Metodi Metodiev - Academia.edu (original) (raw)

Papers by Metodi Metodiev

Journal of experimental botany, Jun 18, 2017

Carbon-concentrating mechanisms (CCMs) enable efficient photosynthesis and growth in CO2-limiting... more Carbon-concentrating mechanisms (CCMs) enable efficient photosynthesis and growth in CO2-limiting environments, and in eukaryotic microalgae localisation of Rubisco to a microcompartment called the pyrenoid is key. In the model green alga Chlamydomonas reinhardtii, Rubisco preferentially relocalises to the pyrenoid during CCM induction and pyrenoid-less mutants lack a functioning CCM and grow very poorly at low CO2. The aim of this study was to investigate the CO2 response of pyrenoid-positive (pyr+) and pyrenoid-negative (pyr-) mutant strains to determine the effect of pyrenoid absence on CCM induction and gene expression. Shotgun proteomic analysis of low-CO2-adapted strains showed reduced accumulation of some CCM-related proteins, suggesting that pyr- has limited capacity to respond to low-CO2 conditions. Comparisons between gene transcription and protein expression revealed potential regulatory interactions, since Rubisco protein linker (EPYC1) protein did not accumulate in pyr-...

Hormones & cancer, Feb 1, 2017

Cancer progression is driven by genome instability incurred rearrangements such as transmembrane ... more Cancer progression is driven by genome instability incurred rearrangements such as transmembrane protease, serine 2 (TMPRSS2)/v-ets erythroblastosis virus E26 oncogene (ERG) that could possibly turn some of the tumor suppressor micro-RNAs into pro-oncogenic ones. Previously, we found dualistic miR-204 effects, acting either as a tumor suppressor or as an oncomiR in ERG fusion-dependent manner. Here, we provided further evidence for an important role of miR-204 for TMPRSS2/ERG and androgen receptor (AR) signaling modulation and fine tuning that prevents TMPRSS2/ERG overexpression in prostate cancer. Based on proximity-based ligation assay, we designed a novel method for detection of TMPRSS2/ERG protein products. We found that miR-204 is TMPRSS2/ERG oncofusion negative regulator, and this was mediated by DNA methylation of TMPRSS2 promoter. Transcriptional factors runt-related transcription factor 2 (RUNX2) and ETS proto-oncogene 1 (ETS1) were positive regulators of TMPRSS2/ERG expres...

Free radical biology & medicine, Feb 20, 2016

Covalent hemoglobin binding to membranes leads to band 3 (AE1) clustering and the removal of eryt... more Covalent hemoglobin binding to membranes leads to band 3 (AE1) clustering and the removal of erythrocytes from the circulation; it is also implicated in blood storage lesions. Damaged hemoglobin, with the heme being in a redox and oxygen-binding inactive hemichrome form, has been implicated as the binding species. However, previous studies used strong non-physiological oxidants. In vivo hemoglobin is constantly being oxidised to methemoglobin (ferric), with around 1% of hemoglobin being in this form at any one time. In this study we tested the ability of the natural oxidised form of hemoglobin (methemoglobin) in the presence or absence of the physiological oxidant hydrogen peroxide to initiate membrane binding. The higher the oxidation state of hemoglobin (from Fe(III) to Fe(V)) the more binding was observed, with approximately 50% of this binding requiring reactive sulphydryl groups. The hemoglobin bound was in a high molecular weight complex containing spectrin, ankyrin and band 4...

EBioMedicine, 2016

SCRIB is a polarity regulator known to be abnormally expressed in cancer at the protein level. He... more SCRIB is a polarity regulator known to be abnormally expressed in cancer at the protein level. Here we report that, in breast cancer, an additional and hidden dimension of deregulations exists: an unexpected SCRIB exon usage pattern appears to mark a more malignant tumor phenotype and significantly correlates with survival. Conserved exons encoding the leucine-rich repeats tend to be overexpressed while others are underused. Mechanistic studies revealed that the underused exons encode part of the protein necessary for interaction with Vimentin and Numa1, a protein which is required for proper positioning of the mitotic spindle. Thus, the inclusion/exclusion of specific SCRIB exons is a mechanistic hallmark of breast cancer, which could potentially be exploited to develop more efficient diagnostics and therapies.

International journal of molecular sciences, Jan 4, 2016

Neoadjuvant chemoradiation (nCRT) is an established procedure in stage union internationale contr... more Neoadjuvant chemoradiation (nCRT) is an established procedure in stage union internationale contre le cancer (UICC) II/III rectal carcinomas. Around 53% of the tumours present with good tumor regression after nCRT, and 8%-15% are complete responders. Reliable selection markers would allow the identification of poor or non-responders prior to therapy. Tumor biopsies were harvested from 20 patients with rectal carcinomas, and stored in liquid nitrogen prior to therapy after obtaining patients' informed consent (Erlangen-No.3784). Patients received standardized nCRT with 5-Fluoruracil (nCRT I) or 5-Fluoruracil ± Oxaliplatin (nCRT II) according to the CAO/ARO/AIO-04 protocol. After surgery, regression grading (Dworak) of the tumors was performed during histopathological examination of the specimens. Tumors were classified as poor (Dworak 1 + 2) or good (Dworak 3 + 4) responders. Laser capture microdissection (LCM) for tumor enrichment was performed on preoperative biopsies. Differen...

