Clinical implications of nutritional interventions reducing calories, a systematic scoping review.

BACKGROUND & AIMS: Caloric restriction (CR) constitutes a dietary approach of (temporarily) reducing calorie intake thereby inducing resilience and resistance mechanisms and promoting health. While CR's feasibility and safety have been proven in human trials, its full benefits and translation to different study populations warrants further exploration. METHODS: We here conducted a systematic scoping review adhering to Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA) guidelines. RESULTS: Our search resulted in 3745 individual records, of which 40 were included. We showed that all studies consistently demonstrated the feasibility and safety of CR-like interventions. The specific effects of nutritional preconditioning vary, further underscoring the need for carefully crafted strategies, according to the intended effect, patient population, and logistical limitations. CONCLUSIONS: CR-like interventions (long-term CR or short-term fasting) are feasible in a broad range of patient populations. Whether it has clinical benefit, f.i. reducing treatment-induced side effects and enhancing therapy efficacy, has to be investigated further.

Fasting before living-kidney donation: effect on donor well-being and postoperative recovery: study protocol of a multicenter randomized controlled trial.

BACKGROUND: One of the main effectors on the quality of life of living-kidney donors is postoperative fatigue. Caloric restriction (CR) and short-term fasting (STF) are associated with improved fitness and increased resistance to acute stress. CR/STF increases the expression of cytoprotective genes, increases immunomodulation via increased anti-inflammatory cytokine production, and decreases the expression of pro-inflammatory markers. As such, nutritional preconditioning by CR or STF represents a non-invasive and cost-effective method that could mitigate the effects of acute surgery-induced stress and postoperative fatigue. To investigate whether preoperative STF contributes to a reduction in fatigue after living-kidney donation, a randomized clinical trial is indicated. METHODS: We aim to determine whether 2.5 days of fasting reduces postoperative fatigue score in subjects undergoing living-kidney donation. In this randomized study, the intervention group will follow a preoperative fasting regime for 2.5 days with a low-dose laxative, while the control group will receive standard care. The main study endpoint is postoperative fatigue, 4 weeks after living-kidney donation. Secondary endpoints include the effect of preoperative fasting on postoperative hospital admission time, the feasibility of STF, and the postoperative recovery of donor and recipient kidney function. This study will provide us with knowledge of the feasibility of STF and confirm its effect on postoperative recovery. DISCUSSION: Our study will provide clinically relevant information on the merits of caloric restriction for living-kidney donors and recipients. We expect to reduce the postoperative fatigue in living-kidney donors and improve the postoperative recovery of living-kidney recipients. It will provide evidence on the clinical merits and potential caveats of preoperative dietary interventions. TRIAL REGISTRATION: Netherlands Trial Register NL9262 . EudraCT 2020-005445-16 . MEC Erasmus MC MEC-2020-0778. CCMO NL74623.078.21.

De novo variants in CDK13 associated with syndromic ID/DD: Molecular and clinical delineation of 15 individuals and a further review.

De novo variants in the gene encoding cyclin-dependent kinase 13 (CDK13) have been associated with congenital heart defects and intellectual disability (ID). Here, we present the clinical assessment of 15 individuals and report novel de novo missense variants within the kinase domain of CDK13. Furthermore, we describe 2 nonsense variants and a recurrent frame-shift variant. We demonstrate the synthesis of 2 aberrant CDK13 transcripts in lymphoblastoid cells from an individual with a splice-site variant. Clinical characteristics of the individuals include mild to severe ID, developmental delay, behavioral problems, (neonatal) hypotonia and a variety of facial dysmorphism. Congenital heart defects were present in 2 individuals of the current cohort, but in at least 42% of all known individuals. An overview of all published cases is provided and does not demonstrate an obvious genotype-phenotype correlation, although 2 individuals harboring a stop codons at the end of the kinase domain might have a milder phenotype. Overall, there seems not to be a clinically recognizable facial appearance. The variability in the phenotypes impedes an à vue diagnosis of this syndrome and therefore genome-wide or gene-panel driven genetic testing is needed. Based on this overview, we provide suggestions for clinical work-up and management of this recently described ID syndrome.

