A mucus production programme promotes classical pancreatic ductal adenocarcinoma.

OBJECTIVE: The optimal therapeutic response in cancer patients is highly dependent upon the differentiation state of their tumours. Pancreatic ductal adenocarcinoma (PDA) is a lethal cancer that harbours distinct phenotypic subtypes with preferential sensitivities to standard therapies. This study aimed to investigate intratumour heterogeneity and plasticity of cancer cell states in PDA in order to reveal cell state-specific regulators. DESIGN: We analysed single-cell expression profiling of mouse PDAs, revealing intratumour heterogeneity and cell plasticity and identified pathways activated in the different cell states. We performed comparative analysis of murine and human expression states and confirmed their phenotypic diversity in specimens by immunolabeling. We assessed the function of phenotypic regulators using mouse models of PDA, organoids, cell lines and orthotopically grafted tumour models. RESULTS: Our expression analysis and immunolabeling analysis show that a mucus production programme regulated by the transcription factor SPDEF is highly active in precancerous lesions and the classical subtype of PDA - the most common differentiation state. SPDEF maintains the classical differentiation and supports PDA transformation in vivo. The SPDEF tumour-promoting function is mediated by its target genes AGR2 and ERN2/IRE1ß that regulate mucus production, and inactivation of the SPDEF programme impairs tumour growth and facilitates subtype interconversion from classical towards basal-like differentiation. CONCLUSIONS: Our findings expand our understanding of the transcriptional programmes active in precancerous lesions and PDAs of classical differentiation, determine the regulators of mucus production as specific vulnerabilities in these cell states and reveal phenotype switching as a response mechanism to inactivation of differentiation states determinants.

Mitochondrial DNA sequencing illuminates genetic diversity and origin of Hunagrian Nonius horse breed and his relatives - Danubian horse and Serbian Nonius.

From a historical perspective, horse breeding in Bulgaria has been very well developed since the time of the Thracians (early Bronze Age c. 3000 BCE). Archaeological discoveries from this era present us with an extremely rich type diversity, including wild and local primitive horses, the prototype of heavy draft horses, and fine riding horses.The objective of this study was to investigate the genetic structure of unexamined populations of three closely related horse breeds - the Danubian Nonius Hungarian Nonius and Serbian Nonius horses. A 608 bp long fragment of the mtDNA D-loop region was amplified and sequenced. The obtained results showed completely different genetic profiles between the investigated breeds. We identified nine of the 17 haplogroups described in modern horses. Most of the obtained sequences fell into M, L, G, and O'P lineages, which reflects the genetic profiles of the ancestral mares that were probably used at the initial stages of formation of the breeds. The population of the Danubian horse was characterized by a high prevalence of Central Asian specific haplogroup G (45%), followed by Western Eurasian specific haplogroups L and M (both about 21%). In contrast to the Danubian horse, in the Nonius breed the highest frequency of Western Eurasian haplogroup M (43.5%) was found, followed by Middle Eastern haplogroups O'P (26.1%) Central Asian specific E (13.0%) and G (13.1%). The Serbian Nonius horse showed a completely different genetic profile with a prevalence of the rare for Europe haplogroup D (66.7%), followed by Central Asian specific G (16.7%). The high mitochondrial haplotype diversity (Hd = 0.886) found in the investigated samples is evidence for multiple maternal origins in all populations.In conclusion, the obtained results demonstrated a high percentage of haplogroup sharing especially in the Danubian and Hungarian Nonius horse breeds, which reflects the possible common origins of the two breeds. In contrast to these breeds, the Serbian Nonius, despite the small number of investigated animals, showed a specific genetic profile, which could be explained by different and independent origins.

Genetic Diversity and Structure of the Main Danubian Horse Paternal Genealogical Lineages Based on Microsatellite Genotyping.

