Characterization of kefir yeasts with antifungal capacity against Aspergillus species.

Kefir is a fermented probiotic drink obtained by placing kefir granules in a suitable substrate. The kefir granules are a consortium of bacteria and yeasts embedded in a exopolysaccharide matrix. The aim of this research was the isolation and identification of yeasts from kefir of different origin, the evaluation of their antifungal capacity against Aspergillus spp., and the characterization of virulence related traits. Using RFLP of ITS1/ITS4 region, D1/D2 region sequencing, and RAPD techniques, 20 kefir isolates were identified as Geotrichum candidum, Pichia kudriavzevii, Pichia membranifaciens, Saccharomyces cerevisiae, and Candida ethanolica. Their antifungal capacity was evaluated by their conidia germination reduction, which allowed the selection of eight isolates with high to moderate conidia germination reduction against Aspergillus flavus and Aspergillus parasiticus. Furthermore, these selected isolates showed growth inhibition on contact in the dual culture assay for both Aspergillus species and 3 of them-belonging to S. cerevisiae and P. kudriavzevii species-generated volatile organic compounds which significantly affected the growth of both fungi. For the evaluation of virulence-related traits, growth at high temperatures, enzymatic activities, and the adhesion to Caco-2 cells were analyzed. The isolates did not present more than one positive virulence-related trait simultaneously. In particular, it is important to highlight that the adhesion capacity to the model of intestinal barrier was extremely low for all of them. According to the results obtained, further studies would be of interest for the possible use of these promising yeasts as biocontrol agents against fungi in food.

Selective Exoenzymatic Labeling of Lipooligosaccharides of Neisseria gonorrhoeae with α2,6-Sialoside Analogues.

The interactions between bacteria and their host often rely on recognition processes that involve host or bacterial glycans. Glycoengineering techniques make it possible to modify and study the glycans on the host's eukaryotic cells, but only a few are available for the study of bacterial glycans. Here, we have adapted selective exoenzymatic labeling (SEEL), a chemical reporter strategy, to label the lipooligosaccharides of the bacterial pathogen Neisseria gonorrhoeae, using the recombinant glycosyltransferase ST6Gal1, and three synthetic CMP-sialic acid derivatives. We show that SEEL treatment does not affect cell viability and can introduce an α2,6-linked sialic acid with a reporter group on the lipooligosaccharides by Western blot, flow cytometry and fluorescent microscopy. This new bacterial glycoengineering technique allows for the precise modification, here with α2,6-sialoside derivatives, and direct detection of specific surface glycans on live bacteria, which will aid in further unravelling the precise biological functions of bacterial glycans.

The SARS-CoV-2 Spike Glycoprotein Directly Binds Exogeneous Sialic Acids: A NMR View.

The interaction of the SARS CoV2 spike glycoprotein with two sialic acid-containing trisaccharides (α2,3 and α2,6 sialyl N-acetyllactosamine) has been demonstrated by NMR. The NMR-based distinction between the signals of those sialic acids in the glycans covalently attached to the spike protein and those belonging to the exogenous α2,3 and α2,6 sialyl N-acetyllactosamine ligands has been achieved by synthesizing uniformly 13 C-labelled trisaccharides at the sialic acid and galactose moieties. STD-1 H,13 C-HSQC NMR experiments elegantly demonstrate the direct interaction of the sialic acid residues of both trisaccharides with additional participation of the galactose moieties, especially for the α2,3-linked analogue. Additional experiments with the spike protein in the presence of a specific antibody for the N-terminal domain and with the isolated receptor binding and N-terminal domains of the spike protein unambiguously show that the sialic acid binding site is located at the N-terminal domain.

Selective 13 C-Labels on Repeating Glycan Oligomers to Reveal Protein Binding Epitopes through NMR: Polylactosamine Binding to Galectins.

