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Presents a response from M. Blaszczyk to a letter to the editor about his article "Substrate Specificity of Antinuclear Antibodies in Scleroderma,"published in the 68th vol. of the "Journal of Investigative Dermatology."

The effect of zinc on the biotransformation of phosphogypsum, COD reduction and growth rate (μmax day-1) of an SRB community and Desulfotomaculum ruminis in media with sodium lactate or ethanol was examined. Depending on the form of zinc (Zn3(PO4)2 x 4H2O, ZnSO4 x7H2O, ZnCl2, Zn(NO3)2 x 6H2O) and its initial concentration (0-80 mg Zn2+/l) lower sulphate reduction and COD reduction was observed. The effect of Zn2+ also depended on the composition of the studied populations and carbon source in the medium. The lowest inhibition of specific growth rate was determined in cultures of the pure strain and in medium with zinc phosphate (with lactate or ethanol IC50 = 63 or 75 mg Zn2+/l, respectively) and the highest in cultures of sulphate-reducing bacterial communities in medium with zinc nitrate (with lactate or ethanol IC50 = 35 or 20 mg Zn2+/l, respectively). [ABSTRACT FROM AUTHOR]

Dynamic assessment of myocardial blood flow (MBF) and myocardial flow reserve (MFR) provides additional information that can improve diagnostic accuracy of radionuclide myocardial perfusion imaging in some clinical situations. This study assessed processing repeatability of these parameters calculated using two models-net retention (RET) and one compartment (1CM) in dynamic SPECT studies, using the latest version of Corridor 4DM software (v2024). Data of 107 patients were analyzed retrospectively (57 of whom were assessed in our previous study using 4DM v2015). Dynamic SPECT studies were carried out using a routine two-day rest-dipyridamole protocol. Data was processed in 4DM v2024 twice by one operator and once by another operator. Automatic heart image positioning during post-processing in 4DM v2024 was significantly improved compared to v2015, reducing the number of studies requiring extensive manual corrections from 41 to 12%. This significantly improved interobserver processing repeatability of MFR values in RCA territory compared to our previous study using v2015-from r = 0.67 to 0.85 (p = 0.0034). Interobserver processing repeatability of MBF and MFR in all 107 patients was significantly better in RET model compared to 1CM model. In conclusion, RET model is more reliable for calculating MBF and MFR values based on dynamic SPECT studies., (© 2024. The Author(s).)

The coenzyme A (CoA) biosynthesis pathway has attracted attention as a potential target for much-needed novel antimicrobial drugs, including for the treatment of tuberculosis (TB), the lethal disease caused by Mycobacterium tuberculosis ( Mtb ). Seeking to identify inhibitors of Mtb phosphopantetheine adenylyltransferase ( Mtb PPAT), the enzyme that catalyses the penultimate step in CoA biosynthesis, we performed a fragment screen. In doing so, we discovered three series of fragments that occupy distinct regions of the Mtb PPAT active site, presenting a unique opportunity for fragment linking. Here we show how, guided by X-ray crystal structures, we could link weakly-binding fragments to produce an active site binder with a K D <20 μM and on-target anti- Mtb activity, as demonstrated using CRISPR interference. This study represents a big step toward validating Mtb PPAT as a potential drug target and designing a Mtb PPAT-targeting anti-TB drug., Competing Interests: The authors declare no conflict of interest., (© 2023 The Authors. Angewandte Chemie published by Wiley-VCH GmbH.)

Introduction: In this paper, we have analysed all hand glomangioma cases referred to our clinic in the context of symptoms, time to diagnosis, and the role of surgical resection of the lesion., Material and Methods: We have collected the following data: the presence of risk factors, manifestation, time to diagnosis, the treatment applied, and follow-up of patients., Results: We have collected medical records from six patients, three males and three females. The median age was 45 (IQR: 29.5-65.75). The main symptom in all patients was severe pain and tenderness. The first-choice physician(s) were: general practitioners, general surgeons, and neurologists. The median time to diagnosis was 7 (IQR: 5-10) years. The main complaint of our patients was severe pain - 9 (IQR: 9-10) on the VAS scale, which was significantly alleviated after surgical treatment - 0 (IQR: 0-0; p = 0.043)., Conclusions: Extremely long times to final diagnosis, and excellent outcomes of surgical treatment, highlight the necessity of raising awareness of glomangiomas among clinicians.

Anti-microbial resistance is a rising global healthcare concern that needs urgent attention as growing number of infections become difficult to treat with the currently available antibiotics. This is particularly true for mycobacterial infections like tuberculosis and leprosy and those with emerging opportunistic pathogens such as Mycobacterium abscessus , where multi-drug resistance leads to increased healthcare cost and mortality. M. abscessus is a highly drug-resistant non-tuberculous mycobacterium which causes life-threatening infections in people with chronic lung conditions such as cystic fibrosis. In this study, we explore M. abscessus phosphopantetheine adenylyl transferase (PPAT), an enzyme involved in the biosynthesis of Coenzyme A, as a target for the development of new antibiotics. We provide structural insights into substrate and feedback inhibitor binding modes of M. abscessus PPAT, thereby setting the basis for further chemical exploration of the enzyme. We then utilize a multi-dimensional fragment screening approach involving biophysical and structural analysis, followed by evaluation of compounds from a previous fragment-based drug discovery campaign against M. tuberculosis PPAT ortholog. This allowed the identification of an early-stage lead molecule exhibiting low micro molar affinity against M. abscessus PPAT (K d 3.2 ± 0.8 µM) and potential new ways to design inhibitors against this enzyme. The resulting crystal structures reveal striking conformational changes and closure of solvent channel of M. abscessus PPAT hexamer providing novel strategies of inhibition. The study thus validates the ligandability of M. abscessus PPAT as an antibiotic target and identifies crucial starting points for structure-guided drug discovery against this bacterium., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Thomas, McCarthy, El Bakali, Brown, Kim, Blaszczyk, Mendes, Abell, Floto, Coyne and Blundell.)

