Background We explore longitudinal trajectories of clinical indicators, patient-reported outcomes, and hospitalizations, in the years preceding death in a population of older patients with advanced... Show moreBackground We explore longitudinal trajectories of clinical indicators, patient-reported outcomes, and hospitalizations, in the years preceding death in a population of older patients with advanced chronic kidney disease (CKD). Methods The EQUAL study is a European observational prospective cohort study with an incident eGFR Results We included 661 decedents with a median time to death of 2.0 years (IQR 0.9-3.2). During the years preceding death, eGFR, Subjective Global Assessment score, and blood pressure declined, with accelerations seen at 6 months preceding death. Serum hemoglobin, hematocrit, cholesterol, calcium, albumin, and sodium values declined slowly during follow-up, with accelerations observed between 6 and 12 months preceding death. Physical and mental quality of life declined linearly throughout follow-up. The number of reported symptoms was stable up to 2 years prior to death, with an acceleration observed at 1 year prior to death. The rate of hospitalization was stable at around one hospitalization per person year, increasing exponentially at 6 months preceding death. Conclusions We identified clinically relevant physiological accelerations in patient trajectories that began similar to 6 to 12 months prior to death, which are likely multifactorial in nature, but correlate with a surge in hospitalizations. Further research should focus on how to effectively use this knowledge to inform patient and family expectations, to benefit the planning of (end-of-life) care, and to establish clinical alert systems. Show less
Sun, J.; Chikunova, A.; Boyle, A.L.; Voskamp, P.; Timmer, M.; Ubbink, M. 2023
The interplay between three-dimensional chromosome organisation and genomic processes such as replication and transcription necessitates in vivo studies of chromosome dynamics. Fluorescent organic... Show moreThe interplay between three-dimensional chromosome organisation and genomic processes such as replication and transcription necessitates in vivo studies of chromosome dynamics. Fluorescent organic dyes are often used for chromosome labelling in vivo. The mode of binding of these dyes to DNA cause its distortion, elongation, and partial unwinding. The structural changes induce DNA damage and interfere with the binding dynamics of chromatin-associated proteins, consequently perturbing gene expression, genome replication, and cell cycle progression. We have developed a minimally-perturbing, genetically encoded fluorescent DNA label consisting of a (photo-switchable) fluorescent protein fused to the DNA-binding domain of H-NS - a bacterial nucleoid-associated protein. We show that this DNA label, abbreviated as HI-NESS (H-NS-based indicator for nucleic acid stainings), is minimally-perturbing to genomic processes and labels chromosomes in eukaryotic cells in culture, and in zebrafish embryos with preferential binding to AT-rich chromatin. Show less
Rashid, F.Z.M.; Mahlandt, E.; Vaart M, van der; Boer, D.E.C.; Varela Alvarez, M.; Henneman, B.; ... ; Dame, R.T. 2021
The interplay between three-dimensional chromosome organisation and genomic processes such as replication and transcription necessitates in vivo studies of chromosome dynamics. Fluorescent organic... Show moreThe interplay between three-dimensional chromosome organisation and genomic processes such as replication and transcription necessitates in vivo studies of chromosome dynamics. Fluorescent organic dyes are often used for chromosome labelling in vivo. The mode of binding of these dyes to DNA cause its distortion, elongation, and partial unwinding. The structural changes induce DNA damage and interfere with the binding dynamics of chromatin-associated proteins, consequently perturbing gene expression, genome replication, and cell cycle progression. We have developed a minimally-perturbing, genetically encoded fluorescent DNA label consisting of a (photo-switchable) fluorescent protein fused to the DNA-binding domain of H-NS - a bacterial nucleoid-associated protein. We show that this DNA label, abbreviated as HI-NESS (H-NS-based indicator for nucleic acid stainings), is minimally-perturbing to genomic processes and labels chromosomes in eukaryotic cells in culture, and in zebrafish embryos with preferential binding to AT-rich chromatin. Show less
