Differential regulation of horizontally acquired and core genome genes by the bacterial modulator H-NS

Horizontal acquisition of DNA by bacteria dramatically increases genetic diversity and hence successful bacterial colonization of several niches, including the human host. A relevant issue is how this newly acquired DNA interacts and integrates in the regulatory networks of the bacterial cell. The global modulator H-NS targets both core genome and HGT genes and silences gene expression in response to external stimuli such as osmolarity and temperature. Here we provide evidence that H-NS discriminates and differentially modulates core and HGT DNA. As an example of this, plasmid R27-encoded H-NS protein has evolved to selectively silence HGT genes and does not interfere with core genome regulation. In turn, differential regulation of both gene lineages by resident chromosomal H-NS requires a helper protein: the Hha protein. Tight silencing of HGT DNA is accomplished by H-NS-Hha complexes. In contrast, core genes are modulated by H-NS homoligomers. Remarkably, the presence of Hha-like proteins is restricted to the Enterobacteriaceae. In addition, conjugative plasmids encoding H-NS variants have hitherto been isolated only from members of the family. Thus, the H-NS system in enteric bacteria presents unique evolutionary features. The capacity to selectively discriminate between core and HGT DNA may help to maintain horizontally transmitted DNA in silent form and may give these bacteria a competitive advantage in adapting to new environments, including host colonization.

Differential regulation of horizontally acquired and core genome genes by the bacterial modulator H-NS

Horizontal acquisition of DNA by bacteria dramatically increases genetic diversity and hence successful bacterial colonization of several niches, including the human host. A relevant issue is how this newly acquired DNA interacts and integrates in the regulatory networks of the bacterial cell. The global modulator H-NS targets both core genome and HGT genes and silences gene expression in response to external stimuli such as osmolarity and temperature. Here we provide evidence that H-NS discriminates and differentially modulates core and HGT DNA. As an example of this, plasmid R27-encoded H-NS protein has evolved to selectively silence HGT genes and does not interfere with core genome regulation. In turn, differential regulation of both gene lineages by resident chromosomal H-NS requires a helper protein: the Hha protein. Tight silencing of HGT DNA is accomplished by H-NS-Hha complexes. In contrast, core genes are modulated by H-NS homoligomers. Remarkably, the presence of Hha-like proteins is restricted to the Enterobacteriaceae. In addition, conjugative plasmids encoding H-NS variants have hitherto been isolated only from members of the family. Thus, the H-NS system in enteric bacteria presents unique evolutionary features. The capacity to selectively discriminate between core and HGT DNA may help to maintain horizontally transmitted DNA in silent form and may give these bacteria a competitive advantage in adapting to new environments, including host colonization.

An affected core drives network integration deficits of the structural connectome in 22q11.2 deletion syndrome.

Chromosome 22q11.2 deletion syndrome (22q11DS) is a genetic disease known to lead to cerebral structural alterations, which we study using the framework of the macroscopic white-matter connectome. We create weighted connectomes of 44 patients with 22q11DS and 44 healthy controls using diffusion tensor magnetic resonance imaging, and perform a weighted graph theoretical analysis. After confirming global network integration deficits in 22q11DS (previously identified using binary connectomes), we identify the spatial distribution of regions responsible for global deficits. Next, we further characterize the dysconnectivity of the deficient regions in terms of sub-network properties, and investigate their relevance with respect to clinical profiles. We define the subset of regions with decreased nodal integration (evaluated using the closeness centrality measure) as the affected core (A-core) of the 22q11DS structural connectome. A-core regions are broadly bilaterally symmetric and consist of numerous network hubs - chiefly parietal and frontal cortical, as well as subcortical regions. Using a simulated lesion approach, we demonstrate that these core regions and their connections are particularly important to efficient network communication. Moreover, these regions are generally densely connected, but less so in 22q11DS. These specific disturbances are associated to a rerouting of shortest network paths that circumvent the A-core in 22q11DS, "de-centralizing" the network. Finally, the efficiency and mean connectivity strength of an orbito-frontal/cingulate circuit, included in the affected regions, correlate negatively with the extent of negative symptoms in 22q11DS patients, revealing the clinical relevance of present findings. The identified A-core overlaps numerous regions previously identified as affected in 22q11DS as well as in schizophrenia, which approximately 30-40% of 22q11DS patients develop.

