Quantification of Dendritic Spines Remodeling under Physiological Stimuli and in Pathological Conditions.

Numerous brain diseases are associated with abnormalities in morphology and density of dendritic spines, small membranous protrusions whose structural geometry correlates with the strength of synaptic connections. Thus, the quantitative analysis of dendritic spines remodeling in microscopic images is one of the key elements towards understanding mechanisms of structural neuronal plasticity and bases of brain pathology. In the following article, we review experimental approaches designed to assess quantitative features of dendritic spines under physiological stimuli and in pathological conditions. We compare various methodological pipelines of biological models, sample preparation, data analysis, image acquisition, sample size, and statistical analysis. The methodology and results of relevant experiments are systematically summarized in a tabular form. In particular, we focus on quantitative data regarding the number of animals, cells, dendritic spines, types of studied parameters, size of observed changes, and their statistical significance.

RAD5a ubiquitin ligase is involved in ubiquitination of Arabidopsis thaliana proliferating cell nuclear antigen.

The proliferating cell nuclear antigen (PCNA) is post-translationally modified by ubiquitin in yeast and mammalian cells. It is widely accepted that in yeast mono- and polyubiquitinated PCNA is involved in distinct pathways of DNA postreplication repair. This study showed an interaction between plant ubiquitin and PCNA in the plant cell. Using different approaches, it was demonstrated that Arabidopsis RAD5a ubiquitin ligase is involved in the post-translational modification of plant PCNA. A detailed analysis of the properties of selected Arabidopsis ubiquitin-conjugating enzymes (AtUBC) has shown that a plant homologue of yeast RAD6 (AtUBC2) is sufficient to monoubiquitinate AtPCNA in the absence of ubiquitin ligase. Using different combinations of selected AtUBC proteins together with AtRAD5a, it was demonstrated that plants have potential to use different pathways to ubiquitinate PCNA. The analysis of Arabidopsis PCNA1 and PCNA2 did not demonstrate substantial differences in the ubiquitination pattern between these two proteins. The major ubiquitination target of Arabidopsis PCNA, conserved in eukaryotes, is lysine 164. Taken together, the presented results clearly demonstrate the involvement of Arabidopsis UBC and RAD5a proteins in the ubiquitination of plant PCNA at lysine 164. The data show the complexity of the plant ubiquitination system and open new questions about its regulation in the plant cell.

[Problems with identification of beta-hemolytic streptococcus resistant to bacitracin isolated from patients with pharyngitis]. / Problemy z identyfikacja paciorkowców beta-hemolizujacych opornych na bacytracyne izolowanych od chorych z zapaleniem gardla.

INTRODUCTION: The genus Streptococcus comprises a number of species characterized by a differential pathogenic potential. These bacteria can be considered as members of microbial physiological flora but they can also cause mild infections or severe, life threatening conditions. The majority of infections of streptococcal etiology are caused by beta-hemolysing species. The predominant causative agent of bacterial pharyngitis is Streptococcus pyogenes. This species usually doesn't give rise to any identification difficulties due to the introduction the well determined diagnostic schemes. Problems concerning laboratory identification can be, however, associated with other species of beta-hemolysing streptococci isolated from patients with pharyngitis. These streptococci can demonstrate features similar to those of S. pyogenes and share the group antygen A, such as some strains of Streptococcus anginosus and Streptococcus dysgalactiae subsp. equisimilis. The determination of sensitivity to bacitracin, which is a feature typical of S. pyogenes, is the basic test useful for its preliminary identification. Nevertheless, the identification of some strains by this test can give rise to incompatibility. The aim of the study was characterisation of beta-hemolysing streptococci resistant to bacitracin isolated from patients with pharyngitis. The examined bacterial strains caused identification problems by the use of routine diagnostic methods. METHODS: The material included 14 streptococcal strains resistant to bacitracin which were isolated from adult patients suffering from pharyngitis. The bacteria were cultured on media dedicated for the species. The following routine diagnostic tests were used for the bacterial identification: sensitivity to bacitracin (0.04 U/disc), CAMP test, determination of the group antigens A, B, C, D, F and G (Slidex Strepto-Kit), and determination of biochemical features by the API 20 STREP test (bioMèrieux). The sensitivity of streptococcal isolates to antibiotics (penicillin, clindamycin, erythromycin, tetracycline, vancomycin, ofloxacin) and trimethoprim/sulfamethoxazole, was determined by the disc diffusion method on the Mueller-Hinton agar with 5% sheep blood (the inoculum-0.5 McFarland). RESULTS: Among the 14 isolates resistant to bacitracin, 6 isolates of S. pyogenes, 6 isolates of S. constellatus, and 2 isolates of S. dysgalactiae subsp. equisimilis were identified. All isolates were sensitive to penicillin and vancomycin. One isolate ofS. pyogenes demonstrated constitutive MLSB resistance mechanism. Seven isolates were resistant to tetracycline: S. dysgalactiae subsp. equisimilis (3 isolates), S. constellatus (3), and S. pyogenes (1). The number of isolates resistant to trimethoprim/sulfamethoxazole was as follows: S. pyogenes (6) and S. dysgalactiae subsp. equisimilis (1), whereas four isolates were resistant to ofloxacin. CONCLUSIONS: The bacitracin test cannot be used as the only test for the laboratory identification of S. pyogenes even if it is combined with the determination of the Lancefield group antigen due to the existence of bacitracin resistant S. pyogenes strains. Therefore, it is necessary to perform biochemical commercial tests in addition to routine phenotypic tests. Isolation of beta-hemolysing streptococci other than S. pyogenes from patients with pharyngitis confirms their role in the etiology of this infection. Taken into account the significance of determination of sensitivity to bacitracin for the preliminary identification of S. pyogenes, it is necessary to standardize this test, which will make obtaining of the comparability of results possible.

