Knockdown of NeuroD2 leads to seizure-like behaviour, brain neuronal hyperactivity and a leaky blood-brain barrier in a Xenopus laevis tadpole model of DEE75.

Developmental and Epileptic Encephalopathies (DEE) are a genetically diverse group of severe, early onset seizure disorders. DEE are normally identified clinically in the first six months of life by the presence of frequent, difficult to control seizures and accompanying stalling or regression of development. DEE75 results from de novo mutations of the NEUROD2 gene that result in loss of activity of the encoded transcription factor, and the seizure phenotype was shown to be recapitulated in Xenopus tropicalis tadpoles. We used CRISPR/Cas9 to make a DEE75 model in Xenopus laevis, to further investigate the developmental aetiology. NeuroD2.S CRISPR/Cas9 edited tadpoles were more active, swam faster on average, and had more seizures (C-shaped contractions resembling unprovoked C-start escape responses) than their sibling controls. Live imaging of Ca2+ signalling revealed prolongued, strong signals sweeping through the brain, indicative of neuronal hyperactivity. While the resulting tadpole brain appeared grossly normal, the blood-brain barrier was found to be leakier than that of controls. Additionally, the TGFß antagonist Losartan was shown to have a short-term protective effect, reducing neuronal hyperactivity and reducing permeability of the blood-brain barrier. Treatment of NeuroD2 CRISPant tadpoles with 5 mM Losartan decreased seizure events by more than fourfold compared to the baseline. Our results support a model of DEE75 resulting from reduced NeuroD2 activity during vertebrate brain development, and indicate that a leaky blood-brain barrier contributes to epileptogenesis.

Characterisation and automated quantification of induced seizure-related behaviours in Xenopus laevis tadpoles.

Epilepsy, a clinical diagnosis characterised by paroxysmal episodes known as seizures, affects 1% of people worldwide. Safe and patient-specific treatment is vital and can be achieved by the development of rapid pre-clinical models of for identified epilepsy genes. Epilepsy can result from either brain injury or gene mutations, and can also be induced chemically. Xenopus laevis tadpoles could be a useful model for confirmation of variants of unknown significance found in epilepsy patients, and for drug re-purposing screens that could eventually lead to benefits for patients. Here, we characterise and quantify seizure-related behaviours in X. laevis tadpoles arrayed in 24-well plates. To provoke acute seizure behaviours, tadpoles were chemically induced with either pentylenetetrazole (PTZ) or 4-aminopyridine (4-AP). To test the capacity to adapt this method for drug testing, we also exposed induced tadpoles to the anti-seizure drug valproate (VPA). Four induced seizure-like behaviours were described and manually quantified, and two of these (darting, circling) could be accurately detected automatically, using the video analysis software TopScan. Additionally, we recorded swimming trajectories and mean swimming velocity. Automatic detection showed that either PTZ or 4-AP induced darting behaviour and increased mean swimming velocity compared to untreated controls. Both parameters were significantly reduced in the presence of VPA. In particular, darting behaviour was a shown to be a sensitive measure of epileptic seizure activity. While we could not automatically detect the full range of seizure behaviours, this method shows promise for future studies since X. laevis is a well-characterised and genetically tractable model organism.

Gene expression analysis of the Xenopus laevis early limb bud proximodistal axis.

BACKGROUND: Limb buds develop as bilateral outgrowths of the lateral plate mesoderm and are patterned along three axes. Current models of proximal to distal patterning of early amniote limb buds suggest that two signals, a distal organizing signal from the apical epithelial ridge (AER, Fgfs) and an opposing proximal (retinoic acid [RA]) act early on pattern this axis. RESULTS: Transcriptional analysis of stage 51 Xenopus laevis hindlimb buds sectioned along the proximal-distal axis showed that the distal region is distinct from the rest of the limb. Expression of capn8.3, a novel calpain, was located in cells immediately flanking the AER. The Wnt antagonist Dkk1 was AER-specific in Xenopus limbs. Two transcription factors, sall1 and zic5, were expressed in distal mesenchyme. Zic5 has no described association with limb development. We also describe expression of two proximal genes, gata5 and tnn, not previously associated with limb development. Differentially expressed genes were associated with Fgf, Wnt, and RA signaling as well as differential cell adhesion and proliferation. CONCLUSIONS: We identify new candidate genes for early proximodistal limb patterning. Our analysis of RA-regulated genes supports a role for transient RA gradients in early limb bud in proximal-to-distal patterning in this anamniote model organism.