Modeling of cancellous bone has important applications in the detection and treatment of fatigue fractures and diseases like osteoporosis. In this paper, we present a fully coupled multiscale approach considering mechanical, electric and magnetic effects by using the multiscale finite element method and a two-phase material model on the microscale. We show numerical results for both scales, including calculations for a femur bone, comparing a healthy bone to ones affected by different stages of osteoporosis. Here, the magnetic field strength resulting from a small mechanical impact decreases drastically for later stages of the disease, confirming experimental research., (© 2021. The Author(s).)

The Coronavirus disease 2019 (COVID-19) pandemic, immigrant status and being a member of the LGBT+ community are all independent factors associated with increased stress levels. Few studies provide more complex analysis on this issue, and there has been no research on the cumulative burden of perceived stress that people belonging to both minorities experience in the current epidemiological situation. The aim of this study was to assess the ability to deal with an external situation during the third wave of the COVID-19 pandemic in Poland in the following groups with different stress levels (total sample n = 370): Polish heterosexual men ( n = 202), heterosexual men from Ukraine ( n = 131) and homo- and bisexual men (men who have sex with men-MSM) from Ukraine ( n = 37). A Perceived Stress Scale (PSS-10) was used. The analysis of the survey did not show statistically significant differences between the three study groups in the general level of perceived stress (24.71, 24.77 and 26.49 points, respectively, p = 0.551), but it revealed numerous differences in coping with various aspects of everyday functioning between these groups. Negative assessment of one's own health proved to be the main factor negatively affecting the level of perceived stress, however specific health risks, medical history or the participants' previous experience have not been taken into account in the study. Our research shows differences in the needs, resources and methods of coping with stress between men who are Polish citizens and migrants from Ukraine, both heterosexual and belonging to the MSM group. Proper identification and addressing of these needs, taking into account different availability of health services, could be the responsibility of NGOs or insurance providers. This should result in the reduction of mental health burdens and the risk of developing serious mental disorders, and consequently in better functioning of persons belonging to minorities and in a reduced burden on the health care system.

Coenzyme A (CoA) is a fundamental co-factor for all life, involved in numerous metabolic pathways and cellular processes, and its biosynthetic pathway has raised substantial interest as a drug target against multiple pathogens including Mycobacterium tuberculosis. The biosynthesis of CoA is performed in five steps, with the second and third steps being catalysed in the vast majority of prokaryotes, including M. tuberculosis, by a single bifunctional protein, CoaBC. Depletion of CoaBC was found to be bactericidal in M. tuberculosis. Here we report the first structure of a full-length CoaBC, from the model organism Mycobacterium smegmatis, describe how it is organised as a dodecamer and regulated by CoA thioesters. A high-throughput biochemical screen focusing on CoaB identified two inhibitors with different chemical scaffolds. Hit expansion led to the discovery of potent and selective inhibitors of M. tuberculosis CoaB, which we show to bind to a cryptic allosteric site within CoaB.

Modeling and simulation have quickly become equivalent pillars of research along with traditional theory and experimentation. The growing realization that most complex phenomena of interest span many orders of spatial and temporal scales has led to an exponential rise in the development and application of multiscale modeling and simulation over the past two decades. In this perspective, the associate editors of the International Journal for Multiscale Computational Engineering and their co-workers illustrate current applications in their respective fields spanning biomolecular structure and dynamics, civil engineering and materials science, computational mechanics, aerospace and mechanical engineering, and more. Such applications are highly tailored, exploit the latest and ever-evolving advances in both computer hardware and software, and contribute significantly to science, technology, and medical challenges in the 21st century.

CABS-dock is a computational method for protein-peptide molecular docking that does not require predefinition of the binding site. The peptide is treated as fully flexible, while the protein backbone undergoes small fluctuations and, optionally, large-scale rearrangements. Here, we present a specific CABS-dock protocol that enhances the docking procedure using fragmentary information about protein-peptide contacts. The contact information is used to narrow down the search for the binding peptide pose to the proximity of the binding site. We used information on a single-chosen and randomly chosen native protein-peptide contact to validate the protocol on the peptiDB benchmark. The contact information significantly improved CABS-dock performance. The protocol has been made available as a new feature of the CABS-dock web server (at http://biocomp.chem.uw.edu.pl/CABSdock/)., Short Abstract: CABS-dock is a tool for flexible docking of peptides to proteins. In this article, we present a protocol for CABS-dock docking driven by information about protein-peptide contact(s). Using information on individual protein-peptide contacts allows to improve the accuracy of CABS-dock docking., (© The Author(s) 2018. Published by Oxford University Press.)

