Analytical validation of quantitative immunohistochemical assays of tumor infiltrating lymphocyte biomarkers

Tumor infiltrating lymphocytes (TIL), especially T-cells, have both prognostic and therapeutic applications. The presence of CD8+ effector T-cells and the ratio of CD8+ cells to FOXP3+ regulatory T-cells have been used as biomarkers of disease prognosis to predict response to various immunotherapies. Blocking the interaction between inhibitory receptors on T-cells and their ligands with therapeutic antibodies including atezolizumab, nivolumab, pembrolizumab and tremelimumab increases the immune response against cancer cells and has shown significant improvement in clinical benefits and survival in several different tumor types. The improved clinical outcome is presumed to be associated with a higher tumor infiltration; therefore, it is thought that more accurate methods for measuring the amount of TIL could assist prognosis and predict treatment response. We have developed and validated quantitative immunohistochemistry (IHC) assays for CD3, CD8 and FOXP3 for immunophenotyping T-lymphocytes in tumor tissue. Various types of formalin fixed, paraffin embedded (FFPE) tumor tissues were immunolabeled with anti-CD3, anti-CD8 and anti-FOXP3 antibodies using an IHC autostainer. The tumor area of stained tissues, including the invasive margin of the tumor, was scored by a pathologist (visual scoring) and by computer-based quantitative image analysis. Two image analysis scores were obtained for the staining of each biomarker: the percent positive cells in the tumor area and positive cells/mm² tumor area. Comparison of visual vs. image analysis scoring methods using regression analysis showed high correlation and indicated that quantitative image analysis can be used to score the number of positive cells in IHC stained slides. To demonstrate that the IHC assays produce consistent results in normal daily testing, we evaluated the specificity, sensitivity and reproducibility of the IHC assays using both visual and image analysis scoring methods. We found that CD3, CD8 and FOXP3 IHC assays met the fit-for-purpose analytical acceptance validation criteria and that they can be used to support clinical studies.     

Evolution of the 28S ribosomal RNA gene in anurans: regions of variability and their phylogenetic implications

Fifteen restriction sites were mapped to the 28S ribosomal RNA gene of individuals representing 54 species of frogs, two species of salamanders, a caecilian, and a lungfish. Eight of these sites were present in all species examined, and two were found in all but one species. Alignment of these conserved restriction sites revealed, among anuran 28S rRNA genes, five regions of major length variation that correspond to four of 12 previously identified divergent domains of this gene. One of the divergent domains (DD8) consists of two regions of length variation separated by a short segment that is conserved at least throughout tetrapods. Most of the insertions, deletions, and restriction-site variations identified in the 28S gene will require sequence-level analysis for a detailed reconstruction of their history. However, an insertion in DD9 that is coextensive with frogs in the suborder Neobatrachia, a BstEII site that is limited to representatives of two leptodactylid subfamilies, and a deletion in DD10 that is found only in three ranoid genera are probably synapomorphies.      

Peroxisome proliferator-activated receptorα (PPARα) agonists down-regulate α2-macroglobulin expression by a PPARα-dependent mechanism

Fibrates are peroxisome proliferator-activated receptor alpha (PPARα) ligands used to normalize lipid and glucose parameters and exert anti-inflammatory effects. The acute-phase response (APR) is an important inflammatory process. One of the most important acute-phase proteins in rats is α2-macroglobulin (A2Mg). Whereas normal adult rats present low serum levels, pregnant rats display high amounts. Therefore, we used pregnant rats to detect the effect of fenofibrate on hepatic A2Mg expression by RT-PCR and Northern blot. Virgin rats were used as controls. The expression of other APR genes, a known fibrate-responder gene, gamma-chain fibrinogen (γ-Fib), and one gene from the same family as A2Mg, complement component 3 (C3), were also measured in liver. In order to determine whether the fibrate-effects were mediated by PPARα, wild-type mice and PPARα-null mice were also used and treated with WY-14,643 (WY) or di-2-ethylhexyl phthalate (DEHP). Fenofibrate depressed A2Mg expression in virgin rats, but expression was decreased more sharply in pregnant rats. Expression of C3 and γ-Fib was diminished after treatment only in pregnant rats. On the other hand, WY, but not DEHP, reduced A2Mg and γ-Fib expression in the livers of wild-type mice, without any effect in PPARα-null mice. WY or DEHP did not affect C3 expression. Therefore, A2Mg expression is modified by PPARα agonists not only in pregnant rats under augmented APR protein synthesis, but also in virgin rats and mice under basal conditions. Interestingly, our results also identify A2Mg as a novel PPARα agonist-regulated gene.     