Noggin proteins are multifunctional extracellular regulators of cell signaling.

Noggin is an extracellular cysteine knot protein that plays a crucial role in vertebrate dorsoventral patterning. Noggin binds and inhibits the activity of bone morphogenetic proteins via a conserved N-terminal clip domain. Noncanonical orthologs of Noggin that lack a clip domain ("Noggin-like" proteins) are encoded in many arthropod genomes and are thought to have evolved into receptor tyrosine kinase ligands that promote Torso/receptor tyrosine kinase signaling rather than inhibiting bone morphogenic protein signaling. Here, we examined the molecular function of noggin/noggin-like genes (ApNL1 and ApNL2) from the arthropod pea aphid using the dorso-ventral patterning of Xenopus and the terminal patterning system of Drosophila to identify whether these proteins function as bone morphogenic protein or receptor tyrosine kinase signaling regulators. Our findings reveal that ApNL1 from the pea aphid can regulate both bone morphogenic protein and receptor tyrosine kinase signaling pathways, and unexpectedly, that the clip domain is not essential for its antagonism of bone morphogenic protein signaling. Our findings indicate that ancestral noggin/noggin-like genes were multifunctional regulators of signaling that have specialized to regulate multiple cell signaling pathways during the evolution of animals.

Manipulating the microbiome alters regenerative outcomes in Xenopus laevis tadpoles via lipopolysaccharide signalling.

Xenopus laevis tadpoles can regenerate functional tails, containing the spinal cord, notochord, muscle, fin, blood vessels and nerves, except for a brief refractory period at around 1 week of age. At this stage, amputation of the tadpole's tail may either result in scarless wound healing or the activation of a regeneration programme, which replaces the lost tissues. We recently demonstrated a link between bacterial lipopolysaccharides and successful tail regeneration in refractory stage tadpoles and proposed that this could result from lipopolysaccharides binding to Toll-like receptor 4 (TLR4). Here, we have used 16S rRNA sequencing to show that the tadpole skin microbiome is highly variable between sibships and that the community can be altered by raising embryos in the antibiotic gentamicin. Six Gram-negative genera, including Delftia and Chryseobacterium, were over-represented in tadpoles that underwent tail regeneration. Lipopolysaccharides purified from a commensal Chryseobacterium spp. XDS4, an exogenous Delftia spp. or Escherichia coli, could significantly increase the number of antibiotic-raised tadpoles that attempted regeneration. Conversely, the quality of regeneration was impaired in native-raised tadpoles exposed to the antagonistic lipopolysaccharide of Rhodobacter sphaeroides. Editing TLR4 using CRISPR/Cas9 also reduced regeneration quality, but not quantity, at the level of the cohort. However, we found that the editing level of individual tadpoles was a poor predictor of regenerative outcome. In conclusion, our results suggest that variable regeneration in refractory stage tadpoles depends at least in part on the skin microbiome and lipopolysaccharide signalling, but that signalling via TLR4 cannot account for all of this effect.

Bacterial lipopolysaccharides can initiate regeneration of the Xenopus tadpole tail.

Tadpoles of the frog Xenopus laevis can regenerate tails except for a short "refractory" period in which they heal rather than regenerate. Rapid and sustained production of ROS by NADPH oxidase (Nox) is critical for regeneration. Here, we show that tail amputation results in rapid, transient activation of the ROS-activated transcription factor NF-κB and expression of its direct target cox2 in the wound epithelium. Activation of NF-κB is also sufficient to rescue refractory tail regeneration. We propose that bacteria on the tadpole's skin could influence tail regenerative outcomes, possibly via LPS-TLR4-NF-κB signaling. When raised in antibiotics, fewer tadpoles in the refractory stage attempted regeneration, whereas addition of LPS rescued regeneration. Short-term activation of NF-κB using small molecules enhanced regeneration of tadpole hindlimbs, but not froglet forelimbs. We propose a model in which host microbiome contributes to creating optimal conditions for regeneration, via regulation of NF-κB by the innate immune system.

Characterization of deaths and therapeutic itineraries investigated by the Porto Alegre AIDS Mortality Committee, Brazil, in 2015. / Caracterização dos óbitos e dos itinerários terapêuticos investigados pelo Comitê Municipal de Mortalidade por Aids de Porto Alegre em 2015.

