Knee mechanics: a review of past and present techniques to determine in vivo loads

This review article evaluates various techniques that have been used to determine in vivo loads in the human knee. Two main techniques that have been used are telemetry, which is an experimental approach, and mathematical modeling, which is a theoretical approach. Telemetric analyses have previously been used to determine the in vivo loading of the human hip and more recently evaluated in the determination of in vivo knee loads. Mathematical modeling approaches can be categorized two ways; those that use optimization techniques to solve an indeterminate system and those that utilize a reduction method that minimizes the number of unknowns, keeping the system solvable as the number of equations of motion are equal to the number of unknown quantities. More recently, we have developed an approach that relies fully on the use of in vivo data from fluoroscopy, CT scanning, magnetic resonant imaging and a revised motion analysis technique that involves only two markers on each rigid body. A review of all techniques revealed a wide range of forces at the human knee, ranging from 1.9 to 7.2 times body weight during level walking.     

Revision surgical hair restoration: repair of undesirable results

Surgical hair restoration has been performed as a treatment for male pattern hair loss for more than 40 years. Although techniques have changed dramatically over the past several years, making it possible to achieve natural-appearing results, there are still many patients with unacceptable outcomes. These patients may have had procedures performed in the past with antiquated techniques or performed recently with substandard techniques. The causes of unfavorable results can be classified into one of three categories: technical errors, poor planning, or complications. The results in these patients can be dramatically improved through a number of different reparative surgical techniques. The majority of these techniques can be performed in an office outpatient setting. More than 40 patients unsatisfied with previous surgical hair restoration have been treated with the different techniques reviewed in this article. All patients had successful outcomes with significant improvement in appearance. Despite the increased challenges when performing reparative surgery, outcomes were favorable in all patients, with small to significant improvements in appearance achieved. Some of these challenges include the limited supply of donor hairs, reduced scalp laxity, and theoretically reduced vascularity due to scarring and transected blood vessels, and patient skepticism. Furthermore, the few complications that occurred were minor and correctable, including one case each of poor hair growth associated with extensive small graft (consisting of one to four hairs) transplanting, and of scalp scarring associated with the removal and primary closure of a large number of "plug" grafts (typically grafts 3 to 4 mm in size consisting of seven or more hairs) in a single procedure.     

Estimating quantitative genetic parameters using sibships reconstructed from marker data

Previous techniques for estimating quantitative genetic parameters, such as heritability in populations where exact relationships are unknown but are instead inferred from marker genotypes, have used data from individuals on a pairwise level only. At this level, families are weighted according to the number of pairs within which each family appears, hence by size rather than information content, and information from multiple relationships is lost. Estimates of parameters are therefore not the most efficient achievable. Here, Markov chain Monte Carlo techniques have been used to partition the population into complete sibships, including, if known, prior knowledge of the distribution of family sizes. These pedigrees have then been used with restricted maximum likelihood under an animal model to estimate quantitative genetic parameters. Simulations to compare the properties of parameter estimates with those of existing techniques indicate that the use of sibship reconstruction is superior to earlier methods, having lower mean square errors and showing nonsignificant downward bias. In addition, sibship reconstruction allows the estimation of population allele frequencies that account for the relationships within the sample, so prior knowledge of allele frequencies need not be assumed. Extensions to these techniques allow reconstruction of half sibships when some or all of the maternal genotypes are known.     

The limited scar mastopexy: current concepts and approaches to correct breast ptosis

The literature on short scar mastopexy was reviewed, with a focus on the different techniques. Currently four techniques have been described: the periareolar, the vertical, the inverted-T, and the L-shaped scar. The different techniques were evaluated with regard to patient selection, operative techniques, scar length, and complications. A large number of techniques have been published for minimal ptosis, whereas for significant ptosis, the number of surgical options is limited. It is evident that limited scar techniques can be applied to all grades of ptosis, but there is no one technique that can satisfactorily correct all degrees of ptosis. Plastic surgeons should weigh the advantages and limitations of each technique to correctly address breast ptosis. This article reviews an algorithmic approach to correct all degrees of ptosis with mastopexy.     

