Undifferentiated Carcinoma with Osteoclast-like Giant Cells of the Pancreas: Molecular Genetic Analysis of 13 Cases.

Undifferentiated carcinoma with osteoclast-like giant cells (UCOGC) of the pancreas is a rare malignancy regarded as a subvariant of pancreatic ductal carcinoma (PDAC) characterized by variable prognosis. UCOGC shows a strikingly similar spectrum of oncogenic DNA mutations to PDAC. In the current work, we analyzed the landscape of somatic mutations in a set of 13 UCOGC cases via next-generation sequencing (NGS). We detected a spectrum of pathogenic or likely pathogenic mutations similar to those observed in PDAC following previously published results (10 KRAS, 9 TP53, 4 CDKN2A, and 1 SMAD4, CIC, GNAS, APC, ATM, NF1, FBXW7, ATR, and FGFR3). Our results support the theory that UCOGC is a variant of PDAC, despite its unique morphology; however, a UCOGC-specific genomic signature as well as predictive markers remain mainly unknown. Programmed death ligand 1 (PD-L1) status remains an important predictive marker based on previous studies.

Ultrastructure of macromolecular assemblies contributing to bacterial spore resistance revealed by in situ cryo-electron tomography.

Bacterial spores owe their incredible resistance capacities to molecular structures that protect the cell content from external aggressions. Among the determinants of resistance are the quaternary structure of the chromosome and an extracellular shell made of proteinaceous layers (the coat), the assembly of which remains poorly understood. Here, in situ cryo-electron tomography on lamellae generated by cryo-focused ion beam micromachining provides insights into the ultrastructural organization of Bacillus subtilis sporangia. The reconstructed tomograms reveal that early during sporulation, the chromosome in the forespore adopts a toroidal structure harboring 5.5-nm thick fibers. At the same stage, coat proteins at the surface of the forespore form a stack of amorphous or structured layers with distinct electron density, dimensions and organization. By analyzing mutant strains using cryo-electron tomography and transmission electron microscopy on resin sections, we distinguish seven nascent coat regions with different molecular properties, and propose a model for the contribution of coat morphogenetic proteins.

Characterization of insulin crystalline form in isolated ß-cell secretory granules.

Insulin is stored in vivo inside the pancreatic ß-cell insulin secretory granules. In vitro studies have led to an assumption that high insulin and Zn2+ concentrations inside the pancreatic ß-cell insulin secretory granules should promote insulin crystalline state in the form of Zn2+-stabilized hexamers. Electron microscopic images of thin sections of the pancreatic ß-cells often show a dense, regular pattern core, suggesting the presence of insulin crystals. However, the structural features of the storage forms of insulin in native preparations of secretory granules are unknown, because of their small size, fragile character and difficult handling. We isolated and investigated the secretory granules from MIN6 cells under near-native conditions, using cryo-electron microscopic (Cryo-EM) techniques. The analysis of these data from multiple intra-granular crystals revealed two different rhomboidal crystal lattices. The minor lattice has unit cell parameters (a ≃ b ≃ 84.0 Å, c ≃ 35.2 Å), similar to in vitro crystallized human 4Zn2+-insulin hexamer, whereas the largely prevalent unit cell has more than double c-axis (a ≃ b ≃ c ≃ 96.5 Å) that probably corresponds to two or three insulin hexamers in the asymmetric unit. Our experimental data show that insulin can be present in pancreatic MIN6 cell granules in a microcrystalline form, probably consisting of 4Zn2+-hexamers of this hormone.

Structure of Human Enterovirus 70 and Its Inhibition by Capsid-Binding Compounds.

