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Theses reviewed in this issue include “Engineering Efficient and Safe In Situ Genome Regulation via CRISPR-Cas9 for Enabling Gene Therapies,” “Investigating the Mechanism of LINE-1 Retrotranspositi...

Theses reviewed in this issue include "Alpha-Synuclein Oligomers: Cellular Mechanisms and Aspects of Antibody Treatment," "Cx3cr1/cx3cl1 Axis Drives the Migration and Maturation of Oligodendroglia in the Central Nervous System," "Genome-Wide Expression Profiling of Human Circulating Monocytes and Macrophages Identifies Diagnostic and Prognostic Signatures for Cancer Outcomes," "Lysosomal Oxidation of Low Density Lipoproteins," and "The Senescence-Associated Secretory Phenotype Induced by ID1-p16 Axis Contributes to Sorafenib Resistance in Hepatocellular Carcinoma."

Theses reviewed in this issue include Characterization and Modeling of Metabolic Stress Responses in Cellular Aging; Engineering Immunity: Enhancing T Cell Vaccines and Combination Immunotherapies for the Treatment of Cancer; Extracellular Inflammatory Signaling from Dysfunctional Telomeres; High-Throughput Microfluidic Labyrinth for the Label-Free Isolation of circulating tumor cells for Single-Cell Gene Expression Profiling; Oxygen Nanobubbles for Ultrasound-guided Targeting of Cancer Hypoxia; and The Eye as a Window to the Alzheimer's Disease Brain.

Theses reviewed in this issue include "Computational Pathology for Quantifying Spatial Heterogeneity in Digital Images of Tissue Sections from Solid Tumors," "Molecular Analysis of Amyotrophic Lateral Sclerosis and Frontotemporal Degeneration Brain Tissue Identifies Disease Mechanisms Associated with Repetitive DNA Elements," "Neuroprotective Potential of the N-Terminal Beta Amyloid Peptide Fragment in the Neurodegeneration, Synaptic Dysfunction and Memory Deficits in Models of Alzheimer's Disease," "pH-triggered Self-Assembly of a PEGylated Peptide Amphiphilic Contrast Agent," "Quantitative Approaches for Profiling the T Cell Receptor Repertoire in Human Tissues," and "Regulation and Repair of Neural Stem Cells and the Neurogenic Niche."

Theses reviewed in this issue include "Bowties, Barcodes, and DNA Origami: A Novel Approach for Paired-Chain Immune Receptor Repertoire Analysis," "Development of Canine Chimeric Antigen Receptor T Cell Therapy for TreatmentTranslation," "Endocytic Vesicle Rupture in the Pathogenesis and Propagation of Neurodegenerative Proteinopathies," "Exploring Mechanisms of Metastasis Suppression in Metastatic Melanoma," "Polymer and Nucleic Acid Self-Assemblies: Properties and Applications at the Biological Interface," and "Towards a Scalable, Biomimetic, Antibacterial Coating."

Can memory be retained after cryopreservation? Our research has attempted to answer this long-standing question by using the nematode worm Caenorhabditis elegans, a well-known model organism for biological research that has generated revolutionary findings but has not been tested for memory retention after cryopreservation. Our study's goal was to test C. elegans' memory recall after vitrification and reviving. Using a method of sensory imprinting in the young C. elegans, we establish that learning acquired through olfactory cues shapes the animal's behavior and the learning is retained at the adult stage after vitrification. Our research method included olfactory imprinting with the chemical benzaldehyde (C6H5CHO) for phase-sense olfactory imprinting at the L1 stage, the fast-cooling SafeSpeed method for vitrification at the L2 stage, reviving, and a chemotaxis assay for testing memory retention of learning at the adult stage. Our results in testing memory retention after cryopreservation show that the mechanisms that regulate the odorant imprinting (a form of long-term memory) in C. elegans have not been modified by the process of vitrification or by slow freezing.

Theses reviewed in this issue include "Atrophied Thymus, a Tumor Reservoir for Harboring Melanoma Cells," "Evolutionary Adaptations in Developmental Signaling Pathways Underlie Regenerative Scar-Free Wound Repair in African Spiny Mouse (Genus Acomys)," "Integrated Immunoassays on Paper/Polymer Hybrid Microfluidic Devices for Low-Cost Detection of Disease Biomarkers," "RNA Regulation in the Nervous System: CircRNA Expression Changes During Aging, and Function of the Calm1 Extended 3' UTR Isoform," "The Role of Amylin in Alzheimer's Disease," and "Therapeutic Monoclonal Antibodies to Detect and Halt ATTR Cardiac Amyloidosis and Neuropathy."

