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© 2016 Bannard et al.Antibody affinity maturation occurs in germinal centers (GCs) through iterative rounds of somatic hypermutation and selection. Selection involves B cells competing for T cell help based on the amount of antigen they capture and present

Cellular senescence is a key driver of the aging process and contributes to tissue dysfunction and age-related pathologies. Senolytics have emerged as a promising therapeutic intervention to extend healthspan and treat age-related diseases. Through a senescent cell-based phenotypic drug screen, we identified a class of conjugated polyunsaturated fatty acids, specifically α-eleostearic acid and its methyl ester derivative, as novel senolytics that effectively killed a broad range of senescent cells, reduced tissue senescence, and extended healthspan in mice. Importantly, these novel lipids induced senolysis through ferroptosis, rather than apoptosis or necrosis, by exploiting elevated iron, cytosolic PUFAs and ROS levels in senescent cells. Mechanistic studies and computational analyses further revealed their key targets in the ferroptosis pathway, ACSL4, LPCAT3, and ALOX15, important for lipid-induced senolysis. This new class of ferroptosis-inducing lipid senolytics provides a novel approach to slow aging and treat age-related disease, targeting senescent cells that are primed for ferroptosis., Competing Interests: DECLARATION OF INTERESTS LJN and PDR are cofounders of Itasca Therapeutics. LJZ, LJN, and PDR have filed a provisional patent on the application of lipid senolytics as a strategy to treat age-related diseases.

The aim of this work was to identify the underlying genetic cause in a four-generation family segregating an unusual phenotype comprising a severe form of skeletal Class II malocclusion with gingival hyperplasia. SNP array identified a copy number gain on chromosome 1 (chr1); however, this chromosomal region did not segregate correctly in the extended family. Exome sequencing also failed to identify a candidate causative variant but highlighted co-segregating genetic markers on chr17 and chr19. Short- and long-read genome sequencing allowed us to pinpoint and characterize at nucleotide-level resolution a chromothripsis-like complex rearrangement (CR) inserted into the chr17 co-segregating region at the KCNJ2-SOX9 locus. The CR involved the gain of five different regions from chr1 that are shuffled, chained, and inserted as a single block (∼828 kb) at chr17q24.3. The inserted sequences contain craniofacial enhancers that are predicted to interact with KCNJ2/KCNJ16 through neo-topologically associating domain (TAD) formation to induce ectopic activation. Our findings suggest that the CR inserted at chr17q24.3 is the cause of the severe skeletal Class II malocclusion with gingival hyperplasia in this family and expands the panoply of phenotypes linked to variation at the KCNJ2-SOX9 locus. In addition, we highlight a previously overlooked potential role for misregulation of the KCNJ2/KCNJ16 genes in the pathomechanism of gingival hyperplasia associated with deletions and other rearrangements of the 17q24.2-q24.3 region (MIM 135400)., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Introduction: KCTD15 encodes an oligomeric BTB domain protein reported to inhibit neural crest formation through repression of Wnt/beta-catenin signalling, as well as transactivation by TFAP2. Heterozygous missense variants in the closely related paralogue KCTD1 cause scalp-ear-nipple syndrome., Methods: Exome sequencing was performed on a two-generation family affected by a distinctive phenotype comprising a lipomatous frontonasal malformation, anosmia, cutis aplasia of the scalp and/or sparse hair, and congenital heart disease. Identification of a de novo missense substitution within KCTD15 led to targeted sequencing of DNA from a similarly affected sporadic patient, revealing a different missense mutation. Structural and biophysical analyses were performed to assess the effects of both amino acid substitutions on the KCTD15 protein., Results: A heterozygous c.310G>C variant encoding p.(Asp104His) within the BTB domain of KCTD15 was identified in an affected father and daughter and segregated with the phenotype. In the sporadically affected patient, a de novo heterozygous c.263G>A variant encoding p.(Gly88Asp) was present in KCTD15. Both substitutions were found to perturb the pentameric assembly of the BTB domain. A crystal structure of the BTB domain variant p.(Gly88Asp) revealed a closed hexameric assembly, whereas biophysical analyses showed that the p.(Asp104His) substitution resulted in a monomeric BTB domain likely to be partially unfolded at physiological temperatures., Conclusion: BTB domain substitutions in KCTD1 and KCTD15 cause clinically overlapping phenotypes involving craniofacial abnormalities and cutis aplasia. The structural analyses demonstrate that missense substitutions act through a dominant negative mechanism by disrupting the higher order structure of the KCTD15 protein complex., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY. Published by BMJ.)

