BACKGROUND: Cell cycle progression and leukemia development are tightly regulated processes in which even a small imbalance in the expression of cell cycle regulatory molecules and microRNAs (miRNAs) can lead to an increased risk of cancer/leukemia development. Here, we focus on the study of a ubiquitous, multifunctional, and oncogenic miRNA-hsa-miR-155-5p (miR-155, MIR155HG), which is overexpressed in malignancies including chronic lymphocytic leukemia (CLL). Nonetheless, the precise mechanism of how miR-155 regulates the cell cycle in leukemic cells remains the subject of extensive research. METHODS: We edited the CLL cell line MEC-1 by CRISPR/Cas9 to introduce a short deletion within the MIR155HG gene. To describe changes at the transcriptome and miRNome level in miR-155-deficient cells, we performed mRNA-seq/miRNA-seq and validated changes by qRT-PCR. Flow cytometry was used to measure cell cycle kinetics. A WST-1 assay, hemocytometer, and Annexin V/PI staining assessed cell viability and proliferation. RESULTS: The limited but phenotypically robust miR-155 modification impaired cell proliferation, cell cycle, and cell ploidy. This was accompanied by overexpression of the negative cell cycle regulator p21/CDKN1A and Cyclin D1 (CCND1). We confirmed the overexpression of canonical miR-155 targets such as PU.1, FOS, SHIP-1, TP53INP1 and revealed new potential targets (FCRL5, ISG15, and MX1). CONCLUSIONS: We demonstrate that miR-155 deficiency impairs cell proliferation, cell cycle, transcriptome, and miRNome via deregulation of the MIR155HG/TP53INP1/CDKN1A/CCND1 axis. Our CLL model is valuable for further studies to manipulate miRNA levels to revert highly aggressive leukemic cells to nearly benign or non-leukemic types.
- MeSH
- Leukemia, Lymphocytic, Chronic, B-Cell * genetics pathology MeSH
- Cyclin D1 genetics metabolism MeSH
- Cyclin-Dependent Kinase Inhibitor p21 * genetics metabolism MeSH
- Cell Cycle Checkpoints * genetics MeSH
- Humans MeSH
- MicroRNAs * genetics metabolism MeSH
- Cell Line, Tumor MeSH
- Cell Proliferation genetics MeSH
- Heat-Shock Proteins MeSH
- Gene Expression Regulation, Leukemic MeSH
- Carrier Proteins genetics metabolism MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
AIMS: CIC-rearranged sarcomas (CRS) are clinically aggressive undifferentiated round cell sarcomas (URCS), commonly driven by CIC::DUX4. Due to the repetitive nature of DUX4 and the variability of the fusion breakpoints, CIC::DUX4 fusion may be missed by molecular testing. Immunohistochemical (IHC) stains have been studied as surrogates for the CIC::DUX4 fusion. We aim to assess the performance of DUX4 IHC in the work-up of CRS and its expression in non-CRS round cell or epithelioid neoplasms. METHODS AND RESULTS: Cases of molecularly confirmed CRS (n = 48) and non-CRS (n = 105) were included. CRS cases consisted of 35 females and 13 males, with ages ranging from less than 1 year to 67 years (median = 41 years). Among the molecularly confirmed non-CRS cases, C-terminal DUX4 expression was investigated in Ewing sarcomas (38 cases), alveolar rhabdomyosarcomas (18 cases), desmoplastic small round cell tumours (12 cases) and synovial sarcomas (n = five), as well as in non-mesenchymal neoplasms such as SMARCA4/SMARCB1-deficient tumours (n = five), carcinomas of unknown primary (n = three) and haematolymphoid neoplasms (four cases). DUX4 IHC was considered positive when strong nuclear expression was detected in more than 50% of neoplastic cells. When used as a surrogate for the diagnosis of CRS, the sensitivity and specificity of DUX4 IHC was 98 and 100%, respectively. Only one CRS case was negative for DUX4 IHC and harboured a CIC::FOXO4 fusion. CONCLUSIONS: DUX4 IHC is a highly sensitive and specific surrogate marker for the presence of CIC::DUX4 fusion, demonstrating its utility in establishing a diagnosis of CRS.
