Two new Paramecium strains were isolated from an artificial pond on a university campus located in Tokyo, Japan. Morphological observations and molecular phylogenetic analyses based on partial sequences of heat shock protein 70 and cytochrome b gene sequences identified both strains as Paramecium pentaurelia , one of the sibling species within the Paramecium aurelia species complex. This represents the first confirmed record of P . pentaurelia in Japan. Although this species has rarely been reported on a worldwide basis, the phylogenetic analyses showed that the Japanese strains shared identical or nearly identical sequences with multiple P . pentaurelia strains from geographically distant regions, suggesting that this species may have a wider, possibly cosmopolitan, distribution than has currently been recognised. The limited number of records may reflect the infrequent occurrence of this species in environmental samples, which may hinder its detection.
Exogenous bacterial species, which mainly populate the intestines of Helix pomatia snails, also affect various biological processes that are crucial to the survival of the host. However, the microbiota undergoes changes during overwintering, as feeding is suspended, leading to serious health consequences for the host. This study aims to investigate whether the food supplementation of snails with a microbiological inoculation containing bacterial strains isolated from the intestines of overwintering snails could improve their cold tolerance in summer. The experiment was conducted on two groups: a control group without supplementation; and an experimental group given the microbiological inoculation. The supercooling point (SCP) of their body fluids was measured to assess the effects of supplementation on the cold tolerance of wild snails in the summer. The results showed that the SCP for the snails in the experimental group was statistically significantly lower than for the snails in the control group, with a mean SCP value of −1.73°C compared to −0.97°C (p < 0.05). These findings confirm that the microbiota has a significant impact on cold tolerance in snails, and the microbiological inoculation had a positive impact on lowering the supercooling temperature of H . pomatia in the summer. This sheds new light on the mechanism of this species’ adaptation to cold, which still remains a mystery.
Delayed fracture healing (DFH) poses a considerable challenge in the clinical treatment of fractures, imposing substantial negative impacts on patients. This study intends to determine the diagnostic significance of miR-339-3p in DFH and further analyses its molecular mechanism in osteoblast differentiation. A total of 98 patients with DFH and 106 patients with normal fracture healing provided serum samples and baseline data for this study. The impact and diagnostic value of miR-339-3p in DFH were respectively assessed via logistic regression analysis and ROC curve. Employing MC3T3-E1 as the model cells for osteogenic differentiation, cell experiments were carried out. The expression levels of insulin-like growth factor 2, miR-339-3p and osteogenic differentiation markers were detected using the RT-qPCR method and Western blot assays. Cell viability and apoptosis were examined using the CCK8 assay and flow cytometry. The target genes of miR-339-3p were verified through dual-luciferase reporter assays. In patients with DFH, the expression of miR-339-3p was up-regulated, whereas the expression level of IGF2 was down-regulated. miR-339-3p served as a potential biomarker for DFH and exhibited a negative correlation with IGF2 . IGF2 was identified as the target of miR-339-3p. Inhibition of miR-339-3p could up-regulate IGF2 expression, enhance the activity and differentiation of MC3T3-E1 cells, and suppress their apoptosis. Promoting miR-339-3p exerted the opposite effect. On this foundation, an increase in IGF2 could reverse this tendency. miR-339-3p demonstrates diagnostic potential for DFH. Inhibiting the expression of miR-339-3p can enhance cell activity and osteogenic differentiation through negative regulation of IGF2 , thus suppressing the onset of DFH.
Land-use changes are a major driver of biodiversity shifts in Southeast Asia, particularly within human modified landscapes. Understanding how migratory species use habitats during the non-breeding season is important for assessing their tolerance to environmental changes. We examined the non-breeding habitat associations of the Brown Shrike ( Lanius cristatus Linnaeus, 1758) in Laos by analysing 139 field-detected occurrence sites and comparing them with an equal number of randomly selected background points. Three landscape variables were most strongly associated with the detection of shrikes: road area (positive relationship), and the extent of built-up areas and forest cover (both negative relationships). Brown Shrikes were most frequently recorded in semi-open environments within agricultural mosaics, where structural elements such as perches and open foraging spaces were maintained. Our findings suggest that modified landscapes can provide suitable conditions for the species when structural habitat elements are retained. Our study provides the first fine-scale assessment of the non-breeding habitat associations of Brown Shrikes in mainland Southeast Asia and highlights the importance of maintaining mosaic agricultural landscapes for migratory insectivorous birds.