Trehalose is an essential disaccharide for mycobacteria and a key constituent of several cell wall glycolipids with fundamental roles in pathogenesis. Mycobacteria possess two pathways for trehalose biosynthesis. However, only the OtsAB pathway was found to be essential in Mycobacterium tuberculosis , with marked growth and virulence defects of OtsA mutants and strict essentiality of OtsB2. Here, we report the first mycobacterial OtsA structures from Mycobacterium thermoresistibile in both apo and ligand-bound forms. Structural information reveals three key residues in the mechanism of substrate preference that were further confirmed by site-directed mutagenesis. Additionally, we identify 2-oxoglutarate and 2-phosphoglycerate as allosteric regulators of OtsA. The structural analysis in this work strongly contributed to define the mechanisms for feedback inhibition, show different conformational states of the enzyme, and map a new allosteric site. IMPORTANCE Mycobacterial infections are a significant source of mortality worldwide, causing millions of deaths annually. Trehalose is a multipurpose disaccharide that plays a fundamental structural role in these organisms as a component of mycolic acids, a molecular hallmark of the cell envelope of mycobacteria. Here, we describe the first mycobacterial OtsA structures. We show mechanisms of substrate preference and show that OtsA is regulated allosterically by 2-oxoglutarate and 2-phosphoglycerate at an interfacial site. These results identify a new allosteric site and provide insight on the regulation of trehalose synthesis through the OtsAB pathway in mycobacteria., (Copyright © 2019 Mendes et al.)

Alpha-1 antitrypsin (A1AT) deficiency is one of the most common genetic disorders in Caucasian population. There is a link between granulomatosis with polyangiitis (GPA) and most frequent variants of SERPINA1 gene encoding severe alpha-1 antitripsin deficiency. However, the potential effect of Pi*Z, Pi*S as well as other SERPINA1 variants on clinical course of vasculitis are not well understood. The aim of the study was to analyze the potential effect of A1AT protein phenotype representing the SERPINA1 gene variants on the clinical course of GPA. The study group consisted of 64 subjects with GPA, stratified according to the disease severity: patients in active phase (group I, n = 12), patients during remission on treatment (group II, n = 40) or untreated (group III, n = 12). Normal Pi*MM SERPINA1 genotype was detected by means of real-time polymerase chain reaction (PCR) or direct sequencing in 59 patients, Pi*MZ genotype in 2, and Pi*IM, Pi*MS or Pi*SZ in 1 patient respectively. The patients with abnormal Pi*Z, Pi*S, or Pi*I allele constituted 17% in group I, 5% in group II, and 8% in group III. The serum content of A1AT and high sensitivity C-reactive protein (hsCRP) assessed by nephelometry did not differ between the groups. Interestingly, the mean serum antiPR3-antibodies level detected by Elisa method was significantly greater in the GPA patients with Pi*Z, Pi*S, or Pi*I SERPINA1 variants than in the Pi*MM homozygotes. In summary, heterozygous Pi*MZ, Pi*MS, and Pi*SZ genotype was detected in 7.8% of total group of GPA patients, and in 10.5% of those with lung lesions. The abnormal alleles of Pi*S and Pi*Z may affect the clinical course of the disease.

Mycobacterium phenotypic hits are a good reservoir for new chemotypes for the treatment of tuberculosis. However, the absence of defined molecular targets and modes of action could lead to failure in drug development. Therefore, a combination of ligand-based and structure-based chemogenomic approaches followed by biophysical and biochemical validation have been used to identify targets for Mycobacterium tuberculosis phenotypic hits. Our approach identified EthR and InhA as targets for several hits, with some showing dual activity against these proteins. From the 35 predicted EthR inhibitors, eight exhibited an IC 50 below 50 μM against M. tuberculosis EthR and three were confirmed to be also simultaneously active against InhA. Further hit validation was performed using X-ray crystallography yielding eight new crystal structures of EthR inhibitors. Although the EthR inhibitors attain their activity against M. tuberculosis by hitting yet undefined targets, these results provide new lead compounds that could be further developed to be used to potentiate the effect of EthA activated pro-drugs, such as ethionamide, thus enhancing their bactericidal effect.

Interest in applications of protein crystallography to medicine was evident, as the first high-resolution structures emerged in the 50s and 60s. In Cambridge, Max Perutz and John Kendrew sought to understand mutations in sickle cell and other genetic diseases related to hemoglobin, while in Oxford, the group of Dorothy Hodgkin became interested in long-lasting zinc-insulin crystals for treatment of diabetes and later considered insulin redesign, as synthetic insulins became possible. The use of protein crystallography in structure-guided drug discovery emerged as enzyme structures allowed the identification of potential inhibitor-binding sites and optimization of interactions of hits using the structure of the target protein. Early examples of this approach were the use of the structure of renin to design antihypertensives and the structure of HIV protease in design of AIDS antivirals. More recently, use of structure-guided design with fragment-based drug discovery, which reduces the size of screening libraries by decreasing complexity, has improved ligand efficiency in drug design and has been used to progress three oncology drugs through clinical trials to FDA approval. We exemplify current developments in structure-guided target identification and fragment-based lead discovery with efforts to develop new antimicrobials for mycobacterial infections., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: The characterization of protein-peptide interactions is a challenge for computational molecular docking. Protein-peptide docking tools face at least two major difficulties: (1) efficient sampling of large-scale conformational changes induced by binding and (2) selection of the best models from a large set of predicted structures. In this paper, we merge an efficient sampling technique with external information about side-chain contacts to sample and select the best possible models., Methods: In this paper we test a new protocol that uses information about side-chain contacts in CABS-dock protein-peptide docking. As shown in our recent studies, CABS-dock enables efficient modeling of large-scale conformational changes without knowledge about the binding site. However, the resulting set of binding sites and poses is in many cases highly diverse and difficult to score., Results: As we demonstrate here, information about a single side-chain contact can significantly improve the prediction accuracy. Importantly, the imposed constraints for side-chain contacts are quite soft. Therefore, the developed protocol does not require precise contact information and ensures large-scale peptide flexibility in the broad contact area., Conclusions: The demonstrated protocol provides the extension of the CABS-dock method that can be practically used in the structure prediction of protein-peptide complexes guided by the knowledge of the binding interface.

