Whole Genome Sequence of a Treponema pallidum Strain From a Formalin-Fixed, Paraffin-Embedded Fine Needle Aspirate of a Cervical Lymph Node.

ABSTRACT: A patient with unilateral cervical lymphadenopathy suspicious for malignancy underwent a fine needle aspiration. Histology demonstrated mixed inflammatory infiltrates with abundant spirochetes. Sufficient spirochete DNA was extracted from paraffin-embedded tissue sections to obtain the near-complete genome sequence of a macrolide-resistant strain belonging to the SS14 omega strain of Treponema pallidum .

Solution structure of the N-terminal extension domain of a Schistosoma japonicum asparaginyl-tRNA synthetase.

Several secreted proteins from helminths (parasitic worms) have been shown to have immunomodulatory activities. Asparaginyl-tRNA synthetases are abundantly secreted in the filarial nematode Brugia malayi (BmAsnRS) and the parasitic flatworm Schistosoma japonicum (SjAsnRS), indicating a possible immune function. The suggestion is supported by BmAsnRS alleviating disease symptoms in a T-cell transfer mouse model of colitis. This immunomodulatory function is potentially related to an N-terminal extension domain present in eukaryotic AsnRS proteins but few structure/function studies have been done on this domain. Here we have determined the three-dimensional solution structure of the N-terminal extension domain of SjAsnRS. A protein containing the 114 N-terminal amino acids of SjAsnRS was recombinantly expressed with isotopic labelling to allow structure determination using 3D NMR spectroscopy, and analysis of dynamics using NMR relaxation experiments. Structural comparisons of the N-terminal extension domain of SjAsnRS with filarial and human homologues highlight a high degree of variability in the ß-hairpin region of these eukaryotic N-AsnRS proteins, but similarities in the disorder of the C-terminal regions. Limitations in PrDOS-based intrinsically disordered region (IDR) model predictions were also evident in this comparison. Empirical structural data such as that presented in our study for N-SjAsnRS will enhance the prediction of sequence-homology based structure modelling and prediction of IDRs in the future.Communicated by Ramaswamy H. Sarma.

Interconversion between two unrelated protein folds in the lymphotactin native state.

Proteins often have multiple functional states, which might not always be accommodated by a single fold. Lymphotactin (Ltn) adopts two distinct structures in equilibrium, one corresponding to the canonical chemokine fold consisting of a monomeric three-stranded beta-sheet and carboxyl-terminal helix. The second Ltn structure solved by NMR reveals a dimeric all-beta-sheet arrangement with no similarity to other known proteins. In physiological solution conditions, both structures are significantly populated and interconvert rapidly. Interconversion replaces long-range interactions that stabilize the chemokine fold with an entirely new set of tertiary and quaternary contacts. The chemokine-like Ltn conformation is a functional XCR1 agonist, but fails to bind heparin. In contrast, the alternative structure binds glycosaminoglycans with high affinity but fails to activate XCR1. Because each structural species displays only one of the two functional properties essential for activity in vivo, the conformational equilibrium is likely to be essential for the biological activity of lymphotactin. These results demonstrate that the functional repertoire and regulation of a single naturally occurring amino acid sequence can be expanded by access to a set of highly dissimilar native-state structures.

Evaluation of the angiogenic properties of Brugia malayi asparaginyl-tRNA synthetase and its mutants: A study on the molecular target for antifilarial drug development.

Brugia malayi asparaginyl-tRNA synthetase (BmAsnRS) has been identified as an immunodominant antigen and a physiocrine that mimics Interleukin-8 (IL-8) to induce chemotaxis and angiogenesis in endothelial cells. Computational analyses have shown that the N-terminal region of BmAsnRS has a novel fold, a lysine rich ß-hairpin α-helix, (FLIRTKKDGKQIWE) which is similar to that present in IL-8 chemokine, CXCR1. This novel fold is involved in tRNA binding and is integral for the manifestation of the disease, lymphatic filariasis (LF). Drug discovery programmes carried out so far for LF have not been successful because of the target (BmAsnRS) resistance due to the disease-associated mutation. Mutations in AARS targets have been shown to correlate with several diseases. However, no disease-associated mutational studies have been carried out for LF. BmAsnRS has been an established target for LF. It was proposed, therefore, to study the effect of single point mutations in BmAsnRS so as to elucidate the molecular target. An understanding of the molecular consequences of mutations will provide insight into how resistance develops in addition to the identification of the likely resistance-conferring mutations. Three mutants were, therefore, generated by site-directed mutagenesis using CUPSAT server and their angiogenic properties evaluated. Cytometric analysis of the mutants on endothelial cell cycle was also carried out. CUPSAT prediction of protein stability upon point mutations reveal that two mutants generated are likely resistance-conferring mutations. All the three mutants show significant reduction in their angiogenic properties and reduction in the DNA content in the cells of S and G2/M phases thus showing altered function of the gene encoding the drug target. The resistance- conferring mutants, however, show angiogenic properties nearer to the wild type protein, BmAsnRS. Future work on designing newer drugs may take into consideration these drug resistance-conferring mutations.

