Effect of polymorphisms in folate-related genes on in vitro methotrexate sensitivity in pediatric acute lymphoblastic leukemia

We studied whether common polymorphisms in genes involved in folate metabolism affect methotrexate (MTX) sensitivity. Ex vivo MTX sensitivity of lymphoblasts obtained from pediatric patients with acute lymphoblastic leukemia (ALL; n = 157) was determined by the in situ thymidylate synthase inhibition assay after either continuous (21 hours; TSI(50, cont)) or short-term (3 hours; TSI(50, short)) MTX exposure. DNA was isolated from lymphoblasts obtained from cytospin slides. Polymorphisms in methylenetetrahydrofolate reductase (MTHFR 677C>T, MTHFR 1298A>C), methionine synthase (MTR 2756A>G), methionine synthase reductase (MTRR 66A>G), methylenetetrahydrofolate dehydrogenase (MTHFD1 1958G>A), serine hydroxymethyl transferase (SHMT1 1420C>T), thymidylate synthase (TS 2R3R), and the reduced folate carrier (RFC 80G>A) were detected by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) or real-time PCR. Patients with the MTHFR 1298AC variant or the MTRR 66 G-allele showed decreased in vitro MTX sensitivity measured under both test conditions. SHMT1 1420TT homozygotes only showed decreased MTX sensitivity in the TSI(50, cont). In conclusion, polymorphisms in the folate-related genes MTHFR, MTRR, and SHMT1 are related to MTX resistance in pediatric patients with ALL

Folates provoke cellular efflux and drug resistance of substrates of the multidrug resistance protein 1 (MRP1)

Cellular folate concentration was earlier reported to be a critical factor in the activity and expression of the multidrug resistance protein MRP1 (ABCC1). Since MRP1 mediates resistance to a variety of therapeutic drugs, we investigated whether the cellular folate concentration inf

Drought alters the spatial distribution, grazing patterns, and radula morphology of a fungal-farming salt marsh snail

Contains fulltext : 208086.pdf (publisher's version ) (Open Access

Repetitive desiccation events weaken a salt marsh mutualism

1. Salt marshes suffered large‐scale degradation in recent decades. Extreme events such as hot and dry spells contributed significantly to this, and are predicted to increase not only in intensity, but also in frequency under future climate scenarios. Such repetitive extreme events may generate cumulative effects on ecosystem resilience. It is therefore important to elucidate how marsh vegetation responds to repetitive stress, and whether changes in key species interactions can modulate vegetation resilience.2. In this study, we investigated how moderate but repetitive desiccation events, caused by the combined effects of drought and high temperatures, affect cordgrass (<i>Spartina alterniflora</i>), the dominant habitat‐forming grass in southeastern US salt marshes. In a 4‐month field experiment, we simulated four consecutive desiccation events by periodically excluding tidal flooding and rainfall, while raising temperature. We crossed this desiccation treatment with the presence/absence of ribbed mussels (<i>Geukensia demissa</i>) – a mutualist of cordgrass known to enhance its desiccation resilience – and with grazing pressure by the marsh periwinkle (<i>Littoraria irrorate</i>) that is known to suppress cordgrass’ desiccation resilience. 3. We found that each subsequent desiccation event deteriorated sediment porewater conditions, resulting in high salinity (53 ppt), low pH‐levels (3.7) and increased porewater Al and Fe concentrations (≈800 μmol/L and ≈1,500 μmol/L) upon rewetting. No effects on porewater chemistry were found as a result of snail grazing, while ribbed mussels strongly mitigated desiccation effects almost to control levels and increased cordgrass biomass by approximately 128%. Importantly, although cordgrass generally appeared healthy above‐ground at the end of the experiment, we found clear negative responses of the repetitive desiccation treatment on cordgrass below‐ground biomass, on proline (osmolyte) levels in shoots and on the number of tillers (−40%), regardless of mussel and/or snail presence.4. <i>Synthesis</i>. Even though the mutualism with mussels strongly mitigated chemical effects in the sediment porewater throughout the experiment, mussels could not buffer the adverse ecophysiological effects observed in cordgrass tissue. Our results therefore suggest that although mussels may alleviate desiccation stress, the predicted increased frequency and intensity of hot dry spells may eventually affect saltmarsh resilience by stressing the mutualism beyond its buffering capacity

Guidelines for resident training in veterinary clinical pathology. IV: Laboratory quality management—teaching domains, competencies, and suggested learning outcomes

