Structural characterization of meningococcal vaccine antigen NadA and of its transcriptional regulator NadR in ligand-bound and free forms.

Serogroup B Neisseria meningitidis (MenB) is the cause of the invasive meningococcal disease (IMD). Bexsero is the first genome-derived vaccine against MenB. Neisserial adhesin A (NadA) is one of the three protein antigens included in Bexsero. The main aim of this work was to obtain detailed insights into the structure of vaccine NadA variant 3 (NadAv3) and into the molecular mechanisms governing its transcriptional regulation by NadR (Neisseria adhesin A Regulator). A deep understanding of nadA expression is important for understanding the contribution of NadA to vaccine-induced protection against meningococcal disease. NadA expression is regulated by the ligand-responsive transcriptional repressor NadR. The functional, biochemical and high-resolution structural characterization of NadR is presented in the first part of the thesis (Part One). These studies provide detailed insights into how small molecule ligands, such as hydroxyphenylacetate derivatives, found in relevant host niches, modulate the structure and activity of NadR, by ‘conformational selection’ of inactive forms. In the second part of the thesis (Part Two), strategies involving both protein engineering and crystal manipulation to increase the likelihood of solving the crystal structure of NadAv3 are described. The first approach was the rational design of new constructs of NadAv3, based on the recently solved crystal structure of a close sequence variant (NadAv5). Then, a comprehensive set of biochemical, biophysical and structural techniques were applied to investigate all the generated NadAv3 constructs. The well-characterized trimeric NadAv3 constructs represented a set of high quality reagents which were validated as probes for functional studies and as a platform for continued attempts for protein crystallization. Mutagenesis studies and screenings to identify a new crystal form of NadAv3 were performed to improve crystal quality; structure determination is ongoing. The atomic resolution structure of NadAv3 will help to understand its biological role as both an adhesin and a vaccine antigen

NadA3 structures reveal undecad coiled coils and LOX1 binding regions competed by meningococcus B vaccine-elicited human antibodies

Neisseria meningitidis serogroup B (MenB) is a major cause of sepsis and invasive meningococcal disease. A multicomponent vaccine, 4CMenB, is approved for protection against MenB. Neisserial adhesin A (NadA) is one of the main vaccine antigens, acts in host cell adhesion, and may influence colonization and invasion. Six major genetic variants of NadA exist and can be classified into immunologically distinct groups I and II. Knowledge of the crystal structure of the 4CMenB vaccine component NadA3 (group I) would improve understanding of its immunogenicity, folding, and functional properties and might aid antigen design. Here, X-ray crystallography, biochemical, and cellular studies were used to deeply characterize NadA3. The NadA3 crystal structure is reported; it revealed two unexpected regions of undecad coiled-coil motifs and other conformational differences from NadA5 (group II) not predicted by previous analyses. Structure-guided engineering was performed to increase NadA3 thermostability, and a second crystal structure confirmed the improved packing. Functional NadA3 residues mediating interactions with human receptor LOX-1 were identified. Also, for two protective vaccine-elicited human monoclonal antibodies (5D11, 12H11), we mapped key NadA3 epitopes. These vaccine-elicited human MAbs competed binding of NadA3 to LOX-1, suggesting their potential to inhibit host-pathogen colonizing interactions. The data presented provide a significant advance in the understanding of the structure, immunogenicity and function of NadA, one of the main antigens of the multicomponent meningococcus B vaccine.IMPORTANCE The bacterial microbe Neisseria meningitidis serogroup B (MenB) is a major cause of devastating meningococcal disease. An approved multicomponent vaccine, 4CMenB, protects against MenB. Neisserial adhesin A (NadA) is a key vaccine antigen and acts in host cell-pathogen interactions. We investigated the 4CMenB vaccine component NadA3 in order to improve the understanding of its immunogenicity, structure, and function and to aid antigen design. We report crystal structures of NadA3, revealing unexpected structural motifs, and other conformational differences from the NadA5 orthologue studied previously. We performed structure-based antigen design to engineer increased NadA3 thermostability. Functional NadA3 residues mediating interactions with the human receptor LOX-1 and vaccine-elicited human antibodies were identified. These antibodies competed binding of NadA3 to LOX-1, suggesting their potential to inhibit host-pathogen colonizing interactions. Our data provide a significant advance in the overall understanding of the 4CMenB vaccine antigen NadA

