Fanconi anemia (FA) develops due to a mutation in one of the FANC genes that are involved in the repair of interstrand crosslinks (ICLs). FANCG, a member of the FA core complex, is essential for ICL ...repair. Previous FANCG-deficient mouse models were generated with drug-based selection cassettes in mixed mice backgrounds, leading to a disparity in the interpretation of genotype-related phenotype. We created a
-KO (KO) mouse model using CRISPR/Cas9 to exclude these confounders. The entire
locus was targeted and maintained on the immunological well-characterized C57BL/6J background. The intercrossing of heterozygous mice resulted in sub-Mendelian numbers of homozygous mice, suggesting the loss of FANCG can be embryonically lethal. KO mice displayed infertility and hypogonadism, but no other developmental problems. Bone marrow analysis revealed a defect in various hematopoietic stem and progenitor subsets with a bias towards myelopoiesis. Cell lines derived from
-KO mice were hypersensitive to the crosslinking agents cisplatin and Mitomycin C, and
-KO mouse embryonic fibroblasts (MEFs) displayed increased γ-H2AX upon cisplatin treatment. The reconstitution of these MEFs with
cDNA corrected for the ICL hypersensitivity. This project provides a new, genetically, and immunologically well-defined
-KO mouse model for further in vivo and in vitro studies on FANCG and ICL repair.
Within the Dutch Acute HCV in HIV Study, a surveillance system was initiated to estimate the incidence of hepatitis C virus (HCV) infections in 2014. Following the Dutch HIV treatment guidelines, ...HIV-positive men having sex with men (MSM) in 19 participating centers were screened. Ninety-nine acute HCV infections were reported, which resulted in a mean incidence of 11 per 1000 patient-years of follow-up. Unfortunately, the HCV epidemic among Dutch HIV-positive MSM is not coming to a halt.
The development and differentiation of B cells is intimately linked to cell proliferation and the generation of diverse immunoglobulin gene (
) repertoires. The ubiquitin E3 ligase HUWE1 controls ...proliferation, DNA damage responses, and DNA repair, including the base excision repair (BER) pathway. These processes are of crucial importance for B-cell development in the bone marrow, and the germinal center (GC) response, which results in the clonal expansion and differentiation of B cells expressing high affinity immunoglobulins. Here, we re-examined the role of HUWE1 in B-cell proliferation and
gene diversification, focusing on its involvement in somatic hypermutation (SHM) and class switch recombination (CSR). B-cell-specific deletion of
resulted in impaired development, differentiation and maturation of B cells in the bone marrow and peripheral lymphoid organs. HUWE1 deficiency diminished SHM and CSR by impairing B-cell proliferation and AID expression upon activation
and
, and was unrelated to the HUWE1-dependent regulation of the BER pathway. Interestingly, we found that HUWE1-deficient B cells showed increased mRNA expression of Myc target genes upon
activation despite diminished proliferation. Our results confirm that the E3 ligase HUWE1 is an important contributor in coordinating the rapid transition of antigen naïve, resting B cells into antigen-activated B cells and regulates mutagenic processes in B cells by controlling AID expression and the post-transcriptional output of Myc target genes.
DNA damage threatens genomic integrity and instigates stem cell failure. To bypass genotoxic lesions during replication, cells employ DNA damage tolerance (DDT), which is regulated via PCNA ...ubiquitination and REV1. DDT is conserved in all domains of life, yet its relevance in mammals remains unclear. Here, we show that inactivation of both PCNA-ubiquitination and REV1 results in embryonic and adult lethality, and the accumulation of DNA damage in hematopoietic stem and progenitor cells (HSPCs) that ultimately resulted in their depletion. Our results reveal the crucial relevance of DDT in the maintenance of stem cell compartments and mammalian life in unperturbed conditions.
The limited preservation duration of organs has contributed to the shortage of organs for transplantation. Recently, a tripling of the storage duration was achieved with supercooling, which relies on ...temperatures between -4 and -6 °C. However, to achieve deeper metabolic stasis, lower temperatures are required. Inspired by freeze-tolerant animals, we entered high-subzero temperatures (-10 to -15 °C) using ice nucleators to control ice and cryoprotective agents (CPAs) to maintain an unfrozen liquid fraction. We present this approach, termed partial freezing, by testing gradual (un)loading and different CPAs, holding temperatures, and storage durations. Results indicate that propylene glycol outperforms glycerol and injury is largely influenced by storage temperatures. Subsequently, we demonstrate that machine perfusion enhancements improve the recovery of livers after freezing. Ultimately, livers that were partially frozen for 5-fold longer showed favorable outcomes as compared to viable controls, although frozen livers had lower cumulative bile and higher liver enzymes.
The Swift Gamma Ray Burst satellite routinely provides prompt positions for GRBs and their afterglows on timescales of a few hundred seconds. However, with a pointing accuracy of only a few ...arcminutes, and a systematic uncertainty on the star-tracker solutions to the World Coordinate System of 3–4 arcsec, the precision of the early XRT positions is limited to 3–4 arcsec at best. This is significant because operationally, the XRT detects >95% of all GRBs, while the UVOT detects only the optically brightest bursts, ~30% of all bursts detected by BAT; thus early and accurate XRT positions are important because for the majority of bursts they provide the best available information for the initial ground-based follow-up campaigns. Here we describe an autonomous way of producing more accurate prompt XRT positions for GRBs and their afterglows, based on UVOT astrometry and a detailed mapping between the XRT and UVOT detectors. The latter significantly reduces the dominant systematic error – the star-tracker solution to the World Coordinate System. This technique, which is limited to times when there is significant overlap between UVOT and XRT PC-mode data, provides a factor of 2 improvement in the localisation of XRT refined positions on timescales of less than a few hours. Furthermore, the accuracy achieved is superior to astrometrically corrected XRT PC mode images at early times (for up to 24 h), for the majority of bursts, and is comparable to the accuracy achieved by astrometrically corrected X-ray positions based on deep XRT PC-mode imaging at later times.