A combined chemo-enzymatic synthesis/NMR-based methodology is presented to identify, in unambiguous manner, the distinctive binding epitope within repeating sugar oligomers when binding to protein receptors. The concept is based on the incorporation of 13 C-labels at specific monosaccharide units, selected within a repeating glycan oligomeric structure. No new chemical tags are added, and thus the chemical entity remains the same, while the presence of the 13 C-labeled monosaccharide breaks the NMR chemical shift degeneracy that occurs in the non-labeled compound and allows the unique identification of the different components of the oligomer. The approach is demonstrated by a proof-of-concept study dealing with the interaction of a polylactosamine hexasaccharide with five different galectins that display distinct preferences for these entities.

[A REVIEW ABOUT SPONTANEOUS MOVEMENTS DISORDERS IN SCHIZOPHRENIA]. / Una revisión sobre los Trastornos Espontáneos del Movimiento (TEM) en la esquizofrenia.

Spontaneous movements disorders are frequently observed in schizophrenia. Together with the negative and cognitive symptoms, they comprise manifestations more linked to the damage of this complex condition, although they are insufficient to make the diagnosis. When the psychotic symptoms express these phenomena, they have probably been causing deleterious effects for years. Within these intrinsic components, motor disorders are the most delayed, paradoxically, as a consequence of the progress of phar-macotherapy. Treatment with antipsychotics modified the clinical symptoms of schizophrenia by developing improvements in positive symptoms, disorganization and agitation, but also produced a change of another order, with the side effects that, many times, had difficulty in being distinguished from the pathology itself. Above all, the specific neuroleptics share to a greater or lesser extent of the production of extrapyramidal symptoms, classically expressed in motor alterations. Therefore, these abnormal movements are often mistakenly associated with antipsychotic medication in an exclusive manner, overshadowing primary motor manifestations.The objective of this work is to highlight the characteristics, importance and relationship with the conventional therapy of spon-taneous movements disorders in schizophrenia.

Wheat storage proteins: changes on the glutenins after wheat infection with different isolates of Fusarium graminearum.

Wheat gluten proteins are decisive for the industrial properties of flour, so alterations resulting from grain infection with Fusarium graminearum produce changes in the glutenin content that affect the baking properties. This work analyzes the high-molecular-weight glutenin changes from wheat flour with different degrees of F. graminearum infection at field, since these proteins are determinant for the quality properties of flour. Wheat cultivars-on field trials-infected with F. graminearum isolates of diverse aggressiveness showed severity values between 9.1 and 42.58% and thousand kernel weight values between 28.12 and 32.33 g. Negative correlations between severity and protein content and positive correlations between yield and protein content were observed, employing reversed-phase high-performance liquid chromatography and polyacrylamide gel electrophoresis. Furthermore, the protein signal changes were in agreement for both methodological approaches. Also, the degree of disease observed and the protein changes on infected wheat cultivars varied in relation with the aggressiveness of the isolate responsible for the infection. The principal component analysis showed a close arrangement among protein values obtained by HPLC. For each cultivar, two principal components were obtained, which explained 80.85%, 88.48%, and 93.33% of the total variance (cultivars Sy200, AGP Fast, and Klein Tigre respectively). To our knowledge, the approaches employed for the analysis of protein changes according to the degree of disease, as well as the thorough statistical analysis, are novel for the study of Fusarium Head Blight.

Quantifying Siglec-sialylated ligand interactions: a versatile 19F-T2 CPMG filtered competitive NMR displacement assay.

