Search Results (115)

Background: Acute lung injury (ALI) and its severe form, acute respiratory distress syndrome (ARDS), are critical conditions lacking effective pharmacologic therapies. Lipopolysaccharide (LPS), a bacterial endotoxin, is a well-established trigger of ALI. Emerging evidence suggests that heparin derivatives may attenuate lung injury, but their mechanisms remain unclear. Methods: This study evaluated the protective effects of 2-O, 3-O desulfated heparin (ODSH) in a murine model of LPS-induced ALI. Mice received LPS intratracheally with or without ODSH pre-treatment. Lung injury was assessed by bronchoalveolar lavage fluid (BALF) analysis, Evans blue dye albumin EBDA) extravasation, and histopathology. Results: ODSH treatment significantly reduced BALF protein concentration, inflammatory cell infiltration, and EBDA leakage. ODSH preserved endothelial barrier function in vitro, as evidenced by transendothelial electrical resistance (TER) measurements in human lung microvascular endothelial cell (HLMVEC) monolayers. Histological assessment (H&E staining) and myeloperoxidase (MPO) staining demonstrated reduced lung injury and neutrophil infiltration in the ODSH group. ODSH also downregulated pro-inflammatory mediators (NF-κB, IL-6, p38 MAPK) and upregulated the anti-inflammatory cytokine IL-10. Conclusions: ODSH mitigates LPS-induced ALI by reducing vascular permeability, neutrophilic inflammation, and pro-inflammatory signaling while enhancing IL-10 expression. These findings suggest ODSH may offer a novel therapeutic approach for treating ALI. Full article

Primary ciliary dyskinesia (PCD) is a genetically heterogeneous disorder characterized by impaired mucociliary clearance due to defects in motile cilia. This study investigates the impact of loss-of-function mutations in the CFAP300 gene on the ciliary structure and function in three PCD patients. Using a multimodal approach, we integrated molecular genetic testing, transmission electron microscopy, the high-speed video microscopy assay and immunofluorescence staining to analyze ciliary motility and protein expression in both ex vivo and in vitro-obtained ciliary cells. Our results revealed that the pathogenic variant c.198_200delinsCC (p.Phe67ProfsTer10) in CFAP300 led to the absence of the functional CFAP300 protein, the complete loss of outer and inner dynein arms and immotile cilia. Air–liquid interface (ALI)-cultured cells from patients exhibited no ciliary beating, contrasting with healthy controls. Immunostaining confirmed the absence of CFAP300 in patient-derived cilia, underscoring its critical role in dynein arm assembly. These findings highlight the diagnostic utility of ALI cultures combined with functional and protein analyses for PCD, offering a clinically actionable framework that can be readily incorporated into standard diagnostic workflows. Full article

Introduction: Subcutaneous panniculitis-like T-cell lymphoma (SPTCL) is a very rare subtype of cutaneous T-cell lymphoma. It is characterized by the neoplastic infiltration of subcutaneous adipose tissue. Its clinical presentation, including subcutaneous nodules, fever, and systemic symptoms, often mimics inflammatory panniculitis, making diagnosis difficult. Case Presentation: This case report describes a 14-year-old female presenting with fever, limb pain, swelling, and subcutaneous nodules, who was ultimately diagnosed with SPTCL via punch biopsy and BIOMED-2 clonality assays, confirming positive T-cell receptor-γ chain gene rearrangement. Positron emission tomography–computed tomography revealed diffuse subcutaneous involvement across multiple body regions. Methylprednisolone and cyclosporine A treatment rapidly resolved her symptoms, with laboratory parameters, including ferritin and inflammatory markers, showing significant improvement. Next-generation sequencing identified a heterozygous C9 gene mutation (c.346C>T, p.Arg116Ter), adding a novel genetic dimension to the case. Following a tapered discontinuation of immunosuppressive therapy, the patient achieved sustained remission without relapse for over 1 year. Conclusions: We report a case of adolescent SPTCL treated with immunosuppressive therapy and suggest that immunosuppressive therapy should be considered before chemotherapy in pediatric patients with SPTCL but without HLH. Full article

