Numerous genomic-based studies have provided insight to the physiological and evolutionary processes involved in developmental and environmental processes of model plants such as arabidopsis and rice. However, far fewer efforts have been attempted to use genomic resources to study physiological and evolutionary processes of weedy plants. Genomics-based tools such as extensive EST databases and microarrays have been developed for a limited number of weedy species, although application of information and resources developed for model plants and crops are possible and have been exploited. These tools have just begun to provide insights into the response of these weeds to herbivore and pathogen attack, survival of extreme environmental conditions, and interaction with crops. The potential of these tools to illuminate mechanisms controlling the traits that allow weeds to invade novel habitats, survive extreme environments, and that make weeds difficult to eradicate have potential for both improving crops and developing novel methods to control weeds. VariousDe novo whole genome sequencing is the holy grail of genomics for any organism, especially for non-model plants such as weeds [cis-acting regulatory sequences (non-protein coding sequences surrounding genes that often contain the information needed for turning the genes on and off). Whole genome sequencing also provides a detailed physical map of gene order and distance. However, the cost of such a project makes it outside the reach of most researchers studying weeds and other non-model plants. Fortunately, the advent of new technologies such as long read 454 Titanium platforms, Solexa paired end pyrosequencing, and SOLiD 3 can produce large amounts of sequence data for significantly less cost than traditional Sanger sequencing methods. For example, the Titanium 454 FLX technology produces read lengths greater than 500 bases and can also generate ~ up to 600 Mb of sequence data for under $8,000. The various paired end read technologies such as SOLEXA can produce up to 20 gigabases (Gb) of random 75 – 100 base sequences (enough to provide a 10X coverage of the ~2Gb leafy spurge genome) for under $400 per Gb. Even greater cost reductions are achieved by the SOLiD 3 technology that boasts the generation of a Gb for under $300. These reduce the cost of such endeavors and bring the possibility of at least partial genome sequencing within the grasp of weed scientists [de novo genome sequencing. as weeds . The datAlthough full genome sequencing is ideal, obtaining genomic sequences of gene-rich regions could provide a more economical approach to provide important regulatory sequences without wasting resources on sequencing vast stretches of repetitive DNA. There are several approaches that could be used to isolate gene-rich regions of the genome. These include preparing genomic libraries using bacterial strains that destroy hypermethylated DNA sequences . Most acSequencing of transcribed regions of the genome is the most common approach for developing genomics data and tools for non-model plants . This isCollections of sequenced cDNAs also allow the production of microarrays. Microarrays are glass slides on which thousands of individual cDNAs or fragments of cDNAs (referred to as probes) are spotted in a grid pattern . FluoresIn cases where whole genome sequences are lacking, high density genetic and physical maps compliment and rely on other genomic resources. Simple sequence repeats (SSRs), microsatilites, amplified fragment length polymorphisms (ALFPs), and inter-simple sequence repeats (ISSRs) are polymerase chain reaction (PCR)- based genetic markers that can be developed without prior knowledge of the genomic sequences, and have been a mainstay for researchers working in non-model plants and weeds . The dev2.2.1.Oryza ssp.), wild sunflowers (Helianthus ssp.), and perennial ryegrass (Lolium perenne)) have close crop relatives. Amaranthus ssp., are related to grain amaranth (Amaranthus caudatus) which is a minor crop species in Latin America and was an important crop in the pre-columbian period [Euphorbia esua), dodder (Cuscuta ssp.), starthistle , and spotted knapweed  do not have close crop relatives. The use of genomic information for studies in weed science is still in its infancy in most cases. There are currently eight weeds with well developed genomic resources and significant research efforts. Three of these  [th of them. The experiment also generated sequence data for nearly the entire chloroplast genome of this species. Consequently numerous markers, and candidate genes, including genes for herbicide resistance, are now available for waterhemp. The information generated by this pilot study should augment the usefulness of a BAC library resources developed for a related grain amaranth species [As yet, the only weedy species for which significant amount of genomic sequence data is available is waterhemp (culatus) . 454 tec species . et al. 2002 [et al. 2005 [There are growing numbers of EST databases for numerous weeds including but not limited to leafy spurge, starthistle, and ryegrass, spotted knapweed, and the parasitic weed dodder -27. Thesal. 2002 , used thal. 2005 , used thSenecio ssps. [As noted above, EST databases can be used to develop high density microarrays to assess global changes in gene expression . However, leafy spurge is the only completely weedy species  for which high density microarrays have been developed . There aio ssps. . Arabidopsis thaliana) cDNA arrays and other genomic resources to follow changes in gene expression between shoots and mature leaves of wild oat and leafy spurge [Abutilon theophrasti) in response to crop competition [et al. 2008 [The earliest reports of using non-weed microarrays to study weed transcriptomes described the use of arabidopsis  have been produced [et al. 2007 [There are several well developed libraries available for several weedy species. BAC libraries have been produced for grain amaranth , and sevproduced  [Andersoal. 2007 , have pr2.3.Oryza sativa) has been fully sequenced, and can cross-hybridize to numerous weedy rice species, molecular markers for cultivated rice have been used identify and in some cases clone genes for weedy characteristics such as shattering [The use of high density maps and PCR-based markers has been extensively used in wild sunflowers to identify changes associated with interspecies hybridization , selectiattering , dormancattering , and seeattering . In addiattering -55 amongAmaranthus palmeri). Given the impact and ubiquity of CNV in animals development, physiology, and evolution, it seems likely that similar processes will be illuminated by genomic studies on these aspects of weeds as well. One of the surprising findings from animal genomics projects is that there appears to be various sizable sections of genomes that have undergone duplication. These duplications have been associated with specific diseases or sub-populations of species, or even in specific tissues within an organism . The dis3.Solanum tuberosum), corn (Zea maize), and model species such as arabidopsis and poplar (populus ssp.). Since many genes are reasonably conserved, it is often possible to use sequence data from model and crop species to amplify and clone candidate genes from non-model and weedy species. Such approaches have been highly successful in identifying herbicide resistance genes from numerous weedy species. For example, the target sites of action for ALS inhibiting herbicides was first cloned from arabidopsis [SHOOTMERISTEMLESS gene which is likely involved in production and growth of underground shoot buds on roots of leafy spurge was cloned by identifying conserved sequences from this gene and developing primers that would amplify a portion of the gene which was sued as a probe to isolate the full length gene form cDNA and genomic libraries [Although there are limited resources for genomics research in weedy species, there is exceptional potential for utilizing genomic resources developed for crops such as potato (bidopsis . These sibraries . Using aFundamental questions of concern to weed scientists such as the evolution and nature of invasiveness and weediness, response to competition and control measures, and origin of weed populations has until recently been unanswerable through conventional studies and techniques. However, the introduction of genomic-based tools and technologies could begin to illuminate the mechanisms and biological processes that make weeds so difficult to control. Plans are underway in several laboratories to use microarray analysis to compare transcriptomes of various invasive weeds in their native and introduced ranges and under common garden conditions to help identify gene expression differences related to invasiveness. Identification of the genes and physiological processes required for weediness and invasiveness should allow the development of methods to help control weeds. Additionally, it is possible that some of the genes that make weeds so competitive may provide targets or information needed to or modify crops and/or management practices to make crops more competitive and able to thrive in less than optimal growing conditions.


Polyphenolics are a large group of related substances. Many of these, in             fact much of that found in food, is composed of processing-derived substances             too complex for complete identification. Recent studies have suggested likely             benefits for diets high in polyphenols, particular in reducing heart disease mortality,             but other benefits have also been suggested. A consumer label based on             the major polyphenolic classes is both manageable and fairly informative as             most foods do not contain all possible classes. Differences between class             member can be significant, but data on individual substances is impractical             and no data is certainly less informative. Equivalency scales may be useful but             may skew content of many foods towards the high-equivalency substances, even             while the full beneficial effects of each individual substance is poorly described.


