SORS: A Universal One-Round PCR-Based Method for Site-Directed Mutagenesis

We have developed a novel protocol for site-directed mutagenesis of double-stranded DNA. The procedure, termed SORS (named because it undergoes the sequential procedure of segmentation-overhang creating PCR-reannealing-splicing) mutagenesis, is exemplified by a substitution, a deletion, and an insertion of nucleotide(s) in target genes. The template DNA is PCR-amplified into two separate segments divided at the prospective mutation site, and each segment is amplified in two parallel PCRs using primers introducing the mutation. The primers are designed to be able to create protruding bases upon pooling, denaturing, and reannealing the two parallel reactions. The protruding bases at the prospective junction of the two segments are mutually complementary; therefore, the two segments can be re-spliced together to generate the mutated gene. Compared to previously published protocols, this procedure is rapid, restriction-independent and ensures higher success rate and lower potential to produce second-site mutations.     

A Hemodilution Technique for Open-Heart Surgery: A Preliminary Clinical Report

With the extension of open-heart procedures and the increasing success of open-heart surgery, a great strain has been placed on many hospital laboratories and on the Red Cross for the maintenance of equipment and the supply of blood. In order to decrease both of these needs, hemodilution techniques combined with disposable oxygenators have been introduced. The authors have used a disposable oxygenator with low molecular weight dextran prime, along with moderate hypothermia, in a consecutive series of 15 patients operated upon for congenital and acquired heart lesions. In this consecutive series there has been one death and this could not be attributed to the perfusion technique but to the surgical procedure carried out. It is felt that this combination not only broadens the horizons of open-heart surgery but makes the procedure more readily available and safer, as well as being economical of blood donors and time.     

Estimating reproductive success of primates and other animals with long and incompletely known reproductive life spans

Lifetime reproductive success is difficult to obtain for adequate numbers of wild, long-lived animals because of incomplete knowledge of reproductive life histories and fates of offspring. A procedure is described that provides a standard estimate of lifetime reproductive success from either complete or incomplete reproductive life spans. For this procedure, reproductive success is defined as the following ratio: the number of offspring living to a criterion age in a given time period divided by the number expected for that age and period. The number expected is based upon the mean inter-birth interval of the population, the length of the time period, the age criterion of reproductive success chosen by the researcher (e.g., the average age of menarche), and the probability that an offspring will live to the criterion age. The effect of error upon the estimation of parameters is analyzed using the yellow baboons of Mikumi National Park as an example. The method can be used for both sexes, any litter (clutch) size, any mating system, any mean length of interbirth interval, any age criterion of reproductive success, and any study length that allows a reasonably sized sample of individuals for each of whom a substantial proportion of their reproductive life span is known. © 1992 Wiley-Liss, Inc.     

When Do Environment–recruitment Correlations Work?

I review the role of environmental variability in the survival of juvenile fish and shellfish by examining the success of previously published environment–recruitment correlations when tested with new data. The proportion of published correlations that have been verified upon retest is low. There is one generalization that stands out: correlations for populations at the limit of a species' geographical range have often remained statistically significant when re-examined. An examination of environment–recruitment correlations that were reviewed 13 years ago by Shepherd and co-workers shows that only 1 out of 47 reviewed studies is currently used in the estimation of recruitment in routine assessments. The results suggest that future progress will require testing general hypotheses using data from many populations.     

Fluorometric enzyme assay for choline and acetylcholine

A sensitive and specific assay for choline and ACh which may be applied directly to brain extracts is described. The method is based upon enzymic coupling to the oxidation of fluorescent NADH. The following enzymic sequence is utilized: acetylcholinesterase, choline phosphokinase, pyruvate kinase, and lactate dehydrogenase. The method detects as little as 0.1 mμmole of choline or ACh, which is the amount of metabolite present in 1 mg or 8 mg of whole rat brain, respectively. The specificity of the method is such that only choline and ACh of tissue samples react. Extraction of whole brain samples by either heating at pH 4 or by chloroform/methanol or perchloric acid were compared in order to find a single procedure which was useful for extraction of both ACh and free choline from brain samples. Perchloric acid extraction proved to be the most efficient of the three methods for extraction of the two constituents. By this procedure the ACh content of whole rat brain was found to be 11.5 mμmoles/gm and the choline content of the same samples was 107 mμmoles/gm. Both values are in agreement with other published results.     