Enterovirus 70 (EV70) is a human pathogen belonging to the family Picornaviridae. EV70 is transmitted by eye secretions and causes acute hemorrhagic conjunctivitis, a serious eye disease. Despite the severity of the disease caused by EV70, its structure is unknown. Here, we present the structures of the EV70 virion, altered particle, and empty capsid determined by cryo-electron microscopy. The capsid of EV70 is composed of the subunits VP1, VP2, VP3, and VP4. The partially collapsed hydrophobic pocket located in VP1 of the EV70 virion is not occupied by a pocket factor, which is commonly present in other enteroviruses. Nevertheless, we show that the pocket can be targeted by the antiviral compounds WIN51711 and pleconaril, which block virus infection. The inhibitors prevent genome release by stabilizing EV70 particles. Knowledge of the structures of complexes of EV70 with inhibitors will enable the development of capsid-binding therapeutics against this virus. IMPORTANCE Globally distributed enterovirus 70 (EV70) causes local outbreaks of acute hemorrhagic conjunctivitis. The discharge from infected eyes enables the high-efficiency transmission of EV70 in overcrowded areas with low hygienic standards. Currently, only symptomatic treatments are available. We determined the structures of EV70 in its native form, the genome release intermediate, and the empty capsid resulting from genome release. Furthermore, we elucidated the structures of EV70 in complex with two inhibitors that block virus infection, and we describe the mechanism of their binding to the virus capsid. These results enable the development of therapeutics against EV70.

Preparation and Cryo-FIB micromachining of Saccharomyces cerevisiae for Cryo-Electron Tomography.

Today, cryo-electron tomography (cryo-ET) is the only technique that can provide near-atomic resolution structural data on macromolecular complexes in situ. Owing to the strong interaction of an electron with the matter, high-resolution cryo-ET studies are limited to specimens with a thickness of less than 200 nm, which restricts the applicability of cryo-ET only to the peripheral regions of a cell. A complex workflow that comprises the preparation of thin cellular cross-sections by cryo-focused ion beam micromachining (cryo-FIBM) was introduced during the last decade to enable the acquisition of cryo-ET data from the interior of larger cells. We present a protocol for the preparation of cellular lamellae from a sample vitrified by plunge freezing utilizing Saccharomyces cerevisiae as a prototypical example of a eukaryotic cell with wide utilization in cellular and molecular biology research. We describe protocols for vitrification of S. cerevisiae into isolated patches of a few cells or a continuous monolayer of the cells on a TEM grid and provide a protocol for lamella preparation by cryo-FIB for these two samples.

Cryo-Focused Ion Beam Lamella Preparation Protocol for in Situ Structural Biology.

The advances in electron cryo-microscopy have enabled high-resolution structural studies of vitrified macromolecular complexes in situ by cryo-electron tomography (cryo-ET). Since utilization of cryo-ET is generally limited to the specimens with thickness < 500 nm, a complex sample preparation protocol to study larger samples such as single eukaryotic cells by cryo-ET was developed and optimized over the last decade. The workflow is based on the preparation of a thin cellular lamella by cryo-focused ion beam milling (cryo-FIBM) from the vitrified cells. The sample preparation protocol is a multi-step process which includes utilization of several high-end instruments and comprises sample manipulation prone to sample deterioration. Here, we present a workflow for preparation of three different model specimens that was optimized to provide high-quality lamellae for cryo-ET or electron diffraction tomography with high reproducibility. Preparation of lamellae from large adherent mammalian cells, small suspension eukaryotic cell line, and protein crystals of intermediate size is described which represents examples of the most frequently studied samples used for cryo-FIBM in life sciences.

Capsid opening enables genome release of iflaviruses.

The family Iflaviridae includes economically important viruses of the western honeybee such as deformed wing virus, slow bee paralysis virus, and sacbrood virus. Iflaviruses have nonenveloped virions and capsids organized with icosahedral symmetry. The genome release of iflaviruses can be induced in vitro by exposure to acidic pH, implying that they enter cells by endocytosis. Genome release intermediates of iflaviruses have not been structurally characterized. Here, we show that conformational changes and expansion of iflavirus RNA genomes, which are induced by acidic pH, trigger the opening of iflavirus particles. Capsids of slow bee paralysis virus and sacbrood virus crack into pieces. In contrast, capsids of deformed wing virus are more flexible and open like flowers to release their genomes. The large openings in iflavirus particles enable the fast exit of genomes from capsids, which decreases the probability of genome degradation by the RNases present in endosomes.