The Alps are recognised as a hotspot of biodiversity and have historically been extensively studied. However, despite the number of studies devoted to the biodiversity of the Alps, large knowledge gaps are still present. The Southwestern Alps are a biodiverse region known for hosting many endemic animals and plants. To increase the faunistic knowledge of the Southwestern Alps, we have produced the first survey of the tardigrades from Monviso. Notably, we sequenced the mitogenome of the rarely found Arctic-alpine Acanthechiniscus victor (Ehrenberg, 1853), which is an important addition to the existing knowledge as it is the second mitogenome to be sequenced for the tardigrade superfamily Echiniscoidea. The findings of this study highlight how continued explorations of the European mountain ecosystem are still needed to fill in the knowledge gaps regarding its biodiversity.
Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune condition that causes patients pain and seriously affects their quality of life. Therefore, finding a diagnostic marker is of great significance for the prediction and early diagnosis of RA. This study included 105 patients with RA and 90 healthy volunteers. miR-383-5p was quantified by RT-qPCR. The diagnostic potential of miR-383-5p for RA was evaluated by the ROC curve. Chi-square analysis was performed to evaluate correlations between clinicopathological parameters and the miR-383-5p level in RA. Fibroblast-like synoviocytes (FLS) were isolated from synovial tissue. miR-383-5p was over-expressed or inhibited by transfection in FLS. ELISA was used to detect inflammatory factor expression. Pearson correlation analysis was utilized to detect the correlations. miR-383-5p was decreased in RA and may have a diagnostic value in RA. In patients with RA, miR-383-5p was closely related to erythrocyte sedimentation rate, C-reactive protein, anti-cyclic citrullinated peptide antibody positivity and rheumatoid factor. In addition, miR-383-5p was negatively correlated with inflammatory factors in RA. In FLS of RA, the over-expression of miR-383-5p decreased the inflammatory factors, while the inhibition of miR-383-5p increased the inflammatory factors. miR-383-5p may be associated with RA by involvement in the inflammatory response in FLS. Thus, miR-383-5p may show diagnostic potential in RA.
Hepatitis B virus-infected pregnant women often develop liver dysfunction. Although the NLRP3 inflammasome drives HBV-related liver injury, its predictive role in this group remains unclear. Participants were categorized into normal liver function (NLF, N = 73), abnormal liver function (ALF, N = 66) and control (N = 40) groups. Reverse transcription quantitative polymerase chain reaction was used to measure NLRP3 mRNA expression in peripheral blood mononuclear cells, and serum levels of interleukin 1 beta (IL-1β) and interleukin 18 (IL-18) were detected using enzyme-linked immunosorbent assay. Correlations between inflammatory markers and liver function parameters were analysed, and multivariate logistic regression and receiver operating characteristic curve analyses were performed to evaluate predictive performance. The ALF group showed significantly elevated NLRP3 mRNA expression and higher serum levels of IL-1β and IL-18 compared to the NLF and control groups (all P < 0.001). These inflammatory markers were positively correlated with ALT, AST and HBV-DNA levels (all P < 0.001). Multivariate analysis identified NLRP3, IL-1β, IL-18, ALT and HBV-DNA as independent risk factors for liver dysfunction. The combined model of NLRP3, IL-1β, IL-18 and HBV-DNA demonstrated superior predictive performance (AUC = 0.954) compared to any single indicator. This research shows that elevated NLRP3 expression and increased levels of IL-1β and IL-18 in early pregnancy are associated with liver dysfunction in HBV-infected pregnant women. The combination of these inflammatory markers with HBV-DNA provides a highly accurate diagnostic model for early identification of high-risk individuals, offering a potential strategy for early intervention and improved management.
