There is an urgent medical need to differentiate active tuberculosis (ATB) from latent tuberculosis infection (LTBI) and prevent undertreatment and overtreatment. The aim of this study was to ...identify biomarker profiles that may support the differentiation between ATB and LTBI and to validate these signatures.
The discovery cohort included adult individuals classified in four groups: ATB (n = 20), LTBI without prophylaxis (untreated LTBI; n = 20), LTBI after completion of prophylaxis (treated LTBI; n = 20), and healthy controls (HC; n = 20). Their sera were analyzed for 40 cytokines/chemokines and activity of adenosine deaminase (ADA) isozymes. A prediction model was designed to differentiate ATB from untreated LTBI using sparse partial least squares (sPLS) and logistic regression analyses. Serum samples of two independent cohorts (national and international) were used for validation.
sPLS regression analyses identified C-C motif chemokine ligand 1 (CCL1), C-reactive protein (CRP), C-X-C motif chemokine ligand 10 (CXCL10), and vascular endothelial growth factor (VEGF) as the most discriminating biomarkers. These markers and ADA(2) activity were significantly increased in ATB compared to untreated LTBI (p ≤ 0.007). Combining CCL1, CXCL10, VEGF, and ADA2 activity yielded a sensitivity and specificity of 95% and 90%, respectively, in differentiating ATB from untreated LTBI. These findings were confirmed in the validation cohort including remotely acquired untreated LTBI participants.
The biomarker signature of CCL1, CXCL10, VEGF, and ADA2 activity provides a promising tool for differentiating patients with ATB from non-treated LTBI individuals.
Coevolution between hosts and pathogens is thought to occur between interacting molecules of both species. This results in the maintenance of genetic diversity at pathogen antigens (or so-called ...effectors) and host resistance genes such as the major histocompatibility complex (MHC) in mammals or resistance (R) genes in plants. In plant-pathogen interactions, the current paradigm posits that a specific defense response is activated upon recognition of pathogen effectors via interaction with their corresponding R proteins. According to the "Guard-Hypothesis," R proteins (the "guards") can sense modification of target molecules in the host (the "guardees") by pathogen effectors and subsequently trigger the defense response. Multiple studies have reported high genetic diversity at R genes maintained by balancing selection. In contrast, little is known about the evolutionary mechanisms shaping the guardee, which may be subject to contrasting evolutionary forces. Here we show that the evolution of the guardee RCR3 is characterized by gene duplication, frequent gene conversion, and balancing selection in the wild tomato species Solanum peruvianum. Investigating the functional characteristics of 54 natural variants through in vitro and in planta assays, we detected differences in recognition of the pathogen effector through interaction with the guardee, as well as substantial variation in the strength of the defense response. This variation is maintained by balancing selection at each copy of the RCR3 gene. Our analyses pinpoint three amino acid polymorphisms with key functional consequences for the coevolution between the guardee (RCR3) and its guard (Cf-2). We conclude that, in addition to coevolution at the "guardee-effector" interface for pathogen recognition, natural selection acts on the "guard-guardee" interface. Guardee evolution may be governed by a counterbalance between improved activation in the presence and prevention of auto-immune responses in the absence of the corresponding pathogen.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Proteolytic cascades regulate immunity and development in animals, but these cascades in plants have not yet been reported. Here we report that the extracellular immune protease Rcr3 of tomato is ...activated by P69B and other subtilases (SBTs), revealing a proteolytic cascade regulating extracellular immunity in solanaceous plants. Rcr3 is a secreted papain-like Cys protease (PLCP) of tomato that acts both in basal resistance against late blight disease (Phytophthora infestans) and in gene-for-gene resistance against the fungal pathogen Cladosporium fulvum (syn. Passalora fulva). Despite the prevalent model that Rcr3-like proteases can activate themselves at low pH, we found that catalytically inactive proRcr3 mutant precursors are still processed into mature mRcr3 isoforms. ProRcr3 is processed by secreted P69B and other Aspselective SBTs in solanaceous plants, providing robust immunity through SBT redundancy. The apoplastic effector EPI1 of P. infestans can block Rcr3 activation by inhibiting SBTs, suggesting that this effector promotes virulence indirectly by preventing the activation of Rcr3(-like) immune proteases. Rcr3 activation in Nicotiana benthamiana requires a SBT from a different subfamily, indicating that extracellular proteolytic cascades have evolved convergently in solanaceous plants or are very ancient in the plant kingdom. The frequent incidence of Asp residues in the cleavage region of Rcr3-like proteases in solanaceous plants indicates that activation of immune proteases by SBTs is a general mechanism, illuminating a proteolytic cascade that provides robust apoplastic immunity.