The interplay of seizures-induced axonal sprouting and transcription-dependent Bdnf repositioning in the model of temporal lobe epilepsy.

The Brain-Derived Neurotrophic Factor is one of the most important trophic proteins in the brain. The role of this growth factor in neuronal plasticity, in health and disease, has been extensively studied. However, mechanisms of epigenetic regulation of Bdnf gene expression in epilepsy are still elusive. In our previous work, using a rat model of neuronal activation upon kainate-induced seizures, we observed a repositioning of Bdnf alleles from the nuclear periphery towards the nuclear center. This change of Bdnf intranuclear position was associated with transcriptional gene activity. In the present study, using the same neuronal activation model, we analyzed the relation between the percentage of the Bdnf allele at the nuclear periphery and clinical and morphological traits of epilepsy. We observed that the decrease of the percentage of the Bdnf allele at the nuclear periphery correlates with stronger mossy fiber sprouting-an aberrant form of excitatory circuits formation. Moreover, using in vitro hippocampal cultures we showed that Bdnf repositioning is a consequence of transcriptional activity. Inhibition of RNA polymerase II activity in primary cultured neurons with Actinomycin D completely blocked Bdnf gene transcription and repositioning occurring after neuronal excitation. Interestingly, we observed that histone deacetylases inhibition with Trichostatin A induced a slight increase of Bdnf gene transcription and its repositioning even in the absence of neuronal excitation. Presented results provide novel insight into the role of BDNF in epileptogenesis. Moreover, they strengthen the statement that this particular gene is a good candidate to search for a new generation of antiepileptic therapies.

Anti-CD44 DNA Aptamers Selectively Target Cancer Cells.

CD44 is a type I transmembrane glycoprotein interacting with a number of extracellular components, including hyaluronic acid (HA). CD44-HA axis is involved in a variety of processes, including adhesion, migration, differentiation, trafficking, and others. CD44 is overexpressed in several cancers where binding of HA induces signal transduction leading to activation of antiapoptotic proteins and factors linked to drug resistance. As such, CD44 has been implicated in cancer growth, progression, and metastasis. It has been convincingly demonstrated that blocking CD44-HA interaction decreases cancer cell survival and metastasis. In this study, using in vitro selection, we have developed DNA aptamers recognizing a HA-binding domain of CD44 with high affinity and specificity. The aptamers bind to CD44 with nanomolar affinities and efficiently inhibit the growth of leukemic cancer cells characterized by high expression of CD44. The selectivity is demonstrated by an irrelevant effect on cells characterized by low CD44 levels. The obtained aptamers broaden the existing landscape of potential approaches to the development of antitumor strategies based on inhibition of the CD44 axis.

Entosis: From Cell Biology to Clinical Cancer Pathology.

Entosis is a phenomenon, in which one cell enters a second one. New clinico-histopathological studies of entosis prompted us to summarize its significance in cancer. It appears that entosis might be a novel, independent prognostic predictor factor in cancer histopathology. We briefly discuss the biological basis of entosis, followed by a summary of published clinico-histopathological studies on entosis significance in cancer prognosis. The correlation of entosis with cancer prognosis in head and neck squamous cell carcinoma, anal carcinoma, lung adenocarcinoma, pancreatic ductal carcinoma and breast ductal carcinoma, is shown. Numerous entotic figures are associated with a more malignant cancer phenotype and poor prognosis in many cancers. We also showed that some anticancer drugs could induce entosis in cell culture, even as an escape mechanism. Thus, entosis is likely beneficial for survival of malignant cells, i.e., an entotic cell can hide from unfavourable factors in another cell and subsequently leave the host cell remaining intact, leading to failure in therapy or cancer recurrence. Finally, we highlight the potential relationship of cell adhesion with entosis in vitro, based on the model of the BxPc3 cells cultured in full adhesive conditions, comparing them to a commonly used MCF7 semiadhesive model of entosis.