Germinal centers (GCs) form in lymph nodes after immunization or infection to facilitate antibody affinity maturation and memory and plasma cell (PC) development. PC differentiation is thought to involve stringent selection for GC B cells expressing the highest-affinity antigen receptors, but how this plays out during complex polyclonal responses is unclear. We combine temporal lineage tracing with antibody characterization to gain a snapshot of PCs developing during influenza infection. GCs co-mature B cell clones with antibody affinities spanning multiple orders of magnitude; however, each generates PCs with similar efficiencies, including weak binders. Within lineages, PC selection is not restricted to variants with the highest-affinity antibodies. Differentiation is commonly associated with proliferative expansion to produce "nodes" of identical PCs. Immunization-induced GCs generate fewer PCs but still of low- and high-antibody affinities. We propose that generating low-affinity antibody PCs reflects an evolutionary compromise to facilitate diverse serum antibody responses., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Whole genome sequencing is increasingly being used for the diagnosis of patients with rare diseases. However, the diagnostic yields of many studies, particularly those conducted in a healthcare setting, are often disappointingly low, at 25-30%. This is in part because although entire genomes are sequenced, analysis is often confined to in silico gene panels or coding regions of the genome., Methods: We undertook WGS on a cohort of 122 unrelated rare disease patients and their relatives (300 genomes) who had been pre-screened by gene panels or arrays. Patients were recruited from a broad spectrum of clinical specialties. We applied a bioinformatics pipeline that would allow comprehensive analysis of all variant types. We combined established bioinformatics tools for phenotypic and genomic analysis with our novel algorithms (SVRare, ALTSPLICE and GREEN-DB) to detect and annotate structural, splice site and non-coding variants., Results: Our diagnostic yield was 43/122 cases (35%), although 47/122 cases (39%) were considered solved when considering novel candidate genes with supporting functional data into account. Structural, splice site and deep intronic variants contributed to 20/47 (43%) of our solved cases. Five genes that are novel, or were novel at the time of discovery, were identified, whilst a further three genes are putative novel disease genes with evidence of causality. We identified variants of uncertain significance in a further fourteen candidate genes. The phenotypic spectrum associated with RMND1 was expanded to include polymicrogyria. Two patients with secondary findings in FBN1 and KCNQ1 were confirmed to have previously unidentified Marfan and long QT syndromes, respectively, and were referred for further clinical interventions. Clinical diagnoses were changed in six patients and treatment adjustments made for eight individuals, which for five patients was considered life-saving., Conclusions: Genome sequencing is increasingly being considered as a first-line genetic test in routine clinical settings and can make a substantial contribution to rapidly identifying a causal aetiology for many patients, shortening their diagnostic odyssey. We have demonstrated that structural, splice site and intronic variants make a significant contribution to diagnostic yield and that comprehensive analysis of the entire genome is essential to maximise the value of clinical genome sequencing., (© 2023. Crown.)

Cardiomyopathy is a progressive disease of the myocardium leading to impaired contractility. Genotoxic cancer therapies are known to be potent drivers of cardiomyopathy, whereas causes of spontaneous disease remain unclear. To test the hypothesis that endogenous genotoxic stress contributes to cardiomyopathy, we deleted the DNA repair gene Ercc1 specifically in striated muscle using a floxed allele of Ercc1 and mice expressing Cre under control of the muscle-specific creatinine kinase (Ckmm) promoter or depleted systemically (Ercc1 -/D mice). Ckmm-Cre +/- ;Ercc1 -/fl mice expired suddenly of heart disease by 7 months of age. As young adults, the hearts of Ckmm-Cre +/- ;Ercc1 -/fl mice were structurally and functionally normal, but by 6-months-of-age, there was significant ventricular dilation, wall thinning, interstitial fibrosis, and systolic dysfunction indicative of dilated cardiomyopathy. Cardiac tissue from the tissue-specific or systemic model showed increased apoptosis and cardiac myocytes from Ckmm-Cre +/- ;Ercc1 -/fl mice were hypersensitive to genotoxins, resulting in apoptosis. p53 levels and target gene expression, including several antioxidants, were increased in cardiac tissue from Ckmm-Cre +/- ;Ercc1 -/fl and Ercc1 -/D mice. Despite this, cardiac tissue from older mutant mice showed evidence of increased oxidative stress. Genetic or pharmacologic inhibition of p53 attenuated apoptosis and improved disease markers. Similarly, overexpression of mitochondrial-targeted catalase improved disease markers. Together, these data support the conclusion that DNA damage produced endogenously can drive cardiac disease and does so mechanistically via chronic activation of p53 and increased oxidative stress, driving cardiac myocyte apoptosis, dilated cardiomyopathy, and sudden death., (© 2023 The Authors. Aging Cell published by Anatomical Society and John Wiley & Sons Ltd.)

Age is the greatest risk factor for the development of type 2 diabetes mellitus (T2DM). Age-related decline in organ function is attributed to the accumulation of stochastic damage, including damage to the nuclear genome. Islets of T2DM patients display increased levels of DNA damage. However, whether this is a cause or consequence of the disease has not been elucidated. Here, we asked if spontaneous, endogenous DNA damage in β-cells can drive β-cell dysfunction and diabetes, via deletion of Ercc1 , a key DNA repair gene, in β-cells. Mice harboring Ercc1 -deficient β-cells developed adult-onset diabetes as demonstrated by increased random and fasted blood glucose levels, impaired glucose tolerance, and reduced insulin secretion. The inability to repair endogenous DNA damage led to an increase in oxidative DNA damage and apoptosis in β-cells and a significant loss of β-cell mass. Using electron microscopy, we identified β-cells in clear distress that showed an increased cell size, enlarged nuclear size, reduced number of mature insulin granules, and decreased number of mitochondria. Some β-cells were more affected than others consistent with the stochastic nature of spontaneous DNA damage. Ercc1 -deficiency in β-cells also resulted in loss of β-cell function as glucose-stimulated insulin secretion and mitochondrial function were impaired in islets isolated from mice harboring Ercc1 -deficient β-cells. These data reveal that unrepaired endogenous DNA damage is sufficient to drive β-cell dysfunction and provide a mechanism by which age increases the risk of T2DM., Competing Interests: The authors have declared that no conflict of interest exists. Declaration of interests The authors declare no competing interests.

