Role of CIP2A in carcinogenesis

Worldwide and notably in the developed countries, cancer is an increasing cause of morbidity and mortality, being the second most common cause of death after ischemic heart disease. Now and in the future new cancer cases need to be diagnosed earlier. Prognostic factors may be helpful in recognizing and handling those patients who need more aggressive therapy, and it is also desirable to predict treatment response accurately. Cancerous inhibitor of protein phosphatase 2A (CIP2A) is an oncoprotein predominantly expressed in malignant tissues and inhibiting protein phosphatase 2A (PP2A) activity; it is a promising target for cancer therapy. The aim of this thesis was to evaluate the prognostic role of CIP2A in solid cancers, and for this purpose to explore expression of CIP2A, and investigating regulation of CIP2A in order to gain insight into signalling pathways leading to alteration in prognosis. Patients diagnosed with gastric, serous ovarian, tongue, or colorectal cancer at Helsinki University Central Hospital were included. Tumour tissue microarrays assembled from specimens from these patients were prepared and stained immunohistochemically for CIP2A protein expression. Associations with clinicopathologic parameters and other biomarkers were explored, and survival analyses were done according to the Kaplan-Meier method. Study of the role of CIP2A in intracellular signalling in vitro involved gastric, ovarian, and tongue cancer cell lines. We found CIP2A to be highly expressed in gastric, ovarian, tongue, and colorectal cancer specimens. CIP2A was associated with clinicopathologic parameters characterizing an aggressive disease, namely advanced stage, high grade, p53 immunopositivity, and high proliferation index. CIP2A led to recognition of gastric, ovarian, and tongue cancer patients with poor prognosis, however, with a cancer type-specific cut-off level for prognostic significance. In tongue cancer, it served as an independent prognostic marker. In contrast, in colorectal cancer, CIP2A provided no prognostic value. In cancer cell lines, CIP2A was highly expressed at both protein and mRNA levels, and promoted cell proliferation and anchorage-independent growth. In gastric cancer, we demonstrated with a MYCER construct in mouse embryo fibroblasts that activation of MYC led to increased CIP2A mRNA expression, and hence we suggested that a positive feedback mechanism between CIP2A and MYC may potentiate and prolong the oncogenic activity of these proteins. We demonstrated in ovarian cancer an association between CIP2A and EGFR protein overexpression and EGFR gene amplification. In ovarian and tongue cancer cells we showed that depletion of EGFR downregulates CIP2A expression. In conclusion, high CIP2A expression occurred frequently among patients with aggressive disease. CIP2A may serve as a prognostic marker in gastric, ovarian, and tongue cancer and thus may help in tailoring therapy for cancer patients. The positive feedback mechanism between CIP2A and MYC, as well as the positive regulation of CIP2A by EGFR, are a few signalling pathways regulating and regulated by CIP2A. These and other mechanisms need to be studied further, however. CIP2A is a potential target for therapy, and its potential role as predictive marker and as a tumour marker in serum requires exploration.

Structure and activity cyclase activated of OK-GC: A kidney receptor-guanylate cyclase activated by guanylin peptides

Uroguanylin, guanylin, and lymphoguanylin are small peptides that activate renal and intestinal receptor guanylate cyclases (GC). They are structurally similar to bacterial heat-stable enterotoxins (ST) that cause secretory diarrhea. Uroguanylin, guanylin, and ST elicit natriuresis, kaliuresis, and diuresis by direct actions on kidney GC receptors. A 3,762-bp cDNA characterizing a uroguanylin/guanylin/ST receptor was isolated from opossum kidney (OK) cell RNA/cDNA. This kidney cDNA (OK-GC) encodes a mature protein containing 1,049 residues sharing 72.4�75.8% identity with rat, human, and porcine forms of intestinal GC-C receptors. COS or HEK-293 cells expressing OK-GC receptor protein were activated by uroguanylin, guanylin, or ST13 peptides. The 3.8-kb OK-GC mRNA transcript is most abundant in the kidney cortex and intestinal mucosa, with lower mRNA levels observed in urinary bladder, adrenal gland, and myocardium and with no detectable transcripts in skin or stomach mucosa. We propose that OK-GC receptor GC participates in a renal mechanism of action for uroguanylin and/or guanylin in the physiological regulation of urinary sodium, potassium, and water excretion. This renal tubular receptor GC may be a target for circulating uroguanylin in an endocrine link between the intestine and kidney and/or participate in an intrarenal paracrine mechanism for regulation of kidney function via the intracellular second messenger, cGMP.