DNA damage-induced histone H1 ubiquitylation is mediated by HUWE1 and stimulates the RNF8-RNF168 pathway.

The DNA damage response (DDR), comprising distinct repair and signalling pathways, safeguards genomic integrity. Protein ubiquitylation is an important regulatory mechanism of the DDR. To study its role in the UV-induced DDR, we characterized changes in protein ubiquitylation following DNA damage using quantitative di-Gly proteomics. Interestingly, we identified multiple sites of histone H1 that are ubiquitylated upon UV-damage. We show that UV-dependent histone H1 ubiquitylation at multiple lysines is mediated by the E3-ligase HUWE1. Recently, it was shown that poly-ubiquitylated histone H1 is an important signalling intermediate in the double strand break response. This poly-ubiquitylation is dependent on RNF8 and Ubc13 which extend pre-existing ubiquitin modifications to K63-linked chains. Here we demonstrate that HUWE1 depleted cells showed reduced recruitment of RNF168 and 53BP1 to sites of DNA damage, two factors downstream of RNF8 mediated histone H1 poly-ubiquitylation, while recruitment of MDC1, which act upstream of histone H1 ubiquitylation, was not affected. Our data show that histone H1 is a prominent target for ubiquitylation after UV-induced DNA damage. Our data are in line with a model in which HUWE1 primes histone H1 with ubiquitin to allow ubiquitin chain elongation by RNF8, thereby stimulating the RNF8-RNF168 mediated DDR.

Vitamin E Supplementation Reduces Cellular Loss in the Brain of a Premature Aging Mouse Model.

BACKGROUND: Aging is a highly complex biological process driven by multiple factors. Its progression can partially be influenced by nutritional interventions. Vitamin E is a lipid-soluble anti-oxidant that is investigated as nutritional supplement for its ability to prevent or delay the onset of specific aging pathologies, including neurodegenerative disorders. PURPOSE: We aimed here to investigate the effect of vitamin E during aging progression in a well characterized mouse model for premature aging. METHOD: Xpg-/- animals received diets with low (~2.5 mg/kg feed), medium (75 mg/kg feed) or high (375 mg/kg feed) vitamin E concentration and their phenotype was monitored during aging progression. Vitamin E content was analyzed in the feed, for stability reasons, and in mouse plasma, brain, and liver, for effectiveness of the treatment. Subsequent age-related changes were monitored for improvement by increased vitamin E or worsening by depletion in both liver and nervous system, organs sensitive to oxidative stress. RESULTS: Mice supplemented with high levels of vitamin E showed a delayed onset of age-related body weight decline and appearance of tremors when compared to mice with a low dietary vitamin E intake. DNA damage resulting in liver abnormalities such as changes in polyploidy, was considerably prevented by elevated amounts of vitamin E. Additionally, immunohistochemical analyses revealed that high intake of vitamin E, when compared with low and medium levels of vitamin E in the diet, reduces the number of p53-positive cells throughout the brain, indicative of a lower number of cells dying due to DNA damage accumulated over time. CONCLUSIONS: Our data underline a neuroprotective role of vitamin E in the premature aging animal model used in this study, likely via a reduction of oxidative stress, and implies the importance of improved nutrition to sustain health.

A signature of renal stress resistance induced by short-term dietary restriction, fasting, and protein restriction.