Background: Pathogenic heterozygous SIX1 variants (predominantly missense) occur in branchio-otic syndrome (BOS), but an association with craniosynostosis has not been reported., Methods: We investigated probands with craniosynostosis of unknown cause using whole exome/genome (n=628) or RNA (n=386) sequencing, and performed targeted resequencing of SIX1 in 615 additional patients. Expression of SIX1 protein in embryonic cranial sutures was examined in the Six1 nLacZ/+ reporter mouse., Results: From 1629 unrelated cases with craniosynostosis we identified seven different SIX1 variants (three missense, including two de novo mutations, and four nonsense, one of which was also present in an affected twin). Compared with population data, enrichment of SIX1 loss-of-function variants was highly significant (p=0.00003). All individuals with craniosynostosis had sagittal suture fusion; additionally four had bilambdoid synostosis. Associated BOS features were often attenuated; some carrier relatives appeared non-penetrant. SIX1 is expressed in a layer basal to the calvaria, likely corresponding to the dura mater, and in the mid-sagittal mesenchyme., Conclusion: Craniosynostosis is associated with heterozygous SIX1 variants, with possible enrichment of loss-of-function variants compared with classical BOS. We recommend screening of SIX1 in craniosynostosis, particularly when sagittal±lambdoid synostosis and/or any BOS phenotypes are present. These findings highlight the role of SIX1 in cranial suture homeostasis., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY. Published by BMJ.)

Constitutive NF-κB activation is associated with cellular senescence and stem cell dysfunction and rare variants in NF-κB family members are enriched in centenarians. We recently identified a novel small molecule (SR12343) that inhibits IKK/NF-κB activation by disrupting the association between IKKβ and NEMO. Here we investigated the therapeutic effects of SR12343 on senescence and aging in three different mouse models. SR12343 reduced senescence-associated beta-galactosidase (SA-β-gal) activity in oxidative stress-induced senescent mouse embryonic fibroblasts as well as in etoposide-induced senescent human IMR90 cells. Chronic administration of SR12343 to the Ercc1 -/ ∆ and Zmpste24 -/- mouse models of accelerated aging reduced markers of cellular senescence and SASP and improved multiple parameters of aging. SR12343 also reduced markers of senescence and increased muscle fiber size in 2-year-old WT mice. Taken together, these results demonstrate that IKK/NF-κB signaling pathway represents a promising target for reducing markers of cellular senescence, extending healthspan and treating age-related diseases., (© 2021 The Authors. Aging Cell published by Anatomical Society and John Wiley & Sons Ltd.)

Heterozygous intragenic loss-of-function mutations of ERF, encoding an ETS transcription factor, were previously reported to cause a novel craniosynostosis syndrome, suggesting that ERF is haploinsufficient. We describe six families harboring heterozygous deletions including, or near to, ERF, of which four were characterized by whole-genome sequencing and two by chromosomal microarray. Based on the severity of associated intellectual disability (ID), we identify three categories of ERF-associated deletions. The smallest (32 kb) and only inherited deletion included two additional centromeric genes and was not associated with ID. Three larger deletions (264-314 kb) that included at least five further centromeric genes were associated with moderate ID, suggesting that deletion of one or more of these five genes causes ID. The individual with the most severe ID had a more telomerically extending deletion, including CIC, a known ID gene. Children found to harbor ERF deletions should be referred for craniofacial assessment, to exclude occult raised intracranial pressure., (© 2021 The Authors. Human Mutation published by Wiley Periodicals LLC.)

Ageing of the immune system, or immunosenescence, contributes to the morbidity and mortality of the elderly 1,2 . To define the contribution of immune system ageing to organism ageing, here we selectively deleted Ercc1, which encodes a crucial DNA repair protein 3,4 , in mouse haematopoietic cells to increase the burden of endogenous DNA damage and thereby senescence 5-7 in the immune system only. We show that Vav-iCre +/- ;Ercc1 -/fl mice were healthy into adulthood, then displayed premature onset of immunosenescence characterized by attrition and senescence of specific immune cell populations and impaired immune function, similar to changes that occur during ageing in wild-type mice 8-10 . Notably, non-lymphoid organs also showed increased senescence and damage, which suggests that senescent, aged immune cells can promote systemic ageing. The transplantation of splenocytes from Vav-iCre +/- ;Ercc1 -/fl or aged wild-type mice into young mice induced senescence in trans, whereas the transplantation of young immune cells attenuated senescence. The treatment of Vav-iCre +/- ;Ercc1 -/fl mice with rapamycin reduced markers of senescence in immune cells and improved immune function 11,12 . These data demonstrate that an aged, senescent immune system has a causal role in driving systemic ageing and therefore represents a key therapeutic target to extend healthy ageing.

