The human umbilical cord (hUC) is the lifeline that connects the fetus to the mother. Hypercoiling of the hUC is associated with pre- and perinatal morbidity and mortality. We investigated the... Show moreThe human umbilical cord (hUC) is the lifeline that connects the fetus to the mother. Hypercoiling of the hUC is associated with pre- and perinatal morbidity and mortality. We investigated the origin of hUC hypercoiling using state-of-the-art imaging and omics approaches. Macroscopic inspection of the hUC revealed the helices to originate from the arteries rather than other components of the hUC. Digital reconstruction of the hUC arteries showed the dynamic alignment of two layers of muscle fibers in the tunica media aligning in opposing directions. We observed that genetically identical twins can be discordant for hUC coiling, excluding genetic, many environmental, and parental origins of hUC coiling. Comparing the transcriptomic and DNA methylation profile of the hUC arteries of four twin pairs with discordant cord coiling, we detected 28 differentially expressed genes, but no differentially methylated CpGs. These genes play a role in vascular development, cell–cell interaction, and axis formation and may account for the increased number of hUC helices. When combined, our results provide a novel framework to understand the origin of hUC helices in fetal development. Show less
Krift, F. van der; Zijlmans, D.W.; Shukla, R.; Javed, A.; Koukos, P.I.; Schwarz, L.L.; ... ; Maurice, M.M. 2023
Cancer cells make extensive use of the folate cycle to sustain increased anabolic metabolism. Multiple chemotherapeutic drugs interfere with the folate cycle, including methotrexate and 5... Show moreCancer cells make extensive use of the folate cycle to sustain increased anabolic metabolism. Multiple chemotherapeutic drugs interfere with the folate cycle, including methotrexate and 5-fluorouracil that are commonly applied for the treatment of leukemia and colorectal cancer (CRC), respectively. Despite high success rates, therapy-induced resistance causes relapse at later disease stages. Depletion of folylpolyglutamate synthetase (FPGS), which normally promotes intracellular accumulation and activity of natural folates and methotrexate, is linked to methotrexate and 5-fluorouracil resistance and its association with relapse illustrates the need for improved intervention strategies. Here, we describe a novel antifolate (C1) that, like methotrexate, potently inhibits dihydrofolate reductase and downstream one-carbon metabolism. Contrary to methotrexate, C1 displays optimal efficacy in FPGS-deficient contexts, due to decreased competition with intracellular folates for interaction with dihydrofolate reductase. We show that FPGS-deficient patient-derived CRC organoids display enhanced sensitivity to C1, whereas FPGS-high CRC organoids are more sensitive to methotrexate. Our results argue that polyglutamylation-independent antifolates can be applied to exert selective pressure on FPGS-deficient cells during chemotherapy, using a vulnerability created by polyglutamylation deficiency. Show less
Czukiewska, S.M.; Fan, X.Y.; Mulder, A.A.; Helm, T. van der; Hillenius, S.; Meeren, L. van der; ... ; Lopes, S.M.C.D. 2023
Gametogenesis is a complex and sex-specific multistep process during which the gonadal somatic niche plays an essential regulatory role. One of the most crucial steps during human female... Show moreGametogenesis is a complex and sex-specific multistep process during which the gonadal somatic niche plays an essential regulatory role. One of the most crucial steps during human female gametogenesis is the formation of primordial follicles, the functional unit of the ovary that constitutes the pool of follicles available at birth during the entire reproductive life. However, the relation between human fetal germ cells (hFGCs) and gonadal somatic cells during the formation of the primordial follicles remains largely unexplored. We have discovered that hFGCs can form multinucleated syncytia, some connected via interconnecting intercellular bridges, and that not all nuclei in hFGC–syncytia were synchronous regarding meiotic stage. As hFGCs progressed in development, pre-granulosa cells formed protrusions that seemed to progressively constrict individual hFGCs, perhaps contributing to separate them from the multinucleated syncytia. Our findings highlighted the cell–cell interaction and molecular dynamics between hFGCs and (pre)granulosa cells during the formation of primordial follicles in humans. Knowledge on how the pool of primordial follicle is formed is important to understand human infertility. Show less
The modification of substrates with ADP-ribose (ADPr) is im-portant in, for example, antiviral immunity and cancer. Recently, several reagents were developed to detect ADP-ribosylation; however, it... Show moreThe modification of substrates with ADP-ribose (ADPr) is im-portant in, for example, antiviral immunity and cancer. Recently, several reagents were developed to detect ADP-ribosylation; however, it is unknown whether they recognise ADPr, specific amino acid-ADPr linkages, or ADPr with the surrounding protein backbone. We first optimised methods to prepare extracts con-taining ADPr-proteins and observe that depending on the amino acid modified, the modification is heatlabile. We tested the re-activity of available reagents with diverse ADP-ribosylated pro-tein and RNA substrates and observed that not all reagents are equally suited for all substrates. Next, we determined cross -reactivity with adenylylated RNA, AMPylated proteins, and me-tabolites, including NADH, which are detected by some reagents. Lastly, we analysed ADP-ribosylation using confocal microscopy, where depending on the fixation method, either mitochondrion, nucleus, or nucleolus is stained. This study allows future work dissecting the function of ADP-ribosylation in cells, both on protein and on RNA substrates, as we optimised sample prepa-ration methods and have defined the reagents suitable for specific methods and substrates. Show less