Background: Many protein-protein interactions are mediated by a short linear motif. Usually, amino acid sequences of those motifs are known or can be predicted. It is much harder to experimentally characterize or predict their structure in the bound form. In this work, we test a possibility of using flexible docking of a short linear motif to predict the interaction interface of the EphB4-EphrinB2 complex (a system extensively studied for its significance in tumor progression)., Methods: In the modeling, we only use knowledge about the motif sequence and experimental structures of EphB4-EphrinB2 complex partners. The proposed protocol enables efficient modeling of significant conformational changes in the short linear motif fragment during molecular docking simulation. For the docking simulations, we use the CABS-dock method for docking fully flexible peptides to flexible protein receptors (available as a server at http://biocomp.chem.uw.edu.pl/CABSdock/ ). Based on the docking result, the protein-protein complex is reconstructed and refined., Results: Using this novel protocol, we obtained an accurate EphB4-EphrinB2 interaction model., Conclusions: The results show that the CABS-dock method may be useful as the primary docking tool in specific protein-protein docking cases similar to EphB4-EphrinB2 complex-that is, where a short linear motif fragment can be identified.

Small-molecule inhibitors of the mycobacterial transcriptional repressor EthR have previously been shown to act as boosters of the second-line antituberculosis drug ethionamide. Fragment-based drug discovery approaches have been used in the past to make highly potent EthR inhibitors with ethionamide boosting activity both in vitro and ex vivo. Herein, we report the development of fragment-sized EthR ligands with nanomolar minimum effective concentration values for boosting the ethionamide activity in Mycobacterium tuberculosis whole-cell assays.

EthR is a transcriptional repressor that increases Mycobacterium tuberculosis resistance to ethionamide. In this study, the EthR-DNA interaction has been investigated by native electrospray-ionization mass spectrometry for the first time. The results show that up to six subunits of EthR are able to bind to its operator.

Fever of unknown origin (FUO) remains one of the most difficult diagnostic challenges. The causes of FUO can be various diseases located in different organs. The aim of the study was to determine the prevalence and nature of pulmonary lesions during FUO. One hundred and sixty one patients with FUO participated in this prospective study. We performed a detailed comprehensive history, physical examination, and a wide spectrum of tests. The most common causes of FUO were infections (39%), autoimmune conditions (28%), and neoplasms (17%). Lung lesions were found in 30% of patients. In this group 35% were infections, 30% autoimmune diseases, and 4% cancer. Among patients with respiratory infections, there were cases of tuberculosis, atypical pneumonia, lung abscess, and bronchiectases. Autoimmune pulmonary lesions were observed during vasculitis and systemic lupus. The causes of FUO in the group of patients with lung lesions were also pulmonary embolism, sarcoidosis, and pulmonary fibrosis. Chest CT played an important role in the diagnosis of the causes of FUO with pulmonary manifestations. Pulmonary lesions are a common cause of FUO. Most FUO with pulmonary lesions are recognized during infections and autoimmune diseases. An important part of diagnosing FUO is a detailed evaluation of the respiratory system.

With the ever-increasing instances of resistance to frontline TB drugs there is the need to develop novel strategies to fight the worldwide TB epidemic. Boosting the effect of the existing second-line antibiotic ethionamide by inhibiting the mycobacterial transcriptional repressor protein EthR is an attractive therapeutic strategy. Herein we report the use of a fragment based drug discovery approach for the structure-guided systematic merging of two fragment molecules, each binding twice to the hydrophobic cavity of EthR from M. tuberculosis. These together fill the entire binding pocket of EthR. We elaborated these fragment hits and developed small molecule inhibitors which have a 100-fold improvement of potency in vitro over the initial fragments.

The osteoclast-associated receptor (OSCAR) is a collagen-binding immune receptor with important roles in dendritic cell maturation and activation of inflammatory monocytes as well as in osteoclastogenesis. The crystal structure of the OSCAR ectodomain is presented, both free and in complex with a consensus triple-helical peptide (THP). The structures revealed a collagen-binding site in each immunoglobulin-like domain (D1 and D2). The THP binds near a predicted collagen-binding groove in D1, but a more extensive interaction with D2 is facilitated by the unusually wide D1-D2 interdomain angle in OSCAR. Direct binding assays, combined with site-directed mutagenesis, confirm that the primary collagen-binding site in OSCAR resides in D2, in marked contrast to the related collagen receptors, glycoprotein VI (GPVI) and leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1). Monomeric OSCAR D1D2 binds to the consensus THP with a KD of 28 µM measured in solution, but shows a higher affinity (KD 1.5 μM) when binding to a solid-phase THP, most likely due to an avidity effect. These data suggest a 2-stage model for the interaction of OSCAR with a collagen fibril, with transient, low-affinity interactions initiated by the membrane-distal D1, followed by firm adhesion to the primary binding site in D2., (© 2016 by The American Society of Hematology.)