Motivation: The 3D structure of chromatin in the nucleus is important for gene expression and regulation. Chromosome conformation capture techniques, such as Hi-C, generate large amounts of data showing interaction points on the genome but these are hard to interpret using standard tools., Results: We have developed CSynth, an interactive 3D genome browser and real-time chromatin restraint-based modeller to visualize models of any chromosome conformation capture (3C) data. Unlike other modelling systems, CSynth allows dynamic interaction with the modelling parameters to allow experimentation and effects on the model. It also allows comparison of models generated from data in different tissues/cell states and the results of third-party 3D modelling outputs. In addition, we include an option to view and manipulate these complicated structures using Virtual Reality (VR) so scientists can immerse themselves in the models for further understanding. This VR component has also proven to be a valuable teaching and a public engagement tool., Availabilityand Implementation: CSynth is web based and available to use at csynth.org., Supplementary Information: Supplementary data are available at Bioinformatics online., (© The Author(s) 2020. Published by Oxford University Press.)

Aging drives progressive loss of the ability of tissues to recover from stress, partly through loss of somatic stem cell function and increased senescent burden. We demonstrate that bone marrow-derived mesenchymal stem cells (BM-MSCs) rapidly senescence and become dysfunctional in culture. Injection of BM-MSCs from young mice prolonged life span and health span, and conditioned media (CM) from young BM-MSCs rescued the function of aged stem cells and senescent fibroblasts. Extracellular vesicles (EVs) from young BM-MSC CM extended life span of Ercc1 -/- mice similarly to injection of young BM-MSCs. Finally, treatment with EVs from MSCs generated from human ES cells reduced senescence in culture and in vivo, and improved health span. Thus, MSC EVs represent an effective and safe approach for conferring the therapeutic effects of adult stem cells, avoiding the risks of tumor development and donor cell rejection. These results demonstrate that MSC-derived EVs are highly effective senotherapeutics, slowing the progression of aging, and diseases driven by cellular senescence., (© 2021 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.)

Background: Type 2 diabetes (T2DM) is an age-associated disease characterized by hyperglycemia due to insulin resistance and decreased beta-cell function. DNA damage accumulation has been associated with T2DM, but whether DNA damage plays a role in the pathogenesis of the disease is unclear. Here, we used mice deficient for the DNA excision-repair gene Ercc1 to study the impact of persistent endogenous DNA damage accumulation on energy metabolism, glucose homeostasis and beta-cell function., Methods: ERCC1-XPF is an endonuclease required for multiple DNA repair pathways and reduced expression of ERCC1-XPF causes accelerated accumulation of unrepaired endogenous DNA damage and accelerated aging in humans and mice. In this study, energy metabolism, glucose metabolism, beta-cell function and insulin sensitivity were studied in Ercc1 d/- mice, which model a human progeroid syndrome., Results: Ercc1 d/- mice displayed suppression of the somatotropic axis and altered energy metabolism. Insulin sensitivity was increased, whereas, plasma insulin levels were decreased in Ercc1 d/- mice. Fasting induced hypoglycemia in Ercc1 d/- mice, which was the result of increased glucose disposal. Ercc1 d/- mice exhibit a significantly reduced beta-cell area, even compared to control mice of similar weight. Glucose-stimulated insulin secretion in vivo was decreased in Ercc1 d/- mice. Islets isolated from Ercc1 d/- mice showed increased DNA damage markers, decreased glucose-stimulated insulin secretion and increased susceptibility to apoptosis., Conclusion: Spontaneous DNA damage accumulation triggers an adaptive response resulting in improved insulin sensitivity. Loss of DNA repair, however, does negatively impacts beta-cell survival and function in Ercc1 d/- mice., Competing Interests: Declaration of competing interest There is no potential conflict of interest for all authors., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

An insulin-like growth factor-1 receptor (IGF1R) variant in exon 6 (Arg-407-His) in Ashkenazi Jewish centenarians was previously found to be associated with reduced IGF1R activity. To further study this longevity associated IGF1R variant, we generated a novel mouse line carrying the R407H variant in exon 6 of the Igf1r gene by employing CRISPR/Cas9 genome editing technology. Here, we show that the Igf1r gene can be edited in mouse embryos by zygotic electroporation of Cas9 protein and a single-guide RNAs together with a single stranded oligonucleotide donor containing the desired key nucleotide changes at the Igf1r locus. Sequence analysis of F0 and F1 mice following targeted editing demonstrated the robustness of this approach in mice using CRISPR/Cas9 directed homologous recombination (HDR). Western blot analysis indicates that mice heterozygous for the variant have a significant decrease in IGF1R phosphorylation in various tissues, including skeletal muscle, compared to wildtype. In addition, depletion of IGF1R signaling specifically in skeletal muscle of progeroid Ercc1 -/Δ mice resulted in extended health span and median lifespan providing the rationale for long term lifespan studies in Igf1r hR407H variant mice. This mouse line will be a valuable genetic tool to help determine the impact of IGF1R signaling on aging and longevity. The CRISPR editing approach represents a prototype for generating additional longevity associated gene variant mouse lines to study relevance to human exceptional longevity., Competing Interests: Competing interests The authors declare there are no competing interests.