The Danubian horse, together with the Pleven and the Eastern Bulgarian horse breeds, is one of the modern breeds in Bulgaria. The objective of this study was to compare the genetic structure and genetic diversity of six paternal genealogical lineages of the Danubian horse breed (Zdravko, NONIUS XVII-30, Torpedo, Lider, Kalifa, and Hrabar). In total, 166 individuals from the six genealogical lines were investigated, based on 15 STR markers (short tandem repeats, also known as microsatellites). In total, 184 alleles were found in the six populations, using 15 microsatellite loci. The mean number of alleles, the effective number of alleles, and the polymorphic information content (PIC) values per locus were 12.28, 9.48, and 0.73, respectively. In a comparison of the allelic diversity among sire lineages, the highest genetic diversity (Na) was observed in Lider and Kalifa (14.60 ± 0.21), while the lowest value of this parameter was observed in the Zdravko lineage 4.20 ± 0.35. The largest genetic diversity was found in loci HMS3 and HMS7, with 13 alleles, and the smallest polymorphism was noted for the locus ASB17, with 10 alleles. The level of observed heterozygosity was in the range of 0.65 ± 0.069 for the Zdravko lineage to 0.93 ± 0.01 for the Torpedo lineage. The expected heterozygosity level range was from 0.57 ± 0.048 to 0.91 ± 0.01 for all horse lineages. Structure analysis revealed three main gene pools in the study population. The first pool included the Zdravko lineage; the second had the NONIUS XVII-30, Torpedo, Lider, and Kalifa lineages; and the third defined the Hrabar lineage, which was significantly differentiated from the other genealogical lineages.

Morphology of blood microbiota in healthy individuals assessed by light and electron microscopy.

Introduction: The blood microbiome is still an enigma. The existence of blood microbiota in clinically healthy individuals was proven during the last 50 years. Indirect evidence from radiometric analysis suggested the existence of living microbial forms in erythrocytes. Recently targeted nucleic acid sequencing demonstrated rich microbial biodiversity in the blood of clinically healthy individuals. The morphology and proliferation cycle of blood microbiota in peripheral blood mononuclear cells (PBMC) isolated from freshly drawn and cultured whole blood are obscure. Methods: To study the life cycle of blood microbiota we focused on light, and electron microscopy analysis. Peripheral blood mononuclear cells isolated from freshly drawn blood and stress-cultured lysed whole blood at 43°C in presence of vitamin K from healthy individuals were studied. Results: Here, we demonstrated that free circulating microbiota in the PMBC fraction possess a well-defined cell wall and proliferate by budding or through a mechanism similar to the extrusion of progeny bodies. By contrast, stress-cultured lysed whole blood microbiota proliferated as cell-wall deficient microbiota by forming electron-dense or electron-transparent bodies. The electron-dense bodies proliferated by fission or produce in chains Gram-negatively stained progeny cells or enlarged and burst to release progeny cells of 180 - 200 nm size. On the other hand, electron-transparent bodies enlarged and emitted progeny cells through the membrane. A novel proliferation mechanism of blood microbiota called by us "a cell within a cell" was observed. It combines proliferation of progeny cells within a progeny cell which is growing within the "mother" cell. Discussion: The rich biodiversity of eukaryotic and prokaryotic microbiota identified in blood by next-generation sequencing technologies and our microscopy results suggest different proliferation mechanisms in whole and cultured blood. Our documented evidence and conclusions provide a more comprehensive view of the existence of normal blood microbiota in healthy individuals.

Sequence analysis of the mitochondrial D-loop region throws a new light on the origin of Hungarian Nonius, Danubian Horse and Serbian Nonius.

The objective of our study was to investigate the genetic structure of yet uninvestigated populations of three closely related horse breeds - the Danubian Horse, the Hungarian Nonius and the Serbian Nonius - in order to clarify their origin and genetic diversity. A 640-bp-long fragment of the mtDNA D-loop region was amplified and sequenced. The results showed that the investigated breeds have different genetic profiles although they share some common characteristics. We identified nine of the 17 haplogroups described in modern horses. Most of the obtained sequences fall into the M, L, G, and O'P lineages, which is indicative of the genetic profile of the ancestral mares that had probably been used at the initial stages of the formation of the breeds. The population of the Danubian Horse is characterised by a high prevalence of the Anatolian specific haplogroup G (45%), followed by the Western Eurasian specific haplogroups L and M (both about 21%). In the Hungarian Nonius breed we found the highest frequency of the Western Eurasian haplogroup M (44%), followed by the Middle Eastern O'P (26%) and the Central Asian specific E (13%) and G (13%). The Serbian Nonius showed a distinct genetic profile, characterised by a high prevalence of the rare European haplogroup D (67%), followed by the Central Asian specific haplogroup G (17%). The high percentage of haplogroups shared especially between the Danubian and the Hungarian Nonius indicates the possibility of a common origin of the two breeds. In contrast, the Serbian Nonius showed a specific genetic profile, which can be explained by a different and independent origin.