Post-stroke cognitive impairment (PSCI) is a common sequela that occurs after ischaemic stroke (IS). This study aimed to investigate whether miR-409-3p is related to PSCI. Patients with IS were divided into two subgroups: PSCI and post-stroke cognitive normality (PSCN). The plasma level of miR-409-3p was determined by RT-qPCR. The association between PSCI and miR-409-3p was evaluated through binary logistic regression and by analysing the correlation between miR-409-3p and the MoCA score. In mice with middle cerebral artery occlusion (MCAO), the effects of miR-409-3p on cognitive function were explored through mNSS score and Morris water maze. In OGD/R-induced SH-SY5Y cells, the effects of miR-409-3p on cell viability, apoptosis and neuronal inflammation were evaluated using CCK-8, flow cytometry and ELISA. The content of miR-409-3p in patients with IS and those with PSCI was both increased, and its content showed a significant negative correlation with the MoCA score. The binary logistic regression analysis showed that a high risk of PSCI was associated with miR-409-3p. In MCAO mice, inhibition of miR-409-3p can significantly reduce the mNSS score and shorten the escape latency in the Morris water maze. Further-more, in neurons induced by OGD/R, down-regulation of miR-409-3p can exert a significant protective effect on neurons, manifested by enhanced cell viability, reduced apoptosis rate and inhibition of the synthesis of inflammatory factors. We conclude that miR-409-3p is a risk factor associated with PSCI. In MCAO mice and neurons induced by OGD/R, inhibition of miR-409-3p significantly alleviated neurological deficits and suppres-sed neuronal apoptosis and neuronal inflammation.
This study aims to investigate the predictive value of circ_0092576 for coronary heart disease (CHD) and major adverse cardiac events (MACE) risk, and to elucidate its association with the degree of coronary artery stenosis and inflammatory status. A total of 200 CHD patients and 180 non-CHD controls were recruited. Plasma levels of circ_0092576, IL-1β, TNF-α and IL-10 were measured using RT-qPCR. Coronary artery stenosis severity was assessed using the Gensini score system, and MACE occurrence was recorded during regular follow-up. circ_0092576 levels were significantly higher in CHD patients than in the control group, with an area under the ROC curve (AUC) of 0.885. Furthermore, circ_0092576 showed significant positive correlations with Gensini scores and pro-inflammatory factors IL-1β and TNF-α while exhibiting a negative correlation with the anti-inflammatory factor IL-10. Follow-up revealed significantly elevated circ_0092576 levels in patients who developed MACE. Compared to the low circ_0092576 group, the high circ_0092576 group exhibited a significantly higher cumulative incidence of MACE. Elevated circ_0092576 levels correlate with increased risk of CHD and subsequent MACE. In summary, circ_0092576 is a potential biomarker for CHD risk and prognosis. Elevated levels indicate increased risk of CHD, more severe coronary artery stenosis, stronger inflammation, and are associated with an increased risk of adverse cardiac events.
Osteoporotic fractures (OPF) represent a substantial share of all fracture types, seriously affecting the quality of life. Therefore, it is essential to find a biomarker for early diagnosis and prevention of OPF. Detection of mRNA expression was performed using RT-qPCR. The diagnostic value of miR-335-3p in OPF was examined by the ROC curve. Pearson's correlation analysis was employed to explore the correlation. The rat model of OPF was established by bilateral oophorectomy (OVX) and creation of a fracture in the middle of the left femur. Then, bone marrow-derived mesenchymal stem cells (BMSCs) were isolated from the rats. MiR-335-3p was over-expressed or inhibited by transfection in BMSCs. Adipogenic factors, inflammatory factors, osteogenic markers and cell metabolic activity were detected in BMSCs. MiR-335-3p expression was markedly reduced in patients with OPF, and this molecule exhibited favorable diagnostic efficacy for OPF. In OPF, miR-335-3p was positively correlated with bone mineral density. In BMSCOPF, miR-335-3p reduced inflammatory factors and increased cell metabolic activity. In addition, the over-expression of miR-335-3p reduced adipogenic factors and increased osteogenic markers. In conclusion, miR-335-3p was decreased in OPF and may have high diagnostic potential in OPF. In BMSCOPF, miR-335-3p is downregulated in OPF and possesses promising diagnostic value. It can inhibit adipogenic differentiation and inflammatory responses while promoting osteogenic differentiation in BMSCs. These findings suggest that miR-335-3p may participate in the pathogenesis of OPF and be associated with fracture healing.