Sialic-acid-binding immunoglobulin-like lectins (Siglecs) are integral cell surface proteins crucial for the regulation of immune responses and the maintenance of immune tolerance through interactions with sialic acids. Siglecs recognize sialic acid moieties, usually found at the end of N-glycan and O-glycan chains. However, the different Siglecs prefer diverse presentations of the recognized sialic acid, depending on the type of glycosidic linkage used to link to the contiguous Gal/GalNAc or sialic acid moieties. This fact, together with possible O- or N-substitutions at the recognized glycan epitope significantly influences their roles in various immune-related processes. Understanding the molecular details of Siglec-sialoglycan interactions is essential for unraveling their specificities and for the development of new molecules targeting these receptors. While traditional biophysical methods like isothermal titration calorimetry (ITC) have been utilized to measure binding between lectins and glycans, contemporary techniques such as surface plasmon resonance (SPR), microscale thermophoresis (MST), and biolayer interferometry (BLI) offer improved throughput. However, these methodologies require chemical modification and immobilization of at least one binding partner, which can interfere the recognition between the lectin and the ligand. Since Siglecs display a large range of dissociation constants, depending on the (bio)chemical nature of the interacting partner, a general and robust method that could monitor and quantify binding would be highly welcomed. Herein, we propose the application of an NMR-based a competitive displacement assay, grounded on 19F T2-relaxation NMR and on the design, synthesis, and use of a strategic spy molecule, to assess and quantify sialoside ligand binding to Siglecs. We show that the use of this specific approach allows the quantification of Siglec binding for natural and modified sialosides, multivalent sialosides, and sialylated glycoproteins in solution, which differ in binding affinities in more than two orders of magnitude, thus providing invaluable insights into sialoglycan-mediated interactions.

Natural history and surgical outcomes of Rathke's cleft cysts: a Spanish multicenter study.

Rathke's cleft cysts (RCC) are a common type of lesion found in the sellar or suprasellar area. They are usually monitored clinically, but in some cases, surgery may be required. However, their natural progression is not yet well understood, and the outcomes of surgery are uncertain. The objective of this study is to evaluate the natural history of Rathke's cleft cysts in patients who are clinically monitored without treatment, and to determine the outcomes of surgery and the incidence of recurrences over time. Design and patients: National multicentric study of patients diagnosed of Rathke's cleft cyst (RCC- Spain) from 2000 onwards and followed in 15 tertiary centers of Spain. A total of 177 patients diagnosed of RCC followed for 67.3 months (6-215) and 88 patients who underwent surgery, (81 patients underwent immediate surgery after diagnosis and 7 later for subsequent growth) followed for 68.8 months (3-235). Results: The cyst size remained stable or decreased in 73.5% (133) of the patients. Only 44 patients (24.3%) experienced a cyst increase and 9 of them (5.1%) experienced an increase greater than 3 mm. In most of the patients who underwent surgery headaches and visual alterations improved, recurrence was observed in 8 (9.1%) after a median time of 96 months, and no predictors of recurrence were discovered. Conclusions: Rathke's cleft cysts without initial compressive symptoms have a low probability of growth, so conservative management is recommended. Patients who undergo transsphenoidal surgery experience rapid clinical improvement, and recurrences are infrequent. However, they can occur after a long period of time, although no predictors of recurrence have been identified.

Highly Diastereoselective Multicomponent Synthesis of Spirocyclopropyl Oxindoles Enabled by Rare-Earth Metal Salts.

The synthesis of polysubstituted spirocyclopropyl oxindoles using a series of rare-earth metal (REM) salts is reported. REMs, in particular Sc(OTf)3, allowed access to the target compounds by a multicomponent reaction with high diastereoselectivity (≤94:6:0:0). Density functional theory calculations on the model reaction are consistent with the observed selectivity and revealed that the special coordinating capabilities and the oxophilicity of the metal are key factors in inducing the formation of one main diastereoisomer.

Structural insights into Siglec-15 reveal glycosylation dependency for its interaction with T cells through integrin CD11b.