The induction of allophanate lyase during the vegetative cell cycle in light-synchronized cultures of Chlamydomonas reinhardi.

Allophanate lyase can be induced by urea or acetamide 20-40-fold within 4 h in NH4 + -deprived cultures of Chlamydomonas reinhardi. In light-synchronized cultures, allophanate lyase induction appeared to be limited to the light phase of the cell cycle, provided that culture samples were induced under ongoing illumination conditions (i.e. light induction of light phase cells and dark induction of dark phase cells). However, when culture samples were induced under constant light conditions this cell cycle pattern was abolished. Light was found to be required for allophanate lyase induction and this was shown to be due, in part, to the light requirement for inducer uptake. The relationship between allophanate lyase induction and gametogenesis is discussed.     

Molecular systematics and paleobiogeography of the South American sigmodontine rodents [published erratum appears in Mol Biol Evol 1998 Feb;15(2):224]

The murid rodent subfamily Sigmodontinae contains 79 genera which are distributed throughout the New World. The time of arrival of the first sigmodontines in South America and the estimated divergence time(s) of the different lineages of South American sigmodontines have been controversial due to the lack of a good fossil record and the immense number of extant species. The "early-arrival hypothesis" states that the sigmodontines must have arrived in South America no later than the early Miocene, at least 20 MYA, in order to account for their vast present-day diversity, whereas the "late-arrival hypothesis" includes the sigmodontines as part of the Plio-Pleistocene Great American Interchange, which occurred approximately 3.5 MYA. The phylogenetic relationships among 33 of these genera were reconstructed using mitochondrial DNA (mtDNA) sequence data from the ND3, ND4L, arginine tRNA, and ND4 genes, which we show to be evolving at the same rate. A molecular clock was calibrated for these genes using published fossil dates, and the genetic distances were estimated from the DNA sequences in this study. The molecular clock was used to estimate the dates of the South American sigmodontine origin and the main sigmodontine radiation in order to evaluate the "early-" and "late-arrival" scenarios. We estimate the time of the sigmodontine invasion of South America as between approximately 5 and 9 MYA, supporting neither of the scenarios but suggesting two possible models in which the invading lineage was either (1) ancestral to the oryzomyines, akodonts, and phyllotines or (2) ancestral to the akodonts and phyllotines and accompanied by the oryzomyines. The sigmodontine invasion of South America provides an example of the advantage afforded to a lineage by the fortuitous invasion of a previously unexploited habitat, in this case an entire continent.      

Security Analysis and Improvement of ‘a More Secure Anonymous User Authentication Scheme for the Integrated EPR Information System’

Over the past few years, secure and privacy-preserving user authentication scheme has become an integral part of the applications of the healthcare systems. Recently, Wen has designed an improved user authentication system over the Lee et al.’s scheme for integrated electronic patient record (EPR) information system, which has been analyzed in this study. We have found that Wen’s scheme still has the following inefficiencies: (1) the correctness of identity and password are not verified during the login and password change phases; (2) it is vulnerable to impersonation attack and privileged-insider attack; (3) it is designed without the revocation of lost/stolen smart card; (4) the explicit key confirmation and the no key control properties are absent, and (5) user cannot update his/her password without the help of server and secure channel. Then we aimed to propose an enhanced two-factor user authentication system based on the intractable assumption of the quadratic residue problem (QRP) in the multiplicative group. Our scheme bears more securities and functionalities than other schemes found in the literature.     

BioManager: the use of a bioinformatics web application as a teaching tool in undergraduate bioinformatics training

The completion of the human genome project, and other genome sequencing projects, has spearheaded the emergence of the field of bioinformatics. Using computer programs to analyse DNA and protein information has become an important area of life science research and development. While it is not necessary for most life science researchers to develop specialist bioinformatic skills (including software development), basic skills in the application of common bioinformatics software and the effective interpretation of results are increasingly required by all life science researchers. Training in bioinformatics is increasingly occurring within the university system as part of existing undergraduate science and specialist degrees. One difficulty in bioinformatics education is the sheer number of software programs required in order to provide a thorough grounding in the subject to the student. Teaching requires either a well-maintained internal server with all the required software, properly interfacing with student terminals, and with sufficient capacity to handle multiple simultaneous requests, or it requires the individual installation and maintenance of every piece of software on each computer. In both cases, there are difficult issues regarding site maintenance and accessibility. In this article, we discuss the use of BioManager, a web-based bioinformatics application integrating a variety of common bioinformatics tools, for teaching, including its role as the main bioinformatics training tool in some Australian and international universities. We discuss some of the issues with using a bioinformatics resource primarily created for research in an undergraduate teaching environment.