BACKGROUND : The 2019 ASVCP Education Committee Forum for Discussion, presented at the annual ASVCP/ACVP meeting, identified a need to develop recommendations for teaching laboratory quality management principles in veterinary clinical pathology residency training programs. OBJECTIVES : To present a competency-based framework for teaching laboratory quality management principles in veterinary clinical pathology residency training programs, including entrustable professional activities (EPAs), domains of competence, individual competencies, and learning outcomes. METHODS : A joint subcommittee of the ASVCP Quality Assurance and Laboratory Standards (QALS) and Education Committees executed this project. A draft guideline version was reviewed by the ASVCP membership and shared with selected ACVP committees in early 2022, and a final version was voted upon by the full QALS and Education Committees in late 2022. RESULTS : Eleven domains of competence with relevant individual competencies were identified. In addition, suggested learning outcomes and resource lists were developed. Domains and individual competencies were mapped to six EPAs. CONCLUSIONS : This guideline presents a framework for teaching principles of laboratory quality management in veterinary clinical pathology residency training programs and was designed to be comprehensive yet practical. Guidance on pedagogical terms and possible routes of implementation are included. Recommendations herein aim to improve and support resident training but may require gradual implementation, as programs phase in necessary expertise and resources. Future directions include the development of learning milestones and assessments and consideration of how recommendations intersect with the American College of Veterinary Pathologists training program accreditation and certifying examination.http://www.wileyonlinelibrary.com/journal/vcphj2023Companion Animal Clinical Studie

Restoration of biogeomorphic systems by creating windows of opportunity to support natural establishment processes

In degraded landscapes, recolonization by pioneer vegetation is often halted by the presence of persistent environmental stress. When natural expansion does occur, it is commonly due to the momentary alleviation of a key environmental variable previously limiting new growth. Thus, studying the circumstances in which expansion occurs can inspire new restoration techniques, wherein vegetation establishment is provoked by emulating natural events through artificial means. Using the salt-marsh pioneer zone on tidal flats as a biogeomorphic model system, we explore how locally raised sediment bed forms, which are the result of natural (bio)geomorphic processes, enhance seedling establishment in an observational study. We then conduct a manipulative experiment designed to emulate these facilitative conditions in order to enable establishment on an uncolonized tidal flat. Here, we attempt to generate raised growth-promoting sediment bed forms using porous artificial structures. Flume experiments demonstrate how these structures produce a sheltered hydrodynamic environment in which suspended sediment and seeds preferentially settle. The application of these structures in the field led to the formation of stable, raised sediment platforms and the spontaneous recruitment of salt-marsh pioneers in the following growing season. These recruits were composed primarily of the annual pioneering Salicornia genus, with densities of up to 140 individuals/m2 within the structures, a 60-fold increase over ambient densities. Lower abundances of five other perennial species were found within structures that did not appear elsewhere in the pioneer zone. Furthermore, recruits grew to be on average three times greater in mass inside of the structures than in the neighboring ambient environment. The success of this restoration design may be attributed to the combination of three factors: (1) enhanced seed retention, (2) suppressed mortality, and (3) accelerated growth rates on the elevated surfaces generated by the artificial structures. We argue that restoration approaches similar to the one shown here, wherein the conditions for natural establishment are actively mimicked to promote vegetation development, may serve as promising tools in many biogeomorphic ecosystems, ranging from coastal to arid ecosystems

Effect of folate derivatives on the activity of antifolate drugs used against malaria and cancer

The folate derivatives folic acid (FA) and folinic acid (FNA) decrease the in vivo and in vitro activities of antifolate drugs in Plasmodium falciparum. However, the effects of 5-methyl-tetrahydrofolate (5-Me-THF) and tetrahydrofolate (THF), the two dominant circulating folate forms in humans, have not been explored yet. We have investigated the effects of FA, FNA, 5-Me-THF, and THF on the in vitro activity of the antimalarial antifolates pyrimethamine and chlorcycloguanil and the anticancer antifolates methotrexate (MTX), aminopterin, and trimetrexate (TMX), against P. falciparum. The results indicate that these anticancers are potent against P. falciparum, with IC50 < 50 nM. 5-Me-THF does not significantly decrease the activity of all tested drugs, and none of the tested folate derivatives significantly decrease the activity of these anticancers. Thus, malaria folate metabolism has features different from those in human, and the exploitation of this difference could lead to the discovery of new drugs to treat malaria. For instance, the combination of 5-Me-THF with a low dose of TMX could be used to treat malaria. In addition, the safety of a low dose of MTX in the treatment of arthritis indicates that this drug could be used alone to treat malaria