Development of progenitor B cells (ProB cells) into precursor B cells (PreB cells) is dictated by immunoglobulin heavy chain checkpoint (IgHCC), where the IgHC encoded by a productively rearranged ...Igh allele assembles into a PreB cell receptor complex (PreBCR) to generate signals to initiate this transition and suppressing antigen receptor gene recombination, ensuring that only one productive Igh allele is expressed, a phenomenon known as Igh allelic exclusion. In contrast to a productively rearranged Igh allele, the Igh messenger RNA (mRNA) (IgHR) from a nonproductively rearranged Igh allele is degraded by nonsense-mediated decay (NMD). This fact prohibited firm conclusions regarding the contribution of stable IgHR to the molecular and developmental changes associated with the IgHCC. This point was addressed by generating the IghTer5HΔTM
mouse model from IghTer5H
mice having a premature termination codon at position +5 in leader exon of IghTer5H
allele. This prohibited NMD, and the lack of a transmembrane region (ΔTM) prevented the formation of any signaling-competent PreBCR complexes that may arise as a result of read-through translation across premature Ter5 stop codon. A highly sensitive sandwich Western blot revealed read-through translation of IghTer5H
message, indicating that previous conclusions regarding a role of IgHR in establishing allelic exclusion requires further exploration. As determined by RNA sequencing (RNA-Seq), this low amount of IgHC sufficed to initiate PreB cell markers normally associated with PreBCR signaling. In contrast, the IghTer5HΔTM
knock-in allele, which generated stable IgHR but no detectable IgHC, failed to induce PreB development. Our data indicate that the IgHCC is controlled at the level of IgHC and not IgHR expression.
Guideline adherence is generally high in Dutch general practices. However, the prescription of insulins to type 2 diabetes mellitus patients is often not in line with the guideline, which recommends ...NPH insulin as first choice and discourages newer insulins. This qualitative study aimed to identify the reasons why primary care healthcare professionals prescribe insulins that are not recommended in guidelines.
Digital focus groups with primary care practitioners were organised. A topic list was developed, based on reasons for preferred insulins obtained from literature and a priori expert discussions. The discussions were video and audio-recorded, transcribed verbatim and coded with a combination of inductive and deductive codes. Codes were categorized into an existing knowledge, attitudes and behaviour model for guideline non-adherence.
Four focus groups with eleven general practitioners, twelve practice nurses, six pharmacists, four diabetes nurses and two nurse practitioners were organised. The prescription of non-recommended insulins was largely driven by argumentation in the domain of attitudes. Lack of agreement with the guideline was the most prominent category. Most of those perspectives did not reflect disagreement with the guideline recommendations in general, but were about advantages of non-recommended insulins, which led, according to the healthcare professionals, to better applicability of those insulins to specific patients. The belief that guideline-recommended insulins were less effective, positive experience with other insulins and marketing from pharmaceutical companies were also identified as attitude-related barriers to prescribe guideline-recommended insulins. One additional category in the domain of attitudes was identified, namely the lack of uniformity in policy between healthcare professionals in the same practice. Only a small number of external barriers were identified, focusing on patient characteristics that prevented the use of recommended insulins, the availability of contradictory guidelines and other, mostly secondary care, healthcare providers initiating non-recommended insulins. No knowledge-related barriers were identified.
The prescription of non-recommended insulins in primary care is mostly driven by lack of agreement with the guideline recommendations and different interpretation of evidence. These insights can be used for the development of interventions to stimulate primary care practitioners to prescribe guideline-recommended insulins.
Background
The combination of combined active antiretroviral therapy (cART) with chemotherapy in the treatment of lymphoma in human immunodeficiency virus (HIV)-positive patients has improved the ...overall survival of these patients. However, drug–drug interactions between antineoplastic agents and the antiretroviral agents non-nucleoside reverse transcriptase inhibitors (NNRTIs) and protease inhibitors (PIs) can occur by influencing the activity of the CYP3A4 enzyme. So far, little is known about the clinical relevance of this interaction: the effect on the efficacy and toxicity of the chemotherapy. Also, there is no general consensus which cART is preferable in combination with antineoplastic drugs.
Objective
To compare PI-based with NNRTI-based cART on the efficacy and toxicity of chemotherapy in lymphoma patients.
Setting
The Onze Lieve Vrouwe Gasthuis, located in Amsterdam, The Netherlands.
Method
A retrospective observational cohort study including all patients with HIV and lymphoma over a 10-year period. Clinical outcome (response to chemotherapy and survival) and toxicity of chemotherapy (renal, hepatic and bone marrow toxicity as well as dose reduction, treatment delay and discontinuation) was compared in patients with PI based and NNRTI-based cART. Main outcome measure: Response to chemotherapy and survival.
Results
Patients using PI-based cART (n = 22) had a significantly lower 1 year survival compared to NNRTI-based cART (n = 21). No significant differences were observed in reaching complete remission after chemotherapy. No overall significant differences in toxicity and discontinuation of the chemotherapy were observed. However, there was a trend towards more severe bone-marrow toxicity in patients with PI-based cART. In addition, patients with PI-based cART received earlier dose-reduction and treatment delay, indicating increased toxicity in PI-treated patients.
Conclusion
This retrospective study shows that PI-based cART is inferior in combination with chemotherapy to NNRTI-based cART: a lower 1 year survival is observed and dose-reduction and treatment delay occur earlier, possibly based on an earlier onset of toxicity.
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