The nucleoside analog cytarabine (Ara-C) is an essential component of primary and salvage chemotherapy regimens for acute myeloid leukemia (AML). After cellular uptake, Ara-C is converted into its ...therapeutically active triphosphate metabolite, Ara-CTP, which exerts antileukemic effects, primarily by inhibiting DNA synthesis in proliferating cells. Currently, a substantial fraction of patients with AML fail to respond effectively to Ara-C therapy, and reliable biomarkers for predicting the therapeutic response to Ara-C are lacking. SAMHD1 is a deoxynucleoside triphosphate (dNTP) triphosphohydrolase that cleaves physiological dNTPs into deoxyribonucleosides and inorganic triphosphate. Although it has been postulated that SAMHD1 sensitizes cancer cells to nucleoside-analog derivatives through the depletion of competing dNTPs, we show here that SAMHD1 reduces Ara-C cytotoxicity in AML cells. Mechanistically, dGTP-activated SAMHD1 hydrolyzes Ara-CTP, which results in a drastic reduction of Ara-CTP in leukemic cells. Loss of SAMHD1 activity-through genetic depletion, mutational inactivation of its triphosphohydrolase activity or proteasomal degradation using specialized, virus-like particles-potentiates the cytotoxicity of Ara-C in AML cells. In mouse models of retroviral AML transplantation, as well as in retrospective analyses of adult patients with AML, the response to Ara-C-containing therapy was inversely correlated with SAMHD1 expression. These results identify SAMHD1 as a potential biomarker for the stratification of patients with AML who might best respond to Ara-C-based therapy and as a target for treating Ara-C-refractory AML.
•Efficient chemical and thermal inactivation of lentiviruses and lentiviral vectors.•Efficient chemical inactivation of lentivirus-infected cells.•Pseudotyping and complex buffers do not alter the ...efficacy of inactivating agents.
Lentiviral vectors and lentiviruses are important tools for basic and applied biomedical research. Yet, biosafety regulations from legal authorities have to be fulfilled when transferring BSL-2 to -3 vectors/viruses to facilities with lower biosafety level. Here, we (re-)evaluated different chemical and thermal approaches to inactivate vesicular stomatitis virus G-protein (VSV-G) pseudotyped lentiviral vectors and either wildtype or VSV-G pseudotyped human immunodeficiency viruses (HIV). Aldehydes, detergents and alcohols were as effective as thermal inactivation procedures to efficiently inactivate purified lentiviral vectors and replication-competent HIV. In addition, no residual infectivity was detected when inactivating HIV-infected TZM-bl reporter cells with selected detergents and aldehydes. Thus, our established inactivation protocols can be used by other laboratories working with lentiviral vectors or infectious lentiviruses and provide a template for viruses with similar physicochemical properties.
Early after entry into monocytes, macrophages, dendritic cells, and resting CD4 T cells, HIV encounters a block, limiting reverse transcription (RT) of the incoming viral RNA genome. In this context, ...dNTP triphosphohydrolase SAM domain and HD domain-containing protein 1 (SAMHD1) has been identified as a restriction factor, lowering the concentration of dNTP substrates to limit RT. The accessory lentiviral protein X (Vpx) proteins from the major simian immunodeficiency virus of rhesus macaque, sooty mangabey, and HIV-2 (SIVsmm/SIVmac/HIV-2) lineage packaged into virions target SAMHD1 for proteasomal degradation, increase intracellular dNTP pools, and facilitate HIV cDNA synthesis. We find that virion-packaged Vpx proteins from a second SIV lineage, SIV of red-capped mangabeys or mandrills (SIVrcm/mnd-2), increased HIV infection in resting CD4 T cells, but not in macrophages, and, unexpectedly, acted in the absence of SAMHD1 degradation, dNTP pool elevation, or changes in SAMHD1 phosphorylation. Vpx rcm/mnd-2 virion incorporation resulted in a dramatic increase of HIV-1 RT intermediates and viral cDNA in infected resting CD4 T cells. These analyses also revealed a barrier limiting HIV-1 infection of resting CD4 T cells at the level of nuclear import. Single amino acid changes in the SAMHD1-degrading Vpx mac239 allowed it to enhance early postentry steps in a Vpx rcm/mnd-2–like fashion. Moreover, Vpx enhanced HIV-1 infection of SAMHD1-deficient resting CD4 T cells of a patient with Aicardi-Goutières syndrome. These results indicate that Vpx, in addition to SAMHD1, overcomes a previously unappreciated restriction for lentiviruses at the level of RT that acts independently of dNTP concentrations and is specific to resting CD4 T cells.