Protein-peptide interactions play essential functional roles in living organisms and their structural characterization is a hot subject of current experimental and theoretical research. Computational modeling of the structure of protein-peptide interactions is usually divided into two stages: prediction of the binding site at a protein receptor surface, and then docking (and modeling) the peptide structure into the known binding site. This paper presents a comprehensive CABS-dock method for the simultaneous search of binding sites and flexible protein-peptide docking, available as a user's friendly web server. We present example CABS-dock results obtained in the default CABS-dock mode and using its advanced options that enable the user to increase the range of flexibility for chosen receptor fragments or to exclude user-selected binding modes from docking search. Furthermore, we demonstrate a strategy to improve CABS-dock performance by assessing the quality of models with classical molecular dynamics. Finally, we discuss the promising extensions and applications of the CABS-dock method and provide a tutorial appendix for the convenient analysis and visualization of CABS-dock results. The CABS-dock web server is freely available at http://biocomp.chem.uw.edu.pl/CABSdock/., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

GlgE, an enzyme of the pathway that converts trehalose to α-glucans, is essential for Mycobacterium tuberculosis. Inhibition of GlgE, which transfers maltose from a maltose-1-phosphate donor to α-glucan/maltooligosaccharide chain acceptor, leads to a toxic accumulation of maltose-1-phosphate that culminates in cellular death. Here we describe the first high-resolution mycobacterial GlgE structure from Mycobacterium thermoresistibile at 1.96 Å. We show that the structure resembles that of M. tuberculosis and Streptomyces coelicolor GlgEs, reported before, with each protomer in the homodimer comprising five domains. However, in M. thermoresistibile GlgE we observe several conformational states of the S domain and provide evidence that its high flexibility is important for enzyme activity. The structures here reported shed further light on the interactions between the N-terminal domains and the catalytic domains of opposing chains and how they contribute to the catalytic reaction. Importantly this work identifies a useful surrogate system to aid the development of GlgE inhibitors against opportunistic and pathogenic mycobacteria.

How is information communicated both within and between cells of living systems with high signal to noise? We discuss transmembrane signaling models involving two receptor tyrosine kinases: the fibroblast growth factor receptor (FGFR) and the MET receptor. We suggest that simple dimerization models might occur opportunistically giving rise to noise but cooperative clustering of the receptor tyrosine kinases observed in these systems is likely to be important for signal transduction. We propose that this may be a more general prerequisite for high signal to noise in transmembrane receptor signaling., (Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Protein-peptide interactions play a key role in cell functions. Their structural characterization, though challenging, is important for the discovery of new drugs. The CABS-dock web server provides an interface for modeling protein-peptide interactions using a highly efficient protocol for the flexible docking of peptides to proteins. While other docking algorithms require pre-defined localization of the binding site, CABS-dock does not require such knowledge. Given a protein receptor structure and a peptide sequence (and starting from random conformations and positions of the peptide), CABS-dock performs simulation search for the binding site allowing for full flexibility of the peptide and small fluctuations of the receptor backbone. This protocol was extensively tested over the largest dataset of non-redundant protein-peptide interactions available to date (including bound and unbound docking cases). For over 80% of bound and unbound dataset cases, we obtained models with high or medium accuracy (sufficient for practical applications). Additionally, as optional features, CABS-dock can exclude user-selected binding modes from docking search or to increase the level of flexibility for chosen receptor fragments. CABS-dock is freely available as a web server at http://biocomp.chem.uw.edu.pl/CABSdock., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The CABS-fold web server provides tools for protein structure prediction from sequence only (de novo modeling) and also using alternative templates (consensus modeling). The web server is based on the CABS modeling procedures ranked in previous Critical Assessment of techniques for protein Structure Prediction competitions as one of the leading approaches for de novo and template-based modeling. Except for template data, fragmentary distance restraints can also be incorporated into the modeling process. The web server output is a coarse-grained trajectory of generated conformations, its Jmol representation and predicted models in all-atom resolution (together with accompanying analysis). CABS-fold can be freely accessed at http://biocomp.chem.uw.edu.pl/CABSfold.

Objective: Activation of Smad1 signaling has recently been implicated in the development of fibrosis. The goal of the present study was to gain further insights into activation of the Smad1 pathway in fibrosis in systemic sclerosis (SSc) and to determine whether this pathway is targeted by the antifibrotic drug imatinib mesylate., Methods: Levels of phosphorylated Smad1 and total Smad1 were examined in SSc and control skin biopsy samples by immunohistochemistry and in cultured fibroblasts by Western blotting. Activity of the CCN2 promoter was examined by a luciferase reporter gene assay. Interactions of Smad1 with the CCN2 promoter were examined by in vitro and in vivo DNA binding assays. Expression of the nonreceptor tyrosine kinase c-Abl and Smad1 was blocked using respective small interfering RNA., Results: Total and phosphorylated Smad1 levels were significantly elevated in SSc skin biopsy samples and in cultured SSc fibroblasts and correlated with elevated CCN2 protein and CCN2 promoter activity. DNA binding assays demonstrated that Smad1 was a direct activator of the CCN2 gene. Small interfering RNA-mediated depletion of Smad1 in SSc fibroblasts normalized the production of CCN2 and collagen. Imatinib mesylate blocked activation of the Smad1 pathway in transforming growth factor beta-stimulated control fibroblasts and reversed activation of this pathway in SSc fibroblasts. Likewise, blockade of c-Abl abrogated activation of the Smad1 pathway in SSc fibroblasts., Conclusion: Our findings demonstrate that activation of Smad1 signaling occurs in a subset of SSc patients and contributes to persistent activation of SSc fibroblasts. Demonstration that the Smad1/CCN2 pathway is blocked by imatinib mesylate further clarifies the mechanism of the antifibrotic effects of this compound. This study suggests that SSc patients with activated Smad1 signaling may benefit from imatinib mesylate treatment.