Mammalian Target of Rapamycin Complex 2 Controls CD8 T Cell Memory Differentiation in a Foxo1-Dependent Manner.

Upon infection, antigen-specific naive CD8 T cells are activated and differentiate into short-lived effector cells (SLECs) and memory precursor cells (MPECs). The underlying signaling pathways remain largely unresolved. We show that Rictor, the core component of mammalian target of rapamycin complex 2 (mTORC2), regulates SLEC and MPEC commitment. Rictor deficiency favors memory formation and increases IL-2 secretion capacity without dampening effector functions. Moreover, mTORC2-deficient memory T cells mount more potent recall responses. Enhanced memory formation in the absence of mTORC2 was associated with Eomes and Tcf-1 upregulation, repression of T-bet, enhanced mitochondrial spare respiratory capacity, and fatty acid oxidation. This transcriptional and metabolic reprogramming is mainly driven by nuclear stabilization of Foxo1. Silencing of Foxo1 reversed the increased MPEC differentiation and IL-2 production and led to an impaired recall response of Rictor KO memory T cells. Therefore, mTORC2 is a critical regulator of CD8 T cell differentiation and may be an important target for immunotherapy interventions.

The fusion protein SS18-SSX1 employs core Wnt pathway transcription factors to induce a partial Wnt signature in synovial sarcoma.

Expression of the SS18/SYT-SSX fusion protein is believed to underlie the pathogenesis of synovial sarcoma (SS). Recent evidence suggests that deregulation of the Wnt pathway may play an important role in SS but the mechanisms whereby SS18-SSX might affect Wnt signaling remain to be elucidated. Here, we show that SS18/SSX tightly regulates the elevated expression of the key Wnt target AXIN2 in primary SS. SS18-SSX is shown to interact with TCF/LEF, TLE and HDAC but not β-catenin in vivo and to induce Wnt target gene expression by forming a complex containing promoter-bound TCF/LEF and HDAC but lacking β-catenin. Our observations provide a tumor-specific mechanistic basis for Wnt target gene induction in SS that can occur in the absence of Wnt ligand stimulation.

Delivery of antigen to nasal-associated lymphoid tissue microfold cells through secretory IgA targeting local dendritic cells confers protective immunity.

BACKGROUND: Transmission of mucosal pathogens relies on their ability to bind to the surfaces of epithelial cells, to cross this thin barrier, and to gain access to target cells and tissues, leading to systemic infection. This implies that pathogen-specific immunity at mucosal sites is critical for the control of infectious agents using these routes to enter the body. Although mucosal delivery would ensure the best onset of protective immunity, most of the candidate vaccines are administered through the parenteral route. OBJECTIVE: The present study evaluates the feasibility of delivering the chemically bound p24gag (referred to as p24 in the text) HIV antigen through secretory IgA (SIgA) in nasal mucosae in mice. RESULTS: We show that SIgA interacts specifically with mucosal microfold cells present in the nasal-associated lymphoid tissue. p24-SIgA complexes are quickly taken up in the nasal cavity and selectively engulfed by mucosal dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin-positive dendritic cells. Nasal immunization with p24-SIgA elicits both a strong humoral and cellular immune response against p24 at the systemic and mucosal levels. This ensures effective protection against intranasal challenge with recombinant vaccinia virus encoding p24. CONCLUSION: This study represents the first example that underscores the remarkable potential of SIgA to serve as a carrier for a protein antigen in a mucosal vaccine approach targeting the nasal environment.