The study aimed to evaluate the therapeutic effects of mesenchymal stem cells and their by-products, including conditioned medium, extract and exosomes, on intraocular inflammation in experimental autoimmune uveitis. Uveitis was induced in the C57BL/6J mouse strain by administration of interphotoreceptor retinoid-binding protein, complete Freund's adjuvant and pertussis toxin. Mesenchymal stem cell-derived products (conditioned medium, extract and exosomes) were prepared and administered intravitreally at varying time points post-induction, primarily on day 14. Retinal inflammatory changes were monitored using fundus imaging, flow cytometry and RT-PCR analysis to evaluate clinical manifestation of inflammation, immune cell infiltration and gene expression of inflammatory markers. In vivo, administration of mesenchymal stem cell by-products did not ameliorate experimental autoimmune uveitis. On the contrary, treated eyes exhibited exacerbated inflammation, including increased immune cell infiltration and up-regulated pro-inflammatory gene expression (e.g., Gfap, Iba1, Il1b and Il17). In vitro studies suggested a trend towards anti-inflammatory effects of the conditioned medium, but these findings were not replicated in vivo. Control experiments on healthy eyes indicated that intravitreal trauma alone significantly contributed to inflammatory responses, irrespective of the substance injected. Intravitreal application of mesenchymal stem cell by-products did not demonstrate therapeutic benefits in the experimental autoimmune uveitis model and instead promoted inflammation. The results highlight the impact of administration-induced trauma and suggest that alternative delivery methods, such as systemic administration, may be more effective for mesenchymal stem cell-based therapies.
Crohn's disease (CD) is a chronic and recurrent inflammatory bowel disease, often accompanied by symptoms of malnutrition. MicroRNA is stably present in faeces and plays a significant role in CD. This study aimed to explore the expression of miR-26b-5p in the faeces of CD patients and its association with disease severity and nutritional status. The GEO2R online tool was used to analyse the datasets GSE144535 and GSE102134. The subjects' data were collected. The ability of miR-26b-5p to distinguish healthy individuals from CD patients was evaluated using the receiver operating characteristic curve. The subjects were grouped based on the Crohn's disease activity index, simple endoscopic score for Crohn's disease (SES-CD), malnutrition universal screening tool (MUST) and nutrition risk screening 2002 (NRS 2002) scales, and the expression levels of miR-26b-5p in the faeces of CD patients were detected. The correlation between miR-26b-5p and MUST and NRS 2002 scores was analysed by Spearman's test. The target genes were predicted by miRDB and TargetScan databases, and the biological processes and pathways involved in these genes were identified through GO/KEGG analysis. MiR-26b-5p was significantly down-regulated in the CD dataset and patient faeces, negatively correlated with the disease activity and severity of inflammation, and its expression was lower in patients with poorer nutritional status. GO/KEGG analysis revealed that the target genes of miR-26b-5p were involved in inflammation and nutrient metabolism pathways. We conclude that miR-26b-5p is a potential biomarker for CD and is negatively correlated with the severity of the disease and the degree of malnutrition.