The Brief Strategic Treatment of Cardiophobia: A Clinical Case Study

AbstractMany individuals presenting to medical settings with heart-related symptoms for which no medical explanation is found might suffer from cardiophobia, but this condition is still poorly identified and addressed. This article presents a case of cardiophobia treated in an outpatient cardiac rehabilitation unit and, for the first time, describes the application of brief strategic therapy for the treatment of this condition. In the case reported, the first therapeutic encounter and the key elements of the strategic approach are described in detail with the aim to explain how brief strategic therapy works and how it can be used to identify and address cardiophobia-related behaviors. A 64-year-old male presented to cardiac rehabilitation reporting intense anxiety-provoking heart palpitations, and believing he was at risk of dying from a heart attack. After 3 sessions, an overall improvement in heart-related bodily sensations followed a decrease in the patient's continuous checking of his heartbeat and seeking reassurance—factors that were largely responsible for the persistence of the problem. Moreover, quantitative evaluation showed increased scores of mood state at the end of treatment. This improvement persisted at the 18-month follow-up. This case is an interesting example of how brief strategic therapy can contribute to the development of a new conceptual model for the diagnosis and treatment of cardiophobia. Still, more systematic research in the field is needed to prove the efficacy and effectiveness of this therapeutic approach on symptoms of heart-focused anxiety

Correlation of NM23-H1 cytoplasmic expression with metastatic stage in human prostate cancer tissue

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Epithelioid variant of pleomorphic liposarcoma as potential mimic of metastatic carcinoma

We report a case of epithelioid variant of pleomorphic liposarcoma (EPL) found in the the infrapatellar fat pad of Hoffa of a 31-year old male. Histologically, the predominant population was formed by epithelioid cells with eosinophilic or clear cytoplasm admixed with rare pleomorphic lipoblasts. The immunohistochemical panel was not helpful in the diagnosis. FISH analysis using the locus-specific indicator CHOP (12q13) dual color break apart was applied to representative formalin-fixed, paraffin-embedded tissue sections. The result of FISH indicated a rearranged CHOP (DDIT3) gene and confirmed the diagnosis of EPL. The EPL should be differentiated from a metastatic carcinoma or other type of sarcoma. In these cases a clinicopathological correlation and an exhaustive sampling of the specimen for demonstration of lipogenic areas or pleomorphic lipoblasts is always necessary. FISH with demonstration of CHOP gene rearrangement is useful in providing specific ancillary information for the difficult differential diagnosis of this case

Contributi per una flora vascolare di Toscana. VIII (440-506)

New localities and/or confirmations concerning 67 specific and subspecific plant taxa of Tuscan vascular flora, belonging to 59 genera and 37 families are presented: Alisma (Alismataceae), Amaranthus (Amaranthaceae), Leucojum, Sternbergia, Tristagma (Amaryllidaceae), Aloe (Asphodelaceae), Erigeron, Galinsoga, Hieracium, Rhagadiolus, Silybum, Soliva, Taraxacum (Asteraceae), Impatiens (Balsaminaceae), Berberis (Berberidaceae), Cardamine (Brassicaceae), Opuntia (Cactaceae), Cephalaria, Sixalix, Succisa (Caprifoliaceae), Silene (Caryophyllaceae), Convolvulus, Ipomoea (Convolvulaceae), Aeonium (Crassulaceae), Scirpus (Cyperaceae), Equisetum (Equisetaceae), Euphorbia (Euphorbiaceae), Astragalus, Trifolium (Fabaceae), Quercus (Fagaceae), Crocus (Iridaceae), Juncus (Juncaceae), Utricularia (Lentibulariaceae), Peplis (Lythraceae), Maclura (Moraceae), Nymphaea (Nymphaeaceae), Oenothera (Onagraceae), Anacamptis, Orchis (Orchidaceae), Orobanche (Orobanchaceae), Callitriche, Veronica (Plantaginaceae), Alopecurus, Eleusine, Glyceria, Phleum (Poaceae), Persicaria, Polygonum (Polygonaceae), Groenlandia (Potamogetonaceae), Clematis, Pulsatilla, Ranunculus (Ranunculaceae), Rhamnus (Rhamnaceae), Fragaria, Potentilla, Pyracantha (Rosaceae), Galium (Rubiaceae), Sparganium (Typhaceae), Vitis (Vitaceae). In the end, the conservation status of the units and eventual protection of the cited biotopes are discussed