Purpose: Enrichment of heterozygous missense and truncating SMAD6 variants was previously reported in nonsyndromic sagittal and metopic synostosis, and interaction of SMAD6 variants with a common polymorphism nearBMP2 (rs1884302) was proposed to contribute to inconsistent penetrance. We determined the occurrence of SMAD6 variants in all types of craniosynostosis, evaluated the impact of different missense variants on SMAD6 function, and tested independently whether rs1884302 genotype significantly modifies the phenotype., Methods: We performed resequencing of SMAD6 in 795 unsolved patients with any type of craniosynostosis and genotyped rs1884302 in SMAD6-positive individuals and relatives. We examined the inhibitory activity and stability of SMAD6 missense variants., Results: We found 18 (2.3%) different rare damaging SMAD6 variants, with the highest prevalence in metopic synostosis (5.8%) and an 18.3-fold enrichment of loss-of-function variants comparedwith gnomAD data (P < 10 -7 ). Combined with eight additional variants, ≥20/26 were transmitted from an unaffected parent but rs1884302 genotype did not predict phenotype., Conclusion: Pathogenic SMAD6 variants substantially increase the risk of both nonsyndromic and syndromic presentations of craniosynostosis, especially metopic synostosis. Functional analysis is important to evaluate missense variants. Genotyping of rs1884302 is not clinically useful. Mechanisms to explain the remarkable diversity of phenotypes associated with SMAD6 variants remain obscure.

Previously, we reported that persistent DNA damage accelerates ageing of the spine, but the mechanisms behind this process are not well understood. Ataxia telangiectasia mutated (ATM) is a protein kinase involved in the DNA damage response, which controls cell fate, including cell death. To test the role of ATM in the human intervertebral disc, we exposed human nucleus pulposus (hNP) cells directly to the DNA damaging agent cisplatin. Cisplatin-treated hNP cells exhibited rapid phosphorylation of ATM and subsequent increased NF-κB activation, aggrecanolysis, decreased total proteoglycan production and increased expression of markers of senescence, including p21, γH 2 AX and SA-ß-gal. Treating cisplatin-exposed hNP cells with an ATM-specific inhibitor negated these effects. In addition, genetic reduction of ATM reduced disc cellular senescence and matrix proteoglycan loss in the progeroid Ercc1 -/∆ mouse model of accelerated ageing. These findings suggest that activation of ATM signalling under persistent genotoxic stress promotes disc cellular senescence and matrix homeostatic perturbation. Thus, the ATM signalling pathway represents a therapeutic target to delay the progression of age-associated spine pathologies., (© 2020 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.)

NF-κB is a transcription factor activated in response to inflammatory, genotoxic and oxidative stress and important for driving senescence and aging. Ataxia-telangiectasia mutated (ATM) kinase, a core component of DNA damage response signaling, activates NF-κB in response to genotoxic and oxidative stress via post-translational modifications. Here we demonstrate that ATM is activated in senescent cells in culture and murine tissues from Ercc1 -deficient mouse models of accelerated aging, as well as naturally aged mice. Genetic and pharmacologic inhibition of ATM reduced activation of NF-κB and markers of senescence and the senescence-associated secretory phenotype (SASP) in senescent Ercc1 -/- MEFs. Ercc1 -/Δ mice heterozygous for Atm have reduced NF-κB activity and cellular senescence, improved function of muscle-derived stem/progenetor cells (MDSPCs) and extended healthspan with reduced age-related pathology especially age-related bone and intervertebral disc pathologies. In addition, treatment of Ercc1 -/∆ mice with the ATM inhibitor KU-55933 suppressed markers of senescence and SASP. Taken together, these results demonstrate that the ATM kinase is a major mediator of DNA damage-induced, NF-κB-mediated cellular senescence, stem cell dysfunction and aging and thus represents a therapeutic target to slow the progression of aging.

Senescent cells accumulate with age in vertebrates and promote aging largely through their senescence-associated secretory phenotype (SASP). Many types of stress induce senescence, including genotoxic stress. ERCC1-XPF is a DNA repair endonuclease required for multiple DNA repair mechanisms that protect the nuclear genome. Humans or mice with reduced expression of this enzyme age rapidly due to increased levels of spontaneous, genotoxic stress. Here, we asked whether this corresponds to an increased level of senescent cells. p16 Ink4a and p21 Cip1 mRNA were increased ~15-fold in peripheral lymphocytes from 4- to 5-month-old Ercc1 -/∆ and 2.5-year-old wild-type (WT) mice, suggesting that these animals exhibit a similar biological age. p16 Ink4a and p21 Cip1 mRNA were elevated in 10 of 13 tissues analyzed from 4- to 5-month-old Ercc1 -/∆ mice, indicating where endogenous DNA damage drives senescence in vivo. Aged WT mice had similar increases of p16 Ink4a and p21 Cip1 mRNA in the same 10 tissues as the mutant mice. Senescence-associated β-galactosidase activity and p21 Cip1 protein also were increased in tissues of the progeroid and aged mice, while Lamin B1 mRNA and protein levels were diminished. In Ercc1 -/Δ mice with a p16 Ink4a luciferase reporter, bioluminescence rose steadily with age, particularly in lung, thymus, and pancreas. These data illustrate where senescence occurs with natural and accelerated aging in mice and the relative extent of senescence among tissues. Interestingly, senescence was greater in male mice until the end of life. The similarities between Ercc1 -/∆ and aged WT mice support the conclusion that the DNA repair-deficient mice accurately model the age-related accumulation of senescent cells, albeit six-times faster., (© 2020 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.)