- MeSH
- Child MeSH
- Adult MeSH
- Oncogene Proteins, Fusion * genetics MeSH
- Gene Rearrangement MeSH
- Homeodomain Proteins * metabolism genetics MeSH
- Immunohistochemistry * MeSH
- Infant MeSH
- Middle Aged MeSH
- Humans MeSH
- Adolescent MeSH
- Young Adult MeSH
- Biomarkers, Tumor * metabolism analysis genetics MeSH
- Soft Tissue Neoplasms diagnosis pathology genetics metabolism MeSH
- Child, Preschool MeSH
- Sarcoma * diagnosis pathology genetics metabolism MeSH
- Aged MeSH
- Sensitivity and Specificity MeSH
- Check Tag
- Child MeSH
- Adult MeSH
- Infant MeSH
- Middle Aged MeSH
- Humans MeSH
- Adolescent MeSH
- Young Adult MeSH
- Male MeSH
- Child, Preschool MeSH
- Aged MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
A novel Gram-stain-negative, strictly aerobic, rod-shaped, light-yellow-pigmented, and chemo-organoheterotrophic bacterium, designated DF-77T, was isolated from dense mats of filamentous algae collected in March 2004 at Okinawa in Japan. The microorganism grew at 0-2.0% NaCl concentrations (w/v), pH 6.0-9.0, and 20-30 °C. The 16S rRNA gene sequence-based phylogenetic tree demonstrated that the strain DF-77T is a novel member of the family Flavobacteriaceae and was greatly related to Flagellimonas nanhaiensis SM1704T with sequence similarity of 95.5%. The main fatty acids were iso-C15:1 G, iso-C15:0, and iso-C17:0 3-OH, and the only isoprenoid quinone was menaquinone-6. The dominant polar lipids were phosphatidylethanolamine, two unidentified aminolipids, an unidentified phosphoaminolipid, and four unidentified lipids. The genome size of strain DF-77T was 3.60 Mbp with a DNA G + C content of 47.5%. The average nucleotide identity (ANI) value between the genomes of strain DF-77T and its closely related species was 69.8-70.7%. The digital DNA - DNA hybridization (dDDH) value of strain DF-77T with the strain of F. nanhaiensis SM1704T was 16.8%. The genome of the strain DF-77T revealed that it encoded several genes involved in bio-macromolecule degradation, indicating a high potential for producing industrially useful enzymes. Consequently, the strain is described as a new species in the genus Flagellimonas, for which the name Flagellimonas algarum sp. nov., is proposed with the type strain DF-77T (= KCTC 72791T = NBRC 114251T).
- MeSH
- DNA, Bacterial genetics chemistry MeSH
- Flavobacteriaceae * classification isolation & purification genetics MeSH
- Phospholipids analysis MeSH
- Phylogeny MeSH
- Genome, Bacterial MeSH
- Nucleic Acid Hybridization MeSH
- Fatty Acids analysis MeSH
- RNA, Ribosomal, 16S genetics MeSH
- Sequence Analysis, DNA MeSH
- Bacterial Typing Techniques MeSH
- Vitamin K 2 analysis analogs & derivatives MeSH
- Base Composition MeSH
- Publication type
- Journal Article MeSH
- Geographicals
- Japan MeSH
BACKGROUND: During human foetal development, the liver is the primary site of blood cell production, but this activity declines in the third trimester and postnatally as haematopoiesis shifts to bone marrow. In humans, this postnatal decline is not well characterized due to the scarcity of appropriate samples. OBJECTIVE: To characterize the effect of (i) gestational age at birth and (ii) length of survival after birth on hepatic haematopoiesis across various cell lineages involved. METHODS: Liver autopsy samples from 25 born-alive infants, predominantly extremely preterm newborns who died mainly between 1 day and 3 weeks after birth, were analysed. Haematopoiesis was characterized using immunohistochemical staining of established cell type-specific protein markers. RNA-sequencing data from our previous study using the same samples were also explored. RESULTS: Haematopoiesis negatively correlates with both the duration of prenatal development and the length of postnatal survival. The effect of these two factors varies across different haematopoietic cell lineages. Prenatally and early postnatally, erythropoietic cells dominated hepatic haematopoiesis but were rapidly suppressed within three days after birth. Granulopoietic activity declined more gradually after birth. Analysis of the gene expression data revealed the possible involvement of several transcription factors in lineage-specific regulatory mechanisms. CONCLUSION: This study enhances our understanding of the postnatal decline of hepatic haematopoiesis in human newborns, highlighting the differential regulation of erythropoiesis and granulopoiesis after birth. These factors bring new in-depth knowledge about the biological processes critical for postnatal adaptation of human newborns.