Urinary retention (UR) can be defined as inability to achieve complete bladder emptying by voluntary micturition, and categorized as acute UR, chronic UR or incomplete bladder emptying. UR is common in elderly men but symptomatic UR is unusual in women. The epidemiology of female UR is not well documented. There are numerous causes now recognized in women, broadly categorized as infective, pharmacological, neurological, anatomical, myopathic and functional; labeling symptoms as having a “psychogenic basis” should be avoided. Detrusor failure is often an underlying factor that complicates interpretation. Initial management includes bladder drainage (intermittent or indwelling catheterization) if the woman is symptomatic or at risk of complications, and correcting likely causes. Investigations should be focused on identifying the underlying etiology and any reversible factor. A detailed history, general and pelvic examination are needed; urine dipstick analysis, routine microscopy and culture, and pelvic and renal ultrasound are suitable baseline investigations. Urodynamic tests are required in specific situations. Urethral dilatation has a limited role, but it should be considered if there is urethral stenosis. Definitive management requires correction of cause where possible and symptom management where no correctable cause is detected. Follow-up is needed for monitoring response to treatment, detection of complications and symptom control. Fowler’s syndrome is a specific group diagnosed on urethral sphincter electromyogram, representing a very challenging clinical scenario. Physiologically, voiding requires a coordinated (synergic) bladder contraction and outlet relaxation, with sustained detrusor contraction achieving complete bladder emptying. Clinical problems can give rise to voiding lower urinary tract symptoms (LUTS) including poor urinary stream, intermittent flow, terminal dribbling and post-micturition dribbling. Urinary det Qmax ) and the concurrent maximum flow rate (Qmax ) to derive the BOO index (BOOI).[For male patients, particularly in older age groups, bladder outlet obstruction (BOO) is comparatively common, usually as a consequence of benign prostate enlargement (BPE). Diagnostic criteria for male BOO have been agreed based on the relationship between the detrusor pressure at maximum flow (Px (BOOI). The samex (BOOI).For women, UR is a contrasting situation, resulting from a much more diverse set of conditions. Accordingly, epidemiological research is difficult and the natural history of the various underlying conditions is only minimally understood. The incidence of UR in women is not well documented. One Scandinavian study revealed an incidence of AUR in women of 7 per 100,000 population per year; the male to female ratio was 13:1. In generIn this review article we summarize what has been published on the subject relating to the anatomical basis, assessment and management of the female BOO, with particular emphasis on iatrogenic obstruction, postoperative urinary retention and Fowler’s syndrome.Although there are no known universally accepted or standardized criteria for BOO in women, several useful studies have examined the question. Diokno and colleagues defined BOO in women in 1984 on the basis of videourodynamic studies. They defNitti and colleagues derived criteria from evaluation of the videourodynamic studies of 261 women for non-neurogenic voiding function and defined BOO as radiographic evidence of obstruction between the bladder neck and the distal urethra in the presence of a sustained detrusor contraction of any magnitude, usually associated with reduced flow rate or delayed flow.[max ≤ 12 ml/sec in repeated free flow studies, combined with a sustained detrusor contraction and Pdet Qmax ≥ 20 cm H2O in a pressure flow study, or presence of obvious radiographic evidence of BOO with a sustained detrusor contraction of at least 20 cm of water and poor Qmax - regardless of free Qmax or inability of void with the transurethral catheter in place despite a sustained detrusor contraction as above. Their nomograms plotted parameters from two separate voids – the maximum flow rate from a free (uncatheterized) flow on the X-axis and the detrusor pressure at maximum flow measured during voiding cystometry on the Y-axis. Whilst use of two separate voids is counter-intuitive, the presence of a urodynamic catheter in the urethral lumen implicitly must alter the gauge of the outlet available for urine flow, potentially confounding the interpretation of the outlet gauge. This nomogram enables differentiation not only between obstructed and non-obstructed patients but also between various degrees  of BOO.Blaivas and Groutz derived a nomogram from studying 50 women with somewhat diverse etiologies, who were concluded to be obstructed on clinical grounds. Their stmax and Pdet Qmax, comparing women with anatomical outlet obstruction versus women with stress urinary incontinence.[max 15 ml/s or less and Pdet Qmax 20 cm H2 O or more are reasonable pressure-flow parameters to define BOO in women . These criteria were revised in subsequent publications.[92 O represented cutoff parameters with the highest sensitivity and specificity for BOO in women.[Chassange and colleagues derived the relationship between Qntinence. It was ccations.9 De FreitThe various approaches to diagnosing BOO in women were compared by Akikwala and colleagues, studying 91 patients, in whom obstruction was suspected clinically in 25 women. All the det.iso ).[A key issue in diagnosing BOO is the difficulty gauging the contractility of the bladder during voiding. Impairment of bladder contractility appears to be a feature of the ageing bladder and can et.iso ). Where thThe Detailed history, abdominal, pelvic and neurological examination should be carried out. Immediate management of AUR requires bladder decompression with catheterization, either indwelling or intermittent catheterization (IC). IC not only avoids potential morbidity with an indwelling catheter but also allows monitoring of return of voiding function, which is usually presaged by a reduction in PVR.22 Any urThe utility of flexible cystoscopy under local anesthesia is debatable, but in a minority of cases it can provide additional anatomical information, for example, presence of intraluminal foreign body. Many women undergo urethral dilatation as a part of management, though there is no evidence to support it.24 HoweveUR is a poorly-understood yet well-recognized complication postoperatively in both men and women. Contributing factors include traumatic instrumentation, bladder over-distention, reduced contractility of bladder, increased outlet resistance, nociceptive inhibitory effect, pharmaceutical influences, preexisting outlet pathology and decreased micturition reflex activity. Various studies have shown that specific types of anesthesia and analgesia can increase the risk of postoperative urinary retention. In a review of more than 3000 obstetric deliveries Olofsson and colleagues demonstrated that patients who received epidural anesthesia had an increased risk of UR, Another Many women have partial UR in the early stages after SUI surgery. Presumably, several factors will be involved, such as the anesthetic agents employed; local discomfort or the analgesic agents used to treat the discomfort; edema or hematoma formation; altered voiding dynamics as a consequence of outlet realignment. Voiding usually recovers comparatively swiftly, but close surveillance is needed to check resolution of symptoms and PVR, and ensure that progression to full AUR and emergency issues do not ensue.Full AUR is rare postoperatively, but needs intensive attention at the outset to ensure bladder overdistension injury does not occur. Timing of trial without catheter has to be individualized, dependent on resolution of identifiable remediable factors. If spontaneous voiding does not result, an early decision needs to be taken on management strategy. Delaying intervention beyond one month may result in a permanent impairment a-s a consequence of fibrotic in-growth into the tape interstices. Factors in the history comprise whether urinary retention is complete or partial and whether it is improving with time. Key features of past medical history should be re-evaluated. For example, any current or previous neurological problem can alter the neural control of the urinary tract, leading to temporary or permanent difficulty with voiding, unrelated to tape placement. It is also valuable to review the preoperative urodynamics to re-evaluate voiding function, and to review technical problems during the operative placement or subsequent complications, such as hematoma. Physica-l examination is crucial; for some women with over-tight mid-urethral sling placement it is possible to feel the mid-urethral compression during vaginal examination. In these patients tape incision has a realistic chance of restoring voiding. Less commonly, it is possible to feel the anterior vaginal wall drawn up behind the pubis, suggesting that incision of the tape alone is unlikely to restore voiding and that additional per-operative attention to address the abdominal retropubic component of the tape will be needed to re-establish voiding. Finally, when physical examination reveals that the anterior vaginal wall lies in an entirely undisturbed location, it can be anticipated that impaired voiding is not a consequence of the urethral compression by the tape; tape incision or excision is then unlikely to achieve re-establishment of voiding. In this last group it is appropriate to consider proceeding to formal voiding cystometry.Various publications have now described the outcomes of releasing mid-urethral tapes surgically, including the restoration of voiding and the recurrence of incontinence as a consequence. If proceeding to cut a mid-urethral tape, cystoscopy should be undertaken at the same time to ensure that no other potential tape-related complications have also arisen. Patients have to be evaluated on an individual basis, rather than simply proceeding to tape incision based purely on failure to achieve normal voiding within a certain time frame. Nonetheless, it is important to proceed in a timely manner if the assessment does suggest that the tape is causing the obstruction, since ongoing mesh fibrosis, potentially compounded by decompensation of detrusor function, may lead to permanent impairment if avoidable delay intervenes. Hong et Other forms of SUI have also been associated with BOO. Colposuspension can lead to urethral compression or distortion, particulSome forms of surgical management of refractory urgency urinary incontinence (UUI) or overactive bladder (OAB) aim to enhance urinary reservoir function by reducing bladder contractions during the storage phase. This can also impair bladder contraction needed for voiding. Thus, patients undergoing successful augmentation cystoplasty or detrusor myectomy will have a substantial PVR in a high proportion of cases.42 IntravFowler’s Syndrome affects young women after the menarche, who develop painless retention at high bladder volumes, often following apparently unconnected precipitating events, such as minor surgery. Often, hDiagnostic criteria include: UR of at least 1 liter on at least one occasion; exclusion of other causative factors; raised maximum urethral closure pressure on urethral pressure profilometry; increased sphincter volume on ultrasound or MRI assessment; and; a characteristic urethral sphincter EMG. Difficulties with IC can be profound—insertion of the catheter can be straightforward but then discomfort may develop, as if the sphincter were gripping the catheter, leading to consequent difficulty on catheter withdrawal. Flow rate patterns tend to be interrupted. Small volumes often are passed by micturition, leaving substantial PVR.The most specific diagnostic test for Fowler’s Syndrome is a urethral sphincter EMG (USEMG), which differs from the pelvic floor EMG generally used for neurourological patients. In USEMG, the EMG needle is placed to one side of midline in the anterior vaginal wall, at the mid-urethral point, and advanced on to the dorsal aspect of the urethra. The neurophysiologist undertaking the test has to pay special attention to the audio signal being generated by the EMG, which confirms successful entry into the sphincter zone. The diagnostic parameter for Fowler’s Syndrome is an audio signal likened to the sound of whale noises in the ocean. Pelvic floor EMG often is non-diagnostic in this patient group.Management of Fowler’s syndrome is specialized, and a sympathetic approach and consideration of psychological elements are essential. Strong efforts should be made to reduce the polypharmacy that many of these patients have, particularly attempting to discourage use of opiate drugs. For those patients manifesting the characteristic EMG signal who are unable to tolerate IC, the treatment of choice is sacral nerve stimulation (SNS), which caBladder outlet obstruction is uncommon in women. Careful evaluation including detailed history, physical examination and urodynamics is required to find out any reversible pathology; formal urodynamic criteria have been proposed, but no standardized approach has been achieved. IC may be necessary for the management of BOO. Surgical treatment in women should be approached with caution as it carries significant risk of injury to sphincter and incontinence, and intervention requires an individualized approach based on careful evaluation. Fowler’s syndrome is a specific group representing a very challenging clinical scenario.


