Extended specificity studies of mRNA assays used to infer human organ tissues and body fluids

Messenger RNA (mRNA) profiling is a technique increasingly applied for the forensic identification of body fluids and skin. More recently, an mRNA‐based organ typing assay was developed which allows for the inference of brain, lung, liver, skeletal muscle, heart, kidney, and skin tissue. When applying this organ typing system in forensic casework for the presence of animal, rather than human, tissue is an alternative scenario to be proposed, for instance that bullets carry cell material from a hunting event. Even though mRNA profiling systems are commonly in silico designed to be primate specific, physical testing against other animal species is generally limited. In this study, human specificity of the organ tissue inferring system was assessed against organ tissue RNAs of various animals. Results confirm human specificity of the system, especially when utilizing interpretation rules considering multiple markers per cell type. Besides, we cross‐tested our organ and body fluid mRNA assays against the target types covered by the other assay. Marker expression in the nontarget organ tissues and body fluids was observed to a limited extent, which emphasizes the importance of involving the case‐specific context of the forensic samples in deciding which mRNA profiling assay to use and when for interpreting results.     

The evolution of catalytic function

It is very likely that the main driving force of enzyme evolution is the requirement to improve catalytic and regulatory efficiency which results from the intrinsic performance as well as from the spatial and functional organization of enzymes in living cells.Kinetic co-operativity may occur in simple monomeric proteins if they display “slow” conformational transitions, at the cost of catalytic efficiency. Oligomeric enzymes on the other hand can be both efficient and co-operative. We speculate that the main reason for the emergence of co-operative oligomeric enzymes is the need for catalysts that are both cooperative and efficient. As it is not useful for an enzyme to respond to a change of substrate concentration in a complex kinetic way, the emergence of symmetry has its probable origin in a requirement for “functional simplicity”.In a living cell, enzyme are associated with other macromolecules and membranes. The fine tuning of their activity may also be reached through mutations of the microenvironment. Our hypothesis is that these mutations are related to the vectorial transport of molecules, to achieve the hysteresis loops of enzyme reactions generated by the coupling of reaction and diffusion, through the co-operativity brought about by electric interactions between a charged substrate and a membrane, and last but not least, through oscillations. As the physical origins of these effects are very simple and do not require complex molecular devices, it is very likely that the functional advantage generated by the spatial and functional organization of enzyme molecules within the cell have appeared in prebiotic catalysis or very early during the primeval stages of biological evolution.We shall began this paper by presenting the nature of the probable earliest catalysts in the RNA world.     

The diagnostic accuracy of MRI for the detection of partial- and full-thickness rotator cuff tears in adults

This study assessed the diagnostic test accuracy of magnetic resonance imaging (MRI) in the detection of partial- and full-thickness rotator cuff tears in the adult population. A systematic review was conducted of the following electronic databases: Cochrane Central Register of Controlled Trials, Medline, Embase, CINAHL, AMED, ISI Web of Science, Current Controlled Trials, National Technical Information Service, the National Institute for Health Research Portfolio, the UK National Research Register Archive and WHO International Clinical Trials Registry Platform database and reference lists of articles. All studies assessing the sensitivity and/or specificity of MRI for adult patients with suspected rotator cuff tear where surgical procedures were the reference standard were included in the study. A meta-analysis was performed to calculate pooled sensitivity, specificity, likelihood and diagnostic odds ratio values, and summary receiver operating characteristic plots were constructed. Forty-four studies were included. These included 2751 shoulders in 2710 patients. For partial-thickness rotator cuff tears, the pooled sensitivity and specificity values were 0.80 [95% confidence interval (CI): 0.79-0.84] and 0.95 (95% CI: 0.94-0.97), respectively. For full-thickness tears, the sensitivity and specificity values were 0.91 (95% CI: 0.86-0.94) and 0.97 (95% CI: 0.96-0.98), respectively. While there was no substantial difference in diagnostic test accuracy between MRIs reviewed by general radiologists and those reviewed by musculoskeletal radiologists, higher-field-strength (3.0 T) MRI systems provided the greatest diagnostic test accuracy.     

