Geometric versus topological clustering: An insight into conformation mapping

Clustering molecular conformations into “families” is a common procedure in conformational analysis of molecular systems. An implicit assumption which often underlies this clustering approach is that the resulting geometric families reflect the energetic structure of the system's potential energy surface. In a broader context we address the question whether structural similarity is correlated with energy basins, i.e., whether conformations that belong to the same energy basin are also geometrically similar. “Topological mapping” and principal coordinate projections are used here to address this question and to assess the quality of the “family clustering” procedure. Applying the analysis to a small tetrapeptide it was found that the general correlation that exists between energy basins and structural similarity is not absolute. Clusters generated by the geometric “family clustering” procedure do not always reflect the underlying energy basins. In particular it was found that the “family tree” that is generated by the “family clustering” procedure is completely inconsistent with its real topological counterpart, the “disconnectivity” graph of this system. It is also demonstrated that principal coordinate analysis is a powerful visualization technique which, at least for this system, works better when distances are measured in dihedral angle space rather than in cartesian space. © 1997 Wiley-Liss, Inc.     

An iterative approach from the standpoint of the additive hypothesis to the dendrogram problem posed by molecular data sets

The problem of constructing a dendrogram depicting phylogenetic relationships for a collection of contemporary species is considered. An approach was developed based on the additive hypothesis in which each “length” between two species can be described by the shortest sum of lengths for the individual links on the dendrogram topology which connect the two species. The additive hypothesis holds equally well if the dendro gram is replaced by its corresponding (rootless) network. Network topologies are defined set theoretically in terms of the initial, contemporary species, and a coefficient is defined for each point of any conceivable network. It is proved mathematically that each point of an additive network gives a coefficient value of zero, whereas each point not belonging to an additive network gives a coefficient value greater than zero. This suggests an iterative procedure in which “false” network points are replaced by “true” ones, or more generally in which “very false” network points are replaced by “nearly true” ones. The first procedure follows from the mathematical proof and the second is confirmed by simulation. Since most real data sets are not additive in the strict sense, a real data example was presented in which the iterative procedure produced a plausible network topology.     

Cell wall glycoproteins and polysaccharides of mature runner beans

A novel use of chlorite-HOAc treatment (delignification procedure) for the isolation of hydroxyproline (HP) rich “glycoproteins” from the depectinated cell wall material of mature runner beans is described. This procedure can be used for the isolation of wall proteins even from heavily lignified tissues. Its main disadvantage is that some of the constituent amino acids are either destroyed or modified; the nature of these changes was studied using gelatine, lysozyme and “cytoplasmic proteins” of mature beans. The main amino acids to be affected were tyrosine, cystine, methionine and lysine. The chlorite-HOAc solubilized proteins were separated by PhOH-H₂O fractionation into two distinct “glycoprotein fractions”. The major fraction (isolated from the aqueous layer) contained most of the HP of the solubilized proteins. The sugars obtained on hydrolysis of both “glycoproteins” were galactose, arabinose, glucose, xylose, rhamnose and uronic acid. Most of the proteins remaining in the holocellulose could readily be extracted with cold alkali and were relatively poor in HP.     

Outgroup sampling in phylogenetics: Severity of test and successive outgroup expansion

Outgroup sampling is a fundamental step in the design of phylogenetic analyses, independent of optimality criterion, taxonomic group, or source of evidence. Studies have demonstrated the efficient analysis of many thousands of terminals, all of which could be included in any empirical investigation, yet outgroup samples typically include only a small number of terminals. Most discussion of outgroup sampling centers on employing “correct” or “appropriate” outgroup terminals to increase “accuracy” or “reliability” by preventing “errors” such as long branch attraction and “incorrect” ingroup rooting. As an alternative, I develop a theory of outgroup sampling grounded in the logic of scientific discovery, whereby the objective is to test nested hypotheses of ingroup topology and character‐state transformation as severely as possible by incorporating outgroup terminals in unconstrained, simultaneous analysis, using background knowledge to select the terminals that have the greatest chance of refuting those hypotheses. This framework provides a logical basis for selecting outgroup taxa but does not provide grounds for limiting the outgroup sample, given that, ceteris paribus, testability and explanatory power increase with the inclusion of additional terminals. Therefore, I propose the ancillary procedure of successively expanding the outgroup sample until ingroup hypotheses become stable (insensitive) to increased sampling, with each expansion guided by the scientific objectives of outgroup sampling. This is a heuristic procedure that does not prevent more outgroup terminals from being sampled or guarantee that ingroup hypotheses will remain insensitive to further outgroup expansion, and it has no bearing on the objective support of a given hypothesis. Nevertheless, it provides an objective, empirical basis for limiting outgroup sampling in a given research cycle. I illustrate this procedure by examining the effect of successive outgroup expansion on the relationships among the poison frog genera Adelphobates, Dendrobates, and Oophaga.     