Since Szilard's seminal 1959 article, the role of accumulating nuclear DNA (nDNA) damage – whether as mutations, i.e. changes to sequence, or as epimutations, i.e. adventitious but persistent alterations to methylation and other decorations of nDNA and histones – has been widely touted as likely to contribute substantially to the aging process throughout the animal kingdom. Such damage certainly accumulates with age and is central to one of the most prevalent age-related causes of death in mammals, namely cancer. However, its role in contributing to the rates of other aspects of aging is less clear. Here I argue that, in animals prone to cancer, evolutionary pressure to postpone cancer will drive the fidelity of nDNA maintenance and repair to a level greatly exceeding that needed to prevent nDNA damage from reaching levels during a normal lifetime that are pathogenic other than via cancer or, possibly, apoptosis resistance. I term this the “protagonistic pleiotropy of chromosomal damage” (PPCD) hypothesis, because this interaction of cancer-related and -unrelated damage is the converse of the well-known “antagonistic pleiotropy” phenomenon. I then consider a selection of recent data on the rate of accumulation of nDNA damage in the context of this hypothesis, and conclude that all presently available evidence is consistent with it. If this conclusion is correct, the implications for the feasibility of greatly postponing mammalian (and eventually human) aging and age-related pathology are far-reaching.

Recent studies have demonstrated that transgenic mice with an increased rate of somatic point mutations in mitochondrial DNA (mtDNA mutator mice) display a premature aging phenotype reminiscent of human aging. These results are widely interpreted as implying that mtDNA mutations may be a central mechanism in mammalian aging. However, the levels of mutations in the mutator mice typically are more than an order of magnitude higher than typical levels in aged humans. Furthermore, most of the aging-like features are not specific to the mtDNA mutator mice, but are shared with several other premature aging mouse models, where no mtDNA mutations are involved. We conclude that, although mtDNA mutator mouse is a very useful model for studies of phenotypes associated with mtDNA mutations, the aging-like phenotypes of the mouse do not imply that mtDNA mutations are necessarily involved in natural mammalian aging. On the other hand, the fact that point mutations in aged human tissues are much less abundant than those causing premature aging in mutator mice does not mean that mtDNA mutations are not involved in human aging. Thus, mtDNA mutations may indeed be relevant to human aging, but they probably differ by origin, type, distribution, and spectra of affected tissues from those observed in mutator mice.

Several major diseases of old age, including atherosclerosis, macular degeneration and neurodegenerative diseases are associated with the intracellular accumulation of substances that impair cellular function and viability. Moreover, the accumulation of lipofuscin, a substance that may have similarly deleterious effects, is one of the most universal markers of aging in postmitotic cells. Reversing this accumulation may thus be valuable, but has proven challenging, doubtless because substances resistant to cellular catabolism are inherently hard to degrade. We suggest a radically new approach: augmenting humans' natural catabolic machinery with microbial enzymes. Many recalcitrant organic molecules are naturally degraded in the soil. Since the soil in certain environments - graveyards, for example - is enriched in human remains but does not accumulate these substances, it presumably harbours microbes that degrade them. The enzymes responsible could be identified and engineered to metabolise these substances in vivo. Here, we survey a range of such substances, their putative roles in age-related diseases and the possible benefits of their removal. We discuss how microbes capable of degrading them can be isolated, characterised and their relevant enzymes engineered for this purpose and ways to avoid potential side-effects.