Structure and activity of OK-GC: a kidney receptor guanylate cyclase activated by guanylin peptides

Uroguanylin, guanylin, and lymphoguanylin are small peptides that activate renal and intestinal receptor guanylate cyclases (GC). They are structurally similar to bacterial heat-stable enterotoxins (ST) that cause secretory diarrhea. Uroguanylin, guanylin, and ST elicit natriuresis, kaliuresis, and diuresis by direct actions on kidney GC receptors. A 3,762-bp cDNA characterizing a uroguanylin/guanylin/ST receptor was isolated from opossum kidney (OK) cell RNA/cDNA. This kidney cDNA (OK-GC) encodes a mature protein containing 1,049 residues sharing 72.4-75.8% identity with rat, human, and porcine forms of intestinal GC-C receptors. COS or HEK-293 cells expressing OK-GC receptor protein were activated by uroguanylin, guanylin, or ST13 peptides. The 3.8-kb OK-GC mRNA transcript is most abundant in the kidney cortex and intestinal mucosa, with lower mRNA levels observed in urinary bladder, adrenal gland, and myocardium and with no detectable transcripts in skin or stomach mucosa. We propose that OK-GC receptor GC participates in a renal mechanism of action for uroguanylin and/or guanylin in the physiological regulation of urinary sodium, potassium, and water excretion. This renal tubular receptor GC may be a target for circulating uroguanylin in an endocrine link between the intestine and kidney and/or participate in an intrarenal paracrine mechanism for regulation of kidney function via the intracellular second messenger, cGMP.

Intranasal Delivery of Plasma and Platelet Growth Factors Using PRGF-Endoret System Enhances Neurogenesis in a Mouse Model of Alzheimer’s Disease

[EN] Neurodegeneration together with a reduction in neurogenesis are cardinal features of Alzheimer’s disease (AD) induced by a combination of toxic amyloid-β peptide (Aβ) and a loss of trophic factor support. Amelioration of these was assessed with diverse neurotrophins in experimental therapeutic approaches. The aim of this study was to investigate whether intranasal delivery of plasma rich in growth factors (PRGF-Endoret), an autologous pool of morphogens and proteins, could enhance hippocampal neurogenesis and reduce neurodegeneration in an amyloid precursor protein/presenilin-1 (APP/PS1) mouse model. Neurotrophic and neuroprotective actions were firstly evident in primary neuronal cultures, where cell proliferation and survival were augmented by Endoret treatment. Translation of these effects in vivo was assessed in wild type and APP/PS1 mice, where neurogenesis was evaluated using 5-bromodeoxyuridine (BdrU), doublecortin (DCX), and NeuN immunostaining 5 weeks after Endoret administration. The number of BrdU, DCX, and NeuN positive cell was increased after chronic treatment. The number of degenerating neurons, detected with fluoro Jade-B staining was reduced in Endoret-treated APP/PS1 mice at 5 week after intranasal administration. In conclusion, Endoret was able to activate neuronal progenitor cells, enhancing hippocampal neurogenesis, and to reduce Aβ-induced neurodegeneration in a mouse model of AD.

Prevalência da síndrome metabólica em adolescentes de 06 Escolas na Cidade de São Luís/MA Brasil

