Animal models of liver diseases and their application in experimental surgery.

Both acute and chronic liver diseases are frequent and potentially lethal conditions. Development of new therapeutic strategies and drugs depends on understanding of liver injury pathogenesis and progression, which can be studied on suitable animal models. Due to the complexity of liver injury, the understanding of underlying mechanisms of liver diseases and their treatment has been limited by the lack of satisfactory animal models. SO far, a wide variety of animals has been used to mimic human liver disease, however, none of the models include all its clinical aspects seen in humans. Rodents, namely rats and mice, represent the largest group of liver disease models despite their limited resemblance to human. On the other hand, large animal models like pigs, previously used mostly in acute liver failure modeling, are now playing an important role in studying various acute and chronic liver diseases. Although significant progress has been made, the research in hepatology should continue to establish animal models anatomically and physiologically as close to human as possible to allow for translation of the experimental results to human medicine. This review presents various approaches to the study of acute and chronic liver diseases in animal models, with special emphasis on large animal models and their role in experimental surgery.

ASXL1 gene alterations in patients with isolated 20q deletion.

Deletion 20q is a recurrent abnormality in myeloid malignancies. In our previous study, we identified fusion of the additional sex combs-like 1 (ASXL1) and teashirt zinc finger homeobox 2 genes in a patient with myelodysplastic syndrome. The objective of this study was to determine the frequency of ASXL1 breakpoints in a cohort of 36 patients with deletion 20q as the sole cytogenetic aberration. A combination of molecular cytogenetic methods was used to confirm ASXL1 gene alterations in 19 of the 36 patients, and the determination of ASXL1 gene changes in patients with deletion 20q revealed clinical and prognostic impacts.

Separation of replication and transcription domains in nucleoli.

In mammalian cells, active ribosomal genes produce the 18S, 5.8S and 28S RNAs of ribosomal particles. Transcription levels of these genes are very high throughout interphase, and the cell needs a special strategy to avoid collision of the DNA polymerase and RNA polymerase machineries. To investigate this problem, we measured the correlation of various replication and transcription signals in the nucleoli of HeLa, HT-1080 and NIH 3T3 cells using a specially devised software for analysis of confocal images. Additionally, to follow the relationship between nucleolar replication and transcription in living cells, we produced a stable cell line expressing GFP-RPA43 (subunit of RNA polymerase I, pol I) and RFP-PCNA (the sliding clamp protein) based on human fibrosarcoma HT-1080 cells. We found that replication and transcription signals are more efficiently separated in nucleoli than in the nucleoplasm. In the course of S phase, separation of PCNA and pol I signals gradually increased. During the same period, separation of pol I and incorporated Cy5-dUTP signals decreased. Analysis of single molecule localization microscopy (SMLM) images indicated that transcriptionally active FC/DFC units (i.e. fibrillar centers with adjacent dense fibrillar components) did not incorporate DNA nucleotides. Taken together, our data show that replication of the ribosomal genes is spatially separated from their transcription, and FC/DFC units may provide a structural basis for that separation.

Frequent chromatin rearrangements in myelodysplastic syndromes--what stands behind?

Myelodysplastic syndromes (MDS) represent a clinically and genetically heterogeneous group of clonal haematopoietic diseases characterized by a short survival and high rate of transformation to acute myeloid leukaemia (AML). In spite of this variability, MDS is associated with typical recurrent non-random cytogenetic defects. Chromosomal abnormalities are detected in the malignant bone-marrow cells of approximately 40-80 % of patients with primary or secondary MDS. The most frequent chromosomal rearrangements involve chromosomes 5, 7 and 8. MDS often shows presence of unbalanced chromosomal changes, especially large deletions [del(5), del(7q), del(12p), del(18q), del(20q)] or losses of whole chromosomes (7 and Y). The most typical cytogenetic abnormality is a partial or complete deletion of 5q- that occurs in roughly 30 % of all MDS cases either as the sole abnormality or in combination with other aberrations as a part of frequently complex karyotypes. The mechanisms responsible for the formation of MDS-associated recurrent translocations and complex karyotypes are unknown. Since some of the mentioned aberrations are characteristic for several haematological malignancies, more general cellular conditions could be expected to play a role. In this article, we introduce the most common rearrangements linked to MDS and discuss the potential role of the non-random higher-order chromatin structure in their formation. A contribution of the chromothripsis - a catastrophic event discovered only recently - is considered to explain how complex karyotypes may occur (during a single event).