Aging is not simply an accumulation of damage or inappropriatehigher-order signaling, though it does secondarily involveboth of these subsidiary mechanisms. Rather, aging occursbecause of the extensive absence of adaptive genomic informationrequired for survival to, and function at, later adult ages,due to the declining forces of natural selection during adultlife. This absence of information then secondarily leadsto misallocations and damage at every level of biologicalorganization. But the primary problem is a failure of adaptationat later ages. Contemporary proposals concerning means by whichhuman aging can be ended or cured which are based on simplesignaling or damage theories will thus reliably fail.Strategies based on reverse-engineering age-extended adaptationusing experimental evolution and genomics offer the prospectof systematically greater success. If I may begin on a personal note, it hastaken me more than 30 years to understand why the evolutionary biology of agingis not adequately understood by the majority of gerontologists, to say nothingof the overwhelming majority of the non-biologists who are interested in theproblem of aging. It's not that we evolutionists haven't tried. In my owncase, I have repeatedly tried to convey the evolutionary approach to thegerontological community, in both books and articles, across a variety oflevels and venues. During the last three decades, I have developed someunderstanding of the great conceptual gulf between gerontology and evolutionarybiology. In the last few years, as the evolutionary biology of aging has beenrevolutionized by a deeper understanding of the Hamiltonian foundations ofaging and late life , I have But I think I have now found a possible solution to this communicationproblem, a solution that might bridge the gulf between evolutionary biologistsand the gerontological community. This solution is the concern of the presentarticle; ideally it will help resolve a "two cultures" problem that hasafflicted, and indeed impaired, gerontology for more than a century. Inaddition, as a secondary issue, I believe that the explanatory breakthroughthat I offer here should help the gerontological community see the cogency ofthe intuitive disquiet that many of them feel about recent attempts to "end" or"cure" aging, such as the SENS proposal of de Grey . I will reduction in the adaptive genomic information requiredfor survival and reproduction at those ages. This reduction in genomicinformation then leads to innumerable and pervasive failures of physiologicaltuning at later ages, including dysfunctional allocative signaling andcumulative cell damage, among many other types of adaptive failure. Sinceadaptation involves the careful tuning of functions at every level, frommolecule to cell to tissue to organ to overall bodily integration, the failureof adaptation necessarily will take place at all of these levels aswell. This is what the absence of the required adaptive genomic informationnecessarily produces at later adult ages.Species that have distinguishable adults enjoy early adult healthbecause of adaptations built by natural selection acting over entire genomes,adaptations that are the cumulative product of many millions of years ofevolution building vast libraries of adaptive genomic information. Theseadaptations are fine-tuned according to the recent evolutionary histories ofpopulations of these species, where this fine-tuning involves thousands ofnucleotide base-pairs distributed across entire genomes with effects that aretypically both pleiotropic and epistatic across component adaptive functions.When forces of natural selection acting on the physiological processes thatunderlie adaptation progressively weaken at later adult ages, as they must inall organisms with ovigerous reproduction according to Hamiltonian theory,4, thereNonetheless, it is easy for evolution by naturalselection to build the adaptive genomic information-base required to sustainhealthspan radically. There are no absolute physiological barriers to it doingso. This is dramatically revealed by the following: (i) the non-aging speciesin which the forces of natural selection never fall; (ii) the non-aging germlines of all species that are not undergoing evolutionary genomic meltdown; and(iii) the ease with which experimental evolution can slow aging when the forcesof natural selection are manipulated in the laboratory. More accurately,these statements are respectively better rendered as follows: (i) adaptationis maintained at all ages in species in which the forces of natural selectionnever fall; (ii) germ lines are indefinitely sustainable in species that arenot undergoing evolutionary genomic meltdown; and (iii) the genomic informationrequired for prolonged age-specific adaptation is easily produced by outbredlaboratory populations in which the forces of natural selection areartificially sustained at high levels at later ages.I will now set about explaining theseideas step-by-step, in effect supplying a brief tutorial in adaptationalgenomics with particular relevance to the problem of aging.The chief way in which biologists first learn about the genetic basisof evolutionary change is with simple examples like the substitution of melanicalleles at pigmentation loci in the case of Lepidopteran industrial melanism.I have used this pedagogical cliché in an evolution textbook myself . ThisexDrosophila melanogaster, theselective mechanisms that underlie such polymorphism are often remarkablyobscure. That is, unlike the textbook cases of industrial melanism andsickle-cell anemia, the relationship between natural selection and the genomeis characteristically "many-to-many," with numerous loci and complex phenotypiceffects of selection being the norm. This is why the study of the geneticbasis of evolutionary change is so difficult .(In thes [vid. . Unfortuperiment , the levperiment . This seDrosophila agingwith the entire human genome give estimates of orthology that are quite high,well over 80% [Drosophila evolutionary genomics to humans in the case of aging. This may be becausethe many loci which must evolve in order to sustain adaptation at later agesare involved in common "housekeeping" functions, such as energetic metabolism -which has been extensively implicated in the evolution of slowed aging in fruitflies [The advent of whole-genome technologies, or"genomics," circa the year 2000 changed the situation substantially. Mostimportantly, it was realized that there was far more orthology among the genesof metazoan genomes than had been anticipated by evolutionary biologists,particularly within the segmented bilaterian group, which includes both insectsand mammals. Genome-wide studies comparing the loci involved in over 80% , suggestitflies , rather itflies -39.Drosophila, particularlybecause such organisms already exist and the physiological changes associatedwith their slowed aging have already been studied extensively.Naturally, any such genomic short-cut toreverse-engineering the evolution of slowed aging from fruit flies to humans isfraught with potential for error. Such evolutionarily deep orthologies aresure to supply incomplete information. The originally proposed evolutionarily slowed-agingmouse remains the best model organism for a project of this kind. But creatingit will be a demanding project, requiring years to produce fruitful results,and substantial resources ,39. For This review evidently proposes that both the scientific foundations ofgerontology and the systematic, rather than adventitious, slowing of humanaging would benefit from the use of evolutionary theory and experimentalevolution augmented with genomics. This is because aging is properly understoodas the fading out of adaptation with adult age. As evolutionary biologistshave been studying adaptation for 150 years, and because we have an extensivetool kit for such research , we evolNor should this be construed as an argument that gerontology can dowithout any of the skills that are deployed so resourcefully by mainstreamgerontologists. Evolutionary biologists have a long history, indeed as old asCharles Darwin's original publications, of making use of all elements ofbiology, both as sources of information and as sources of useful tools for theirown experiments. Evolutionary research on aging has been no exception to thisoverall pattern.But evolutionists are generally united in their impatience withgerontological theories that are incompatible with evolutionary theory. And wechiefly feel scorn toward "anti-aging medicine" that is based on presumptionsprecisely contrary to evolutionary biology, just as we feel towardcreationism. We offer the formal foundation necessary for mathematicallysorting out much gerontological theorizing. We can supply useful experimentalstrategies from our longstanding tradition of studying adaptation, and we candemonstrably produce model organisms with extended healthspan at will. We feelthat gerontology can prosper if it makes use of these substantive contributions.I hope that I have managed to explain to a few more gerontologists whythey should make material use of evolutionary thinking in their theoretical andexperimental research. It provides concrete foundations, not windowdressings.