Na população pediátrica tem aumentado a prevalência da Síndrome Metabólica. No Brasil, poucos estudos foram realizados em relação a sua prevalência, critérios de diagnósticos, principalmente no Norte/Nordeste. Apesar de não termos uma definição oficial para a Síndrome Metabólica em adolescentes, utilizamos critérios da NCEP ATP III modificados que consistiram em: Obesidade Abdominal >= 90 percentil, HDL-colesterol <= 40 mg/dl, Triglicerídeo > = 110 mg/dl, Glicemia em Jejum >= 100 mg/dl, Pressão Arterial >= 90 percentil ajustável para idade e gênero. Participaram deste estudo 468 adolescentes sendo 168 (40,2%) do gênero masculino e 280 (59,8%) do gênero feminino de 06 (seis) escolas públicas e particulares, da cidade de São Luís/MA, durante o ano de 2012. A prevalência da Síndrome Metabólica foi de 12,2% sendo 30 pacientes do gênero masculino e 27 do gênero feminino. Houve uma predominância no gênero masculino e a faixa etária mais acometida foi 16-17 anos, seguido da faixa de 13-14 anos. Em relação aos componentes da Síndrome Metabólica, a Hipertensão Arterial foi o componente dominante (100% dos casos), seguida do HDL-colesterol diminuída (94,7%), obesidade (79%), triglicerídeos (71,9%), proteína C reativa média e alto risco em 28,1% dos casos, e glicemia em jejum alterada não foi encontrada em nenhum caso, porém, evidenciamos HOMA-IR alterado em 75,4% dos casos. Apesar de termos apena 01 referência na literatura brasileira na padronização sobre a medida da circunferência abdominal, foi realizado a sua medida na população estudada e a sua relação com a altura. Utilizamos como ponto de corte >= 0,5 para avaliar a adiposidade visceral nos pacientes estudados com Síndrome Metabólica encontramos relação Circunferência Abdominal e Altura aumentada em 66,7% dos casos. Em relação aos antecedentes mórbidos familiares dos adolescentes portadores de Síndrome Metabólica, a prevalência de obesidade foi de 22,8%, seguido de diabetes e hipertensão arterial em 17,5%, diabetes, hipertensão arterial e obesidade foi de 15,8%.

An immunoregulatory peptide from salivary glands of the horsefly, Hybomitra atriperoides

Horseflies are economically important blood-feeding arthropods and also a nuisance for humans, and vectors for filariasis. They rely heavily on the pharmacological propriety of their saliva to get blood meat and suppress immune reactions of hosts. Little information is available on horsefly immune suppressants. By high-performance liquid chromatography (HPLC) purification coupling with pharmacological testing, an immunoregulatory peptide named immunoregulin HA has been identified and characterized from salivary glands of the horsefly of Hybomitra atriperoides (Diptera, Tabanidae). Immunoregulin HA could inhibit the secretion of interferon-gamma (IFN-gamma) and monocyte chemoattractant protein (MCP-1) and increase the secretion of interteukin-10 (IL-10) induced by lipopolysaccharide (LIPS) in rat splenocytes. IL-10 is a suppressor cytokine of T-cell proliferative and cytokine responses. IL-10 can inhibit the elaboration of pro-inflammatory cytokines. Immunoregulin HA possibly unregulated the IL-10 production to inhibit IFN-gamma and MCP-1 secretion in the current experiments. This immunosuppression may facilitate the blood feeding of this horsefly. The current works will facilitate to understand the molecular mechanisms of the ectoparasite-host relationship. 2008 Elsevier Ltd. All rights reserved.

Androgen receptor targets NFKB and TSPI to suppress prostate tumor growth in vivo

The androgen role in the maintenance of prostate epithelium is subject to conflicting opinions. While androgen ablation drives the regression of normal and cancerous prostate, testosterone may cause both proliferation and apoptosis. Several investigators note decreased proliferation and stronger response to chemotherapy of the prostate cancer cells stably expressing androgen receptor (AR), however no mechanistic explanation was offered. In this paper we demonstrate in vivo anti-tumor effect of the AR on prostate cancer growth and identify its molecular mediators. We analyzed the effect of AR on the tumorigenicity of prostate cancer cells. Unexpectedly, the AR-expressing cells formed tumors in male mice at a much lower rate than the AR-negative controls. Moreover, the AR-expressing tumors showed decreased vascularity and massive apoptosis. AR expression lowered the angiogenic potential of cancer cells, by increasing secretion of an anti-angiogenic protein, thrombospondin-1. AR activation caused a decrease in RelA, a subunit of the pro-survival transcription factor NF kappa B, reduced its nuclear localization and transcriptional activity. This, in turn, diminished the expression of its anti-apoptotic targets, Bcl-2 and IL-6. Increased apoptosis within AR-expressing tumors was likely due to the NF kappa B suppression, since it was restricted to the cells lacking nuclear (active) NF kappa B. Thus we for the first time identified combined decrease of NF kappa B and increased TSP1 as molecular events underlying the AR anti-tumor activity in vivo. Our data indicate that intermittent androgen ablation is preferable to continuous withdrawal, a standard treatment for early-stage prostate cancer. (C) 2007 Wiley-Liss, Inc.

