Somatic Evolution in Non-neoplastic IBD-Affected Colon.

Inflammatory bowel disease (IBD) is a chronic inflammatory disease associated with increased risk of gastrointestinal cancers. We whole-genome sequenced 446 colonic crypts from 46 IBD patients and compared these to 412 crypts from 41 non-IBD controls from our previous publication on the mutation landscape of the normal colon. The average mutation rate of affected colonic epithelial cells is 2.4-fold that of healthy colon, and this increase is mostly driven by acceleration of mutational processes ubiquitously observed in normal colon. In contrast to the normal colon, where clonal expansions outside the confines of the crypt are rare, we observed widespread millimeter-scale clonal expansions. We discovered non-synonymous mutations in ARID1A, FBXW7, PIGR, ZC3H12A, and genes in the interleukin 17 and Toll-like receptor pathways, under positive selection in IBD. These results suggest distinct selection mechanisms in the colitis-affected colon and that somatic mutations potentially play a causal role in IBD pathogenesis.

Clonal dynamics of haematopoiesis across the human lifespan.

Age-related change in human haematopoiesis causes reduced regenerative capacity1, cytopenias2, immune dysfunction3 and increased risk of blood cancer4-6, but the reason for such abrupt functional decline after 70 years of age remains unclear. Here we sequenced 3,579 genomes from single cell-derived colonies of haematopoietic cells across 10 human subjects from 0 to 81 years of age. Haematopoietic stem cells or multipotent progenitors (HSC/MPPs) accumulated a mean of 17 mutations per year after birth and lost 30 base pairs per year of telomere length. Haematopoiesis in adults less than 65 years of age was massively polyclonal, with high clonal diversity and a stable population of 20,000-200,000 HSC/MPPs contributing evenly to blood production. By contrast, haematopoiesis in individuals aged over 75 showed profoundly decreased clonal diversity. In each of the older subjects, 30-60% of haematopoiesis was accounted for by 12-18 independent clones, each contributing 1-34% of blood production. Most clones had begun their expansion before the subject was 40 years old, but only 22% had known driver mutations. Genome-wide selection analysis estimated that between 1 in 34 and 1 in 12 non-synonymous mutations were drivers, accruing at constant rates throughout life, affecting more genes than identified in blood cancers. Loss of the Y chromosome conferred selective benefits in males. Simulations of haematopoiesis, with constant stem cell population size and constant acquisition of driver mutations conferring moderate fitness benefits, entirely explained the abrupt change in clonal structure in the elderly. Rapidly decreasing clonal diversity is a universal feature of haematopoiesis in aged humans, underpinned by pervasive positive selection acting on many more genes than currently identified.

TRIM28 inactivation in epithelial nephroblastoma is frequent and often associated with predisposing TRIM28 germline variants.

Wilms tumors (WTs) are histologically diverse childhood cancers with variable contributions of blastema, stroma, and epithelia. A variety of cancer genes operate in WTs, including the tripartite-motif-containing-28 gene (TRIM28). Case reports and small case series suggest that TRIM28 mutations are associated with epithelial morphology and WT predisposition. Here, we systematically investigated the prevalence of TRIM28 inactivation and predisposing mutations in a cohort of 126 WTs with >2/3 epithelial cells, spanning 20 years of biobanking in the German SIOP93-01/GPOH and SIOP2001/GPOH studies. Overall, 44.4% (56/126) cases exhibited loss of TRIM28 by immunohistochemical staining. Of these, 48 could be further analyzed molecularly, revealing TRIM28 sequence variants in each case - either homozygous (~2/3) or heterozygous with epigenetic silencing of the second allele (~1/3). The majority (80%) of the mutations resulted in premature stops and frameshifts. In addition, we detected missense mutations and small deletions predicted to destabilize the protein through interference with folding of key structural elements such as the zinc-binding clusters of the RING, B-box-2, and PHD domains or the central coiled-coil region. TRIM28-mutant tumors otherwise lacked WT-typical IGF2 alterations or driver events, except for rare TP53 progression events that occurred with expected frequency. Expression profiling identified TRIM28-mutant tumors as a homogeneous subset of epithelial WTs that mostly present with stage I disease. There was a high prevalence of perilobar nephrogenic rests, putative precursor lesions, that carried the same biallelic TRIM28 alterations in 7/7 cases tested. Importantly, 46% of the TRIM28 mutations were present in blood cells or normal kidney tissue, suggesting germline events or somatic mosaicism, partly supported by family history. Given the high prevalence of predisposing variants in TRIM28-driven WT, we suggest that immunohistochemical testing of TRIM28 be integrated into diagnostic practice as the management of WT in predisposed children differs from that with sporadic tumors. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Immune signatures correlate with L1 retrotransposition in gastrointestinal cancers.