The vicious cycle theory postulates that typical mitochondrial DNA (mtDNA) mutations cause their host mitochondria to generate more superoxide and other reactive oxygen species (ROS) than do normal mitochondria, thereby promoting the occurrence of additional mtDNA mutations at an ever-accelerating rate. However, nearly all the loss-of-function mtDNA mutations seen in vivo are large deletions, which (as the original statement of the theory indeed noted, though this has been widely overlooked) should not trigger a vicious cycle because they will prevent the assembly of the potentially superoxide-generating enzyme complexes. Consistent with this is the observation that each cell exhibiting loss of mtDNA-encoded function in vivo contains copies of a single, evidently clonally expanded, mutant mtDNA species, whereas the vicious cycle theory predicts a spectrum of mutant forms in each cell. Two recent papers, however, unveil a way in which mtDNA mutations could indeed promote ROS production of their host mitochondria. MtDNA mutations probably shift the intramitochondrial NAD(+)/NADH redox couple towards NADH, and this is now shown in vitro to cause ROS production by alpha-ketoglutarate dehydrogenase, an essential enzyme of the TCA cycle. This does not revive the vicious cycle theory, but it has complex implications for the two most plausible more recent theories, known as "survival of the slowest" and "crippled mitochondria." It may also prove to explain other recent observations in mitochondrially mutant cells in vivo.

Much research interest, and recently even commercial interest, has been predicated on the assumption that reasonably closely-related species – humans and mice, for example – should, in principle, respond to ageing-retarding interventions with an increase in maximum lifespan roughly proportional to their control lifespan (that without the intervention). Here, it is argued that the best-studied life-extending manipulations of mice are examples of a category that is highly unlikely to follow this rule, and more likely to exhibit only a similar absolute increase in maximum lifespan from one species to the next, independent of the species’ control lifespan. That category – reduction in dietary calories or in the organism’s ability to metabolize or sense them – is widely recognized to extend lifespan as an evolutionary adaptation to transient starvation in the wild, a situation which alters the organism’s optimal partitioning of resources between maintenance and reproduction. What has been generally overlooked is that the extent of the evolutionary pressure to maintain adaptability to a given duration of starvation varies with the frequency of that duration, something which is – certainly for terrestrial animals and less directly for others – determined principally by the weather. The pattern of starvation that the weather imposes is suggested here to be of a sort that will tend to cause all terrestrial animals, even those as far apart phylogenetically as nematodes and mice, to possess the ability to live a similar maximum absolute (rather than proportional) amount longer when food is short than when it is plentiful. This generalization is strikingly in line with available data, leading (given the increasing implausibility of further extending human mean but not maximum lifespan in the industrialized world) to the biomedically and commercially sobering conclusion that interventions which manipulate caloric intake or its sensing are unlikely ever to confer more than 2 or 3 years’ increase in human mean or maximum lifespan at the most.

The very low abundance of mitochondrial DNA (mtDNA) mutations in nearly all mammalian tissues even in old age has led most mitochondriologists to reject the idea that such mutations might have a causal role in aging, despite (1) the strong circumstantial (e. g., interspecies) evidence that they do have such a role, (2) the promulgation since 1998 of two detailed mechanisms whereby low levels of mtDNA mutations could be harmful, and (3) the report of a transgenic mouse with cardiomyopathy apparently caused by artificially high levels of mtDNA mutations in the heart. A recent report of a mouse with ubiquitously accelerated accumulation of mtDNA mutations and an array of phenotypes reminiscent of aging has abruptly overturned this consensus, with not only the authors but also many other expert commentators suggesting that the mtDNA mutation theory of aging has risen from the ashes. However, there are compelling reasons to doubt the relevance of this mouse to normal mammalian aging, and thus to seek further testing of specific mechanistic hypotheses for how mtDNA mutations could cause age-related dysfunction.