[Molecular cytogenetic analysis of chromosomal aberrations in cells of low grade gliomas and its contribution for tumour classification]. / Molekulárne cytogenetická analýza chromozomových aberací v bunkách nízkostupnových gliomu a její prínos pro klasifikaci nádoru.

BACKGROUND: Low-grade gliomas represent a heterogeneous group of primary brain malignancies. The current diagnostics of these tumors rely strongly on histological classification. With the development of molecular cytogenetic methods several genetic markers were described, contributing to a better distinction of glial subtypes. The aim of this study was to assess the frequency of acquired chromosomal aberrations in lowgrade gliomas and to search for new genomic changes associated with higher risk of tumor progression. PATIENTS AND METHODS: We analysed biopsy specimens from 41 patients with histological dia-gnosis of low-grade glioma using interphase fluorescence in situ hybridization (I FISH) and single nucleotide polymorphism (SNP) array techniques (19 females and 22 males, medium age 42 years). RESULTS: Besides notorious and most frequent finding of combined deletion of 1p/ 19q (81.25% patients) several other recurrent aberrations were described in patients with oligodendrogliomas: deletions of p and q arms of chromosome 4 (25% patients), deletions of the short arms of chromosome 9 (18.75% patients), deletions of the long arms of chromosome 13 and monosomy of chromosome 18 (18.75% patients). In bio-psy specimens from patients with astrocytomas, we often observed deletion of 1p (24% patients), amplification of the long arms of chromosome 7 (16% patients), deletion of the long arm of chromosome 13 (20% patients), segmental uniparental disomy (UPD) of the short arms of chromosome 17 (60% patients) and deletion of the long arms of chromosome 19 (28% patients). In one patient we detected a shuttered chromosome 10 resulting from chromothripsis. CONCLUSION: Using a combination of I FISH and SNP array, we detected not only known chromosomal changes but also new or less frequent recur-rent aberrations. Their role in cancer  cell progression and their impact on low grade gliomas classification remains to be elucidated in a larger cohort of patients.

The effect of allogeneic stem cell transplantation on high risk chronic lymphocytic leukaemia: a single centre retrospective analysis.

In recent years, many studies have confirmed that allogeneic stem cell transplantation (allo-SCT) can provide long-term disease control and possible cure in selected patients with chronic lymphocytic leukaemia (CLL), including those with a biologically highly unfavourable risk profile. A retrospective analysis of allo-SCT in 30 patients with CLL whose risk profile was unfavourable and who were treated in the years 2000-2009 was performed. The aim was to compare the results of allo-SCT by prognostic factors and conditioning type and evaluate the results of unrelated transplantation. The median age was 54 years. Donors were 8 HLA-matched siblings and 22 unrelated volunteers, 11 of whom were mismatched. Eighteen patients were treated with reduced intensity conditioning. Twelve patients received myeloablative conditioning. Estimated overall survival (OS) at 3 years was 78%, progression-free survival (PFS) 71%, relapse incidence 10% and non-relapse mortality (NRM) 16%, respectively, with a median follow-up of 35 months. According to molecular/cytogenetic characteristics, OS and PFS for the high risk group (17p- or 11q-) were 89 and 77%, respectively, not significantly different from those with standard risk. Graft-versus-host disease (GVHD) was associated with greater toxicity; significantly higher NRM for patients with aGVHD (p = 0.04) and worse PFS for patients with cGVHD (p = 0.04). Our results for the refractory disease group (77% responses) indicate that chemoresistance may be overcome by the GVL effect. Transplants from unrelated donors may be considered comparable to those from related donors.

The assessment of array comparative genomic hybridization in complex karyotype analyses.

