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Background: Targeted capture of genomic regions reduces sequencing cost while generating higher coverage by allowing biomedical researchers to focus on specific loci of interest, such as exons. Targeted capture also has the potential to facilitate the gene

Purpose of Review: The tibial tubercle osteotomy (TTO) is a versatile surgical technique used to treat a range of patellofemoral disorders, including patellar instability, painful malalignment, focal chondral defects, and patellar maltracking that have failed conservative therapies. TTO is a personalized procedure that can be tailored to the pathoanatomy of the patient based on physical examination and imaging. The complication rate associated with TTO strongly depends on the indication for surgery, the severity of the patient's condition, and the surgical approach. Despite the literature on TTO, to our knowledge, no single source has addressed the indications, techniques, outcomes, and complications of this procedure. The purpose of this article is to serve as such a valuable resource., Recent Findings: Highlights from recent studies we would like to emphasize are two-fold. First, maintaining a distal cortical hinge yields lower complication rates than osteotomies involving complete tubercle detachment with classic or standard techniques. Second, based on current evidence, TTO consistently provides symptomatic relief, and most patients can return to work or sport at their pre-operative level within 3 and 6 months, respectively. TTO is a personalizable surgical technique that may be utilized for multiple patellofemoral disorders and is associated with good outcomes., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Introduction: Giant cell tumor of tendon sheath (GCTTS), also known as tenosynovial giant cell tumor or pigmented villonodular tenosynovitis, is a rare benign soft-tissue tumor with an unclear cause. It is the second most frequent soft-tissue tumor in the hand after ganglion cyst., Case Report: We described a female patient, age 19, who has had a 3 cm × 2 cm firm swelling on the palmer aspect of the right second metacarpal region for 7 years. The bulge developed spontaneously and moved quite slowly. It is required to do a histological and radiographic evaluation to determine whether or not to pursue additional treatment. Excision surgery was done, and the tumor was entirely removed. According to histopathology, this mass was compatible with GCTTS without being malignant., Conclusion: It is an uncommon instance of GCTTS at the hand, to sum up. The tumor should be entirely excised due to its high risk of recurrence to lower the likelihood of recurrence and restore hand function., Competing Interests: Conflict of Interest: Nil, (Copyright: © Indian Orthopaedic Research Group.)

Diabetic nephropathy is a leading cause of end-stage renal disease, characterized by endothelial dysfunction and a compromised glomerular permeability barrier. Dysregulation of the angiopoietin 1 (ANGPT1)/angiopoietin 2 (ANGPT2) signaling axis is implicated in disease progression. We recently described the discovery of an IgG 1 antibody, O010, with therapeutic potential to elevate circulating endogenous ANGPT1, a tyrosine kinase with Ig and epidermal growth factor (EGF) homology domains-2 (TIE2) agonist. Studies are described that detail the effect of various ANGPT1-elevating strategies to limit progression of renal dysfunction in diabetic-obese (db/db) mice. Results demonstrate that adeno-associated virus- or DNA minicircle-directed overexpression of ANGPT1 elicits a reduction in albuminuria (56%-73%) and an improvement in histopathology score (18% reduction in glomerulosclerosis). An improved acetylcholine response in isolated aortic rings was also observed indicative of a benefit on vascular function. In separate pharmacokinetic studies, an efficacious dose of the ANGPT1 DNA minicircle increased circulating levels of the protein by >80%, resulting in a concomitant suppression of ANGPT2. At a dose of O010-producing maximal elevation of circulating ANGPT1 achievable with the molecule (60% increase), no suppression of ANGPT2 was observed in db/db mice, suggesting insufficient pathway engagement; no reduction in albuminuria or improvement in histopathological outcomes were observed. To pinpoint the mechanism resulting in lack of efficacy, we demonstrate, using confocal microscopy, an interference with TIE2 translocation to adherens junctions, resulting in a loss of protection against vascular permeability normally conferred by ANGPT1. Results demonstrated the essential importance of ANGPT1 to maintain the glomerular permeability barrier, and, due to interference of O010 with this process, led to the discontinuation of the molecule for clinical development. SIGNIFICANCE STATEMENT: This body of original research demonstrates that elevation of systemic angiopoietin 1 (ANGPT1) is protective against diabetic nephropathy. However, using a novel biotherapeutic approach to elevate systemic ANGPT1 renoprotection was not observed; we demonstrate that protection was lost due to interference of the therapeutic with ANGPT1/ tyrosine kinase with Ig and EGF homology domains-2 translocation to adherens junctions. Thus, the clinical development of the antibody was terminated., (Copyright © 2022 by The American Society for Pharmacology and Experimental Therapeutics.)