- Publication type
- Journal Article MeSH
Despite the significance of neck muscles in musculoskeletal disorders, their microscopic anatomy remains poorly characterized. This study examined the splenius capitis muscle, focusing on its fiber-type composition, fiber size, and capillary network characteristics. For comparison and validation, the vastus lateralis muscle was also analyzed. Muscle samples from 13 young male subjects (mean age ± SD: 35.7 ± 8.6 years) were collected within 24-h post-mortem during autopsy. Myosin heavy chain (MyHC) isoform expression was characterized immunohistochemically in 10 μm sections, while the capillary network architecture was assessed in 100 μm sections. Immunofluorescence staining, confocal microscopy, and 3D image analysis were employed to quantify capillary tortuosity, anisotropy, branch density (Br dens), and the length of capillaries per muscle volume (LV), per muscle fiber length (LL), per fiber surface area (LS), and per fiber volume (LVf). Compared to the vastus lateralis muscle, the splenius capitis muscle had a higher percentage of type 1 fibers (51.2% vs 39.7%), fewer type 2a fibers (16.2% vs 31.4%), and smaller fiber diameters (35.5-40.9 μm vs 47-56.1 μm). It also displayed lower Br dens (P = 0.0069), higher anisotropy (P = 0.0004), and lower LL (P < 0.0001) but higher LVf (P = 0.0486). In the splenius capitis muscle, body mass index (BMI) negatively correlated with LV (P = 0.0155), LS (P = 0.0091), LVf (P = 0.0137), and anisotropy (P = 0.0425), and positively correlated with tortuosity (P = 0.0473), indicating a reduction in the capillary network. In the vastus lateralis muscle, only LV (P = 0.0161) decreased with high BMI. This study characterized the fiber-type composition, fiber size, and 3D capillary network of the splenius capitis muscle, establishing a baseline for investigations into pathological muscle alterations.
Extracellular vesicles can play an important role in the processes occurring after stem cell transplantation, preventing cell apoptosis, stimulating immunological processes, and promoting the synthesis of extracellular matrix. Human follicular fluid (FF) can be a source of a subpopulation of cells with mesenchymal stem cells (MSCs) properties. Moreover these subpopulations of FF cells can differentiate into osteoblasts. In presented studies flow cytometry of ovarian FF cells confirmed positive expression of MSCs markers such as: CD44, CD90, CD105, CD73 and negative expression of a hematopoietic marker: CD45. The CD90+, CD105+, CD45- cell subpopulation has been obtained during magnetic separation using appropriate antibodies conjugated with microbeads. The extracellular vesicles (EVs) secreted by the cells during osteodifferentiation process differed from those secreted by cells culture in the basal medium. Based on the previous and current electron microscopy research, changes in size, number, and shape would support the notion that released EVs could be crucial to the ovarian FF cell subpopulation differentiation process. Osteogenic differentiation has been confirmed via Alizarin red staining. Therefore, follicular fluid (FF) can be a new source of a cell subpopulation with MSC properties, with the cells capable of differentiating into the osteogenic lineage. EVs could play a key role as mediators in tissue regeneration, especially bone tissue regeneration.