Mitochondrial Profiles of the East Bulgarian and the Pleven Horse Breeds.

It is well known that horse breeding in Bulgaria is a cultural heritage in Bulgaria, dating from prehistoric and historic times. Until now, molecular data on Bulgarian horses from the plain regions of the country were not available. Therefore, for the first time, we have collected genetic information about some modern horse breeds from the plain regions in Bulgaria. A total of 50 horses originating from different families from two different breeds were investigated: the first one was the Pleven horse (n = 11, breeding in the Danubian Plain), and the second one was the East Bulgarian horse breed (n = 39, Shumen district). These breeds were genotyped according to the mitochondrial D-loop region. The results showed that the Pleven horse particularly carries the European haplogroup L (45.45%), followed by the Middle East haplogroup C (27.27%). In contrast to the Pleven horse, the East Bulgarian horse breed revealed almost equal frequencies of the European specific haplogroup L (33.33%) and the Central Asiatic haplogroup Q (35.90%). Analyses of these two horse breeds revealed a specific genetic profile, but it is obvious that the East Bulgarian horse showed an unusual, mixed profile-a massive admixture with the Asiatic-type haplogroup Q and a high haplogroup diversity. In conclusion, the differences in genetic structure of the two plain horse breeds may be explained with the various horse breeds involved in their formation.

Oncogenic KRAS Induces NIX-Mediated Mitophagy to Promote Pancreatic Cancer.

Activating KRAS mutations are found in nearly all cases of pancreatic ductal adenocarcinoma (PDAC), yet effective clinical targeting of oncogenic KRAS remains elusive. Understanding of KRAS-dependent PDAC-promoting pathways could lead to the identification of vulnerabilities and the development of new treatments. We show that oncogenic KRAS induces BNIP3L/NIX expression and a selective mitophagy program that restricts glucose flux to the mitochondria and enhances redox capacity. Loss of Nix restores functional mitochondria to cells, increasing demands for NADPH reducing power and decreasing proliferation in glucose-limited conditions. Nix deletion markedly delays progression of pancreatic cancer and improves survival in a murine (KPC) model of PDAC. Although conditional Nix ablation in vivo initially results in the accumulation of mitochondria, mitochondrial content eventually normalizes via increased mitochondrial clearance programs, and pancreatic intraepithelial neoplasia (PanIN) lesions progress to PDAC. We identify the KRAS-NIX mitophagy program as a novel driver of glycolysis, redox robustness, and disease progression in PDAC. SIGNIFICANCE: NIX-mediated mitophagy is a new oncogenic KRAS effector pathway that suppresses functional mitochondrial content to stimulate cell proliferation and augment redox homeostasis. This pathway promotes the progression of PanIN to PDAC and represents a new dependency in pancreatic cancer.This article is highlighted in the In This Issue feature, p. 1143.

Mitochondrial diversity in mountain horse population from the South-Eastern Europe.

It is believed that population structure of mountain horse breeds is preserved from any genetic introgression, because of their geographical isolation and specific semi-wild life style of husbandry. Till date there are no molecular data for the Balkan horses. In this study we try to give information about some autochthonous mountain horse breeds from Bulgaria. A total of 121 horses from three different mountain massifs are presented: Stara Planina (the Balkan mountain), the Rhodopes and Rila-Pirin massif were genotyped according to mitochondrial D-loop region. The results showed huge diversity of all known haplogroups with exception of C, F and R. West Eurasian haplogroups B, D, M and L were with the highest frequencies. Haplogroups A, J, I, O'P and Q were also observed with the highest frequencies, but not equally distributed among the three populations. Analyses of the horse breeds reveal preserved genetic profile of the Balkan and the Rhodopes mountains populations. In contrast, a Rila-Pirin breed unexpectedly showed mixed profile - a massive genetic introgression with an Asiatic-type haplogroups. A similar mixed Euro-Asiatic haplotype profile possessed the Carpathian mountain pony, although both populations are separated geographically and historically. The genetic pool of three Bulgarian mountain horse populations is highly heterogenic and because of that these breeds should be preserved.

Epirubicin loading in poly(butyl cyanoacrylate) nanoparticles manifests via altered intracellular localization and cellular response in cervical carcinoma (HeLa) cells.