Background: Infections by several DNA viruses can severely impact outcomes in paediatric immunocompromised patients. Current testing, which is generally limited to singleplex qPCR assays, can miss both common and rarer viruses if they are not targeted., Objectives: To evaluate the performance of the Galileo Viral Panel (Galileo), a sample-to-result shotgun metagenomics platform for the detection and quantification of 12 DNA viruses, compared to standard of care qPCR assays., Study Design: A clinical performance evaluation was carried out using 43 prospectively collected EDTA plasma samples positive for one or more DNA viruses. Agreement between assays was assessed by overall, positive, and negative percent agreement, as well as quantitative agreement by linear regression and Bland-Altman analysis., Results: Overall positive percent agreement was 84% (95% CI: 76%-90%), and negative percent agreement was 95% (95% CI: 92%-97%). There was a high correlation between Galileo and qPCR for ADV, CMV, EBV, and VZV ( R 2  = 0.91) and a mean difference by Bland Altman of -0.43 log 10 IU or cp/ml (95% limits of agreement, -1.37 to 0.51). In addition, there was a high correlation between Galileo Signal Score and qPCR for TTV ( R 2  = 0.85)., Conclusion: We observed high qualitative and quantitative agreement between qPCR and Galileo. Galileo identified additional viruses that were not tested with routine qPCR and could impact clinical outcomes., Competing Interests: MLC and GW are current or former employees of Arc Bio, LLC. JB is a clinical advisor for Arc Bio., (© 2022 The Authors. Published by Elsevier Ltd.)

Infections with DNA viruses are frequent causes of morbidity and mortality in transplant recipients. This study describes the analytical and clinical performance characteristics of the Arc Bio Galileo Pathogen Solution, an all-inclusive metagenomic next-generation sequencing (mNGS) reagent and bioinformatics pipeline that allows the simultaneous quantitation of 10 transplant-related double-stranded DNA (dsDNA) viruses (adenovirus [ADV], BK virus [BKV], cytomegalovirus [CMV], Epstein-Barr virus [EBV], human herpesvirus 6A [HHV-6A], HHV-6B, herpes simplex virus 1 [HSV-1], HSV-2, JC virus [JCV], and varicella-zoster virus [VZV]). The mNGS 95% limit of detection ranged from 14 copies/ml (HHV-6) to 191 copies/ml (BKV), and the lower limit of quantitation ranged from 442 international units (IU)/ml (EBV) to 661 copies/ml (VZV). An evaluation of 50 residual plasma samples with at least one DNA virus detected in prior clinical testing showed a total percent agreement of mNGS and quantitative PCR (qPCR) of 89.2% (306/343), with a κ statistic of 0.725. The positive percent agreement was 84.9% (73/86), and the negative percent agreement was 90.7% (233/257). Furthermore, mNGS detected seven subsequently confirmed coinfections that were not initially requested by qPCR. Passing-Bablok regression revealed a regression line of y  = 0.953 x  + 0.075 (95% confidence interval [CI] of the slope, 0.883 to 1.011; intercept, -0.100 to 0.299), and Bland-Altman analysis (mNGS - qPCR) showed a slight positive bias (0.28 log 10 concentration; 95% limits of agreement, -0.62 to 1.18). In conclusion, the mNGS-based Galileo pipeline demonstrates analytical and clinical performance comparable to that of qPCR for transplant-related DNA viruses., (Copyright © 2019 Carpenter et al.)

Patagonia was the last region of the Americas reached by humans who entered the continent from Siberia ∼15,000-20,000 y ago. Despite recent genomic approaches to reconstruct the continental evolutionary history, regional characterization of ancient and modern genomes remains understudied. Exploring the genomic diversity within Patagonia is not just a valuable strategy to gain a better understanding of the history and diversification of human populations in the southernmost tip of the Americas, but it would also improve the representation of Native American diversity in global databases of human variation. Here, we present genome data from four modern populations from Central Southern Chile and Patagonia ( n = 61) and four ancient maritime individuals from Patagonia (∼1,000 y old). Both the modern and ancient individuals studied in this work have a greater genetic affinity with other modern Native Americans than to any non-American population, showing within South America a clear structure between major geographical regions. Native Patagonian Kawéskar and Yámana showed the highest genetic affinity with the ancient individuals, indicating genetic continuity in the region during the past 1,000 y before present, together with an important agreement between the ethnic affiliation and historical distribution of both groups. Lastly, the ancient maritime individuals were genetically equidistant to a ∼200-y-old terrestrial hunter-gatherer from Tierra del Fuego, which supports a model with an initial separation of a common ancestral group to both maritime populations from a terrestrial population, with a later diversification of the maritime groups., Competing Interests: The authors declare no conflict of interest.