95 THE CONCEPT of therapeutic cloning, proposed by Gurdon and Colman,1 entails the synthesis of tissues or organs for transplantation into a patient starting from embryonic stem (ES) cells that are genetically identical to that patient, due to having been derived from an oocyte whose nucleus was replaced by one taken from the patient (a process termed somatic cell nuclear transfer, SCNT). It remains the leading proposal for exploiting stem cell technology in the clinic in a manner that avoids the severe drawback of immune rejection. However, it presently faces three formidable hurdles. One, our ability to manipulate the differentiation of ES cells along a desired lineage, is progressively being overcome. The others, unfortunately, are more sociological than technical and are thus, perhaps, even greater. These are (a) the profound ethical concerns voiced by highly influential policy-makers and (b) the inadequate supply of human oocytes to permit synthesis of autologous tissues and organs at a rate comparable with demand. Two recent papers2,3 would seem to cast doubt on the feasibility of the only currently available proposal for escaping both these latter problems. In this perspective, I will explain that proposal, the problem with it that is highlighted by these papers, and two possible ways to avoid that problem. First it is appropriate to mention two other proposals, each of which avoids one of the sociological issues mentioned above but not the other. The supply problem might in principle be met by isolating large numbers of oocytes from females who have died in infancy, since the infant ovary contains many times more oocytes than in adulthood. However, this seems likely to encounter even more ardent ethical opposition than the use of oocytes donated by consenting adults. Conversely, the ethical problem may in principle be met by our recently developed ability to differentiate ES cells into oocytes,4 but the limited availability and variety of the cell lines presently authorised for federally funded work is potentially a barrier to high-volume production of such oocytes. The proposal that meets both objections is to use oocytes from non-human species. The idea here is simple. A non-human oocyte is made of non-human proteins, but when its nucleus is replaced by a human one and development is begun, those non-human proteins are progressively diluted and degraded, so that the ES cells that are the source of the eventual tissue or organ are entirely human in composition. Well, almost entirely—but not, in this protocol, quite. The problem is that not all our proteins are encoded in the nucleus: a paltry 13 of them are encoded in the mitochondrial DNA. Paltry in number but not in function: these proteins are essential subunits of the respiratory chain, without which the cell cannot use oxy

Despite enormous effort, progress in reducing mortality from cancer remains modest. Can a true cancer "cure" ever be developed, given the vast versatility that tumors derive from their genomic instability? Here we consider the efficacy, feasibility, and safety of a therapy that, unlike any available or in development, could never be escaped by spontaneous changes of gene expression: the total elimination from the body of all genetic potential for telomere elongation, combined with stem cell therapies administered about once a decade to maintain proliferative tissues despite this handicap. We term this therapy WILT, for whole-body interdiction of lengthening of telomeres. We first argue that a whole-body gene-deletion approach, however bizarre it initially seems, is truly the only way to overcome the hypermutation that makes tumors so insidious. We then identify the key obstacles to developing such a therapy and conclude that, while some will probably be insurmountable for at least a decade, none is a clear-cut showstopper. Hence, given the absence of alternatives with comparable anticancer promise, we advocate working toward such a therapy.

The feasibility of reversing human aging within a matter of decades has traditionally been dismissed by all professional biogerontologists, on the grounds that not only is aging still poorly understood, but also many of those aspects that we do understand are not reversible by any current or foreseeable therapeutic regimen. This broad consensus has recently been challenged by the publication, by five respected experimentalists in diverse subfields of biogerontology together with three of the present authors, of an article (Ann NY Acad Sci 959, 452–462) whose conclusion was that all the key components of mammalian aging are indeed amenable to substantial reversal (not merely retardation) in mice, with technology that has a reasonable prospect of being developed within about a decade. Translation of that panel of interventions to humans who are already alive, within a few decades thereafter, was deemed potentially feasible (though it was not claimed to be likely). If the prospect of controlling human aging within the foreseeable future cannot be categorically rejected, then it becomes a matter of personal significance to most people presently alive. Consequently, we suggest that serious public debate on this subject is now warranted, and we survey here several of the biological, social and political issues relating to it. BioEssays 24:667–676, 2002. © 2002 Wiley Periodicals, Inc.

Aging is a three-stage process: metabolism, damage, and pathology. The biochemical processes that sustain life generate toxins as an intrinsic side effect. These toxins cause damage, of which a small proportion cannot be removed by any endogenous repair process and thus accumulates. This accumulating damage ultimately drives age-related degeneration. Interventions can be designed at all three stages. However, intervention in metabolism can only modestly postpone pathology, because production of toxins is so intrinsic a property of metabolic processes that greatly reducing that production would entail fundamental redesign of those processes. Similarly, intervention in pathology is a "losing battle" if the damage that drives it is accumulating unabated. By contrast, intervention to remove the accumulating damage would sever the link between metabolism and pathology, and so has the potential to postpone aging indefinitely. We survey the major categories of such damage and the ways in which, with current or foreseeable biotechnology, they could be reversed. Such ways exist in all cases, implying that indefinite postponement of aging--which we term "engineered negligible senescence"--may be within sight. Given the major demographic consequences if it came about, this possibility merits urgent debate.