Ultrastructural visualization of 3D chromatin folding using volume electron microscopy and DNA in situ hybridization.

The human genome is extensively folded into 3-dimensional organization. However, the detailed 3D chromatin folding structures have not been fully visualized due to the lack of robust and ultra-resolution imaging capability. Here, we report the development of an electron microscopy method that combines serial block-face scanning electron microscopy with in situ hybridization (3D-EMISH) to visualize 3D chromatin folding at targeted genomic regions with ultra-resolution (5 × 5 × 30 nm in xyz dimensions) that is superior to the current super-resolution by fluorescence light microscopy. We apply 3D-EMISH to human lymphoblastoid cells at a 1.7 Mb segment of the genome and visualize a large number of distinctive 3D chromatin folding structures in ultra-resolution. We further quantitatively characterize the reconstituted chromatin folding structures by identifying sub-domains, and uncover a high level heterogeneity of chromatin folding ultrastructures in individual nuclei, suggestive of extensive dynamic fluidity in 3D chromatin states.

Astrocytes determine conditioned response to morphine via glucocorticoid receptor-dependent regulation of lactate release.

To date, neurons have been the primary focus of research on the role of glucocorticoids in the regulation of brain function and pathological behaviors, such as addiction. Astrocytes, which are also glucocorticoid-responsive, have been recently implicated in the development of drug abuse, albeit through as yet undefined mechanisms. Here, using a spectrum of tools (whole-transcriptome profiling, viral-mediated RNA interference in vitro and in vivo, behavioral pharmacology and electrophysiology), we demonstrate that astrocytes in the nucleus accumbens (NAc) are an important locus of glucocorticoid receptor (GR)-dependent transcriptional changes that regulate rewarding effects of morphine. Specifically, we show that targeted knockdown of the GR in the NAc astrocytes enhanced conditioned responses to morphine, with a concomitant inhibition of morphine-induced neuronal excitability and plasticity. Interestingly, GR knockdown did not influence sensitivity to cocaine. Further analyses revealed GR-dependent regulation of astroglial metabolism. Notably, GR knockdown inhibited induced by glucocorticoids lactate release in astrocytes. Finally, lactate administration outbalanced conditioned responses to morphine in astroglial GR knockdown mice. These findings demonstrate a role of GR-dependent regulation of astrocytic metabolism in the NAc and a key role of GR-expressing astrocytes in opioid reward processing.

Three-Dimensional Segmentation and Reconstruction of Neuronal Nuclei in Confocal Microscopic Images.

The detailed architectural examination of the neuronal nuclei in any brain region, using confocal microscopy, requires quantification of fluorescent signals in three-dimensional stacks of confocal images. An essential prerequisite to any quantification is the segmentation of the nuclei which are typically tightly packed in the tissue, the extreme being the hippocampal dentate gyrus (DG), in which nuclei frequently appear to overlap due to limitations in microscope resolution. Segmentation in DG is a challenging task due to the presence of a significant amount of image artifacts and densely packed nuclei. Accordingly, we established an algorithm based on continuous boundary tracing criterion aiming to reconstruct the nucleus surface and to separate the adjacent nuclei. The presented algorithm neither uses a pre-built nucleus model, nor performs image thresholding, which makes it robust against variations in image intensity and poor contrast. Further, the reconstructed surface is used to study morphology and spatial arrangement of the nuclear interior. The presented method is generally dedicated to segmentation of crowded, overlapping objects in 3D space. In particular, it allows us to study quantitatively the architecture of the neuronal nucleus using confocal-microscopic approach.

Imidazole-free purification of His3-tagged recombinant proteins using ssDNA aptamer-based affinity chromatography.

Immobilized metal ion affinity chromatography (IMAC) is widely used for the purification of many different His6-tagged recombinant proteins. On the one hand, it is a powerful technique but on the other hand it has its disadvantages. In this report, we present the development of a unique ssDNA aptamer for the purification of His3-tagged recombinant proteins. Our study shows that stability of the His3-tag/H3T aptamer complex can be controlled by the sodium ion concentration. Based on this feature, we demonstrate that H3T aptamer resin was successfully employed for the purification of three out of four tested His3-tagged recombinant proteins from an E. coli total protein extract using imidazole-free buffers. Finally, we show that the purity of His3-tagged proteins is superior when purified with the help of the H3T aptamer in comparison with Ni-NTA resin.