Molecular-cytogenetic methods were used to analyse and specify complex genome rearrangements in malignant cells. Twelve samples of bone marrow cells were collected from patients with myelodysplastic syndromes (MDS). The complex karyotypes were examined by multicolour fluorescence in situ hybridization (mFISH), high-resolution multicolour banding (mBAND) and array comparative genomic hybridization (aCGH). For aCGH, DNA was isolated from fixed bone marrow cells in methanol and acetic acid and amplified by whole-genome amplification. Three samples were analysed by the oligonucleotide array NimbleGen on the basis of full service. BAC-based Haematochips (BlueGnome) were used for the other nine samples. Sensitivity and detection limits of both methods were compared. The results obtained by mFISH/mBAND were in most cases confirmed by the microarray technique. aCGH detected 43 unbalanced chromosomal changes that were also identified by classical cytogenetics and FISH. Moreover, aCGH discovered 14 additional changes. Cryptic amplifications and deletions were characterized with a resolution of 0.5 Mb. In one bone marrow sample with suspected monosomy 5 detected by conventional cytogenetic analysis, aCGH revealed a 22.3 Mb region of chromosome 5 inserted in another autosome within the complex karyotype. Amplified DNA was successfully used for aCGH in 11 out of 12 cases, improving resolution of unbalanced chromosomal aberrations. The combination of both approaches brought more detailed description of complex karyotypes and is highly recommended.

Prognostic factors and treatment outcome in 1,516 adult patients with de novo and secondary acute myeloid leukemia in 1999-2009 in 5 hematology intensive care centers in the Czech Republic.

Acute myeloid leukemia (AML) is a severe condition with a high mortality. When making decisions about the optimal tailor-made therapy, numerous prognostic factors are considered. The study represents a detailed analysis of the role of these factors and treatment outcomes based on a long-term follow-up of patients treated in 5 hematology intensive care centers in the Czech Republic.The studied group comprised 1,188 patients with de novo AML and 328 patients with secondary AML. The latter were significantly older, had more unfavorable cytogenetic changes and less frequently received curative therapy. Curatively treated patients achieved fewer complete remissions and relapsed more often than those with de novo AML. Patients with secondary AML had lower rates of allogeneic transplantation as part of consolidation therapy and a significantly shorter median overall survival. A lower proportion of the patients were alive at the time of analysis. However, the treatment outcome of de novo AML patients is not satisfactory, the only exception being those with acute promyelocytic leukemia. The analysis, which did not evaluate the intention-to-treat criteria and was without randomization, found allogeneic stem cell transplantation to be the most effective modality of consolidation therapy in both groups of patients. .

The role of telomeres and telomerase complex in haematological neoplasia: the length of telomeres as a marker of carcinogenesis and prognosis of disease.

Human telomeres (discovery of telomere structure and function has been recently awarded The Nobel Prize) consist of approximately 5-12 kb of tandem repeated sequences (TTAGGG)n and associated proteins capping chromosome ends which prevent degradation, loss of genetic information, end-to-end fusion, senescence and apoptosis. Due to the end-replication problem, telomere repeats are lost with each cell division, eventually leading to genetic instability and cellular senescence when telomeres become critically short. Stabilization of the telomeric DNA through telomerase activation, unique reverse transcriptase, or activation of the alternative mechanism of telomere maintenance is essential if the cells are to survive and proliferate indefinitely. Telomerase is expressed during early development and remains fully active in specific germline cells, but is undetectable in most normal somatic cells. High level of telomerase activity is detected in almost 90% of human tumours and immortalized cell lines. The hematopoietic compartment may develop genetic instability as a consequence of telomere erosion, resulting in aplastic anaemia (AA) and increased risk of myelodysplastic syndrome (MDS) and acute myeloid leukaemia (AML). Genetic instability associated with telomere dysfunction (i.e. short telomeres) is an early event in carcinogenesis. The molecular cytogenetic method telomere/centromere fluorescence in situ hybridization (T/C-FISH) can be used to characterize the telomere length of hematopoietic cells. This review describes recent advances in the molecular characterization of telomere system, the regulation of telomerase activity in cancer pathogenesis and shows that the telomeric length could be a potential clinical marker of hematologic neoplasia and prognosis of disease.

Dasatinib in imatinib-resistant or -intolerant CML patients: data from the clinical practice of 6 hematological centers in the Czech Republic.