Scientific Reports, 2016

Sertoli cells, can function as non-professional tolerogenic antigen-presenting cells and sustain ... more Sertoli cells, can function as non-professional tolerogenic antigen-presenting cells and sustain the blood-testis barrier formed by their tight junctions. The NOD-like receptor family members and the NALP3 inflammasome play a key role in pro-inflammatory innate immunity signalling pathways. Limited data exist on NOD1 and NOD2 expression in human and mouse Sertoli cells. Currently, there is no data on inflammasome expression or function in Sertoli cells. We found that in primary pre-pubertal Sertoli cells and in adult Sertoli line, TLR4\NOD1 and NOD2 crosstalk converged in NFκB activation and elicited a NALP3 activation, leading to de novo synthesis and inflammasome priming. This led to caspase-1 activation and IL-1β secretion. We demonstrated this process was controlled by mechanisms linked to autophagy. NOD1 promoted pro-IL-1β restriction and autophagosome maturation arrest, while NOD2 promoted caspase-1 activation, IL-1β secretion and autophagy maturation. NALP3 modulated NOD1 and...

Neoplasia, 2013

The γ subunit of the major histocompatibility complex (MHC) class II complex, CD74, is overexpres... more The γ subunit of the major histocompatibility complex (MHC) class II complex, CD74, is overexpressed in a significant proportion of metastatic breast tumors, but the mechanistic foundation and biologic significance of this phenomenon are not fully understood. Here, we show that when CD74 is overexpressed in human cancer and noncancerous epithelial cells, it interacts and interferes with the function of Scribble, a product of a well-known tumor suppressor gene. Furthermore, using epithelial cell lines expressing CD74 under the control of tetracycline-inducible promoter and quantitative high-resolution mass spectrometry, we demonstrate that, as a result of CD74 overexpression, the phosphorylation pattern of the C-terminal part of Scribble undergoes specific changes. This is accompanied with a translocation of the protein from the sites of cell-to-cell contacts at the plasma membrane to the cytoplasm, which is likely to effectively enhance the motility and invasiveness of the cancer cells.

The basic structures of placentas and tumors have many similarities. Both have an abundance of ac... more The basic structures of placentas and tumors have many similarities. Both have an abundance of actively dividing cells; trophoblasts in the placenta and cancer cells in tumors. In both cases, the vasculature is supported by the infiltration of mononuclear cells, in particular, the macrophage and NK cells. We are interested in 2 proteins in cancer and pregnancy; a2 V-ATPase and IL-22. Both of these proteins are involved in angiogenesis and the accompanying immune regulation that promotes novo growth. The a2V-ATPase directly correlates with Zygote expression and pregnancy success. In pregnancy, a2V-ATPase is expressed first in the sperm (and not the Ova). After fertilization, it is dramatically increased in the pre-implantation Zygote. Its concentration in the placenta cells including both the white blood cells and trophoblasts increases until mid-gestation and then level off until parturition. Its confirmed that a2V-ATPase is invlovled, in part, in bothangiogenesis and in "immune suppression". We have previously shown that tumor associated a2V-ATPase enhances angiogenesis and metastasis of tumors by causing the increased secretion of VEGF and MMP-9 from resident macrophages. Also it was shown that tumor associated a2V-ATPase stimulates macrophages to create sterile non-destructive inflammation that is required for establishment of cancer by inducing the expression of IL-10 and CCl-22 (TGF inducing cytokine. The expression of a2V-ATPase appears to involve the leading edge of tumors and trophoblast, which is no doubt induced by the lack of nutrients and decrease in the pH of the leading edge of the marauding (implanting cell) and. The next factor we have investigated is IL-22. This factor is abundant in the placentas from midgestation through parturition. The knocking down or inhibition of IL-22 causesdramatic preterm birth and can be prevented by the addition of recombinant IL-22. Probably the most interesting possibility of the proposed relationship between cancer and pregnancy is that in humans it is regularly cured after~38 weeks. This can provide unique possibilities which can lead not onlytocures for cancer but treatments for recurrent miscarriage and preterm birth.

Environmental Microbiology, 2015

Limitation of marine primary production by the availability of nitrogen or phosphorus is common. ... more Limitation of marine primary production by the availability of nitrogen or phosphorus is common. Emiliania huxleyi, a ubiquitous phytoplankter that plays key roles in primary production, calcium carbonate precipitation and production of dimethyl sulfide, often blooms in mid-latitude at the beginning of summer when inorganic nutrient concentrations are low. To understand physiological mechanisms that allow such blooms, we examined how the proteome of E. huxleyi (strain 1516) responds to N and P limitation. We observed modest changes in much of the proteome despite large physiological changes (e.g. cellular biomass, C, N and P) associated with nutrient limitation of growth rate. Acclimation to nutrient limitation did however involve significant increases in the abundance of transporters for ammonium and nitrate under N limitation and for phosphate under P limitation. More notable were large increases in proteins involved in the acquisition of organic forms of N and P, including urea and amino acid/polyamine transporters and numerous C-N hydrolases under N limitation and a large upregulation of alkaline phosphatase under P limitation. This highly targeted reorganization of the proteome towards scavenging organic forms of macronutrients gives unique insight into the molecular mechanisms that underpin how E. huxleyi has found its niche to bloom in surface waters depleted of inorganic nutrients.