Conditional Immortalization of Human B Cells by CD40 Ligation

It is generally assumed that human differentiated cells have a limited life-span and proliferation capacity in vivo, and that genetic modifications are a prerequisite for their immortalization in vitro. Here we readdress this issue, studying the long-term proliferation potential of human B cells. It was shown earlier that human B cells from peripheral blood of healthy donors can be efficiently induced to proliferate for up to ten weeks in vitro by stimulating their receptor CD40 in the presence of interleukin-4. When we applied the same stimuli under conditions of modified cell number and culture size, we were surprised to find that our treatment induced B cells to proliferate throughout an observation period of presently up to 1650 days, representing more than 370 population doublings, which suggested that these B cells were immortalized in vitro. Long-term CD40-stimulated B cell cultures could be established from most healthy adult human donors. These B cells had a constant phenotype, were free from Epstein-Barr virus, and remained dependent on CD40 ligation. They had constitutive telomerase activity and stabilized telomere length. Moreover, they were susceptible to activation by Toll-like receptor 9 ligands, and could be used to expand antigen-specific cytotoxic T cells in vitro. Our results indicate that human somatic cells can evade senescence and be conditionally immortalized by external stimulation only, without a requirement for genetic manipulation or oncoviral infection. Conditionally immortalized human B cells are a new tool for immunotherapy and studies of B cell oncogenesis, activation, and function

High susceptibility of c-KIT+CD34+ precursors to prolonged doxorubicin exposure interferes with Langerhans cell differentiation in a human cell line model

As neoadjuvant and adjuvant chemotherapy schedules often consist of multiple treatment cycles over relatively long periods of time, it is important to know what effects protracted drug administration can have on the immune system. Here, we studied the long-term effects of doxorubicin on the capacity of dendritic cell (DC) precursors to differentiate into a particular DC subset, the Langerhans cells (LC). In order to achieve high telomerase activity as detected in hematological stem cells, precursor cells from the acute-myeloid leukemia (AML)-derived cell line MUTZ3 were stably transduced with human telomerase reverse transcriptase (hTERT) to facilitate their growth potential, while preventing growth, and drug-induced senescence, and preserving their unique capacity for cytokine-dependent DC and LC differentiation. The hTERT-MUTZ3 cells were selected with increasing concentrations of the anthracyclin doxorubicin. After 1–2 months of selection with 30–90 nM doxorubicin, the cells completely lost their capacity to differentiate into LC. This inhibition turned out to be reversible, as the cells slowly regained their capacity to differentiate after a 3- to 4-month drug-free period and with this became capable again of priming allogeneic T cells. Of note, the loss and gain of this capacity to differentiate coincided with the loss and gain of a subpopulation within the CD34+ proliferative compartment with surface expression of the stem cell factor receptor (SCF-R/CD117/c-Kit). These data are in favor of cytostatic drug-free intervals before applying autologous DC-based vaccination protocols, as specific DC precursors may need time to recover from protracted chemotherapy treatment and re-emerge among the circulating CD34+ hematopoietic stem and precursor cells

Potent interaction of flavopiridol with MRP1

The multidrug resistance protein 1 (MRP1) is an ATP-dependent transport protein for organic anions, as well as neutral or positively charged anticancer agents. In this study we show that flavopiridol, a synthetic flavonoid currently studied in phase 1 trials for its anti-proliferative characteristics, interacts with MRP1 in a potent way. Flavopiridol, as well as other (iso)flavonoids stimulate the ATPase activity of MRP1 in a dose-dependent way at low micromolar concentrations. A new specific monoclonal antibody against MRP1 (MIB6) inhibits the (iso)flavonoid-induced ATPase activity of plasma membrane vesicles prepared from the MRP1 overexpressing cell line GLC4/ADR. The accumulation of daunorubicin in GLC4/ADR cells is increased by flavopiridol and by other non-glycosylated (iso)flavonoids that interact with MRP1 ATPase activity. However, flavopiridol is the only tested compound that affects the daunorubicin accumulation when present at concentrations below 1 μM. Glycosylated (iso)flavonoids do not affect MRP1-mediated transport or ATPase activity. Finally, MRP1 overexpressing and transfected cells are resistant to flavopiridol, but not to other (iso)flavonoids tested. These findings may be of relevance for the development of anticancer therapies with flavopiridol. © 1999 Cancer Research Campaig