Saethre-Chotzen syndrome (SCS), one of the most common forms of syndromic craniosynostosis (premature fusion of the cranial sutures), results from haploinsufficiency of TWIST1, caused by deletions of the entire gene or loss-of-function variants within the coding region. To determine whether non-coding variants also contribute to SCS, we screened 14 genetically undiagnosed SCS patients using targeted capture sequencing, and identified novel single nucleotide variants (SNVs) in the 5' untranslated region (UTR) of TWIST1 in two unrelated SCS cases. We show experimentally that these variants, which create translation start sites in the TWIST1 leader sequence, reduce translation from the main open reading frame (mORF). This is the first demonstration that non-coding SNVs of TWIST1 can cause SCS, and highlights the importance of screening the 5' UTR in clinically diagnosed SCS patients without a coding mutation. Similar 5' UTR variants, particularly of haploinsufficient genes, may represent an under-ascertained cause of monogenic disease., (© 2018 The Authors. Human Mutation published by Wiley Periodicals, Inc.)

Background: Senescence is a tumor suppressor mechanism activated in stressed cells to prevent replication of damaged DNA. Senescent cells have been demonstrated to play a causal role in driving aging and age-related diseases using genetic and pharmacologic approaches. We previously demonstrated that the combination of dasatinib and the flavonoid quercetin is a potent senolytic improving numerous age-related conditions including frailty, osteoporosis and cardiovascular disease. The goal of this study was to identify flavonoids with more potent senolytic activity., Methods: A panel of flavonoid polyphenols was screened for senolytic activity using senescent murine and human fibroblasts, driven by oxidative and genotoxic stress, respectively. The top senotherapeutic flavonoid was tested in mice modeling a progeroid syndrome carrying a p16 INK4a -luciferase reporter and aged wild-type mice to determine the effects of fisetin on senescence markers, age-related histopathology, disease markers, health span and lifespan. Human adipose tissue explants were used to determine if results translated., Findings: Of the 10 flavonoids tested, fisetin was the most potent senolytic. Acute or intermittent treatment of progeroid and old mice with fisetin reduced senescence markers in multiple tissues, consistent with a hit-and-run senolytic mechanism. Fisetin reduced senescence in a subset of cells in murine and human adipose tissue, demonstrating cell-type specificity. Administration of fisetin to wild-type mice late in life restored tissue homeostasis, reduced age-related pathology, and extended median and maximum lifespan., Interpretation: The natural product fisetin has senotherapeutic activity in mice and in human tissues. Late life intervention was sufficient to yield a potent health benefit. These characteristics suggest the feasibility to translation to human clinical studies. FUND: NIH grants P01 AG043376 (PDR, LJN), U19 AG056278 (PDR, LJN, WLL), R24 AG047115 (WLL), R37 AG013925 (JLK), R21 AG047984 (JLK), P30 DK050456 (Adipocyte Subcore, JLK), a Glenn Foundation/American Federation for Aging Research (AFAR) BIG Award (JLK), Glenn/AFAR (LJN, CEB), the Ted Nash Long Life and Noaber Foundations (JLK), the Connor Group (JLK), Robert J. and Theresa W. Ryan (JLK), and a Minnesota Partnership Grant (AMAY-UMN#99)-P004610401-1 (JLK, EAA)., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