New Phytologist, 2013

Optimality principles are often applied in theoretical studies of microalgal ecophysiology to pre... more Optimality principles are often applied in theoretical studies of microalgal ecophysiology to predict changes in allocation of resources to different metabolic pathways, and optimal acclimation is likely to involve changes in the proteome, which typically accounts for > 50% of cellular nitrogen (N). We tested the hypothesis that acclimation of the microalga Emiliania huxleyi CCMP 1516 to suboptimal vs supraoptimal light involves large changes in the proteome as cells rebalance the capacities to absorb light, fix CO 2 , perform biosynthesis and resist photooxidative stress. Emiliania huxleyi was grown in nutrient-replete continuous culture at 30 (LL) and 1000 lmol photons m À2 s À1 (HL), and changes in the proteome were assessed by LC-MS/MS shotgun proteomics. Changes were most evident in proteins involved in the light reactions of photosynthesis; the relative abundance of photosystem I (PSI) and PSII proteins was 70% greater in LL, light-harvesting fucoxanthin-chlorophyll proteins (Lhcfs) were up to 500% greater in LL and photoprotective LI818 proteins were 300% greater in HL. The marked changes in the abundances of Lhcfs and LI818s, together with the limited plasticity in the bulk of the E. huxleyi proteome, probably reflect evolutionary pressures to provide energy to maintain metabolic capabilities in stochastic light environments encountered by this species in nature.

New Phytologist, 2013

Mechanistic understanding of the costs and benefits of photoacclimation requires knowledge of how... more Mechanistic understanding of the costs and benefits of photoacclimation requires knowledge of how photophysiology is affected by changes in the molecular structure of the chloroplast. We tested the hypothesis that changes in the light dependencies of photosynthesis, nonphotochemical quenching and PSII photoinactivation arises from changes in the abundances of chloroplast proteins in Emiliania huxleyi strain CCMP 1516 grown at 30 (Low Light; LL) and 1000 (High Light; HL) lmol photons m À2 s À1 photon flux densities. Carbon-specific light-saturated gross photosynthesis rates were not significantly different between cells acclimated to LL and HL. Acclimation to LL benefited cells by increasing biomass-specific light absorption and gross photosynthesis rates under low light, whereas acclimation to HL benefited cells by reducing the rate of photoinactivation of PSII under high light. Differences in the relative abundances of proteins assigned to light-harvesting (Lhcf), photoprotection (LI818-like), and the photosystem II (PSII) core complex accompanied differences in photophysiology: specifically, Lhcf:PSII was greater under LL, whereas LI818:PSII was greater in HL. Thus, photoacclimation in E. huxleyi involved a trade-off amongst the characteristics of light absorption and photoprotection, which could be attributed to changes in the abundance and composition of proteins in the light-harvesting antenna of PSII.

Journal of Cell Science, 2013

Mating yeast cells interpret complex pheromone gradients and polarize their growth in the directi... more Mating yeast cells interpret complex pheromone gradients and polarize their growth in the direction of the closest partner. Chemotropic growth depends on both the pheromone receptor and its associated G-protein. Upon activation by the receptor, Gα dissociates from Gβγ and Gβ is subsequently phosphorylated. Free Gβγ signals to the nucleus via a MAPK cascade and recruits Far1-Cdc24 to the incipient growth site. It is not clear how the cell establishes and stabilizes the axis of polarity, but this process is thought to require local signal amplification via the Gβγ-Far1-Cdc24 chemotropic complex, as well as communication between this complex and the activated receptor. Here we show that a mutant form of Gβ that cannot be phosphorylated confers defects in directional sensing and chemotropic growth. Our data suggest that phosphorylation of Gβ plays a role in localized signal amplification and in the dynamic communication between the receptor and the chemotropic complex, which underlie gr...

Clinical Cancer Research, 2006

Purpose: Brother of the regulator of imprinted sites (BORIS) is a novel member of the cancer-test... more Purpose: Brother of the regulator of imprinted sites (BORIS) is a novel member of the cancer-testis antigen gene family. These genes are normally expressed only in spermatocytes but abnormally activated in different malignancies, including breast cancer. The aim of this study was to investigate the expression of BORIS in the leukocytes of breast cancer patients and the correlation between BORIS levels and clinical/pathologic variables. Experimental Design: Leukocytes were obtained from whole blood of 87 breast cancer patients and 52 donors not diagnosed with cancer. BORIS protein was detected in leukocytes by immunohistochemical staining; the immunoreactivity score (IRS) of each sample was determined. Additionally, BORIS expression was assessed by Western blot analysis and real-time reverse transcription-PCR. Results: We describe significantly high levels of BORIS (IRS = 4.25 ± 0.034) in a subpopulation of leukocytes, the neutrophil polymorphonuclear granulocytes, in 88.5% of breast...

Science signaling, Jan 12, 2016

Gradient-directed cell migration (chemotaxis) and growth (chemotropism) are processes that are es... more Gradient-directed cell migration (chemotaxis) and growth (chemotropism) are processes that are essential to the development and life cycles of all species. Cells use surface receptors to sense the shallow chemical gradients that elicit chemotaxis and chemotropism. Slight asymmetries in receptor activation are amplified by downstream signaling systems, which ultimately induce dynamic reorganization of the cytoskeleton. During the mating response of budding yeast, a model chemotropic system, the pheromone receptors on the plasma membrane polarize to the side of the cell closest to the stimulus. Although receptor polarization occurs before and independently of actin cable-dependent delivery of vesicles to the plasma membrane (directed secretion), it requires receptor internalization. Phosphorylation of pheromone receptors by yeast casein kinase 1 or 2 (Yck1/2) stimulates their internalization. We showed that the pheromone-responsive Gβγ dimer promotes the polarization of the pheromone ...