OBJECTIVE: Drug loading into nanocarriers is used to facilitate drug delivery to target cells and organs. We have previously reported a change in cellular localization of epirubicin after loading to poly(butyl cyanoacrylate) (PBCA) nanoparticles. We aimed to further investigate the altered cellular localization and cellular responses to the described drug formulation. MATERIALS AND METHODS: HeLa cells were treated with epirubicin-loaded PBCA nanoparticles prepared by the pre-polymerization method. A systematic study was performed to evaluate the formulation cytotoxicity. Cellular localization and uptake of the formulation as well as cellular response to the treatment were evaluated. RESULTS: Our studies revealed decreased cytotoxicity of the nanoparticle-formulated epirubicin compared to the free drug as well as a noticeable change in the drug's intracellular localization. Epirubicin-loaded nanoparticles were internalized via endocytosis, accumulated inside endosomal vesicles and induced a two-fold stronger pro-apoptotic signal when compared to the free drug. The level of the tumor suppressor protein p53 in HeLa cells increased significantly upon treatment with free epirubicin, but remained relatively unchanged when cells were treated with equivalent dose of nanoparticle-loaded drug, suggesting a possible shift from p53-dependent DNA/RNA intercalation-based induction of cytotoxicity by free epirubicin to a caspase 3-induced cell death by the epirubicin-loaded PBCA formulation.

Hb G-Waimanalo [A1] or α64(E13)Asp→Asn (α1) (HBA1: c.193G>A) Observed in a Bulgarian Family.

The abnormal hemoglobin (Hb) with an aspartic acid to asparagine substitution at α64 has been found on both the α2- and α1-globin genes. It has been described in many different populations under different names, but never in Bulgaria. Using the recently proposed nomenclature, Hb G-Waimanalo [A1] refers to the HBA1: c.193G > A, while Hb G-Waimanalo [A2] refers to the HBA2: c.193G > A mutation. Here, we present the first family from Bulgaria with Hb G-Waimanalo [A1].

Organoid models of human and mouse ductal pancreatic cancer.

Pancreatic cancer is one of the most lethal malignancies due to its late diagnosis and limited response to treatment. Tractable methods to identify and interrogate pathways involved in pancreatic tumorigenesis are urgently needed. We established organoid models from normal and neoplastic murine and human pancreas tissues. Pancreatic organoids can be rapidly generated from resected tumors and biopsies, survive cryopreservation, and exhibit ductal- and disease-stage-specific characteristics. Orthotopically transplanted neoplastic organoids recapitulate the full spectrum of tumor development by forming early-grade neoplasms that progress to locally invasive and metastatic carcinomas. Due to their ability to be genetically manipulated, organoids are a platform to probe genetic cooperation. Comprehensive transcriptional and proteomic analyses of murine pancreatic organoids revealed genes and pathways altered during disease progression. The confirmation of many of these protein changes in human tissues demonstrates that organoids are a facile model system to discover characteristics of this deadly malignancy.

Dysfunctional Effects of a Conflict in a Healthcare Organization.

UNLABELLED: Conflicts in healthcare settings are quite common events because of the continuous changes and transformations today's healthcare organizations are undergoing and the vigorous interaction between the medical professionals working in them. AIM: To survey the opinions of medical professionals about the possible destructive effects of conflicts on them in the workplace. MATERIALS AND METHODS: We conducted a direct individual survey of 279 medical employees at four general hospitals. We used a set of questions that reflect the negative effects and consequences of conflict on healthcare professionals as direct or indirect participants. All data were analysed using the descriptive statistics and non-parametric analysis at a significance level for the null hypothesis of p < 0.05. RESULTS: Workplace conflicts contribute a lot to the stress, psychological tension and emotional exhaustion medical professionals are exposed to. The confrontation the conflict brings the participants into acts as a catalyst of the conflict and enhances the manifestation of hostile actions. A conflict generates a situation which has an impact on the behaviour of all participants involved in it giving rise to emotional states such as anger, aggression and reproaches. The destructive consequences resulting from a conflict are seen in the reduced work satisfaction and demotivation to perform the work activity. The contradictions that arise as a result affect negatively the team cooperation and obstruct the collaborative efforts in solving the problems in the healthcare setting. CONCLUSION: A conflict in a healthcare setting exerts a considerable destructive effect on an employee, therefore it requires prompt identification and effective intervention to minimise its unfavourable outcomes.