Impact of T cell receptor affinity on the molecular mechanisms modulating CD8 T cell signalling and function against NY-ESO-1 tumour antigen

It is well established that cytotoxic T lymphocytes play a pivotal role in the protection against intracellular pathogens and tumour cells. Such protective immune responses rely on the specific T cell receptor (TCR)-mediated recognition by CD8 T cells of small antigenic peptides presented in the context of class-I Major Histocompatibility Complex molecules (pMHCs) on the surface of infected or malignant cells. The strength (affinity/avidity) of this interaction is a major correlate of protection. Although tumour-reactive CD8 T cells can be observed in cancer patients, anti-tumour immune responses are often ineffective in controlling or eradicating the disease due to the relative low TCR affinity of these cells. To overcome this limitation, tumour-specific CD8 T cells can be genetically modified to express TCRs of improved binding strength against a defined tumour antigen before adoptive cell transfer into cancer patients. We previously generated a panel of TCRs specific for the cancer-testis antigen NY-ESO-l,57.165 with progressively increased affinities for the pMHC complex, thus providing us with a unique tool to investigate the causal link between the surface expression of such TCRs and T cell activation and function. We recently demonstrated that anti-tumour CD8 T cell reactivity could only be improved within physiological affinity limits, beyond which drastic functional declines were observed, suggesting the presence of multiple regulatory mechanisms limiting T cell activation and function in a TCR affinity-dependent manner. The overarching goal of this thesis was (i) to assess the precise impact of TCR affinity on T cell activation and signalling at the molecular level and (ii) to gain further insights on the mechanisms that regulate and delimitate maximal/optimized CD8 T cell activation and signalling. Specifically, by combining several technical approaches we characterized the activation status of proximal (i.e. CD3Ç, Lek, and ZAP-70) and distal (i.e. ERK1/2) signalling molecules along the TCR affinity gradient. Moreover, we assessed the extent of TCR downmodulation, a critical step for initial T cell activation. CD8 T cells engineered with the optimal TCR affinity variants showed increased activation levels of both proximal and distal signalling molecules when compared to the wild-type T cells. Our analyses also highlighted the "paradoxical" status of tumour-reactive CD8 T cells bearing very high TCR affinities, which retained strong proximal signalling capacity and TCR downmodulation, but were unable to propagate signalling distally (i.e. pERKl/2), resulting in impaired cell-mediated functions. Importantly, these very high affinity T cells displayed maximal levels of SHP-1 and SHP-2 phosphatases, two negative regulatory molecules, and this correlated with a partial pERKl/2 signalling recovery upon pharmacological SHP-l/SHP-2 inhibition. These findings revealed the putative presence of inhibitory regulators of the TCR signalling cascade acting very rapidly following tumour-specific stimulation. Moreover, the very high affinity T cells were only able to transiently express enhanced proximal signalling molecules, suggesting the presence of an additional level of regulation that operates through the activation of negative feedback loops over time, limiting the duration of the TCR-mediated signalling. Overall, the determination of TCR-pMHC binding parameters eliciting optimal CD8 T cell activation, signalling, and effector function while guaranteeing high antigen specificity, together with the identification of critical regulatory mechanisms acting proximally in the TCR signalling cascade, will directly contribute to optimize and support the development of future TCR-based adoptive T cell strategies for the treatment of malignant diseases. -- Les lymphocytes T CD8 cytotoxiques jouent un rôle prédominant dans la protection contre les pathogènes intracellulaires et les cellules tumorales. Ces réponses immunitaires dépendent de la spécificité avec