaminergic neurons in the substantia nigra. This neuronal loss and intracellular protein aggregates, termed Lewy bodies (LBs), are pathological characteristics of PD. With disease progression, a spread of LBs through the brain can be observed which mainly follows axonal projections. Understanding the mechanisms of this progressive spread could be central to discovering the underlying molecular pathogenesis of the disease. As LBs mainly consist of alpha-synuclein (-syn), a prion-like spreading of -syn was suggested and is now widely accepted as a component in the PD pathogenesis. New dopaminergic model systems to study the exact mechanisms underlying -syn spread are urgently needed. As PD is a human disease, in vitro models should be derived from humans. Lund human mesencephalic (LUHMES) cells are a suitable alternative to other, mostly non-human, dopaminergic cell lines. However, difficulties cultivating them in microfluidics devices has made them thus far inaccessible for co-cultivation studies in the field of PD spreading. In the first part of this thesis, a human dopaminergic cell model system for studying the spreading of -syn fibrils is presented. First, the well-characterized LUHMES cell line was tested for suitability of PD research on prion-like spreading, as no data is currently available on this matter. For the analysis, immunofluorescence light microscopy was employed. An extended period of differentiation aimed for a high degree of neuronal maturity and long neurites to facilitate the connectivity of spatially-separated cell populations. Seeding experiments with -syn fibrils revealed a weak toxicity against these assemblies, even at prolonged differentiation. Second, to study the transmission of -syn fibrils via neuronal projections, we developed a light microscopy-compatible microfluidic co-culturing device, to maintain two LUHMES cell populations in separate cell compartments for up to two weeks of differentiation. During this time, a neurite network is formed which connects the fluidically isolated cell growth compartments. The ability to cultivate cells with neurites and soma in an isolated environment enabled seeding and transmission experiments in anterograde and retrograde directions. In the second part of this thesis, implementation strategies of the microfluidic co-culturing chip for alternative analysis methods are discussed. Firstly, the accessibility of the cells in the co-culturing device using a single-cell lysis instrument is evaluated. The tool allows for targeted lysis of individual adherent cells. Preliminary tests point in a promising direction, while LUHMES single cell lysate was successfully transferred to different analysis techniques. However, direct access to the channels of the microfluidic co-culturing chip was problematic and needs further modifications. Secondly, an implementation of the microfluidic device aiming for co-cultivation of LUHMES cells on electron microscopy grids to study neurite architecture was pursued. Thereby, microfluidic devices harbor only cell soma, but neurites can grow onto an electron microscopy grid, as only they are thin enough to be visualized by cryo-electron microscopy. Proof-of-concept experiments demonstrate the direct visualization of LUHMES cell neurites in a near-native, frozen-hydrated state

Prelamin A Influences a Program of Gene Expression In Regulation of Cell Cycle Control

The A-type lamins are intermediate filament proteins that constitute a major part of the eukaryotic nuclear lamina—a tough, polymerized, mesh lining of the inner nuclear membrane, providing shape and structural integrity to the nucleus. Lamin A (LA) filaments also permeate the nucleoplasm, providing additional structural support, but also scaffolding numerous tethered molecules to stabilize, organize, and facilitate molecular interactions to accomplish critical functions of cellular metabolism. Over the past 2 decades, much attention has been focused on roles of LA in maintenance of nuclear structural integrity. Only since the late 1990s have scientists discovered the devastating effects of LA gene (LMNA) mutations, as they have associated hundreds of LMNA mutations to a large group of diseases, called laminopathies, with a broad spectrum of phenotypes, ranging from skeletal, muscular, and neurological defects, to defective lipid storage, to accelerated aging phenotypes in diseases called progerias. Recent advances demonstrate LA regulatory functions include cell signaling, cell cycle regulation, transcription, chromatin organization, viral egress, and DNA damage repair. Amidst the flurry of fascinating research, only recently have researchers begun to focus attention on the different isoforms that exist for LA, a precursor form among them. LA is initially synthesized as Prelamin A (PreA), and undergoes a series of modifications that truncate the protein to produce “mature” LA. Existence of the precursor form, and its complex maturation pathway, have puzzled researchers since their realization. With a pattern of expression related to cell cycle phase, we hypothesized a role for PreA in cell cycle control. To investigate, we have performed array studies to assess gene expression effects at the levels of transcript expression, protein expression, and phosphorylation modification status. Here, we present evidence for a PreA-mediated program of cell cycle regulatory gene and protein expression modulation. Implicated pathways include RB-E2F, p53, p27Kip1, FoxOs, p300, and the Cyclins, with additional evidence indicating a role for the Pin1 prolyl isomerase in mediating PreA regulation of the cell cycle

Polyomavirus BK-specific cellular immune response in Kidney transplant recipients