Negative prognostic impact of PD-L1 expression in tumor cells of undifferentiated (anaplastic) carcinoma with osteoclast-like giant cells of the pancreas: study of 13 cases comparing ductal pancreatic carcinoma and review of the literature.

Pancreatic carcinoma remains one of the leading cancer-related causes of death worldwide and is generally characterized by a dismal prognosis and limited potential for oncologic treatment. A rare subvariant of pancreatic cancer, undifferentiated carcinoma with osteoclast-like giant cells (UCOGC), has an unpredictable prognosis according to many previous studies, with unexpectedly long survival in individual cases. In this study, we collected, retrospectively, 13 cases of well-documented UCOGCs and performed immunohistochemistry focused on the expression of the programmed death-ligand 1 (PD-L1) and several other potential therapeutic and predictive markers (PanTRK, p53, MSH2, PMS2, and the number of tumor-infiltrating lymphocytes), to explore their correlation with the follow-up of the patients. As a control group, we examined 24 cases of conventional pancreatic ductal adenocarcinoma (PDAC). In our results, PanTRK was negative in all 24 cases. P53 did not show any significant differences between UCOGCs and PDACs, and the entire cohort was MSH2, MLH1, PMS2, and MSH6 positive. Significant differences were present in the analysis of PD-L1: UCOGCs were found to express PD-L1 significantly more frequently and have a higher number of tumor-infiltrating lymphocytes than PDAC. The expression of PD-L1 was related to significantly shorter survival in patients with UCOGC and in the entire cohort. Patients with PD-L1 negative UCOGCs displayed surprisingly long survival in comparison to PD-L1 positive UCOGCs and PDACs (both PD-L1+ and PD-L1-). We compared our results with previously published data, and, after statistical analysis, we were able to identify PD-L1 as an effective prognostic marker of UCOGC and suggest a strong need for a clinical trial of immune checkpoint immunotherapy in patients with advanced PD-L1 positive UCOGC.

Enterovirus particles expel capsid pentamers to enable genome release.

Viruses from the genus Enterovirus are important human pathogens. Receptor binding or exposure to acidic pH in endosomes converts enterovirus particles to an activated state that is required for genome release. However, the mechanism of enterovirus uncoating is not well understood. Here, we use cryo-electron microscopy to visualize virions of human echovirus 18 in the process of genome release. We discover that the exit of the RNA from the particle of echovirus 18 results in a loss of one, two, or three adjacent capsid-protein pentamers. The opening in the capsid, which is more than 120 Å in diameter, enables the release of the genome without the need to unwind its putative double-stranded RNA segments. We also detect capsids lacking pentamers during genome release from echovirus 30. Thus, our findings uncover a mechanism of enterovirus genome release that could become target for antiviral drugs.

Virion structure and genome delivery mechanism of sacbrood honeybee virus.

Infection by sacbrood virus (SBV) from the family Iflaviridae is lethal to honey bee larvae but only rarely causes the collapse of honey bee colonies. Despite the negative effect of SBV on honey bees, the structure of its particles and mechanism of its genome delivery are unknown. Here we present the crystal structure of SBV virion and show that it contains 60 copies of a minor capsid protein (MiCP) attached to the virion surface. No similar MiCPs have been previously reported in any of the related viruses from the order Picornavirales. The location of the MiCP coding sequence within the SBV genome indicates that the MiCP evolved from a C-terminal extension of a major capsid protein by the introduction of a cleavage site for a virus protease. The exposure of SBV to acidic pH, which the virus likely encounters during cell entry, induces the formation of pores at threefold and fivefold axes of the capsid that are 7 Å and 12 Å in diameter, respectively. This is in contrast to vertebrate picornaviruses, in which the pores along twofold icosahedral symmetry axes are currently considered the most likely sites for genome release. SBV virions lack VP4 subunits that facilitate the genome delivery of many related dicistroviruses and picornaviruses. MiCP subunits induce liposome disruption in vitro, indicating that they are functional analogs of VP4 subunits and enable the virus genome to escape across the endosome membrane into the cell cytoplasm.