laquelle les récepteurs T (TCR) des lymphocytes CD8 reconnaissent les peptides antigéniques présentés par les molécules du complexe Majeur de Histocompatibilité de classe I (pCMH) à la surface des cellules infectées ou malignes. La force (ou affinité/avidité) de l'interaction du TCR-pCMH est un corrélat majeur de protection. Les réponses immunitaires sont cependant souvent inefficaces et ne permettent pas de contrôler ou d'éliminer les cellules tumorales chez les patients atteint du cancer, et ce à cause de la relative faible reconnaissance des TCRs exprimés par les lymphocytes T CD8 envers les antigènes tumoraux. Afin de surmonter cette limitation, les cellules T anti-tumorales peuvent être génétiquement modifiées en les dotant de TCRs préalablement optimisés afin d'augmenter leur reconnaissance ou affinité contre les antigènes tumoraux, avant leur ré¬infusion dans le patient. Nous avons récemment généré des cellules T CD8 exprimant un panel de TCRs spécifiques pour l'antigène tumoral NY-ESO-l157.16J avec des affinités croissantes, permettant ainsi d'investiguer la causalité directe entre l'affinité du TCR-pCMH et la fonction des cellules T CD8. Nous avons démontré que la réactivité anti-tumorale pouvait être améliorée en augmentant l'affinité du TCR dans une intervalle physiologique, mais au delà duquel nous observons un important déclin fonctionnel. Ces résultats suggèrent la présence de mécanismes de régulation limitant l'activation des cellules T de manière dépendante de l'affinité du TCR. Le but de cette thèse a été (i) de définir l'impact précis de l'affinité du TCR sur l'activation et la signalisation des cellules T CD8 au niveau moléculaire et (ii) d'acquérir de nouvelles connaissances sur les mécanismes qui régulent et délimitent l'activation et la signalisation maximale des cellules T CD8 optimisées. Spécifiquement, en combinant plusieurs approches technologiques, nous avons caractérisé l'état d'activation de différentes protéines de la voie de signalisation proximale (CD3Ç, Lek et ZAP-70) et distale (ERK1/2) le long du gradient d'affinité du TCR, ainsi que l'internalisation du TCR, une étape clef dans l'activation initiale des cellules T. Les lymphocytes T CD8 exprimant des TCRs d'affinité optimale ont montré des niveaux d'activation augmentés des molécules proximales et distales par rapport aux cellules de type sauvage (wild-type). Nos analyses ont également mis en évidence un paradoxe chez les cellules T CD8 équipées avec des TCRs de très haute affinité. En effet, ces cellules anti-tumorales sont capables d'activer leurs circuits biochimiques au niveau proximal et d'internaliser efficacement leur TCR, mais ne parviennent pas à propager les signaux biochimiques dépendants du TCR jusqu'au niveau distal (via phospho-ERKl/2), avec pour conséquence une limitation de leur capacité fonctionnelle. Finalement, nous avons démontré que SHP-1 et SHP-2, deux phosphatases avec des propriétés régulatrices négatives, étaient majoritairement exprimées dans les cellules T CD8 de très hautes affinités. Une récupération partielle des niveaux d'activation de ERK1/2 a pu être observée après l'inhibition pharmacologique de ces phosphatases. Ces découvertes révèlent la présence de régulateurs moléculaires qui inhibent le complexe de signalisation du TCR très rapidement après la stimulation anti-tumorale. De plus, les cellules T de très hautes affinités ne sont capables d'activer les molécules de la cascade de signalisation proximale que de manière transitoire, suggérant ainsi un second niveau de régulation via l'activation de mécanismes de rétroaction prenant place progressivement au cours du temps et limitant la durée de la signalisation dépendante du TCR. En résumé, la détermination des paramètres impliqués dans l'interaction du TCR-pCMH permettant l'activation de voies de signalisation et des fonctions effectrices optimales ainsi que l'identification des mécanismes de régulation au niveau proximal de la cascade de signalisation du TCR contribuent directement à l'optimisation et au développement de stratégies anti-tumorales basées sur l'ingénierie des TCRs pour le traitement des maladies malignes.