Background: Pemphigus vulgaris is a potentially life-threatening, autoimmune bullous disease of the skin and mucous membranes. Most commonly, the disease is treated with prednisone in combination with an immunosuppressant agent, frequently referred to as adjuvant drug. However, there is no consensus regarding the first-choice adjuvant drug for the treatment of pemphigus vulgaris or the recommended dosage., Objective: To evaluate the efficacy and safety of prednisone as monotherapy and in combination with the three most popular adjuvant agents - azathioprine, cyclosporine (ciclosporin), and cyclophosphamide - in the treatment of pemphigus vulgaris., Methods: This was a retrospective study with a follow-up of 7-21 years. The study was conducted in an academic hospital with an outpatient division for patients with bullous diseases. A total of 101 patients with moderate-to-severe mucocutaneous pemphigus vulgaris were included in the study. For assessment of disease severity a 'pemphigus score,' based on the percentage of involved skin or oral mucous membranes, was developed. At treatment initiation the average pemphigus score was comparable in all treated groups of patients. Four treatment regimens were evaluated: oral prednisone at an initial dose of 100mg (1.1-1.5 mg/kg) per day as monotherapy, and prednisone combined with adjuvant drugs, i.e. oral azathioprine at a dose of 100mg (1.1-1.5 mg/kg) per day; cyclosporine (ciclosporin) at a dose of 2.5-3 mg/kg/day; or cyclophosphamide at a dose of 100mg (1.1-1.5 mg/kg) per day. The main outcome measures were average time to clinical remission, average time to immunologic remission (non-detectable circulating pemphigus vulgaris antibodies), proportion of patients who remained free of clinical relapse within 5 years after discontinuation of therapy, time from treatment discontinuation until first relapse, and incidence of adverse effects., Results: The average (+/- SD) time to clinical remission was 7.2 +/- 13.1 months in patients who received prednisone monotherapy, 6.8 +/- 10.5 months in patients receiving additional azathioprine, 8.1 +/- 11.8 months in the cyclosporine group, and 4.9 +/- 6.9 months (which was significantly shorter than all other treatment groups, p < 0.05) in patients receiving cyclophosphamide. The average (+/- SD) times to immunologic remission were 33 +/- 27 months, 28 +/- 24 months, 30 +/- 21 months, and 23 +/- 17 months for prednisone monotherapy, azathioprine, cyclosporine, and cyclophosphamide, respectively. The proportions of patients who remained free of clinical relapse within 5 years after discontinuation of therapy were 55%, 50%, 43%, and 69% for prednisone monotherapy, azathioprine, cyclosporine, and cyclophosphamide, respectively. In patents who experienced relapse, the average (+/- SD) time from treatment discontinuation to clinical relapse was 10.50 +/- 6.86 months in patients receiving prednisone monotherapy, 16.40 +/- 17.36 months in the azathioprine group, 12.44 +/- 6.48 months in the cyclosporine group, and 21.16 +/- 20.13 months in the cyclophosphamide group. The safety profiles of all treatment regimens were comparable., Conclusion: Oral prednisone with cyclophosphamide is the most effective treatment for pemphigus vulgaris. All therapy regimens had a similar safety profile. In our opinion, cyclophosphamide at a dose of 1.1-1.5 mg/kg/day should be the adjuvant drug of choice in the treatment of moderate-to-severe pemphigus vulgaris.

Long-range chromosome organization is known to influence nuclear function. Budding yeast centromeres cluster near the spindle pole body, whereas telomeres are grouped in five to eight perinuclear foci. Using live microscopy, we examine the relative positions of right and left telomeres of several yeast chromosomes. Integrated lac and tet operator arrays are visualized by their respective repressor fused to CFP and YFP in interphase yeast cells. The two ends of chromosomes 3 and 6 interact significantly but transiently, forming whole chromosome loops. For chromosomes 5 and 14, end-to-end interaction is less frequent, yet telomeres are closer to each other than to the centromere, suggesting that yeast chromosomes fold in a Rabl-like conformation. Disruption of telomere anchoring by deletions of YKU70 or SIR4 significantly compromises contact between two linked telomeres. These mutations do not, however, eliminate coordinated movement of telomere (Tel) 6R and Tel6L, which we propose stems from the territorial organization of yeast chromosomes.