Viral discovery and diversity in trypanosomatid protozoa with a focus on relatives of the human parasite Leishmania.

Knowledge of viral diversity is expanding greatly, but many lineages remain underexplored. We surveyed RNA viruses in 52 cultured monoxenous relatives of the human parasite Leishmania (Crithidia and Leptomonas), as well as plant-infecting PhytomonasLeptomonas pyrrhocoris was a hotbed for viral discovery, carrying a virus (Leptomonas pyrrhocoris ostravirus 1) with a highly divergent RNA-dependent RNA polymerase missed by conventional BLAST searches, an emergent clade of tombus-like viruses, and an example of viral endogenization. A deep-branching clade of trypanosomatid narnaviruses was found, notable as Leptomonas seymouri bearing Narna-like virus 1 (LepseyNLV1) have been reported in cultures recovered from patients with visceral leishmaniasis. A deep-branching trypanosomatid viral lineage showing strong affinities to bunyaviruses was termed "Leishbunyavirus" (LBV) and judged sufficiently distinct to warrant assignment within a proposed family termed "Leishbunyaviridae" Numerous relatives of trypanosomatid viruses were found in insect metatranscriptomic surveys, which likely arise from trypanosomatid microbiota. Despite extensive sampling we found no relatives of the totivirus Leishmaniavirus (LRV1/2), implying that it was acquired at about the same time the Leishmania became able to parasitize vertebrates. As viruses were found in over a quarter of isolates tested, many more are likely to be found in the >600 unsurveyed trypanosomatid species. Viral loss was occasionally observed in culture, providing potentially isogenic virus-free lines enabling studies probing the biological role of trypanosomatid viruses. These data shed important insights on the emergence of viruses within an important trypanosomatid clade relevant to human disease.

AFLP reveals low genetic diversity of the bryozoan Pectinatella magnifica (Leidy, 1851) in the Czech Republic.

BACKGROUND: Non-native species have aroused scientific interest because of their ability to successfully colonise areas to which they have been introduced, despite their sometimes limited genetic variation compared to their native range. These species establish themselves with the aid of some pre-existing features favouring them in the new environment. Pectinatella magnifica (Leidy, 1851), the freshwater magnificent bryozoan, is non-native in Europe and Asia. This study was designed to determine the genetic diversity and population structure of P. magnifica colonies collected from the Protected Landscape Area (PLA) and UNESCO Biosphere Reserve Trebonsko (the Czech Republic) in the 2009 and 2011-2014 periods using Amplified Fragment Length Polymorphism (AFLP). FINDINGS: The vast majority of the examined non-native colonies, except three colonies sampled in 2012, expressed very low levels of genetic variation, not differentiating from the USA native colony. The Bayesian clustering approach grouped the 28 accessions into two genetically different populations. CONCLUSIONS: The data suggest relatively low gene diversity within all colonies, which might reflect the recent expansion of P. magnifica in the Czech Republic.

Simplifying procedure for prediction of resistance risk in CML patients - Test of sensitivity to TKI ex vivo.

Tyrosine kinase inhibitors (TKIs) targeting BCR-ABL have dramatically improved chronic myeloid leukemia therapy. While imatinib remains to be the first line therapy, about 30% of patients develop resistance or intolerance to this drug and are recommended to switch to other TKIs. Nilotinib and dasatinib are currently implemented into the first line therapy and other inhibitors have already entered the clinical practice. This opens further questions on how to select the best TKI for each patient not only during the therapy but also at diagnosis. The individualized therapy concept requires a reliable establishment of prognosis and prediction of response to the available TKIs. We tested the ex vivo sensitivity of patient primary leukocytes to imatinib, nilotinib and dasatinib - two concentrations of each inhibitor for 48h incubation - and we evaluated the usefulness of such tests for the clinical practice. Besides reflecting the actual sensitivity to the therapy, our optimized simple tests were able to predict the outcome in 90/87% of patients, for the next 12/24months, respectively. According to these results, the presented ex vivo testing could help clinicians to select the appropriate drug for each patient at diagnosis and also at any time of the therapy.