Comparison and analysis of objective functions in flux balance analysis

Flux balance analysis (FBA) is currently one of the most important and used techniques for estimation of metabolic reaction rates (fluxes). This mathematical approach utilizes an optimization criterion in order to select a distribution of fluxes from the feasible space delimited by the metabolic reactions and some restrictions imposed over them, assuming that cellular metabolism is in steady state. Therefore, the obtained flux distribution depends on the specific objective function used. Multiple studies have been aimed to compare distinct objective functions at given conditions, in order to determine which of those functions produces values of fluxes closer to real data when used as objective in the FBA; in other words, what is the best objective function for modeling cell metabolism at a determined environmental condition. However, these comparative studies have been designed in very dissimilar ways, and in general, several factors that can change the ideal objective function in a cellular condition have not been adequately considered. Additionally, most of them have used only one dataset for representing one condition of cell growth, and different measuring techniques have been used. For these reasons, a rigorous study on the effect of factors such as the quantity of used data, the number and type of fluxes utilized as input data, and the selected classification of growth conditions, are required in order to obtain useful conclusions for these comparative studies, allowing limiting clearly the application range on any of those results. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:985–991, 2014     

TeloTool: a new tool for telomere length measurement from terminal restriction fragment analysis with improved probe intensity correction

Telomeres comprise the protective caps of natural chromosome ends and function in the suppression of DNA damage signaling and cellular senescence. Therefore, techniques used to determine telomere length are important in a number of studies, ranging from those investigating telomeric structure to effects on human disease. Terminal restriction fragment (TRF) analysis has for a long time shown to be one of the most accurate methods for quantification of absolute telomere length and range from a number of species. As this technique centers on standard Southern blotting, telomeric DNA is observed on resulting autoradiograms as a heterogeneous smear. Methods to accurately determine telomere length from telomeric smears have proven problematic, and no reliable technique has been suggested to obtain mean telomere length values. Here, we present TeloTool, a new program allowing thorough statistical analysis of TRF data. Using this new method, a number of methodical biases are removed from previously stated techniques, including assumptions based on probe intensity corrections. This program provides a standardized mean for quick and reliable extraction of quantitative data from TRF autoradiograms; its wide application will allow accurate comparison between datasets generated in different laboratories.     

Geometric morphometrics: recent applications to the study of evolution and development

The field of morphometrics is developing quickly and recent advances allow for geometric techniques to be applied easily to many zoological problems. This paper briefly introduces geometric morphometric techniques and then reviews selected areas where those techniques have been applied to questions of general interest. This paper is relevant to non-specialists looking for an entry into geometric morphometric methods and for ideas of how to incorporate them into the study of variation within and between species, the measurement of developmental stability, the role of development in shaping evolution and the special problem of measuring the shape of fossil specimens that are deformed from their original shape.     

Genetic Analysis of Voltage-Dependent Calcium Channels

Molecular cloning of calcium channel subunit genes has identified an unexpectedly large number of genes and splicing variants, and a central problem of calcium channel biology is to now understand the functional significance of this genetic complexity. While electrophyisological, pharmacological, and molecular cloning techniques are providing one level of understanding, a complete understanding will require many additional kinds of studies, including genetic studies done in intact animals. In this regard, an intriguing variety of episodic diseases have recently been identified that result from defects in calcium channel genes. A study of these diseases illustrates the kind of insights into calcium channel function that can be expected from this method of inquiry.     

Isolation and enrichment of murine spermatogonial stem cells using rhodamine 123 mitochondrial dye