Genetic markers are important resources for individual identification and parentage assessment. Although short tandem repeats (STRs) have been the traditional DNA marker, technological advances have led to single nucleotide polymorphisms (SNPs) becoming an attractive alternative. SNPs can be highly multiplexed and automatically scored, which allows for easier standardization and sharing among laboratories. Equine parentage is currently assessed using STRs. We obtained a publicly available SNP dataset of 729 horses representing 32 diverse breeds. A proposed set of 101 SNPs was analyzed for DNA typing suitability. The overall minor allele frequency of the panel was 0.376 (range 0.304-0.419), with per breed probability of identities ranging from 5.6 × 10 -35 to 1.86 × 10 -42 . When one parent was available, exclusion probabilities ranged from 0.9998 to 0.999996, although when both parents were available, all breeds had exclusion probabilities greater than 0.9999999. A set of 388 horses from 35 breeds was genotyped to evaluate marker performance on known families. The set included 107 parent-offspring pairs and 101 full trios. No horses shared identical genotypes across all markers, indicating that the selected set was sufficient for individual identification. All pairwise comparisons were classified using ISAG rules, with one or two excluding markers considered an accepted parent-offspring pair, two or three excluding markers considered doubtful and four or more excluding markers rejecting parentage. The panel had an overall accuracy of 99.9% for identifying true parent-offspring pairs. Our developed marker set is both present on current generation SNP chips and can be highly multiplexed in standalone panels and thus is a promising resource for SNP-based DNA typing., (© 2017 Stichting International Foundation for Animal Genetics.)

Between 1500 and 1850, more than 12 million enslaved Africans were transported to the New World. The vast majority were shipped from West and West-Central Africa, but their precise origins are largely unknown. We used genome-wide ancient DNA analyses to investigate the genetic origins of three enslaved Africans whose remains were recovered on the Caribbean island of Saint Martin. We trace their origins to distinct subcontinental source populations within Africa, including Bantu-speaking groups from northern Cameroon and non-Bantu speakers living in present-day Nigeria and Ghana. To our knowledge, these findings provide the first direct evidence for the ethnic origins of enslaved Africans, at a time for which historical records are scarce, and demonstrate that genomic data provide another type of record that can shed new light on long-standing historical questions.

Genome sequencing of the 5,300-year-old mummy of the Tyrolean Iceman, found in 1991 on a glacier near the border of Italy and Austria, has yielded new insights into his origin and relationship to modern European populations. A key finding of that study was an apparent recent common ancestry with individuals from Sardinia, based largely on the Y chromosome haplogroup and common autosomal SNP variation. Here, we compiled and analyzed genomic datasets from both modern and ancient Europeans, including genome sequence data from over 400 Sardinians and two ancient Thracians from Bulgaria, to investigate this result in greater detail and determine its implications for the genetic structure of Neolithic Europe. Using whole-genome sequencing data, we confirm that the Iceman is, indeed, most closely related to Sardinians. Furthermore, we show that this relationship extends to other individuals from cultural contexts associated with the spread of agriculture during the Neolithic transition, in contrast to individuals from a hunter-gatherer context. We hypothesize that this genetic affinity of ancient samples from different parts of Europe with Sardinians represents a common genetic component that was geographically widespread across Europe during the Neolithic, likely related to migrations and population expansions associated with the spread of agriculture.