Chronic obstructive pulmonary disease (COPD) involves persistent airflow limitation and chronic inflammation. To define the clinical and mechanistic role of the miR-532-5p/CXCL1 axis in COPD, this study used bioinformatic analysis of the GEO dataset GSE70080 to identify miR-532-5p, which was significantly down-regulated in COPD. Multiple databases predicted CXCL1 as its potential target gene. The clinical study included 90 subjects (52 with COPD, 38 controls). We detected serum levels of miR-532-5p and CXCL1 mRNA using RT-qPCR and evaluated their diagnostic value through ROC curves and logistic regression. Inflammatory factors (TNF-α, IL-1β) and oxidative stress indicators (SOD, MDA) were measured by ELISA and colorimetry. A dual-luciferase reporter gene assay verified that miR-532-5p directly targets CXCL1. In BEAS-2B cells treated with cigarette smoke extract (CSE), we conducted functional recovery experiments by transfecting miR-532-5p mimics and CXCL1 over-expression vectors to explore their regulatory role in cell injury. The study found that the expression of miR-532-5p in the serum of COPD patients was down-regulated, and its level was correlated with the decline of lung function and the enhancement of inflammation. It also had a diagnostic value for COPD (AUC = 0.8229) and was an independent protective factor. Mechanistically, CXCL1 was confirmed to be a direct target of miR-532-5p; in the cell smoke injury model; miR-532-5p inhibited CXCL1 to alleviate inflammation and oxidative stress. This study identifies miR-532-5p as a potential protective factor in COPD that acts by targeting CXCL1 to mitigate inflammation and oxidative stress, suggesting its diagnostic and therapeutic relevance.
Cyclin-dependent kinase 4/6 (CDK4/6) inhibitors such as palbociclib have improved the treatment of hormone receptor-positive, luminal A breast cancer; however, therapeutic resistance remains a major challenge. Wild-type p53-induced phosphatase 1 (Wip1) is a negative regulator of the p53 pathway and is often over-expressed in luminal breast cancers. This study aimed to determine whether Wip1 inhibition enhances the anti-proliferative and pro-apoptotic effects of the CDK4/6 inhibitor palbociclib in luminal A breast cancer cells and to elucidate the underlying cell cycle-related mechanisms. MCF-7 cells were treated with palbociclib and Wip1 inhibitor GSK2830371, alone or in combination. Cell viability was assessed using the WST-1 assay, cell cycle distribution was analysed by flow cytometry, and apoptosis was evaluated using Annexin V/7-aminoactinomycin D staining. Expression of cell cycle regulators (CDK2, CDK4, CDK6, cyclin D1/D3, Rb, phospho-Rb) and p53-related proteins (p53, phospho-p53 Ser15, p21, p27) was determined by Western blot analysis. The combined treatment produced a concentration-dependent reduction in cell viability and a marked increase in both early and late apoptotic populations compared with monotherapies. While palbociclib alone induced G1 arrest, co-treatment with GSK2830371 shifted cells toward G2 accumulation. This was accompanied by enhanced phosphorylation of p53, up-regulation of p21 and p27, and dephosphorylation of Rb, indicating dual checkpoint engagement. These findings demonstrate that Wip1 inhibition augments palbociclib-mediated cell cycle arrest and apoptosis through modulation of the p53-Rb axis. The dual blockade of Wip1 and CDK4/6 may represent a promising therapeutic strategy for p53-proficient luminal A breast cancer.
Heat stress disrupts cellular homeostasis in cells by inducing protein misfolding, activating endoplasmic reticulum stress pathways and impairing cell survival mechanisms. The present study aimed to evaluate the cytoprotective effects of tauroursodeoxycholic acid (TUDCA) against heat stress-induced cellular damage in an in vitro spermatogonial GC1 cell model. The IC50 concentration of TUDCA was determined by the MTT assay. GC1 cells were divided into four groups: control (GC1C), TUDCA-treated (GC1TUDCA), heat stress model (HSM; GC1HSM) and TUDCA-treated HSM (GC1HSMTUDCA). Standard culture conditions were used for GC1C. HSM group cells were incubated for 60 minutes at 43 °C. TUDCA groups of cells were treated with 200 µM TUDCA in culture media for 24 hours. Following the experiments, all groups were subjected to immuno-cytochemical staining with phosphorylated PERK (p-PERK) and C/EBP homologous protein (CHOP) antibodies, and immunoreactivity was evaluated using the H-Score system. The MTT assay determined the IC50 value of TUDCA as 558 µM for GC1 cells, with 200 µM selected for subsequent experiments due to minimal cytotoxicity. Heat stress exposure was associated with a reduction in GC1 cell viability and an increase in p-PERK and CHOP expression compared with the GC1C group. In heat-stressed cells, TUDCA treatment was associated with a significant decrease in p-PERK and CHOP expression relative to the GC1HSM group. These results suggest that TUDCA might reduce the cellular reactions related to endoplasmic reticulum stress in heat-stressed GC1 spermatogonial cells under in vitro conditions.