During kidney transplantation, ischemia-reperfusion injury (IRI) induces oxidative stress. Short-term preoperative 30% dietary restriction (DR) and 3-day fasting protect against renal IRI. We investigated the contribution of macronutrients to this protection on both phenotypical and transcriptional levels. Male C57BL/6 mice were fed control food ad libitum, underwent two weeks of 30%DR, 3-day fasting, or received a protein-, carbohydrate- or fat-free diet for various periods of time. After completion of each diet, renal gene expression was investigated using microarrays. After induction of renal IRI by clamping the renal pedicles, animals were monitored seven days postoperatively for signs of IRI. In addition to 3-day fasting and two weeks 30%DR, three days of a protein-free diet protected against renal IRI as well, whereas the other diets did not. Gene expression patterns significantly overlapped between all diets except the fat-free diet. Detailed meta-analysis showed involvement of nuclear receptor signaling via transcription factors, including FOXO3, HNF4A and HMGA1. In conclusion, three days of a protein-free diet is sufficient to induce protection against renal IRI similar to 3-day fasting and two weeks of 30%DR. The elucidated network of common protective pathways and transcription factors further improves our mechanistic insight into the increased stress resistance induced by short-term DR.

Restricted diet delays accelerated ageing and genomic stress in DNA-repair-deficient mice.

Mice deficient in the DNA excision-repair gene Ercc1 (Ercc1∆/-) show numerous accelerated ageing features that limit their lifespan to 4-6 months. They also exhibit a 'survival response', which suppresses growth and enhances cellular maintenance. Such a response resembles the anti-ageing response induced by dietary restriction (also known as caloric restriction). Here we report that a dietary restriction of 30% tripled the median and maximal remaining lifespans of these progeroid mice, strongly retarding numerous aspects of accelerated ageing. Mice undergoing dietary restriction retained 50% more neurons and maintained full motor function far beyond the lifespan of mice fed ad libitum. Other DNA-repair-deficient, progeroid Xpg-/- (also known as Ercc5-/-) mice, a model of Cockayne syndrome, responded similarly. The dietary restriction response in Ercc1∆/- mice closely resembled the effects of dietary restriction in wild-type animals. Notably, liver tissue from Ercc1∆/- mice fed ad libitum showed preferential extinction of the expression of long genes, a phenomenon we also observed in several tissues ageing normally. This is consistent with the accumulation of stochastic, transcription-blocking lesions that affect long genes more than short ones. Dietary restriction largely prevented this declining transcriptional output and reduced the number of γH2AX DNA damage foci, indicating that dietary restriction preserves genome function by alleviating DNA damage. Our findings establish the Ercc1∆/- mouse as a powerful model organism for health-sustaining interventions, reveal potential for reducing endogenous DNA damage, facilitate a better understanding of the molecular mechanism of dietary restriction and suggest a role for counterintuitive dietary-restriction-like therapy for human progeroid genome instability syndromes and possibly neurodegeneration in general.

Targeted inhibition of metastatic melanoma through interference with Pin1-FOXM1 signaling.

Melanoma is the most lethal form of skin cancer and successful treatment of metastatic melanoma remains challenging. BRAF/MEK inhibitors only show a temporary benefit due to rapid occurrence of resistance, whereas immunotherapy is mainly effective in selected subsets of patients. Thus, there is a need to identify new targets to improve treatment of metastatic melanoma. To this extent, we searched for markers that are elevated in melanoma and are under regulation of potentially druggable enzymes. Here, we show that the pro-proliferative transcription factor FOXM1 is elevated and activated in malignant melanoma. FOXM1 activity correlated with expression of the enzyme Pin1, which we found to be indicative of a poor prognosis. In functional experiments, Pin1 proved to be a main regulator of FOXM1 activity through MEK-dependent physical regulation during the cell cycle. The Pin1-FOXM1 interaction was enhanced by BRAF(V600E), the driver oncogene in the majority of melanomas, and in extrapolation of the correlation data, interference with\ Pin1 in BRAF(V600E)-driven metastatic melanoma cells impaired both FOXM1 activity and cell survival. Importantly, cell-permeable Pin1-FOXM1-blocking peptides repressed the proliferation of melanoma cells in freshly isolated human metastatic melanoma ex vivo and in three-dimensional-cultured patient-derived melanoids. When combined with the BRAF(V600E)-inhibitor PLX4032 a robust repression in melanoid viability was obtained, establishing preclinical value of patient-derived melanoids for prognostic use of drug sensitivity and further underscoring the beneficial effect of Pin1-FOXM1 inhibitory peptides as anti-melanoma drugs. These proof-of-concept results provide a starting point for development of therapeutic Pin1-FOXM1 inhibitors to target metastatic melanoma.