Long interspersed nuclear element-1 (LINE-1 or L1) retrotransposons are normally suppressed in somatic tissues mainly due to DNA methylation and antiviral defense. However, the mechanism to suppress L1s may be disrupted in cancers, thus allowing L1s to act as insertional mutagens and cause genomic rearrangement and instability. Whereas the frequency of somatic L1 insertions varies greatly among individual tumors, much remains to be learned about underlying genetic, cellular, or environmental factors. Here, we report multiple correlates of L1 activity in stomach, colorectal, and esophageal tumors through an integrative analysis of cancer whole-genome and matched RNA-sequencing profiles. Clinical indicators of tumor progression, such as tumor grade and patient age, showed positive association. A potential L1 expression suppressor, TP53, was mutated in tumors with frequent L1 insertions. We characterized the effects of somatic L1 insertions on mRNA splicing and expression, and demonstrated an increased risk of gene disruption in retrotransposition-prone cancers. In particular, we found that a cancer-specific L1 insertion in an exon of MOV10, a key L1 suppressor, caused exon skipping and decreased expression of the affected allele due to nonsense-mediated decay in a tumor with a high L1 insertion load. Importantly, tumors with high immune activity, for example, those associated with Epstein-Barr virus infection or microsatellite instability, tended to carry a low number of L1 insertions in genomes with high expression levels of L1 suppressors such as APOBEC3s and SAMHD1 Our results indicate that cancer immunity may contribute to genome stability by suppressing L1 retrotransposition in gastrointestinal cancers.

Pericentromeric satellite repeat expansions through RNA-derived DNA intermediates in cancer.

Aberrant transcription of the pericentromeric human satellite II (HSATII) repeat is present in a wide variety of epithelial cancers. In deriving experimental systems to study its deregulation, we observed that HSATII expression is induced in colon cancer cells cultured as xenografts or under nonadherent conditions in vitro, but it is rapidly lost in standard 2D cultures. Unexpectedly, physiological induction of endogenous HSATII RNA, as well as introduction of synthetic HSATII transcripts, generated cDNA intermediates in the form of DNA/RNA hybrids. Single molecule sequencing of tumor xenografts showed that HSATII RNA-derived DNA (rdDNA) molecules are stably incorporated within pericentromeric loci. Suppression of RT activity using small molecule inhibitors reduced HSATII copy gain. Analysis of whole-genome sequencing data revealed that HSATII copy number gain is a common feature in primary human colon tumors and is associated with a lower overall survival. Together, our observations suggest that cancer-associated derepression of specific repetitive sequences can promote their RNA-driven genomic expansion, with potential implications on pericentromeric architecture.

A novel semi-automatic snake robot for natural orifice transluminal endoscopic surgery: preclinical tests in animal and human cadaver models (with video).

BACKGROUND AND STUDY AIMS: Natural orifice transluminal endoscopic surgery (NOTES) is an emerging surgical technique. We aimed to design, create, and evaluate a new semi-automatic snake robot for NOTES. MATERIALS AND METHODS: The snake robot employs the characteristics of both a manual endoscope and a multi-segment snake robot. This robot is inserted and retracted manually, like a classical endoscope, while its shape is controlled using embedded robot technology. The feasibility of a prototype robot for NOTES was evaluated in animals and human cadavers. RESULTS: The transverse stiffness and maneuverability of the snake robot appeared satisfactory. It could be advanced through the anus as far as the peritoneal cavity without any injury to adjacent organs. Preclinical tests showed that the device could navigate the peritoneal cavity. CONCLUSIONS: The snake robot has advantages of high transverse force and intuitive control. This new robot may be clinically superior to conventional tools for transanal NOTES.

Fabrication of highly stable silica coated ZnCuInS nanocrystals monolayer via layer by layer deposition for LED application.