Performance of the first protein docking server ClusPro in CAPRI rounds 3-5

To evaluate the current status of the protein-protein docking field, the CAPRI experiment came to life. Researchers are given the receptor and ligand 3-dimensional (3D) coordinates before the cocrystallized complex is published. Human predictions of the complex structure are supposed to be submitted within 3 weeks, whereas the server ClusPro has only 24 h and does not make use of any biochemical information. From the 10 targets analyzed in the second evaluation meeting of CAPRI, ClusPro was able to predict meaningful models for 5 targets using only empirical free energy estimates. For two of the targets, the server predictions were assessed to be among the best in the field. Namely, for Targets 8 and 12, ClusPro predicted the model with the most accurate binding-site interface and the model with the highest percentage of nativelike contacts, among 180 and 230 submissions, respectively. After CAPRI, the server has been further developed to predict oligomeric assemblies, and new tools now allow the user to restrict the search for the complex to specific regions on the protein surface, significantly enhancing the predictive capabilities of the server. The performance of ClusPro in CAPRI Rounds 3-5 suggests that clustering the low free energy (i.e., desolvation and electrostatic energy) conformations of a homogeneous conformational sampling of the binding interface is a fast and reliable procedure to detect protein-protein interactions and eliminate false positives. Not including targets that had a significant structural rearrangement upon binding, the success rate of ClusPro was found to be around 71%.     

Analysis of recursive gene selection approaches from microarray data

MOTIVATION: Finding a small subset of most predictive genes from microarray for disease prediction is a challenging problem. Support vector machines (SVMs) have been found to be successful with a recursive procedure in selecting important genes for cancer prediction. However, it is not well understood how much of the success depends on the choice of the specific classifier and how much on the recursive procedure. We answer this question by examining multiple classifers [SVM, ridge regression (RR) and Rocchio] with feature selection in recursive and non-recursive settings on three DNA microarray datasets (ALL-AML Leukemia data, Breast Cancer data and GCM data). RESULTS: We found recursive RR most effective. On the AML-ALL dataset, it achieved zero error rate on the test set using only three genes (selected from over 7000), which is more encouraging than the best published result (zero error rate using 8 genes by recursive SVM). On the Breast Cancer dataset and the two largest categories of the GCM dataset, the results achieved by recursive RR are also very encouraging. A further analysis of the experimental results shows that different classifiers penalize redundant features to different extent and this property plays an important role in the recursive feature selection process. RR classifier tends to penalize redundant features to a much larger extent than the SVM does. This may be the reason why recursive RR has a better performance in selecting genes.     

Emergency Preservation and Resuscitation Trial: A Philosophical Justification for Non‐Voluntary Enrollment

In a current clinical trial for Emergency Preservation and Resuscitation (EPR), Dr. Samuel Tisherman of the University of Maryland aims to induce therapeutic hypothermia in order to ‘buy time’ for operating on victims of severe exsanguination. While recent publicity has framed this controversial procedure as ‘killing a patient to save his life’, the US Army and Acute Care Research appear to support the study on the grounds that such patients already face low chances of survival. Given that enrollment in the trial must be non‐voluntary, the study has received an exemption from federal standards for obtaining informed consent. How exactly, if at all, is non‐voluntary enrollment morally justifiable? In this essay, I appeal to the notable work of Hans Jonas in an effort to defend the EPR trial's use of non‐voluntary enrollment. It is often thought and, as I show, it may appear that Jonas has called for the end of experimental medical practice. Still, I derive from Jonas a principle of double‐effect upon which physicians may be seen as morally permitted to pursue innovations in emergency medicine but only as a byproduct of pursuing therapeutic success. With this position, I argue that the EPR trial can be granted a stronger philosophical justification than simply waiving the requirement of obtaining informed consent. The double‐effect justification would obtain, perhaps regardless of the success of such innovative procedures as therapeutic hypothermia.     