Sialic acid-binding Ig-like lectin 15 (Siglec-15) is an immune modulator and emerging cancer immunotherapy target. However, limited understanding of its structure and mechanism of action restrains the development of drug candidates that unleash its full therapeutic potential. In this study, we elucidate the crystal structure of Siglec-15 and its binding epitope via co-crystallization with an anti-Siglec-15 blocking antibody. Using saturation transfer-difference nuclear magnetic resonance (STD-NMR) spectroscopy and molecular dynamics simulations, we reveal Siglec-15 binding mode to α(2,3)- and α(2,6)-linked sialic acids and the cancer-associated sialyl-Tn (STn) glycoform. We demonstrate that binding of Siglec-15 to T cells, which lack STn expression, depends on the presence of α(2,3)- and α(2,6)-linked sialoglycans. Furthermore, we identify the leukocyte integrin CD11b as a Siglec-15 binding partner on human T cells. Collectively, our findings provide an integrated understanding of the structural features of Siglec-15 and emphasize glycosylation as a crucial factor in controlling T cell responses.

Mortality in Acromegaly Diagnosed in Older Individuals in Spain Is Higher in Women Compared to the General Spanish Population.

CONTEXT: There are no data on mortality of acromegaly diagnosed in older individuals. OBJECTIVE: This work aimed to compare clinical characteristics, growth hormone-related comorbidities, therapeutic approaches, and mortality rate of patients diagnosed before or after 2010 and to assess overall mortality rate compared with the general Spanish population. METHODS: A retrospective evaluation was conducted among Spanish tertiary care centers of 118 patients diagnosed with acromegaly at age 65 or older. Kaplan-Meier curves were constructed to trace survival, and Cox proportional hazard models were used to assess the risk factors associated with mortality. We also compared mortality with that of the Spanish population by using age- and sex-adjusted standardized mortality ratios (SMRs). RESULTS: No differences were found in first-line treatment or biochemical control, between both periods except for faster biochemical control after 2010. Twenty-nine (24.6%) patients died, without differences between groups, and had a median of follow-up 8.6 years (103, [72.3] months). Overall SMR was 1.02 (95% CI, 0.57-1.54), (0.60; 95% CI, 0.35-1.06) for men and (1.80; 95% CI, 1.07-2.94) for women. The most common cause of death was cardiovascular disease (CVD). CONCLUSION: The mortality in patients with acromegaly diagnosed in older individuals was no different between both periods, and there was no overall SMR difference compared with the general Spanish population. However, the SMR was higher in women. As CVD is the leading cause of mortality, it seems advisable to initiate an intense CVD protective treatment as soon as acromegaly is diagnosed, particularly in women, in addition to tight acromegaly control to prevent excess mortality.

Factors associated with therapeutic response in acromegaly diagnosed in the elderly in Spain.

Context: Some reports suggest that acromegaly in elderly patients has a more benign clinical behavior and could have a better response to first-generation long-acting somatostatin receptor ligands (SRL). However, there is no specific therapeutic protocol for this special subgroup of patients. Objective: This study aimed at identifying predictors of response to SRL in elderly patients. Design: Multicentric retrospective nationwide study of patients diagnosed with acromegaly at or over the age of 65 years. Results: One-hundred and eighteen patients (34 men, 84 women, mean age at diagnosis 71.7 ± 5.4 years old) were included. Basal insulin-like growth factor type 1 (IGF-1) above the upper limit of normal (ULN) and growth hormone (GH) levels (mean ± SD) were 2.7 ± 1.4 and 11.0 ± 11.9 ng/ml, respectively. The mean maximal tumor diameter was 12.3 ± 6.4 mm, and up to 68.6% were macroadenoma. Seventy-two out of 118 patients (61.0%) underwent surgery as primary treatment. One-third of patients required first-line medical treatment due to a rejection of surgical treatment or non-suitability because of high surgical risk. After first-line surgery, 45/72 (63.9%) were in disease remission, and 16/34 (46.7%) of those treated with SRL had controlled disease. Patients with basal GH at diagnosis ≤6 ng/ml had lower IGF-1 levels and had smaller tumors, and more patients in this group reached control with SRL (72.7% vs. 33.3%; p < 0.04) [OR: 21.3, IC: 95% (2.4-91.1)], while male patients had a worse response [OR: 0.09, IC 95% (0.01-0.75)]. The predictive model curve obtained for SRL response showed an AUC of 0.82 CI (0.71-0.94). Conclusions: The most frequent phenotype in newly diagnosed acromegaly in the elderly includes small adenomas and moderately high IGF-1 levels. GH at diagnosis ≤6 ng/ml and female gender, but not age per se, were associated with a greater chance of response to SRL.