Histidyl-tRNA synthetase and asparaginyl-tRNA synthetase, autoantigens in myositis, activate chemokine receptors on T lymphocytes and immature dendritic cells.

Autoantibodies to histidyl-tRNA synthetase (HisRS) or to alanyl-, asparaginyl-, glycyl-, isoleucyl-, or threonyl-tRNA synthetase occur in approximately 25% of patients with polymyositis or dermatomyositis. We tested the ability of several aminoacyl-tRNA synthetases to induce leukocyte migration. HisRS induced CD4(+) and CD8(+) lymphocytes, interleukin (IL)-2-activated monocytes, and immature dendritic cells (iDCs) to migrate, but not neutrophils, mature DCs, or unstimulated monocytes. An NH(2)-terminal domain, 1-48 HisRS, was chemotactic for lymphocytes and activated monocytes, whereas a deletion mutant, HisRS-M, was inactive. HisRS selectively activated CC chemokine receptor (CCR)5-transfected HEK-293 cells, inducing migration by interacting with extracellular domain three. Furthermore, monoclonal anti-CCR5 blocked HisRS-induced chemotaxis and conversely, HisRS blocked anti-CCR5 binding. Asparaginyl-tRNA synthetase induced migration of lymphocytes, activated monocytes, iDCs, and CCR3-transfected HEK-293 cells. Seryl-tRNA synthetase induced migration of CCR3-transfected cells but not iDCs. Nonautoantigenic aspartyl-tRNA and lysyl-tRNA synthetases were not chemotactic. Thus, autoantigenic aminoacyl-tRNA synthetases, perhaps liberated from damaged muscle cells, may perpetuate the development of myositis by recruiting mononuclear cells that induce innate and adaptive immune responses. Therefore, the selection of a self-molecule as a target for an autoantibody response may be a consequence of the proinflammatory properties of the molecule itself.

Crystallization and preliminary X-ray diffraction analysis of human cytosolic seryl-tRNA synthetase.

Human cytosolic seryl-tRNA synthetase (hsSerRS) is responsible for the covalent attachment of serine to its cognate tRNA(Ser). Significant differences between the amino-acid sequences of eukaryotic, prokaryotic and archaebacterial SerRSs indicate that the domain composition of hsSerRS differs from that of its eubacterial and archaebacterial analogues. As a consequence of an N-terminal insertion and a C-terminal extra-sequence, the binding mode of tRNA(Ser) to hsSerRS is expected to differ from that in prokaryotes. Recombinant hsSerRS protein was purified to homogeneity and crystallized. Diffraction data were collected to 3.13 Šresolution. The structure of hsSerRS has been solved by the molecular-replacement method.

Postgraduate programs in biochemistry and molecular biology: A parallel session at the IUBMB/PSBMB 2019 "Harnessing Interdisciplinary Education in Biochemistry and Molecular Biology" conference.

The conference session on Postgraduate Education in Biochemistry and Molecular Biology consisted of wide-ranging presentations and discussions. Approaches, issues, and solutions for postgraduate education and training in countries ranging from the Philippines to Mongolia and the United States were covered.

Student evaluation of medical school curriculum transformation in Iraq.

This article was migrated. The article was marked as recommended. Introduction: Decades of political and social unrest negatively impacted medical education in Iraq. Recently, new opportunities arose for medical schools to engage international education organizations and the World Health Organization to implement medical school curriculum changes, replacing older discipline-based, teacher-focused systems with a systems-based, student-focused reformed curriculum. Methods: A descriptive, cross sectional quantitative study was designed to survey medical students near the beginning (years 2-3) and at near the end (years 5-6) of their six-year program at the Al Kindy College of Medicine, University of Baghdad, Iraq. Results: A validated questionnaire collected data on thirty-two issues, including student perception of learning, student perception of teachers, academic self-perception and student self-perception. Seven of the thirty-two questions included in this survey resulted in significantly different responses from group 1 (second and third year) vs group 2 (fifth and sixth year) students. Conclusions: This study concluded that the two student groups were significantly different in their awareness of the need for curriculum change, but that student self-perception in both groups was less than ideal at present. In the future, studies are planned to assess student confidence in their professional development, as teaching institutions advance toward broader accreditation and thus opportunities for their students.

