Molecular cloning and functional characterisation of a glucose transporter, CaHGT1, of Candida albicans

We have cloned the first glucose transporter CaHGT1 (Candida albicanshigh-affinity glucose transporter) of a pathogenic yeast, Candida albicans. The DNA sequence (GenBank accession number Y16834) analysis revealed an ORF encoding a novel protein of 545 amino acids with a predicted molecular mass of 60.67 kDa. The putative protein with 12 transmembrane domains has 51% identity with Kluyveromyces lactis high-affinity glucose transporter, HGT1. The protein signatures which are conserved and distinctive of the sugar transporters belonging to the major facilitator superfamily (MFS) were also found in CaHgt1p. When heterologously expressed, the ORF functionally complemented a mutant strain of Saccharomyces cerevisiae RE700A which was deleted in seven hexose transporter genes and thus was unable to grow or transport glucose. The expression of CaHGT1 in C. albicans showed a transcript of 1.6 kb which was enhanced in response to the human steroid hormone progesterone. Interestingly, the transcript levels were also enhanced in the presence of drugs, e.g. cycloheximide, chloramphenicol and benomyl. The results suggest that CaHGT1, which encodes a MFS protein, could be linked to the drug resistance phenomenon in C. albicans

Case-control diagnostic accuracy study of a non-sputum CD38-based TAM-TB test from a single milliliter of blood

Background: CD4 T cell phenotyping-based blood assays have the potential to meet WHO target product profiles (TPP) of non-sputum-biomarker-based tests to diagnose tuberculosis (TB). Yet, substantial refinements are required to allow their implementation in clinical settings. This study assessed the real time performance of a simplified T cell activation marker (TAM)-TB assay to detect TB in adults from one millilitre of blood with a 24h turnaround time. Methods: We recruited 479 GeneXpert® positive cases and 108 symptomatic but GeneXpert® negativecontrols from presumptive adult TB patients in the Temeke District of Dar-es-Salaam, Tanzania. TAM-TB assay accuracy was assessed by comparison with a composite reference standard comprising GeneXpert® and solid culture. A single millilitre of fresh blood was processed to measure expression of CD38 or CD27 by CD4 T cells producing INF-γ and/or TNF-α in response to a synthetic peptide pool covering the sequences of Mycobacterium tuberculosis (Mtb) ESAT-6, CFP-10 and TB10.4 antigens on a 4-color FACSCalibur apparatus. Results: Significantly superior to CD27 in accurately diagnosing TB, the CD38-based TAM-TB assay specificity reached 93.4% for a sensitivity of 82.2% with an area under the receiver operating characteristics curve of 0.87 (95% CI 0.84-0.91). The assay performance was not significantly affected by HIV status. Conclusions: Wesuccessfully implemented TAM-TB immunoassay routine testing with a 24h turnaround time at district level in a resource limited setting. Starting from one millilitre of fresh blood and being not influenced by HIV status, TAM-TB assay format and performance appears closely compatible with the optimal TPP accuracy criteria defined by WHO for a non-sputum confirmatory TB test

Bone Morphogenetic Protein 4 Gene Therapy in Mice Inhibits Myeloma Tumor Growth, But Has a Negative Impact on Bone.

Multiple myeloma is characterized by accumulation of malignant plasma cells in the bone marrow. Most patients suffer from an osteolytic bone disease, caused by increased bone degradation and reduced bone formation. Bone morphogenetic protein 4 (BMP4) is important for both pre- and postnatal bone formation and induces growth arrest and apoptosis of myeloma cells. BMP4-treatment of myeloma patients could have the potential to reduce tumor growth and restore bone formation. We therefore explored BMP4 gene therapy in a human-mouse model of multiple myeloma where humanized bone scaffolds were implanted subcutaneously in RAG2-/- γC-/-mice. Mice were treated with adeno-associated virus serotype 8 BMP4 vectors (AAV8-BMP4) to express BMP4 in the liver. When mature BMP4 was detectable in the circulation, myeloma cells were injected into the scaffolds and tumor growth was examined by weekly imaging. Strikingly, the tumor burden was reduced in AAV8-BMP4 mice compared with the AAV8-CTRL mice, suggesting that increased circulating BMP4 reduced tumor growth. BMP4-treatment also prevented bone loss in the scaffolds, most likely due to reduced tumor load. To delineate the effects of BMP4 overexpression on bone per se, without direct influence from cancer cells, we examined the unaffected, non-myeloma femurs by μCT. Surprisingly, the AAV8-BMP4 mice had significantly reduced trabecular bone volume, trabecular numbers, as well as significantly increased trabecular separation compared with the AAV8-CTRL mice. There was no difference in cortical bone parameters between the two groups. Taken together, BMP4 gene therapy inhibited myeloma tumor growth, but also reduced the amount of trabecular bone in mice. Our data suggest that care should be taken when considering using BMP4 as a therapeutic agent. © 2019 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research