Epirubicin loaded to pre-polymerized poly(butyl cyanoacrylate) nanoparticles: preparation and in vitro evaluation in human lung adenocarcinoma cells.

This article describes the preparation of epirubicin-loaded nanoparticles, prepared by loading of the drug in pre-polymerized poly(butyl cyanoacrylate) nanoparticles, their physicochemical characterization and in vitro evaluation on human lung adenocarcinoma (A549) cells. Nanoparticles were also coated in aqueous dispersions with two different non-ionic surfactants (Pluronic F68 and Polysorbate 80). All particles were spherical in shape, with monomodal size distributions. The zeta-potentials at pH 7.4 increased with augmentation of the particle drug content. The increased drug content was found to correlate with the initial concentration of the drug, used for the particle preparation. In vitro studies on A549 cells showed that the drug-loaded nanoparticles, as well as the combinations of free drug and empty nanoparticles, exhibited higher cytotoxicity than the free drug alone. The presence of surfactants also resulted in increased cytotoxicity. Fluorescent imaging of epirubicin internalization by the adenocarcinoma cells revealed that the free drug was predominantly localized in the cell nucleus, while a cytoplasmic localization was observed for the nanoparticle-bound drug formulations, suggesting the probability of nanoparticle endocytosis. Thus the hereby presented results could be useful for development of nanoparticle-based anthracycline formulations for treatment of lung adenocarcinoma.

Colloidal formulations of etoposide based on poly(butyl cyanoacrylate) nanoparticles: preparation, physicochemical properties and cytotoxicity.

This article describes the preparation, physicochemical characterization and cytotoxicity assessment of novel colloidal formulations of etoposide based on poly(butyl cyanoacrylate) nanoparticles. Nanoparticles were prepared by controlled emulsion polymerization of butyl cyanoacrylate in aqueous medium using two different non-ionic colloidal stabilizers (pluronic F68 and polysorbate 80). The nanoparticles were spherical in shape, with average size ranging from 110-150 nm (empty nanoparticles) to 170-260 nm (drug-loaded nanoparticles), monomodal size distributions, and negative zeta-potentials at pH 7.4. Drug loading efficiency was around 63-68%. More than 80% of the drug was released from the formulations within 6-7h of dialysis experiments. Pluronic-coated nanoparticles possessed lower magnitude of the ζ-potentials (around -4 mV) in comparison with the polysorbate-coated ones (around -12 mV). All tested etoposide formulations induced apoptosis in adenocarcinoma human epithelial (A549) cells, as evident from condensation of chromatin and fragmentation of nuclei. It was found that etoposide formulated with poly(butyl cyanoacrylate) nanoparticles and polysorbate 80 exhibited the highest cytotoxicity toward adenocarcinoma cells.

Entrapment of epirubicin in poly(butyl cyanoacrylate) colloidal nanospheres by nanoprecipitation: formulation development and in vitro studies on cancer cell lines.

This report describes the preparation of poly(butyl cyanoacrylate) nanospheres loaded with epirubicin by nanoprecipitation, their characterization and in vitro evaluation of the drug uptake and cytotoxicity on cancer cell lines. The epirubicin-loaded nanospheres were prepared by nanoprecipitation using presynthesized polymer and dextran 40 as a colloidal stabilizer at different pH and initial drug concentrations. The nanospheres were characterized for particle morphology, size distribution, zeta-potential and drug loading. Epirubicin-loaded particles with diameters around 350 nm were obtained. Drug loading depended on the pH and epirubicin concentration. Epirubicin was more cytotoxic when loaded in nanospheres. Drug release was studied by dialysis method. Cytotoxicity and drug uptake experiments were performed on HeLa and A549 cell lines. It was found that addition of polysorbate 80 could increase cytotoxicity. The cytotoxicity was found to correlate with the drug uptake by cells. The findings reported here demonstrate that epirubicin-loaded nanospheres of poly(butyl cyanoacrylate) can be successfully prepared by the nanoprecipitation approach as alternative to the well-known polymerization-based methods. It is found that the epirubicin-loaded nanospheres are more cytotoxic than the free drug to human carcinoma cell lines in vitro. The higher cytotoxicity of the obtained epirubicin formulations, compared with the free drug, is due to enhanced cellular internalization of epirubicin.