Global change biology, Jan 19, 2015

The advent of genomic, transcriptomic and proteomic-based approaches has revolutionised our abili... more The advent of genomic, transcriptomic and proteomic-based approaches has revolutionised our ability to describe marine microbial communities, including biogeography, metabolic potential and diversity, mechanisms of adaptation, and phylogeny and evolutionary history. New inter-disciplinary approaches are needed to move from this descriptive level to improved quantitative, process-level understanding of the roles of marine microbes in biogeochemical cycles, and of the impact of environmental change on the marine microbial ecosystem. To link studies at levels from the genome to the organism, to ecological strategies and organism and ecosystem response, requires new modelling approaches. Key to this will be a fundamental shift in modelling scale that represents microorganisms from the level of their macromolecular components. This will enable contact with omics datasets, and allow acclimation and adaptive response at the phenotype level (i.e. traits) to be simulated as a combination of ...

New Phytologist, 2013

Optimality principles are often applied in theoretical studies of microalgal ecophysiology to pre... more Optimality principles are often applied in theoretical studies of microalgal ecophysiology to predict changes in allocation of resources to different metabolic pathways, and optimal acclimation is likely to involve changes in the proteome, which typically accounts for > 50% of cellular nitrogen (N).

Science (New York, N.Y.), Jan 24, 2002

The mating response of the budding yeast Saccharomyces cerevisiae is mediated by a prototypical h... more The mating response of the budding yeast Saccharomyces cerevisiae is mediated by a prototypical heterotrimeric GTP-binding protein (G protein) and mitogen-activated protein kinase (MAPK) cascade. Although signal transmission by such pathways has been modeled in detail, postreceptor down-regulation is less well understood. The pheromone-responsive G protein alpha subunit (Galpha) of yeast down-regulates the mating signal, but its targets are unknown. We have found that Galpha binds directly to the mating-specific MAPK in yeast cells responding to pheromone. This interaction contributes both to modulation of the mating signal and to the chemotropic response, and it demonstrates direct communication between the top and bottom of a Galpha-MAPK pathway.

New Phytologist, 2013

Mechanistic understanding of the costs and benefits of photoacclimation requires knowledge of how... more Mechanistic understanding of the costs and benefits of photoacclimation requires knowledge of how photophysiology is affected by changes in the molecular structure of the chloroplast.

Molecular Carcinogenesis, 2015

During cancer progression, the genome instability incurred rearrangement could possibly turn some... more During cancer progression, the genome instability incurred rearrangement could possibly turn some of the tumor suppressor micro-RNAs into pro-oncogenic ones. We aimed to investigate miR-204 in the context of prostate cancer progression using a cell line model of different levels of genome instability (LNCaP, PC3, VCaP and NCI H660), as demonstrated by the availability of ERG fusion. We studied the effect of miR-204 modulation on master transcription factors important for lineage development, cell differentiation and prostate cancer bone marrow metastasis. We followed c-MYB, ETS1 and RUNX2 transcript and protein expression and the miR-204 affected global proteome. We further investigated if these transcription factors exert an effect on miR-204 expression (qPCR, luciferase reporter assay) by silencing them using esiRNA. We found dualistic miR-204 effects, either acting as a tumor suppressor on c-MYB, or as an oncomiR on ETS1. RUNX2 and ETS1 regulation by miR-204 was ERG fusion dependent, demonstrating regulatory circuitry disruption in advanced metastatic models. miR-204 also differentially affected mRNA splicing and protein stability. miR-204 levels were found dependent on cancer hypermethylation and supported by positive feedback induced by all three transcription factors. In this regulatory circuitry among miR-204, c-MYB, RUNX2 and ETS1, the c-MYB was found to induce all three other members, but its expression was differentially affected by the methylation status in lymph node vs. bone metastasis. We demonstrate that not only tumor suppressor micro-RNA loss, but also significant genome rearrangement-driven regulatory loop perturbations play a role in the advanced cancer progression, conferring better pro-survival and metastatic potential. © 2015 Wiley Periodicals, Inc.

Molecular biology of the cell, 2009

As a budding yeast cell elongates toward its mating partner, cytoplasmic microtubules connect the... more As a budding yeast cell elongates toward its mating partner, cytoplasmic microtubules connect the nucleus to the cell cortex at the growth tip. The Kar3 kinesin-like motor protein is then thought to stimulate plus-end depolymerization of these microtubules, thus drawing the nucleus closer to the site where cell fusion and karyogamy will occur. Here, we show that pheromone stimulates a microtubule-independent interaction between Kar3 and the mating-specific Galpha protein Gpa1 and that Gpa1 affects both microtubule orientation and cortical contact. The membrane localization of Gpa1 was found to polarize early in the mating response, at about the same time that the microtubules begin to attach to the incipient growth site. In the absence of Gpa1, microtubules lose contact with the cortex upon shrinking and Kar3 is improperly localized, suggesting that Gpa1 is a cortical anchor for Kar3. We infer that Gpa1 serves as a positional determinant for Kar3-bound microtubule plus ends during m...