Xiao, X.S.; Willemse, J.; Voskamp, P.; Li, X.M.; Prota, A.E.; Lamers, M.; ... ; Wezel, G.P. van 2021
In most bacteria, cell division begins with the polymerization of the GTPase FtsZ at mid-cell, which recruits the division machinery to initiate cell constriction. In the filamentous bacterium... Show moreIn most bacteria, cell division begins with the polymerization of the GTPase FtsZ at mid-cell, which recruits the division machinery to initiate cell constriction. In the filamentous bacterium Streptomyces, cell division is positively controlled by SsgB, which recruits FtsZ to the future septum sites and promotes Z-ring formation. Here, we show that various amino acid (aa) substitutions in the highly conserved SsgB protein result in ectopically placed septa that sever spores diagonally or along the long axis, perpendicular to the division plane. Fluorescence microscopy revealed that between 3.3% and 9.8% of the spores of strains expressing SsgB E120 variants were severed ectopically. Biochemical analysis of SsgB variant E120G revealed that its interaction with FtsZ had been maintained. The crystal structure of Streptomyces coelicolor SsgB was resolved and the key residues were mapped on the structure. Notably, residue substitutions (V115G, G118V, E120G) that are associated with septum misplacement localize in the alpha 2-alpha 3 loop region that links the final helix and the rest of the protein. Structural analyses and molecular simulation revealed that these residues are essential for maintaining the proper angle of helix alpha 3. Our data suggest that besides altering FtsZ, aa substitutions in the FtsZ-recruiting protein SsgB also lead to diagonally or longitudinally divided cells in Streptomyces. Show less
Xiao, X.; Willemse, J.J.; Voskamp, P.; Li, X.; Prota, A.E.; Lamers, M.; ... ; Wezel, G.P. van 2021
Background-People with reduced glomerular filtration rate (GFR) often have elevated cardiac troponin T (cTnT) levels. It remains unclear how cTnT levels develop over time in those with chronic... Show moreBackground-People with reduced glomerular filtration rate (GFR) often have elevated cardiac troponin T (cTnT) levels. It remains unclear how cTnT levels develop over time in those with chronic kidney disease (CKD). The aim of this study was to prospectively study the association between cTnT and GFR over time in older advanced-stage CKD patients not on dialysis.Methods and Results-The EQUAL (European Quality Study) study is an observational prospective cohort study in stage 4 to 5 CKD patients aged >= 65 years not on dialysis (incident estimated GFR, <20 mL/min/1.73 m(2)). The EQUAL cohort used for the purpose of this study includes 171 patients followed in Sweden between April 2012 and December 2018. We used linear mixed models, adjusted for important groups of confounders, to investigate the effect of both measured GFR and estimated GFR on high-sensitivity cTnT (hs-cTnT) trajectory over 4 years. Almost all patients had at least 1 hs-cTnT measurement elevated above the 99th percentile of the general reference population (<= 14 ng/L). On average, hs-cTnT increased by 16%/year (95% CI, 13-19; P<0.0001). Each 15 mL/min/1.73 m(2) lower mean estimated GFR was associated with a 23% (95% CI, 14-31; P<0.0001) higher baseline hs-cTnT and 9% (95% CI, 5-13%; P<0.0001) steeper increase in hs-cTnT. The effect of estimated GFR on hs-cTnT trajectory was somewhat lower than a previous myocardial infarction (15%), but higher than presence of diabetes mellitus (4%) and male sex (5%).Conclusions-In CKD patients, hs-cTnT increases over time as renal function decreases. Lower CKD stage (each 15 mL/min/1.73 m2 lower) is independently associated with a steeper hs-cTnT increase over time in the same range as other established cardiovascular risk factors. Show less
Boyle, A.L.; Rabe, M.; Crone, N.S.A.; Rhys, G.G.; Soler, N.; Voskamp, P.; ... ; Kros, A. 2019