Stem cells possess enormous therapeutic potential in tissue replacement. To study stem cells further, they must be isolated. Techniques are available for enrichment and study of hematopoietic stems cells, but thus far, techniques for purification of spermatogonial stem cells have not been described. Enrichment techniques for hematopoietic stem cells include the use of fluorescence-activated cell sorter analysis with Hoechst 33342 and rhodamine 123 (Rho) dyes. Use of Hoechst dye to isolate spermatogonial stem cells has been unsuccessful in our laboratory, and our results have conflicted with those from other laboratories. Taking advantage of the differential staining of the Rho dye, we report a novel method to enrich murine spermatogonial stem cells. Testicular cells are harvested from cryptorchid ROSA26 male mice. Populations of these cells are then stained with the Hoechst and Rho dyes, allowing them to be sorted by flow cytometry into a side population (SP) of Hoechst low-intensity cells and populations of low (Rho(low)) or high (Rho(hi)) fluorescent intensity. Sterile recipients, W/W(v) mice, with an intrinsic germ cell deficiency were transplanted with the Hoechst SP cells, Rho(low), Rho(hi), and nonsorted donor cells. No spermatogonial stem cell colonies were derived from the Hoechst SP cells. The number of spermatogonial stem cell colonies from transplanted Rho(low) cells showed a 17- and 20-fold enrichment over those of Rho(hi) and nonsorted cells, respectively.     

Differences in RNA patterns of influenza A viruses.

Analysis of the segmented RNAs of influenza A viruses by electrophoresis on polyacrylamide urea slab gels has provided a method for sharper resolution of the number and migration rates of different segments than previously has been possible. Using this system, the RNA genome of influenza A/WSN (HON1) virus can be separated into seven to nine separate bands, depending on whether virus is obtained after high or low multiplicity of infection, and the genome of influenza A/PR/8 (HON1) virus can be resolved into eight bands, six of which migrate differently from comparable RNA bands of WSN virus. Comparision of the RNA patterns produced by influenza A/PR/8 (HON1) and A/England/42/72 (H8n2) virus also reveals major differences in migration speeds of different bands, and analysis of the RNAs of the RNAs of an HON2 recombinant virus derived from these two strains permits the identification of RNA segments which have been derived from one particular parent. By extension of these techniques, it may be possible to define which RNA segment codes for each viral protein and to analyze recombinant strains to identify which genes have been derived from each of its parents.     

EcoRI activity: Enzyme modification or activation of accompanying endonuclease?

A study has been made of the factors and mechanism leading to appearance of the so-called EcoRI activity described by Polisky et al. (1975) in the restrictase EcoRI preparations. The preparations of purified restrictase EcoRI, precipitated at 0.9 ammonium sulphate saturation, as well as that obtained using standard techniques have been found to contain an admixture of an endonuclease which at neutral pH and high ionic strength multiply cleaves those DNAs which normally have only one recognition site for EcoRI. Under the standard conditions for EcoRI digestion this activity is found only when large amounts of freshly isolated enzyme are added to the incubation mixture and it is sharply enhanced by replacement of Mg2+ with Mn2+. The number and size of DNA fragments produced under such conditions practically do not differ from those found under the so-called EcoRI conditions, that is for alkaline pH values and low ionic strength. The optimum incubation mixture for the EcoRI activity has been found to be 10 mM Tris . HCl buffer (pH 8.8) + 2 mM Mn2+. Similar activity is induced also by addition to EcoRI solution of 40--50% glycerol or a number of organic solvents (dimethylacetamide (DMA), dimethylformamide (DMF), dimethylsulphoxide (DMSO), sulphalane (SP) in concentrations from 1 to 6%. The EcoRI activity induced by 50% glycerol or at alkaline pH values and low ionic strength is suppressed or sharply inhibited by 2--3 mM parachloromercuribenzoate (PCMB), while EcoRI is not sensitive to this agent. The DNA fragments cleaved by EcoRI have cohesive termini and can be easily ligated. It is suggested that the EcoRI activity can be due not only (or largely not) to modification of the "recognizing capacity" of the EcoRI restrictase but not activation of a latent specific endonuclease which is present in the restrictase preparation as an impurity.     

An experimental demonstration of the effect of group size on cultural accumulation

Cumulative culture is thought to have played a major role in hominin evolution, and so an understanding of the factors that affect cultural accumulation is important for understanding human evolution. Population size may be one such factor, with larger populations thought to be able to support more complex cultural traits. This hypothesis has been suggested by mathematical models and empirical studies of small-scale societies. However, to date there have been few experimental demonstrations of an effect of population size on cultural accumulation. Here we provide such a demonstration using a novel task, solving jigsaw puzzles. 80 participants divided into ten transmission chains solved puzzles in one of two conditions: one in which participants had access to one semi-completed puzzle from the previous generation, and the other in which participants simultaneously saw three semi-completed puzzles from the previous generation. As predicted, the mean number of pieces solved increased over time in the three-puzzle-per-generation condition, but not in the one-puzzle-per-generation condition. Thus, our experiment provides support for a hypothesized relationship between population size and cultural accumulation. In particular, our results suggest that the ability to simultaneously learn from multiple cultural models, and combine the knowledge of those multiple models, is most likely to allow larger groups to support more complex culture.     