化学发光免疫测定技术在检测肿瘤患者CEA中的应用

目的将化学发光免疫测定(Chemiluminescence immunoassay)方法应用于血清CEA测定.方法以L9(34)正交设计确定化学发光免疫测定方法检测血清CEA的最适实验条件,并对方法的特异性、敏感性和重复性进行评价.结果化学发光免疫测定方法测定血清CEA的最适实验条件为:CEA McAb包被质量浓度10mg/L,ABEI-CEAMcAb结合物使用发光强度为1.0×104mV,Hemin浓度为5μmol/L,H2O2质量分数为0.3%.结论化学发光免疫测定方法检测血清CEA特异性强、重复性好、敏感性超过ELISA和RIA方法,且与ELISA和RIA方法高度相关.     

Two-mode Fluorescent Detection of Cyanide by a Simple AIE-based Chemosensor with Red Emission

TPE-TCF, a simple TPE-derivative with red-emission was used to detect cyanide in the condition of single dispersion as well as under aggregate state. It could be found that TPE-TCF exhibited excellent fluorescent response to cyanide in both situations, and the mechanism was supposed to be the reaction between cyanide and the double bond in TPE-TCF as well as the aggregation induced emission property of the reacted TPE-TCF molecules. What's more, TPE-TCF could distinguish cyanide with other species, such as common anions and biotiol well, which indicated it as a potential indicator for cyanide with good selectivity and specificity.     

Obtaining a family of high-affinity,high-specificity protein inhibitors of plasmin and plasma kallikrein

Summary Human lipoprotein-associated coagulation inhibitor (LACI) is a serum protein containing three Kunitz domains. We displayed the first domain (LACI-D1) on the III protein of phage M13 and made libraries of this domain. We iteratively varied 13 residues in the region corresponding to the BPTI-trypsin interface and selected for binding to human plasmin (PLA) and human plasma kallikrein (pKAL). For PLA, our first-round best binder, EPI-P211, had K_D=2 nM. Using information from the first selection, we made a PLA-biased library containing 500 000 proteins and selected from these a protein, EPI-P302, having a K_D for PLA of 87 pM. EPI-P302 inhibits pKAL with K_D=250 nM (2800-fold higher than for PLA) and K_D values for other proteases are higher yet. From the same initial LACI-D1 library, we selected an inhibitor of pKAL, EPI-K401, with a K_D for pKAL of 287 pM. We used information from this selection to construct a pKAL-biased library from which we selected EPI-K502, which has a K_D for pKAL of 40 pM. EPI-K502 inhibits PLA with K_D20 nM (500-fold higher than for pKAL); K_D values for other proteases are much higher. For both targets and for both selections, there are families of proteins having a few differences and a range of affinities for their targets. These proteins are candidate drugs and imaging agents for indications involving excess PLA or pKAL. Structure-activity relationships of PLA and pKAL binders will allow design of small molecules that are specific for these targets.Abbreviations APP-I-D1 Kunitz domain of amyloid precursor protein-I - BPTI bovine pancreatic trypsin inhibitor - F.VII_a Factor VII_a - F.XI_a Factor XI_a - FIR fraction of input recovered - hNE human neutrophil elastase - LACI-D1 lipoprotein-associated coagulation inhibitor, first Kunitz domain - pKAL human plasma kallikrein - PLA human plasmin - RT room temperature - THBN human thrombin - Vg variegated - w.t. wild-type - %RIA percent residual inhibitory activity - :: fusion (gene or protein)     

Characterization of Multihapten Antigens on Antibody Sensitivity and Specificity for Parathion

Four multihapten antigens were conjugated to a single protein to obtain broad-specific polyclonal antibody characterization. The sensitivity and specificity of the polyclonal antibodies for parathion were evaluated with differences due to the structure of the determinant or the number of multihapten antigens. The sensitivity of immunoassay may decrease with a corresponding increase in the number of antigens in a multihapten immunogen or with an increase in complexity of the structure. The specificity of other analytes may broaden when the determinants of multihapten have similar structures.     