Evaluation of 5-year imatinib treatment of 458 patients with CP-CML in routine clinical practice and prognostic impact of different BCR-ABL cutoff levels.

We evaluated responses to the treatment and long-term outcomes of chronic myeloid leukemia patients treated with imatinib as first-line treatment in routine clinical setting from two countries with centralized tyrosine kinase inhibitors (TKIs) treatment. We assessed prognostic significance of European LeukemiaNet (ELN) 2006- and 2009-defined responses and the prognostic value of molecular responses at defined time points on 5-year survivals. Among the cumulative rates of incidence of hematologic, cytogenetic, and molecular responses and all important survival parameters, we evaluated the prognostic significance of different BCR-ABL transcript-level ratios (≤1%; >1%-≤10%; >10%) at 3, 6, 12, and 18 months (n = 199). The ELN optimal response criteria and their predictive role were significantly beneficial for event-free survival at all given time points. We found significant improvement in survivals of patients with BCR-ABL lower than 10% in the 6th and 12th months. Significantly better outcome was found in patients who achieved major molecular response (MMR) in the 12th month. The cumulative incidences of complete cytogenetic response (CCyR) and MMR were significantly associated with the molecular response in the 3rd month. The ELN response criteria and their predictive role were helpful at given time points; however, the 2009 definition did not significantly alter the prognostic accuracy compared with that of the 2006 definition. The significant value was observed for cytogenetic responses at the 6th and 12th month. Moreover, progression-free and event-free survivals were improved with MMR at the 12th month.

Hsp90 - a potential prognostic marker in CML.

Heat shock proteins (Hsp) are important for the stability and function of cell proteins and thus for cell survival under physiological as well as stress conditions. Hsps were also reported to play an important role in tumorogenesis including leukemias. In this study we followed up Hsp70 and 90 protein levels in samples from patients with chronic myeloid leukemia (CML) to evaluate these Hsps with regard to their ability to characterize the disease status and disease prognosis. We analyzed 68 samples of total leukocytes of CML patients with different response to therapy with tyrosine kinase inhibitors. The results of Western blot analyses showed that the level of Hsp70 did not change in the course of the disease and did not correlate with response to therapy. In contrast, Hsp90 levels showed good correlation with the disease state. Patients with good response to therapy (major molecular response-MMR, molecular remission-CMR) had low expression levels of Hsp90, similar to those in healthy individuals. High Hsp90 levels were found in patients with resistance to therapy (hematological relapse-HR, accelerated phase or blast crisis), and in leukemic cell line K562. The results of the study suggested that not the kinetics but the particular level of Hsp90 at any time point since therapy start is of prognostic value: Hsp90 level above 0.27 significantly predicted poor response to TKI therapy (relapse, progression) and the level below 0.085 good response (MMR, CMR). In conclusion, Hsp90 level in total leukocytes could serve as a risk factor at diagnosis as well as during therapy and might help in clinical decision making especially in cases where BCR-ABL monitoring is of low predictive value. Our data suggest that high expression of HSP90 contributes to the aggressivity of the disease and should be considered as an important target for specialized CML therapy.

Expression of four major WT1 splicing variants in acute and chronic myeloid leukemia patients analyzed by newly developed four real-time RT PCRs.