The anticipated biological and clinical utility of biomarkers has attracted significant interest recently. Aging and early cancer detection represent areas active in the search for predictive and prognostic biomarkers. While applications differ, overlapping biological features, analytical technologies and specific biomarker analytes bear comparison. Mitochondrial DNA (mtDNA) as a biomarker in both biological models has been evaluated. However, it remains unclear whether mtDNA changes in aging and cancer represent biological relationships that are causal, incidental, or a combination of both. This article focuses on evaluation of mtDNA-based biomarkers, emerging strategies for quantitating mtDNA admixtures, and how current understanding of mtDNA in aging and cancer evolves with introduction of new technologies. Whether for cancer or aging, lessons from mtDNA based biomarker evaluations are several. Biological systems are inherently dynamic and heterogeneous. Detection limits for mtDNA sequencing technologies differ among methods for low-level DNA sequence admixtures in healthy and diseased states. Performance metrics of analytical mtDNA technology should be validated prior to application in heterogeneous biologically-based systems. Critical in evaluating biomarker performance is the ability to distinguish measurement system variance from inherent biological variance, because it is within the latter that background healthy variability as well as high-value, disease-specific information reside. Biomarker discussions have dominated applied clinical research programs in recent years with the promise of significant clinical utility. Early cancer detectionIn the drug discovery domain, biomarker validation and qualification discussions have led to the “Fitness-For-Purpose” validation model.9As with prior clinical trial paradigms, the phases of discovery and validation in early cancer detection for screening biomarkers have been recognized.12Despite efforts in many laboratories, disappointingly few candidate biomarkers have been brought to clinical application for early detection of solid tumors. Discovery work narrows the universe of candidate biomarker analytes to those for which data support a link with outcomes in independent specimens. Subsequent application studies are focused on the performance metrics of candidate biomarkers and their measurement technologies for definitive evaluation and classification accuracy in a specified clinical application for a defined clinical purpose. In the final stage of biomarker evaluation, net benefit to patients is determined after utilizing biomarker assay results for clinical intervention.At biomarker discovery phase, challenges include discovering and quantitating valuable analytes within dynamic and biologically complex matrices such as blood, urine, and sputum. Further challenges include the paucity of biomarker measurement data in healthy populations , and the related issue of appropriate specimen controls. Preanalytical challenges include instability of bioanalytes, RNA and serum proteins for example, and measurements in common archival specimen formats such as formalin-fixed, paraffin embedded (FFPE) tissue blocks. In practice, validation and qualification efforts suffer if the dimensions of the bioanalyte space, normal population variation and bioanalyte stabilities are not addressed fully within the protocols at both the discovery and application phase.Study design and bias have also been problematic for cancer biomarker validation and qualification. This is illustrated in recent prostate cancer early detection proteomics with surface enhanced laser desorption-ionization based mass spectrometry (SELDI-MS), where biased specimen collection and storage has hampered progress.Over the past two decades, the research community targeting aging has also sought predictive and diagnostic biomarkers for physiological aging and age-linked diseases.23Although there are a few functional human age-related phenotypes on which metrics might be based , there are still many unknowns in human aging phenotypes and how these compare with animal models.In cancer biomarker work, the goal is to define the precision and reproducibility with which a measured analyte serves a useful clinical function .Sir2 and eliciting a CR-like physiological change.Sir2 ortholog) may repress repetitive DNA and genes, and re-localizes to DNA breaks in a manner reminiscent of the yeast system.Among the most promising interventions in aging has been dietary caloric restriction (CR). Caloric restriction has reproducible, favorable effects on lifespan and morbidity in a number of metazoan systems.Challenges arising from normal individual biological variation and the difficulties in understanding the relative contribution of aging to disease processes have been debated for years by biologists and gerontologists seeking validated biomarkers of physiological aging.8Clearly, in addition to preanalytical processing,33Since initial searches for aging biomarkers, attention has now turned to high-throughput and rapid, technology-based strategies for revisiting the discovery and validation of biomarkers of aging with new strategies based on aging phenotypes that characterize age-linked functional and degenerative processes. In addition, recent progress in cancer biomarker validation study designSince the initial search for biomarkers in aging two decades ago,Thus, it is timely given these strides forward to revisit the discovery and validation process that incorporates high-throughput tools for discovering and validating panels of biomarkers, and how these might apply to the development of biomarkers for physiological aging, with special reference to mtDNA.Comprehensive biological analysis that borrows from the principles of systems engineering has been termed “systems biology”. In this approach to understanding biology, high-dimensional biological measurements are integrated with computational computer models to predict how perturbations in any part of the system will impact the whole. Applications in medicine, drug discovery and engineering have been reviewed.48Aging can be viewed as a system of metabolic or genetic pathways with branch points of importance at many levels of biological organization. The biological basis of cellular senescence and its relationship to organismal aging has been explored.49For example, the roles of specific pathways in aging have been reported, including the CDKN2a pathway,For decades, mitochondrial dysfunction and anomalies of oxidative phosphorylation (mutagenetic effects of reactive oxygen species (ROS) production, for example) in aging and cancer have been the focus of numerous studies. Although a mitochondrial theory of aging has emerged recently,The role of epigenetic events such as DNA methylation studies in various nuclear genes in cancer64Sir2 gene) may be involved in chromatin organization while inhibiting the initiation of DNA replication, and may have a significant role in the biology of longevity and aging.Studies of other epigenetic changes such as histone acetylation status, have revealed that the mouse SIRT1 gene  that differ from the reference mtDNA sequence,Acute needs for mtDNA analysis include high-throughput, deep sequencing, resolution of mtDNA sequence and variants, amounts and heterogeneity at the single-cell level of resolution. A better understanding of normal mtDNA variability as a function of age, tissue type and nuclear genotype should be developed. To differing degrees, there has been recent technical and scientific progress in each area, although a comprehensive picture of the biology of normal mtDNA and its relationships to disease processes is still emerging.Why are mtDNA copy number determinations important, and how does the biology of copy number control differ when nuclear genes and mitochondrial genes are compared? To evaluate copy number and sequence changes in disease processes, variability in normal human mtDNA is important to establish, especially as reflected in the design of experimental controls.Most metazoan organisms including human, are represented by haploid and diploid genomic phases that, in terms of genomic copy number, have been considered roughly comparable between genders for autosomal loci. In contrast, mtDNA copy numbers become highly asymmetrical when male and female gametogenesis and early zygote development are compared, with significantly higher and possibly exclusive reliance on maternally derived mtDNA species after fertilization.68Detailed human genomic resequencing has recently uncovered a surprisingly high incidence of nuclear genomic copy number variation (CNV) among phenotypically healthy individualsWhen considering mtDNA metrics as biomarkers for aging, it is appropriate to bear in mind which areas of mtDNA biology remain under active investigation and what facts have been established thus far. With more obscure genetic distribution mechanisms to daughter cells than nuclear chromosomal genes, mitochondria and their genomes are a superb illustration of systems biology interrelatedness at the level of the cell, and represent a highly integrated cellular organelle system with inherent as well as interactive functions. Mitochondria should be viewed both as discrete organelles each containing a genomic complement comprised of many mtDNAs, as well as a subsystem integral to broader cell functioning in critical cellular processes such as bioenergetics72Nuclear and mitochondrially-encoded genes both contribute protein components to mitochondrial function, with the vast majority of mitochondrial proteins arising from nuclear genes. Analysis combining mass spectrometry, GFP-tagging, and machine learning, has defined a compendium of 1098 genes and their expression across more than a dozen C57BL/6J strain mouse tissues to define the murine mitochondrial proteome (“mitochondriome”) at an unprecedented level of resolution.49O human somatic celll lines81Compared with nuclear genomic loci that follow Mendelian inheritance, normal mitochondrial gene copy variation represents a less tractable system for whole-animal or somatic cell genetic analysis. In the research laboratory, the availability of mutant ρAlthough much has been made of the high mutation rates and lack of DNA repair mechanisms within mitochondria in mature mammalian cells and tissues, the early stages of oogenesis appear to have mechanisms for restricting mtDNA genotype.nucleoid, an intra-mitochondrial particle consisting of a few defined proteins encoded by nuclear genes  as well as those associated with 2–10 mtDNA molecules that share spatial and temporal functions.83At the level of single cells, recent experiments emphasize that intracellular mtDNA populations are not randomly distributed within cells.Several independently developing areas of biological research support the idea of biological cross-talk between nuclear and mitochondrial genes. That nucleoid function is genetically and biologically regulated by nuclear genes, is implied by analysis of mtDNA heteroplasmy in centenariansIn mice, the presence of specific genetic controls and biases against intraspecific paternal mtDNA is also suggested by paternal mtDNA leakage at the FDrosophila in which mitochondrial genotype is varied while controlling for nuclear genotype, implies that mitochondrial genotype has significant effects on longevity of flies, and that the effects may be modulated by the nuclear genetic background.27In support of the biological idea that longevity may involve mitochondrial-nuclear genome cross-talk, recent experimental work in Thus, before concluding that mtDNA heteroplasmy and mutations in solid tumors are aging-related, disease-specific or normal variability, more work is needed on the normal course of heteroplasmy development and control among aging human individuals and in