Anthrax toxin lethal factor domain 3 is highly mobile and responsive to ligand binding.

The secreted anthrax toxin consists of three components: the protective antigen (PA), edema factor (EF) and lethal factor (LF). LF, a zinc metalloproteinase, compromises the host immune system primarily by targeting mitogen-activated protein kinase kinases in macrophages. Peptide substrates and small-molecule inhibitors bind LF in the space between domains 3 and 4 of the hydrolase. Domain 3 is attached on a hinge to domain 2 via residues Ile300 and Pro385, and can move through an angular arc of greater than 35° in response to the binding of different ligands. Here, multiple LF structures including five new complexes with co-crystallized inhibitors are compared and three frequently populated LF conformational states termed `bioactive', `open' and `tight' are identified. The bioactive position is observed with large substrate peptides and leaves all peptide-recognition subsites open and accessible. The tight state is seen in unliganded and small-molecule complex structures. In this state, domain 3 is clamped over certain substrate subsites, blocking access. The open position appears to be an intermediate state between these extremes and is observed owing to steric constraints imposed by specific bound ligands. The tight conformation may be the lowest-energy conformation among the reported structures, as it is the position observed with no bound ligand, while the open and bioactive conformations are likely to be ligand-induced.

Monitoring of Coxiella burnetii in the Iberian lynx (Lynx pardinus).

Coxiella burnetii is a multi-host bacterium of major public and animal health concern. This pathogen circulates among several wild species in the Iberian Peninsula, however, the role of the Iberian lynx (Lynx pardinus) in the epidemiology of this emerging pathogen is still unknown. The objective of this work was to assess the circulation of C. burnetii in Iberian lynx populations from the Iberian Peninsula and to study the molecular characterisation of this pathogen in lynxes and their feeding ticks. A total of 922 lynxes, including free-ranging and captive individuals, were sampled between 2010 and 2022 for the collection of sera (n = 543), spleen samples (n = 390) and ticks (n = 357 from 61 lynxes). The overall seroprevalence was 7.7 % (42/543; 95 %CI: 5.5-10.0 %), with age being significantly associated with the C. burnetii exposure in free-ranging lynxes. A longitudinal study was also carried out to assess the dynamics of the circulation of C. burnetii in this wild host, revealing that 7 of the 37 longitudinally surveyed individuals seroconverted during the study period. The PCR prevalence was 4.4 % (17/390, 95 %CI: 2.3-6.4 %) for spleen samples and 1.1 % (4/357; 95 % CI: 0.0-2.2) in ticks. This is the first study to evaluate the circulation of C. burnetii in the Iberian lynx and to confirm the infection in this felid. The results obtained show a moderate, wide, homogeneous, and endemic circulation of this bacterium in the Iberian lynx populations.

Exposure to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the endangered Iberian lynx (Lynx pardinus).

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an emerging zoonotic virus of public and animal health concern, of which felids have been suggested as potential reservoirs. Although SARS-CoV-2 exposure has been detected in domestic and wild captive animals belonging to Felidae family, surveillance has not been carried out in free-ranging wild felids so far. The aim of the present study was to assess SARS-CoV-2 exposure in the Iberian lynx (Lynx pardinus), the most endangered felid in the world. Between 2019 and 2022, we conducted a seroepidemiological study of SARS-CoV-2 in 276 free-ranging and captive Iberian lynxes. Our results evidenced limited (0.4%; 95%CI: 0.0-1.1) but not negligible exposure to this emerging virus in this endangered felid species, increasing the SARS-CoV-2 host range. The circulation of this virus in wildlife evidences the need of integrated European wildlife monitoring.

Observational Study to Compare Biological Drug Concentration Quantification Techniques and Immunogenicity in Patients with Immune-Mediated Diseases.