In this study, non-toxic and highly stable silica coated ZnCuInS NCs were synthesized by a reverse microemulsion method. The single NCs were uniformly encapsulated in a silica shell with a diameter of ~30 nm. Although hydrolyzed TEOS caused a QY reduction, and a 12.5 nm red shift occurred after silica coating, the photo and thermal stabilities were extremely improved. For LED application, the silica coated ZnCuInS NCs phosphor layer was arrayed on the InGaN LED surface by layer-by-layer deposition utilizing electrostatic attraction. When the ZnCuInS/SiO2 NCs single monolayer was fabricated, 6.73% high color conversion efficiency was achieved.

Identification of high-confidence somatic mutations in whole genome sequence of formalin-fixed breast cancer specimens.

The utilization of archived, formalin-fixed paraffin-embedded (FFPE) tumor samples for massive parallel sequencing has been challenging due to DNA damage and contamination with normal stroma. Here, we perform whole genome sequencing of DNA isolated from two triple-negative breast cancer tumors archived for >11 years as 5 µm FFPE sections and matched germline DNA. The tumor samples show differing amounts of FFPE damaged DNA sequencing reads revealed as relatively high alignment mismatch rates enriched for C · G > T · A substitutions compared to germline samples. This increase in mismatch rate is observable with as few as one million reads, allowing for an upfront evaluation of the sample integrity before whole genome sequencing. By applying innovative quality filters incorporating global nucleotide mismatch rates and local mismatch rates, we present a method to identify high-confidence somatic mutations even in the presence of FFPE induced DNA damage. This results in a breast cancer mutational profile consistent with previous studies and revealing potentially important functional mutations. Our study demonstrates the feasibility of performing genome-wide deep sequencing analysis of FFPE archived tumors of limited sample size such as residual cancer after treatment or metastatic biopsies.

Suggestion of Follow-Up Period in Nonfunctioning Pituitary Incidentaloma Based on MRI Characteristics.

BACKGROUND: For patients diagnosed with asymptomatic, non-functional pituitary incidentaloma (PI), periodic follow-up is generally proposed. However, the recommended follow-up period differs among existing guidelines and consensus is lacking. Thus, this study aimed to suggest follow-up periods for PI based on MRI characteristics. METHODS: Between 2007 and 2023, 245 patients who were diagnosed with PI were retrospectively assessed. Their mean clinical and neuroradiological follow-up periods were 74.2 and 27.3 months, respectively. Their baseline clinical and neuroradiological characteristics were analyzed. These 245 patients were divided into two groups: those with PI size progression and those without PI size progression. Additionally, neuroradiological features of each group were analyzed according to presumptive diagnoses of PI. RESULTS: PI size increased in 33 of 245 patients. For the remaining 212 patients, PI size decreased or stayed unchanged. Of the 33 patients with PI size progression, ten underwent surgery. Stalk deviation (p<0.001) and lesion enhancement (p=0.001) were significantly more observed in those with PI size progression than in those without PI size progression. MRI morphological factors were not related to changes in PI size in the presumptive Rathke's cleft cyst group. In the presumptive pituitary adenoma group, absence of tumor enhancement (p<0.001) and stalk deviation (p<0.001) were significantly associated with tumor reduction and progression, respectively. CONCLUSION: Our findings support an additional guideline for patients with asymptomatic non-functional PI without stalk deviation and enhancement. For these patients, the clinical and neuroradiological follow-up periods could be reduced.

Sex Differences in Heart Rate Variability and Vascular Function Following High-Intensity Interval Training in Young Adults.