Adaptive immunity involves the development of bespoke antibodies in germinal centers (GCs) through immunoglobulin somatic hypermutation (SHM) in GC dark zones (DZs) and clonal selection in light zones (LZs). Accurate selection requires that cells fully replace surface B cell receptors (BCRs) following SHM, but whether this happens before LZ entry is not clear. We found that most GC B cells degrade pre-SHM receptors before leaving the DZ, and that B cells acquiring crippling mutations during SHM rarely reached the LZ. Instead, apoptosis was triggered preferentially in late G1, a stage wherein cells with functional BCRs re-entered cell cycle or reduced surface expression of the chemokine receptor CXCR4 to enable LZ migration. Ectopic expression of the anti-apoptotic gene Bcl2 was not sufficient for cells with damaging mutations to reach the LZ, suggesting that BCR-dependent cues may actively facilitate the transition. Thus, BCR replacement and pre-screening in DZs prevents the accumulation of clones with non-functional receptors and facilitates selection in the LZ., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Lipid peroxidation (LPO) products are relatively stable and abundant metabolites, which accumulate in tissues of mammals with aging, being able to modify all cellular nucleophiles, creating protein and DNA adducts including crosslinks. Here, we used cells and mice deficient in the ERCC1-XPF endonuclease required for nucleotide excision repair and the repair of DNA interstrand crosslinks to ask if specifically LPO-induced DNA damage contributes to loss of cell and tissue homeostasis. Ercc1 -/- mouse embryonic fibroblasts were more sensitive than wild-type (WT) cells to the LPO products: 4-hydroxy-2-nonenal (HNE), crotonaldehyde and malondialdehyde. ERCC1-XPF hypomorphic mice were hypersensitive to CCl 4 and a diet rich in polyunsaturated fatty acids, two potent inducers of endogenous LPO. To gain insight into the mechanism of how LPO influences DNA repair-deficient cells, we measured the impact of the major endogenous LPO product, HNE, on WT and Ercc1 -/- cells. HNE inhibited proliferation, stimulated ROS and LPO formation, induced DNA base damage, strand breaks, error-prone translesion DNA synthesis and cellular senescence much more potently in Ercc1 -/- cells than in DNA repair-competent control cells. HNE also deregulated base excision repair and energy production pathways. Our observations that ERCC1-deficient cells and mice are hypersensitive to LPO implicates LPO-induced DNA damage in contributing to cellular demise and tissue degeneration, notably even when the source of LPO is dietary polyunsaturated fats., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Accumulation of senescent cells over time contributes to aging and age-related diseases. However, what drives senescence in vivo is not clear. Here we used a genetic approach to determine if spontaneous nuclear DNA damage is sufficient to initiate senescence in mammals. Ercc1 -/∆ mice with reduced expression of ERCC1-XPF endonuclease have impaired capacity to repair the nuclear genome. Ercc1 -/∆ mice accumulated spontaneous, oxidative DNA damage more rapidly than wild-type (WT) mice. As a consequence, senescent cells accumulated more rapidly in Ercc1 -/∆ mice compared to repair-competent animals. However, the levels of DNA damage and senescent cells in Ercc1 -/∆ mice never exceeded that observed in old WT mice. Surprisingly, levels of reactive oxygen species (ROS) were increased in tissues of Ercc1 -/∆ mice to an extent identical to naturally-aged WT mice. Increased enzymatic production of ROS and decreased antioxidants contributed to the elevation in oxidative stress in both Ercc1 -/∆ and aged WT mice. Chronic treatment of Ercc1 -/∆ mice with the mitochondrial-targeted radical scavenger XJB-5-131 attenuated oxidative DNA damage, senescence and age-related pathology. Our findings indicate that nuclear genotoxic stress arises, at least in part, due to mitochondrial-derived ROS, and this spontaneous DNA damage is sufficient to drive increased levels of ROS, cellular senescence, and the consequent age-related physiological decline., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

Sequencing the genomes of individual cells enables the direct determination of genetic heterogeneity amongst cells within a population. We have developed an injection-moulded valveless microfluidic device in which single cells from colorectal cancer derived cell lines (LS174T, LS180 and RKO) and fresh colorectal tumors have been individually trapped, their genomes extracted and prepared for sequencing using multiple displacement amplification (MDA). Ninety nine percent of the DNA sequences obtained mapped to a reference human genome, indicating that there was effectively no contamination of these samples from non-human sources. In addition, most of the reads are correctly paired, with a low percentage of singletons (0.17 ± 0.06%) and we obtain genome coverages approaching 90%. To achieve this high quality, our device design and process shows that amplification can be conducted in microliter volumes as long as the lysis is in sub-nanoliter volumes. Our data thus demonstrates that high quality whole genome sequencing of single cells can be achieved using a relatively simple, inexpensive and scalable device. Detection of genetic heterogeneity at the single cell level, as we have demonstrated for freshly obtained single cancer cells, could soon become available as a clinical tool to precisely match treatment with the properties of a patient's own tumor.

Nuclear factor κB (NF-κB) is a transcription factor important for regulating innate and adaptive immunity, cellular proliferation, apoptosis, and senescence. Dysregulation of NF-κB and its upstream regulator IκB kinase (IKK) contributes to the pathogenesis of multiple inflammatory and degenerative diseases as well as cancer. An 11-amino acid peptide containing the NF-κB essential modulator (NEMO)-binding domain (NBD) derived from the C-terminus of β subunit of IKK, functions as a highly selective inhibitor of the IKK complex by disrupting the association of IKKβ and the IKKγ subunit NEMO. A structure-based pharmacophore model was developed to identify NBD mimetics by in silico screening. Two optimized lead NBD mimetics, SR12343 and SR12460, inhibited tumor necrosis factor α (TNF-α)- and lipopolysaccharide (LPS)-induced NF-κB activation by blocking the interaction between IKKβ and NEMO and suppressed LPS-induced acute pulmonary inflammation in mice. Chronic treatment of a mouse model of Duchenne muscular dystrophy (DMD) with SR12343 and SR12460 attenuated inflammatory infiltration, necrosis and muscle degeneration, demonstrating that these small-molecule NBD mimetics are potential therapeutics for inflammatory and degenerative diseases., Competing Interests: The authors have declared that no competing interests exist.