Although the mechanism of action of leukemic oncogene Wilms' tumor gene 1 (WT1) remains unclear, WT1 has already been used in monitoring of patients with acute myeloid leukemia (AML) and it is being tested for immunotherapy. More detailed understanding of the role of WT1 in leukemia may improve its utilization. At least 36 isoforms may be produced. Four major variants denoted as -5/-KTS, -5/+KTS, +5/-KTS and +5/+KTS are produced by combining splicing of exon 5 and KTS sequence. In this study, we report applicability of newly developed real-time RT PCRs enabling for the first time full quantification of the four major WT1 splicing variants. Following careful optimization and testing of quantification reliability of four assays, we analyzed 34 samples of patients with AML and 12 samples of patients with chronic myeloid leukemia (CML) at the time of diagnosis. Analyses of five more CML patients provided insight into WT1 variants expression kinetics. We found predominance of +5/+KTS in both diagnoses. Comparison of WT1 variant expression in AML and CML patients' groups differing in response to therapy suggested possible importance of particular WT1 variant levels as markers of further disease course.

Modulation of metabolic activity of phagocytes by antihistamines.

The purpose of the study was to investigate the effects of H(1)-antihistamines of the 1(st) generation (antazoline, bromadryl, brompheniramine, dithiaden, cyclizine, chlorcyclizine, chlorpheniramine, clemastine) and the 2(nd) generation (acrivastine, ketotifen, and loratadine) on the respiratory burst of phagocytes. Reactive oxygen species generation in neutrophils isolated from rat blood was measured using luminol-enhanced chemiluminescence. Changes in nitrite formation and iNOS protein expression by RAW 264.7 macrophages were analysed using Griess reaction and Western blotting. The antioxidative properties of drugs in cell-free systems were detected spectrophotometrically, luminometrically, fluorimetrically, and amperometrically. The majority of the H(1)-antihistamines tested (bromadryl, brompheniramine, chlorcyclizine, chlorpheniramine, clemastine, dithiaden, and ketotifen) exhibited a significant inhibitory effect on the chemiluminescence activity of phagocytes. H(1)-antihistamines did not show significant scavenging properties against superoxide anion and hydroxyl radical, thus this could not contribute to the inhibition of chemiluminescence. H(1)-antihistamines had a different ability to modulate nitric oxide production by LPS-stimulated macrophages. Bromadryl, clemastine, and dithiaden were the most effective since they inhibited iNOS expression, which was followed by a significant reduction in nitrite levels. H(1)-antihistamines had no scavenging activity against nitric oxide. It can be concluded that the effects observed in the H(1)-antihistamines tested are not mediated exclusively via H(1)-receptor pathway or by direct antioxidative properties. Based on our results, antihistamines not interfering with the microbicidal mechanisms of leukocytes (antazoline, acrivastine and cyclizine) could be used preferentially in infections. Other antihistamines should be used, under pathological conditions accompanied by the overproduction of reactive oxygen species.

Expression patterns of microRNAs associated with CML phases and their disease related targets.

BACKGROUND: MicroRNAs are important regulators of transcription in hematopoiesis. Their expression deregulations were described in association with pathogenesis of some hematological malignancies. This study provides integrated microRNA expression profiling at different phases of chronic myeloid leukemia (CML) with the aim to identify microRNAs associated with CML pathogenesis. The functions of in silico filtered targets are in this report annotated and discussed in relation to CML pathogenesis. RESULTS: Using microarrays we identified differential expression profiles of 49 miRNAs in CML patients at diagnosis, in hematological relapse, therapy failure, blast crisis and major molecular response. The expression deregulation of miR-150, miR-20a, miR-17, miR-19a, miR-103, miR-144, miR-155, miR-181a, miR-221 and miR-222 in CML was confirmed by real-time quantitative PCR. In silico analyses identified targeted genes of these miRNAs encoding proteins that are involved in cell cycle and growth regulation as well as several key signaling pathways such as of mitogen activated kinase-like protein (MAPK), epidermal growth factor receptor (EGFR, ERBB), transforming growth factor beta (TGFB1) and tumor protein p53 that are all related to CML. Decreased levels of miR-150 were detected in patients at diagnosis, in blast crisis and 67% of hematological relapses and showed significant negative correlation with miR-150 proved target MYB and with BCR-ABL transcript level. CONCLUSIONS: This study uncovers microRNAs that are potentially involved in CML and the annotated functions of in silico filtered targets of selected miRNAs outline mechanisms whereby microRNAs may be involved in CML pathogenesis.