Epigenetic mechanisms silence the HM mating-type loci in budding yeast. These loci are tightly linked to telomeres, which are also repressed and held together in clusters at the nuclear periphery, much like mammalian heterochromatin. Yeast telomere anchoring can occur in the absence of silent chromatin through the DNA end binding factor Ku. Here we examine whether silent chromatin binds the nuclear periphery independently of telomeres and whether silencing persists in the absence of anchorage. HMR was excised from the chromosome by inducible site-specific recombination and tracked by real-time fluorescence microscopy. Silent rings associate with the nuclear envelope, while nonsilent rings move freely throughout the nucleus. Silent chromatin anchorage requires the action of either Ku or Esc1. In the absence of both proteins, rings move throughout the nucleoplasm yet remain silent. Thus, transcriptional repression can be sustained without perinuclear anchoring.

Objective: Aberrant transforming growth factor beta (TGFbeta) signaling has been implicated in the pathogenesis of scleroderma (systemic sclerosis [SSc]), but the contribution of specific components in this pathway to SSc fibroblast phenotype remains unclear. This study was undertaken to delineate the role of TGFbeta receptor type I (TGFbetaRI) and TGFbetaRII in collagen overexpression by SSc fibroblasts., Methods: Primary dermal fibroblasts from SSc patients and healthy adults were studied (n = 10 matched pairs). Adenoviral vectors were generated for TGFbetaRI (AdTGFbetaRI), TGFbetaRII (AdTGFbetaRII), and kinase-deficient TGFbetaRII (AdDeltakRII). TGFbetaRI basal protein levels were analyzed by (35)S-methionine labeling/immunoprecipitation and immunohistochemistry. Type I collagen and TGFbetaRII basal protein levels were analyzed by Western blot and newly secreted collagen by (3)H-proline incorporation assay., Results: Analysis of endogenous TGFbetaRI and TGFbetaRII protein levels revealed that SSc TGFbetaRI levels were increased 1.7-fold (P = 0.008; n = 7) compared with levels in healthy controls, while TGFbetaRII levels were decreased by 30% (P = 0.03; n = 7). This increased TGFbetaRI:TGFbetaRII ratio correlated with SSc collagen overexpression. To determine the consequences of altered TGFbetaRI:TGFbetaRII ratio on collagen expression, healthy fibroblasts were transduced with AdTGFbetaRI or AdTGFbetaRII. Forced expression of TGFbetaRI in the range corresponding to elevated SSc TGFbetaRI levels increased basal collagen expression in a dose-dependent manner, while similar TGFbetaRII overexpression had no effect, although transduction of fibroblasts at higher multiplicities of infection led to a marked reduction of basal collagen levels. Blockade of TGFbeta signaling via AdDeltakRII resulted in approximately 50% inhibition of basal collagen levels in healthy fibroblasts and in 5 of 9 SSc cell lines. A subset of SSc fibroblasts (4 of 9 cell lines) was resistant to this treatment. SSc fibroblasts with the highest levels of TGFbetaRI were the least responsive to collagen inhibition via DeltakRII., Conclusion: This study indicates that an increased TGFbetaRI:TGFbetaRII ratio may underlie aberrant TGFbeta signaling in SSc and contribute to elevated basal collagen production, which is insensitive to TGFbeta signaling blockade via DeltakRII.

The aim of the study was to compare alterations of various regions of the basement membrane zone (BMZ) in lichen sclerosus (LS) using laser scanning confocal microscopy. The study included three cases of bullous LS, one case of bullous LS that developed in the course of graft-versus-host disease (GVHD), and six cases of non-bullous LS. Three cases of morphea served as a control. Biopsies from patients' skin and control biopsies from normal human skin were cut into 30-microm thick slides and labeled with antibodies against beta4-intergin (lamina lucida marker), collagen IV, and the N-terminal end of collagen VII (lamina densa markers) and the C-terminal end of collagen VII (sublamina densa marker) using routine immunofluorescence (IF). Three-dimensional (3D) reconstruction of various regions of the BMZ showed a decrease in the number and size of the dermal papillae in LS and morphea as compared with normal skin. In LS numerous invaginations and holes were present in the BMZ at the level of the lamina lucida and lamina densa. Computer animation of 3D projections revealed that the thickness of the lamina densa observed under the light microscopy is an optical artifact dependent on periodical tortion of the lamina densa along its axis. Torsions and invaginations of the BMZ are equally responsible for the phenomenon of artificial reduplication of the lamina densa observed at the ultrastructural level. IF labeling with antibody against the N-terminal end of collagen VII disclosed the presence of a large hole (up to 25 microm) in the lamina densa and the presence of granular material in deep dermis suggestive of partial degradation of lamina densa at the level of anchoring fibers. An IF mapping study showed blister formation below the lamina densa in three patients with bullous LS, whereas in a case of LS associated with GVHD, a blister formed through the basal layer of the epidermis. In morphea, there was flattening of BMZ at the level of lamina lucida, lamina densa, and sublamina densa but the continuity of BMZ was preserved. Three-dimensional reconstruction of dermal-epidermal junction in LS revealed alterations of the BMZ, most pronounced at the level of the lamina densa and sublamina densa.