HO2*, usually termed either hydroperoxyl radical or perhydroxyl radical, is the protonated form of superoxide; the protonation/deprotonation equilibrium exhibits a pK(a) of around 4.8. Consequently, about 0.3% of any superoxide present in the cytosol of a typical cell is in the protonated form. This ratio is rather accurately reflected by the published literature on the two species, as identified by a PubMed search; at the time of writing only 28 articles mention "HO2," "hydroperoxyl" or "perhydroxyl" in their titles, as against 9228 mentioning superoxide. Here it is argued that this correlation is not justifiable: that HO2*'s biological and biomedical importance far exceeds the attention it has received. Several key observations of recent years are reviewed that can be explained much more economically when the participation of HO2* is postulated. It is suggested that a more widespread appreciation of the possible role of HO2* in biological systems would be of considerable benefit to biomedical research.

Caloric restriction (CR) of laboratory rodents, which extends their maximum lifespan, only transiently reduces the specific metabolic rate of highly oxidative tissues. However, superoxide production by mitochondria of those tissues is greatly reduced by CR. This is probably a major contributor to the slowed aging seen in CR, but its mechanism is unknown. Here it is proposed that the major metabolic shift enabling reduced superoxide production is a diversion of much of the electron flux generated by glycolysis and the TCA cycle away from its usual destination, Complex I, and to the plasma membrane redox system. The cell's ATP synthesis capacity is thereby diminished, but so is its ATP demand, due to reduced turnover of the Na + /K + –ATPase. Direct tests of this hypothesis are proposed.

Mitochondrial DNA (mtDNA) mutations underlie many rare diseases and might also contribute to human ageing. Gene therapy is a tempting future possibility for intervening in mitochondriopathies. Expression of the 13 mtDNA-encoded proteins from nuclear transgenes (allotopic expression) might be the most effective gene-therapy strategy. Its only confirmed difficulty is the extreme hydrophobicity of these proteins, which prevents their import into mitochondria from the cytosol. Inteins (self-splicing 'protein introns') might offer a solution to this problem: their insertion into such transgenes could greatly reduce the encoded proteins' hydrophobicity, enabling import, with post-import excision restoring the natural amino acid sequence.

A series of studies over many years has conclusively disproved the hypothesis that longevity in warm-blooded animals (homeotherms) correlates with high levels of antioxidant enzymes: in fact, these...

Twenty years have passed since Kadenbach and Muller-Hocker first proposed that the age-related accumulation of mutant mitochondrial DNA is caused by its clonal expansion, rather than by a “vicious cycle” of de novo mutational events caused by the disruptive metabolic impact of prior ones. Proof that they were correct emerged rapidly (though recognition of this was much slower); however, the mechanism underlying this selective advantage remained obscure. Numerous hypotheses were advanced during the 1990s, but proved hard to test. A wealth of data has been published very recently that bears on this question. While these reports surely bring us closer to an understanding of this phenomenon, and thus probably to a better understanding of how it might be combated or even reversed, they currently raise more questions than they answer.

Most phenotypes of aging in vertebrates may be caused by a progressive decline in the ability of antioxidant defences to maintain cellular and systemic homeostasis. This is due both to a diminished efficacy of those defences and to an enhanced level of pro-oxidant toxicity; the imbalance between the two has been termed oxidative stress. However, the cause of this increasing imbalance remains obscure. This article proposes a mechanism by which spontaneously mutant mitochondrial DNA (mtDNA), despite being present only in very small quantities in the body, may be the main generator of oxidative stress. Mutant mtDNA is distributed very unevenly within a tissue: some cells apparently contain no wild-type mtDNA whatever. Those cells must rely on glycolysis for ATP production; furthermore, they require a system to stabilize their NAD+/NADH ratio. This can only be achieved by an efflux of electrons from the cell, most probably mediated by the plasma membrane oxidoreductase (PMOR). It is proposed that the required...

In this viewpoint, we describe the strategies for engineered negligible senescence (SENS) concept - a simple and appealing model for the design of therapeutic interventions able to meaningfully and persistently reverse the deleterious effects of aging. We go on to outline how current or foreseeable biotechnologies could feasibly be employed to repair every currently identified category of pathogenic damage that accumulates over a human lifespan. Then, briefly, we explain why this goal is not only ethically sound, but can in fact be considered to verge on an ethical obligation. Finally, we review recent progress in some key areas of the SENS platform, including proof-of-concept research sponsored by the SENS Foundation, a charity based in California.