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BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
About 10-15% of couples who want to conceive suffer from subfertility, while in 30% of these cases, a male factor plays a role. Levels of particular microRNAs in seminal plasma, including those ...involved in spermatogenesis, may serve as an indicative parameter for subfertility. We first optimized a protocol for acquiring microRNAs from seminal plasma. Next, using a test-validation strategy in a male cohort, we aimed to identify microRNAs of which the levels are related to semen motility and concentration. By qPCR, 742 microRNAs were profiled in three normozoospermic samples, three seminal samples with a low semen motility (asthenozoospermia), and three with a low semen concentration (oligozoospermia). MicroRNAs showing significant differences between groups were further validated in a second cohort consisting of 40 samples with normozoospermia (control group), 47 samples with asthenozoospermia, and 19 samples with oligozoospermia (of which 74% also low motility). Highest microRNA yields were obtained with the Biofluids RNA extraction kit, with inclusion of MS2 RNA carrier and proteinase K treatment to the protocol, and when 50 µL of seminal plasma was used as input. Exosome isolation prior to RNA extraction did not lead to enhanced yields. In the test cohort, 236 microRNAs could be detected, of which 54 microRNAs showed a difference between groups. Five microRNAs were analyzed in the validation cohort. MiR-34b-5p levels in the control group were significantly higher compared to the asthenozoospermia group (
< 0.05) and compared to the oligozoospermia group (
< 0.001). We optimized microRNA acquirement from seminal plasma and identified microRNA levels in relation to semen concentration and motility. As recent human and mouse studies show that the miR-34 family is a marker of low semen concentration and is crucial in spermatogenesis, seminal plasma miR-34b-5p may represent a suitable candidate to study further as a marker of male subfertility.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
To successfully colonize the host, phytopathogens have developed a large repertoire of components to both combat the host plant defense mechanisms and to survive in adverse environmental conditions. ...Microbial proteases are predicted to be crucial components of these systems. In the present work, we aimed to identify active secreted proteases from the oomycete
, which causes root rot diseases on legumes. Genome mining and expression analysis highlighted an overrepresentation of microbial tandemly repeated proteases, which are upregulated during host infection. Activity Based Protein Profiling and mass spectrometry (ABPP-MS) on apoplastic fluids isolated from pea roots infected by the pathogen led to the identification of 35 active extracellular microbial proteases, which represents around 30% of the genes expressed encoding serine and cysteine proteases during infection. Notably, eight of the detected active secreted proteases carry an additional C-terminal domain. This study reveals novel active modular extracellular eukaryotic proteases as potential pathogenicity factors in
genus.
Many preclinical and clinical studies of hematopoietic stem cell-based gene therapy (GT) are based on the use of lentiviruses as the vector of choice. Assessment of the vector titer and transduction ...efficiency of the cell product is critical for these studies. Efficacy and safety of the modified cell product are commonly determined by assessing the vector copy number (VCN) using qPCR. However, this optimized and well-established method in the GT field is based on bulk population averages, which can lead to misinterpretation of the actual VCN per transduced cell. Therefore, we introduce here a single cell-based method that allows to unmask cellular heterogeneity in the GT product, even when antibodies are not available. We use Invitrogen's flow cytometry-based PrimeFlow™ RNA Assay with customized probes to determine transduction efficiency of transgenes of interest, promoter strength, and the cellular heterogeneity of murine and human stem cells. The assay has good specificity and sensitivity to detect the transgenes, as shown by the high correlations between PrimeFlow™-positive cells and the VCN. Differences in promoter strengths can readily be detected by differences in percentages and fluorescence intensity. Hence, we show a customizable method that allows to determine the number of transduced cells and the actual VCN per transduced cell in a GT product. The assay is suitable for all therapeutic genes for which antibodies are not available or too cumbersome for routine flow cytometry. The method also allows co-staining of surface markers to analyze differential transduction efficiencies in subpopulations of target cells.