Lung adenocarcinoma is a prevalent cancer worldwide. MicroRNAs (miRNAs) are key regulators in various cancers, with miR-200a-5p emerging as a potential candidate due to its possible role in lung adenocarcinoma. However, its exact mechanisms of action remain unclear. We performed transcriptome sequencing of 32 clinical pairs to identify differentially expressed miRNAs. miR-200a-5p expression was validated in tissues and cell lines (H1975, A549) using bioinformatics and RT-qPCR. Biological effects were assessed via CCK8, EdU, Transwell, scratch assays, flow cytometry, and Western blotting (phosphorylated transducer and activator of transcription 3, fibronectin, matrix metallopeptidase 9, proliferating cell nuclear antigen, and Wnt pathway-related proteins). A protein-protein interaction network predicted downstream targets, focusing on the Wnt pathway. In vivo experiments, immunohistochemistry, and Western blotting evaluated tumour growth and Ki-67/Wnt protein expression. We found that miR-200a-5p was significantly up-regulated in lung adenocarcinoma tissues and cells, correlating with poor prognosis. Up-regulation enhanced proliferation, migration and invasion while inhibiting apoptosis in vitro. Down-regulation produced opposite effects. In vivo, miR-200a-5p promoted tumour growth via Wnt pathway activation. We conclude that miR-200a-5p acts as an oncogenic factor in lung adenocarcinoma by facilitating progression through the Wnt pathway. These findings suggest its potential as a therapeutic target and prognostic biomarker.
Nigella sativa black cumin oil (BCO) exhibits well-documented anti-inflammatory and antioxidant properties; however, the impact of extraction-related compositional variation on its cellular and molecular effects in stem cell systems remains insufficiently characterized. This study investigated the effects of cold-pressed (CP) and supercritical CO2 (ScCO2)-extracted BCO on the viability, inflammatory response and angiogenic potential of adipose-derived mesenchymal stem cells (ASCs). The oil composition and antioxidant activity were assessed using GC/MS and DPPH assays, respectively. ASCs were treated with different volumes of BCO, and cell viability was evaluated at 24, 48 and 72 h using the MTT assay. Anti-inflammatory effects were evaluated by real-time PCR analysis of IL6 and IL10 mRNA expression, while angiogenesis-related molecular responses were evaluated based on VEGF mRNA expression. Both CP and ScCO2-extracted BCO significantly increased ASC viability in a volume-dependent manner, with the highest viability consistently observed at 100 µl (P < 0.05). Treatment with 50 µl and 100 µl of either oil significantly reduced IL6 expression and concomitantly increased IL10 expression at 24 and 48 h (P < 0.05). VEGF expression was also significantly up-regulated at these time points, with ScCO2-extracted BCO inducing a more pronounced and sustained angiogenic response (P < 0.05). These findings indicate that BCO enhances ASC via-bility and molecular responses in a volume-dependent manner, while differences associated with extraction-related compositional variation may modulate inflammation- and angiogenesis-related molecular signalling in ASCs.