Clinical and genetic characteristics in patients under 30 years with sporadic pituitary adenomas.

OBJECTIVE: Pituitary adenomas (PA) are rare in young patients, and additional studies are needed to fully understand their pathogenesis in this population. We describe the clinical and genetic characteristics of apparently sporadic PA in a cohort of young patients. DESIGN: Clinical and molecular analysis of 235 patients (age ≤ 30 years) with PA. Clinicians from several Spanish and Chilean hospitals provided data. METHODS: Genetic screening was performed via next-generation sequencing and comparative genomic hybridization array. Clinical variables were compared among paediatric, adolescent (<19 years) and young adults' (≥19-30 years) cohorts and types of adenomas. Phenotype-genotype associations were examined. RESULTS: Among the total cohort, mean age was 17.3 years. Local mass effect symptoms were present in 22.0%, and prolactinomas were the most frequent (44.7%). Disease-causing germline variants were identified in 22 individuals (9.3%), more exactly in 13.1 and 4.7% of the populations aged between 0-19 and 19-30 years, respectively; genetically positive patients were younger at diagnosis and had larger tumour size. Healthy family carriers were also identified. CONCLUSIONS: Variants in genes associated with syndromic forms of PAs were detected in a large cohort of apparently sporadic pituitary tumours. We have identified novel variants in well-known genes and set the possibility of incomplete disease penetrance in carriers of MEN1 alterations or a limited clinical expression of the syndrome. Despite the low penetrance observed, screening of AIP and MEN1 variants in young patients and relatives is of clinical value.

Noninferiority of heart failure nurse titration versus heart failure cardiologist titration. ETIFIC multicenter randomized trial.

INTRODUCTION AND OBJECTIVES: Beta-blockers, angiotensin-converting enzyme inhibitors (ACE inhibitors), angiotensin-II-receptor-blockers (ARB), and mineralocorticoid-receptor antagonists decrease mortality and heart failure (HF) hospitalizations in HF patients with reduced left ventricular ejection fraction. The effect is dose-dependent. Careful titration is recommended. However, suboptimal doses are common in clinical practice. This study aimed to compare the safety and efficacy of dose titration of the aforementioned drugs by HF nurses vs HF cardiologists. METHODS: ETIFIC was a multicenter (n=20) noninferiority randomized controlled open label trial. A total of 320 hospitalized patients with new-onset HF, reduced ejection fraction and New York Heart Association II-III, without beta-blocker contraindications were randomized 1:1 in blocks of 4 patients each stratified by hospital: 164 to HF nurse titration vs 156 to HF cardiologist titration (144 vs 145 analyzed). The primary endpoint was the beta-blocker mean relative dose (% of target dose) achieved at 4 months. Secondary endpoints included ACE inhibitors, ARB, and mineralocorticoid-receptor antagonists mean relative doses, associated variables, adverse events, and clinical outcomes at 6 months. RESULTS: The mean±standard deviation relative doses achieved by HF nurses vs HF cardiologists were as follows: beta-blockers 71.09%±31.49% vs 56.29%±31.32%, with a difference of 14.8% (95%CI, 7.5-22.1), P <.001; ACE inhibitors 72.61%±29.80% vs 56.13%±30.37%, P <.001; ARB 44.48%±33.47% vs 43.51%±33.69%, P=.93; and mineralocorticoid-receptor antagonists 71%±32.12% vs 70.47%±29.78%, P=.86; mean±standard deviation visits were 6.41±2.82 vs 2.81±1.58, P <.001, while the number (%) of adverse events were 34 (23.6) vs 30 (20.7), P=.55; and at 6 months HF hospitalizations were 1 (0.69) vs 9 (5.51), P=.01. CONCLUSIONS: ETIFIC is the first multicenter randomized trial to demonstrate the noninferiority of HF specialist-nurse titration vs HF cardiologist titration. Moreover, HF nurses achieved higher beta-blocker/ACE inhibitors doses, with more outpatient visits and fewer HF hospitalizations. Trial registry number: NCT02546856.

