Encyclopedia of CLL Subsets - a Unique Bioinformatics Tool and Database for Analysis of Subsets of Stereotypical B-Cell Receptors in CLL.

BACKGROUND: Chronic lymphocytic leukemia (CLL) is clinically and biologically highly variable disease which is closely related with multiple cellular and molecular markers, including sequence motifs of B-cell receptors. These motifs are highly similar (stereotyped) within one third of CLL patients and create homogeneous groups called stereotyped CLL subsets. The homogeneity is reflected also in clinical and biological characteristics of the disease. To facilitate access to the information about individual subsets, we have created a publicly available web-based tool Encyclopedia of CLL Subsets. MATERIALS AND METHODS: The Encyclopedia of CLL subsets belongs to our bioinformatics platform Antigen Receptor Research Tool (ARResT) developed for analysis, clustering, and annotation of immunoglobulin sequences. To gather primary knowledge about the subsets, we have analyzed a dataset of 7,500 CLL patients published by Agathangelidis et al in 2012 [1]. We have created an overview of major stereotyped subsets and their characteristics. Additional clinical and cytogenomic information about individual subsets has been obtained by machine text processing of available literature from server PubMed and is regularly updated. RESULTS: We have created a unique web-based application Encyclopedia of CLL Subsets available from http: //arrest.tools/subsets for an interactive access to the information about stereotyped CLL subsets. A user can obtain and compare basic information about the major subsets including their clinical and cytogenomic characteristics. These have been manually curated from machine processed results from PubMed database by experts in CLL research. Through the Encyclopedias user interface, user can also directly use our published tool ARResT/AssignSubsets to assign new immunoglobulin sequences to the major subsets. CONCLUSION: The Encyclopedia of CLL Subsets is a publicly available online tool facilitating access to the most recent research knowledge about stereotyped CLL subsets and enabling analysis of own data and interpretation of the results. This gives the Encyclopedia a great potential for its use in clinical routine. This work was supported by Czech Ministry of Health grant No. 34272A. All rights reserved. The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers. Submitted: 1. 3. 2019 Accepted: 4. 3. 2019.

GvL effects in T-prolymphocytic leukemia: evidence from MRD kinetics and TCR repertoire analyses.

Allogeneic stem cell transplantation (alloSCT) is used for treating patients with T-prolymphocytic leukemia (T-PLL). However, direct evidence of GvL activity in T-PLL is lacking. We correlated minimal residual disease (MRD) kinetics with immune interventions and T-cell receptor (TCR) repertoire diversity alterations in patients after alloSCT for T-PLL. Longitudinal quantitative MRD monitoring was performed by clone-specific real-time PCR of TCR rearrangements (n=7), and TCR repertoire diversity assessment by next-generation sequencing (NGS; n=3) Although post-transplant immunomodulation (immunosuppression tapering or donor lymphocyte infusions) resulted in significant reduction (>1 log) of MRD levels in 7 of 10 occasions, durable MRD clearance was observed in only two patients. In all three patients analyzed by TCR-NGS, MRD responses were reproducibly associated with a shift from a clonal, T-PLL-driven profile to a polyclonal signature. Novel clonotypes that could explain a clonal GvL effect did not emerge. In conclusion, TCR-based MRD quantification appears to be a suitable tool for monitoring and guiding treatment interventions in T-PLL. The MRD responses to immune modulation observed here provide first molecular evidence for GvL activity in T-PLL which, however, may be often only transient and reliant on a poly-/oligoclonal rather than a monoclonal T-cell response.

[Bioinformatics and Next -generation Sequencing]. / Bioinformatika a sekvenování nové generace.

Next-generation sequencing technologies are currently well-established in the research field and progressively find their way towards clinical applications. Sequencers produce vast amounts of data and therefore bioinformatics methods are needed for processing. Without computational methods, sequencing would not be able to produce relevant biological information. In this review, we introduce the basics of common NGS-related bioinformatics methods used in oncological research. We also state some of the common problems complicating data processing and interpretation of the results.

In vitro approaches to evaluation of Sun Protection Factor.

The efficacy of sunscreen products has been recognized as an important public health issue. Adequate methods for assessment of the level of protection should be developed and standardised. While the SPF COLIPA testing method in vivo has been used for years, preference should be given to in vitro testing methods as in vivo methods raise ethical concern. The present study aims to assess possible in vitro approaches based on diffuse transmission spectroscopy, published previously by Diffey, and two methods based on measurements of UVB transmission through a defined layer of a sunscreen product applied on various UV-transparent substrates. The attenuated UVB intensity, using different UV light sources, is detected radiometrically and transformed to real SPF value by means of a calibration curve, which is based on an extensive number of measurements performed using both in vivo and in vitro method The outcome of the three in vitro methods employed in the study showed great differences in the obtained SPF values in comparison with reference SPF determined by means of the COLIPA method in vivo. The high variability of in vitro results suggests that main attention should be focused on substrate selection simulating the human skin surface and homogenous product application. The in vitro screening methods may represent a fast and reasonable tool reducing the number of in vivo experiments and risks related to UV exposure of human subjects, when the technical test parameters are adjusted and optimized.

Asparagine, valine, and threonine in the third extracellular loop of muscarinic receptor have essential roles in the positive cooperativity of strychnine-like allosteric modulators.