Next-generation sequencing is a powerful tool for the discovery of genes related to neurodevelopmental disorders (NDDs). Here, we report the identification of a distinct syndrome due to de novo or inherited heterozygous mutations in Tousled-like kinase 2 (TLK2) in 38 unrelated individuals and two affected mothers, using whole-exome and whole-genome sequencing technologies, matchmaker databases, and international collaborations. Affected individuals had a consistent phenotype, characterized by mild-borderline neurodevelopmental delay (86%), behavioral disorders (68%), severe gastro-intestinal problems (63%), and facial dysmorphism including blepharophimosis (82%), telecanthus (74%), prominent nasal bridge (68%), broad nasal tip (66%), thin vermilion of the upper lip (62%), and upslanting palpebral fissures (55%). Analysis of cell lines from three affected individuals showed that mutations act through a loss-of-function mechanism in at least two case subjects. Genotype-phenotype analysis and comparison of computationally modeled faces showed that phenotypes of these and other individuals with loss-of-function variants significantly overlapped with phenotypes of individuals with other variant types (missense and C-terminal truncating). This suggests that haploinsufficiency of TLK2 is the most likely underlying disease mechanism, leading to a consistent neurodevelopmental phenotype. This work illustrates the power of international data sharing, by the identification of 40 individuals from 26 different centers in 7 different countries, allowing the identification, clinical delineation, and genotype-phenotype evaluation of a distinct NDD caused by mutations in TLK2., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

In the era of genome-wide association studies (GWAS) and personalized medicine, predicting the impact of single nucleotide polymorphisms (SNPs) in regulatory elements is an important goal. Current approaches to determine the potential of regulatory SNPs depend on inadequate knowledge of cell-specific DNA binding motifs. Here, we present Sasquatch, a new computational approach that uses DNase footprint data to estimate and visualize the effects of noncoding variants on transcription factor binding. Sasquatch performs a comprehensive k -mer-based analysis of DNase footprints to determine any k -mer's potential for protein binding in a specific cell type and how this may be changed by sequence variants. Therefore, Sasquatch uses an unbiased approach, independent of known transcription factor binding sites and motifs. Sasquatch only requires a single DNase-seq data set per cell type, from any genotype, and produces consistent predictions from data generated by different experimental procedures and at different sequence depths. Here we demonstrate the effectiveness of Sasquatch using previously validated functional SNPs and benchmark its performance against existing approaches. Sasquatch is available as a versatile webtool incorporating publicly available data, including the human ENCODE collection. Thus, Sasquatch provides a powerful tool and repository for prioritizing likely regulatory SNPs in the noncoding genome., (© 2017 Schwessinger et al.; Published by Cold Spring Harbor Laboratory Press.)

Multiple cytokines, including interleukin 6 (IL-6), IL-11, IL-27, oncostatin M (OSM), and leukemia inhibitory factor (LIF), signal via the common GP130 cytokine receptor subunit. In this study, we describe a patient with a homozygous mutation of IL6ST (encoding GP130 p.N404Y) who presented with recurrent infections, eczema, bronchiectasis, high IgE, eosinophilia, defective B cell memory, and an impaired acute-phase response, as well as skeletal abnormalities including craniosynostosis. The p.N404Y missense substitution is associated with loss of IL-6, IL-11, IL-27, and OSM signaling but a largely intact LIF response. This study identifies a novel immunodeficiency with phenotypic similarities to STAT3 hyper-IgE syndrome caused by loss of function of GP130., (© 2017 Schwerd et al.)

Aging is the main risk factor for many chronic degenerative diseases and cancer. Increased senescent cell burden in various tissues is a major contributor to aging and age-related diseases. Recently, a new class of drugs termed senolytics were demonstrated to extending healthspan, reducing frailty and improving stem cell function in multiple murine models of aging. To identify novel and more optimal senotherapeutic drugs and combinations, we established a senescence associated β-galactosidase assay as a screening platform to rapidly identify drugs that specifically affect senescent cells. We used primary Ercc1 -/- murine embryonic fibroblasts with reduced DNA repair capacity, which senesce rapidly if grown at atmospheric oxygen. This platform was used to screen a small library of compounds that regulate autophagy, identifying two inhibitors of the HSP90 chaperone family as having significant senolytic activity in mouse and human cells. Treatment of Ercc1 -/∆ mice, a mouse model of a human progeroid syndrome, with the HSP90 inhibitor 17-DMAG extended healthspan, delayed the onset of several age-related symptoms and reduced p16 INK4a expression. These results demonstrate the utility of our screening platform to identify senotherapeutic agents as well as identified HSP90 inhibitors as a promising new class of senolytic drugs.The accumulation of senescent cells is thought to contribute to the age-associated decline in tissue function. Here, the authors identify HSP90 inhibitors as a new class of senolytic compounds in an in vitro screening and show that administration of a HSP90 inhibitor reduces age-related symptoms in progeroid mice.