- MeSH
- Cell Differentiation * MeSH
- Extracellular Vesicles * ultrastructure metabolism MeSH
- Follicular Fluid * cytology metabolism MeSH
- Cells, Cultured MeSH
- Humans MeSH
- Mesenchymal Stem Cells * cytology metabolism MeSH
- Osteoblasts cytology metabolism MeSH
- Osteogenesis * MeSH
- Flow Cytometry MeSH
- Check Tag
- Humans MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
Cyanobacterial harmful blooms (CyanoHABs) pose a global ecological problem, and their lipopolysaccharides (LPS) are among the bioactive compounds they release. Previous studies on CyanoHAB-LPS from single cyanobacterial species have shown varying bioactivities in different in vitro cell models. In this study, we isolated LPS from 19 CyanoHAB samples collected at 18 water bodies in the Czech Republic over two consecutive seasons. The proportions of cyanobacteria, Gram-negative bacteria (G-), and other bacteria in the biomass were determined by qPCR, while the cyanobacterial genera were identified using light microscopy. In vitro models of keratinocytes (HaCaT), the intestinal epithelium (co-culture of differentiated Caco-2 cells and peripheral blood mononuclear cells - PBMC), and PBMC alone were treated with isolated LPS at concentrations of 50, 100, and 1 μg/ml, respectively. The endotoxin activities of these concentrations were within the range measured in the aquatic environment. Approximately 85-90% of the samples displayed biological activity. However, the potency of individual LPS effects and response patterns varied across the different in vitro models. Furthermore, the observed activities did not exhibit a clear correlation with the taxonomic composition of the phytoplankton community, the relative share of microbial groups in the biomass, endotoxin activity of the LPS, or LPS migration and staining pattern in SDS-PAGE. These findings suggest that the effects of CyanoHAB-LPS depend on the specific composition and abundance of various LPS structures within the complex environmental sample and their interactions with cellular receptors.
- MeSH
- Biomass MeSH
- Caco-2 Cells MeSH
- Leukocytes, Mononuclear MeSH
- Humans MeSH
- Lipopolysaccharides * toxicity MeSH
- Cyanobacteria * MeSH
- Harmful Algal Bloom MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
Three closely related, aerobic, Gram-stain-negative, motile, rod-shaped bacterial strains (PS-2T, PS-17, and PS-19) were isolated from the skin of freshwater pufferfish (Tetraodon cutcutia). Colonies are pinkish-colored. The optimum growth occurred at 28-30 °C, and the pH was 6.5-7. The major cellular fatty acids were C16:1 ω7c, iso-C15.0, C17:1 ω8c, C18:1 ω7c, and C16:0. The predominant polar lipids were phosphatidylglycerol, phosphatidylethanolamine, and amino lipids. The genome size of strain PS-2T is 4.8 Mbp, and the G + C content was 46.0%. The major fraction of genes were associated with biological processes (45.64%), followed by molecular function (29.86%) and cellular components (24.49%). The unique genes identified in strain PS-2T secreted cyanophycinase, UDP-N-acetylglucosamine 2-epimerase, methyltransferase, kynureninase, ADA regulatory protein, biphenyl degradation, thermostable carboxypeptidase 1, tetrathionate respiration, etc. In addition, alanine and glutamate racemases were present. The 16S rRNA gene sequences shared 98.83-99.24% similarity with the closely related type strains of Shewanella. The ANI and AAI of strain PS-2T with reference type strains of the genus Shewanella were below 95-96%, and the corresponding dDDH values were below 70%. A phylogenetic tree based on 16S rRNA gene sequences and genome-wide core genes revealed that strain PS-2T clustered with Shewanella oneidensis LMG 19005T in both phylogenetic trees. Based on the polyphasic analysis, the new isolates (PS-2T, PS-17, and PS-19) represent a novel species of Shewanella, for which Shewanella cutis sp. nov. is proposed. The type strain is PS-2T (= TBRC 15838T = NBRC 115342T).