Background: This study evaluated a single-centre experience with endovascular repair of traumatic arteriovenous fistula in the cervicothoracic region., Methods: Endovascular repair of 27 traumatic cervicothoracic arteriovenous fistulas was attempted between August 1998 and December 2001. Patients with active bleeding or end-organ ischaemia were excluded. Follow-up was accomplished with clinical, duplex Doppler and arteriographic evaluation after 1 month and then every 3 months., Results: Twelve patients with a major vessel injury were treated by stent-graft placement. Vessels involved were the subclavian (eight), common carotid (three) and internal carotid (one) arteries. Subclavian artery side branches were embolized in three of the eight patients. Four patients developed early type 4 endoleaks but all resolved. Treatment with stent-grafts was ultimately successful in all 12 patients. Three patients were lost to follow-up. During mean follow-up of 21 (range 3-36) months, one of the remaining patients developed a graft stenosis. Fifteen patients with minor vessel injuries were treated with arterial embolization. Vessels embolized were subclavian artery branches (four), external carotid artery and branches (seven) and vertebral arteries (four). Successful embolization was accomplished in ten of 15 patients., Conclusion: Endovascular therapy is a promising alternative to surgery for selected patients with cervicothoracic arteriovenous fistula., (Copyright 2003 British Journal of Surgery Society Ltd. Published by John Wiley & Sons, Ltd.)

Objective: To investigate the relationship of progressive facial hemiatrophy (PFH) and scleroderma en coup de sabre by establishing the presence and type of central nervous system (CNS) involvement in both diseases and the possible coexistence of PFH with scleroderma in other body sites., Methods: We divided 19 cases of PFH into 2 groups: group 1 in which atrophies were preceded by cutaneous indurations (n = 10) and group 2 with no precedent indurations (n = 9). The third group consisted of 7 cases of scleroderma en coup de sabre with no PFH features. Clinical and laboratory investigations included indirect immunofluorescence for antinuclear antibodies, and routine neurological examination involved electroencephalography, magnetic resonance imaging (MRI) before and after contrast application to evaluate the integrity of blood-brain barrier, angio-MRI to evaluate intracranial blood vessel anomalies, and 99mTc-HM-PAO-SPECT to evaluate regional cerebral blood flow (CBF)., Results: We found similar anomalies in all 3 groups. MRI did not show abnormality in 2 out of 9 PFH cases preceded by indurations, in 5 out of 9 cases not preceded by indurations, and in all 7 cases of scleroderma en coup de sabre, including 5 patients, in whom the CBF was found to be diminished. In single cases of groups 1 and 2, SPECT was normal despite some MRI abnormalities. Angio-MRI was not contributory since the same abnormalities of Willis circle were found in normal controls. In single cases of both PFH groups, MRI with contrast disclosed some damage of the blood-brain barrier., Conclusion: Our results suggest frequent CNS involvement in PFH cases, regardless of the time of presentation of cutaneous indurations, with or without coexistent plaques of localized scleroderma in other locations. This indicates a close relationship between PFH and scleroderma en coup de sabre. The detection of abnormal SPECT by normal MRI in some cases of PFH and scleroderma en coup de sabre is of practical importance. This indicates the usefulness of SPECT in studying both PFH and scleroderma en coup de sabre.

The molecular and cellular mechanisms that maintain proper collagen homeostasis in healthy human skin and are responsible for the dysregulated collagen synthesis in scleroderma remain primarily unknown. This study demonstrates that Fli1 is a physiological negative regulator of collagen gene expression in dermal fibroblasts in vitro and in human skin in vivo. This conclusion is supported by the analyses of mouse embryonic fibroblasts from Fli1(-/-), Fli1(+/-), and Fli1(+/+) mice. In cultured human and mouse fibroblasts Fli1 expression levels are inversely correlated with the collagen type I expression levels. These in vitro observations were validated in vivo. In healthy human skin Fli1 protein is expressed in fibroblasts and endothelial cells. Significantly, absence of Fli1 expression in individual fibroblasts correlates with elevated collagen synthesis. In contrast to healthy skin, Fli1 protein is consistently absent from fibroblasts and significantly reduced in endothelial cells in clinically involved scleroderma skin, which correlates with enhanced collagen synthesis in systemic sclerosis skin. This study supports the role of Fli1 as a suppressor of collagen transcription in human skin in vivo. Persistent down-regulation of Fli1 in scleroderma fibroblasts in vivo may directly contribute to uncontrolled matrix deposition in scleroderma skin.

Fibrodysplasia ossificans progressiva, a rare genetic disabling disease characterized by heterotopic bone formation, is of special interest for general medicine since the bone morphogenetic proteins (especially BMP-4) involved in its pathogenesis are known to play a role in skeletal morphogenesis, and the gene antagonist to BMP-4 noggin might be useful in preventing lamellar bone formation. We present two cases with characteristic musculo-skeletal abnormalities and histopathological features (inflammatory infiltrates which destroyed muscle tissue replacing it by proliferating fibroblasts). In one patient due to high activity of fibroblasts, the histopathologic pattern appeared to be atypical and the lesion was recognized by a general pathologist as sarcoma. The other patient, due to the symmetrical induration of sternocleidomastoid muscles, was primarily recognized as scleredema. We stress the diagnostic importance of skeletal abnormalities (hallux valgus and others), and discuss differentiation from progressive osseous heteroplasia (POH) and congenital or acquired localized cutaneous and muscle ossifications which have a much better prognosis, as well as Albright's hereditary osteodystrophy, which differs by the presence of various systemic abnormalities. A study of FOP might provide an important clue to the genetic molecular mechanism of bone formation, development of heterotopic bone and a possible prevention by molecular manipulation with the gene responsible for bone morphogenetic proteins and their antagonists.