Collapsin response mediator proteins (CRMPs) are key for brain development and function. Here, we link CRMP1 to a neurodevelopmental disorder. We report heterozygous de novo variants in the
gene in ...three unrelated individuals with muscular hypotonia, intellectual disability, and/or autism spectrum disorder. Based on in silico analysis these variants are predicted to affect the CRMP1 structure. We further analyzed the effect of the variants on the protein structure/levels and cellular processes. We showed that the human
variants impact the oligomerization of CRMP1 proteins. Moreover, overexpression of the
variants affect neurite outgrowth of murine cortical neurons. While altered CRMP1 levels have been reported in psychiatric diseases, genetic variants in
gene have never been linked to human disease. We report for the first-time variants in the
gene and emphasize its key role in brain development and function by linking directly to a human neurodevelopmental disease.
Ruminants such as cattle and sheep depend on the breakdown of carbohydrates from plant-based feedstuff, which is accomplished by the microbial community in the rumen. Roughly 40% of the members of ...the rumen microbiota belong to the family
, which ferments sugars to organic acids such as acetate, propionate, and succinate. These substrates are important nutrients for the ruminant. In a metaproteome analysis of the rumen of cattle, proteins that are homologous to the Na
-translocating NADH:quinone oxidoreductase (NQR) and the quinone:fumarate reductase (QFR) were identified in different
species. Here, we show that fumarate reduction to succinate in anaerobically growing Prevotella bryantii is coupled to chemiosmotic energy conservation by a supercomplex composed of NQR and QFR. This
odium-translocating
ADH:
umarate oxido
eductase (SNFR) supercomplex was enriched by blue native PAGE (BN-PAGE) and characterized by in-gel enzyme activity staining and mass spectrometry. High NADH oxidation (850 nmol min
mg
), quinone reduction (490 nmol min
mg
), and fumarate reduction (1,200 nmol min
mg
) activities, together with high expression levels, demonstrate that SNFR represents a charge-separating unit in
. Absorption spectroscopy of SNFR exposed to different substrates revealed intramolecular electron transfer from the flavin adenine dinucleotide (FAD) cofactor in NQR to heme
cofactors in QFR. SNFR catalyzed the stoichiometric conversion of NADH and fumarate to NAD
and succinate. We propose that the regeneration of NAD
in
is intimately linked to the buildup of an electrochemical gradient which powers ATP synthesis by electron transport phosphorylation.
Feeding strategies for ruminants are designed to optimize nutrient efficiency for animals and to prevent energy losses like enhanced methane production. Key to this are the fermentative reactions of the rumen microbiota, dominated by
spp. We show that succinate formation by
is coupled to NADH oxidation and sodium gradient formation by a newly described supercomplex consisting of Na
-translocating NADH:quinone oxidoreductase (NQR) and fumarate reductase (QFR), representing the
odium-translocating
ADH:
umarate oxido
eductase (SNFR) supercomplex. SNFR is the major charge-separating module, generating an electrochemical sodium gradient in
. Our findings offer clues to the observation that use of fumarate as feed additive does not significantly increase succinate production, or decrease methanogenesis, by the microbial community in the rumen.
Mental disorders (MDs) are one of the leading causes for workforce sickness absence and disability worldwide. The burden, costs and challenges are enormous for the individuals concerned, employers ...and society at large. Although most MDs are characterised by a high risk of relapse after treatment or by chronic courses, interventions that link medical-psychotherapeutic approaches with work-directed components to facilitate a sustainable return to work (RTW) are rare. This protocol describes the design of a study to evaluate the (cost-)effectiveness and implementation process of a multimodal, clinical and work-directed intervention, called RTW-PIA, aimed at employees with MDs to achieve sustainable RTW in Germany.
The study consists of an effectiveness, a health-economic and a process evaluation, designed as a two-armed, multicentre, randomised controlled trial, conducted in German psychiatric outpatient clinics. Sick-listed employees with MDs will receive either the 18-month RTW-PIA treatment in conjunction with care as usual, or care as usual only. RTW-PIA consists of a face-to-face individual RTW support, RTW aftercare group meetings, and web-based aftercare. Assessments will be conducted at baseline and 6, 12, 18 and 24 months after completion of baseline survey. The primary outcome is the employees´ achievement of sustainable RTW, defined as reporting less than six weeks of working days missed out due to sickness absence within 12 months after first RTW. Secondary outcomes include health-related quality of life, mental functioning, RTW self-efficacy, overall job satisfaction, severity of mental illness and work ability. The health-economic evaluation will be conducted from a societal and public health care perspective, as well as from the employer's perspective in a cost-benefit analysis. The design will be supplemented by a qualitative effect evaluation using pre- and post-interviews, and a multimethod process evaluation examining various predefined key process indicators from different stakeholder perspectives.
By applying a comprehensive, multimethodological evaluation design, this study captures various facets of RTW-PIA. In case of promising results for sustainable RTW, RTW-PIA may be integrated into standard care within German psychiatric outpatient clinics.
The study was prospectively registered with the German Clinical Trials Register ( DRKS00026232 , 1 September 2021).
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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