- MeSH
- DNA, Bacterial * genetics MeSH
- Phospholipids analysis MeSH
- Phylogeny * MeSH
- Genome, Bacterial * MeSH
- Genomics MeSH
- Skin microbiology MeSH
- Fatty Acids * analysis MeSH
- RNA, Ribosomal, 16S * genetics MeSH
- Sequence Analysis, DNA MeSH
- Shewanella * genetics isolation & purification classification MeSH
- Fresh Water microbiology MeSH
- Bacterial Typing Techniques MeSH
- Tetraodontiformes * microbiology MeSH
- Base Composition * MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
- Publication type
- Journal Article MeSH
Pathogenic mutations in the genes associated with tuberous sclerosis complex (TSC)/mTOR pathway are linked to histologically diverse renal cell neoplasms, including eosinophilic solid and cystic renal cell carcinoma (ESC RCC), low grade oncocytic tumor (LOT), eosinophilic vacuolated tumor (EVT), and xanthomatous giant cell renal cell carcinoma (XGC RCC). Here, we validate a TSC2 immunohistochemistry (IHC) assay by comparison to genomic data in these neoplasms. Automated TSC2 IHC was performed on formalin-fixed paraffin embedded (FFPE) tissues from 38 genetically-confirmed TSC/mTOR-associated renal tumors (6 ESCs, 16 EVTs, 13 LOTs, 2 XGC and 1 clear cell RCC) and visually scored in a semi-dichotomous fashion compared to internal control tissue. The positive predictive value (PPV) of TSC2 protein loss for underlying pathogenic mutation in TSC2 was 92% (11/12), while the negative predictive value (NPV) of intact TSC2 by IHC for lack of underlying pathogenic mutation in TSC2 was 81% (21/26). Intact TSC2 by IHC was 95% (21/22) specific for absence of underlying pathogenic TSC2 mutation. All the cases lacking TSC2 mutation with intact TSC2 protein had an underlying mutation in TSC1, MTOR or PIK3CA. Loss of TSC2 was 77% (10/13) sensitive for underlying TSC2 truncation mutations and 33% (1/3) sensitive for underlying TSC2 missense mutations. Overall, 73% (8/11) tumors with TSC2 IHC loss and underlying pathogenic alterations in TSC2 showed heterogeneous protein loss, with rare interspersed positively staining tumor cells. These data support TSC2 IHC as a potentially useful assay for the diagnostic workup of renal tumors suspected to belong to the TSC/mTOR-associated subgroups.
- MeSH
- Immunohistochemistry * MeSH
- Carcinoma, Renal Cell * genetics pathology MeSH
- Humans MeSH
- Mutation MeSH
- Biomarkers, Tumor * genetics analysis MeSH
- Tumor Suppressor Proteins * analysis genetics MeSH
- Kidney Neoplasms * genetics pathology MeSH
- Predictive Value of Tests MeSH
- Reproducibility of Results MeSH
- Signal Transduction MeSH
- TOR Serine-Threonine Kinases * metabolism MeSH
- Tuberous Sclerosis Complex 2 Protein * genetics MeSH
- Tuberous Sclerosis genetics pathology diagnosis MeSH
- Check Tag
- Humans MeSH
- Male MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
- Validation Study MeSH
We report a very unusual case of melanocytic neoplasm appearing clinically as a 0.5-cm dome-shaped pigmented papule on the chest of a 63-year-old man. Microscopically, it was an asymmetric, entirely dermally based neoplasm characterized by a multinodular, vaguely plexiform architecture composed of moderately pleomorphic spindled melanocytes with ample, dusty pigmented cytoplasm and scattered multinucleated cells. The tumor cells were strongly positive for Melan-A, HMB45, S100, and PRAME, whereas p16 showed diffuse nuclear loss. β-catenin presented a strong and diffuse cytoplasmic staining, while nuclei were negative. Despite an increased cellularity, mitotic count was low (1/mm 2 ). Fluorescence in situ hybridization revealed no copy number alteration in melanoma-related genes ( CDKN2A, MYB, MYC, CCND1 and RREB1 ). DNA and RNA sequencing identified KIT c.2458G>T and APC c.6709C>T mutations. No further genetic alteration was detected including TERT-promoter (TERT-p ) hot-spot mutation. A re-excision was performed. A sentinel lymph node biopsy was negative. Clinical investigations revealed no extracutaneous involvement. The patient is disease-free after a follow-up period of 8 months. Given the peculiar morphologic and molecular findings, we hypothesize the lesion may represent a novel subtype of an intermediate grade melanocytic tumor (melanocytoma).
- MeSH
- Antigens, Neoplasm MeSH
- Sentinel Lymph Node Biopsy MeSH
- In Situ Hybridization, Fluorescence MeSH
- Middle Aged MeSH
- Humans MeSH
- Melanocytes pathology MeSH
- Melanoma * pathology MeSH
- Mutation MeSH
- Skin Neoplasms * pathology MeSH
- Check Tag
- Middle Aged MeSH
- Humans MeSH
- Male MeSH
- Publication type
- Journal Article MeSH
- Case Reports MeSH