Current status of the organ replacement approach for malignancies and an overture for organ bioengineering and regenerative medicine

Significant achievements in the organ replacement approach for malignancies over the last 2 decades opened new horizons, and the age of “Transplant Oncology” has dawned. The indications of liver transplantation for malignancies have been carefully expanded by a strict patient selection to assure comparable outcomes with non-malignant diseases. Currently, the Milan criteria, gold standard for hepatocellular carcinoma, are being challenged by high-volume centers worldwide. Neoadjuvant chemoradiation therapy and liver transplantation for unresectable hilar cholangiocarcinoma has been successful in specialized institutions. For other primary and metastatic liver tumors, clinical evidence to establish standardized criteria is lacking. Intestinal and multivisceral transplantation is an option for low-grade neoplasms deemed unresectable by conventional surgery. However, the procedure itself is in the adolescent stage. Solid organ transplantation for malignancies inevitably suffers from “triple distress,” i.e., oncological, immunological, and technical. Organ bioengineering and regenerative medicine should serve as the “triple threat” therapy and revolutionize “Transplant Oncology.”     

Electron microscopy and restriction enzyme mapping reveal additional intervening sequences in the chicken ovalbumin split gene

The Eco RI fragment “b” of chicken DNA (Breathnach, Mandel and Chambon, 1977), which contains the sequences coding for the 5′ quarter of ovalbumin mRNA (ov mRNA), has been isolated by molecular cloning using a “shotgun” approach. Electron microscopy and restriction enzyme analysis have revealed that the sequences coding for the 5′ quarter (～500 nucleotides) of ov mRNA are split into four regions separated by three intervening sequences. The cloning procedure seems to be reliable, since the restriction enzyme pattern of the cloned Eco RI fragment “b” is similar to that of the corresponding chromosomal DNA fragment. There is no evidence supporting the existence of a 150–200 nucleotide long sequence at the 5′ end of the ov mRNA similar to the “leader” sequences found at the 5′ end of some adenovirus and SV40 mRNAs.     

Purification of Uricase by Biospecific Adsorption-Desorption

A simple procedure for the purification of uricase from bovine kidney is described. The procedure involves the following steps: 1) processing of kidney mince by borate/butanol, 2) ammonium sulphate precipitation, and 3) biospecific adsorption-desorption. The adsorbents were prepared by chemical attachment of urate or xanthine to agarose gel beads. The desorption was performed by a xanthine solution. The adsorption-desorption procedure resulted in an 11 000–12 000-fold purification. The specific activity of the purified uricase was 19.8 U/mg using either “urate” or “xanthine” adsorbent. The recovery was about 70%.The adsorbents were also used for the purification of commercial uricase preparations from hog liver. In this case the purified uricase also possessed a specific activity of 19.8 U/mg. The products were homogenous as judged by gradipore electrophoresis and gel filtration.     

Radioimmunoassay of met-enkephalin in microdissected areas of paraformaldehyde-fixed rat brain

We studied the effect of various sample preparation procedures on rat brain met-enkephalin content, measured by radioimmunoassay. Whole brain met-enkephalin content of rats killed by decapitation followed by immediate tissue freezing was similar to that of rats killed by microwave irradiation and to those of rats anesthetized with pentobarbital or halothane before killing, whether previously perfused with paraformaldehyde or not. In contrast, a decrease (up to 80%) in met-enkephalin concentrations was observed when brain samples were frozen and thawed to mimic the procedure utilized in the “punch” technique for analysis of discrete brain nuclei. This decrease was totally prevented by paraformaldehyde perfusion of the brain prior to sacrifice. Brain perfusion did not alter the amount of immunoassayable met-enkephalin extracted from tissue or its profile after Sephadex chromatography. Paraformaldehyde perfusion results in better morphological tissue preservation and facilitates the “punch” dissecting technique. Paraformaldehyde perfusion may be the procedure of choice for the measurement of neuropeptides in specific brain nuclei dissected by the “punch” technique.     