Background: Targeted capture of genomic regions reduces sequencing cost while generating higher coverage by allowing biomedical researchers to focus on specific loci of interest, such as exons. Targeted capture also has the potential to facilitate the generation of genomic data from DNA collected via saliva or buccal cells. DNA samples derived from these cell types tend to have a lower human DNA yield, may be degraded from age and/or have contamination from bacteria or other ambient oral microbiota. However, thousands of samples have been previously collected from these cell types, and saliva collection has the advantage that it is a non-invasive and appropriate for a wide variety of research., Results: We demonstrate successful enrichment and sequencing of 15 South African KhoeSan exomes and 2 full genomes with samples initially derived from saliva. The expanded exome dataset enables us to characterize genetic diversity free from ascertainment bias for multiple KhoeSan populations, including new exome data from six HGDP Namibian San, revealing substantial population structure across the Kalahari Desert region. Additionally, we discover and independently verify thirty-one previously unknown KIR alleles using methods we developed to accurately map and call the highly polymorphic HLA and KIR loci from exome capture data. Finally, we show that exome capture of saliva-derived DNA yields sufficient non-human sequences to characterize oral microbial communities, including detection of bacteria linked to oral disease (e.g. Prevotella melaninogenica). For comparison, two samples were sequenced using standard full genome library preparation without exome capture and we found no systematic bias of metagenomic information between exome-captured and non-captured data., Conclusions: DNA from human saliva samples, collected and extracted using standard procedures, can be used to successfully sequence high quality human exomes, and metagenomic data can be derived from non-human reads. We find that individuals from the Kalahari carry a higher oral pathogenic microbial load than samples surveyed in the Human Microbiome Project. Additionally, rare variants present in the exomes suggest strong population structure across different KhoeSan populations.

The protozoan parasite Giardia intestinalis (also known as Giardia lamblia) is a major waterborne pathogen. During its life cycle, Giardia alternates between the actively growing trophozoite, which has two diploid nuclei with low levels of allelic heterozygosity, and the infectious cyst, which has four nuclei and a tough outer wall. Although the formation of the cyst wall has been studied extensively, we still lack basic knowledge about many fundamental aspects of the cyst, including the sources of the four nuclei and their distribution during the transformation from cyst into trophozoite. In this study, we tracked the identities of the nuclei in the trophozoite and cyst using integrated nuclear markers and immunofluorescence staining. We demonstrate that the cyst is formed from a single trophozoite by a mitotic division without cytokinesis and not by the fusion of two trophozoites. During excystation, the cell completes cytokinesis to form two daughter trophozoites. The non-identical nuclear pairs derived from the parent trophozoite remain associated in the cyst and are distributed to daughter cells during excystation as pairs. Thus, nuclear sorting (such that each daughter cell receives a pair of identical nuclei) does not appear to be a mechanism by which Giardia reduces heterozygosity between its nuclei. Rather, we show that the cyst nuclei exchange chromosomal genetic material, perhaps as a way to reduce heterozygosity in the absence of meiosis and sex, which have not been described in Giardia. These results shed light on fundamental aspects of the Giardia life cycle and have implications for our understanding of the population genetics and cell biology of this binucleate parasite.

Genome sequences of diverse free-living protists are essential for understanding eukaryotic evolution and molecular and cell biology. The free-living amoeboflagellate Naegleria gruberi belongs to a varied and ubiquitous protist clade (Heterolobosea) that diverged from other eukaryotic lineages over a billion years ago. Analysis of the 15,727 protein-coding genes encoded by Naegleria's 41 Mb nuclear genome indicates a capacity for both aerobic respiration and anaerobic metabolism with concomitant hydrogen production, with fundamental implications for the evolution of organelle metabolism. The Naegleria genome facilitates substantially broader phylogenomic comparisons of free-living eukaryotes than previously possible, allowing us to identify thousands of genes likely present in the pan-eukaryotic ancestor, with 40% likely eukaryotic inventions. Moreover, we construct a comprehensive catalog of amoeboid-motility genes. The Naegleria genome, analyzed in the context of other protists, reveals a remarkably complex ancestral eukaryote with a rich repertoire of cytoskeletal, sexual, signaling, and metabolic modules., ((c) 2010 Elsevier Inc. All rights reserved.)

We used translation-blocking morpholinos to reduce protein levels in Giardia intestinalis. Twenty-four hours after electroporation with morpholinos targeting either green fluorescent protein or kinesin-2b, levels of these proteins were reduced by 60%. An epitope-tagged transgene can also be used as a reporter for morpholino efficacy with targets lacking specific antibodies.

As arguably the simplest free-living animals, placozoans may represent a primitive metazoan form, yet their biology is poorly understood. Here we report the sequencing and analysis of the approximately 98 million base pair nuclear genome of the placozoan Trichoplax adhaerens. Whole-genome phylogenetic analysis suggests that placozoans belong to a 'eumetazoan' clade that includes cnidarians and bilaterians, with sponges as the earliest diverging animals. The compact genome shows conserved gene content, gene structure and synteny in relation to the human and other complex eumetazoan genomes. Despite the apparent cellular and organismal simplicity of Trichoplax, its genome encodes a rich array of transcription factor and signalling pathway genes that are typically associated with diverse cell types and developmental processes in eumetazoans, motivating further searches for cryptic cellular complexity and/or as yet unobserved life history stages.