Protein glycosylation analysis with capillary-based electromigrative separation techniques

An impressive complexity is associated with glycoproteins due to the microheterogeneity of glycosylation as posttranslational modification giving rise to a vast number of isoforms. The full characterization of glycoproteins is difficult to achieve, and a number of analytical methods have to be combined for a detailed understanding of glycosylation. In this review, we focus on capillary electromigrative separation techniques in the formats capillary electrophoresis, micellar electrokinetic chromatography, and capillary sieving electrophoresis. These separation techniques can be applied to all levels of glycosylation analysis including intact glycoproteins, glycopeptides, and released glycans. We here discuss the separation characteristics for each method and the information that they can provide for each level. Detection issues, especially laser-induced fluorescence detection and mass spectrometry are taken into account. In addition, tables provide an overview on the achievements made from the very beginning of glycosylation research by electromigrative separation techniques. From the literature presented here it is clear, that glycosylation analysis by electromigrative separation techniques is on the edge of transition of basic research and method development towards applications. First proof-of-principle studies for in-depth glycoprotein characterization and clinical diagnosis are described. However, this overview also shows that many basic aspects of separation have not yet been fully understood and more research is necessary to be able to fully use the capabilities of electromigrative separation techniques.     

Current concepts in membrane protein reconstitution

The reconstitution of integral proteins into artificial lipid vesicles is largely prompted by the complexity of most biological membranes and the inherent difficulty of studying individual components in situ. Ideally, therefore, the reconstituted system should consist of a single protein in a lipid matrix which mimics the native membrane in all but its diversity. While such an approach allows individual components of a complex system to be studied in isolation it should also be sufficiently versatile to permit the generation of increasingly sophisticated multicomponent models. From the considerable number of reconstitution techniques which have been developed I have tried in this review to identify those characteristics of a particular system which maximise both the information it can provide and its versatility.     

Structural proteomics by NMR spectroscopy

Structural proteomics is one of the powerful research areas in the postgenomic era, elucidating structure-function relationships of uncharacterized gene products based on the 3D protein structure. It proposes biochemical and cellular functions of unannotated proteins and thereby identifies potential drug design and protein engineering targets. Recently, a number of pioneering groups in structural proteomics research have achieved proof of structural proteomic theory by predicting the 3D structures of hypothetical proteins that successfully identified the biological functions of those proteins. The pioneering groups made use of a number of techniques, including NMR spectroscopy, which has been applied successfully to structural proteomics studies over the past 10 years. In addition, advances in hardware design, data acquisition methods, sample preparation and automation of data analysis have been developed and successfully applied to high-throughput structure determination techniques. These efforts ensure that NMR spectroscopy will become an important methodology for performing structural proteomics research on a genomic scale. NMR-based structural proteomics together with x-ray crystallography will provide a comprehensive structural database to predict the basic biological functions of hypothetical proteins identified by the genome projects.     

Engineering clinically relevant volumes of vascularized bone

Vascularization remains one of the most important challenges that must be overcome for tissue engineering to be consistently implemented for reconstruction of large volume bone defects. An extensive vascular network is needed for transport of nutrients, waste and progenitor cells required for remodelling and repair. A variety of tissue engineering strategies have been investigated in an attempt to vascularize tissues, including those applying cells, soluble factor delivery strategies, novel design and optimization of bio‐active materials, vascular assembly pre‐implantation and surgical techniques. However, many of these strategies face substantial barriers that must be overcome prior to their ultimate translation into clinical application. In this review recent progress in engineering vascularized bone will be presented with an emphasis on clinical feasibility.