The purification of peptides which contain methionine residues

A novel procedure for isolating peptides which contain methionine is described. It relies upon the reversible increase in charge which occurs upon the alkylation of methionine by iodoacetamide. A digest of the protein is reacted with lodo[¹⁴C]acetamide under conditions which direct the reaction exclusively to the methionine residues. In this way, methionine-containing peptides are rendered radioactive and gain one positive charge per methionine simultaneously. The digest is then separated on a cation exchange column, the peptides are located by their radioactivity, and they are separately collected. The carboxyamidomethylation is reversed by thiolysis, which eliminates the extra positive charge which each methionine-containing peptide bore, decreasing their charge selectively. A second chromatographic separation, performed on the same cation exchange column, is sufficient to produce the desired peptides in a high state of purity. Equine myoglobin and bovine ribonuclease were used as models to demonstrate the feasibility of this approach. Methionine-containing tryptic peptides were purified from digests of these proteins in yields which were equivalent to those of previously reported separations. The present procedure, however, is applicable to peptide mixtures of far greater complexity than those which were derived from the model compounds and can be applied with the same success to digests of very large proteins containing many methionine residues.     

The Scientific Impact of Nations: Journal Placement and Citation Performance

International collaboration is becoming increasingly important for the advancement of science. To gain a more precise understanding of how factors such as international collaboration influence publication success, we divide publication success into two categories: journal placement and citation performance. Analyzing all papers published between 1996 and 2012 in eight disciplines, we find that those with more countries in their affiliations performed better in both categories. Furthermore, specific countries vary in their effects both individually and in combination. Finally, we look at the relationship between national output (in papers published) and input (in citations received) over the 17 years, expanding upon prior depictions by also plotting an expected proportion of citations based on Journal Placement. Discrepancies between this expectation and the realized proportion of citations illuminate trends in performance, such as the decline of the Global North in response to rapidly developing countries, especially China. Yet, most countries'' show little to no discrepancy, meaning that, in most cases, citation proportion can be predicted by Journal Placement alone. This reveals an extreme asymmetry between the opinions of a few reviewers and the degree to which paper acceptance and citation rates influence career advancement.     

Energetic constraints on clutch size and time of breeding in temperate zone birds

Summary A model for the energy budget of a breeding pair of Great Tits is synthesized from published data. The model is used to investigate the relationship between temperature, clutch size, onset of laying, and male feeding on breeding success. Two periods of energetic stress are found, one at the end of the incubation period which is independent of the clutch size, and one at the end of the fledging period which does depend upon the clutch size. This energetic bottleneck theory is easily testable and is in agreement with existing data. The model also provides a consistent explanation of the effects of reduced male feeding, and changes in clutch size and nest box insulation on breeding success.     

Determination of N-acetyl-L-glutamate using high-performance liquid chromatography

Acetylglutamate in HClO4 tissue extracts is first separated from glutamate by ion exchange. It is then deacylated with aminoacylase, and the resulting glutamate, after adsorption to and elution from an AG 50 column, is quantitated by a fast-HPLC method using o-phthaldialdehyde precolumn derivatization, separation in a C18 reverse-phase column, and fluorescence detection. A linear response is obtained up to 2 nmol, the detection limit is 5 pmol, and the method is suitable for assay in 1 mg liver tissue and thus for needle biopsies. When samples were analyzed by this procedure and by earlier procedures based upon detection of glutamate with glutamate dehydrogenase or upon activation of carbamoyl phosphate synthetase the results were similar. The method, which is highly specific, compares favorably in sensitivity, precision, and accuracy with all other published procedures. Using this assay, no acetylglutamate has been found in chicken liver and rat kidney.     