The diplomonad parasite Giardia intestinalis contains two functionally equivalent nuclei that are inherited independently during mitosis. Although presumed to be asexual, Giardia has low levels of allelic heterozygosity, indicating that the two nuclear genomes may exchange genetic material. Fluorescence in situ hybridization performed with probes to an episomal plasmid suggests that plasmids are transferred between nuclei in the cyst, and transmission electron micrographs demonstrate fusion between cyst nuclei. Green fluorescent protein fusions of giardial homologs of meiosis-specific genes localized to the nuclei of cysts, but not the vegetative trophozoite. These data suggest that the fusion of nuclei, or karyogamy, and subsequently somatic homologous recombination facilitated by the meiosis gene homologs, occur in the giardial cyst.

During replication and recombination, two DNA duplexes lie side by side. We have developed reagents that might be used to probe structure during these critical processes; they contain two intercalating groups connected by a rigid linker that forces those groups to point in opposite directions. If their stereochemistry proves appropriate, such structure-specific agents should intercalate specifically into adjacent duplexes in the Y- and X-shaped structures (i.e. 3- and 4-way junctions, now known as 3H and 4H junctions) found at replication and recombination sites. We prepared DNA structures in which four duplexes were arranged in all possible combinations around 2- and 4-way junctions and then probed the accessibility to DNase I of all their phosphodiester bonds. In the absence of any bis-intercalators, 7-9 nucleotides (nt) in each of the strands in 4-way junctions were protected from attack; protected regions were significantly offset to the 3' side of the junction in continuous strands, but only slightly offset, if at all, in exchanging strands. All the intercalators decreased accessibility throughout the structure, but none did so at specific points in the two adjacent arms of 4-way junctions. However, one bis-intercalator--but not its sister with a shorter linker--strikingly increased access to a particular CpT bond that lay 9 nt away from the centre of some 4-way junctions without reducing access to neighbouring bonds. Binding was both sequence and structure specific, and depended on complementary stereochemistry between bis-intercalator and junction.

The binding of polyamines, including spermidine (1) and spermine (2), to poly[d(G-C).d(G-C)] was probed using spectroscopic studies of anthracene-9-carbonyl-N1-spermine (3); data from normal absorption, linear dichroism (LD), and circular dichroism (CD) are reported. Ligand LD and CD for transitions located in the DNA region of the spectrum were used. The data show that 3 binds to DNA in a manner characteristic of both its amine and polycyclic aromatic parts. With poly[(dG-dC).(dG-dC)], binding modes are occupied sequentially and different modes correspond to different structural perturbations of the DNA. The most stable binding mode for 3 with poly[d(G-C).d(G-C)] has a site size of 6 +/- 1 bases, and an equilibrium binding constant of (2.2 +/- 1.1) x 10(7) M-1 with the anthracene moiety intercalated. It dominates the spectra from mixing ratios of approximately 133:1 until 6:1 DNA phosphate: 3 is reached. The analogous data for poly[d(A-T).d(A-T)] between mixing ratios 36:1 and 7:1 indicates a site size of 8.3 +/- 1.1 bases and an equilibrium binding constant of (6.6 +/- 3.3) x 10(5) M-1. Thus, 3 binds preferentially to poly[d(G-C).d(G-C)] at these concentrations.

We have studied the interaction of the GC-specific, minor groove-binding ligand, mithramycin, with cloned DNA inserts containing isolated GC and CG sites flanked by regions of (AT)n and An.Tn using DNase I and hydroxyl radical footprinting. We find that mithramycin binds to GC better than CG and that AGCT is a better site than TGCA. Sites flanked by (AT)n appear to be bound better than those surrounded by An.Tn. Although no footprints are produced at T9GCA9 and T15CGA15, DNase I cleavage is enhanced within the GC sites suggesting that there is some interaction with the ligand. Mithramycin also alters the DNase I cleavage of (GA)n.(CT)n.