The olive oil-production sector engages with the environment on multiple levels, and the valorization of olive pomace (OP) has emerged as a key strategy to improve the entire system’s sustainability. Numerous studies have investigated the biological effects of OP phenolic fraction for nutraceutical applications, highlighting its antioxidant properties. This study aimed to assess the effect of an OP extract (OPE) and its phenolic content on ferroptosis induced by RAS-selective lethal 3 (RSL3), an inhibitor of glutathione peroxidase 4. After characterization of OPE phenolic composition, its antioxidant properties were confirmed through the Fenton reaction assay. Subsequently, we examined the effect of OPE on ter-butyl hydroperoxide-induced ROS generation and lipid peroxidation in TPH-1 and HIECs cells and found that OPE reduced ROS and lipid peroxidation. RSL3 decreased the number of vital cells, which was associated with an elevation in ROS and lipid peroxidation, and a reduction in GSH. Interestingly, all these detrimental effects were reversed by OPE. Furthermore, OPE was also found to significantly increase GSH and the GSH/GSSG ratio per se. In conclusion, the fact that OPE decreases ROS and lipid peroxidation induced by RSL3 and augments GSH and cell viability suggests that OPE has potential as a ferroptosis inhibitor. Full article

Background/Objectives: Chronic pancreatitis (CP) is a progressive inflammatory condition of the pancreas that leads to irreversible changes in pancreatic structure. The pancreatic α and β cells secrete hormones such as insulin and glucagon into the bloodstream. The pancreatic acinar cells secrete digestive enzymes that break down macromolecules. When these digestive enzymes do not function properly, maldigestion, malabsorption, and malnutrition may result. Presented here is a case study of an individual newly diagnosed with chronic pancreatitis, along with a genetic analysis of his son and an in-silico analysis of two of the variant proteins. Methods: This study was conducted using human subjects, namely, the proband (father) and his son. Medical genetic testing of the proband (father) identified the presence of two variants in the cystic fibrosis transmembrane receptor gene (CFTR): variant rs213950, resulting in a single amino acid change (p. Val470Met), and variant rs74767530, a nonsense variant (Arg1162Ter) with known pathogenicity for cystic fibrosis. Medical testing also revealed an additional missense variant, rs515726209 (Ala73Thr), in the CTRC gene. Cheek cell DNA was collected from both the proband and his son to determine the inheritance pattern and identify any additional variants. A variant in the human leukocyte antigen (rs7454108), which results in the HLA-DQ8 haplotype, was examined in both the proband and his son due to its known association with autoimmune disease, a condition also linked to chronic pancreatitis. In silico tools were subsequently used to examine the impact of the identified variants on protein function. Results: Heterozygosity for all variants originally identified through medical genetic testing was confirmed in the proband and was absent in the son. Both the proband and his son were found to have the DRB1*0301 (common) haplotype for the HLA locus. However, the proband was also found to carry a linked noncoding variant, rs2647088, which was absent in the son. In silico analysis of variant rs213950 (Val470Met) in CFTR and rs515726209 (Ala73Thr) in CTRC revealed distinct changes in predicted ligand binding for both proteins, which may affect protein function and contribute to the development of CP. Conclusions: This case study of a proband and his son provides additional evidence for a polygenic inheritance pattern in CP. The results also highlight new information on the role of the variants on protein function, suggesting additional testing of ligand binding for these variants should be done to confirm the functional impairments. Full article

High-grade B-cell lymphoma (HGBL), not otherwise specified (NOS), is a rare entity within the spectrum of B-cell lymphomas. HGBL, NOS remains a diagnosis of exclusion with limited data available on the optimal clinical approach. We report a case of a 67-year-old man with HGBL, NOS with a germinal center B-cell (GCB) immunophenotype. The disease was characterized by an aggressive clinical course, refractory to multiple lines of cytotoxic chemotherapy, immunotargeted treatment, therapy with a PD-1 inhibitor, and haploidentical hematopoietic stem cell transplantation (haplo-HSCT). Ultimately, the disease progression led to the patient’s death nine months post-diagnosis. A FISH assay identified a sole genetic rearrangement: BCL2/IGH. Whole-exome sequencing revealed a number of significant somatic mutations, such as TP53 p.C238G, B2M p.L12R, STAT6 p.D419G, STAT3 p.S614R, TREX1 p.T49fs, and CREBBP p.C367Ter, as well as a high focal amplification of the MUC3A gene and the deletion of the short arm of chromosome 17 (del(17p)). An inactivating somatic mutation in the TREX1 gene (p.T49fs) has not been previously described in patients with non-Hodgkin lymphomas. Additionally, our analysis uncovered a key cancer hallmark: tumor genomic instability, manifested as a high tumor mutational burden, which likely contributed to the aggressive disease course. Full article