Identification and characterization of a cathepsin L-like cysteine protease from Taenia solium metacestode

Taenia solium metacestode, a larval pork tapeworm, is a causative agent of neurocysticercosis, one of the most common parasitic diseases in the human central nervous system. In this study, we identified a cDNA encoding for a cathepsin L-like cysteine protease from the T solium metacestode (TsCL-1) and characterized the biochemical properties of the recombinant enzyme. The cloned cDNA of 1216 bp encoded 339 amino acids with an approximate molecular weight of 37.6 kDa which containing a typical signal peptide sequence (17 amino acids), a pro-domain (106 amino acids), and a mature domain (216 amino acids). Sequence alignments of TsCL-1 showed low sequence similarity of 27.3-44.6 to cathepsin L-like cysteine proteases from other helminth parasites, but the similarity was increased to 35.9-55.0 when compared to mature domains. The bacterially expressed recombinant protein (rTsCL-1) did not show enzyme activity; however, the rTsCL-1 expressed in Pichia pastoris showed typical biochemical characteristics of cysteine proteases. It degraded human immunoglobulin G (IgG) and bovine serum albumin (BSA), but not collagen. Western blot analysis of the rTsCL-1 showed antigenicity against the sera from patients with cysticercosis, sparganosis or fascioliasis, but weak or no antigenicity against the sera from patients with paragonimiasis or clonorchiasis. (c) 2006 Published by Elsevier B.V.

PURIFICATION AND CHARACTERIZATION OF A KINASE SPECIFIC FOR THE SERINE-RICH AND ARGININE-RICH PRE-MESSENGER-RNA SPLICING FACTORS

Members of the SR family of pre-mRNA splicing factors are phosphoproteins that share a phosphoepitope specifically recognized by monoclonal antibody (mAb) 104. Recent studies have indicated that phosphorylation may regulate the activity and the intracellular localization of these splicing factors. Here, we report the purification and kinetic properties of SR protein kinase 1 (SRPK1), a kinase specific for SR family members. We demonstrate that the kinase specifically recognizes the SR domain, which contains serine/arginine repeats. Previous studies have shown that dephosphorylated SR proteins did not react with mAb 104 and migrated faster in SDS gels than SR proteins from mammalian cells. We show that SRPK1 restores both mobility and mAB 104 reactivity to a SR protein SF2/ASF (splicing factor 2/alternative splicing factor) produced in bacteria, suggesting that SRPK1 is responsible for the generation of the mAb 104-specific phosphoepitope in vivo. Finally, we have correlated the effects of mutagenesis in the SR domain of SF2/ASF on splicing with those on phosphorylation of the protein by SRPK1, suggesting that phosphorylation of SR proteins is required for splicing.

A colorimetric assay for screening microcystin class compounds in aquatic systems

Secondary metabolites produced by water-blooming cyanobacteria in eutrophic waters include some potent hepatotoxins, These compounds also have tumour-promoting properties, attributable to their inhibition and activation of protein phosphatases and kinases respectively. The inhibitory effect of these toxins on protein phosphatases have been employed in a commonly used radiometric assay, involving the use of a P-32-labeled substrate, for the detection and quantitation of these compounds. This paper investigates and describes a colorimetric method in which the activity of protein phosphatase 2A is determined by measuring the rate of colour production from the release of yellow p-nitrophenol using p-nitrophenyl phosphate as the substrate. Results of this study suggest that the colorimetric protein phosphatase inhibition assay is a simple, inexpensive tool for screening substances that may have tumour-promoting characteristics in aquatic systems. The detection limit of the colorimetric method is comparable to the radiometric assay. (C) 1998 Elsevier Science Ltd. All rights reserved.