Journal of experimental botany, Jun 18, 2017

Carbon-concentrating mechanisms (CCMs) enable efficient photosynthesis and growth in CO2-limiting... more Carbon-concentrating mechanisms (CCMs) enable efficient photosynthesis and growth in CO2-limiting environments, and in eukaryotic microalgae localisation of Rubisco to a microcompartment called the pyrenoid is key. In the model green alga Chlamydomonas reinhardtii, Rubisco preferentially relocalises to the pyrenoid during CCM induction and pyrenoid-less mutants lack a functioning CCM and grow very poorly at low CO2. The aim of this study was to investigate the CO2 response of pyrenoid-positive (pyr+) and pyrenoid-negative (pyr-) mutant strains to determine the effect of pyrenoid absence on CCM induction and gene expression. Shotgun proteomic analysis of low-CO2-adapted strains showed reduced accumulation of some CCM-related proteins, suggesting that pyr- has limited capacity to respond to low-CO2 conditions. Comparisons between gene transcription and protein expression revealed potential regulatory interactions, since Rubisco protein linker (EPYC1) protein did not accumulate in pyr-...

Hormones & cancer, Feb 1, 2017

Cancer progression is driven by genome instability incurred rearrangements such as transmembrane ... more Cancer progression is driven by genome instability incurred rearrangements such as transmembrane protease, serine 2 (TMPRSS2)/v-ets erythroblastosis virus E26 oncogene (ERG) that could possibly turn some of the tumor suppressor micro-RNAs into pro-oncogenic ones. Previously, we found dualistic miR-204 effects, acting either as a tumor suppressor or as an oncomiR in ERG fusion-dependent manner. Here, we provided further evidence for an important role of miR-204 for TMPRSS2/ERG and androgen receptor (AR) signaling modulation and fine tuning that prevents TMPRSS2/ERG overexpression in prostate cancer. Based on proximity-based ligation assay, we designed a novel method for detection of TMPRSS2/ERG protein products. We found that miR-204 is TMPRSS2/ERG oncofusion negative regulator, and this was mediated by DNA methylation of TMPRSS2 promoter. Transcriptional factors runt-related transcription factor 2 (RUNX2) and ETS proto-oncogene 1 (ETS1) were positive regulators of TMPRSS2/ERG expres...

Free radical biology & medicine, Feb 20, 2016

Covalent hemoglobin binding to membranes leads to band 3 (AE1) clustering and the removal of eryt... more Covalent hemoglobin binding to membranes leads to band 3 (AE1) clustering and the removal of erythrocytes from the circulation; it is also implicated in blood storage lesions. Damaged hemoglobin, with the heme being in a redox and oxygen-binding inactive hemichrome form, has been implicated as the binding species. However, previous studies used strong non-physiological oxidants. In vivo hemoglobin is constantly being oxidised to methemoglobin (ferric), with around 1% of hemoglobin being in this form at any one time. In this study we tested the ability of the natural oxidised form of hemoglobin (methemoglobin) in the presence or absence of the physiological oxidant hydrogen peroxide to initiate membrane binding. The higher the oxidation state of hemoglobin (from Fe(III) to Fe(V)) the more binding was observed, with approximately 50% of this binding requiring reactive sulphydryl groups. The hemoglobin bound was in a high molecular weight complex containing spectrin, ankyrin and band 4...

EBioMedicine, 2016

SCRIB is a polarity regulator known to be abnormally expressed in cancer at the protein level. He... more SCRIB is a polarity regulator known to be abnormally expressed in cancer at the protein level. Here we report that, in breast cancer, an additional and hidden dimension of deregulations exists: an unexpected SCRIB exon usage pattern appears to mark a more malignant tumor phenotype and significantly correlates with survival. Conserved exons encoding the leucine-rich repeats tend to be overexpressed while others are underused. Mechanistic studies revealed that the underused exons encode part of the protein necessary for interaction with Vimentin and Numa1, a protein which is required for proper positioning of the mitotic spindle. Thus, the inclusion/exclusion of specific SCRIB exons is a mechanistic hallmark of breast cancer, which could potentially be exploited to develop more efficient diagnostics and therapies.

International journal of molecular sciences, Jan 4, 2016

Neoadjuvant chemoradiation (nCRT) is an established procedure in stage union internationale contr... more Neoadjuvant chemoradiation (nCRT) is an established procedure in stage union internationale contre le cancer (UICC) II/III rectal carcinomas. Around 53% of the tumours present with good tumor regression after nCRT, and 8%-15% are complete responders. Reliable selection markers would allow the identification of poor or non-responders prior to therapy. Tumor biopsies were harvested from 20 patients with rectal carcinomas, and stored in liquid nitrogen prior to therapy after obtaining patients' informed consent (Erlangen-No.3784). Patients received standardized nCRT with 5-Fluoruracil (nCRT I) or 5-Fluoruracil ± Oxaliplatin (nCRT II) according to the CAO/ARO/AIO-04 protocol. After surgery, regression grading (Dworak) of the tumors was performed during histopathological examination of the specimens. Tumors were classified as poor (Dworak 1 + 2) or good (Dworak 3 + 4) responders. Laser capture microdissection (LCM) for tumor enrichment was performed on preoperative biopsies. Differen...