model animal systems, and its sequence spectrum among healthy tissues. Compared with nuclear genes, control over copy number variants among mtDNAs is not well established in metazoan species. At the very least, the rules of mtDNA copy number constraint are as yet not well understood for mammalian cells. These biological variabilities and uncertainties could compromise claims of mtDNA changes as biomarkers of aging or cancer.In order to evaluate abnormal mtDNA changes in aging or early cancer, a thorough understanding of normal variability range prior to comparison is critical. The degree to which biological as well as technical variables (measurement and protocol uncertainties) may be confounded, is illustrated by an ongoing debate among experts. These issues are highly relevant to two opposing views of biomarker discovery and validation.Among some clinicians doing translational work, the molecular identification and details of biomarkers associated with disease and physiological status, may be considered a lower priority than the biomarker’s utility for clinical associations. This point of view held sway in early serum proteomics studies in which a surface-enhanced laser desorption-ionization mass spectrometry (SELDI-MS) pattern was the measured biomarker. In early studies, patterns were validated as disease biomarkers prior to identifying the physical analyte or protein species represented.An opposing point of view held by molecular biologists is that the physical identity of the candidate biomarker analyte  is essential to establish improved and sensitive assays, should the initial discovery need a better analytical platform for widespread use. The results of SELDI screening of clinical sera would seem to argue in favor of establishing scientific and biological details on candidate analytes, and most laboratories have adopted peptide identification methods orthogonal to mass spectrometry to address this issue. This further emphasizes the importance of appropriate normal controls and a working knowledge of the dynamic range and concentration limits of normal bioanalyte values. Such lessons should also increase caution in the evaluation of candidate biomarkers based on analytes other than serum proteins.The confusion and controversy over the physical and chemical identity of biomarkers, have underscored the importance of establishing a molecular basis for biomarkers of all types, but should also bring attention to the previously under appreciated importance of rigorous standard operating procedures (SOPs) for specimen collection, preparation, storage and analysis of biomarker specimens. It is likely that optimization of specimen collection and biobanking will be necessary before the preanalytical variables are better understood, especially as the newest high-resolution, high-dimensional, high-throughput technologies come into clinical research use. Such experiences should inform the path forward as new candidate biomarkers are discovered and evaluated in different areas of application, whether it is early cancer detection, drug development or aging.The biology of mtDNA sequence, content, distribution and heteroplasmy in mammalian cells (and tissues) is complex, and the factors that control cell content and spectrum of mtDNA are not well understood. In addition, the adequacy of appropriate controls for cancer and aging studies remains under discussion because the background values for mtDNA copy number and sequence heterogeneity are for the most part incomplete, or unknown, in healthy human populations worldwide, and in different tissues within the same individuals.Given the biological complexity, it is of interest to review technology and platform performance metrics, and the proportion of measurement variance that might be attributed to the analytical tools and platforms used to develop the current picture of mtDNA mutation and heteroplasmy in normal aging and disease. The biology of existing mtDNA sequence and heteroplasmy data in mouse and man is difficult to parse from the point of view of technology, given performance differences in analytical DNA sequencing and quantitation technologies.Several levels of mtDNA analysis have contributed to the current understanding of mtDNA variability in human and mammalian model systems. A clear understanding of the biology of mtDNA is important because mtDNA analysis may constitute the basis of critical decisions with significant social impact such as paternity, legal culpability, and identification of human remains.99The biological variability of human mtDNA is measured against the revised Cambridge reference sequence (rCRS).104To add to the complexity of mtDNA sequence analysis and its interpretation, the genome also contains several hundred incomplete DNA copies of mtDNA sequence integrated into genomic nuclear DNA in humans and many other species. Such sequences have differing degrees of sequence homology with bona fide mtDNA.http://phenome.jax.org).113In model systems such as inbred mice,In addition to inter-individual mtDNA sequence at a single time point or age, many studies document mtDNA sequence changes with increasing age in healthy inbred mice,118Since mtDNA sequence varies normally among healthy individuals in human and murine populations, and within individuals from tissues to tissue, differences detected in association with disease states warrant attention to appropriate controls. For example, whether peripheral blood is the most biologically appropriate control for tissues like lung or bladder tumors might be further investigated.Despite data that micetwinkle locus (a nuclear gene encoding mtDNA helicase protein located in the mitochondrion) mutations result in mtDNA deletions and late-onset mitochondrial disease.mtDNA sequence changes found in various normal human postmortemEarly work identified point mutations in colorectal, bladder, head/neckRecent analysis of larger patient and healthy populations with MitoChips demonstrates that the extent of normal heteroplasmy and polymorphism in mtDNA has not been fully appreciated.114The technology of DNA sequencing and mutation detection is rapidly evolving. Methods such as denaturing gradient gel electrophoresis (DGGE)The physical reference mtDNA sequence and its revision130Intermediate in resolution are the resequencing methods such as MitoChip Versions 1 and 2113Thus, DNA sequencing methods characterized by high resolving power for low levels of admixture, as well as high accuracy nucleotide-level sequencing, are of great interest. The most promising are the so-call next generation (NGS) sequencing methods exemplified by the 454 (Roche), Gene Analyzer II (Solexa/Illumina), ABI and Helicos Systems. The power of the 454 system has been demonstrated in recent success with complete sequencing of the degraded mtDNA sequence of 38,000 year old human Neandertal tissues.As DNA sequencing methods move toward lower cost coupled with high throughput, increased resolution and improved limits of quantitation, what is accepted as the normal biological situation for the presence and degree of mtDNA heteroplasmy, may change and evolve with improvements in data. Before disease-related changes in mtDNA sequence can be established, the normal biological background of mtDNA sequence variability must be established.Although not addressing the issue of mtDNA sequence change, measurement of the absolute number of mtDNA molecules in cells is also relevant to biomarker evaluation studies. Ensemble methods based on realtime PCR have been recently reported.118in situ hybridization. mtDNA methods utilizing DNA intercalating dyes are available, such as DAPI in fixed cellsin situ PCRin situ hybridization.Direct visualization of mtDNA nucleoids in cells by microscopy has been possible by detection with DNA dyes, and by a priori, it will be rather difficult to establish and interpret fundamental healthy biological variability. In the long run, it would make more sense and be more cost-effective to work out details through pilot studies prior to planning extensive analytical validations in clinical specimens, or clinical trials.Better data are needed on the breadth and depth of normal variation in mtDNA sequence and copy number as a function of normal aging and tissue types in mammalian systems. Until normal variation is determined, associations of mtDNA change in somatic cells with diseases will be difficult to evaluate with confidence. Running parallel, the limits of detection (LOD) and limits of quantitation (LOQ) for the technologies should be characterized on healthy specimens prior to disease biomarker studies. Without valid data on the analytical systems established Areas that would profit from such technology validation and normal biological studies include early cancer detection. In addition, mtDNA quantity is an important metric in the clinical management of AIDs patientsThe importance of good analytical validation and physical standards,Each of these considerations comes into play in designing better biomarker pilot studies to verify adequate preanalytical processing and technology performance metrics early when studies are of limited scope and cost.In aging research, useful composite biomarkers or panels might be valuable if one could apply them as predictive or diagnostic markers. Another intended use for such a panel would be to assign biological age, and finally, a further goal might be to predict rate of aging and overall longevity. Obviously, longevity and disease risk are not wholly independent endpoints.Once the purpose or application of the biomarker measurement is defined, the analytical validation of panels consisting of multiple biomarkers of the same type (multiplex biomarker panel), or panels of biomarkers of various types , is the next step. It is clear there will be few if any single-analyte biomarkers for early cancer detection of solid tumors with the possible exception of specific, rare Mendelian mutations. In drug development work, increasing attention has been paid to the value of biomarkers that are theranostics (therapeutic + diagnostic), or targets in which the druggable target and diagnostic are the same. With the fairly recent realization that single biomarkers may not prove effective, the metrology of how multiple biomarkers can best be applied, is emerging. How such panels will be analytically validated and evaluated to obtain the optimal “fitness-for-use” with a minimum of independent biomarkers or assays, is a novel area of biomarker metrology with which there is limited experience at present. In parallel with novel candidate biomarkers emerging in many areas such as microRNA diagnostics,