CO2升高对土壤微生物群落影响的模拟研究

大气CO2浓度升高可以通过植物间接影响土壤生态系统。土壤生态系统的结构和功能改变将影响有机质矿化和营养物质循环，进而可能对CO2浓度升高产生正反馈或负反馈。微生物是土壤生态系统的主体，在对CO2浓度升高的反馈中起着至关重要的作用。本研究以开顶箱系统为平台，采用微生物分子生态学技术和现代酶学技术，通过对长期接受500 ppm CO2的红松幼树、长白赤松幼树和蒙古栎幼树非根际土壤连续两个生长季的测定，系统研究了高浓度CO2对温带森林土壤微生物群落的生物量和微生物活性的影响，检测了土壤微生物群落的结构和功能以及土壤化学性质变化，主要结论如下： （1）高浓度CO2处理提高了土壤有机碳含量。与对照组相比较，红松幼树土壤有机碳含量提高9.4%；长白赤松幼树土壤提高0.6%；蒙古栎幼树土壤提高1.3%。 （2）高浓度CO2处理使土壤磷酸酶（phosphatase）、几丁质酶（1,4-β-acetylglucosaminidase, 1,4-β-NAG）和多酚氧化酶（phenol oxidase）活性发生了显著变化，高浓度CO2使红松土壤 1,4-β-NAG活性提高7-25%，长白松土壤1,4-β-NAG平均活性降低14%，蒙古栎土壤1,4-β-NAG平均活性提高31%。 同时研究还发现，过氧化物酶（peroxidase）和多酚氧化酶(phenol oxidase)活性与微生物量碳和微生物量氮呈显著的正相关。相关分析还显示，土壤湿度与1,4-α-葡萄糖苷酶（1,4-α-glucosidase）活性、 微生物生物量碳和微生物生物量氮呈显著的正相关。 高浓度CO2在不同程度上改变了土壤转化酶活性和脱氢酶活性。高浓度CO2显著提高了红松和长白赤松土壤硝化酶活性；而显著降低反硝化酶活性。 （3）研究发现三种树土壤的真菌和细菌群落存在着季节性演替，并且高浓度CO2熏蒸处理使真菌群落结构发生了显著的变化，表现为一些种群优势度下降，另一些升高。虽然，细菌群落没有如真菌群落变化的明显，但研究中也发现高浓度CO2的确使个别细菌种群的优势度发生了显著改变。 亲缘关系与Calocybe carnea，Magmatodrilus obscurus密切的真菌是红松土壤优势种群，与Humicola fuscoatra关系相近的是长白松土壤的优势种群，并且此三种真菌的季节性变化不显著。研究发现高浓度CO2使红松土壤中亲缘关系与Pachyella clypeata，Cochlonema euryblastum，Lepiota cristata，Eimeriidae sp.， Trichoderma sp.相近的种群的丰富度显著提高，使蒙古栎土壤中亲缘关系与Serendipita vermifera，Calocybe carnea种群丰富度显著下降，使蒙古栎土壤中与Candida sp.，Magmatodrilus obscurus和Pachyella clypeata亲缘关系密切种群的丰富度显著提高。 （4）三种幼树叶的原位分解培养429天结果显示，红松和长白松凋落物的β-葡萄糖苷酶（1,4-β-glucosidase）和木糖苷酶（1,4-β-xylosidase）活性随着分解而逐渐增加，而这两种酶在蒙古栎凋落物分解过程中保持相对恒定；高浓度CO2显著影响叶凋落物分解磷酸酶（phosphatase），纤维二糖酶（cellobiohydrolase）, 几丁质酶(1,4-β-NAG)，多酚氧化酶（phenol oxidase）和过氧化物酶（peroxidase）的活性。研究发现，凋落物的生物化学性质变化能引起分解的微生物群落发生变化，进而引起分泌的胞外酶活性变化，科学印证了大气CO2浓度升高“通过影响凋落物质量进而影响分解叶凋落物的微生物群落的结构和功能”的猜测。 不同凋落物之间酶活性差异显著，真菌和细菌群落结构也显著不同。序列与Hyphodiscus hymeniophilus亲缘关系密切的真菌和亲缘关系与Verrucomicrobia bacterium密切的细菌是长白松凋落分解的最优势种群，序列与Lophium mytilinum亲缘关系密切的真菌是红松凋落分解的最优势种群。 另外，研究还发现，高浓度CO2使参与分解红松凋落物Beta proteobacterium OS-15A亲缘关系相近的细菌种群和与Azospirillum amazonense亲缘关系相近的种群丰富度显著降低；使与Luteibactor rhizovicina亲缘关系相近的种群和与Luteibactor rhizovicina亲缘关系相近的种群显著提高。高浓度CO2使定殖于长白松凋落物上Hyphodiscus hymeniophilus亲缘关系相近的种群和与Bionectria pityrodes亲缘关系相近的种群显著提高，而使与Neofabraea malicorticis亲缘关系相近的种群和与Hyphodiscus hymeniophilus亲缘关系相近的种群显著下降。

GUS基因在优化海带表达系统中的应用

本文研究了如何应用GUS基因优化海带表达系统。首先研究了GUS在各种海带材料中的本底值，而后以GUS基因作为报告基因比较了几种基因工程常用启动子在海带中启动瞬间表达的效率。选择表达效率较高的真核藻类启动子与GUS基因融合，在海带中获得了稳定表达的结果。分别用组织化学染色法和荧光分析法对海带雌配子体、雄配子体、孤雌生殖海带幼孢子体和二倍体海带孢子体等四种不同的材料的GUS本底进行了检测到GUS本底。荧光分析法所得数据显示，这四种材料的GUS活性分别为3.4±0.3、5.2±0.8、14.4±1.8、12.5±1.4 (pmol MU mg~(-1) protein min~(-1))，与高等植物类似，说明GUS基因可作为报造基因用于海带基因工程研究。选用真核藻启动子FCP启动子、高等植物基因工程中常用的高效Ubi启动子和CaMV35S启动子，与GUS基因融合构建四种质粒，通过基因松的方法转化孤雌生殖海带幼孢子体，比较GUS基因在海带中的瞬间表达量。实验结果表明，与CaMV35S和FCP融合的GUS基因在海带中表达效率较高。采用基因松的方法用FCP启动子－GUS基因转化海带雌配子体，通过诱导孤雌生殖获得孤雌生殖海带幼孢子体，用组织染色法检测到了GUS活性，并从海带的总DNA扩增到了FCP启动子－GUS基因的特征条带，从而提示了FCP启动子能引导外源基因在海带中稳定表达。为了找寻高效筛选元件进行孤雌生殖海带对草丁膦的敏感性实验，用统计学的方法得出了草丁膦对不同长度孤雌海带的半致死剂量。研究发现海带全长0.5-1.6cm范围内，草丁膦的LD_(50)与海带长度不相关，同时发现浓度在2-5μg/ml狭小范围内的草丁膦对孤雌海带比高浓度具有更为明显的毒性反应。本文结果提示草丁膦抗性基因－bar基因有可能成为海带基因工程更为理想的选择标记，因为海带对草丁膦比对氯霉素和潮霉素更敏感，而后两者是目前采用的筛选压力。本文结果为海带表达系统的优化，提供了有效的报告基因、启动子和选择标记元件。

Investigation of the function of the LNX family of E3 ubiquitin ligases in the nervous system

The Lnx1 (Ligand of Numb protein X 1) and Lnx2 genes belong to a family of PDZ domain-containing RING finger domain E3 ubiquitin ligases. mRNA expression for both genes have been reported in early murine central nervous system. However, there have been limited reports with regards to the expression patterns for both the proteins in vivo. Hence, we have attempted to characterize the significance of these proteins in the context of morphology and physiology of the central nervous system. Through our studies, we have attempted to examine closely the expression of these two genes in the murine central nervous system. We have also looked at novel interacting ligands for both proteins. Interacting partners have been examined with particular relevance to possible roles of their interactions with LNX1 and LNX2 in the functioning of the nervous system. Moreover, we have performed loss-of-function studies by way of creation and characterization of knockout mice.

Desensitization, internalization, and signaling functions of beta-arrestins demonstrated by RNA interference.

Beta-arrestins bind to activated G protein-coupled receptor kinase-phosphorylated receptors, which leads to their desensitization with respect to G proteins, internalization via clathrin-coated pits, and signaling via a growing list of "scaffolded" pathways. To facilitate the discovery of novel adaptor and signaling roles of beta-arrestins, we have developed and validated a generally applicable interfering RNA approach for selectively suppressing beta-arrestins 1 or 2 expression by up to 95%. Beta-arrestin depletion in HEK293 cells leads to enhanced cAMP generation in response to beta(2)-adrenergic receptor stimulation, markedly reduced beta(2)-adrenergic receptor and angiotensin II receptor internalization and impaired activation of the MAP kinases ERK 1 and 2 by angiotensin II. This approach should allow discovery of novel signaling and regulatory roles for the beta-arrestins in many seven-membrane-spanning receptor systems.