Scientific Reports, 2016

Sertoli cells, can function as non-professional tolerogenic antigen-presenting cells and sustain ... more Sertoli cells, can function as non-professional tolerogenic antigen-presenting cells and sustain the blood-testis barrier formed by their tight junctions. The NOD-like receptor family members and the NALP3 inflammasome play a key role in pro-inflammatory innate immunity signalling pathways. Limited data exist on NOD1 and NOD2 expression in human and mouse Sertoli cells. Currently, there is no data on inflammasome expression or function in Sertoli cells. We found that in primary pre-pubertal Sertoli cells and in adult Sertoli line, TLR4\NOD1 and NOD2 crosstalk converged in NFκB activation and elicited a NALP3 activation, leading to de novo synthesis and inflammasome priming. This led to caspase-1 activation and IL-1β secretion. We demonstrated this process was controlled by mechanisms linked to autophagy. NOD1 promoted pro-IL-1β restriction and autophagosome maturation arrest, while NOD2 promoted caspase-1 activation, IL-1β secretion and autophagy maturation. NALP3 modulated NOD1 and...

Neoplasia, 2013

The γ subunit of the major histocompatibility complex (MHC) class II complex, CD74, is overexpres... more The γ subunit of the major histocompatibility complex (MHC) class II complex, CD74, is overexpressed in a significant proportion of metastatic breast tumors, but the mechanistic foundation and biologic significance of this phenomenon are not fully understood. Here, we show that when CD74 is overexpressed in human cancer and noncancerous epithelial cells, it interacts and interferes with the function of Scribble, a product of a well-known tumor suppressor gene. Furthermore, using epithelial cell lines expressing CD74 under the control of tetracycline-inducible promoter and quantitative high-resolution mass spectrometry, we demonstrate that, as a result of CD74 overexpression, the phosphorylation pattern of the C-terminal part of Scribble undergoes specific changes. This is accompanied with a translocation of the protein from the sites of cell-to-cell contacts at the plasma membrane to the cytoplasm, which is likely to effectively enhance the motility and invasiveness of the cancer cells.

The basic structures of placentas and tumors have many similarities. Both have an abundance of ac... more The basic structures of placentas and tumors have many similarities. Both have an abundance of actively dividing cells; trophoblasts in the placenta and cancer cells in tumors. In both cases, the vasculature is supported by the infiltration of mononuclear cells, in particular, the macrophage and NK cells. We are interested in 2 proteins in cancer and pregnancy; a2 V-ATPase and IL-22. Both of these proteins are involved in angiogenesis and the accompanying immune regulation that promotes novo growth. The a2V-ATPase directly correlates with Zygote expression and pregnancy success. In pregnancy, a2V-ATPase is expressed first in the sperm (and not the Ova). After fertilization, it is dramatically increased in the pre-implantation Zygote. Its concentration in the placenta cells including both the white blood cells and trophoblasts increases until mid-gestation and then level off until parturition. Its confirmed that a2V-ATPase is invlovled, in part, in bothangiogenesis and in "immune suppression". We have previously shown that tumor associated a2V-ATPase enhances angiogenesis and metastasis of tumors by causing the increased secretion of VEGF and MMP-9 from resident macrophages. Also it was shown that tumor associated a2V-ATPase stimulates macrophages to create sterile non-destructive inflammation that is required for establishment of cancer by inducing the expression of IL-10 and CCl-22 (TGF inducing cytokine. The expression of a2V-ATPase appears to involve the leading edge of tumors and trophoblast, which is no doubt induced by the lack of nutrients and decrease in the pH of the leading edge of the marauding (implanting cell) and. The next factor we have investigated is IL-22. This factor is abundant in the placentas from midgestation through parturition. The knocking down or inhibition of IL-22 causesdramatic preterm birth and can be prevented by the addition of recombinant IL-22. Probably the most interesting possibility of the proposed relationship between cancer and pregnancy is that in humans it is regularly cured after~38 weeks. This can provide unique possibilities which can lead not onlytocures for cancer but treatments for recurrent miscarriage and preterm birth.

Environmental Microbiology, 2015

Limitation of marine primary production by the availability of nitrogen or phosphorus is common. ... more Limitation of marine primary production by the availability of nitrogen or phosphorus is common. Emiliania huxleyi, a ubiquitous phytoplankter that plays key roles in primary production, calcium carbonate precipitation and production of dimethyl sulfide, often blooms in mid-latitude at the beginning of summer when inorganic nutrient concentrations are low. To understand physiological mechanisms that allow such blooms, we examined how the proteome of E. huxleyi (strain 1516) responds to N and P limitation. We observed modest changes in much of the proteome despite large physiological changes (e.g. cellular biomass, C, N and P) associated with nutrient limitation of growth rate. Acclimation to nutrient limitation did however involve significant increases in the abundance of transporters for ammonium and nitrate under N limitation and for phosphate under P limitation. More notable were large increases in proteins involved in the acquisition of organic forms of N and P, including urea and amino acid/polyamine transporters and numerous C-N hydrolases under N limitation and a large upregulation of alkaline phosphatase under P limitation. This highly targeted reorganization of the proteome towards scavenging organic forms of macronutrients gives unique insight into the molecular mechanisms that underpin how E. huxleyi has found its niche to bloom in surface waters depleted of inorganic nutrients.