We reviewed some of our initial insights about the process of upper-limb behavioral recovery following stroke. Evidence to date indicates that intensity, task specificity, active engagement, and focusing training on motor coordination are key factors enabling efficacious recovery. On modeling, experience with over 400 stroke patients has suggested a working model of recovery similar to implicit motor learning. Ultimately, we plan to apply these insights in the development of customized training paradigms to enhance recovery. Rehabilitation robotics has begun to realize its promise that delivery of high-dosage, guided movement protocols will alter the impairment  phase of modern post-stroke therapy. However, the mechanisms of this motor performance enhancement remain unclear. Recovery, which occurs spontaneously and continues imperfectly, depends on a myriad of biological and social/economic factors: age, gender, physical and mental health, size and location of lesion, family and other support networks, insurance, income, and probably many more. A quantitative, scientific understanding of the mechanisms of the post-stroke recovery process is the key to improving the speed and ultimate level of recovery. By design, robotics provides a reliable, controllable, objective instrument platform from which to deliver high intensity therapy and to characterize recovery at the behavioral level. By controlling the amount of therapy and quantitative characterization of recovery, it will allow us to determine the optimal therapy for a particular patient's needs. It will also enable a richer set of therapies complementary to existing ones and novel cellular, electrophysiological and pharmacological interventions.We recently reviewed some initial insights about the process of upper-limb behavioral recovery following stroke that have emerged from our robotics work . EvidencAt the same time, we recognize that simplistic ideas based on motor adaptation and learning may be insufficient to describe the complex process of recovery after neurological injury. The strongest evidence to support this statement is the ubiquitous incidence of abnormal muscle tone, spasticity, and abnormal synergies during recovery. It seems tritely obvious that unimpaired adults learning a new motor skill do not have to contend with these difficulties, yet they are so common in recovery after neurological injury as to be characteristic of the process. These abnormalities warrant study in their own right. Our work to date has shown that the conventional clinical perception of abnormal synergies – that they are exhibited early in recovery and must be suppressed or "broken" if recovery is to gain momentum – may not stand up to close quantitative scrutiny. Our analysis indicates that synergies are not first expressed and then "broken" during recovery; instead, they are present throughout but with a "gain" or magnitude that varies as recovery proceeds ,9.Nevertheless, we acknowledge the appeal of motor learning as the basis of a theory of motor recovery, though it may need careful refinement to serve as a basis for designing new therapies. Here, we will attempt to refine the idea of motor recovery as a process of motor re-learning and to present a "working model" (admittedly speculative) of the process of neuro-recovery. We will provide neither an overview of our different robots nor a discussion of the multitude of robotic devices designed elsewhere following our pioneering robotic module, MIT-Manus. Comparisons of alternative robotic design philosophies and summaries of past clinical results, including several meta-analyses, can be found elsewhere -14.From the outset we recognized that the successful development of rehabilitation robotics required a multi-disciplinary effort. We had to abandon the "comfort zone" of our academic elitism at engineering laboratories and engage with clinicians and patients at rehabilitation facilities. We recognized that we had to abandon our Ivory Towers and establish well-balanced multidisciplinary collaborations. In fact, we perceive that the single greatest weakness of the plethora of different therapeutic robot designs that have emerged recently – some quite ingenious and technically appealing – is the lack of a truly balanced multi-disciplinary team to establish objectively verified and clinically meaningful target requirements. A similar (though perhaps more recent) weakness is evident in several attempts to apply mathematical modeling and computational neuroscience to describe recovery and prescribe treatment. For example, one very ingenious suggestion is to capitalize on the after-effects of adaptation to novel mechanical environments so as to induce beneficial changes in patients' motor behavior . HoweverSince 1994, we have had the privilege of spending significant time with over 400 stroke patients at multiple institutions. Patients would describe their crisp, clear understanding of the goals of training and the frustrations of being unable to execute them. They understood the "games" used for robotic therapy and their simple objectives, but seethed with frustration and anger at their inability to perform the appropriate movements to accomplish the goals. Unless the impact of stroke is resolved within the initial 24 hours, impairments linger. Yet they appear to be amenable to the acquisition of new skills without awareness of the learned information over repetitive trials. The insightful self-assessment of stroke by Brodal should be required reading for all researchers interested in stroke recovery . Quoted implicit motor learning, in which subjects understand the goals but are unable to comprehend how to activate their muscles to achieve those goals. Our paper will focus on the latter.We believe this expert's insight can be translated into working models of motor recovery. First of all, his description entices further research into models fractionating motor control and how this may be deranged by stroke ,18-20 anIf human learning can be divided into so-called declarative and procedural forms, then declarative or explicit learning and memory refer to the acquisition and retrieval of information accompanied by awareness of the learned information and its influence. Explicit learning is most often put into practice through language functions . ProceduIn previous work, we reported on the integration of robotic technology with functional brain imaging to study whether the unskilled phase of procedural learning of a motor task (early learning) involves areas of the brain distinct from those involved in a more skilled learning phase of the task (late learning) in young healthy right-handed subjects ,29. PET We found during a right-handed task in young unimpaired subjects that early learning activated the right striatum and right parietal area, as well as the left parietal and primary sensory area, and that there was a deactivation of the left premotor area. As subjects became skilled at the motor task (late learning), the pattern of neural activity shifted to the cortico-cerebellar feedback loop, i.e., there was significant activation in the left premotor, left primary motor, and sensory areas, and in the right cerebellar cortex. These results support the notion of different stages of implicit motor learning (early and late implicit learning), occurring in an orderly fashion at different rates. Moreover, these findings indicate that the cortico-striatal loop plays a significant role during early implicit motor learning, whereas the cortico-cerebellar loop plays a significant role during late implicit motor learning . These cWe have assessed the competence of this working model to account for clinical experience with patients recovering from stroke. Here we will present a few selected pieces of data that appear to support the model. Our procedural motor leaning experiments performed with PET metabolic and blood flow information revealed that the cortico-striatal loop played a significant role during early learning and motor plan transition, while the motor execution areas played a significant role during late motor learning (cortico-cerebellar). If motor recovery has similar traits to implicit motor learning, then we speculate that patients with basal ganglia lesions would take longer to start the recovery process . Conversely, patients with lesions in the motor execution areas would recover more slowly during later phases . Of those patients with lesions in the motor execution areas, the smaller the number of structures affected, the better the outcome expected in the late recovery phases.These predictions do not speak to the ultimate potential of recovery but to the pattern of recovery. Intuitively one might expect that larger lesions would lead to slower recovery. However, Miyai and colleagues showed that, in fact, patients with smaller lesions confined to the basal ganglia (CS) have diminished response during the sub-acute rehabilitation period compared to patients with much larger lesions that involve cortical and subcortical territories (CS+) . Miyai s3, CS+ = 95.1 ± 25.2 cm3, p < 0.05). We found that early recovery shows a trend to progress at a slower pace for those with smaller lesions (CS) compared to those with larger lesions (CS+). While non-significant, our clear trend with this small sample size is inline with Miyai's very counter-intuitive result. To our knowledge, prior to his finding, the traditional wisdom had generally been that smaller lesion leads to better outcome. As we showed in Table For example, from our initial study delivering rehabilitation robotic therapy to 20 sub-acute patients, the comparison of outcome for 5 patients with corpus striatum lesions (CS) versus 6 patients with corpus striatum plus cortex (CS+) is shown in Table Our results are also consistent with our working model that during late recovery, lesions in the basal ganglia do not preempt improvement while the converse is true for lesions in the motor execution areas. Note in Table So far our working model justification attempted to establish temporal relationship between implicit motor learning in unimpaired young subjects with lesion foci, between early implicit motor learning with early recovery, and between late implicit motor learning and late recovery. Miyai has demonstrated that patients with large strokes on the middle cerebral artery territories can have quite distinct outcomes depending on whether the pre-motor region was spared or not [ and 44]44]]. Thi3) suggested a bimodal distribution, indicating two distinct classes of patients: one with lesion volumes smaller than 100 cm3 (N = 42) and another with lesions larger than 100 cm3 (N = 14). While an analysis of whether the differences in motor outcome might result from lesion volume alone was unrevealing, of those in the group of 42 patients with smaller lesion volume, who were exposed to an additional 1-hour of high-intensity, task specific robotic sensory-motor training outranked those not exposed to this kind of focused exercise [It is also important to emphasize again the impact of the intensity and task-specificity of robotic rehabilitation on recovery . For thi cm3 N =  and anotWe will conclude briefly discussing our selection of a motor learning model versus a motor adaptation model. Dipietro et al examined in persons with chronic impairment due to stroke whether untrained movements were also characterized by changes similar to trained movements . We enroThe results above should be viewed with appropriate caution, but they support an emerging understanding of motor recovery that provides hope to improve patient outcomes.Experience with over 400 stroke patients has suggested a working model of recovery similar to implicit motor learning. Most strokes preserve the patient's understanding of task goals, but leave an inability to perform the task – even simple tasks. As with implicit learning, recovery occurs without awareness of the learned information. Hence therapy might be more successful if it attempted to inform patients of their progress toward their goals and de-emphasized explicit explanations of the set of muscles or muscle groups that must be activated. While the results presented here are serendipitous in nature, we are testing in very severe to moderate strokes whether motor recovery indeed involves similar brain structures as in implicit motor learning by unimpaired subjects. Ultimately, we plan to apply this knowledge to the design of training paradigms to complement pharmaceutical agents and electrophysiological stimulation that enhance implicit motor learning, potentially opening new routes for greater rehabilitation success.H.I.K and N.H are co-inventors in MIT-held patents for the robotic devices used to treat patients in this work. They hold equity positions in Interactive Motion Technologies, Inc., the company that manufactures this type of technology under license to MIT.This manuscript was drafted by HIK with editorial assistance of BV and NH and it represents shared views amassed during 15 years of close collaboration.