Academic Medicine and Science Diplomacy: Medical Education in Iraq.

Physicians are infrequently selected to serve in the yearlong role of Jefferson Science Fellow (JSF) as senior science advisor for the U.S. Department of State or the United States Agency for International Development. The authors discuss the role of a JSF in promoting "science diplomacy" from the perspective of one alumnus, who sites examples of ongoing, long-term collaborative projects including one focusing on medical education reform in Iraq that would not have been possible without the JSF program. More than 3 decades of political and social unrest, terrorism, the internal displacement of millions of Iraqi citizens, the arrival of Syrian refugees, international sanctions, and the effect of fluctuating oil prices that fund education in Iraq have all led to a standstill in modernizing educational systems and the need for reform. In 2016, new opportunities for international collaboration and travel allowed faculty from the Al Kindy College of Medicine, University of Baghdad, Iraq, to visit the United States, explore U.S. medical school teaching methods, interact with U.S. faculty and students, and then implement their vision of a revised medical curriculum. These faculty members shared their observations with the Iraqi Council of Deans, which represents all 30 Iraqi medical schools. Structural changes in curriculum content and teaching methods have been implemented with the goal of restoring quantifiable excellence in education and health care in Iraq. The authors close with lessons learned to encourage other physicians to participate in the JSF program, which offers a unique opportunity to influence foreign policy through science diplomacy.

Immune Response to Brugia malayi Asparaginyl-tRNA Synthetase in Balb/c Mice and Human Clinical Samples of Lymphatic Filariasis.

Background: Lymphatic filariasis (LF) is a global health problem, with a peculiar nature of parasite-specific immunosuppression that promotes long-term pathology and disability. Immune modulation in the host by parasitic antigens is an integral part of this disease. The current study attempts to dissect the immune responses of aminoacyl-tRNA synthetases (AARS) with emphasis on Brugia malayi asparaginyl-tRNA synthetase (BmAsnRS), since it is one among the highly expressed excretory/secretory proteins expressed in all stages of the parasite life cycle, whereas its role in filarial pathology has not been elaborately studied. Methods and Results: In this study, recombinant BmAsnRS (rBmAsnRS) immunological effects were studied in semipermissive filarial animal model Balb/c mice and on clinically defined human samples for LF. In mice study, humoral responses showed considerable titer levels with IgG2a isotype followed by IgG2b and IgG1. Immunoreactivity studies with clinical samples showed significant humoral responses especially in endemic normal with marked levels of IgG1 and IgG2 followed by IgG3. The cell-mediated immune response, evaluated by splenocytes and peripheral blood mononuclear cells proliferation, did not yield significant difference when compared with control groups. Cytokine profiling and qRT-PCR analysis of mice samples immunized with rBmAsnRS showed elevated levels of IFN-γ, IL-10, inhibitory factor-cytotoxic T lymphocyte-associated protein-A (CTLA-4) and Treg cell marker-Forkhead Box P3 (FoxP3). Conclusions: These observations suggest that rBmAsnRS has immunomodulatory effects with modified Th2 response along with suppressed cellular proliferation indicating the essence of this molecule for immune evasion by the parasite.

Mental health indicators in APEC.

BACKGROUND: The World Economic Forum (2011) concluded that the economic impact of mental illness is the single most important contributor among all non-communicable diseases to loss of productivity. The 21 economies represented by Asia Pacific Economic Cooperation (APEC) responded to that information with efforts to address mental health as an integral component of economic development. AIM: In order to help assess the progress of APEC region compared to other regions of the world, the World Health Organization (WHO) granted us access to a subset of the 2014 Mental Health Atlas database containing health indicators from all 21 APEC economies. METHODS: APEC-specific data were extracted using the same format used by WHO in its Mental Health Atlas to compare/contrast data in APEC versus the six WHO regions of the world. RESULTS: It was observed that mental health workforces in APEC include a higher number of psychiatrist providers compared with WHO regions. Suicide rates reported in three APEC economies are among the highest in the world. All APEC economies continue their individual and coordinated efforts to support their ' APEC Roadmap to Promote Mental Wellness in a Healthy Asia Pacific (2014-2020)'. CONCLUSION: Significant challenges for APEC members exist to coordinate regional efforts to improve mental health due to highly variable income levels, existing health infrastructures and social preferences. The findings in this report may serve as a helpful baseline for measuring success within the APEC region by 2020, the year in which progress in support of economic development will be reassessed.