Batte, A.; Horst, S.C. van der; Tittel-Elmer, M.; Sun, S.M.; Sharma, S.; Leeuwen, J. van; ... ; Attikum, H. van 2022
Eukaryotic cells have evolved a replication stress response that helps to overcome stalled/collapsed replication forks and ensure proper DNA replication. The replication checkpoint protein Mrc1... Show moreEukaryotic cells have evolved a replication stress response that helps to overcome stalled/collapsed replication forks and ensure proper DNA replication. The replication checkpoint protein Mrc1 plays important roles in these processes, although its functional interactions are not fully understood. Here, we show that MRC1 negatively interacts with CHL1, which encodes the helicase protein Chl1, suggesting distinct roles for these factors during the replication stress response. Indeed, whereas Mrc1 is known to facilitate the restart of stalled replication forks, we uncovered that Chl1 controls replication fork rate under replication stress conditions. Chl1 loss leads to increased RNR1 gene expression and dNTP levels at the onset of S phase likely without activating the DNA damage response. This in turn impairs the formation of RPA-coated ssDNA and subsequent checkpoint activation. Thus, the Chl1 helicase affects RPA-dependent checkpoint activation in response to replication fork arrest by ensuring proper intracellular dNTP levels, thereby controlling replication fork progression under replication stress conditions. Show less
Blight, J.; Sala, A.K.; Atcheson, E.; Kramer, H.; El-Turabi, A.; Real, E.; ... ; Baum, J. 2021
Progress towards a protective vaccine against malaria remains slow. To date, only limited protection has been routinely achieved following immunisation with either whole-parasite (sporozoite) or... Show moreProgress towards a protective vaccine against malaria remains slow. To date, only limited protection has been routinely achieved following immunisation with either whole-parasite (sporozoite) or subunit-based vaccines. One major roadblock to vaccine progress, and to pre-erythrocytic parasite biology in general, is the continued reliance on manual salivary gland dissection for sporozoite isolation from infected mosquitoes. Here, we report development of a multi-step method, based on batch processing of homogenised whole mosquitoes, slurry, and density-gradient filtration, which combined with free -flow electrophoresis rapidly produces a pure, infective sporozoite inoculum. Human-infective Plasmodium falciparum and rodent-infective Plasmodium berghei sporozoites produced in this way are two-to threefold more infective than salivary gland dissection sporozoites in in vitro hepatocyte infection assays. In an in vivo rodent malaria model, the same P. berghei sporozoites confer sterile protection from mosquito-bite challenge when immunisation is delivered intravenously or 60-70% protection when delivered intramuscularly. By improving purity, infectivity, and immunogenicity, this method represents a key advancement in capacity to produce research grade sporozoites, which should impact delivery of a whole parasite based malaria vaccine at scale in the future. Show less
Alvelos, M.I.; Bruggemann, M.; Sutandy, F.X.R.; Juan-Mateu, J.; Colli, M.L.; Busch, A.; ... ; Castela, A. 2021
In pancreatic beta-cells, the expression of the splicing factor SRSF6 is regulated by GLIS3, a transcription factor encoded by a diabetes susceptibility gene. SRSF6 down-regulation promotes beta... Show moreIn pancreatic beta-cells, the expression of the splicing factor SRSF6 is regulated by GLIS3, a transcription factor encoded by a diabetes susceptibility gene. SRSF6 down-regulation promotes beta-cell demise through splicing dysregulation of central genes for beta-cells function and survival, but how RNAs are targeted by SRSF6 remains poorly understood. Here, we define the SRSF6 binding landscape in the human pancreatic beta-cell line EndoC-beta H1 by integrating individual-nucleotide resolution UV cross-linking and immuno-precipitation (iCLIP) under basal conditions with RNA sequencing after SRSF6 knockdown. We detect thousands of SRSF6 bindings sites in coding sequences. Motif analyses suggest that SRSF6 specifically recognizes a purine-rich consensus motif consisting of GAA triplets and that the number of contiguous GAA triplets correlates with increasing binding site strength. The SRSF6 positioning determines the splicing fate. In line with its role in beta-cell function, we identify SRSF6 binding sites on regulated exons in several diabetes susceptibility genes. In a proof-of-principle, the splicing of the susceptibility gene LMO7 is modulated by antisense oligonucleotides. Our present study unveils the splicing regulatory landscape of SRSF6 in immortalized human pancreatic beta-cells. Show less