Prevalence and clinical impact of systemic autoimmune rheumatic disease in patients with silicosis.

BACKGROUND: Silicosis is associated with an increased risk of developing systemic autoimmune rheumatic disease (SARD). The prognostic implications of this association are poorly characterized. The aim of this study was to determine the prevalence of SARD and autoimmune markers in a cohort of patients with exposure to silica and assess their impact on prognosis. METHOD: We performed a prospective observational study of all patients attending the dedicated silicosis clinic of our pulmonology unit between 2009 and December 2017. Diagnosis was confirmed by a rheumatologist according to Spanish Rheumatology Society criteria. Autoimmune markers, pulmonary function tests, radiological progression, visits to the emergency department and primary care center, and hospital admissions for respiratory causes, and mortality were analyzed. RESULTS: Overall, 489 cases of silicosis and 95 cases of exposure were studied. In total, 54 (11.0%) patients with silicosis had SARD: 12 (2.4%) rheumatoid arthritis, 10 (2.0%) systemic lupus erythematosus, 10 (2.0%) systemic sclerosis, 3 (0.6%) Sjögren syndrome, 2 (0.4%) vasculitis associated with anti-neutrophil cytoplasmic antibodies (ANCA +), 6 (1.2%) psoriatic arthritis, 3 (0.6%) ankylosing spondylitis, and 8 (1.6%) other autoimmune diseases with no special features. The patients with SARD visited the emergency room more often (63.0% vs. 42.5%; p = 0.004), and progressed more rapidly (22.2 vs. 11.7%; p = 0.030). CONCLUSIONS: The presence of systemic rheumatic autoimmune diseases involves radiological progression and a higher clinical impact.

Prevalence and Clinical Impact of Systemic Autoimmune Rheumatic Disease in Patients with Silicosis. / Prevalencia e impacto clínico de las enfermedades reumatológicas autoinmunitarias sistémicas en pacientes con silicosis.

BACKGROUND: Silicosis is associated with an increased risk of developing systemic autoimmune rheumatic disease (SARD). The prognostic implications of this association are poorly characterized. The aim of this study was to determine the prevalence of SARD and autoimmune markers in a cohort of patients with exposure to silica and assess their impact on prognosis. METHOD: We performed a prospective observational study of all patients attending the dedicated silicosis clinic of our pulmonology unit between 2009 and December 2017. Diagnosis was confirmed by a rheumatologist according to Spanish Rheumatology Society criteria. Autoimmune markers, pulmonary function tests, radiological progression, visits to the emergency department and primary care center, and hospital admissions for respiratory causes, and mortality were analyzed. RESULTS: Overall, 489 cases of silicosis and 95 cases of exposure were studied. In total, 54 (11.0%) patients with silicosis had SARD: 12 (2.4%) rheumatoid arthritis, 10 (2.0%) systemic lupus erythematosus, 10 (2.0%) systemic sclerosis, 3 (0.6%) Sjögren syndrome, 2 (0.4%) vasculitis associated with anti-neutrophil cytoplasmic antibodies (ANCA +), 6 (1.2%) psoriatic arthritis, 3 (0.6%) ankylosing spondylitis, and 8 (1.6%) other autoimmune diseases with no special features. The patients with SARD visited the emergency room more often (63.0 vs. 42.5%; p = 0.004), and progressed more rapidly (22.2 vs. 11.7%; p = 0.030). CONCLUSIONS: The presence of systemic rheumatic autoimmune diseases involves radiological progression and a higher clinical impact.