BACH2: a marker of DNA damage and ageing.

DNA damage and ageing share expression changes involving alterations in many aspects of metabolism, suppression of growth and upregulation of defence and genome maintenance systems. "Omics" technologies have permitted large-scale parallel measurements covering global cellular constituents and aided the identification of specific response pathways that change during ageing and after DNA damage. We have set out to identify genes with highly conserved response patterns through meta-analysis of mRNA expression datasets collected during natural ageing and accelerated ageing caused by a Transcription-Coupled Nucleotide Excision Repair (TC-NER) defect in a diverse set of organs and tissues in mice, and from in vitro UV-induced DNA damage in a variety of murine cells. The identified set of genes that show similar expression patterns in response to organ ageing (accelerated and normal), and endogenously and exogenously induced DNA damage, consists of genes involved in anti-oxidant systems and includes the transcription factor Bach2 as one of the most consistent markers. BACH2 was originally identified as a partner of the small Maf proteins and antagonist of the NRF2 anti-oxidant defence pathway and has been implicated in B-cell differentiation and immune system homeostasis. Although BACH2 has never before been associated with UV-induced damage or ageing, it shows a strong downregulation in both conditions. We have characterized the dynamics of Bach2 expression in response to DNA damage and show that it is a highly sensitive responder to transcription-blocking DNA lesions. Gene expression profiling using Affymetrix microarray analysis after siRNA-mediated silencing of Bach2 identified cell cycle and transcription regulation as the most significantly altered processes consistent with a function as transcription factor affecting proliferation.

DNA damage leads to progressive replicative decline but extends the life span of long-lived mutant animals.

Human-nucleotide-excision repair (NER) deficiency leads to different developmental and segmental progeroid symptoms of which the pathogenesis is only partially understood. To understand the biological impact of accumulating spontaneous DNA damage, we studied the phenotypic consequences of DNA-repair deficiency in Caenorhabditis elegans. We find that DNA damage accumulation does not decrease the adult life span of post-mitotic tissue. Surprisingly, loss of functional ERCC-1/XPF even further extends the life span of long-lived daf-2 mutants, likely through an adaptive activation of stress signaling. Contrariwise, NER deficiency leads to a striking transgenerational decline in replicative capacity and viability of proliferating cells. DNA damage accumulation induces severe, stochastic impairment of development and growth, which is most pronounced in NER mutants that are also impaired in their response to ionizing radiation and inter-strand crosslinks. These results suggest that multiple DNA-repair pathways can protect against replicative decline and indicate that there might be a direct link between the severity of symptoms and the level of DNA-repair deficiency in patients.

MLL2 mutation detection in 86 patients with Kabuki syndrome: a genotype-phenotype study.

Recently, pathogenic variants in the MLL2 gene were identified as the most common cause of Kabuki (Niikawa-Kuroki) syndrome (MIM#147920). To further elucidate the genotype-phenotype correlation, we studied a large cohort of 86 clinically defined patients with Kabuki syndrome (KS) for mutations in MLL2. All patients were assessed using a standardized phenotype list and all were scored using a newly developed clinical score list for KS (MLL2-Kabuki score 0-10). Sequencing of the full coding region and intron-exon boundaries of MLL2 identified a total of 45 likely pathogenic mutations (52%): 31 nonsense, 10 missense and four splice-site mutations, 34 of which were novel. In five additional patients, novel, i.e. non-dbSNP132 variants of clinically unknown relevance, were identified. Patients with likely pathogenic nonsense or missense MLL2 mutations were usually more severely affected (median 'MLL2-Kabuki score' of 6) as compared to the patients without MLL2 mutations (median 'MLL2-Kabuki score' of 5), a significant difference (p < 0.0014). Several typical facial features such as large dysplastic ears, arched eyebrows with sparse lateral third, blue sclerae, a flat nasal tip with a broad nasal root, and a thin upper and a full lower lip were observed more often in mutation positive patients.