New Phytologist, 2013

Optimality principles are often applied in theoretical studies of microalgal ecophysiology to pre... more Optimality principles are often applied in theoretical studies of microalgal ecophysiology to predict changes in allocation of resources to different metabolic pathways, and optimal acclimation is likely to involve changes in the proteome, which typically accounts for > 50% of cellular nitrogen (N). We tested the hypothesis that acclimation of the microalga Emiliania huxleyi CCMP 1516 to suboptimal vs supraoptimal light involves large changes in the proteome as cells rebalance the capacities to absorb light, fix CO 2 , perform biosynthesis and resist photooxidative stress. Emiliania huxleyi was grown in nutrient-replete continuous culture at 30 (LL) and 1000 lmol photons m À2 s À1 (HL), and changes in the proteome were assessed by LC-MS/MS shotgun proteomics. Changes were most evident in proteins involved in the light reactions of photosynthesis; the relative abundance of photosystem I (PSI) and PSII proteins was 70% greater in LL, light-harvesting fucoxanthin-chlorophyll proteins (Lhcfs) were up to 500% greater in LL and photoprotective LI818 proteins were 300% greater in HL. The marked changes in the abundances of Lhcfs and LI818s, together with the limited plasticity in the bulk of the E. huxleyi proteome, probably reflect evolutionary pressures to provide energy to maintain metabolic capabilities in stochastic light environments encountered by this species in nature.

New Phytologist, 2013

Mechanistic understanding of the costs and benefits of photoacclimation requires knowledge of how... more Mechanistic understanding of the costs and benefits of photoacclimation requires knowledge of how photophysiology is affected by changes in the molecular structure of the chloroplast. We tested the hypothesis that changes in the light dependencies of photosynthesis, nonphotochemical quenching and PSII photoinactivation arises from changes in the abundances of chloroplast proteins in Emiliania huxleyi strain CCMP 1516 grown at 30 (Low Light; LL) and 1000 (High Light; HL) lmol photons m À2 s À1 photon flux densities. Carbon-specific light-saturated gross photosynthesis rates were not significantly different between cells acclimated to LL and HL. Acclimation to LL benefited cells by increasing biomass-specific light absorption and gross photosynthesis rates under low light, whereas acclimation to HL benefited cells by reducing the rate of photoinactivation of PSII under high light. Differences in the relative abundances of proteins assigned to light-harvesting (Lhcf), photoprotection (LI818-like), and the photosystem II (PSII) core complex accompanied differences in photophysiology: specifically, Lhcf:PSII was greater under LL, whereas LI818:PSII was greater in HL. Thus, photoacclimation in E. huxleyi involved a trade-off amongst the characteristics of light absorption and photoprotection, which could be attributed to changes in the abundance and composition of proteins in the light-harvesting antenna of PSII.

Journal of Cell Science, 2013

Mating yeast cells interpret complex pheromone gradients and polarize their growth in the directi... more Mating yeast cells interpret complex pheromone gradients and polarize their growth in the direction of the closest partner. Chemotropic growth depends on both the pheromone receptor and its associated G-protein. Upon activation by the receptor, Gα dissociates from Gβγ and Gβ is subsequently phosphorylated. Free Gβγ signals to the nucleus via a MAPK cascade and recruits Far1-Cdc24 to the incipient growth site. It is not clear how the cell establishes and stabilizes the axis of polarity, but this process is thought to require local signal amplification via the Gβγ-Far1-Cdc24 chemotropic complex, as well as communication between this complex and the activated receptor. Here we show that a mutant form of Gβ that cannot be phosphorylated confers defects in directional sensing and chemotropic growth. Our data suggest that phosphorylation of Gβ plays a role in localized signal amplification and in the dynamic communication between the receptor and the chemotropic complex, which underlie gr...

Clinical Cancer Research, 2006

Purpose: Brother of the regulator of imprinted sites (BORIS) is a novel member of the cancer-test... more Purpose: Brother of the regulator of imprinted sites (BORIS) is a novel member of the cancer-testis antigen gene family. These genes are normally expressed only in spermatocytes but abnormally activated in different malignancies, including breast cancer. The aim of this study was to investigate the expression of BORIS in the leukocytes of breast cancer patients and the correlation between BORIS levels and clinical/pathologic variables. Experimental Design: Leukocytes were obtained from whole blood of 87 breast cancer patients and 52 donors not diagnosed with cancer. BORIS protein was detected in leukocytes by immunohistochemical staining; the immunoreactivity score (IRS) of each sample was determined. Additionally, BORIS expression was assessed by Western blot analysis and real-time reverse transcription-PCR. Results: We describe significantly high levels of BORIS (IRS = 4.25 ± 0.034) in a subpopulation of leukocytes, the neutrophil polymorphonuclear granulocytes, in 88.5% of breast...

Science signaling, Jan 12, 2016

Gradient-directed cell migration (chemotaxis) and growth (chemotropism) are processes that are es... more Gradient-directed cell migration (chemotaxis) and growth (chemotropism) are processes that are essential to the development and life cycles of all species. Cells use surface receptors to sense the shallow chemical gradients that elicit chemotaxis and chemotropism. Slight asymmetries in receptor activation are amplified by downstream signaling systems, which ultimately induce dynamic reorganization of the cytoskeleton. During the mating response of budding yeast, a model chemotropic system, the pheromone receptors on the plasma membrane polarize to the side of the cell closest to the stimulus. Although receptor polarization occurs before and independently of actin cable-dependent delivery of vesicles to the plasma membrane (directed secretion), it requires receptor internalization. Phosphorylation of pheromone receptors by yeast casein kinase 1 or 2 (Yck1/2) stimulates their internalization. We showed that the pheromone-responsive Gβγ dimer promotes the polarization of the pheromone ...

