No Evidence for “Break-Induced Replication” in a Higher Plant – But Break-Induced Conversion May Occur

“Break-induced replication” (BIR) is considered as one way to repair DNA double-strand breaks (DSBs). BIR is defined as replication of the proximal break-ends up to the end of the broken chromosome using an undamaged (homologous) double-stranded template and mimicking a non-reciprocal translocation. This phenomenon was detected by genetic experiments in yeast. BIR is assumed to occur also in mammals, but experimental evidence is not yet at hand. We have studied chromosomes of the field bean, Vicia faba L., with respect to the occurrence of BIR after DSB induction during S and G2 phase. Simultaneous incorporation of the base analog ethynyldeoxyuridine (EdU) revealed no chromosomal replication pattern indicative of BIR. Thus, if occurring at all, BIR does not play a major role in DSB repair in higher plants with large chromosome arms. However, the frequency of interstitial asymmetric EdU incorporation within heterochromatic regions, visible on metaphase chromosomes, increased after chromosome breakage during S and G2 phase. Such asymmetric labeling could be interpreted as conservative replication up to the next replicon, circumventing a DSB, and yielding an interstitial conversion-like event

A gradual depth-dependent change of connectivity features of supragranular pyramidal cells in rat barrel cortex

Recent experimental evidence suggests a finer genetic, structural and functional subdivision of the layers which form a cortical column. The classical layer II/III (LII/III) of rodent neocortex integrates ascending sensory information with contextual cortical information for behavioral read-out. We systematically investigated to which extent regular-spiking supragranular pyramidal neurons, located at different depths within the cortex, show different input-output connectivity patterns. Combining glutamate-uncaging with whole-cell recordings and biocytin filling, we revealed a novel cellular organization of LII/III: (i) “Lower LII/III” pyramidal cells receive a very strong excitatory input from lemniscal LIV and much fewer inputs from paralemniscal LVa. They project to all layers of the home column, including a feedback projection to LIV whereas transcolumnar projections are relatively sparse. (ii) “Upper LII/III” pyramidal cells also receive their strongest input from LIV, but in addition, a very strong and dense excitatory input from LVa. They project extensively to LII/III as well as LVa and Vb of their home and neighboring columns, (iii) “Middle LII/III” pyramidal cell show an intermediate connectivity phenotype that stands in many ways in-between the features described for lower versus upper LII/III. “Lower LII/III” intracolumnarly segregates and transcolumnarly integrates lemniscal information whereas “upper LII/III” seems to integrate lemniscal with paralemniscal information. This suggests a finegrained functional subdivision of the supragranular compartment containing multiple circuits without any obvious cytoarchitectonic, other structural or functional correlate of a laminar border in rodent barrel cortex

Tandem repetitive transgenes and fluorescent chromatin tags alter local interphase chromosome arrangement in Arabidopsis thaliana

Fluorescent protein chromatin tagging as achieved by the lac operator/lac repressor system is useful to trace distinct chromatin domains in living eukaryotic nuclei. To interpret the data correctly, it is important to recognize influences of the tagging system on nuclear architecture of the host cells. Within an Arabidopsis line that carries lac operator/lac repressor/GFP transgenes, the transgene loci frequently associate with each other and with heterochromatic chromocenters. Accumulation of tagged fusion protein further enhances the association frequency. Independent experiments with a transgenic plant carrying another multi-copy transgene also revealed, independent of its transcriptional state, unusually high frequencies of association with each other and with heterochromatin. From these results we conclude that the lac operator/lac repressor chromatin tagging system may alter the spatial chromatin organization in the host nuclei (in particular when more than one insertion locus is present) and also that loci of homologous transgenic repeats associate more often with each other and with endogenous heterochromatin than normal euchromatic regions

Sphingosine kinase 1/S1P receptor signaling axis is essential for cellular uptake of Neisseria meningitidis in brain endothelial cells

Invasion of brain endothelial cells (BECs) is central to the pathogenicity of Neisseria meningitidis infection. Here, we established a key role for the bioactive sphingolipid sphingosine-1-phosphate (S1P) and S1P receptor (S1PR) 2 in the uptake process. Quantitative sphingolipidome analyses of BECs infected with N. meningitidis revealed elevated S1P levels, which could be attributed to enhanced expression of the enzyme sphingosine kinase 1 and its activity. Increased activity was dependent on the interaction of meningococcal type IV pilus with the endothelial receptor CD147. Concurrently, infection led to increased expression of the S1PR2. Blocking S1PR2 signaling impaired epidermal growth factor receptor (EGFR) phosphorylation, which has been shown to be involved in cytoskeletal remodeling and bacterial endocytosis. Strikingly, targeting S1PR1 or S1PR3 also interfered with bacterial uptake. Collectively, our data support a critical role of the SphK/S1P/S1PR axis in the invasion of N. meningitidis into BECs, defining a potential target for adjuvant therapy

NK cells from an AML patient have recovered in remission and reached comparable cytolytic activity to that of a healthy monozygotic twin mediated by the single-chain triplebody SPM-2

Background: The capacity of patient's Natural Killer cells (NKs) to be activated for cytolysis is an important prerequisite for the success of antibody-derived agents such as single-chain triplebodies (triplebodies) in cancer therapy. NKs recovered from AML patients at diagnosis are often found to be reduced in peripheral blood titers and cytolytic activity. Here, we had the unique opportunity to compare blood titers and cytolytic function of NKs from an AML patient with those of a healthy monozygotic twin. The sibling's NKs were compared with the patient's drawn either at diagnosis or in remission after chemotherapy. The cytolytic activities of NKs from these different sources for the patient's autologous AML blasts and other leukemic target cells in conjunction with triplebody SPM-2, targeting the surface antigens CD33 and CD123 on the AML cells, were compared. Methods: Patient NKs drawn at diagnosis were compared to NKs drawn in remission after chemotherapy and a sibling's NKs, all prepared from PBMCs by immunomagnetic beads (MACS). Redirected lysis (RDL) assays using SPM-2 and antibody-dependent cellular cytotoxicity (ADCC) assays using the therapeutic antibody Rituximab (TM) were performed with the enriched NKs. In addition, MACS-sorted NKs were analyzed for NK cell activating receptors (NCRs) by flow cytometry, and the release of TNF-alpha and IFN-gamma from blood samples of both siblings after the addition of the triplebody were measured in ELISA-assays. Results: Patient NKs isolated from peripheral blood drawn in remission produced comparable lysis as NKs from the healthy twin against the patient's autologous bone marrow (BM) blasts, mediated by SPM-2. The NCR receptor expression profiles on NKs from patient and twin were similar, but NK cell titers in peripheral blood were lower for samples drawn at diagnosis than in remission. Conclusions: Peripheral blood NK titers and ex vivo cytolytic activities mediated by triplebody SPM-2 were comparable for cells drawn from an AML patient in remission and a healthy twin. If these results can be generalized, then NKs from AML patients in remission are sufficient in numbers and cytolytic activity to make triplebodies promising new agents for the treatment of AML

Удаление сернистых соединений из дизельных топлив с использованием металлосодержащих ионных жидкостей

Данная статья посвящена проблеме удаления сернистых соединений из дизельных топлив. Представлены характеристики полученных экстракционных систем на основе ионных жидкостей и солей металлов (СuBr[2], CoBr[2], NiBr[2]). Показана возможность использования комплексов ионных жидкостей с солями металлов в качестве экстрагентов для удаления серы из дизельного топлива