Premature aging and cancer in nucleotide excision repair-disorders.

During the past decades, the major impact of DNA damage on cancer as 'disease of the genes' has become abundantly apparent. In addition to cancer, recent years have also uncovered a very strong association of DNA damage with many features of (premature) aging. The notion that DNA repair systems protect not only against cancer but also equally against to fast aging has become evident from a systematic, integral analysis of a variety of mouse mutants carrying defects in e.g. transcription-coupled repair with or without an additional impairment of global genome nucleotide excision repair and the corresponding segmental premature aging syndromes in human. A striking correlation between the degree of the DNA repair deficiency and the acceleration of specific progeroid symptoms has been discovered for those repair systems that primarily protect from the cytotoxic and cytostatic effects of DNA damage. These observations are explained from the perspective of nucleotide excision repair mouse mutant and human syndromes. However, similar principles likely apply to other DNA repair pathways including interstrand crosslink repair and double strand break repair and genome maintenance systems in general, supporting the notion that DNA damage constitutes an important intermediate in the process of aging.

X-chromosome duplications in males with mental retardation: pathogenic or benign variants?

Studies to identify copy number variants (CNVs) on the X-chromosome have revealed novel genes important in the causation of X-linked mental retardation (XLMR). Still, for many CNVs it is unclear whether they are associated with disease or are benign variants. We describe six different CNVs on the X-chromosome in five male patients with mental retardation that were identified by conventional karyotyping and single nucleotide polymorphism array analysis. One deletion and five duplications ranging in size from 325 kb to 12.5 Mb were observed. Five CNVs were maternally inherited and one occurred de novo. We discuss the involvement of potential candidate genes and focus on the complexity of X-chromosomal duplications in males inherited from healthy mothers with different X-inactivation patterns. Based on size and/or the presence of XLMR genes we were able to classify CNVs as pathogenic in two patients. However, it remains difficult to decide if the CNVs in the other three patients are pathogenic or benign.

Extending the phenotype of recurrent rearrangements of 16p11.2: deletions in mentally retarded patients without autism and in normal individuals.

Array CGH (comparative genomic hybridization) screening of large patient cohorts with mental retardation and/or multiple congenital anomalies (MR/MCA) has led to the identification of a number of new microdeletion and microduplication syndromes. Recently, a recurrent copy number variant (CNV) at chromosome 16p11.2 was reported to occur in up to 1% of autistic patients in three large autism studies. In the screening of 4284 patients with MR/MCA with various array platforms, we detected 22 individuals (14 index patients and 8 family members) with deletions in 16p11.2, which are genomically identical to those identified in the autism studies. Though some patients shared a facial resemblance and a tendency to overweight, there was no evidence for a recognizable phenotype. Autism was not the presenting feature in our series. The assembled evidence indicates that recurrent 16p11.2 deletions are associated with variable clinical outcome, most likely arising from haploinsufficiency of one or more genes. The phenotypical spectrum ranges from MR and/or MCA, autism, learning and speech problems, to a normal phenotype.

Transcription-coupled repair and premature ageing.

During the past decades, several cellular pathways have been discovered to be connected with the ageing process. These pathways, which either suppress or enhance the ageing process, include regulation of the insulin/growth hormone axis, pathways involved with caloric restriction, ROS metabolism and DNA repair. In this review, we will provide a comprehensive overview of cancer and/or accelerated ageing pathologies associated with defects in the multi-step nucleotide excision repair pathway. Moreover, we will discuss evidence suggesting that there is a causative link between transcription-coupled repair and ageing.

Scanning confocal fluorescence microscopy for single molecule analysis of nucleotide excision repair complexes.