Histamine immunohistochemistry: a new and highly sensitive method for studying cutaneous mast cells

Summary Mast cells have characteristic granulae containing various glucoseaminoglycans, proteases and amines (predominantly histamine). The conventional histological methods for studying mast cells are based upon acidic ortho-and metachromatic routine stains of the glucoseaminoglycans. However, the success of these procedures is dependent upon both the fixatives and the tissues used. In this study, we wanted to find out whether an immunohistochemical procedure could overcome some of these difficulties. Normal human skin was fixed in five different types of fixative and processed for indirect immunofluorescence, using an antiserum to histamine. Only one, 4% carbodiimide in 0.1m phosphate buffer (pH 7.4), resulted in immunostaining. The quality of the staining was good, with a high signal-to-noise ratio, and was located on the mast cells. The method, made it possible to visualize small structures such as a single secreted granula, the thin cytoplasmatic extension of some cells, and a previously undescribed dendritic morphology of some of the mast cells. We therefore recommend, this procedure for cellular studies of mast cells when accuracy is needed.     

WHY DOES A METHOD THAT FAILS CONTINUE TO BE USED? THE ANSWER

It has been claimed that hundreds of researchers use nested clade phylogeographic analysis (NCPA) based on what the method promises rather than requiring objective validation of the method. The supposed failure of NCPA is based upon the argument that validating it by using positive controls ignored type I error, and that computer simulations have shown a high type I error. The first argument is factually incorrect: the previously published validation analysis fully accounted for both type I and type II errors. The simulations that indicate a 75% type I error rate have serious flaws and only evaluate outdated versions of NCPA. These outdated type I error rates fall precipitously when the 2003 version of single-locus NCPA is used or when the 2002 multilocus version of NCPA is used. It is shown that the tree-wise type I errors in single-locus NCPA can be corrected to the desired nominal level by a simple statistical procedure, and that multilocus NCPA reconstructs a simulated scenario used to discredit NCPA with 100% accuracy. Hence, NCPA is a not a failed method at all, but rather has been validated both by actual data and by simulated data in a manner that satisfies the published criteria given by its critics. The critics have come to different conclusions because they have focused on the pre-2002 versions of NCPA and have failed to take into account the extensive developments in NCPA since 2002. Hence, researchers can choose to use NCPA based upon objective critical validation that shows that NCPA delivers what it promises.     

A membrane model describing the effect of temperature on the water conductivity of erythrocyte membranes at subzero temperatures

Thermodynamic models show that the loss of intracellular water from human erythrocytes during freezing depends heavily upon the water conductivity of the erythrocyte membrane. These calculations, which are based on the simple extrapolation of ambient conductivity data to subzero temperatures, show that more than 95% of cell water is transferable during freezing, whereas experiments show that at least 20% of cell water is retained. A study of the effects of different published values for the membrane water conductivity on cell water retained during freezing shows that this discrepancy may be a consequence of the simple extrapolation procedure.For a homogeneous membrane system, absolute reaction rate theory was used to develop a surface-limited permeation model that includes the resistance to the flow of water not only through the interior region of the membrane but also across possible rate-limiting barriers at the solution-membrane interfaces. The model shows that it is unlikely that a single ratelimiting process dominates water transport in the red cell as it is being cooled from ambient to subzero temperatures. The effective membrane conductivity at subzero temperatures could possibly be much lower than a simple extrapolation of existing data would predict. With the aid of this model analytical predictions of intracellular water during freezing are more consistent with experimental observations.     