High-intensityintervaltraining (HIIT) issuperiortoothertrainingstrategies in both male andfemalehealthyindividuals. Understanding sex-specificdifferences in cardiac auto-regulation maycontributetothe optimal trainingstrategiesfor HIIT. The presentstudyaimedtoidentifysexdifferences in heart rate variability (HRV) andvascularfunctionfollowing HIIT in youngadults. Twenty-fourphysicallyactiveyoung male andfemaleadults (M: 12, F: 12, age: 19.5 yr, BMI: 22.1 kg·m-2) volunteeredtoparticipate in thestudy. Participantsperformed 10 boutsof HIIT including 20 s of high-intensitycycling at 115-130% Wmaxfollowedby 100 s ofrecovery. The cardiac auto-regulationsincluding HRV andvascularfunctionweremeasured at five different time points. The R-R interval, rMSSD, and SDNN wererecoveredfaster in malesthan in females after 15 min of HIIT. Thereweresexdifferences in theautonomicnervoussystemwhereln LF andln HF activitiesalongwithsympathovagalbalance (ln LF/HF) weregreater in femalescomparedwithmalesimmediatelyand 15 min after HIIT. However, nosignificantdifferences in bloodpressureand brachial-ankle pulse wavevelocitywereobservedbetween male andfemaleparticipants. Overall, HRV was moreactivated in femalesthan in malesfollowing HIIT, but theacuteresponse in vascularfunction was not different betweensexes. In futurestudies, sex-specificadaptationsofcardiacautoregulationfollowingrepeated HIIT mayneedtobeperformed.

Haplotype-specific assembly of shattered chromosomes in esophageal adenocarcinomas.

The epigenetic landscape of cancer is regulated by many factors, but primarily it derives from the underlying genome sequence. Chromothripsis is a catastrophic localized genome shattering event that drives, and often initiates, cancer evolution. We characterized five esophageal adenocarcinoma organoids with chromothripsis using long-read sequencing and transcriptome and epigenome profiling. Complex structural variation and subclonal variants meant that haplotype-aware de novo methods were required to generate contiguous cancer genome assemblies. Chromosomes were assembled separately and scaffolded using haplotype-resolved Hi-C reads, producing accurate assemblies even with up to 900 structural rearrangements. There were widespread differences between the chromothriptic and wild-type copies of chromosomes in topologically associated domains, chromatin accessibility, histone modifications, and gene expression. Differential epigenome peaks were most enriched within 10 kb of chromothriptic structural variants. Alterations in transcriptome and higher-order chromosome organization frequently occurred near differential epigenetic marks. Overall, chromothripsis reshapes gene regulation, causing coordinated changes in epigenetic landscape, transcription, and chromosome conformation.

The epidemiology of male lower urinary tract symptoms associated with benign prostatic hyperplasia: Results of 20 years of Korean community care and surveys.

PURPOSE: To investigate the prevalence of lower urinary tract symptoms/benign prostatic hyperplasia in a Korean population. MATERIALS AND METHODS: The Korean Prostate & Voiding Health Association provided free prostate-related community health care and conducted surveys in all regions of Korea from 2001 to 2022 with the cooperation of local government public health centers. A total of 72,068 males older than 50 were surveyed and analyzed. History taking, International Prostate Symptom Score (IPSS), transrectal ultrasonography, prostate-specific antigen (PSA) testing, uroflowmetry, and urine volume testing were performed. RESULTS: The mean prostate volumes in males in their 50s, 60s, 70s, and 80s or above were 24.7 g, 27.7 g, 31 g, and 33.7 g, respectively. The proportion of males with high PSA greater than 3 ng/mL was 3.8% among males in their 50s, 7.7% among males in their 60s, 13.1% among males in their 70s, and 17.9% among males 80 years of age or older. The mean IPSS total scores in males in their 50s, 60s, 70s, and 80s or above were 10.7, 12.7, 14.5, and 16, respectively. Severe symptoms were reported by 27.3% of males, whereas 51.7% reported moderate symptoms. The mean Qmax in males in their 50s, 60s, 70s, and 80s or above were 20 mL/s, 17.4 mL/s, 15.4 mL/s, and 13.8 mL/s, respectively. CONCLUSIONS: In this population-based study, mean prostate volume, IPSS, PSA, and Qmax were 30.6±15.1 g, 14.8±8.2, 1.9±4.7 ng/mL, and 15.6±6.5 mL/s, respectively. Aging was significantly associated with increased prostate volume, PSA levels, and IPSS scores, and with decreased Qmax and urine volume.

Characterization of surface modified ZnCuInS2 nanocrystals and its application to white light-emitting diodes.

Red-emitting ZnCuInS2 semiconductor nanocrystals (NCs) were synthesized and surface modification was performed on the NCs with oleylamine, trioctylphosphine, and 3-mercaptopropionic acid by the ligand exchange strategy. UV-visible spectroscopy, photoluminescence (PL) spectroscopy, time-resolved PL analysis, and attenuated total reflection Fourier transform infrared spectroscopy were used to verify the surface modification of NCs. Additionally, white light-emitting diodes (LEDs) were fabricated using surface modified red-emitting ZnCuInS2 NCs as red phosphor to compensate for the deficiency of red emission in white LEDs, consisting of blue LEDs as excitation sources and YAG:Ce as yellow phosphor.

Clonal selection of hematopoietic stem cells after gene therapy for sickle cell disease.

Gene therapy (GT) provides a potentially curative treatment option for patients with sickle cell disease (SCD); however, the occurrence of myeloid malignancies in GT clinical trials has prompted concern, with several postulated mechanisms. Here, we used whole-genome sequencing to track hematopoietic stem cells (HSCs) from six patients with SCD at pre- and post-GT time points to map the somatic mutation and clonal landscape of gene-modified and unmodified HSCs. Pre-GT, phylogenetic trees were highly polyclonal and mutation burdens per cell were elevated in some, but not all, patients. Post-GT, no clonal expansions were identified among gene-modified or unmodified cells; however, an increased frequency of potential driver mutations associated with myeloid neoplasms or clonal hematopoiesis (DNMT3A- and EZH2-mutated clones in particular) was observed in both genetically modified and unmodified cells, suggesting positive selection of mutant clones during GT. This work sheds light on HSC clonal dynamics and the mutational landscape after GT in SCD, highlighting the enhanced fitness of some HSCs harboring pre-existing driver mutations. Future studies should define the long-term fate of mutant clones, including any contribution to expansions associated with myeloid neoplasms.

APOBEC mutagenesis is a common process in normal human small intestine.

APOBEC mutational signatures SBS2 and SBS13 are common in many human cancer types. However, there is an incomplete understanding of its stimulus, when it occurs in the progression from normal to cancer cell and the APOBEC enzymes responsible. Here we whole-genome sequenced 342 microdissected normal epithelial crypts from the small intestines of 39 individuals and found that SBS2/SBS13 mutations were present in 17% of crypts, more frequent than most other normal tissues. Crypts with SBS2/SBS13 often had immediate crypt neighbors without SBS2/SBS13, suggesting that the underlying cause of SBS2/SBS13 is cell-intrinsic. APOBEC mutagenesis occurred in an episodic manner throughout the human lifespan, including in young children. APOBEC1 mRNA levels were very high in the small intestine epithelium, but low in the large intestine epithelium and other tissues. The results suggest that the high levels of SBS2/SBS13 in the small intestine are collateral damage from APOBEC1 fulfilling its physiological function of editing APOB mRNA.

Fabrication of high color rendering index white LED using Cd-free wavelength tunable Zn doped CuInS2 nanocrystals.

Highly luminescent Cd-free Zn doped CuInS(2) nanocrystals (ZCIS NCs) were synthesized, and their properties were evaluated using X-ray diffraction, Raman, UV, and photoluminescence. The crystal structure of the ZCIS NCs was similar to the zinc blende, and the lattice constant decreased with increasing Zn concentration. By incorporation of Zn, the emission wavelength was tuned from 536 to 637 nm with concomitant enhancement of the quantum yield up to 45%. A white light emitting diodes, integrating dual ZCIS NCs (λ(em) = 567, and 617 nm) and a 460 nm InGaN LED, exhibited a high color rendering index of 84.1 with a warm color temperature of 4256.2K. The CIE-1931 chromaticity coordinates were slightly shifted from (0.3626, 0.3378) at 20 mA to (0.3480, 0.3206) at 50 mA.

Spray pyrolysis prepared yellow to red color tunable Sr1-xCaxSe:Eu²+ phosphors for white LED.

The spherical and submicron size of Sr1-xCaxSe:Eu²âº phosphors were successfully prepared by ultrasonic spray pyrolysis. The phosphors adopted a cubic structure, and the replacement of Sr²âº with Ca²âº decreased the lattice parameter. The Sr1-xCaxSe:Eu²âº showed broad and strong excitation under 420-460 nm blue light, and the emission band could be tuned from 565 to 607 nm by increasing the Ca²âº ratio in the host lattice. In addition, the doping of Zn²âº into Sr²âº or Ca²âº enhanced the emission intensity with a small red shift due to the change in crystal field strength and nephelauxetic effects. The warm and high CRI of white LED was achieved using blue LED pumped with blending phosphors of 612 nm emitting Ca0.98Zn0.02Se:Eu²âº and 565 nm emitting YAG. The correlated color temperatures and CRI were 4719.2K, and 86.3, respectively, and an acceptable color variation was also observed at operating currents ranging from 20 to 70 mA.

Fabrication of white light-emitting diodes based on UV light-emitting diodes with conjugated polymers-(CdSe/ZnS) quantum dots as hybrid phosphors.

White light-emitting diodes (LEDs) were fabricated using GaN-based 380-nm UV LEDs precoated with the composite of blue-emitting polymer (poly[(9,9-dihexylfluorenyl-2,7-diyl)-alt-co-(2-methoxy-5-{2-ethylhexyloxy)-1 ,4-phenylene)]), yellow green-emitting polymer (poly[(9,9-dioctylfluorenyl-2,7-diyl)-co-(1,4-benzo-{2,1',3}-thiadiazole)]), and 605-nm red-emitting quantum dots (QDs). CdSe cores were obtained by solvothermal route using CdO, Se precursors and ZnS shells were synthesized by using diethylzinc, and hexamethyldisilathiane precursors. The optical properties of CdSe/ZnS QDs were characterized by UV-visible and photoluminescence (PL) spectra. The structural data and composition of the QDs were transmission electron microscopy (TEM), and EDX technique. The quantum yield and size of the QDs were 58.7% and about 6.7 nm, respectively. Three-band white light was generated by hybridizing blue (430 nm), green (535 nm), and red (605 nm) emission. The color-rendering index (CRI) of the device was extremely improved by introducing the QDs. The CIE-1931 chromaticity coordinate, color temperature, and CRI of a white LED at 20 mA were (0.379, 0.368), 3969 K, and 90, respectively.

Sr0.95Zn0.05Se:Eu2+ and CdSe/ZnS nanocrystals hybrid phosphors for enhancing color rendering index of white light emitting diode.

In this study, the yellow emitting cubic structure of Sr0.95Zn0.05Se:Eu2+ phosphors were prepared by high temperature solid state reaction. The Sr0.95Zn0.05Se:Eu2+ phosphors exhibited strong excitation intensity under 400-460 nm region, and broad band emission appeared at around 545-600 nm due to the d-f transition of Eu2+. To enhance the red emission, HDA/TOP/TOPO capped CdSe/ZnS NCs were synthesized via fast nucleation and slow growth method. The narrow emission peak was located at 615 nm with 69% of high quantum yield. Bright white emission was generated by combining a 460 nm InGaN LED chip with CdSe/ZnS NCs and Sr0.95Zn0.05Se:Eu2+ hybrid phosphors. The fabricated white LEDs showed warm white light with acceptable CIE chromaticity coordinate variation from (0.343, 0.255) at 20 mA to (0.335, 0.250) at 50 mA. The addition of CdSe/ZnS NCs contributed to the extension of white light spectrum by supplement of the red region. The color rendering index was largely enhanced from 41.7 to 79.7 compared to the Sr0.95Zn0.05Se:Eu2+ based phosphors white LED.

Inherited MUTYH mutations cause elevated somatic mutation rates and distinctive mutational signatures in normal human cells.

Cellular DNA damage caused by reactive oxygen species is repaired by the base excision repair (BER) pathway which includes the DNA glycosylase MUTYH. Inherited biallelic MUTYH mutations cause predisposition to colorectal adenomas and carcinoma. However, the mechanistic progression from germline MUTYH mutations to MUTYH-Associated Polyposis (MAP) is incompletely understood. Here, we sequence normal tissue DNAs from 10 individuals with MAP. Somatic base substitution mutation rates in intestinal epithelial cells were elevated 2 to 4-fold in all individuals, except for one showing a 31-fold increase, and were also increased in other tissues. The increased mutation burdens were of multiple mutational signatures characterised by C > A changes. Different mutation rates and signatures between individuals are likely due to different MUTYH mutations or additional inherited mutations in other BER pathway genes. The elevated base substitution rate in normal cells likely accounts for the predisposition to neoplasia in MAP. Despite ubiquitously elevated mutation rates, individuals with MAP do not display overt evidence of premature ageing. Thus, accumulation of somatic mutations may not be sufficient to cause the global organismal functional decline of ageing.