Persistence of Schistosoma japonicum DNA in a Kidney-Liver Transplant Recipient.

Mitochondrial genome analysis of Schistosoma japonicum suggests that diversity of intermediate host snails drove intra-species divergence during its expansion in Asia. We applied the knowledge of this genomic variation to study an unusual patient we recently diagnosed with schistosomiasis. The patient had not visited any schistosomiasis-endemic countries for more than 35 years and had no idea where she became infected. Unusual clinical features of this patient included the absence of egg granulomas in tissue and persistent noncalcified eggs despite multiple praziquantel (PZQ) treatments over 7 years. A digital droplet polymerase chair reaction (PCR) assay that specifically targets the schistosome 1,4 dihydronicotinamide adenine dinucleotide-1 (NADH1) dehydrogenase-1 mitochondrial gene successfully amplified parasite DNA extracted from colon biopsies. DNA sequence analysis of parasite DNA revealed that it was a Philippine strain of S. japonicum. Future molecular studies using stored DNA from patients such as this may provide new insight into why some persons do not respond well to PZQ treatment.

Identification of diadenosine triphosphate in Brugia malayi by reverse phase high performance liquid chromatography and MALDI mass spectrometry.

The presence of diadenosine oligophosphates (ApnA) in eukaryotic pathogens has been difficult technically to assess and thus is often overlooked. ApnA are a family of intercellular and intracellular signaling molecules and their biological activities differ relative to the number of phosphate moieties. The application of mass spectrometry to differentiate nucleotide phosphates has been limited by the high salt content in tissue extracts, enzymatic reactions or high performance liquid chromatography (HPLC) buffers, as well as the potential for sample loss when processing and desalting small biological samples. To address this problem a simple reverse phase HPLC (RP-HPLC) method using volatile organic buffers at low pH was developed to create elution profiles of adenosine and diadenosine phosphates. To test this method on a eukaryotic pathogen, small intravascular human filarial parasites (Brugia malayi) were extracted in phosphate buffered saline and a nucleotide phosphate profile was visualized by RP-HPLC. A major peak eluting at 10.4 min was analyzed directly by mass spectrometry and this confirmed the presence of significant quantities of diadenosine triphosphate, Ap3A. Application of this simplified RP-HPLC method will facilitate research on the normal and pathophysiological effects of ApnA particularly in situations when analysis of small biological samples is required.

Field-Effect Transistor Biosensor for Rapid Detection of Ebola Antigen.

The Ebola virus transmits a highly contagious, frequently fatal human disease for which there is no specific antiviral treatment. Therefore, rapid, accurate, and early diagnosis of Ebola virus disease (EVD) is critical to public health containment efforts, particularly in developing countries where resources are few and EVD is endemic. We have developed a reduced graphene oxide-based field-effect transistor method for real-time detection of the Ebola virus antigen. This method uses the attractive semiconductor characteristics of graphene-based material, and instantaneously yields highly sensitive and specific detection of Ebola glycoprotein. The feasibility of this method for clinical application in point-of-care technology is evaluated using Ebola glycoprotein suspended in diluted PBS buffer, human serum, and plasma. These results demonstrate the successful fabrication of a promising field-effect transistor biosensor for EVD diagnosis.

Correction: Brugia malayi Asparaginyl-tRNA Synthetase Stimulates Endothelial Cell Proliferation, Vasodilation and Angiogenesis.

[This corrects the article DOI: 10.1371/journal.pone.0146132.].

Brugia malayi Asparaginyl-tRNA Synthetase Stimulates Endothelial Cell Proliferation, Vasodilation and Angiogenesis.

A hallmark of chronic infection with lymphatic filarial parasites is the development of lymphatic disease which often results in permanent vasodilation and lymphedema, but all of the mechanisms by which filarial parasites induce pathology are not known. Prior work showed that the asparaginyl-tRNA synthetase (BmAsnRS) of Brugia malayi, an etiological agent of lymphatic filariasis, acts as a physiocrine that binds specifically to interleukin-8 (IL-8) chemokine receptors. Endothelial cells are one of the many cell types that express IL-8 receptors. IL-8 also has been reported previously to induce angiogenesis and vasodilation, however, the effect of BmAsnRS on endothelial cells has not been reported. Therefore, we tested the hypothesis that BmAsnRS might produce physiological changes in endothelial by studying the in vitro effects of BmAsnRS using a human umbilical vein cell line EA.hy926 and six different endothelial cell assays. Our results demonstrated that BmAsnRS produces consistent and statistically significant effects on endothelial cells that are identical to the effects of VEGF, vascular endothelial growth factor. This study supports the idea that new drugs or immunotherapies that counteract the adverse effects of parasite-derived physiocrines may prevent or ameliorate the vascular pathology observed in patients with lymphatic filariasis.