OBJECTIVE: to characterize AIDS deaths eligible for Porto Alegre AIDS Mortality Committee (AIDSMC) investigation, Brazil, in 2015, and their therapeutic itineraries. METHODS: this was a descriptive study using secondary data from surveillance information systems and AIDSMC investigation forms. RESULTS: out of 336 deaths from AIDS-related causes, 113 (33.6%) were considered avoidable, of which 52 were analyzed by AIDSMC; there was predominance of males (30/52), low schooling level (29/52 incomplete elementary education), and less than 2 years between HIV infection diagnosis and death (28/52); tuberculosis was the most frequent cause of death (17/52); and in 50/52 cases at least one therapeutic itinerary inadequacy was identified. CONCLUSION: avoidable deaths of people with AIDS occurred mostly in men, those with low education level, those with recent HIV diagnosis and most deaths were due to tuberculosis.

Caracterização dos óbitos e dos itinerários terapêuticos investigados pelo Comitê Municipal de Mortalidade por Aids de Porto Alegre em 2015 / Caracterización de óbitos e itinerario terapéutico investigados por el Comité de Mortalidad por Sida de Porto Alegre, Brasil, en 2015 / Characterization of deaths and therapeutic itineraries investigated by the Porto Alegre AIDS Mortality Committee, Brazil, in 2015

Objetivo: caracterizar os óbitos por aids elegíveis para investigação pelo Comitê Municipal de Mortalidade por Aids (CMAids) de Porto Alegre, Brasil, em 2015, e seus itinerários terapêuticos. Métodos: estudo descritivo, com dados secundários de sistemas de informações de vigilância e das fichas de investigação do CMAids. Resultados: entre 336 óbitos por causas relacionadas à aids, 113 (33,6%) foram considerados evitáveis, dos quais 52 foram investigados pelo CMAids; verificou-se predomínio do sexo masculino (30/52), baixa escolaridade (29/52 casos até a 8ª série incompleta) e tempo de até 2 anos entre o diagnóstico da infecção pelo HIV e a morte (28/52); a tuberculose foi a causa de morte mais frequente (17/52); em 50/52 casos, identificou-se pelo menos uma falha no itinerário terapêutico. Conclusão: os óbitos evitáveis de pessoas com aids ocorreram, majoritariamente, em pessoas do sexo masculino, de baixa escolaridade, com diagnóstico recente de HIV e em decorrência da tuberculose.

Objetivo: caracterizar los óbitos por sida elegibles para investigación por el Comité de Mortalidad por Sida (CMSida) de Porto Alegre, Brasil, en 2015, y sus itinerarios terapéuticos. Métodos: estudio descriptivo con datos secundarios de los sistemas de información de vigilancia y formulario de investigación de CMSida. Resultados entre 336 muertes por sida, 113 (33,6%) se consideraron evitables, de las cuales 52 fueron investigadas por el CMSida; predominó el sexo masculino (30/52), el bajo nivel de educación (29/52 hasta 8º grado incompleto de primaria) y hasta 2 años entre el diagnóstico de la infección por VIH y la muerte (28/52); la tuberculosis fue la causa más frecuente de muerte (17/52); y en 50/52 casos, se identificó al menos una falla en los itinerarios terapéuticos. Conclusión: las muertes evitables de personas con sida ocurrieron principalmente en hombres, con baja escolaridad, diagnóstico reciente de VIH y debido a la tuberculosis.

Objective: to characterize AIDS deaths eligible for Porto Alegre AIDS Mortality Committee (AIDSMC) investigation, Brazil, in 2015, and their therapeutic itineraries. Methods: this was a descriptive study using secondary data from surveillance information systems and AIDSMC investigation forms. Results: out of 336 deaths from AIDS-related causes, 113 (33.6%) were considered avoidable, of which 52 were analyzed by AIDSMC; there was predominance of males (30/52), low schooling level (29/52 incomplete elementary education), and less than 2 years between HIV infection diagnosis and death (28/52); tuberculosis was the most frequent cause of death (17/52); and in 50/52 cases at least one therapeutic itinerary inadequacy was identified. Conclusion: avoidable deaths of people with AIDS occurred mostly in men, those with low education level, those with recent HIV diagnosis and most deaths were due to tuberculosis.

Bifurcation of Health Policy Regimes: A Study of Sleep Apnea Care and Benefits Coverage in Saskatchewan.