Dasatinib is effective second line treatment for patients with chronic myeloid leukemia (CML) resistant or intolerant to imatinib. We report here the first experiences with dasatinib therapy in 71 CML patients resistant or intolerant to imatinib from the real clinical practice of 6 hematological centers in the Czech Republic. Dose 100 mg daily and 70 mg twice daily was administered to patients with chronic phase (CP) and advanced phases (AP) CML. In chronic phase (n=46), complete hematological reponse (CHR) was achieved in 97%, major cytogenetic reponse (MCgR) in 77% and complete cytogenetic response (CCgR) in 67%. Major molecular reponse (MMR) was achieved in 19/31 patients in median of 10 months. In advanced phase (n=25), CHR was attained in 77%, MCgR in 39%, CCgR in 33% and MMR in 2/18 patients. Eleven different baseline mutations were followed up in 15 patients. Dasatinib eliminated mutations in most of the patients, but 3 patients acquired a new one. Novel mutations were detected under dasatinib therapy in 2 patients. Dasatinib was well tolerated, cytopenias were common and was managed by dose modification. The estimated progression free survival (PFS) at 12 months was 97+/-3% in CP and 62+/-21% in AP. The median time to treatment failure was 605 days in AP while it was not reached in CP patients. Our clinical experiences, described here, confirmed that dasatinib is associated with high response rates especially in imatinib resistant or intolerant CML patients in chronic phase.

Telomere length, molecular cytogenetic findings, and immunophenotypic features in previously untreated patients with B-chronic lymphocytic leukemia.

UNLABELLED: Telomere length was evaluated by terminal repeat fragment method in 66 previously untreated patients with B-chronic lymphocytic leukemia (B-CLL) to ascertain whether telomere shortening was associated with genomic aberrations, immunoglobulin variable heavy chain (IgVH) mutational status, CD38 and ZAP-70 expression, and telomerase activity. Chromosomal aberrations were present in peripheral blood cells of 73% patients (48/66), no difference in telomere length between patients with good and intermediate prognosis according to cytogenetics was found. Association between telomere length and IgVH mutational status, ZAP-70 and CD38 expression was proved as significantly shorter telomeres in patients with unmutated IgVH status (p=0.01) and ZAP-70 positivity (p=0.01) and CD38 positivity (p=0.05) were detected. Telomerase activity was positive in 11 patients out of 21 examined, correlation between telomere length and telomerase activity was found (p=0.05). Telomere length and telomerase activity in combination with other prognostic parameters complete the risk profile of B-CLL patients and might serve for an easy decision on optimal treatment strategy. KEYWORDS: B-chronic lymphocytic leukemia, telomere length, telomerase activity, chromosomal aberrations, prognosis.

[Patients older than 80 years with de novo acute myeloid leukemias without erythroblastic and/or megakaryocytic dysplasia achieve complete remission and longer survival after classical chemotherapy 3 + 7]. / Nemocní starsí 80 let s de novo akutními myeloidními leukemiemi bez dysplazie v erytroblastické 8/nebo megakaryocytární rade dosahují kompletní remise a delsího prezlití po klasické chemoterapii 3+7.

Chemotherapy in most patients with AML over 80 years of age is not recommended because their median survival is about 1 month. The aim of our study was to identify patients in this age group who might achieve complete remission with standard dose chemotherapy. We report 9 consecutive patients with de novo AML diagnosed and treated in 1992-2008. All bone marrow samples were hypercellular, classified as FAB types M2 in 2 cases, M4 in 6, and M5 in one case. Three patients opted for supportive or palliative therapy and survived 1-4 months. Six patients received standard dose chemotherapy. Two patients with a normal karyotype had resistant AML and survived 1.0 and 2.7 months; one patient with a complex karyotype died of septic shock on the 10th day of therapy. All these three patients exhibited erythroblastic and/or megakaryocytic dysplasia (EMD) at presentation (two in more than 26% erythroblasts, all three in a half or more of megakaryocytes). Three remaining patients with AML M4, a normal karyotype but without EMD, achieved complete remission in spite of co-morbidities and a poor performance status. Two of them survived 18.6 and 28 months on maintenance therapy, the third 16.5 months without it. Very elderly AML patients without EMD appear to represent a favorable prognostic biological category (single-lineage AML) that show a good response to standard dose chemotherapy.

Fluorescence in situ hybridization applied to domestic animal cytogenetics.