Step-by-step of ultrasound-guided core-needle biopsy of the breast: review and technique

Ultrasound-guided core-needle biopsy has high sensitivity in the diagnosis of breast cancer. The present study is aimed at detailing the main steps of such procedure, including indications, advantages, limitations, follow-up and description of the technique, besides presenting a checklist including the critical steps required for an appropriate practice of the technique. In the recent years, an increasing number of patients have required breast biopsy, indicating the necessity of a proportional increase in the number of skilled professionals to carry out the procedures and histological diagnoses. A multidisciplinary approach involving the tripod clinical practice-radiology-pathology is responsible for the highest rate of accuracy of the technique and must always be adopted.

Diagnostic underestimation of atypical ductal hyperplasia and ductal carcinoma in situ at percutaneous core needle and vacuum-assisted biopsies of the breast in a Brazilian reference institution

Abstract Objective: To determine the rates of diagnostic underestimation at stereotactic percutaneous core needle biopsies (CNB) and vacuum-assisted biopsies (VABB) of nonpalpable breast lesions, with histopathological results of atypical ductal hyperplasia (ADH) or ductal carcinoma in situ (DCIS) subsequently submitted to surgical excision. As a secondary objective, the frequency of ADH and DCIS was determined for the cases submitted to biopsy. Materials and Methods: Retrospective review of 40 cases with diagnosis of ADH or DCIS on the basis of biopsies performed between February 2011 and July 2013, subsequently submitted to surgery, whose histopathological reports were available in the internal information system. Biopsy results were compared with those observed at surgery and the underestimation rate was calculated by means of specific mathematical equations. Results: The underestimation rate at CNB was 50% for ADH and 28.57% for DCIS, and at VABB it was 25% for ADH and 14.28% for DCIS. ADH represented 10.25% of all cases undergoing biopsy, whereas DCIS accounted for 23.91%. Conclusion: The diagnostic underestimation rate at CNB is two times the rate at VABB. Certainty that the target has been achieved is not the sole determining factor for a reliable diagnosis. Removal of more than 50% of the target lesion should further reduce the risk of underestimation.

The lipopolysaccharide of Aeromonas spp: structure-activity relationships.

Marine microorganisms, including Aeromonas, are a source of compounds for drug development that have generated great expectations in the last decades. Aeromonas infections produce septicaemia, and ulcerative and haemorrhagic diseases in fish. Among the pathogenic factors associated with Aeromonas, the lipopolysaccharides (LPS), a surface glyconconjugate unique to Gram-negative bacteria consisting of lipid A (lipid anchor of the molecule), core oligosaccharide and O-specific polysaccharide (O antigen), are key elicitors of innate immune responses. The chemical structure of these three parts has been characterized in Aeromonas. Based on the high variability of repeated units of O-polysaccharides, a total of 97 O-serogroups have been described in Aeromonas species, of which four of them (O:11; O:16; O:18 and O:34) account for more than 60% of the septicemia cases. The core of LPS is subdivided into two regions, the inner (highly conserved) and the outer core. The inner core of Aeromonas LPS is characterized by the presence of 3-deoxy-D-manno-oct-2-ulosonic (ketodeoxyoctonic) acid (Kdo) and L-glycero-D-manno-Heptoses (L,D-Hep), which are linked to the outer core, characterized by the presence of Glc, GlcN, Gal, and GalNAc (in Aeromonas salmonicida), D,D-Hep (in Aeromonas salmonicida), and L,D-Hep (in Aeromonas hydrophila). The biological relevance of these differences in the distal part of the outer core among these species has not been fully assessed to date. The inner core is attached to the lipid A, a highly conserved structure that confers endotoxic properties to the LPS when the molecule is released in blood from lysed bacteria, thus inducing a major systemic inflammatory response known as septic or endotoxic shock. In Aeromonas salmonicida subsp. salmonicida the Lipid A components contain three major lipid A molecules, differing in acylation patterns corresponding to tetra-, penta- and hexaacylated lipid A species and comprising of 4′-monophosphorylated β-2-amino-2-deoxy-D-glucopyranose-(1→6)-2-amino-2-deoxy-D-glucopyranose disaccharide. In the present review, we discuss the structure-activity relationships of Aeromonas LPS, focusing on its role in bacterial pathogenesis and its possible applications.

Coolant Flow and Heat Transfer in Pebble-Bed Reactor Core

This thesis gathers knowledge about ongoing high-temperature reactor projects around the world. Methods for calculating coolant flow and heat transfer inside a pebble-bed reactor core are also developed. The thesis begins with the introduction of high-temperature reactors including the current state of the technology. Process heat applications that could use the heat from a high-temperature reactor are also introduced. A suitable reactor design with data available in literature is selected for the calculation part of the thesis. Commercial computational fluid dynamics software Fluent is used for the calculations. The pebble-bed is approximated as a packed-bed, which causes sink terms to the momentum equations of the gas flowing through it. A position dependent value is used for the packing fraction. Two different models are used to calculate heat transfer. First a local thermal equilibrium is assumed between the gas and solid phases and a single energy equation is used. In the second approach, separate energy equations are used for the phases. Information about steady state flow behavior, pressure loss, and temperature distribution in the core is obtained as results of the calculations. The effect of inlet mass flow rate to pressure loss is also investigated. Data found in literature and the results correspond each other quite well, considered the amount of simplifications in the calculations. The models developed in this thesis can be used to solve coolant flow and heat transfer in a pebble-bed reactor, although additional development and model validation is needed for better accuracy and reliability.