Contributi per una flora vascolare di Toscana. VIII (440-506) [Contributions for a vascular flora of Tuscany. VIII (440-506)]

Contributions for a vascular flora of Tuscany. VIII (440-506). New localities and/or confirmations concerning 67 specific and subspecific plant taxa of Tuscan vascular flora, belonging to 59 genera and 37 families are presented: Alisma (Alismataceae), Amaranthus (Amaranthaceae), Leucojum, Sternbergia, Tristagma (Amaryllidaceae), Aloe (Asphodelaceae), Erigeron, Galinsoga, Hieracium, Rhagadiolus, Silybum, Soliva, Taraxacum (Asteraceae), Impatiens (Balsaminaceae), Berberis (Berberidaceae), Cardamine (Brassicaceae), Opuntia (Cactaceae), Cephalaria, Sixalix, Succisa (Caprifoliaceae), Silene (Caryophyllaceae), Convolvulus, Ipomoea (Convolvulaceae), Aeonium (Crassulaceae), Scirpus (Cyperaceae), Equisetum (Equisetaceae), Euphorbia (Euphorbiaceae), Astragalus, Trifolium (Fabaceae), Quercus (Fagaceae), Crocus (Iridaceae), Juncus (Juncaceae), Utricularia (Lentibulariaceae), Peplis (Lythraceae), Maclura (Moraceae), Nymphaea (Nymphaeaceae), Oenothera (Onagraceae), Anacamptis, Orchis (Orchidaceae), Orobanche (Orobanchaceae), Callitriche, Veronica (Plantaginaceae), Alopecurus, Eleusine, Glyceria, Phleum (Poaceae), Persicaria, Polygonum (Polygonaceae), Groenlandia (Potamogetonaceae), Clematis, Pulsatilla, Ranunculus (Ranunculaceae), Rhamnus (Rhamnaceae), Fragaria, Potentilla, Pyracantha (Rosaceae), Galium (Rubiaceae), Sparganium (Typhaceae), Vitis (Vitaceae). In the end, the conservation status of the units and eventual protection of the cited biotopes are discussed

Impact of COVID-19 on cardiovascular testing in the United States versus the rest of the world

Objectives: This study sought to quantify and compare the decline in volumes of cardiovascular procedures between the United States and non-US institutions during the early phase of the coronavirus disease-2019 (COVID-19) pandemic. Background: The COVID-19 pandemic has disrupted the care of many non-COVID-19 illnesses. Reductions in diagnostic cardiovascular testing around the world have led to concerns over the implications of reduced testing for cardiovascular disease (CVD) morbidity and mortality. Methods: Data were submitted to the INCAPS-COVID (International Atomic Energy Agency Non-Invasive Cardiology Protocols Study of COVID-19), a multinational registry comprising 909 institutions in 108 countries (including 155 facilities in 40 U.S. states), assessing the impact of the COVID-19 pandemic on volumes of diagnostic cardiovascular procedures. Data were obtained for April 2020 and compared with volumes of baseline procedures from March 2019. We compared laboratory characteristics, practices, and procedure volumes between U.S. and non-U.S. facilities and between U.S. geographic regions and identified factors associated with volume reduction in the United States. Results: Reductions in the volumes of procedures in the United States were similar to those in non-U.S. facilities (68% vs. 63%, respectively; p = 0.237), although U.S. facilities reported greater reductions in invasive coronary angiography (69% vs. 53%, respectively; p < 0.001). Significantly more U.S. facilities reported increased use of telehealth and patient screening measures than non-U.S. facilities, such as temperature checks, symptom screenings, and COVID-19 testing. Reductions in volumes of procedures differed between U.S. regions, with larger declines observed in the Northeast (76%) and Midwest (74%) than in the South (62%) and West (44%). Prevalence of COVID-19, staff redeployments, outpatient centers, and urban centers were associated with greater reductions in volume in U.S. facilities in a multivariable analysis. Conclusions: We observed marked reductions in U.S. cardiovascular testing in the early phase of the pandemic and significant variability between U.S. regions. The association between reductions of volumes and COVID-19 prevalence in the United States highlighted the need for proactive efforts to maintain access to cardiovascular testing in areas most affected by outbreaks of COVID-19 infection