Synthesis and activity of substituted anthraquinones against a human filarial parasite, Brugia malayi.

Lymphatic filariasis (elephantiasis) is a global public health problem caused by the parasitic nematodes Wuchereria bancrofti and Brugia malayi. We have previously reported anthraquinones from daylily roots with potent activity against pathogenic trematode Schistosoma mansoni. Here we report the synthesis of novel anthraquinones A-S and their antifilrarial activity. Anthraquinones A-S were synthesized by a single-step Friedel-Crafts acylation reaction between phthalic anhydrides and substituted benzenes. The antifilarial properties of these synthetic anthraquinones were tested against microfilaria as well as adult male and female worms of B. malayi. The most active anthraquinone was K, which showed 100% mortality within 1, 5, and 3 days, respectively, against microfilaria and adult male and female worms at 5 ppm concentration. Albendazole, an oral drug currently used to treat parasitic infections, was used as a positive control. Methylated products of anthraquinones did not affect the microfilaria. Histological examination of treated adult female parasites showed most of the anthraquinones caused marked effects on intrauterine embryos.

Do tissue levels of autoantigenic aminoacyl-tRNA synthetase predict clinical disease?

The etiologies of most autoimmune diseases are not completely understood. Aminoacyl-tRNA synthetases (AARS) are a family of heterogenous enzymes responsible for protein synthesis and whose secondary functions include a role in autoimmune myositis. A subset of patients with idiopathic inflammatory myopathies demonstrate autoantibody against specific cytoplasmic AARS and the human asparaginyl-tRNA synthetase (AsnRS) has been shown to be a potent chemokine that interacts with CCR3 chemokine receptors. One way in which a chemotactic cytoplasmic enzyme might contribute to tissue inflammation is if it were abundant in a specific injured tissue and thereby released to the microenvironment at times of cellular damage. To test this hypothesis, the relative levels of AsnRS mRNA were studied in six human tissues. A 1.6 kbF RNA probe identified highly variable levels of the corresponding mRNA in Northern blot analysis of human lung, brain, heart, skeletal muscle, pancreas and liver. The highest levels of signal were noted in muscle and pancreas. Polyclonal antibody raised against recombinant human AsnRS identified abundant antigenic material in the pancreas, in particular in islet cells. Thus, the local abundance of an endogenous pro-inflammatory autoantigen may provide one explanation for perpetuation or exacerbation of tissue specific immune-mediated pathologies.

Expression, localization and alternative function of cytoplasmic asparaginyl-tRNA synthetase in Brugia malayi.

Aminoacyl-tRNA synthetases (AARS) are a family of enzymes that exhibit primary and various secondary functions in different species. In Brugia malayi, the gene for asparaginyl-tRNA synthetase (AsnRS), a class II AARS, previously has been identified as a multicopy gene encoding an immunodominant antigen in the serum of humans with lymphatic filariasis. However, the relative level of expression and alternative functions of AARS in nematode parasites is not well understood. We searched the Filarial Genome Project database to identify the number and amino acid specificity of B. malayi AARS cDNAs to gain insight into the role of different AARS in filaria. These data showed that cytoplasmic AsnRS was present in five gene clusters, and is the most frequently represented member of the aminoacyl-tRNA synthetase family in adult B. malayi. The relative level of AsnRS transcribed in adult female B. malayi was compared to the levels of a low abundance and medium abundance AARS by quantitative real-time RT-PCR. By this method, AsnRS cDNA was 11 times greater than arginyl-tRNA synthetase and methionyl-tRNA synthetase cDNA. In situ hybridization using a B. malayi AsnRS-specific oligonucleotide probe identified abundant cytoplasmic mRNA, particularly in the hypodermis of adult B. malayi. In the absence of tRNA, AsnRS synthesizes diadenosine triphosphate, a potent regulator of cell growth in other eukaryotes. These data support the hypothesis that all AARS are not equally expressed in B. malayi and that these enzymes may demonstrate important alternative functions in filaria.