FMS-like tyrosine kinase 3 (FLT-3) mutations represent one of the most common genetic anomalies in acute myeloid leukaemia (AML), particularly in adults. The two most common types of mutations, internal tandem duplications (ITD) and tyrosine kinase domain (TKD) point mutations, facilitate uncontrolled cellular proliferation and unfavourable patient outcomes. These mutations are linked with a high relapse rate and shorter overall survival, highlighting the need for targeted therapies to be used. Recent advances in the discovery of new agents enabled incorporation of FLT-3 inhibitors into the frontline treatment regimen. First-generation inhibitors, such as midostaurin, provided the foundation for targeted therapy, while recently developed agents such as gilteritinib and quizartinib have shown more selectivity and demonstrated superior clinical efficiency and improved tolerability. This review discusses the significance of FLT-3 mutations, the evolution of targeted therapies, current treatment guidelines, and ongoing challenges such as resistance and high relapse rates. We also discuss the emerging combinations of therapies and novel agents currently in clinical trials that aim to overcome resistance and improve long-term outcomes for patients with FLT-3-mutated AML.
The present study was undertaken to provide complementary information on the cell cycle of leukaemic lymphoblasts and myeloblasts based on the computer-assisted maximal nuclear diameter measurements. The measurements were carried out in "one-size cell layer" loci of bone marrow smears of patients suffering from B-cell acute lymphoblastic leukaemia (B-ALL), acute myeloblastic leukaemia with minimal differentiation (M0 AML), acute myeloblastic leukaemia without maturation (M1 AML), acute myeloblastic leukaemia with maturation (M2 AML) and chronic myeloid leukaemia (CML). The maximal nuclear diameter was also measured in peripheral blood mature lymphocytes of B-cell chronic lymphocytic leukaemia (CLL), which are known to be in the G0/G1 phase of the cell cycle. In contrast, the maximal nuclear diameter in lymphoblasts of patients with B-ALL was larger and reflected the S and G2 cell cycle phase. The largest incidence of myeloblasts with the smallest maximal nuclear diameter was in M0 AML. The smaller incidence of such cells was also noted in patients with M1 AML. Myeloblasts with larger nuclear bodies were present in M2 AML and CML. Thus, post-mitotic myeloblasts in G0 and G1 phase were characteristic of M0 and to a smaller extent of M1 AML. Myeloblasts with larger maximal nuclear diameter reflecting the S and G2 phase were dominant in M2 AML and CML. In summary, the nuclear size heterogeneity of leukaemic lymphoblasts and myeloblasts in bone marrow smears depends on various phases of the cell cycle classified according to the maximal nuclear diameter.
Although next-generation RNA sequencing (RNA-seq) is increasingly incorporated into germline cancer predisposition testing, its diagnostic utility is often limited by low expression of many clinically relevant genes. To improve RNA yield and transcript representation for targeted RNA-seq, we optimized a simple protocol based on short-term lymphocyte culture prepared directly from whole blood collected in Li-heparin tubes. We systematically evaluated biological reproducibility and pre-analytical sample handling variability and demonstrated that whole blood can be stored at 4 °C for up to 5 days prior to lymphocyte cultivation without compromising RNA quality/gene expression. Gene expression was comparable for RNA isolated from K2EDTA and Tempus tubes, whereas short-term lymphocyte culture resulted in a substantial increase in expression of clinically important genes including BRCA1, BRCA2, RAD51C, RAD51D, PALB2, CHEK2, and multiple Fanconi anaemia genes otherwise low expressed in whole blood. Cultivation for 3-5 days did not significantly affect lymphocyte gene expression, providing flexibility for routine dia-gnostics. The protocol also enables inhibition of nonsense-mediated decay to facilitate analysis of variants causing premature termination. As a proof of principle, we characterized the splicing impact of FANCA c.2602-3C>G variant (located in intron 27) using cultured lymphocytes from its carrier. The variant causes deletion of six nucleotides in the mature transcript (ΔE28p(-6)/r.2602_2607del), resulting in an in-frame deletion (p. Gln869_Phe870del). This spliceogenic effect was reliably detectable only in cultured lymphocytes preferentially expressing the full-length FANCA transcript. Overall, short-term lymphocyte culture re-presents a simple and flexible RNA source that enhances variant interpretation in clinical RNA-seq analyses.