The flavonoid family shows a high potential for inhibition of xanthine oxidase. Currently, more than 4000 flavonoids are known. The data of this study indicate that a planar structure is necessary ...for high inhibitory activity towards xanthine oxidase. Moreover, the contribution of a hydroxyl conjugate turns out to be a constant factor when the natural logarithm of IC
50 values is taken. This finding allows us to accurately predict the IC
50 value of any given hydroxyl group added to the basic flavone structure towards xanthine oxidase. This new method may provide an important research tool for elucidating the role that flavonoids may have in radical related diseases.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Synapses are a primary pathological target in neurodegenerative diseases. Identifying therapeutic targets at the synapse could delay progression of numerous conditions. The mitochondrial protein ...SFXN3 is a neuronally enriched protein expressed in synaptic terminals and regulated by key synaptic proteins, including α‐synuclein. We first show that SFXN3 uses the carrier import pathway to insert into the inner mitochondrial membrane. Using high‐resolution proteomics on Sfxn3‐KO mice synapses, we then demonstrate that SFXN3 influences proteins and pathways associated with neurodegeneration and cell death (including CSPα and Caspase‐3), as well as neurological conditions (including Parkinson's disease and Alzheimer’s disease). Overexpression of SFXN3 orthologues in Drosophila models of Parkinson's disease significantly reduced dopaminergic neuron loss. In contrast, the loss of SFXN3 was insufficient to trigger neurodegeneration in mice, indicating an anti‐ rather than pro‐neurodegeneration role for SFXN3. Taken together, these results suggest a potential role for SFXN3 in the regulation of neurodegeneration pathways.
SFXN3 is a neuronally enriched mitochondrial protein found at the synapse. Here, we show that SFXN3 influences expression levels of neurodegeneration and cell death‐associated proteins in mice, that its overexpression provides neuroprotection at the synapse in Drosophila models of Parkinson's disease, but that its absence or downregulation is insufficient to trigger neurodegeneration in mice and Drosophila.
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BFBNIB, DOBA, FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, SIK, UILJ, UKNU, UL, UM, UPUK
The plant-specific translation initiation complex eIFiso4F is encoded by three genes in Arabidopsis (
)-genes encoding the cap binding protein eIFiso4E (
) and two isoforms of the large subunit ...scaffolding protein eIFiso4G (
and
). To quantitate phenotypic changes, a phenomics platform was used to grow wild-type and mutant plants (
,
,
,
, and
) under various light conditions. Mutants lacking both eIFiso4G isoforms showed the most obvious phenotypic differences from the wild type. Two-dimensional differential gel electrophoresis and mass spectrometry were used to identify changes in protein levels in plants lacking eIFiso4G. Four of the proteins identified as measurably decreased and validated by immunoblot analysis were two light harvesting complex binding proteins 1 and 3, Rubisco activase, and carbonic anhydrase. The observed decreased levels for these proteins were not the direct result of decreased transcription or protein instability. Chlorophyll fluorescence induction experiments indicated altered quinone reduction kinetics for the double and triple mutant plants with significant differences observed for absorbance, trapping, and electron transport. Transmission electron microscopy analysis of the chloroplasts in mutant plants showed impaired grana stacking and increased accumulation of starch granules consistent with some chloroplast proteins being decreased. Rescue of the
plant growth phenotype and increased expression of the validated proteins to wild-type levels was obtained by overexpression of eIFiso4G1. These data suggest a direct and specialized role for eIFiso4G in the synthesis of a subset of plant proteins.
Background
Treatment choices for individual patients with an inborn bleeding disorder are increasingly challenging due to increasing options and rising costs for society. We have initiated an ...integrated interdisciplinary national research program.
Objectives
The SYMPHONY consortium strives to orchestrate personalized treatment in patients with an inborn bleeding disorder, by unraveling the mechanisms behind interindividual variations of bleeding phenotype.
Patients
The SYMPHONY consortium will investigate patients with an inborn bleeding disorder, both diagnosed and not yet diagnosed.
Results
Research questions are categorized under the themes: (1) diagnosis, (2) treatment, and (3) fundamental research, and consist of work packages addressing specific domains. Importantly, collaborations between patients and talented researchers from different areas of expertise promise to augment the impact of the SYMPHONY consortium, leading to unique interactions and intellectual property.
Conclusions
SYMPHONY will perform research on all aspects of care, treatment individualization in patients with inborn bleeding disorders, as well as diagnostic innovations and results of molecular genetics and cellular model technology with regard to the hemostatic process. We believe that these research investments will lead to health‐care innovations with long‐term clinical and societal impact.
This consortium has been made possible by a governmental, competitive grant from the Netherlands Organization for Scientific Research (NWO) within the framework of the NWA‐ORC Call grant agreement NWA.1160.18.038.
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FZAB, GEOZS, GIS, IJS, IMTLJ, IZUM, KILJ, KISLJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBJE, SBMB, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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