Geographical classification of wine and olive oil by means of classification and influence matrix analysis (CAIMAN)

Classification and influence matrix analysis (CAIMAN) is a new classification method, recently proposed and based on the influence matrix (also called leverage matrix). Depending on the purposes of the classification analysis, CAIMAN can be used in three outlines: (1) D-CAIMAN is a discriminant classification method, (2) M-CAIMAN is a class modelling method allowing a sample to be classified, not classified at all, or assigned to more than one class (confused) and (3) A-CAIMAN deals with the asymmetric case, where only a reference class needs to be modelled.

In this work, the geographic classification of samples of wine and olive oil has been carried out by means of CAIMAN and its results compared with discriminant analysis, by focusing great attention on the model predictive capabilities. The geographic characterization has been carried out on three different datasets: extra virgin olive oils produced in a small area, with a “protected denomination of origin” label, wines with different denominations of origin, but produced in enclosed geographical areas, and olive oils belonging to different production areas.

Final results seem to indicate that the application of CAIMAN to the geographical origin identification offers several advantages: first, it shows – on an average basis – good performances; second, it is able to deal in a simple way classification problems related to tipicity, authenticity, and uniqueness characterization, which are of increasing interest in food quality issues.     

Experimental parameters affecting sensitivity and specificity of a yeast assay for estrogenic compounds: results of an interlaboratory validation exercise

In vitro assays are considered as the first step in a tiered approach to compound screening for hormonal activity. Although many new assays have been developed in recent years, little attention has been paid towards assay validation. Our objective was to identify critical experimental parameters in a yeast estrogen screen (YES) that affect its sensitivity and specificity. We investigated the role of incubation time, solvent type, yeast inoculum growth stage and concentration on the outcome of the YES. Compounds tested included new and established agonists, antagonists and negative controls, and results were evaluated according to prefixed statistical criteria. In addition, we assessed the assay’s performance in a blind interlaboratory validation exercise (IVE). An incubation time of five days was necessary to positively identify the estrogenic properties of all agonists tested, when dissolved in DMSO. Longer incubation times were required when using an ethanol protocol. Similar estrogenic activity was reported for benzyl butyl phthalate, bisphenol-A, methoxychlor, permethrin and genistein in the IVE. One out of the three laboratories did not classify α,β-endosulfan, dissolved in DMSO, as an estrogen. The same was true for 4,4′-DDE and lindane, dissolved in ethanol, a result that might be attributable to an inappropriate yeast start concentration and/or growth stage. These validation experiments show that under appropriate experimental conditions the YES yields sensitive, specific and reliable results. Therefore it fulfills the requirements as a first step screening assay to evaluate the capacity of chemicals to interact with the estrogen receptor.     

Challenging Near InfraRed Spectroscopy discriminating ability for counterfeit pharmaceuticals detection

This study was initiated by the laboratories and control department of the French Health Products Safety Agency (AFSSAPS) as part of the fight against the public health problem of rising counterfeit and imitation medicines. To test the discriminating ability of Near InfraRed Spectroscopy (NIRS), worse cases scenarios were first considered for the discrimination of various pharmaceutical final products containing the same Active Pharmaceutical Ingredient (API) with different excipients, such as generics of proprietary medicinal products (PMP). Two generic databases were explored: low active strength hard capsules of Fluoxetine and high strength tablets of Ciprofloxacin. Then 4 other cases involving suspicious samples, counterfeits and imitations products were treated. In all these cases, spectral differences between samples were studied, giving access to API or excipient contents information, and eventually allowing manufacturing site identification.A chemometric background is developed to explain the optimisation methodology, consisting in the choices of appropriate pretreatments, algorithms for data exploratory analyses (unsupervised Principal Component Analysis), and data classification (supervised cluster analysis, and Soft Independent Modelling of Class Analogy). Results demonstrate the high performance of NIRS, highlighting slight differences in formulations, such as 2.5% (w/w) in API strength, 1.0% (w/w) in excipient and even coating variations (<1%, w/w) with identical contents, approaching the theoretical limits of NIRS sensitivity. All the different generic formulations were correctly discriminated and foreign PMP, constituted of formulations slightly different from the calibration ones, were also all discriminated. This publication addresses the ability of NIRS to detect counterfeits and imitations and presents the NIRS as an ideal tool to master the global threat of counterfeit drugs.