Global change biology, Jan 19, 2015

The advent of genomic, transcriptomic and proteomic-based approaches has revolutionised our abili... more The advent of genomic, transcriptomic and proteomic-based approaches has revolutionised our ability to describe marine microbial communities, including biogeography, metabolic potential and diversity, mechanisms of adaptation, and phylogeny and evolutionary history. New inter-disciplinary approaches are needed to move from this descriptive level to improved quantitative, process-level understanding of the roles of marine microbes in biogeochemical cycles, and of the impact of environmental change on the marine microbial ecosystem. To link studies at levels from the genome to the organism, to ecological strategies and organism and ecosystem response, requires new modelling approaches. Key to this will be a fundamental shift in modelling scale that represents microorganisms from the level of their macromolecular components. This will enable contact with omics datasets, and allow acclimation and adaptive response at the phenotype level (i.e. traits) to be simulated as a combination of ...

New Phytologist, 2013

Optimality principles are often applied in theoretical studies of microalgal ecophysiology to pre... more Optimality principles are often applied in theoretical studies of microalgal ecophysiology to predict changes in allocation of resources to different metabolic pathways, and optimal acclimation is likely to involve changes in the proteome, which typically accounts for > 50% of cellular nitrogen (N).

Science (New York, N.Y.), Jan 24, 2002

The mating response of the budding yeast Saccharomyces cerevisiae is mediated by a prototypical h... more The mating response of the budding yeast Saccharomyces cerevisiae is mediated by a prototypical heterotrimeric GTP-binding protein (G protein) and mitogen-activated protein kinase (MAPK) cascade. Although signal transmission by such pathways has been modeled in detail, postreceptor down-regulation is less well understood. The pheromone-responsive G protein alpha subunit (Galpha) of yeast down-regulates the mating signal, but its targets are unknown. We have found that Galpha binds directly to the mating-specific MAPK in yeast cells responding to pheromone. This interaction contributes both to modulation of the mating signal and to the chemotropic response, and it demonstrates direct communication between the top and bottom of a Galpha-MAPK pathway.

New Phytologist, 2013

Mechanistic understanding of the costs and benefits of photoacclimation requires knowledge of how... more Mechanistic understanding of the costs and benefits of photoacclimation requires knowledge of how photophysiology is affected by changes in the molecular structure of the chloroplast.

Molecular Carcinogenesis, 2015

During cancer progression, the genome instability incurred rearrangement could possibly turn some... more During cancer progression, the genome instability incurred rearrangement could possibly turn some of the tumor suppressor micro-RNAs into pro-oncogenic ones. We aimed to investigate miR-204 in the context of prostate cancer progression using a cell line model of different levels of genome instability (LNCaP, PC3, VCaP and NCI H660), as demonstrated by the availability of ERG fusion. We studied the effect of miR-204 modulation on master transcription factors important for lineage development, cell differentiation and prostate cancer bone marrow metastasis. We followed c-MYB, ETS1 and RUNX2 transcript and protein expression and the miR-204 affected global proteome. We further investigated if these transcription factors exert an effect on miR-204 expression (qPCR, luciferase reporter assay) by silencing them using esiRNA. We found dualistic miR-204 effects, either acting as a tumor suppressor on c-MYB, or as an oncomiR on ETS1. RUNX2 and ETS1 regulation by miR-204 was ERG fusion dependent, demonstrating regulatory circuitry disruption in advanced metastatic models. miR-204 also differentially affected mRNA splicing and protein stability. miR-204 levels were found dependent on cancer hypermethylation and supported by positive feedback induced by all three transcription factors. In this regulatory circuitry among miR-204, c-MYB, RUNX2 and ETS1, the c-MYB was found to induce all three other members, but its expression was differentially affected by the methylation status in lymph node vs. bone metastasis. We demonstrate that not only tumor suppressor micro-RNA loss, but also significant genome rearrangement-driven regulatory loop perturbations play a role in the advanced cancer progression, conferring better pro-survival and metastatic potential. © 2015 Wiley Periodicals, Inc.

Molecular biology of the cell, 2009

As a budding yeast cell elongates toward its mating partner, cytoplasmic microtubules connect the... more As a budding yeast cell elongates toward its mating partner, cytoplasmic microtubules connect the nucleus to the cell cortex at the growth tip. The Kar3 kinesin-like motor protein is then thought to stimulate plus-end depolymerization of these microtubules, thus drawing the nucleus closer to the site where cell fusion and karyogamy will occur. Here, we show that pheromone stimulates a microtubule-independent interaction between Kar3 and the mating-specific Galpha protein Gpa1 and that Gpa1 affects both microtubule orientation and cortical contact. The membrane localization of Gpa1 was found to polarize early in the mating response, at about the same time that the microtubules begin to attach to the incipient growth site. In the absence of Gpa1, microtubules lose contact with the cortex upon shrinking and Kar3 is improperly localized, suggesting that Gpa1 is a cortical anchor for Kar3. We infer that Gpa1 serves as a positional determinant for Kar3-bound microtubule plus ends during m...