We used scanning confocal fluorescence microscopy to observe and analyze individual DNA- protein complexes formed between human nucleotide excision repair (NER) proteins and model DNA substrates. For this purpose human XPA protein was fused to EGFP, purified and shown to be functional. Binding of EGFP-labeled XPA protein to a Cy3.5-labeled DNA substrate, in the presence and absence of RPA, was assessed quantitatively by simultaneous excitation and emission detection of both fluorophores. Co-localization of Cy3.5 and EGFP signals within one diffraction limited spot indicated complexes of XPA with DNA. Measurements were performed on samples in a 1% agarose matrix in conditions that are compatible with protein activity and where reactions can be studied under equilibrium conditions. In these samples DNA alone was freely diffusing and protein-bound DNA was immobile, whereby they could be discriminated resulting in quantitative data on DNA binding. On the single molecule level approximately 10% of XPA co-localized with DNA; this increased to 32% in the presence of RPA. These results, especially the enhanced binding of XPA in the presence of RPA, are similar to those obtained in bulk experiments, validating the utility of scanning confocal fluorescence microscopy for investigating functional interactions at the single molecule level.

Early postnatal ataxia and abnormal cerebellar development in mice lacking Xeroderma pigmentosum Group A and Cockayne syndrome Group B DNA repair genes.

Xeroderma pigmentosum (XP) and Cockayne syndrome (CS) are rare autosomal recessive disorders associated with a defect in the nucleotide excision repair (NER) pathway required for the removal of DNA damage induced by UV light and distorting chemical adducts. Although progressive neurological dysfunction is one of the hallmarks of CS and of some groups of XP patients, the causative mechanisms are largely unknown. Here we show that mice lacking both the XPA (XP-group A) and CSB (CS-group B) genes in contrast to the single mutants display severe growth retardation, ataxia, and motor dysfunction during early postnatal development. Their cerebella are hypoplastic and showed impaired foliation and stunted Purkinje cell dendrites. Reduced neurogenesis and increased apoptotic cell death occur in the cerebellar external granular layer. These findings suggest that XPA and CSB have additive roles in the mouse nervous system and support a crucial role for these genes in normal brain development.

The structure-specific endonuclease Ercc1-Xpf is required for targeted gene replacement in embryonic stem cells.

The Ercc1-Xpf heterodimer, a highly conserved structure-specific endonuclease, functions in multiple DNA repair pathways that are pivotal for maintaining genome stability, including nucleotide excision repair, interstrand crosslink repair and homologous recombination. Ercc1-Xpf incises double-stranded DNA at double-strand/single-strand junctions, making it an ideal enzyme for processing DNA structures that contain partially unwound strands. Here we demonstrate that although Ercc1 is dispensable for recombination between sister chromatids, it is essential for targeted gene replacement in mouse embryonic stem cells. Surprisingly, the role of Ercc1-Xpf in gene targeting is distinct from its previously identified role in removing nonhomologous termini from recombination intermediates because it was required irrespective of whether the ends of the DNA targeting constructs were heterologous or homologous to the genomic locus. Our observations have implications for the mechanism of gene targeting in mammalian cells and define a new role for Ercc1-Xpf in mammalian homologous recombination. We propose a model for the mechanism of targeted gene replacement that invokes a role for Ercc1-Xpf in making the recipient genomic locus receptive for gene replacement.

Sequential assembly of the nucleotide excision repair factors in vivo.

Here, we describe the assembly of the nucleotide excision repair (NER) complex in normal and repair-deficient (xeroderma pigmentosum) human cells, employing a novel technique of local UV irradiation combined with fluorescent antibody labeling. The damage recognition complex XPC-hHR23B appears to be essential for the recruitment of all subsequent NER factors in the preincision complex, including transcription repair factor TFIIH. XPA associates relatively late, is required for anchoring of ERCC1-XPF, and may be essential for activation of the endonuclease activity of XPG. These findings identify XPC as the earliest known NER factor in the reaction mechanism, give insight into the order of subsequent NER components, provide evidence for a dual role of XPA, and support a concept of sequential assembly of repair proteins at the site of the damage rather than a preassembled repairosome.