von Hippel–Lindau syndrome (VHLS) is a hereditary cancer syndrome with CNS hemangioblastomas, clear cell renal carcinoma, pheochromocytoma, retinal angiomas, and a number of other manifestations. VHLS is caused by a mutation in the VHL gene and is inherited in an autosomal dominant manner. However, some cases of VHLS develop de novo, and among them, there are rare patients with a mosaic form of the disease. Genetic testing in mosaic patients is prone to false-negative results due to the low copy number of a mutant allele in DNA isolated from the blood. We describe a case of molecular genetic diagnostics of VHLS in a 39-year-old patient using various methods, including mutation analysis in asynchronous primary tumors and repeated DNA analysis from blood using NGS with high coverage for the mutant position. As a result, the patient was diagnosed with a mosaic form of VHLS caused by the variant c.481C>T (p.Arg161Ter), the proportion of which in the blood DNA was 2%. We also summarized the literature data on the mosaic form of VHLS: the severity of clinical manifestations, the features of differential diagnostics of VHLS with a negative result of routine molecular genetic VHL testing, and specific options of active surveillance and treatment for mutation carriers. Full article

Integrin β4 (ITGB4) mediates lung endothelial cell (EC) inflammation attenuated by simvastatin, an HMG CoA-reductase inhibitor. The cytoplasmic domain of ITGB4 is predicted to bind kindlin-2. Kindlin-2 expression is mediated by SMURF1, an E3 ubiquitin ligase that promotes kindlin-2 ubiquitination and degradation. We hypothesized that increased kindlin-2 expression via the inhibition of SMURF1 mediates EC inflammatory responses relevant to acute lung injury (ALI). To investigate this, human lung ECs were treated with simvastatin (5 µM, 16 h) prior to the immunoprecipitation of kindlin-2 and Western blotting for ITGB4. Next, ECs were treated with a SMURF1 inhibitor, A01, and increased kindlin-2 expression was confirmed. In assays of barrier function, kindlin-2 was silenced (siRNA) in ECs prior to thrombin and measurements of transendothelial resistance (TER) and FITC-dextran transwell flux. Repeat assessments of barrier function were performed in A01-treated ECs. Finally, mice were pretreated with A01 prior to LPS; bronchoalveolar lavage (BAL) fluid was collected, and their lungs were used for histology. Simvastatin increased ITGB4:kindlin-2 association, while A01 increased kindlin-2 expression. Thrombin-induced EC barrier disruption was both increased after kindlin-2 silencing and decreased by A01. Finally, murine ALI was significantly attenuated by A01. Our findings suggest that the augmentation of kindlin-2 may serve as a novel ALI therapeutic strategy. Full article

The aim of this study was to investigate the effects of terpinen-4-ol (TER) supplementation on the intestinal barrier function of pigs. Five groups of fifty 28-day-old piglets with comparable body weights were randomly assigned to the following groups: the control group (CON), the lipopolysaccharide group (LPS), the low TER group (PLT), the middle TER group (PMT), and the high TER group (PHT). The basal diet was given to the CON and LPS groups, and 30, 60, or 90 mg/kg TER was added to the basal diet for the TER groups. After the 21-day trial period, piglets in the LPS and TER groups received an intraperitoneal injection of 100 μg/kg body weight of LPS, whereas the piglets in the CON group received an injection of 0.9% normal saline solution. The results showed that LPS stimulation resulted in a decrease (p < 0.05) in the depth of colonic crypts in piglets, which was greater (p < 0.05) in the TER group. Compared with those in the CON group, the number of goblet cells and MUC2 expression were decreased in the colon of piglets in the LPS group, while these parameters were increased in the PMT group (p < 0.05). The malondialdehyde (MDA) content was greater in the colon of the LPS group than in that of the CON group, while the activities of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), and catalase (CAT) were lower in the colon of the LPS group; conversely, the MDA content was lower in the colons of the PLT and PMT groups than in those of the LPS group (p < 0.05). TER also reduced (p < 0.05) LPS-induced upregulation of IL-1β and TNF-α expression, along with the relative gene expression of NLRP3, ASC, and caspase-1 in the colon of piglets (p < 0.05). Compared with those in the CON group, the abundances of Firmicutes and UCG-005 in the LPS group were lower (p < 0.05), and those in the TER group were significantly greater than those in the LPS group. Compared with those in the CON group, the abundance of Proteobacteria in the LPS group increased (p < 0.05), while the abundance of Actinobacteria and Phascolarctobacterium increased (p < 0.05) in the colon of the PHT group compared with that in the LPS group. In conclusion, TER effectively improved the intestinal barrier function of the colon in weaning piglets. Based on the results of this study, the appropriate dose of TER in the diets of weaning piglets was 60 mg/kg. Full article