The aim of this article is not to present an exhaustive review of molecular cytogenetics applications in domestic animal species, but more to illustrate the considerable contribution of these approaches in diagnostics and research in economically important species. A short presentation of the main applications of molecular cytogenetics in humans points out the domains in which it has become an essential tool and underlines the specificities attached to this species in comparison to farm animals. This article is devoted to outlining the current resources available in domestic species and to some examples of fluorescence in situ hybridization applications in the cattle, pig, horse and avian species. From a clinical point of view, these examples illustrate the advantages of FISH for the study of chromosomal abnormalities (identification, characterization and estimation of their effects). Other applications of molecular cytogenetics are also illustrated, particularly ZOO-FISH, an approach which allows the determination of chromosome homologies between species. Finally, a specific emphasis was placed on the usefulness of molecular cytogenetics for the analysis of species such as poultry, which harbour a complex karyotype.

Ranibizumab treatment for neovascular age-related macular degeneration: from randomized trials to clinical practice.

BACKGROUND: Although randomized clinical trials (ANCHOR and MARINA) have shown excellent results of ranibizumab treatment in patients with neovascular age-related macular degeneration (AMD), it is unclear whether such an outcome is achievable in daily practice. We evaluated the results of ranibizumab treatment for neovascular AMD in clinical practice in Australia. METHODS: A retrospective chart review of patients in four practices injected with ranibizumab in 2006 for AMD. Patients who had been diagnosed with subfoveal choroidal neovascular membrane in the preceding 6 months and had completed at least 6 months follow-up were enrolled. No standard treatment protocols were required. The main outcome measure was visual acuity (VA) at 6 and 12 months. RESULTS: A total of 158 patients fulfilled the entry criteria. The mean baseline VA (decimal) was 0.35+/-0.21 (Snellen equivalent 6/17). At 6 months, the mean VA improved to 0.46+/-0.27 (6/13) and remained stable until month 12 (0.48+/-0.30). The improvement in VA between baseline and months 6 and 12 was statistically significant (P<0.0001). Both the mean and the median number of injections were four in the first 6 months and nine at 12 months. VA results were comparable with those of the ANCHOR and MARINA trials, and were achieved with a lower number of injections (P<0.0001). CONCLUSION: VA results achieved in daily clinical practice using ranibizumab for neovascular AMD are similar to large prospective randomized trials.

Clonal evolution in chronic lymphocytic leukemia studied by interphase fluorescence in-situ hybridization.

The results of repeated interphase fluorescence in-situ hybridization (I-FISH, FISH) examination of 97 CLL patients and correlation of these findings with IgVH hypermutation status, ZAP-70 and CD38 expression are presented. The appearance of new, FISH-detectable, genomic aberrations during disease course, described as clonal evolution (CE), was observed in 26% of patients. The most frequent newly acquired cytogenetic abnormality was 13q deletion in 64% (16/25). In contrast to earlier studies, there was no correlation found between CE and either one of single negative prognostic factors (unmutated IgVH; CD38 positivity; ZAP-70 positivity). However, the combination of all three negative factors correlated with CE highly significantly (p=0.005) and moreover, also with a shift from lower to higher FISH risk category (p=0.010). As the prognostic data were known in all patients, this study represents the complete insight on the association of CE and other risk parameters in CLL.

[Urgent states in hematology: acute promyelocytic leukemia--principles of diagnosis]. / Urgentní stav v hematologii: akutní promyelocytární leukemie--principy diagnostiky.

A review of diagnosis of acute promyelocytic leukemia (APL) is presented. There are still many patients with progressive disease with leukocytosis at presentation. These are at greater risk of early death due to bleeding (often intracranial), or, less frequently, due to thrombotic complications. In Czechia, we have, in some instances, noted an unacceptably long time from the first symptoms to diagnosis and to administration of the highly specific differentiation therapy with tretinoin (ATRA) along with anthracycline chemotherapy. This combination is highly efficient--cures are seen in some 70% of patients. Therefore, we present a diagnostic minimum for each and every internist, and even better for every general practician, to get acquainted with. All cases of pancytopenia and consumption coagulopathy should be suspected of APL and referred to a specialized hematologist without any delay. In the following more detailed review of diagnostic measures, much attention is given to APL morphology, which is the first clue leading to diagnosis. The finding of the typical hypergranular FAB M3 morphology and of cells with bundles of Auer rods ("faggot cells"), along with the HLA-DR, CD33+ immunophenotype, is highly (but not absolutely) specific for APL. In cases of the micro-/hypo-granular variant FAB M3v Form, and whenever APL cannot be ruled out with certainty, a test to prove the presence of the PML/RARalpha fusion gene is indicated, using either RT-PCR or, eventually, immunological demonstration of the specific distribution of the PML protein in the cell nucleus. Given that morphology of APL cases, as defined according to WHO criteria (95% of which carry the PML/RARalpha fusion gene), admits extremely divergent morphological pictures ofthe variant forms, we recommend these investigations to be performed in every case of de novo acute myeloid leukemia. A review of the less frequent morphological, as well as genetic variants is given, and the principles of immunophenotypic, cytogenetic and molecular diagnostics are also reviewed.