Polyomavirus BK is an emerging pathogen in KT recipients. New potent immunosuppressive drugs promote reactivation and replication of BKV and progression towards PVAN. PVAN occurs in up to 10% of the KT recipients with a graft loss in up to 80% of the cases. New potent immunosuppressive drugs, as MMF) and FK506 are risk factors for developing PVAN. As no proven antiviral drugs are available, the only therapy of choice is the reduction of immunosuppressiva in order to regain BKV-replication control (H. H. Hirsch, M. Dickenmann, S. Binggeli, J. Steiger, Schweiz Med Forum 2004; 4:538–541). BKV-specific cellular and humoral immune response is not well characterized. Recent findings have shown that BKV-seropositive patients prior to transplantation are not protected from BKV-replication. In contrast, BKV-specific cellular immune response correlates with the diagnosis of PVAN (P. Comoli, S. Binggeli, F. Ginevri, H. H. Hirsch, Transplant Infectious Disease Jun 2006; 8(2):86-94, Review). The aim of this study was to investigate the interplay of BKV-specific immune response and BKV-replication in blood samples of KT recipients. We examined the BKV-specific immune response by ELISpot assay in KT. PBMC of KT recipients were stimulated with BKV LT-antigen and BKV-VP1 peptide libraries. The BKV-specific immune response was measured by the detection of IFN-γ by ELISpot assay. From the results of a pilot study with eight patients we were able to deduce that the dynamics of viral-replication rather than the viral load correlates with a protective immune response (S. Binggeli, A. Egli, M. Dickenmann, I. Binet, J. Steiger, H. H. Hirsch, American Journal of Transplantation, Sep 2006; 6(9):2218-9). To corroborate this previous observation the BKV-specific cellular immunity in 42 KT recipients and 10 HB were tested. The KT patients were divided into two groups: patient group 1 with an increasing or stable viral load (inc/hi)1 and patient group 2 with a decreasing viral load or after resolved PVAN (dec)2. Indeed patients in group 2 showed a significantly higher immune response upon stimulation with BKV-LT and BKV-VP1 than patients in group 1 (P=0.003, P=0.001, respectively, Wilcoxon, two-sided). Detailed analysis revealed a cut-off of >69 SFU/Mio PBMC for BKV LT-antigen, but not for BKV VP1, with significantly more KT patients from group 2 (dec) than from group 1 (inc/hi). This cut-off has to be validated in a prospective study and also analyzed whether such a cut-off can be used for immunosuppressive reduction guidance. BKV-specific cell expansion was tested in a short-term culture in the presence of either BKV-LT or -VP1. After 9-day culture, PBMC were restimulated with BKV-LT or -VP1 and the responses were then compared with responses to direct stimulation (without prior cultivation). BKV-LT and -VP1 specific cellular immune responses were significantly higher after 9-day cultivation than after direct stimulation (P=0.002, P=0.003, respectively, Wilcoxon, two sided). Due to high sequence homology between JCV and BKV, JCV-LT and -VP1 overlapping peptide pools were used to test PBMC-cross recognition. JCV-LT and -VP1 responses were significantly lower than BKV-mediated response (P=0.008, P<0.001, respectively, Wilcoxon, two-sided). Comparison of JCV- and BKV-specific responses after 9-day culture revealed that the BKV-VP1 response was significantly higher than the JCV-VP1 (P=0.016, Wilcoxon, two sided), but no significant difference was observed for LT-antigen (S. Binggeli, A. Egli, S. Schaub, I. Binet, M. Mayr, J. Steiger, H. H. Hirsch, American Journal of Transplantation, Mar 2007; 7:1-9). Agnoprotein, a late viral protein, is highly expressed upon infection. We investigated whether agnoprotein is able to induce a BKV-specific immune response and whether it may serve as a diagnostic marker. Immunostaining revealed that agnoprotein was highly expressed in the cytoplasm of infected cells and was only seen in combination with BKV-LT which is located in the nucleus. Interestingly, BKV-agnoprotein specific cellular and humoral immune responses were scarcely detected in HB or KT recipients. There are only few published studies concerning BKV-agnoprotein, and further investigations are necessary to fully understand the function of agnoprotein during infection. (D. Leuenberger, P. A. Andresen, R. Gosert, S. Binggeli, E. H. Ström, S. Bodaghi, C Hanssen Rinaldo, H. H. Hirsch, Clinical and Vaccine Immunology, Aug 2007; 14(8): 959-968). As no antiviral treatment is available for BKV, the only therapy is the reduction of immunosuppressive drugs in order to regain immunological control over BKV-replication and PVAN. However reduction of immunosuppressants upon PVAN diagnosis bears the risk of rejection or inflammatory response to BKV. It is difficult to distinguish between these two outcomes because specific markers are yet lacking. Therefore, it is pivotal to record the clinico-pathological course of the KT patient in order to correctly diagnose the problem as the therapies are completely different. Measuring the BKV-specific cellular immune response may support and complement other markers, such as PCR analysis and biopsies, to better distinguish between rejection and BKV-specific immune response. (S. Schaub, M. Mayr, A. Egli, S. Binggeli, B. Descoeudres, J. Steiger, M. J. Mihatsch, H. H. Hirsch, Nephrology Dialysis Transplantation, Aug 2007; 22(8): 2386-90). Finding the optimal immunosuppressive drug level is crucial for preventing rejection (under-immunosuppressed) and viral replication (over-immunosuppressed). Our current study showed a cut-off level of 6.65 ng/ml FK506 drug level in blood, dividing those KT patients with and without BKV-replication control (ROC-curve: AUC=0.897, sensitivity=78%, specificity=86%). If this cut-off is validated by a well designed prospective study, it may serve as a guideline to administrate the optimal drug level. (S. Binggeli, 2007, current results). BKV-specific epitopes have received considerable attention in the last five years. We started with the epitope mapping in a kidney patient with the most common HLA-type: HLAA* 01, HLA-B*08. First screening of BKV-LT revealed ten 15aa long peptides with immunogenic potential. Three of these ten peptides were further investigated for crossrecognition with the homologous JCV-peptides. Even though response to the three JCVpeptides was lower, cellular immune response could be clearly detected. It needs further investigation to find more BKV-specific epitopes and also to test the ability of CD8+ T-cells to kill BKV-antigen presenting cells. (S. Binggeli, 2007, current results)