BACKGROUND: A complex, poorly understood bifurcated health policy regime exists for Canada's First Nations people for extended health benefits coverage. This research adds to a small body of literature on the regime's impact on access and quality of care and its role in perpetuating health inequities in First Nations populations. METHODS: Using a case study of sleep apnea care in Saskatchewan, we identified issues of health service access and coverage through a literature review of extended benefits programs, legislation and policies and through 10 key informant interviews with federal and provincial extended benefit program administrators and sleep medicine physicians. RESULTS: Important access and coverage differences were found for First Nations populations, many of which were recognized by federal and provincial policy makers. Despite these, government respondents recommended few policy ameliorations, perhaps due to system complexities, constitutional constraints or political sensitivities. CONCLUSIONS: We suggest three policy options to ameliorate current hardships wrought by this policy bifurcation.

Xenopus Limb bud morphogenesis.

Xenopus laevis, the South African clawed frog, is a well-established model organism for the study of developmental biology and regeneration due to its many advantages for both classical and molecular studies of patterning and morphogenesis. While contemporary studies of limb development tend to focus on models developed from the study of chicken and mouse embryos, there are also many classical studies of limb development in frogs. These include both fate and specification maps, that, due to their age, are perhaps not as widely known or cited as they should be. This has led to some inevitable misinterpretations- for example, it is often said that Xenopus limb buds have no apical ectodermal ridge, a morphological signalling centre located at the distal dorsal/ventral epithelial boundary and known to regulate limb bud outgrowth. These studies are valuable both from an evolutionary perspective, because amphibians diverged early from the amniote lineage, and from a developmental perspective, as amphibian limbs are capable of regeneration. Here, we describe Xenopus limb morphogenesis with reference to both classical and molecular studies, to create a clearer picture of what we know, and what is still mysterious, about this process.

Exploring policy driven systemic inequities leading to differential access to care among Indigenous populations with obstructive sleep apnea in Canada.

BACKGROUND: In settler societies such as Australia, Canada, New Zealand and the United States, health inequities drive lower health status and poorer health outcomes in Indigenous populations. This research unravels the dense complexity of how historical policy decisions in Canada can influence inequities in health care access in the 21(st) century through a case study on the diagnosis and treatment of obstructive sleep apnea (OSA). In Canada, historically rooted policy regimes determine current discrepancies in health care policy, and in turn, shape current health insurance coverage and physician decisions in terms of diagnosis and treatment of OSA, a clinical condition that is associated with considerable morbidity in Canada. METHODS: This qualitative study was based in Saskatchewan, a Western Canadian province which has proportionately one of the largest provincial populations of an Indigenous subpopulation (status Indians) which is the focus of this study. The study began with determining approaches to OSA care provision based on Canadian Thoracic Society guidelines for referral, diagnosis and treatment of sleep disordered breathing. Thereafter, health policy determining health benefits coverage and program differences between status Indians and other Canadians were ascertained. Finally, respirologists who specialized in sleep medicine were interviewed. All interviews were audio-recorded and the transcripts were thematically analyzed using NVIVO. RESULTS: In terms of access and provision of OSA care, different patient pathways emerged for status Indians in comparison with other Canadians. Using Saskatchewan as a case study, the preliminary evidence suggests that status Indians face significant barriers in accessing diagnostic and treatment services for OSA in a timely manner. CONCLUSIONS: In order to confirm initial findings, further investigations are required in other Canadian jurisdictions. Moreover, as other clinical conditions could share similar features of health care access and provision of health benefits coverage, this policy analysis could be replicated in other provincial and territorial health care systems across Canada, and other settler nations where there are differential health coverage arrangements for Indigenous peoples.

Gremlin1 induces anterior-posterior limb bifurcations in developing Xenopus limbs but does not enhance limb regeneration.

Gremlin1 (grem1) has been previously identified as being significantly up-regulated during regeneration of Xenopus laevis limbs. Grem1 is an antagonist of bone morphogenetic proteins (BMPs) with a known role in limb development in amniotes. It forms part of a self-regulating feedback loop linking epithelial (FGF) and mesenchymal (shh) signalling centres, thereby controlling outgrowth, anterior posterior and proximal distal patterning. Spatiotemporal regulation of the same genes in developing and regenerating Xenopus limb buds supports conservation of this mechanism. Using a heat shock inducible grem1 (G) transgene to created temperature regulated stable lines, we have shown that despite being upregulated in regeneration, grem1 overexpression does not enhance regeneration of tadpole hindlimbs. However, both the regenerating and contralateral, developing limb of G transgenics developed skeletal defects, suggesting that overexpressing grem1 negatively affects limb patterning. When grem1 expression was targeted earlier in limb bud development, we saw dramatic bifurcations of the limbs resulting in duplication of anterior posterior (AP) pattern, forming a phenotypic continuum ranging from duplications arising at the level of the femoral head to digit bifurcations, but never involving the pelvis. Intriguingly, the original limbs have AP pattern inversion due to de-restricted Shh signalling. We discuss a possible role for Grem1 regulation of limb BMPs in regulation of branching pattern in the limbs.