The use of next-generation sequencing (NGS) has recently enabled the discovery of genetic causes of primary ovarian insufficiency (POI) with high genetic heterogeneity. In contrast, the causes of diminished ovarian reserve (DOR) remain poorly understood. Here, we identified by NGS and whole exome sequencing (WES) the cause of isolated DOR in a 14-year-old patient. Two frameshift mutations in BRCA1 (NM_007294.4) were found: in exon 8 (c.470_471del; p.Ser157Ter) and in exon 11 (c.791_794del, p.Ser264MetfsTer33). Unexpectedly, the patient presented no signs of Fanconi anemia (FA), i.e., no developmental abnormalities or indications of bone marrow failure. However, high chromosomal fragility was found in the patient’s cells, consistent with an FA diagnosis. RT-PCR and Western-blot analysis support the fact that the c. 791_794del BRCA1 allele is transcribed and translated into a shorter protein (del11q), while no expression of the full-length BRCA1 protein was found. DNA damage response (DDR) studies after genotoxic agents demonstrate normal activation of the early stages of the DDR and FANC/BRCA pathway. This is consistent with the maintenance of residual repair activity for the del11q BRCA1 isoform. Our observation is the first implication of bi-allelic BRCA1 mutations in isolated ovarian dysfunction or infertility in humans, without clinical signs of FA, and highlights the importance of BRCA1 in ovarian development and function. Full article

Background/Objectives: The non-canonical GC-5′ splice sites (5′ss) are the most common exception (~1%) to the classical GT/AG splicing rule. They constitute weak 5′ss and can be regulated by splicing factors, so they are especially sensitive to genetic variations inducing the misrecognition of their respective exons. We aimed to investigate the GC-5′ss of the breast/ovarian cancer susceptibility genes, ATM (exon 50), BRIP1 (exon 1), and PALB2 (exon 12), and their dysregulation induced by DNA variants. Methods: Splicing assays of the minigenes, mgATM_49-52, mgBRIP1_1-2, and mgPALB2_5-12, were conducted to study the regulation of the indicated GC-5′ss. Results: A functional map of the splicing regulatory elements (SRE) formed by overlapping exonic microdeletions revealed three essential intervals, ATM c.7335_7344del, PALB2 c.3229_3258del, and c.3293_3322del, which are likely targets for spliceogenic SRE-variants. We then selected 14 ATM and 9 PALB2 variants (Hexplorer score < −40) located at these intervals that were assayed in MCF-7 cells. Nine ATM and three PALB2 variants affected splicing, impairing the recognition of exons 50 and 12, respectively. Therefore, these variants likely disrupt the active SREs involved in the inclusion of both exons in the mature mRNA. DeepCLIP predictions suggested the participation of several splicing factors in exon recognition, including SRSF1, SRSF2, and SRSF7, involved in the recognition of other GC sites. The ATM spliceogenic variants c.7336G>T (p.(Glu2446Ter)) and c.7340T>A (p.(Leu2447Ter)) produced significant amounts of full-length transcripts (55–59%), which include premature termination stop codons, so they would inactivate ATM through both splicing disruption and protein truncation mechanisms. Conclusions: ATM exon 50 and PALB2 exon 12 require specific sequences for efficient recognition by the splicing machinery. The mapping of SRE-rich intervals in minigenes is a valuable approach for the identification of spliceogenic variants that outperforms any prediction software. Indeed, 12 spliceogenic SRE-variants were identified in the critical intervals. Full article

Serine and folate metabolism play critical roles in erythroid development in both embryonic and adult mice; however, the precise roles of these metabolic pathways in erythropoiesis and the pathophysiology of anemia remain inadequately characterized in the literature. To delineate the contributions of serine and folate metabolism to erythroid differentiation, we focused on serine hydroxymethyltransferase 2 (SHMT2), a key regulatory enzyme within these metabolic pathways. Using gene-editing techniques, we created fetal and adult mouse models with targeted deletion of Shmt2 in the hematopoietic system. Our findings demonstrated that the deletion of Shmt2 within the hematopoietic system led to the distinctive anemia phenotype in both fetal and adult mice. Detailed progression analysis of anemia revealed that Shmt2 deletion exerts stage-specific effects on the development and maturation of erythroid cells. Specifically, Shmt2 deficiency promoted erythroid differentiation in the R2 (CD71⁺ Ter119⁻) cell population residing in the bone marrow while concurrently inhibiting the proliferation and erythroid differentiation of the R3 (CD71⁺ Ter119⁺) cell population. This disruption resulted in developmental arrest at the R3 stage, significantly contributing to the anemia phenotype observed in the models. This study elucidates the critical role of Shmt2 in erythroid development within the hematopoietic system, highlighting the underlying mechanisms of erythroid developmental arrest associated with Shmt2 loss. Full article