Designing peptides that fold and assemble in response to metal ions tests our understanding of how peptide folding and metal binding influence one another. Here, histidine residues are introduced... Show moreDesigning peptides that fold and assemble in response to metal ions tests our understanding of how peptide folding and metal binding influence one another. Here, histidine residues are introduced into the hydrophobic core of a coiled-coil trimer, generating a peptide that self-assembles upon the addition of metal ions. HisAD, the resulting peptide, is unstructured in the absence of metal and folds selectively to form an α-helical construct upon complexation with Cu(II) and Ni(II) but not Co(II) or Zn(II). The structure, and metal-binding ability, of HisAD is probed using a combination of circular dichroism (CD) spectroscopy, analytical ultracentrifugation (AUC), nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography. These show the peptide is trimeric and binds to both Cu(II) and Ni(II) in a 1 : 1 ratio with the histidine residues involved in the metal coordination, as designed. The X-ray crystal structure of the HisAD-Cu(II) complex reveals the trimeric HisAD peptide coordinates three Cu(II) ions; this is the first example of such a structure. Additionally, HisAD demonstrates an unprecedented discrimination between transition metal ions, the basis of which is likely to be related to the stability of the peptide-metal complexes formed. Show less
Kytidou, K.; Beekwilder, J.; Artola, M.; Meel, E. van; Wilbers, R.H.P.; Moolenaar, G.F.; ... ; Aerts, J.M.F.G. 2018
Immunosuppressive drugs are thought to cause the dramatically increased risk of carcinomas in sun-exposed skin of organ transplant recipients. These drugs differ in local effects on skin. We... Show moreImmunosuppressive drugs are thought to cause the dramatically increased risk of carcinomas in sun-exposed skin of organ transplant recipients. These drugs differ in local effects on skin. We investigated whether this local impact is predictive of skin cancer risk and may thus provide guidance on minimizing the risk. Immunosuppressants (azathioprine, cyclosporine, tacrolimus, mycophenolate mofetil and rapamycin) were assessed on altering the UV induction of apoptosis in human skin models, and of p53 mutant cell clones (putative tumor precursors) and ensuing skin carcinomas (with mutant p53) in the skin of hairless mice. Rapamycin was found to increase apoptosis (3 fold) whereas cyclosporine decreased apoptosis (3 fold). Correspondingly, a 1.5-5 fold reduction (p=0.07) or a 2-3 fold increase (p<0.001) was found in cell clusters overexpressing mutant p53 in chronically UV-exposed skin of mice that had been fed rapamycin or cyclosporine, respectively. Deep sequencing showed, however, that the allelic frequency (~5%) of the hotspot mutations in p53 (codons 270 and 275) remained unaffected. The majority of cells with mutated p53 appeared not to overexpress the mutated protein. Unexpectedly, none of the immunosuppressants admixed in high dosages to the diet accelerated tumor development, and cyclosporine even delayed tumor onset by ~15% (p<0.01). Thus, in contrast to earlier findings, the frequency of p53-mutant cells was not predictive of the incidence of skin carcinoma. Moreover, the lack of any accelerative effect on tumor development suggests that immunosuppressive medication is not the sole cause of the dramatic increase in skin cancer risk in organ transplant recipients. Show less
Because of its antitumor effect, the immunosuppressant rapamycin holds great promise for organ transplant recipients in that it may lower their cancer risk. In a mouse model, we showed previously... Show moreBecause of its antitumor effect, the immunosuppressant rapamycin holds great promise for organ transplant recipients in that it may lower their cancer risk. In a mouse model, we showed previously that rapamycin inhibits the outgrowth of primary skin carcinomas induced by UV radiation. However, the tumors that did grow out showed an altered p53 mutation spectrum. Here, we investigated whether this shift in p53 mutations already occurred in the smallest tumors, which were not affected in onset. We found that rapamycin did not alter the mutational spectrum in small tumors and in preceding microscopic clusters of cells expressing mutant-p53. However, rapamycin did reduce the number of these cell clusters. As this reduction did not affect tumor onset, we subsequently investigated whether rapamycin merely suppressed expression of mutated p53. This was not the case, as we could demonstrate that switching from a diet with rapamycin