Measuring biological drugs' trough concentrations and the concentrations of anti-drug antibodies is a valuable practice for treatment optimization. ELISA techniques are the gold standard for biological drug concentration quantification, but new techniques such as chemiluminescence immunoassays present some advantages. The aim of this unicentric prospective observational study is to compare the infliximab, adalimumab, vedolizumab and ustekinumab trough levels and anti-adalimumab and anti-infliximab antibodies concentrations obtained when using a chemiluminescent instrument (i-TRACK®, Theradiag, Croissy-Beaubourg, France) and an ELISA instrument (TRITURUS®, Griffols, Barcelona, Spain). Linear regression, Pearson or Spearman tests, Bland-Altman plots and the Cohen kappa test were applied for every sample. The correlation was excellent for both assays in the measurement of all drug concentrations. In general, values were lower when measured using i-TRACK than when using TRITURUS, especially when the values were high. Both techniques proved valuable in clinical practice for monitoring adalimumab and infliximab drug concentration. However, the results were modest for ustekinumab and vedolizumab, so caution is recommended and further research is needed. The limited number of anti-drug antibody-positive samples precluded a comparison between the techniques.

The structure of the TOG-like domain of Drosophila melanogaster Mast/Orbit.

Mast/Orbit is a nonmotor microtubule-associated protein (MAP) present in Drosophila melanogaster that reportedly binds microtubules at the plus end and is essential for mitosis. Sequence analysis has shown that the N-terminal domain (Mast-M1) resembles TOG domains from the Dis1-TOG family of proteins and stands as a representative of one of the three subclasses of divergent TOG-like domains (TOGL1) that includes human CLASP1. The crystal structure of Mast-M1 has been determined at 2.0 Å resolution and provides the first detailed structural description of any TOG-like domain. The structure confirms that Mast-M1 adopts a similar fold to the previously described Dis1-TOG domains of microtubule-binding proteins. A comparison with three known TOG-domain structures from XMAP215/Dis1 family members exposes significant differences between Mast-M1 and other TOG-domain structures in key residues at the proposed tubulin-binding edge.

Impact of confinement measures due to the COVID-19 pandemic on people living with dementia and their caregivers in Spain.

The COVID-19 pandemic has particularly affected people living with dementia (PLWD) and their caregivers, who have seen their access to social support services and opportunities for socialisation limited. The objective of the study was to explore the impact of COVID-19 on PLWD and their caregivers in Spain. An online survey was conducted between November 27, 2020, and January 19, 2021, that explored compliance with prevention guidelines, changes at the family level and in access to social support services. Instruments were included to estimate levels of anxiety and depression. The survey was answered by 229 people (161 current caregivers, 54 former caregivers, 13 formal caregivers and 1 person with dementia). Analysis of the current and former caregivers showed that they felt well informed, although they find it difficult for PLWD to comply with prevention guidelines. The use of social support services was reduced and the difficulty of access to social and health services increased, there was a negative impact on the economic situation and family relationships, with an increase in perceived overload. In addition, caregivers of PLWD scored above the cut-off points in the tests used to assess depression and anxiety, although the results of the multiple regression analysis do not allow us to conclude that the loss of resources influences the anxiety and depression scores. The negative impact of the pandemic on caregivers of PLWD is verified. It is necessary to adapt social support services and design strategies to maintain the provision of support to these vulnerable groups.

[Paraesophageal abscess: an uncommon cause of dysphagia in epidermolysis bullosa]. / Absceso paraesofágico: una causa poco frecuente de disfagia en la epidermólisis ampollosa.