We developed a technique that can measure corneal transepithelial electrical resistance (TER) in mice, which was used for evaluating corneal toxicity induced by ophthalmic drugs. We used a tissue culture well and its insert to mount the mouse globe and separated the cornea from the rest of the globe to enable corneal TER measurements to be taken. The explanted mouse eyes were divided into groups, and the corneal epithelia were exposed to different concentrations of BAC. Half of these eyes were fixed for transmission electron microscopy (TEM) examination and the other for ZO-1 immunohistochemical (IHC) evaluation. After exposure to control, 0.1%, 0.2%, and 0.5% BAC, the TER was 100 ± 0%, 91 ± 14%, 83 ± 13%, and 34 ± 12% of the pre-exposure TER at 1 min, respectively, with a statistically significant decrease in the 0.5% group. After 3 min, the TER showed a statistically significant decrease in the 0.2% and 0.5% groups. The TEM examinations showed a loss of epithelial tight junctions between superficial cells in the 0.2% and 0.5% groups. The IHC examination showed decreased ZO-1 staining of the corneal epithelium of the same groups as compared to the control. To the best of our knowledge, we succeeded in developing an innovative technique for corneal TER measurement in mice. Full article

Biallelic variants in USH2A are associated with retinitis pigmentosa (RP) and Type 2 Usher Syndrome (USH2), leading to impaired vision and, additionally, hearing loss in the latter. Although the introduction of next-generation sequencing into clinical diagnostics has led to a significant uplift in molecular diagnostic rates, many patients remain molecularly unsolved. It is thought that non-coding variants or variants of uncertain significance contribute significantly to this diagnostic gap. This study aims to demonstrate the clinical utility of the reverse transcription–polymerase chain reaction (RT-PCR)–Oxford Nanopore Technology (ONT) sequencing of USH2A mRNA transcripts from nasal epithelial cells to determine the splice-altering effect of candidate variants. Five affected individuals with USH2 or non-syndromic RP who had undergone whole genome sequencing were recruited for further investigation. All individuals had uncertain genotypes in USH2A, including deep intronic rare variants, c.8682-654C>G, c.9055+389G>A, and c.9959-2971C>T; a synonymous variant of uncertain significance, c.2139C>T; p.(Gly713=); and a predicted loss of function duplication spanning an intron/exon boundary, c.3812-3_3837dup p.(Met1280Ter). In silico assessment using SpliceAI provided splice-altering predictions for all candidate variants which were investigated using ONT sequencing. All predictions were found to be accurate; however, in the case of c.3812-3_3837dup, the outcome was a complex cryptic splicing pattern with predominant in-frame exon 18 skipping and a low level of exon 18 inclusion leading to the predicted stop gain. This study detected and functionally characterised simple and complex mis-splicing patterns in USH2A arising from previously unknown deep intronic variants and previously reported variants of uncertain significance, confirming the pathogenicity of the variants. Full article

Autosomal dominant optic atrophy (ADOA) is a rare progressive disease mainly caused by mutations in OPA1, a nuclear gene encoding for a mitochondrial protein that plays an essential role in mitochondrial dynamics, cell survival, oxidative phosphorylation, and mtDNA maintenance. ADOA is characterized by the degeneration of retinal ganglion cells (RGCs). This causes visual loss, which can lead to legal blindness in many cases. Nowadays, there is no effective treatment for ADOA. In this article, we have established an isogenic human RGC model for ADOA using iPSC technology and the genome editing tool CRISPR/Cas9 from a previously generated iPSC line of an ADOA plus patient harboring the pathogenic variant NM_015560.3: c.1861C>T (p.Gln621Ter) in heterozygosis in OPA1. To this end, a protocol based on supplementing the iPSC culture media with several small molecules and defined factors trying to mimic embryonic development has been employed. Subsequently, the created model was validated, confirming the presence of a defect of intergenomic communication, impaired mitochondrial respiration, and an increase in apoptosis and ROS generation. Finally, we propose the analysis of OPA1 expression by qPCR as an easy read-out method to carry out future drug screening studies using the created RGC model. In summary, this model provides a useful platform for further investigation of the underlying pathophysiological mechanisms of ADOA plus and for testing compounds with potential pharmacological action. Full article