A simple method to sequentially reveal Q- and C-bands on the same metaphase chromosomes

The Q-C staining procedure, i.e., to treat QM stained preparations with a modified ASG method, conveniently provides Q? and C? (instead of the expected G?) bands on the same metaphase chromosomes. This procedure is especially useful for karyological study of heteroploid cells and interspecific cell hybrids in which extensive chromosomal rearrangements and complex karyotypic compositions are present. A few examples of karyological interest are reported. C-bands, which are either Q+ or Q?, can be divided into two to three subsegments in human chromosomes 1, 9, and 16. These subsegments are connected by a Q?/C?element. Interstitial C-bands could have originated mostly from a C-band without the kinetochore or with the“non-functional” kinetochore.“Double Minutes” are of two kinds, Q+/C+ and Q?/C+. Mechanism for the production of C-bands by the Q-C procedure is briefly discussed.     

The preparation of high molecular weight divalent "antigens" of defined size

A simple, rapid procedure for the synthesis of divalent “antigens” that consist of high molecular weight polyethylene oxide chains bearing dinitrophenyl groups at their ends and for their fractionation to near size-homogeneity is described.     

Mapping species abundance by a spatial zero‐inflated Poisson model: a case study in the Wadden Sea,the Netherlands

The objective of the study was to provide a general procedure for mapping species abundance when data are zero‐inflated and spatially correlated counts. The bivalve species Macoma balthica was observed on a 500×500 m grid in the Dutch part of the Wadden Sea. In total, 66% of the 3451 counts were zeros. A zero‐inflated Poisson mixture model was used to relate counts to environmental covariates. Two models were considered, one with relatively fewer covariates (model “small”) than the other (model “large”). The models contained two processes: a Bernoulli (species prevalence) and a Poisson (species intensity, when the Bernoulli process predicts presence). The model was used to make predictions for sites where only environmental data are available. Predicted prevalences and intensities show that the model “small” predicts lower mean prevalence and higher mean intensity, than the model “large”. Yet, the product of prevalence and intensity, which might be called the unconditional intensity, is very similar. Cross‐validation showed that the model “small” performed slightly better, but the difference was small. The proposed methodology might be generally applicable, but is computer intensive.     

Second-order control of sequence-class equivalences in children

Children learned matching-to-sample tasks to establish two equivalence classes. Then, one member from each class appeared in a sequence procedure, thereby acquiring the ordinal properties “first” and “second”. When the remaining members in the two equivalence classes were placed in the sequence context, subjects responded in appropriate order without additional training. The data suggest a basic mechanism which can account for the production of new sequence behavior which has no explicit history of training.     

改良手工显微切割法获取特定细胞提取高质量RNA

探讨显微切割过程中有效保持RNA完整性的组织固定方法,建立一种简易的手工显微切割法.应用自制“T形板”辅助冰冻切片,100％无水乙醇一次性脱水固定,“排除切割法”获取目的细胞,用TRIzol提取RNA,琼脂糖凝胶电泳和RT-PCR分析RNA质量.“一步法”固定可长时间保存RNA的完整性;从食管癌标本5个特定阶段的细胞中提取的RNA,经电泳和RT-PCR分析均具有较高的质量.无水乙醇“一步法”固定,在显微切割的过程中可有效保持RNA的完整性;T形板和“排除切割法”简化了手工显微切割的操作,提取的RNA质、量均可满足后续分子水平研究的需要.     

A Two-Stage Test for Distinguishing Random,Pseudo-Random and Nonrandom Mating Populations

There is no such an implication that a population in Hardy-Weinberg equilibrium must have undergone random mating. Therefore, it is unequivocal that the usual tests for “Hardy-Weinberg equilibrium” are indeed tests for “random union of gametes” but not for “random mating”. In this paper, utilizing population characteristics expressed in equilibrium state (equilibrium or disequilibrium) and mating behavior (random or nonrandom), a two-stage testing procedure for distinguishing random, pseudo-random and nonrandom mating populations is proposed. At the first stage, a population is tested for Hardy-Weinberg equilibrium. If insignificant result (i.e., in equilibrium) is obtained, then to a second stage the population is further tested for mating behavior. Random mating-pairs data are needed here for analysis instead of random individuals for usual Hardy-Weinberg equilibrium tests. Since distinguishing the three types of mating populations depends on the combined results of two stages, the probability of correct determination of the two-stage tests is discussed by simulation studies.     

A weighted FDR procedure under discrete and heterogeneous null distributions

Multiple testing (MT) with false discovery rate (FDR) control has been widely conducted in the “discrete paradigm” where p-values have discrete and heterogeneous null distributions. However, in this scenario existing FDR procedures often lose some power and may yield unreliable inference, and for this scenario there does not seem to be an FDR procedure that partitions hypotheses into groups, employs data-adaptive weights and is nonasymptotically conservative. We propose a weighted p-value-based FDR procedure, “weighted FDR (wFDR) procedure” for short, for MT in the discrete paradigm that efficiently adapts to both heterogeneity and discreteness of p-value distributions. We theoretically justify the nonasymptotic conservativeness of the wFDR procedure under independence, and show via simulation studies that, for MT based on p-values of binomial test or Fisher's exact test, it is more powerful than six other procedures. The wFDR procedure is applied to two examples based on discrete data, a drug safety study, and a differential methylation study, where it makes more discoveries than two existing methods.     