Integrating atom-based and residue-based scoring functions for protein-protein docking

Most scoring functions for protein-protein docking algorithms are either atom-based or residue-based, with the former being able to produce higher quality structures and latter more tolerant to conformational changes upon binding. Earlier, we developed the ZRANK algorithm for reranking docking predictions, with a scoring function that contained only atom-based terms. Here we combine ZRANK's atom-based potentials with five residue-based potentials published by other labs, as well as an atom-based potential IFACE that we published after ZRANK. We simultaneously optimized the weights for selected combinations of terms in the scoring function, using decoys generated with the protein-protein docking algorithm ZDOCK. We performed rigorous cross validation of the combinations using 96 test cases from a docking benchmark. Judged by the integrative success rate of making 1000 predictions per complex, addition of IFACE and the best residue-based pair potential reduced the number of cases without a correct prediction by 38 and 27% relative to ZDOCK and ZRANK, respectively. Thus combination of residue-based and atom-based potentials into a scoring function can improve performance for protein-protein docking. The resulting scoring function is called IRAD (integration of residue- and atom-based potentials for docking) and is available at http://zlab.umassmed.edu.     

Isolation and characterization of two distinct forms of protein kinase C

Protein kinase C (Ca2+- and phospholipid-dependent protein kinase) has been purified from rat brain by a three-step, 18-h procedure resulting in the isolation of milligram quantities of enzyme. Unlike previous preparations from published protocols, which yield a single polypeptide, this procedure yields a protein which consists of a 78/80-kDa doublet upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The two polypeptides have been characterized with respect to structure and function and are very similar in both regards. However, the two forms can be distinguished immunologically by polyclonal antisera generated against purified protein kinase C. The 78- and 80-kDa proteins do not appear to be related to one another by proteolytic cleavage or by differential phosphorylation, although the two purified proteins do contain stoichiometric amounts of phosphate. The 78- and 80-kDa polypeptides therefore appear to represent two distinct forms of protein kinase C, thus providing evidence for the existence of multiple isozymes of this key regulatory protein.     

Genetic Analysis of Male Reproductive Contributions in Chamaelirium Luteum (L.) Gray (Liliaceae)

Genealogical analysis is a powerful tool for analysis of reproductive performance in both natural and captive populations, but assignment of paternity has always been a stumbling block for this sort of work. Statistical methods for determining paternity have undergone several phases of development, ranging from straightforward genetic exclusion to assignment of paternity based on genetic likelihood criteria. In the present study, we present a genetic likelihood-based iterative procedure for fractional allocation of paternity within a progeny pool and apply this method to a population of Chamaelirium luteum, a dioecious member of the Liliaceae. Results from this analysis clearly demonstrate that different males make unequal contributions to the overall progeny pool, with many males contributing essentially nothing to the next generation. Furthermore, the distribution of paternal success among males shows a highly significant departure from (Poisson) randomness. The results from the present analysis were compared with earlier results obtained from the same data set, using likelihood-based categorical paternity assignments. The general biological pattern revealed by the two analyses is the same, but the estimates of reproductive success are only modestly (though significantly) correlated. The iterative procedure makes more complete use of the data and generates a more sharply resolved distribution of male reproductive success.     

An optimisation approach to increase DNA amplification success of otter faeces

Faeces have proved to be a suitable non-invasive DNA source for microsatellite analysis in wildlife research. For the success of such studies it is essential to obtain the highest possible PCR amplification success rate. These rates are still relatively low in most carnivorous species, especially in the otter (Lutra lutra). We therefore optimised the entire microsatellite genotyping process by combining our findings with results from previous studies to gain a high rate of reliable genotypes. We investigated the influence of otter faecal quality in relation to the quantity of slimy secretions and three levels of storage periods at ?20°C on amplification success. Further, we tested the cost-effective and time-saving Chelex extraction method against the profitable QIAamp^® DNA Stool Kit (Qiagen), and compared three PCR methods - a standard single-step PCR protocol, a single-locus two-step PCR procedure and a multiplex two-step PCR procedure - regarding success rate and genotyping errors. The highest amplification success rate (median: 94%; mean: 78%) was achieved using faecal samples consisting only of jelly extracted with the QIAamp^® DNA Stool Mini Kit (Qiagen) immediately after collection and amplified following the time and cost efficient multiplex two-step PCR protocol. The two-step procedure, also referred to as pre-amplification approach, turned out to be the main improvement as it increases amplification success about 11% and reduces genotyping errors about 53%, most notably allelic dropouts.