We have used hydroxy-radical and deoxyribonuclease-I footprinting to probe the interaction of mithramycin with DNA fragments containing the sequences (AT)10X(AT)10 (X = CCCG, CCGC or CGGC) and A14GCCCT15. As expected the drug produces clear footprints located around the central four GC base pairs. The exact position of the footprint is different for the four sequences; the footprint with CCCG is displayed by two base pairs in the 5' direction relative to GCCC. These variations are explained by suggesting that mithramycin avoids the dinucleotide CG and binds better to GG/CC than GC. Although there is little change in deoxyribonuclease-I cleavage of the surrounding blocks of (AT)n, cleavage by deoxyribonuclease II is markedly enhanced and certain thymines on the 5' side of the ligand-binding site become hyperreactive to hydroxy-radical attack. Adjacent regions of An.Tn show enhanced rates of deoxyribonuclease-I cleavage in the presence of the antibiotic.

Circular dichroism (c.d.) and fluorescence spectroscopy have been used to investigate the interaction of the gene 5 protein of the filamentous bacteriophage Pf1 with single-stranded DNA. The c.d. spectrum of the Pf1 gene 5 protein is consistent with the absence of any significant alpha-helical content. The negative c.d. peak in the region of 210 nm, which arises from the protein, is diminished in the complex with poly(dT). Likewise, the c.d. peak at 265 nm arising from the poly(dT) decreases when the Pf1 gene 5 protein is bound, c.d. titrations of poly(dT) with Pf1 gene 5 protein indicate strong binding with a stoichiometry (n) of four nucleotides per protein subunit. In contrast, when the titrations were done using fluorescence anisotropy or fluorescence spectral shifts to follow binding, apparent stoichiometries between n = 2 and n = 4 were observed, often in the same experiment, depending on precise conditions. The results are interpreted in terms of two distinct modes of binding, in which either one or two subunits of the protein dimer are bound to the polynucleotide lattice, but still retaining the same local interaction with the DNA, with each binding site covering four nucleotides. The apparent stoichiometry of 2 results from the interaction of only one subunit of the dimer with the nucleic acid lattice, when protein is in excess. The second, unfilled, subunit of the dimer is nevertheless incorporated into the complex, resulting in the maximum possible fluorescence change when only half the sites are filled, since the fluorescence properties of the complex arise from protein-protein contacts associated with co-operative binding to the lattice. Further experiments in which the order of addition of components is changed, and the concentration of MgCl2 is varied, show that both of these factors are important in determining the dominant binding mode. In the absence of salt, dissociation and redistribution of the polynucleotide can occur following the addition of excess protein. This transition is suppressed in the presence of greater than 3 mM-MgCl2.

In addition to lead shielding, increased distance between the operator and x-ray source will lower radiation exposure. To utilize this principle, we interposed a 24 in. piece of pressure tubing between the catheter used for coronary angiography and the manifold apparatus. Radiation exposure to the hand of the operator during coronary angiography was compared with and without the extension tubing. When corrected for the differences in exposure time, operator exposure was 5.38 mrem/min without the extension and 4.84 mrem/min with the extension. Although this is a small difference in exposure/min, a substantial reduction in exposure could accumulate over a 1 yr period. Insertion of this extension tube into the catheter system is a simple and safe way to further reduce operator exposure during coronary angiography.

A prospective study was undertaken in order to test the efficacy of intermittent calf compression for prevention of thromboembolism in patients undergoing total hip arthroplasty. The patients were studied pre- and postoperatively by routine Doppler ultrasound examinations in addition to clinical assessment. Statistically significant evidence substantiates the view that intermittent calf compression decreased the incidence of thr thromboembolic problem.

One hundred thirty-four lower extremity amputees were evaluated from six months to 12 years postamputation by means of retrospective questionnaires. Patient population was similar to that of the "Amputee Census" in terms of sex, amputation level and cause of amputation. Information was gathered on activities generally considered essential for daily living, vocation and recreation, living arrangements and adjustments therein, as well as feedback on the patients' beliefs concerning what rehabilitation personnel should be doing to improve amputees' lifestyle. The relationship of functional outcome to age, amputation level, and cause of amputation was also evaluated. Results showed that most amputees did not resume a completely normal lifestyle and many modifications were made. The most popular recreational activities were fishing and swimming. Activities that amputees found most difficult were running and walking long distances. Patients requested better communication between professional staff and themselves. Below-knee amputees were significantly more independent than above-knee and bilateral amputees, but the differences between above-knee and bilateral amputees were statistically insignificant. Tumor patients did better than the other three etiologic groups. As age increased, functional independence decreased.