On a global basis, both potent vaccine efficacy and high vaccine coverage are necessary to control and eliminate vaccine-preventable diseases. Emerging evidence from animal and human studies suggest that neglected tropical diseases (NTDs) significantly impair response to standard childhood immunizations. A review of efficacy and effectiveness studies of vaccination among individuals with chronic parasitic infections was conducted, using PUBMED database searches and analysis of data from the authors' published and unpublished studies. Both animal models and human studies suggest that chronic trematode, nematode, and protozoan infections can result in decreased vaccine efficacy. Among pregnant women, who in developing countries are often infected with multiple parasites, soluble parasite antigens have been shown to cross the placenta and prime or tolerize fetal immune responses. As a result, antenatal infections can have a significant impact on later vaccine responses. Acquired childhood parasitic infections, most commonly malaria, can also affect subsequent immune response to vaccination. Additional data suggest that antiparasite therapy can improve the effectiveness of several human vaccines. Emerging evidence demonstrates that both antenatal and childhood parasitic infections alter levels of protective immune response to routine vaccinations. Successful antiparasite treatment may prevent immunomodulation caused by parasitic antigens during pregnancy and early childhood and may improve vaccine efficacy. Future research should highlight the varied effects that different parasites  can have on human vaccine-related immunity. To optimize vaccine effectiveness in developing countries, better control of chronic NTDs may prove imperative. Since the inception of the Expanded Program on Immunization (EPI) in 1974, many global partners, including the World Health Organization, United Nations Children's Fund, and the Gates Foundation, have joined to support mass global immunization projects that have resulted in a significant drop in child mortality worldwide Vaccines are among the most cost-effective health interventions available for the prevention of life-threatening and disabling infectious diseases. Even so, the overall effectiveness of vaccine strategies requires both adequate coverage among vulnerable populations and induction of a satisfactory protective immune response in each susceptible individual. Although extensive resources are now being committed to improve global childhood vaccination coverage, in developing nations the response to standard vaccination often remains suboptimal The reduced effectiveness of vaccination programs in developing communities often has been blamed on cold-chain lapses and a lack of support infrastructure Several vaccination studies have shown that children from areas of sub-Saharan Africa are less responsive to standard childhood vaccines than children from developed countries. These include vaccine trials of the antituberculosis vaccine BCG  What are the implications of this phenomenon? Recent outbreaks of polio in Africa show us that our “control” of vaccine-preventable diseases is tenuous at best. A short-term failure in vaccine coverage resulted in the rapid and serious resurgence of a vaccine-preventable disease Failure to respond appropriately to vaccination is most often associated with a suite of poverty-related conditions, including both malnutrition and chronic infection. Clinical features of poverty include protein-calorie and micronutrient undernutrition and recurring exposure to parasites (protozoa and helminths) that independently contribute to chronic anemia and poor physical growth and development Plasmodium infections lead to decreased vaccine efficacy and an inability to ward off new infection The deleterious effects of polyparasitism on host immunity may help to explain the poor response to childhood vaccination that is seen in the developing world. Both animal and human studies indicate that parasitic infections can impair long-term responses to vaccination. In addition, multiple concurrent infections are likely to have additive or synergistic effects on immune responses. Studies in both human and animal models have shown that trematode, nematode, and Schistosoma mansoni infection and determined that BCG-vaccinated, schistosome-infected mice have significantly less vaccine-induced protection against virulent tuberculosis challenge than those without schistosome infection. In studies of humans with trematode infection, a decreased response to tetanus toxoid vaccination has been demonstrated in the presence of schistosomiasis, associated with significantly altered Th1- and Th2-type immune responses to tetanus toxoid in vitro Elias et al. Two studies performed in mice show that nematode infection impairs response to malaria vaccination and further illustrate that cure of parasitic infection before immunization, and not after, increases host vaccine response Haemophilus influenzae type b Chronic protozoan infections also have been proven to have harmful effects on vaccination response, with decreased levels of response to tetanus, “Imprinting” of the immune system during fetal development also may play a significant role in reduced vaccine effectiveness in parasite-endemic areas. In developing countries, women of child-bearing age are frequently infected with one or more parasites, as seen in our cohorts in coastal Kenya and others Successful treatment of pregnant mothers for parasitic infections may prevent sensitization to parasitic antigens during pregnancy and may improve vaccination outcomes of the unborn child by diminishing parasite-driven fetal priming. Later primary exposure to parasitic infections during the first years of life also can affect subsequent immune response to vaccination. Although malaria is the most common parasitic infection in young children, schistosomiasis, filariasis, and intestinal helminthic infections also have been shown to occur in infants Studies have clearly established the presence of T and B cell responses by the human fetus to maternal parasitic infections Immune tolerance may be due to clonal deletion of specific cell subsets, which leads to loss of antigen-reactive cells in utero and subsequent lack of recall response during antigen exposure later in infancy. Another possible tolerance mechanism is clonal anergy, which results from a lack of adequate costimulation by antigen-presenting cells to CD4+ cells. Finally, tolerance may be due to the de novo generation of populations of immunoregulatory cells. Prenatal exposure to malaria, schistosomiasis, and filariasis may lead to the acquisition of a subset of immunoregulatory CD4+ T cells. A distinct population of CD4+ regulatory-1 cells (Tr1) has been shown to be able to arise from CD34+ cells in the neonate Trypanosoma cruzi and T. gondii have been shown to enhance the offspring's susceptibility to subsequent infection, and this phenomenon is associated with impaired or altered fetal immune response to parasite antigens. Children born to filarial-infected mothers, for example, have depressed cytokine responses in T cells and lymphocyte proliferation by peripheral blood mononuclear cells to filarial antigens compared to offspring from uninfected mothers. They are also significantly more likely to acquire filarial infection in the first 5 years of life and continue to show altered responses to parasite antigens years after birth In humans, “tolerance”  is likely to contribute to the long-term persistence of many intravascular parasitic infections Not all in utero exposure to maternal helminth infections results in tolerance. Instead, for some newborns, prenatal parasite exposure results in a constant state of antiparasite immune activation that is characterized by a Th2-dominant cytokine profile, high IgE levels, and eosinophilia. Such an immune profile also may have an adverse impact on the efficacy of vaccines by limiting Th1 pathways of immune response to vaccination. By altering the immunologic balance between Th1 and Th2 pathways, chronic parasitic infections appear to alter the immunologic milieu and would also likely impair or suppress the “normal” responses to vaccines that have been described in parasite-free, developed countries.Polyparasitism's effects on immunity are believed to be partly responsible for the increasing virulence of the world's most lethal infections: HIV A growing body of evidence now demonstrates that antenatal and childhood parasitic infections both deleteriously alter responses to routine vaccination. To truly optimize vaccination campaign effectiveness and vaccine efficacy in all areas of the world, parallel efforts are needed to control endemic parasitic infections. More data are needed to identify the multifactorial causes of impaired vaccine efficacy, including research specifically addressing the impact of preventive antiparasite therapy on maternal and infant health outcomes.The immune response to parasites can be beneficial or detrimental. Immune modulation makes it difficult to predict the impact of intermittent parasite treatment. Perhaps a repeating cycle of infection/treatment/reinfection may be worse for vaccine response than low level chronic infection. This area is worthy of further study and must determine the appropriate treatment intervals for these infections. Also, more data are needed to objectively define the optimal timing between deworming and vaccination. Ideally, pregnant women should be free of infection throughout all three trimesters of pregnancy, but perhaps reinfection in the last trimester is particularly detrimental with respect to fetal priming. Ultimately, a carefully designed double-blind, placebo-controlled trial in infected pregnant women would provide the critical evidence to support or refute the need for antenatal parasitic treatment.If antenatal and early child deworming are to be implemented as standard interventions, then the associated changes in vaccine efficacy and effectiveness should be concurrently documented in well-designed, prospective studies. Animal models suggest that the loss of cross-protective or immunomodulatory effects of polyparasitism may mean that some parasitic diseases may become clinically worse before they are fully eradicated. So far, the available evidence indicates that deworming has clear benefits in terms of birth outcomes


Proximal femoral fractures are amongst the most devastating consequences of osteoporosis and injurious accidental falls with 25–35% of patients dying in the first year post-fracture. Effective rehabilitation strategies are evolving however, despite established associations between nutrition, mobility, strength and strength-related functional outcomes; there has been only one small study with older adults immediately following fragility fracture where a combination of both exercise and nutrition have been provided. The aim of the INTERACTIVE trial is to establish whether a six month, individualised exercise and nutrition program commencing within fourteen days of surgery for proximal femur fracture, results in clinically and statistically significant improvements in physical function, body composition and quality of life at an acceptable level of cost and resource use and without increasing the burden of caregivers.This randomised controlled trial will be performed across two sites, a 500 bed acute hospital in Adelaide, South Australia and a 250 bed acute hospital in Sydney, New South Wales. Four hundred and sixty community-dwelling older adults aged > 70 will be recruited after suffering a proximal femoral fracture and followed into the community over a 12-month period. Participants allocated to the intervention group will receive a six month individualised care plan combining resistance training and nutrition therapy commencing within 14 days post-surgery. Outcomes will be assessed by an individual masked to treatment allocation at six and 12 months. To determine differences between the groups at the primary end-point (six months), ANCOVA or logistic regression will be used with models adjusted according to potential confounders.The INTERACTIVE trial is among the first to combine nutrition and exercise therapy as an early intervention to address the serious consequence of rapid deconditioning and weight loss and subsequent ability to regain pre-morbid function in older patients post proximal femoral fracture. The results of this trial will guide the development of more effective rehabilitation programs, which may ultimately lead to reduced health care costs, and improvements in mobility, independence and quality of life for proximal femoral fracture sufferers.Australian Clinical Trials Registry: ACTRN12607000017426. Proximal femoral fracture (PFF) is amongst the most devastating consequences of osteoporosis and injurious accidental falls with 25–35% of patients dying in the first year post-fracture ,2, only For older people who sustain PFF, rapid deconditioning, loss of muscle strength and weight loss often develop. Causes for this may include inadequate