We have investigated allosteric interactions of four closely related strychnine-like substances: Wieland-Gumlich aldehyde (WGA), propargyl Wieland-Gumlich aldehyde, strychnine, and brucine with N-methylscopolamine (NMS) on M(3) subtype of muscarinic receptor genetically modified in the second or the third extracellular loop to corresponding loops of M(2) subtype (M(3)o2 and M(3)o3 chimera). The M(3)o2 chimeric receptor The exhibited no change in either affinity of strychnine, brucine, and WGA or in cooperativity of brucine or WGA, whereas both parameters for propargyl-WGA changed. In contrast, there was a change in affinity of all tested modulators (except for brucine) and in their cooperativity in the M(3)o3 chimera. Directions of affinity changes in both chimeras were always toward values of the donor M(2) subtype, but changes in cooperativity were variable. Compared with the native M(3) receptor, strychnine displayed a slight increase in positive cooperativity and propargyl-WGA a robust decrease in negative cooperativity at M(3)o2 chimera. Similar changes were found in the M(3)o3 chimera. Interestingly, cooperativity of brucine and WGA at the M(3)o3 chimera changed from negative to positive. This is the first evidence of constitution of positive cooperativity of WGA by switching sequences of two parental receptors, both exhibiting negative cooperativity. Gradual replacement of individual amino acids revealed that only three residues (NVT of the o3 loop of the M(2) receptor) are involved in this effect. Data suggest that these amino acids are essential for propagation of a conformation change resulting in positive cooperativity induced by these modulators.

Regulation of signal transduction at M2 muscarinic receptor.

Muscarinic acetylcholine receptors mediate transmission of an extracellular signal represented by released acetylcholine to neuronal or effector cells. There are five subtypes of closely homologous muscarinic receptors which are coupled by means of heterotrimeric G-proteins to a variety of signaling pathways resulting in a multitude of target cell effects. Endogenous agonist acetylcholine does not discriminate among individual subtypes and due to the close homology of the orthosteric binding site the same holds true for most of exogenous agonists. In addition to the classical binding site muscarinic receptors have one or more allosteric binding sites at extracellular domains. Binding of allosteric modulators induces conformational changes in the receptor that result in subtype-specific changes in orthosteric binding site affinity for both muscarinic agonists and antagonists. This overview summarizes our recent experimental effort in investigating certain aspects of M2 muscarinic receptor functioning concerning i) the molecular determinants that contribute to the binding of allosteric modulators, ii) G-protein coupling specificity and subsequent cellular responses and iii) possible functional assays that exploit the unique properties of allosteric modulators for characterization of muscarinic receptor subtypes in intact tissue. A detailed knowledge of allosteric properties of muscarinic receptors is required to permit drug design that will modulate signal transmission strength of specific muscarinic receptor subtypes. Furthermore, allosteric modulation of signal transmission strength is determined by cooperativity rather than concentration of allosteric modulator and thus reduces the danger of overdose.

Changes of cooperativity between N-methylscopolamine and allosteric modulators alcuronium and gallamine induced by mutations of external loops of muscarinic M(3) receptors.

To clarify the involvement of specific domains of muscarinic receptors in the action of allosteric modulators, muscarinic M(3) receptors (on which allosteric interactions are weak) were genetically modified to become more similar to M(2) receptors (on which allosteric interactions are strong) and were expressed in COS-7 cells. Affinity for allosteric modulator gallamine was enhanced 25- to 50-fold by modifications of the third external loop (o3) and the negative effect of gallamine on the affinity for classical antagonist N-[(3)H]methylscopolamine ([(3)H]NMS) was augmented. Affinity for alcuronium became 3-fold higher after the o3 loop of M(3) receptors was made identical with the o3 loop of M(2) receptors, and alcuronium acquired positive influence on the affinity for [(3)H]NMS. This is the first instance of inducing positive cooperativity on muscarinic receptors by genetic manipulation. Transferring whole o2 loop from M(2) to M(3) receptors substantially enhanced affinities for gallamine and alcuronium without augmenting their negative action on [(3)H]NMS binding. In contrast, effects of simply adding two negative charges into the o2 loop of M(3) receptors were small. Removal of Arg from o1 loop abolished the negative effect of gallamine but not of alcuronium on [(3)H]NMS binding at equilibrium. Data point to an important role of o3 loop in the mechanism of the positive and negative cooperativity between [(3)H]NMS and alcuronium and gallamine, respectively, and in the binding of both modulators to M(2) receptors and reveal independence between mutation-induced changes in the affinity for a modulator and in the magnitude and direction of the allosteric effect of the modulator.

Pallister-Hall syndrome and McKusick-Kaufmann syndrome: one entity?

The Pallister-Hall syndrome is characterised by specific facial anomalies, postaxial polydactyly, imperforate anus, and brain anomalies including a diencephalic hamartoblastoma. The hallmarks of the McKusick-Kaufmann syndrome are hydrocolpos owing to vaginal atresia, postaxial polydactyly, imperforate anus, and congenital heart defects. We report a patient with the unique features of hydrocolpos, postaxial polydactyly, and hypothalamic hamartoblastoma and discuss the different aetiological considerations of both syndromes and implications for clinical management.

[Clinical picture and therapy of gastroschisis (author's transl)]. / Zur Klinik und Therapie der Gastroschisis

The results of operative treatment of gastroschisis have been improving in recent years. The survival rate is now between 50 and 60 p.c. It is very important to perform primary repair as soon as possible after birth. According to our own experiences resection of the bowel should be avoided as it gives a poor prognosis. Combined malformations of gastroschisis are very rare and their treatment should be tired since there is no other alternative. An own successfully treated case of combined malformations of gastroschisis with atresia of the small bowel is presented. Stenosis of the duodenum and tubular duplication are described