The relative insensitivity of lifespan to environmental factors constitutes compelling evidence that the physiological decline associated with aging derives primarily from the accumulation of intrinsic molecular and cellular side-effects of metabolism. Here we model that accumulation starting from a biologically based interpretation of the way in which those side-effects interact. We first validate this model by showing that it very accurately reproduces the distribution of ages at death seen in typical populations that are well protected from age-independent causes of death. We then exploit the mechanistic basis of this model to explore the impact on lifespans of interventions that combat aging, with an emphasis on interventions that repair (rather than merely retard) the direct molecular or cellular consequences of metabolism and thus prevent them from accumulating to pathogenic levels. Our results strengthen the case that an indefinite extension of healthy and total life expectancy can be achieved by a plausible rate of progress in the development of such therapies, once a threshold level of efficacy of those therapies has been reached.

Various molecular and cellular alterations to our tissues accumulate throughout life as intrinsic side-effects of metabolism. These alterations are initially harmless, but some, which we may term "damage", are pathogenic when sufficiently abundant. The slowness of their accumulation explains why decline of tissue and organismal function generally does not appear until the age of 40 or older. Aging is thus best viewed as a two-part process in which metabolism causes accumulating damage and sufficiently abundant damage causes pathology. Hence, a promising approach to avoiding age-related pathology is periodically to repair the various types of damage and so maintain them at a sub-pathogenic level. Some examples of such types of damage are intracellular and others extracellular. Several types of intracellular damage are highly challenging--sophisticated cellular and genetic therapies will be needed to combat them, which are surely at least 20 years away and maybe much more. Extracellular damage, by contrast, generally appears more amenable to pharmaceutical repair which may be feasible in a shorter timeframe. In this article, the major types of age-related extracellular damage and promising avenues for their repair are reviewed.

It remains controversial why mitochondria and chloroplasts retain the genes encoding a small subset of their constituent proteins, despite the transfer of so many other genes to the nucleus. Two candidate obstacles to gene transfer, suggested long ago, are that the genetic code of some mitochondrial genomes differs from the standard nuclear code, such that a transferred gene would encode an incorrect amino acid sequence, and that the proteins most frequently encoded in mitochondria are generally very hydrophobic, which may impede their import after synthesis in the cytosol. More recently it has been suggested that both these interpretations suffer from serious "false positives" and "false negatives": genes that they predict should be readily transferred but which have never (or seldom) been, and genes whose transfer has occurred often or early, even though this is predicted to be very difficult. Here I consider the full known range of ostensibly problematic such genes, with particular reference to the sequences of events that could have led to their present location. I show that this detailed analysis of these cases reveals that they are in fact wholly consistent with the hypothesis that code disparity and hydrophobicity are much more powerful barriers to functional gene transfer than any other. The popularity of the contrary view has led to the search for other barriers that might retain genes in organelles even more powerfully than code disparity or hydrophobicity; one proposal, concerning the role of proteins in redox processes, has received widespread support. I conclude that this abandonment of the original explanations for the retention of organellar genomes has been premature. Several other, relatively minor, obstacles to gene transfer certainly exist, contributing to the retention of relatively many organellar genes in most lineages compared to animal mtDNA, but there is no evidence for obstacles as severe as code disparity or hydrophobicity. One corollary of this conclusion is that there is currently no reason to suppose that engineering nuclear versions of the remaining mammalian mitochondrial genes, a feat that may have widespread biomedical relevance, should require anything other than sequence alterations obviating code disparity and causing modest reductions in hydrophobicity without loss of enzymatic function.

Intraneuronal, largely proteinaceous aggregates accumulate in all major neurodegenerative disorders. Lysosomal degradation of proteinaceous and other material declines early in such diseases. This suggests that intraneuronal aggregates consist of material which is normally broken down in the lysosome and thus accumulates when lysosomal degradation fails. This is plausible even though those aggregates are generally non-lysosomal, because lysosomal uptake may be affected. Thus, restoring lysosomal function might eliminate them--and without increasing the concentration of the soluble monomers or oligomers of which they are formed. This approach is therefore unlikely to be harmful and may well be beneficial. How might lysosomes be rejuvenated? Since lysosomal dysfunction is likely to be caused by intralysosomal material that is resistant to lysosomal degradation, normal function might be recovered by augmenting that function to cause the toxin to be degraded. Here, I describe how such augmentation might be achieved with microbial enzymes. Soil microbes display astonishing catabolic diversity, something exploited for decades in the bioremediation industry. Environments enriched in human remains impose selective pressure on the microbial population to evolve the ability to degrade any recalcitrant, energy-rich human material. Thus, microbes may exist that can degrade these lysosomal toxins. If so, it should be possible to isolate the genes responsible and modify them for therapeutic activity in the mammalian lysosome.

Occasionally, experimental biologists obtain results which mystify them so deeply that the paradoxical nature of their finding is acknowledged in the paper reporting it. This constitutes a more-or-less explicit invitation to those who did not perform the experiments - and even those who do not perform experiments at all - to propose explanations that eluded the experimenter. A much more frequent scenario, however, is that the experimenter asserts confidently that his or her data can be explained by a particular model but are at odds with some other model. In such circumstances, it is often overlooked that the stated falsification of the latter model is error-prone: just as the mystified experimenter saw no explanation when in fact there is one, the other experimenter may see only one explanation of the data when there are two. The main reason this phenomenon is neglected is, of course, the fact that here the theoretician (or other experimenter) must take the initiative in critiquing a conclusion that, far from troubling the experimenter, may by the time of its publication be a cornerstone of his or her research program, so whose refutation may be decidedly unwelcome. For precisely this reason, such critiques - especially, perhaps, when they come from those who do not do bench work at all and thus have a complementary approach to the analysis of data - are fundamental to maximising the rate of progress in fields of biology that otherwise risk languishing in ever-better-studied cul-de-sacs for many years. Computational biology, including simulation, plays an especially important role in this, whereas its ability to contribute to biology in other ways is often less than its proponents claim. Here I discuss some representative examples of falsification-falsification, including a previously unpublished analysis of mitochondrial DNA population dynamics in cell culture, in the hope of stimulating more theoreticians - and perhaps also more experimentalists - to engage in it.

Lysosomal degradation of damaged macromolecules is imperfect: many cell types accumulate lysosomal aggregates with age. Some such deposits are known, or are strongly suspected, to cause age-related disorders such as atherosclerosis and neurodegeration. It is possible that they also influence the rate of aging in general. Lysosomal degradation involves extensive cooperation between the participating enzymes: each generates a substrate for others until breakdown of the target material to recyclable units (such as amino acids) is complete. Hence, the age-related accumulation of lysosomal aggregates might be markedly retarded, or even reversed, by introducing just a few bacterial or fungal enzymes –‘xenohydrolases' – that can degrade molecules that our natural machinery cannot. This article examines the feasibility and biomedical potential of such lysosomal enhancement as an approach to retarding or treating age-related physiological decline and disease.

A severe challenge to the idea that mitochondrial DNA mutations play a major role in the aging process in mammals is that clear loss-of-function mutations accumulate only to very low levels (under 1% of total) in almost any tissue, even by very old age. Their accumulation is punctate: some cells become nearly devoid of wild-type mitochondrial DNA and exhibit no activity for the partly mitochondrially encoded enzyme cytochrome c oxidase. Such cells accumulate in number with aging, suggesting that they survive indefinitely, which is itself paradoxical. The reductive hotspot hypothesis suggests that these cells adjust their metabolism to use plasma membrane electron transport as a substitute for the mitochondrial electron transport chain in the reoxidation of reduced dinucleotides, and that, like mitochondrial electron transport, this process is imperfect and generates superoxide as a side-effect. This superoxide, generated on the outside of the cell, can potentially initiate classical free radical chemistry including lipid peroxidation chain reactions in circulating material such as lipoproteins. These, in turn, can be toxic to mitochondrially nonmutant cells that import them to satisfy their cholesterol requirements. Thus, the relatively few cells that have lost oxidative phosphorylation capacity may be toxic to the rest of the body. In this minireview, recent results relevant to this hypothesis are surveyed and approaches to intervening in the proposed process are discussed.