INTRODUCTION: Epidermolysis bullosa encompasses a group of disorders characterized by the development of blisters on the skin and mucous membranes after minimal trauma. Gastrointestinal involvement is almost always present in the recessive dystrophic form, with the esophagus being one of the most frequent sites of extracutaneous manifestations. The most common symptom is dysphagia, which is usually secondary to esophageal blisters that evolve to scar tissue and stenosis. CASE REPORT: We report the case of a 48-year-old woman with recessive dystrophic epidermolysis bullosa who was referred because of dysphagia, with suspected esophageal stenosis. Pediatric gastroscopy was abandoned due to the development of blistering of the hypopharynx caused by the instrument and the apparent presence of extrinsic esophageal compression. To continue the examination, cervical computed tomography was performed, showing an image compatible with a paraesophageal abscess. After evaluating the risk-benefit ratio of performing endoscopic biopsy-drainage, we decided on conservative treatment, achieving favorable results and complete symptom resolution. CONCLUSIONS: We describe a case of paraesophageal abscess associated with epidermolysis bullosa, a rare cause of dysphagia in these patients, which was resolved with antibiotic and steroid treatment. In patients with this disease, invasive procedures, including endoscopy, have a high success rate. Despite the safety of these techniques, the utmost precautions should be taken, an appropriate technique should be used, and other diagnostic options should be considered.

Synergy within structural biology of single crystal optical spectroscopy and X-ray crystallography.

Advances in the adaptation of optical spectroscopy to monitor photo-induced or enzyme-catalyzed reactions in the crystalline state have enabled X-ray crystal structures to be accurately linked with spectroscopically defined intermediates. This, in turn, has led to a deeper understanding of the role protein structural changes play in function. The integration of optical spectroscopy with X-ray crystallography is growing and now extends beyond linking crystal structure to reaction intermediate. Recent examples of this synergy include applications in protein crystallization, X-ray data acquisition, radiation damage, and acquisition of phase information important for structure determination.

Targeting Mycobacterium tuberculosis Biotin Protein Ligase (MtBPL) with Nucleoside-Based Bisubstrate Adenylation Inhibitors.

Mycobacterium tuberculosis (Mtb), responsible for both latent and symptomatic tuberculosis (TB), remains the second leading cause of mortality among infectious diseases worldwide. Mycobacterial biotin protein ligase (MtBPL) is an essential enzyme in Mtb and regulates lipid metabolism through the post-translational biotinylation of acyl coenzyme A carboxylases. We report the synthesis and evaluation of a systematic series of potent nucleoside-based inhibitors of MtBPL that contain modifications to the ribofuranosyl ring of the nucleoside. All compounds were characterized by isothermal titration calorimetry (ITC) and shown to bind potently with K(D)s ≤ 2 nM. Additionally, we obtained high-resolution cocrystal structures for a majority of the compounds. Despite fairly uniform biochemical potency, the whole-cell Mtb activity varied greatly with minimum inhibitory concentrations (MIC) ranging from 0.78 to >100 µM. Cellular accumulation studies showed a nearly 10-fold enhancement in accumulation of a C-2'-α analogue over the corresponding C-2'-ß analogue, consistent with their differential whole-cell activity.

Long-term success of combined kidney-lung transplantation in a patient with cystic fibrosis.

Advanced kidney disease is usually considered an absolute contraindication for lung transplantation due to the difficult management of these patients in the post-operative period. Combined lung-kidney transplantation, however, could offer an opportunity for selected patients with renal and pulmonary dysfunction. This study summarizes the long-term success of a double transplantation in a 38-year-old male patient with cystic fibrosis who presented respiratory and kidney failure. After a complicated post-operative period, the patient currently lives completely independently 46 months after the operation and he enjoys excellent pulmonary and renal function.

Hemoglobin Seville [α2 ß2 81(EF5) Leu→Phe] a silent phenotypic variant that interferes in hemoglobin A1c measurement by ion-exchange HPLC method.

OBJECTIVES: The aim of the study was to investigate hemoglobin (Hb) species in a 61 year-old male with diabetes mellitus type II and a low value of Hb A(1c). DESIGN AND METHODS: Hb species were analyzed by electrophoresis and chromatography methods. Functional properties were determined by oxygen equilibrium studies. ß-globin gene was amplified by PCR and sequenced. RESULTS AND CONCLUSIONS: A novel clinically silent Hb (Hb Seville), that results in falsely low Hb A(1c) measurement, was detected. This Hb variant presented a single base mutation at codon 81 (C→T) of the ß-globin gene. This case points out the necessity of careful inspection of the chromatograms and the use of additional methods to Hb A(1c) measurement when the presence of aberrant peaks is detected.