The 6-Minute Walk Test as a Tool for Determining Exercise Capacity and Prognosis in Patients with Silicosis. / La prueba de la marcha de 6 minutos como factor pronóstico y de capacidad de ejercicio en pacientes con silicosis.

OBJECTIVE: To evaluate exercise capacity using the 6-minute walk test (6MWT) in patients with complicated silicosis, and to determine how results shape the prognostic value of 6MWT for hospitalization and mortality. METHOD: Prospective observational study in 143 patients with complicated silicosis who performed the 6MWT in our outpatient pulmonology clinic between 2009 and the end of 2016. Lung function tests, computed tomography findings and 6MWT parameters (distance walked and oxygen desaturation) were evaluated. RESULTS: Patients with complicated silicosis walked 387±92m. Results deteriorated as silicosis severity increased; category A: 407±97m, B: 370±88m, and C: 357±68m (P=.022). The 6MWT correlated positively with lung function and inversely with dyspnea determined by mMRC (P<.001). Patients with moderate/severe emphysema and greater CT90 presented a worse result on 6MWT. Patients who walked<350m had more hospitalizations (3.7±4.0) than those who walked>350m (1.1±2.9). There was an impact on survival: non-survivors walked 327.9±73.0m, while survivors walked 404.5±89.7m (P<.001). CONCLUSIONS: Spirometry values and category of complicated silicosis correlated with 6MWT and distance walked was a prognostic factor for hospitalization and mortality. The 6MWT is a useful tool in occupational health for monitoring patients with silicosis.

Paramagnetic Tag for Glycosylation Sites in Glycoproteins: Structural Constraints on Heparan Sulfate Binding to Robo1.

An enzyme- and click chemistry-mediated methodology for the site-specific nitroxide spin labeling of glycoproteins has been developed and applied. The procedure relies on the presence of single N-glycosylation sites that are present natively in proteins or that can be engineered into glycoproteins by mutational elimination of all but one glycosylation site. Recombinantly expressing glycoproteins in HEK293S (GnT1-) cells results in N-glycans with high-mannose structures that can be processed to leave a single GlcNAc residue. This can in turn be modified by enzymatic addition of a GalNAz residue that is subject to reaction with an alkyne-carrying TEMPO moiety using copper(I)-catalyzed click chemistry. To illustrate the procedure, we have made an application to a two-domain construct of Robo1, a protein that carries a single N-glycosylation site in its N-terminal domains. The construct has also been labeled with 15N at amide nitrogens of lysine residues to provide a set of sites that are used to derive an effective location of the paramagnetic nitroxide moiety of the TEMPO group. This, in turn, allowed measurements of paramagnetic perturbations to the spectra of a new high affinity heparan sulfate ligand. Calculation of distance constraints from these data facilitated determination of an atomic level model for the docked complex.

Pulmonary vein stenosis: Etiology, diagnosis and management.

Pulmonary vein stenosis (PVS) is rare condition characterized by a challenging diagnosis and unfavorable prognosis at advance stages. At present, injury from radiofrequency ablation for atrial fibrillation has become the main cause of the disease. PVS is characterized by a progressive lumen size reduction of one or more pulmonary veins that, when hemodynamically significant, may raise lobar capillary pressure leading to signs and symptoms such as shortness of breath, cough, and hemoptysis. Image techniques (transesophageal echocardiography, computed tomography, magnetic resonance and perfusion imaging) are essential to reach a final diagnosis and decide an appropriate therapy. In this regard, series from referral centers have shown that surgical and transcatheter interventions may improve prognosis. The purpose of this article is to review the etiology, assessment and management of PVS.