Subcellular localization of 3-methylcrotonyl-coenzyme A carboxylase in bovine kidney

The intracellular localization of 3-methylcrotonyl-CoA carboxylase (MCase), an enzyme of the leucine oxidative pathway, was studied in bovine kidney. Differential centrifugation of kidney homogenates demonstrated that the majority of the enzyme was associated with the mitochondrial and cytosolic fractions. Isopycnic centrifugation of the mitochondrial fraction demonstrated cofractionation of MCase with mitochondrial markers, but not with lysosomal markers, consistent with a mitochondrial location for the enzyme. Using different homogenization techniques and comparing the fractional extraction of MCase and mitochondrial and cytosolic marker enzymes, the appearance of MCase in the “cytosolic” fraction was shown to be due to mitochondrial damage. The intramitochondrial distribution of MCase was determined using a digitonin procedure, and indicated that the enzyme is associated with the inner mitochondrial membrane. Although a fraction of MCase (30–40%) was “solubilized” by homogenization of whole mitochondria, the remaining MCase (60–70%) was tightly associated with the mitochondrial membrane fraction. Release and “solubilization” of this latter fraction was achieved by polyethylene glycol treatment. The “solubilized” MCase was stabilized in a glycerol-containing buffer.     

A Selection Procedure for Selecting Good Exponential Populations

Consider k independent exponential populations with location parameters μ₁,…, μ_k and a common scale parameter or standard deviation θ. Let μ_(k) be the largest of the μ's and define a population to be good if its location parameter exceeds μ_(k) –Δ₁. A selection procedure is proposed to select a subset of the k populations which includes the good populations with probability at least P*, a pre-assigned value. Simultaneous confidence intervals, that can be derived with the proposed selection procedure, are discussed. Moreover, if populations with locations below μ_(k) –δ₂, (δ₂ > δ₁) are “bad”, a selection procedure is proposed and a sample size is determined so that the probability of omitting a “good” population or selecting a “bad” population is at most 1 – P*.     

An improved method for preparing rat small intestine microsomal fractions for studying drug metabolism.

Epithelial cells from isolated rat small intestine were harvested by vibration of the everted intestine in 0.14 m NaCl containing 5 mm EDTA. These cells, which were largely free of mucus contamination, were homogenized in hypotonic (74 mm) sucrose using a Potter-Elvehjem homogeniser. After successively sedimenting a “brush border plus nuclei” and a “mitochondrial” fraction, microsomes were prepared from the postmitochondrial supernatant by ultracentrifugation or by precipitation at pH 5.0. The isolation and fractionation procedure was validated by the distribution of marker enzymes and by light microscopy and found to be largely uncontaminated by other subcellular components or by haemoglobin. The usefulness of this preparation was demonstrated by determining drug-metabolising enzyme activity and by substrate- and metabolite-binding spectra to cytochrome P-450. A comparison of precipitated “acid” and “normal” intestinal microsomes indicated similar apparent K_m and V_max values for a number of drug-metabolising enzymes. The content of components of the microsomal electron transport system were also similar in both preparations while the “acid” microsomes contained approximately 50% more protein.     

De l’innovation au remboursement

Medical devices and processes can be reimbursed by the health insurance system if they bring some added value for the patients. There is, however, a long way to go from innovation to reimbursement. The industry must obtain the CE mark in order to commercialize the product. The “Afssaps” verifies the product quality, the conformity to standards and norms, and follows its impact over time. To be reimbursed, the product application must be submitted to the “Haute Autorité de santé”, either by asking for its acceptation as a medical procedure by the “Commission d’évaluation des actes professionnels” (if approved it will be paid in relation to the subsequent activity) or by requesting the agreement of the “Commission nationale d’évaluation des dispositifs médicaux et technologies de santé”, which will be based on the expected/proved service, therefore authorizing its reimbursement as a medical device. The “Comité économique des produits de santé”, depending of the Health Ministry, after negociation, then gives a final approval and the corresponding price for reimbursement. This whole process can take several years, and guarantees the quality of products and prestations but may also have a negative impact on the innovations.     

Mutants with decreased differentiation to plasmodia in Physarum polycephalum

Molecular Genetics and Genomics - Mutant (“APT”) amoebae that display reduced ability to form plasmodia asexually were isolated by the use of an enrichment procedure. The results of...