Budget impact of using midnight salivary cortisol in the diagnosis of hypercortisolism.

BACKGROUND: A single midnight serum cortisol (MSC) test has been reported to possess the best sensitivity and specificity for diagnosing Cushing's syndrome (CS). However, this test requires patient hospitalization, making it costly. This paper aims to compare the hospital budget impact and accuracy of using midnight salivary cortisol (MSVC), as opposed to MSC, in the diagnosis of hypercortisolism. METHODS: 77 patients with at least two high urinary free cortisol (UFC) values (>360 nmol/24 h) were selected from 611 patients with clinical symptoms of CS. The costs of the method to confirm the diagnosis of hypercortisolism was calculated comparing Option A using MSC (UFCx2, low-dose dexamethasone suppression test [LDDST]) that requires patient hospitalization versus Option B using MSVC (UFCx2, LDDST) in which the evaluation is done outside the Hospital. A budget impact analysis for one year was developed, and a sensitivity analysis in different scenarios was performed. Reproducibility and diagnostic performance of MSVC and MSC were also measured. RESULTS: Salivary cortisol is a sound analytical method for evaluating free serum cortisol due to its classification accuracy, good imprecision, linearity, and stability. AUC(ROC) comparison between MSVC and MSC shows no significant differences. The substitution of the MSC for MSVC in our hospital could save between €16,762 and €132,804 in one year. CONCLUSIONS: The use of MSVC in the diagnosis of hypercortisolism can result in a substantial decrease in the budget impact, without losing diagnosis accuracy and reliability, a significant advantage considering the current emphasis on reducing the financial burden of health care.

Further insights into quinone cofactor biogenesis: probing the role of mauG in methylamine dehydrogenase tryptophan tryptophylquinone formation.

Paracoccus denitrificans methylamine dehydrogenase (MADH) is an enzyme containing a quinone cofactor tryptophan tryptophylquinone (TTQ) derived from two tryptophan residues (betaTrp(57) and betaTrp(108)) within the polypeptide chain. During cofactor formation, the two tryptophan residues become covalently linked, and two carbonyl oxygens are added to the indole ring of betaTrp(57). Expression of active MADH from P. denitrificans requires four other genes in addition to those that encode the polypeptides of the MADH alpha(2)beta(2) heterotetramer. One of these, mauG, has been shown to be involved in TTQ biogenesis. It contains two covalently attached c-type hemes but exhibits unusual properties compared to c-type cytochromes and diheme cytochrome c peroxidases, to which it has some sequence similarity. To test the role that MauG may play in TTQ maturation, the predicted proximal histidine to each heme (His(35) and His(205)) has each been mutated to valine, and wild-type MADH was expressed in the background of these two mauG mutants. The resultant MADH has been characterized by mass spectrometry and electrophoretic and kinetic analyses. The majority species is a TTQ biogenesis intermediate containing a monohydroxylated betaTrp(57), suggesting that this is the natural substrate for MauG. Previous work has shown that MADH mutated at the betaTrp(108) position (the non-oxygenated TTQ partner) is predominantly also this intermediate, and work on these mutants is extended and compared to the MADH expressed in the background of the histidine to valine mauG mutations. In this study, it is unequivocally demonstrated that MauG is required to initiate the formation of the TTQ cross-link, the conversion of a single hydroxyl located on betaTrp(57) to a carbonyl, and the incorporation of the second oxygen into the TTQ ring to complete TTQ biogenesis. The properties of MauG, which are atypical of c-type cytochromes, are discussed in the context of these final stages of TTQ biogenesis.