Development of the vertebrate tailbud.

The anatomical tailbud is a defining feature of all embryonic chordates, including vertebrates that do not end up with a morphological tail. Due to its seamless continuity with trunk tissues, the tailbud is often overlooked as a mere extension of the body axis; however, the formation of the tail from the tailbud undoubtedly involves unique and distinct mechanisms for forming axial tissues, such as the secondary neurulation process that generates the tailbud-derived spinal cord. Tailbud formation in the frog Xenopus laevis has been demonstrated to involve interaction of three posterior regions of the embryo that first come into alignment at the end of gastrulation, and molecular models for tailbud outgrowth and patterning have been proposed. While classical studies of other vertebrate models, such as the chicken, initially appeared to draw incompatible conclusions, molecular studies have subsequently shown the involvement of at least some similar genetic pathways. Finally, there is an emerging consensus that at least some vertebrate tailbud cells are multipotent progenitors with the ability to form tissues normally derived from different germ layers- a trait normally associated with regeneration of complex appendages, or stem-like cells.

Distinct patterns of endosulfatase gene expression during Xenopus laevis limb development and regeneration.

The heparan sulfate 6-O-endosulfatases sulf1 and sulf2 regulate multiple cellular processes and organ development. Sulfs modulate a range of heparan-sulfate-dependent extracellular pathways, including the fibroblast growth factor, bone morphogenetic protein, and wingless/wnt signaling pathways. Known patterns of sulf transcript expression together with functional experiments have implicated the sulfs in chondrogenesis and muscle regeneration in mammals. Here, we describe the expression patterns of Xenopus laevis sulf1 and sulf2 in developing forelimbs and hindlimbs and demonstrate novel expression of the sulf transcripts in the regenerating hindlimbs, with prominent sulf2 expression in the proliferating blastema and transient expression of sulf1 in the redeveloping apical epidermal ridge. These findings further suggest involvement of the sulfs in successful limb regeneration in amphibians.

Distal expression of sprouty (spry) genes during Xenopus laevis limb development and regeneration.

The eukaryotic Sprouty (Spry) proteins are negative regulators of receptor tyrosine kinase signaling pathways, which in turn provide readout for morphogens such as fibroblast growth factors (Fgfs) that are essential for many developmental processes, including limb development. In a transcriptome analysis of early proximo-distal patterning of the Xenopus laevis limb bud, spry1a, 2 and 4 were predicted to be expressed predominantly in the distal third. Expression of all three in the distal limb initially corresponded to the progress zone mesenchyme, adjacent to the fgf8b-positive cryptic apical ectodermal ridge (AER). Spry2 transcripts were also expressed ectodermally, and later localized to the AER. During formation of the autopod, spry1a and spry4 became restricted to the anterior distal mesenchyme. All three spry genes were re-expressed in the mesenchyme cells of the blastema during hindlimb regeneration, with spry2 also overlapping with the region of fgf8b re-expression. However, the anterior bias seen in developing limbs was not recapitulated. We conclude that Spry1a, 2 and 4 have partially overlapping expression in developing and regenerating Xenopus limbs, which correspond with known areas of Fgf signaling. Sprys may therefore be involved in refining of Fgf signal transduction from the AER during development and regeneration.

Viral epidemiology of respiratory infections among children at a tertiary hospital in Southern Brazil.

INTRODUCTION: This study reports the pediatric epidemiology of respiratory syncytial virus (RSV), influenza (IF), parainfluenza (PIV), and adenovirus (ADV) at Hospital de Clínicas de Porto Alegre. METHODS: Cases of infection, hospitalizations in intensive care units (ICUs), nosocomial infections, and lethality rates were collected from 2007 to 2010. RESULTS: RSV accounted for most nosocomial infections. Intensive care units admission rates for ADV and RSV infections were highest in 2007 and 2010. During 2008-2009, H1N1 and ADV had the highest ICU admission rates. ADV had the highest fatality rate during 2007-2009. CONCLUSIONS: Each virus exhibited distinct behavior, causing hospitalization, outbreaks, or lethality.