Nanoparticle Labels in Bioaffinity Assays

Nanoparticles offer adjustable and expandable reactive surface area compared to the more traditional solid phase forms utilized in bioaffinity assays due to the high surface to-volume ratio. The versatility of nanoparticles is further improved by the ability to incorporate various molecular complexes such as luminophores into the core. Nanoparticle labels composed of polystyrene, silica, inorganic crystals doped with high number of luminophores, preferably lanthanide(III) complexes, are employed in bioaffinity assays. Other label species such as semiconductor crystals (quantum dots) or colloidal gold clusters are also utilized. The surface derivatization of such particles with biomolecules is crucial for the applicability to bioaffinity assays. The effectiveness of a coating is reliant on the biomolecule and particle surface characteristics and the selected coupling technique. The most critical aspects of the particle labels in bioaffinity assays are their size-dependent features. For polystyrene, silica and inorganic phosphor particles, these include the kinetics, specific activity and colloidal stability. For quantum dots and gold colloids, the spectral properties are also dependent on particle size. This study reports the utilization of europium(III)-chelate-embedded nanoparticle labels in the development of bioaffinity assays. The experimental covers both the heterogeneous and homogeneous assay formats elucidating the wide applicability of the nanoparticles. It was revealed that the employment of europium(III) nanoparticles in heterogeneous assays for viral antigens, adenovirus hexon and hepatitis B surface antigen (HBsAg), resulted in sensitivity improvement of 10-1000 fold compared to the reference methods. This improvement was attributed to the extreme specific activity and enhanced monovalent affinity of the nanoparticles conjugates. The applicability of europium(III)-chelate-doped nanoparticles to homogeneous assay formats were proved in two completely different experimental settings; assays based on immunological recognition or proteolytic activity. It was shown that in addition to small molecule acceptors, particulate acceptors may also be employed due to the high specific activity of the particles promoting proximity-induced reabsorptive energy transfer in addition to non-radiative energy transfer. The principle of proteolytic activity assay relied on a novel dual-step FRET concept, wherein the streptavidin-derivatized europium(III)-chelate-doped nanoparticles were used as donors for peptide substrates modified with biotin and terminal europium emission compliant primary acceptor and a secondary quencher acceptor. The recorded sensitized emission was proportional to the enzyme activity, and the assay response to various inhibitor doses was in agreement with those found in literature showing the feasibility of the technique. Experiments regarding the impact of donor particle size on the extent of direct donor fluorescence and reabsorptive excitation interference in a FRET-based application was conducted with differently sized europium(III)-chelate-doped nanoparticles. It was shown that the size effect was minimal

Generalized three-sided assignment markets: consistency and the core

A class of three-sided markets (and games) is considered, where value is generated by pairs or triplets of agents belonging to different sectors, as well as by individuals. For these markets we analyze the situation that arises when some agents leave the market with some payoff To this end, we introduce the derived market (and game) and relate it to the Davis and Maschler (1965) reduced game. Consistency with respect to the derived market, together with singleness best and individual anti-monotonicity axiomatically characterize the core for these generalized three-sided assignment markets. These markets may have an empty core, but we define a balanced subclass, where the worth of each triplet is defined as the addition of the worths of the pairs it contains. Keywords: Multi-sided assignment market, Consistency, Core, Nucleolus. JEL Classification: C71, C78

Core communication components along the cancer care process: the perspective of breast cancer patients

This study sought to assess the impact of health care professional (HCP) communication on breast cancer patients across the acute care process as perceived by patients. Methodological approach was based on eight focus groups conducted with a sample of patients (n ¼ 37) drawn from 15 Spanish Regions; thematic analysis was undertaken using the National Cancer Institute (NCI) framework of HCP communication as the theoretical basis. Relevant results of this study were the identification of four main communication components: (1) reassurance in coping with uncertainty after symptom detection and prompt access until confirmed diagnosis; (2) fostering involvement before delivering treatments, by anticipating information on practical and emotional illness-related issues; (3) guidance on the different therapeutic options, through use of clinical scenarios; and, (4) eliciting the feeling of emotional exhaustion after ending treatments and addressing the management of potential treatment-related effects. These communication-related components highlighted the need for a comprehensive approach in this area of cancer care