An optimized whole blood assay measuring expression and activity of NLRP3, NLRC4 and AIM2 inflammasomes

The proinflammatory protease caspase-1 plays pivotal roles in central pathways of innate immunity, thereby contributing to pathogen clearance. Beside its physiological role, dysregulated activity of caspase-1 is known to contribute to an increasing number of diseases. In this study, we optimized and validated a low-volume human whole blood assay facilitating the measurement of caspase-1 activation and inflammasome-related gene expression upon stimulation of the NLRP3, NLRC4 or AIM2 inflammasome. Using the NLRP3 inflammasome specific inhibitor MCC950, we were able to measure the activity of canonical or alternative NLRP3 pathways, AIM2 and NLRC4 inflammasomes in whole blood. Based on our data we assume a superposition of NLRP3 and NLRC4 inflammasome activities in human whole blood following stimulation with S. typhimurium. The optimized whole blood assay may be suitable for diagnostic and research purposes for pediatric patients who can only donate small amounts of blood

Finding a moral homeground: appropriately critical religious education and transmission of spiritual values

Values-inspired issues remain an important part of the British school curriculum. Avoiding moral relativism while fostering enthusiasm for spiritual values and applying them to non-curricular learning such as school ethos or children's home lives are challenges where spiritual, moral, social and cultural (SMSC) development might benefit from leadership by critical religious education (RE). Whether the school's model of spirituality is that of an individual spiritual tradition (schools of a particular religious character) or universal pluralistic religiosity (schools of plural religious character), the pedagogy of RE thought capable of leading SMSC development would be the dialogical approach with examples of successful implementation described by Gates, Ipgrave and Skeie. Marton's phenomenography, is thought to provide a valuable framework to allow the teacher to be appropriately critical in the transmission of spiritual values in schools of a particular religious character as evidenced by Hella's work in Lutheran schools

Ultrasound in managing extrapulmonary tuberculosis: a randomized controlled two-center study

Patients with clinically suspected tuberculosis are often treated empirically, as diagnosis - especially of extrapulmonary tuberculosis - remains challenging. This leads to an overtreatment of tuberculosis and to underdiagnosis of possible differential diagnoses.; This open-label, parallel-group, superiority randomized controlled trial is done in a rural and an urban center in Tanzania. HIV-positive and -negative adults (≥18 years) with clinically suspected extrapulmonary tuberculosis are randomized in a 1:1 ratio to an intervention- or control group, stratified by center and HIV status. The intervention consists of a management algorithm including extended focused assessment of sonography for HIV and tuberculosis (eFASH) in combination with chest X-ray and microbiological tests. Treatment with anti-tuberculosis drugs is started, if eFASH is positive, chest X-ray suggests tuberculosis, or a microbiological result is positive for tuberculosis. Patients in the control group are managed according national guidelines. Treatment is started if microbiology is positive or empirically according to the treating physician. The primary outcome is the proportion of correctly managed patients at 6 months (i.e patients who were treated with anti-tuberculosis treatment and had definite or probable tuberculosis, and patients who were not treated with anti-tuberculosis treatment and did not have tuberculosis). Secondary outcomes are the proportion of symptom-free patients at two and 6 months, and time to death. The sample size is 650 patients.; This study will determine, whether ultrasound in combination with other tests can increase the proportion of correctly managed patients with clinically suspected extrapulmonary tuberculosis, thus reducing overtreatment with anti-tuberculosis drugs.; PACTR, Registration number: PACTR201712002829221, registered December 1st 2017

S1 Constrains S4 in the Voltage Sensor Domain of Kv7.1 K+ Channels

Voltage-gated K+ channels comprise a central pore enclosed by four voltage-sensing domains (VSDs). While movement of the S4 helix is known to couple to channel gate opening and closing, the nature of S4 motion is unclear. Here, we substituted S4 residues of Kv7.1 channels by cysteine and recorded whole-cell mutant channel currents in Xenopus oocytes using the two-electrode voltage-clamp technique. In the closed state, disulfide and metal bridges constrain residue S225 (S4) nearby C136 (S1) within the same VSD. In the open state, two neighboring I227 (S4) are constrained at proximity while residue R228 (S4) is confined close to C136 (S1) of an adjacent VSD. Structural modeling predicts that in the closed to open transition, an axial rotation (∼190°) and outward translation of S4 (∼12 Å) is accompanied by VSD rocking. This large sensor motion changes the intra-VSD S1–S4 interaction to an inter-VSD S1–S4 interaction. These constraints provide a ground for cooperative subunit interactions and suggest a key role of the S1 segment in steering S4 motion during Kv7.1 gating