Evaluation of respiratory syncytial virus group A and B genotypes among nosocomial and community-acquired pediatric infections in Southern Brazil.

BACKGROUND: Respiratory syncytial virus (RSV) is the main cause of lower respiratory tract illness in children worldwide. Molecular analyses show two distinct RSV groups (A and B) that comprise different genotypes. This variability contributes to the capacity of RSV to cause yearly outbreaks. These RSV genotypes circulate within the community and within hospital wards. RSV is currently the leading cause of nosocomial respiratory tract infections in pediatric populations. The aim of this study was to evaluate the G protein gene diversity of RSV amplicons. METHODS: Nasopharyngeal aspirate samples were collected from children with nosocomial or community-acquired infections. Sixty-three RSV samples (21 nosocomial and 42 community-acquired) were evaluated and classified as RSV-A or RSV-B by real-time PCR. Sequencing of the second variable region of the G protein gene was performed to establish RSV phylogenetics. RESULTS: We observed co-circulation of RSV-A and RSV-B, with RSV-A as the predominant group. All nosocomial and community-acquired RSV-A samples were from the same phylogenetic group, comprising the NA1 genotype, and all RSV-B samples (nosocomial and community-acquired) were of the BA4 genotype. Therefore, in both RSV groups (nosocomial and community-acquired), the isolates belonged to only one genotype in circulation. CONCLUSIONS: This is the first study to describe circulation of the NA1 RSV genotype in Brazil. Furthermore, this study showed that the BA4 genotype remains in circulation. Deciphering worldwide RSV genetic variability will aid vaccine design and development.

Attenuation of bone morphogenetic protein signaling during amphibian limb development results in the generation of stage-specific defects.

The vertebrate limb is one of the most intensively studied organs in the field of developmental biology. Limb development in tetrapod vertebrates is highly conserved and dependent on the interaction of several important molecular pathways. The bone morphogenetic protein (BMP) signaling cascade is one of these pathways and has been shown to be crucial for several aspects of limb development. Here, we have used a Xenopus laevis transgenic line, in which expression of the inhibitor Noggin is under the control of the heat-shock promoter hsp70 to examine the effects of attenuation of BMP signaling at different stages of limb development. Remarkably different phenotypes were produced at different stages, illustrating the varied roles of BMP in development of the limb. Very early limb buds appeared to be refractory to the effects of BMP attenuation, developing normally in most cases. Ectopic limbs were produced by overexpression of Noggin corresponding to a brief window of limb development at about stage 49/50, as recently described by Christen et al. (2012). Attenuation of BMP signaling in stage 51 or 52 tadpoles lead to a reduction in the number of digits formed, resulting in hypodactyly or ectrodactyly, as well as occasional defects in the more proximal tibia-fibula. Finally, inhibition at stage 54 (paddle stage) led to the formation of dramatically shortened digits resulting from loss of distal phalanges. Transcriptome analysis has revealed the possibility that more Noggin-sensitive members of the BMP family could be involved in limb development than previously suspected. Our analysis demonstrates the usefulness of heat-shock-driven gene expression as an effective method for inhibiting a developmental pathway at different times during limb development.

Fundamental ratios and logarithmic periodicity in human limb bones.

Fundamental mathematical relationships are widespread in biology yet there is little information on this topic with regard to human limb bone lengths and none related to human limb bone volumes. Forty-six sets of ipsilateral upper and lower limb long bones and third digit short bones were imaged by computed tomography. Maximum bone lengths were measured manually and individual bone volumes calculated from computed tomography images using a stereologic method. Length ratios of femur : tibia and humerus : ulna were remarkably similar (1.21 and 1.22, respectively) and varied little (<7%) between individuals. The volume ratio of femur : tibia was approximately half that of humerus : ulna (1.58 and 3.28, respectively; P < 0.0001). Lower limb bone volume ratios varied much more than upper limb ratios. The relationship between bone length and volume was found to be well described by power laws, with R(2) values ranging from 0.983 to 0.995. The most striking finding was a logarithmic periodicity in bone length moving from distal to proximal up the limb (upper limb λ = 0.72, lower limb λ = 0.93). These novel data suggest that human limb bone lengths and volumes follow fundamental and highly conserved mathematical relationships, which may contribute to our understanding of normal and disordered growth, stature estimation, and biomechanics.