The mitochondrial free radical theory of aging is seriously challenged by the finding that mutant mtDNA never becomes abundant in vivo, a result disputed only in experiments using novel PCR variants whose quantitative accuracy is widely doubted. However, evidence continues to mount that mitochondria are the crucial site of free radical damage in vivo, most notably that mice lacking the nonmitochondrial isoforms of superoxide dismutase are healthy. It is thus important to determine whether a low level of mutant mtDNA could have serious systemic effects. This possibility exists because of the observed mosaic distribution of mutant mtDNA: some cells (or muscle fiber segments) lack any aerobic respiration. Such cells are presumed to satisfy their ATP needs by glycolysis. In vitro, however, NADH recycling by transmembrane pyruvate/lactate exchange does not suffice: cells only survive if they can up-regulate the plasma membrane oxidoreductase (PMOR). The PMOR's physiological electron acceptor is unknown. It was proposed recently (de Grey, A. D. N. J. (1998) J. Anti-Aging Med. 1(1), 53–66) that a prominent in vivo acceptor from these mitochondrially mutant cells may be oxygen, forming extracellular superoxide. The mosaic (“hotspot”) distribution of this superoxide would limit its dismutation by extracellular superoxide dismutase; it may thus reduce transition metals leading to oxidation of circulating material, such as LDL. This would raise systemic oxidative stress, greatly amplifying the damage done by the originating mitochondrially mutant cells. This model, now known as the “reductive hotspot hypothesis,” has recently gained much indirect experimental support; several direct tests of it are also feasible.

A cornerstone of textbook bioenergetics is that oxidative ATP synthesis in mitochondria requires, in normal conditions of internal and external pH, a potential difference (delta psi) of well over 100 mV between the aqueous compartments that the energy-transducing membrane separates. Measurements of delta psi inferred from diffusion of membrane-permeant ions confirm this, but those using microelectrodes consistently find no such delta psi--a result ostensibly irreconcilable with the chemiosmotic theory. Transmembrane hydroxide transport necessarily accompanies mitochondrial ATP synthesis, due to the action of several carrier proteins; this nullifies some of the proton transport by the respiratory chain. Here, it is proposed that these carriers' structure causes the path of this "lost" proton flow to include a component perpendicular to the membrane but within the aqueous phases, so maintaining a steady-state proton-motive force between the water at each membrane surface and in the adjacent bulk medium. The conflicting measurements of delta psi are shown to be consistent with the response of this system to its chemical environment.

Comment on: Capparelli C, et al. Cell Cycle 2012; 11:2272-84 and Capparelli C, et al. Cell Cycle 2012; 11:2285-302.

Lord Kelvin, once President of the Royal Society, notoriously asserted in 1895 that “Heavier‐than‐air flying machines are impossible.” Ignoring such unenviable precedents, in this issue of EMBO reports , Warner and 27 other biogerontologists dismiss strategies for engineered negligible senescence (SENS) as ‘scientifically’ unrealistic (Warner et al , 2005). Like Kelvin, they forget that engineering—of which life extension will be an example, as all medicine is—differs profoundly from science in its goals, methods and skills. Illustrating this, Warner et al accuse me of “[t]reating arguments and proposals that are not backed up by scientific evidence as though they were scientific ideas”, but they are wrong in both fact and logic. Regarding logic, they stress my failure to note that no SENS intervention—in isolation—has ever been shown to extend any organism's lifespan. I do not recall Henry Ford alerting potential customers that the components of a car—in isolation—remain obstinately stationary when burning petrol is poured on them, nor do I recall his being castigated …

The biogerontologist David Sinclair and the bioethicist Leon Kass recently locked horns in a radio debate (http://www.theconnection.org/shows/2004/01/20040106_b_main.asp) on human life extension that was remarkable for one thing: on the key issue, Kass was right and Sinclair wrong. Sinclair suggested, as have other experts, including his mentor Lenny Guarente and the National Institute on Aging advisory council member Elizabeth Blackburn, that Kass and other bioconservatives are creating a false alarm about life extension, because only a modest (say, 30%) increase in human life span is achievable by biomedical intervention, whereas Kass's apprehensions concern extreme or indefinite life extension. Kass retorted that science isn't like that: modest success tends to place the bit between our teeth and can often result in advances far exceeding our expectations.​expectations.