dietary intake, hypermetabolism, and a decline in skeletal muscle mass as a result of a complex interaction between immobility, sarcopenia, wasting and possibly cachexia ,8. MusclExercise programs with community-dwelling, medically stable older adults have been extensively evaluated, the findings suggesting significant reductions in falls -15, injuThe quality of evidence supporting nutritional interventions following PFF is limited and inconsistent . One of Despite established associations and potential interactions between nutrition, mobility, strength and strength-related functional outcomes, there has been only one small study (n = 100) amongst older adults immediately following lower limb fragility fracture where a combination of both exercise and nutrition have been provided . In thisThe aim of this randomised controlled trial (RCT) is to implement a six month, individualised exercise and nutrition program commencing within 14 days of surgery for PFF, that will result in clinically and statistically significant improvements in terms of physical function, body composition and QOL at an acceptable level of cost and resource use and without increasing the burden of caregivers. Comparisons will be drawn with the attention control group.A two-site RCT with masked outcome assessment and 12 month follow up of 460 community-dwelling older adults post PFF. Participants will be randomly allocated to: a) six month individualised exercise and nutrition program (intervention group) or b) attention control (control group). An overview of the recruitment method, randomisation process and follow-up procedures is provided in Figure Participants will be recruited from two acute care settings: Flinders Medical Centre (Adelaide), and Hornsby Ku-ring-gai Hospital (Sydney). Eligible individuals will be all those admitted with a diagnosis of PFF confirmed by radiology report, aged 70 years and over and residing in the community, within existing local service boundaries.2 and 35 kg/m2. A BMI less than 18.5 kg/m2 is considered to be underweight [2 (morbidly obese) [Screening will be carried out for all individuals who meet these inclusion criteria by project personnel in consultation with members of the orthogeriatric team. Potential participants will need to achieve a mini-mental score (MMSE) > or = 18/30, as it is thought that participants with severe cognitive impairment  will be Exclusion criteria include a pathological fracture or malignancy; those residing in residential care, non-English speaking, limited to stand transfers only post surgery or non ambulatory pre fracture, unable to give informed consent or not deemed to be medically stable within fourteen days post surgery. All those individuals who meet the inclusion and exclusion criteria will be invited to participate. When study personnel are satisfied that an individual understands the study information and implications of their involvement, written informed consent will be obtained prior to commencement of baseline measures and randomisation. In the situation where it is felt that a participant does not have the capacity to give informed consent (i.e. post-operative delirium or mild cognitive impairment) third party consent will be obtained from a close relative or immediate caregiver.The Flinders Clinical Research Ethics Committee reviewed and approved the study protocol – Research Application 110/067.Project personnel will assess the selected outcome measures at baseline (prior to randomisation) whilst an individual masked to treatment allocation will assess outcomes at six and 12 months. Testing will coincide with maximum effect of each participant's analgesic regime. Outcome measures have been selected based on suitability for older adults following PFF, strength of the evidence to support validity and reliability, significance to rehabilitation and previous use in the literature.The baseline assessment protocol entails a detailed multi-dimensional health assessment by means of:a) Physical Function, Strength and Balance: Physical autonomy will be measured using a physical and instrumental ADL scale [DL scale . A subjeDL scale  and a 30DL scale , gait spDL scale , balanceDL scale  and gripDL scale .b) Body Composition and Nutrition Measures: Percent weight change will be assessed using calibrated scales. Body composition (fat free mass) will be measured at baseline, six and 12 months using dual-energy x-ray absorptiometry . Lumbar spine and proximal femur DEXA scans will also be performed to assess changes in bone mineral density. Triceps skinfold measures and mid upper arm circumference (MUAC) will also be measured to determine corrected arm muscle area (CAMA) [a (CAMA) . Appetita (CAMA)  and 24 ha (CAMA) .c) Questionnaires: Assessment of Quality of Life Instrument (AQoL) [t (AQoL) , a four t (AQoL)  to detert (AQoL) ,43.Monitoring of participants for six months via weekly home visits will allow for an account of any change in accommodation, falls and injuries and any change in health status. At 12 months participants will be contacted via telephone to organise the follow-up assessment. If there is no response then project personnel will contact the next of kin.Within study analysis will be undertaken based on observed effects and resource use, including hospital inpatient admission and length of stay from medical records, PBS and MBS use from the Health Insurance Commission , as well as direct costs of the proposed intervention . Use of community services will also be estimated based on a weekly survey administered to all participants.Participants will be randomly assigned to either the combined nutrition and exercise therapy group or the attention control group following completion of all baseline measures. Group allocation will be managed externally by the Pharmacy Department at the Repatriation General Hospital. Therapists and participants will be aware of treatment allocation however staff performing the outcome assessments and the data analysis will remain unaware of allocation.Participants allocated to the intervention group will receive a well-integrated, coordinated, individualised care plan combining resistance training and nutrition therapy commencing between day seven to 14 post-surgery and continued over a six month period. Therapists administering the intervention across the two sites will be trained in the established protocols and fidelity to protocols will be addressed through random checks of therapy sessions and fortnightly project staff meetings.The Otago exercise program  has beenAs a basis for developing a nutrition program that will achieve energy balance , the resting energy expenditure (REE) of each participant will be measured using indirect calorimetry. Achieving the shortfall between estimated dietary intake  and measured or recommended requirements, whilst considering risk factors threatening nutritional well-being, will become the focus of the nutrition care plan. Strategies for achieving energy and nutrient requirements will include counselling with attention to timing, size and frequency of meals, recommendations of nutrient dense foods, provision of recipes and referral to community meal programs and providers and supplementation with commercial liquid diets, protein supplements or multi-vitamins where deemed necessary by a dietitian. Intake of any prescribed supplements will be recorded by nursing staff while in hospital, and by the patient or caregiver once discharged home. Dietary intake and requirements will be reviewed on a fortnightly basis by a dietitian and modified as required.Participants allocated to the attention control group will continue therapy as prescribed during hospital admission (acute and rehabilitation) according to current practice. Social visits of equivalent duration will occur to match the active intervention group. The project physiotherapist and dietitian will provide participants with generic nutrition, exercise and falls information in line with Government recommendations to provide some relevant topics for discussion, this information will also be given to the intervention group.Gait speed at six months was chosen as the primary outcome because safe, independent walking is a high priority amongst patients participating in rehabilitation, and gait speed is often recommended as a measure of status and outcome ,47. UsinData will be maintained centrally at Flinders University with double entry of data into SPSS software to verify accuracy. Primary analysis for this study will be undertaken using intention to treat principles. Location of central tendency and distribution will be determined and data presented as appropriate. Independent sample t-tests, Mann-Whitney U tests and Chi-square test of association will be used as appropriate to compare groups at baseline. To determine differences between the groups at the primary end-point (six months), and at 12 month follow up ANCOVA or logistic regression will be used with models adjusted according to potential confounders. The analyses will be undertaken by a statistician blinded to group allocation.This study has a 3 year time frame. Recruitment commenced in June of 2007, follow up assessment will continue until September 2009, after which time data analysis will be performed and a final report will be drafted.The INTERACTIVE trial is among the first to combine nutrition and exercise therapy as an intervention to address the serious consequence of rapid deconditioning and weight loss and subsequent ability to regain pre-morbid function in older patients post PFF. It differs from previous studies in this area in a number of significant ways. Commencement of the intervention will occur sooner than any previous work in an attempt to intercept the rapid deconditioning often observed amongst older adults admitted to hospital. The duration of both components of the study intervention will be longer than any previous work in this area, with weekly follow up over a six month period. This is based on evidence to suggest that weight loss continues for at least 12 weeks following PFF  and thatThe benefits of an exercise program to maintain strength and balance and reduce the risk of falls are well established -17, howeThe Otago program is yet to be evaluated amongst patients recovering from PFF and provides a pragmatic alternative to other exercise programs due to its established effectiveness, ease of implementation and affordability.Through regular supervision attempts will be made to maintain patient motivation, continually set realistic and achievable goals and provide an opportunity to address factors affecting adherence. Another strategy targeted at improving adherence is to involve close relatives or immediate caregivers where appropriate. This comprehensive data collection will provide an opportunity to identify groups most likely to respond to such an intervention, which will then inform future service provision. This trial will also be one of the first to utilise body composition measures obtained through DEXA scans to accurately assess for change in muscle mass following such a comprehensive therapy program administered over six months.Previous studies have failed to consider the need for provision of an adequate amount of energy and protein to address the deficits associated with pre-existing undernutrition, injury and surgical stress and inadequate dietary intakes during recovery from PFF . A comprThis study is also unique in that the impact on caregivers and overall costs and resource use will be carefully measured. Measurement of caregiver strain and burden, quality of life and positive affect over time will provide an opportunity to capture whether the potentially negative impact of frequent visits in the home and possible invasion of privacy are outweighed in the long term by the increased independence and quality of life experienced by the PFF sufferer. The comprehensive analysis relating to both direct and indirect costs of the intervention is also unique and will provide realistic data on the feasibility of the intervention becoming an ongoing service. It is important to provide adequate rehabilitation post PFF to return people as close to premorbid function as possible so that physical decline, hospital re-admission and even residential care admission are avoided. The results of this trial will help to guide the development of more effective rehabilitation programs following PFF, which may ultimately lead to reduced health care costs, and improvements in mobility, independence and quality of life for PFF sufferers.The author(s) declare that they have no competing interests.ST and KH are research higher degree students actively involved in implementation of the study. All other authors wrote the grant application for this project that was funded by National Health and Medical Research Council in 2007. The grant application formed the basis for this manuscript which was jointly drafted by ST and KH with all other authors contributing to its critical review and approving the final draft.The pre-publication history for this paper can be accessed here: