Lower Gastrointestinal Microbiota in Health and Irritable Bowel Syndrome : Characterisation and Effect of Probiotic Intervention

The human gastrointestinal (GI) microbiota is a complex ecosystem that lives in symbiosis with its host. The growing awareness of the importance of the microbiota to the host as well as the development of culture-free laboratory techniques and computational methods has enormously expanded our knowledge of this microbial community. Irritable bowel syndrome (IBS) is a common functional bowel disorder affecting up to a fifth of the Western population. To date, IBS diagnosis has been based on GI symptoms and the exclusion of organic diseases. The GI microbiota has been found to be altered in this syndrome and probiotics can alleviate the symptoms, although clear links between the symptoms and the microbiota have not been demonstrated. The aim of the present work was to characterise IBS related alterations in the intestinal microbiota, their relation to IBS symptoms and their responsiveness to probiotic theraphy. In this thesis research, the healthy human microbiota was characterised by cloning and sequencing 16S rRNA genes from a faecal microbial community DNA pool that was first profiled and fractionated according to its guanine and cytosine content (%G+C). The most noticeable finding was that the high G+C Gram-positive bacteria (the phylum Actinobacteria) were more abundant compared to a corresponding library constructed from the unfractionated DNA pool sample. Previous molecular analyses of the gut microbiota have also shown comparatively low amounts of high G+C bacteria. Furthermore, the %G+C profiling approach was applied to a sample constructed of faecal DNA from diarrhea-predominant IBS (IBS-D) subjects. The phylogenetic microbial community comparison performed for healthy and IBS-D sequence libraries revealed that the IBS-D sample was rich in representatives of the phyla Firmicutes and Proteobacteria whereas Actinobacteria and Bacteroidetes were abundant in the healthy subjects. The family Lachnospiraceae within the Firmicutes was especially prevalent in the IBS-D sample. Moreover, associations of the GI microbiota with intestinal symptoms and the quality of life (QOL) were investigated, as well as the effect of probiotics on these factors. The microbial targets that were analysed with the quantitative real-time polymerase chain reaction (qPCR) in this study were phylotypes (species definition according to 16S rRNA gene sequence similarity) previously associated with either health or IBS. With a set of samples, the presence or abundance of a phylotype that had 94% 16S rRNA gene sequence similarity to Ruminococcus torques (R. torques 94%) was shown to be associated with the severity of IBS symptoms. The qPCR analyses for selected phylotypes were also applied to samples from a six-month probiotic intervention with a mixture of Lactobacillus rhamnosus GG, L. rhamnosus Lc705, Propionibacterium freudenreichii ssp. shermanii JS and Bifidobacterium breve Bb99. The intervention had been previously reported to alleviate IBS symptoms, but no associations with the analysed microbiota representatives were shown. However, with the phylotype-specific assays applied here, the abundance of the R. torques 94% -phylotype was shown to be lowered in the probiotic-receiving group during the probiotic supplementation, whereas a Clostridium thermosuccinogenes 85% phylotype, previously associated with a healthy microbiota, was found to be increased compared to the placebo group. To conclude, with the combination of methods applied, higher abundance of Actinobacteria was detected in the healthy gut than found in previous studies, and significant phylum-level microbiota alterations could be shown in IBS-D. Thus, the results of this study provide a detailed overview of the human GI microbiota in healthy subjects and in subjects with IBS. Furthermore, the IBS symptoms were linked to a particular clostridial phylotype, and probiotic supplementation was demonstrated to alter the GI microbiota towards a healthier state with regard to this and an additional bacterial phylotype. For the first time, distinct phylotype-level alterations in the microbiota were linked to IBS symptoms and shown to respond to probiotic therapy.

Hypertension, kardiotoxicitet och njursvikt i samband med tyrosinkinashämmaren sunitinib

Nybildning av blodkärl från tidigare existerande kärl, angiogenes, är ett väsentligt skede vid tumörtillväxt. Denna process regleras av bland annat tillväxtfaktorer, var av den vaskulära endoteliala tillväxtfaktorn har en central roll. Hämning av angiogenes kan ske antingen extracellulärt med hjälp av humaniserade monoklonala antikroppar eller intracellulärt med hjälp av småmolekylära hämmaren. Sunitinib är en småmolekylär multikinashämmare och inhiberar flera tyrosinkinasreceptorer som påverkar tumörtillväxten och metastasutvecklingen vid cancer. Sunitinibs främsta indikationer är gastrointestinala stromacellstumörer, metastaserad njurcellscancer och neuroendokrina tumörer i bukspottskörteln. Behandling med tyrosinkinashämmare orsakar biverkningar som hypertension, kardiotoxicitet och njursvikt, vilka antas bero på de hämmande effekterna på mål som inte är väsentliga för anti-cancer-aktiviteten (”off-target” biverkningar). Bland annat AMP-aktiverat proteinkinas (AMPK), ett kinas som upprätthåller metabolisk homeostas i hjärtat, inhiberas av sunitinib och antas framkalla kardiovaskulära biverkningar. För att reducera ”off-target” biverkningar strävar man till att hitta alternativ som minskar de skadliga effekterna utan att den terapeutiska aktiviteten försvagas. Bland annat ett begränsat kaloriintag har uppvisat skyddande effekt på hjärtat via mekanismer sammankopplade till ökad resistens mot oxidativ stress, inflammation och mitokondriell dysfunktion, samt avtagande apoptos och autofagi. Detta sker delvis genom aktivering av enzymet Sirt1. Syftet med den här studien var att undersöka ifall kaloribegränsning skyddar mot kardiovaskulära och renala biverkningar inducerade av sunitinib hos råttor. Dessutom studerades vilka signalkedjor i cellen som medverkar. I studien användes 40 spontant hypertensiva råttor samt 10 normotensiva Wistar-Kyoto råttor. Försöksdjuren delades in i fem grupper beroende på behandling; I WKY kontroll, II SHR kontroll, III SHR + kaloribegränsning 70 %, IV SHR + sunitinib 3 mg/kg och V SHR + sunitinib 3 mg/kg + kaloribegränsning 70 %. Behandlingsperioden var åtta veckor. Blodtrycket mättes varje vecka med svansmanchett, urinutsöndringen undersöktes vecka 4 och vecka 8 med metabolismburar, ultraljudsundersökning av hjärtat utfördes sista veckan och blodkärlens respons till acetylkolin och natriumnitroprussid studerades i samband med avlivning. Proteinerna Sirt1 och AMPK analyserades i hjärtat med Western blotting samt förekomsten av makrofagmarkören ED1 i njurarna med immunhistokemi. Studien visade att sunitinibdosen 3 mg/kg är mycket väl tolererbar hos råttor eftersom sunitinib inte orsakade högre blodtryck, kraftigare hypertrofi eller mer omfattande njurskada jämfört med obehandlade SHR- grupper. Utgående från resultaten kan man också konstatera att kaloribegränsningen har positiva kardiovaskulära effekter.

Rule-based modelling of iron homeostasis in tuberculosis

To establish itself within the host system, Mycobacterium tuberculosis (Mtb) has formulated various means of attacking the host system. One such crucial strategy is the exploitation of the iron resources of the host system. Obtaining and maintaining the required concentration of iron becomes a matter of contest between the host and the pathogen, both trying to achieve this through complex molecular networks. The extent of complexity makes it important to obtain a systems perspective of the interplay between the host and the pathogen with respect to iron homeostasis. We have reconstructed a systems model comprising 92 components and 85 protein-protein or protein-metabolite interactions, which have been captured as a set of 194 rules. Apart from the interactions, these rules also account for protein synthesis and decay, RBC circulation and bacterial production and death rates. We have used a rule-based modelling approach, Kappa, to simulate the system separately under infection and non-infection conditions. Various perturbations including knock-outs and dual perturbation were also carried out to monitor the behavioral change of important proteins and metabolites. From this, key components as well as the required controlling factors in the model that are critical for maintaining iron homeostasis were identified. The model is able to re-establish the importance of iron-dependent regulator (ideR) in Mtb and transferrin (Tf) in the host. Perturbations, where iron storage is increased, appear to enhance nutritional immunity and the analysis indicates how they can be harmful for the host. Instead, decreasing the rate of iron uptake by Tf may prove to be helpful. Simulation and perturbation studies help in identifying Tf as a possible drug target. Regulating the mycobactin (myB) concentration was also identified as a possible strategy to control bacterial growth. The simulations thus provide significant insight into iron homeostasis and also for identifying possible drug targets for tuberculosis.

Site-specific N-Linked Glycosylation of Receptor Guanylyl Cyclase C Regulates Ligand Binding, Ligand-mediated Activation and Interaction with Vesicular Integral Membrane Protein 36, VIP36

Guanylyl cyclase C (GC-C) is a multidomain, membrane-associated receptor guanylyl cyclase. GC-C is primarily expressed in the gastrointestinal tract, where it mediates fluid-ion homeostasis, intestinal inflammation, and cell proliferation in a cGMP-dependent manner, following activation by its ligands guanylin, uroguanylin, or the heat-stable enterotoxin peptide (ST). GC-C is also expressed in neurons, where it plays a role in satiation and attention deficiency/hyperactive behavior. GC-C is glycosylated in the extracellular domain, and differentially glycosylated forms that are resident in the endoplasmic reticulum (130 kDa) and the plasma membrane (145 kDa) bind the ST peptide with equal affinity. When glycosylation of human GC-C was prevented, either by pharmacological intervention or by mutation of all of the 10 predicted glycosylation sites, ST binding and surface localization was abolished. Systematic mutagenesis of each of the 10 sites of glycosylation in GC-C, either singly or in combination, identified two sites that were critical for ligand binding and two that regulated ST-mediated activation. We also show that GC-C is the first identified receptor client of the lectin chaperone vesicular integral membrane protein, VIP36. Interaction with VIP36 is dependent on glycosylation at the same sites that allow GC-C to fold and bind ligand. Because glycosylation of proteins is altered in many diseases and in a tissue-dependent manner, the activity and/or glycan-mediated interactions of GC-C may have a crucial role to play in its functions in different cell types.

A Calcium-Dependent Plasticity Rule for HCN Channels Maintains Activity Homeostasis and Stable Synaptic Learning

Theoretical and computational frameworks for synaptic plasticity and learning have a long and cherished history, with few parallels within the well-established literature for plasticity of voltage-gated ion channels. In this study, we derive rules for plasticity in the hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, and assess the synergy between synaptic and HCN channel plasticity in establishing stability during synaptic learning. To do this, we employ a conductance-based model for the hippocampal pyramidal neuron, and incorporate synaptic plasticity through the well-established Bienenstock-Cooper-Munro (BCM)-like rule for synaptic plasticity, wherein the direction and strength of the plasticity is dependent on the concentration of calcium influx. Under this framework, we derive a rule for HCN channel plasticity to establish homeostasis in synaptically-driven firing rate, and incorporate such plasticity into our model. In demonstrating that this rule for HCN channel plasticity helps maintain firing rate homeostasis after bidirectional synaptic plasticity, we observe a linear relationship between synaptic plasticity and HCN channel plasticity for maintaining firing rate homeostasis. Motivated by this linear relationship, we derive a calcium-dependent rule for HCN-channel plasticity, and demonstrate that firing rate homeostasis is maintained in the face of synaptic plasticity when moderate and high levels of cytosolic calcium influx induced depression and potentiation of the HCN-channel conductance, respectively. Additionally, we show that such synergy between synaptic and HCN-channel plasticity enhances the stability of synaptic learning through metaplasticity in the BCM-like synaptic plasticity profile. Finally, we demonstrate that the synergistic interaction between synaptic and HCN-channel plasticity preserves robustness of information transfer across the neuron under a rate-coding schema. Our results establish specific physiological roles for experimentally observed plasticity in HCN channels accompanying synaptic plasticity in hippocampal neurons, and uncover potential links between HCN-channel plasticity and calcium influx, dynamic gain control and stable synaptic learning.

Intestinal Cell Proliferation and Senescence Are Regulated by Receptor Guanylyl Cyclase C and p21

Guanylyl cyclase C (GC-C) is expressed in intestinal epithelial cells and serves as the receptor for bacterial heat-stable enterotoxin (ST) peptides and the guanylin family of gastrointestinal hormones. Activation of GC-C elevates intracellular cGMP, which modulates intestinal fluid-ion homeostasis and differentiation of enterocytes along the crypt-villus axis. GC-C activity can regulate colonic cell proliferation by inducing cell cycle arrest, and mice lacking GC-C display increased cell proliferation in colonic crypts. Activation of GC-C by administration of ST to wild type, but not Gucy2c(-/-), mice resulted in a reduction in carcinogen-induced aberrant crypt foci formation. In p53-deficient human colorectal carcinoma cells, ST led to a transcriptional up-regulation of p21, the cell cycle inhibitor, via activation of the cGMP-responsive kinase PKGII and p38 MAPK. Prolonged treatment of human colonic carcinoma cells with ST led to nuclear accumulation of p21, resulting in cellular senescence and reduced tumorigenic potential. Our results, therefore, identify downstream effectors for GC-C that contribute to regulating intestinal cell proliferation. Thus, genomic responses to a bacterial toxin can influence intestinal neoplasia and senescence.

Homeostasis of functional maps in active dendrites emerges in the absence of individual channelostasis

The maintenance of ion channel homeostasis, or channelostasis, is a complex puzzle in neurons with extensive dendritic arborization, encompassing a combinatorial diversity of proteins that encode these channels and their auxiliary subunits, their localization profiles, and associated signaling machinery. Despite this, neurons exhibit amazingly stereotypic, topographically continuous maps of several functional properties along their active dendritic arbor. Here, we asked whether the membrane composition of neurons, at the level of individual ion channels, is constrained by this structural requirement of sustaining several functional maps along the same topograph. We performed global sensitivity analysis on morphologically realistic conductance-based models of hippocampal pyramidal neurons that coexpressed six well-characterized functional maps along their trunk. We generated randomized models by varying 32 underlying parameters and constrained these models with quantitative experimental measurements from the soma and dendrites of hippocampal pyramidal neurons. Analyzing valid models that satisfied experimental constraints on all six functional maps, we found topographically analogous functional maps to emerge from disparate model parameters with weak pairwise correlations between parameters. Finally, we derived a methodology to assess the contribution of individual channel conductances to the various functional measurements, using virtual knockout simulations on the valid model population. We found that the virtual knockout of individual channels resulted in variable, measurement and location-specific impacts across the population. Our results suggest collective channelostasis as a mechanism behind the robust emergence of analogous functional maps and have significant ramifications for the localization and targeting of ion channels and enzymes that regulate neural coding and homeostasis.

Copper homeostasis in Salmonella is atypical and copper-CueP is a major periplasmic metal complex.

Salmonella enterica sv. typhimurium (S. enterica sv. Typhimurium) has two metal-transporting P(1)-type ATPases whose actions largely overlap with respect to growth in elevated copper. Mutants lacking both ATPases over-accumulate copper relative to wild-type or either single mutant. Such duplication of ATPases is unusual in bacterial copper tolerance. Both ATPases are under the control of MerR family metal-responsive transcriptional activators. Analyses of periplasmic copper complexes identified copper-CueP as one of the predominant metal pools. Expression of cueP was recently shown to be controlled by the same metal-responsive activator as one of the P(1)-type ATPase genes (copA), and copper-CueP is a further atypical feature of copper homeostasis in S. enterica sv. Typhimurium. Elevated copper is detected by a reporter construct driven by the promoter of copA in wild-type S. enterica sv. Typhimurium during infection of macrophages. Double mutants missing both ATPases also show reduced survival inside cultured macrophages. It is hypothesized that elevated copper within macrophages may have selected for specialized copper-resistance systems in pathogenic microorganism such as S. enterica sv. Typhimurium.

Structural Basis for Feed-Forward Transcriptional Regulation of Membrane Lipid Homeostasis in Staphylococcus aureus

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Collision tumour involving a rectal gastrointestinal stromal tumour with invasion of the prostate and a prostatic adenocarcinoma

Background: Gastrointestinal stromal tumours (GISTs) are the most common primary mesenchymal neoplasia in the gastrointestinal tract, although they represent only a small fraction of total gastrointestinal malignancies in adults (<2%). GISTs can be located at any level of the gastrointestinal tract; the stomach is the most common location (60-70%), in contrast to the rectum, which is most rare (4%). When a GIST invades into the adjacent prostate tissue, it can simulate prostate cancer. In this study, we report on a case comprising the unexpected collision between a rectal GIST tumour and a prostatic adenocarcinoma. Findings: We describe the complexity of the clinical, endoscopic and radiological diagnosis, of the differential diagnosis based on tumour biopsy, and of the role of neoadjuvant therapy using imatinib prior to surgical treatment. Conclusions: Although isolated cases of coexisting GISTs and prostatic adenocarcinomas have reviously been described, this is the first reported case in the medical literature of a collision tumour involving a rectal GIST and prostatic adenocarcinoma components.

The E2F2 Transcription Factor Sustains Hepatic Glycerophospholipid Homeostasis in Mice

Increasing evidence links metabolic signals to cell proliferation, but the molecular wiring that connects the two core machineries remains largely unknown. E2Fs are master regulators of cellular proliferation. We have recently shown that E2F2 activity facilitates the completion of liver regeneration after partial hepatectomy (PH) by regulating the expression of genes required for S-phase entry. Our study also revealed that E2F2 determines the duration of hepatectomy-induced hepatic steatosis. A transcriptomic analysis of normal adult liver identified "lipid metabolism regulation" as a major E2F2 functional target, suggesting that E2F2 has a role in lipid homeostasis. Here we use wild-type (E2F2(+/+)) and E2F2 deficient (E2F2(-/-)) mice to investigate the in vivo role of E2F2 in the composition of liver lipids and fatty acids in two metabolically different contexts: quiescence and 48-h post-PH, when cellular proliferation and anabolic demands are maximal. We show that liver regeneration is accompanied by large triglyceride and protein increases without changes in total phospholipids both in E2F2(+/+) and E2F2(-/-) mice. Remarkably, we found that the phenotype of quiescent liver tissue from E2F2(-/-) mice resembles the phenotype of proliferating E2F2(+/+) liver tissue, characterized by a decreased phosphatidylcholine to phosphatidylethanolamine ratio and a reprogramming of genes involved in generation of choline and ethanolamine derivatives. The diversity of fatty acids in total lipid, triglycerides and phospholipids was essentially preserved on E2F2 loss both in proliferating and non-proliferating liver tissue, although notable exceptions in inflammation-related fatty acids of defined phospholipid classes were detected. Overall, our results indicate that E2F2 activity sustains the hepatic homeostasis of major membrane glycerolipid components while it is dispensable for storage glycerolipid balance.

On the Dynamics of the Adenylate Energy System: Homeorhesis vs Homeostasis

Biochemical energy is the fundamental element that maintains both the adequate turnover of the biomolecular structures and the functional metabolic viability of unicellular organisms. The levels of ATP, ADP and AMP reflect roughly the energetic status of the cell, and a precise ratio relating them was proposed by Atkinson as the adenylate energy charge (AEC). Under growth-phase conditions, cells maintain the AEC within narrow physiological values, despite extremely large fluctuations in the adenine nucleotides concentration. Intensive experimental studies have shown that these AEC values are preserved in a wide variety of organisms, both eukaryotes and prokaryotes. Here, to understand some of the functional elements involved in the cellular energy status, we present a computational model conformed by some key essential parts of the adenylate energy system. Specifically, we have considered (I) the main synthesis process of ATP from ADP, (II) the main catalyzed phosphotransfer reaction for interconversion of ATP, ADP and AMP, (III) the enzymatic hydrolysis of ATP yielding ADP, and (IV) the enzymatic hydrolysis of ATP providing AMP. This leads to a dynamic metabolic model (with the form of a delayed differential system) in which the enzymatic rate equations and all the physiological kinetic parameters have been explicitly considered and experimentally tested in vitro. Our central hypothesis is that cells are characterized by changing energy dynamics (homeorhesis). The results show that the AEC presents stable transitions between steady states and periodic oscillations and, in agreement with experimental data these oscillations range within the narrow AEC window. Furthermore, the model shows sustained oscillations in the Gibbs free energy and in the total nucleotide pool. The present study provides a step forward towards the understanding of the fundamental principles and quantitative laws governing the adenylate energy system, which is a fundamental element for unveiling the dynamics of cellular life.

Dose terapêutica de radiação ionizante induz um fenótipo migratório em células de carcinoma de colon humano (caco-2): vias de sinalização envolvidas

O câncer colo-retal é a terceira neoplasia mais frequente em todo o mundo e a recorrência local e neoplasia refratária são desafios no tratamento do câncer colo-retal após a cirurgia convencional. Com o intuito de controlar a recorrência e aumentar a média de sobrevida dos pacientes, uma estratégia multidisciplinar que combina a radioterapia (RT) e a quimioterapia com o processo cirúrgico tem sido protocolo clínico de escolha. Embora esta combinação seja capaz de otimizar o tratamento, nem todos os pacientes são beneficiados com o protocolo quimio-rádio combinado, uma vez que existem os insucessos terapêuticos relacionados com a incidência de neoplasias secundárias tardias em pacientes que foram submetidos à RT para tratamento de neoplasias anteriores. Além da doença refratária, outro agravante da RT são os efeitos colaterais produzidos pela radiação ionizante (IR), em especial àqueles do trato gastrointestinal. Estes efeitos estão relacionados com alterações da homeostase do epitélio intestinal, através da desorganização dos complexos juncionais. Porém, os mecanismos que medeiam estes efeitos ainda não estão elucidados. Este estudo avaliou as vias de sinalização que medeiam os efeitos da IR em células Caco-2. Foi observado que a IR causa uma desorganização da junção aderente via Src, EGFR e MAPK, sendo estas alterações acompanhadas por desorganização do citoesqueleto de actina em todo o volume celular. Src, EGFR e MAPK participam de maneira diferenciada na modulação destes efeitos. Observamos também que a radiação aumenta a motilidade dessas células via Src e MAPK e não induz alteração na proliferação celular até 48 horas após o tratamento. Este é o primeiro trabalho que correlaciona vias de sobrevivência celular como Src, EGFR e MAPK com alterações nas proteínas de junção aderente, alterações do citoesqueleto e migração celular. Estes eventos são relacionados aos efeitos colaterais primários e tardios induzidos pela IR, e podem favorecer à aquisição de um fenótipo maligno herdável durante o fracionamento de doses na RT, favorecendo a progressão tumoral do câncer colo-retal. Logo, além da correlação das vias de sinalização envolvidas nos eventos induzidos pela IR mostrados neste estudo, os resultados também corroboram para um melhor entendimento da atividade farmacológica dos inibidores químicos utilizados, uma vez que muitos deles encontram-se em fase de ensaios pré-clínicos e clínicos.