[Therapy of acute promyelocytic leukemia in Czechia: results and analysis of prognostic factors]. / Lécba akutní promyelorytárni leukemie v Cesku: výsledky a analýza prognostických faktoru.

We have retrospectively evaluated a cohort of 144 patients (including 17 pediatric ones) with de novo acute promyelocytic leukemia registered in databases of institutions cooperating within the CELL group (The Czech Leukemia Study Group for Life). The patients were diagnosed according to WHO criteria from 1989 until 2006. The aim was to check how well fared the patients, the majority of whom was not included into clinical trials, in real life. Of 140 evaluable patients, 97 (69.3%) attained complete remission (CR). The projected overall survival (OS) 4 years after diagnosis was 58.9%, and 55.3% at 6 years. In 8 patients (6.0%), no antileukemic therapy at all was given (either they died shortly after admission to the ward or therapy was not feasible due to their clinical status). Of 125 patients with documented commencement of some kind of therapy, 96 (76.8%) achieved CR. Of 102 patients with induction treatment with a combination of anthracycline and tretinoin (ATRA), 84 individuals (82.4%) attained CR (typically, this cohort might have been subjected to clinical trials). This result was better than that of patients treated by chemotherapy only (n = 15; CR 46.7%; P = 0.003) or by ATRA monotherapy (n = 13; CR 62.5%; P = 0.17). Another parameter with a significant impact on attaining CR was the leukocyte (WBC) count at diagnosis: its median values in patients achieving and not achieving CR were 2.1 and 24.0 x 10(9)/l, respectively (P < 0.0001). The WBC counts affected OS as well (P = 0.0001). However, when only patients after attaining CR were evaluated, the initial WBC counts no longer affected OS (P = 0.18). Achieving CR was also influenced by the performance status (PS) 0-1 (P = 0.005), which was in turn closely correlated to WBC counts (P = 0.0006). Additional factors (most likely connected with leukocytosis) influenced attaining CR with borderline statistical significance: e.g. FAB M3v morphology, LDH serum level, fibrinogen level, presence of internal tandem duplication (ITD) of the FLT3 gene (which was strongly associated with leukocytosis and also with the short PML/RARalpha transcript resulting from the bcr3 break in the PML gene). It may be speculated that FLT3-ITD is just one of the possible factors that lead to leukocytosis. The platelet counts at diagnosis had no impact on entering CR. Thus, we have not validated the current PETHEMA risk stratification in distinguishing intertermediate and low risk patients. Our study points to a significant difference of the results obtained in real life and of the results that could be achieved in patients who were fit to enter clinical trials. Among the prognostic factors, the most important one was the WBC count, the PS (which is highly affected by the WBC count), and feasibility of administration of the most potent induction therapy with anthracyclines and ATRA.

Combined molecular biological and molecular cytogenetic analysis of genomic changes in 146 patients with B-cell chronic lymphocytic leukemia.

Interphase fluorescence in situ hybridization was used to detect common deletions in B-CLL patients as well as trisomy 12 and aberrations of IgH gene complex at 14q32.33 where we evaluated not only translocation-like signal pattern but also deletions. 120 (82%) patients showed genetic changes - del(13)(q14) 95 (62%), deletion of ATM gene 22 (15%), deletion of p53 gene 25 (17%) and trisomy 12 was proved in 18 (12%) cases. IgH rearrangements were detected in 45 (31%), split of the signals in 11 (8%), deletion of 3' segment flanking IgH gene in 5 (3%) and deletions of variable segment in 29 (20%) patients. Although deletions of 3' segment flanking IgH gene complex are supposed to have an adverse prognostic impact and the genetic background of variable segment deletions is believed to be most probably physiological, we assumed a detailed mapping of the 14q32.33 region will be needed to unravel these mysteries.