to one without, or vice versa, did not affect the number of existing mutant-p53 expressing cell clusters. Hence, rapamycin actually reduced the formation of mutant-p53 cell clusters. In wild-type and p53-mutant mice, we could not measure a significant enhancement of UV-induced apoptosis, but we did observe clear enhancement in human skin equivalents. This was associated with a clear suppression of HIF1α accumulation. Thus, we conclude that rapamycin reduces the formation of mutant-p53-expressing cell clusters without affecting tumor onset, suggesting that tumors grow out of a minor subset of cell clusters, the formation of which is not affected by rapamycin. Show less
Skin cancer is a serious problem for many organ transplant recipients. Half of them develop skin cancer within 20 years after the transplantation. The main cause of this increased skin cancer risk... Show moreSkin cancer is a serious problem for many organ transplant recipients. Half of them develop skin cancer within 20 years after the transplantation. The main cause of this increased skin cancer risk is thought to be suppression of the immune system, a necessity to prevent rejection of the transplanted organ. This thesis focuses on the effects of immunosuppressive drugs on the responses of skin cells to UV irradiation and how these altered responses would affect UV-induced skin cancer development. The goal was to compare several immunosuppressants and identify the least hazardous one. Human skin cultures and mice were used as experimental models to study short and long term effects in successive stages of tumor development. These studies have yielded several unexpected findings. Foremost, none of the tested immunosuppressants increased skin cancer development in mice when administrated in the diet; remarkably, cyclos porin even delayed tumor onset. And furthermore, seemingly discordant effects of the drugs on different putative stages of tumor development evoked a new perspective on the step-wise process leading up to skin cancer. More specifically and contrary to the prevailing consensus, this study showed that UV-induced early microscopic clusters of skin cells overexpressing the mutant-p53 tumorsuppressor protein are not early stages or precursors of ensuing skin carcinomas with mutant-p53. Surprisingly, some immunosuppressants affected the number of these clusters without corresponding effects on tumor onset. Show less
Increased skin cancer risk in organ transplant recipients has been experimentally emulated with enhanced UV carcinogenesis from administering conventional immunosuppressants. However, newer... Show moreIncreased skin cancer risk in organ transplant recipients has been experimentally emulated with enhanced UV carcinogenesis from administering conventional immunosuppressants. However, newer generation immunosuppressive drugs, rapamycin (Rapa) and mycophenolate mofetil (MMF), have been shown to impair angiogenesis and outgrowth of tumor implants. To ascertain the overall effect on UV carcinogenesis, Rapa and MMF were admixed into the food pellets of hairless SKH1 mice receiving daily sub-sunburn UV dosages. With immunosuppressive blood levels neither of the drugs affected onset of tumors (<2 mm), but in contrast to MMF, Rapa significantly increased latency of large tumors (>= 4 mm, medians of 190 vs 125 days) and reduced their multiplicity (1.6 vs 4.5 tumors per mouse at 200 days). Interestingly, tumors (>2 mm) from the Rapa-fed group showed a reduction in UV-signature p53 mutations (39% vs 90%) in favor of mutations from putative base oxidation. This shift in mutation spectrum was not essentially linked to the reduction in large tumors because it was absent in large tumors similarly reduced in number when feeding Rapa in combination with MMF, possibly owing to an antioxidant effect of MMF. Significantly fewer tumor cells were Vegf-positive in the Rapa-fed groups, but a correspondingly reduced expression of Hif1 alpha target genes (Vegf, Ldha, Glut1, Pdk1) that would indicate altered glucose metabolism with increased oxidative stress was not found. Remarkably, we observed no effect of the immunosuppressants on UV-induced tumor onset, and with impaired tumor outgrowth Rapa could therefore strongly reduce skin carcinoma morbidity and mortality rates in organ transplant recipients. Show less
