Search Results (14)

The postmortem interval (PMI) is defined as the time interval between physiological death and the examination of the corpse, playing a critical role in forensic investigations. Traditional PMI estimation methods are often influenced by subjective and environmental factors. Circular RNAs (circRNAs), known for their stability, abundance, and conservation in brain tissue, show promise as biomarkers for PMI estimation. However, research on circRNAs in this context remains limited. This study aimed to develop PMI estimation models using circRNAs across multiple temperatures. By employing semi-quantitative reverse transcription-PCR, circTulp4, circSlc8a1, and circStrn3 were identified as reliable biomarkers for mouse brain tissue. Mathematical models were constructed using the reference genes 28S rRNA, mt-co1, and circCDR1as. At 4 °C, most equations had p-values below 0.05, with the equation using circSlc8a1 as a marker exhibiting the highest goodness of fit. Validation results indicated that the equation using circTulp4 as the reference gene had the highest accuracy. When applying the combined aforementioned three circRNAs, the equation using circCDR1as as the reference gene showed better accuracy. At 25 °C, all equations had R² values greater than 0.86, but most cubic equations had p-values above 0.05. Validation results demonstrated that the circTulp4/mt-co1 equation had the highest accuracy. When applying combined circRNAs, the R² values improved, and long-term PMI estimation was more accurate than short-term PMI estimation. At 35 °C, the linear equations had significantly poorer goodness of fit compared to nonlinear equations, and nonlinear equations exhibited better accuracy than linear equations. When applying the combined aforementioned three circRNAs, the accuracy of the three reference genes was similar, and the accuracy of long-term PMI estimation was consistently higher than that of short-term estimation. For the three-dimensional models, all R² values exceeded 0.75 with p-values significantly below 0.0001. Validation results demonstrated higher accuracy at 25 °C and 35 °C, with superior performance for long-term PMI estimation. In summary, this study constructed PMI estimation models under multiple temperature conditions based on highly expressed circRNAs in mouse brain tissue, highlighting circTulp4, circSlc8a1, and circStrn3 as novel biomarkers. These findings offer a complementary tool for PMI estimation, particularly for long-term PMI estimation. Full article

The TULP family was first identified in progressively obese mice, and TULP3, as a member of its family, has been much studied in tumor cells, but studies on its role in adipocytes have not yet been reported. This study found that the expression of TULP3 showed an increasing trend in the differentiation of 3T3-L1 cells, and overexpression of TULP3 enhanced the proliferation and differentiation capacity of the cells, while inhibition caused the opposite result. TULP3 is a negative regulator of the Hedgehog signaling pathway, which can control lipid metabolism in adipose tissues, but whether TULP3 can play a role in adipose tissues through the Hedgehog signaling pathway is not yet known. It was experimentally found that TULP3 could promote adipogenic differentiation of precursor adipocytes by inhibiting the activity of the Hedgehog signaling pathway. Our results elucidate the role of TULP3 in the generation of precursor adipocytes and provide useful information for a deeper understanding of the molecular mechanisms of adipocytogenesis, which will contribute to the improvement of the treatment of adipose tissue dysfunction or uncontrolled adipogenesis-related diseases. Full article

Autoantibodies have been implicated in the pathogenesis of autoimmune diseases, including autoimmune retinopathies. Our study aimed to identify retinal autoantigens recognized by serum autoantibodies (AAbs) in patients with visual disturbance. We evaluated 2453 serum samples for anti-retinal AAbs from patients with or without cancer and complaints of visual loss. Anti-TULP1 AAbs were more prevalent in the subset of women with breast cancer and vision loss. Epitope mapping was determined by ELISA using peptides covering the conservative carboxy terminal of TULP1, revealing major lineal epitopes within the sequences 334–341 and 480–488. We found no significant difference in the main epitope recognition between sera from patients with or without breast cancer. Although we show a correlation of anti-TULP1 AAbs with breast cancer, we found no TULP1 protein expression in breast cells, making this association unclear. In the retina, anti-TULP1 AAbs can disrupt the transport of proteins to outer segments and be involved in the degeneration of photoreceptors in a similar fashion to the degeneration induced by TULP1 gene mutation. Nevertheless, the strong association of detectable anti-TULP1 AAbs in breast cancer patients with vision problems indicates its potential as a biomarker for cancer-associated autoimmune retinopathy. Full article

Background: Leber congenital amaurosis (LCA) is a congenital onset severe form of inherited retinal dystrophy (IRD) and a common cause of pediatric blindness. Disease-causing variants in at least 14 genes are reported to predispose LCA phenotype. LCA is inherited as an autosomal recessive disease. It can be an isolated eye disorder or as part of a syndrome, such as Senior Loken or Joubert syndrome. Sequencing studies from consanguineous populations have proven useful for novel variants identification; thus, the present study aimed to explore the genetic heterogeneity of 15 consanguineous Pakistani families, each segregating a severe IRD phenotype using targeted next generation sequencing. Methods: This study enrolled 15 consanguineous families, each with multiple affected cases of retinal dystrophy phenotype. DNA was extracted from blood samples. Targeted panel sequencing of 344 known genes for IRDs was performed, followed by Sanger sequencing for segregation analysis. Results: Data analysis revealed a total of eight reported (c.316C>T and c.506G>A in RDH12; c.864dup and c.1012C>T in SPATA7, as well as c.1459T>C, c.1062_1068del, c.1495+1G>A, c.998G>A in the CRB1, LCA5, TULP1, and IFT140 genes, respectively) and four novel homozygous (c.720+1G>T in LCA5, c.196G>C in LRAT, c.620_625del in PRPH2, and c.3411_3414del in CRB1) variants segregating with disease phenotype in each respective family. Furthermore, a novel heterozygous variant of CRB1 gene, i.e., c.1935delC in compound heterozygous condition was found segregating with disease phenotype in one large family with multiple consanguinity loops. Conclusion: Comprehensive molecular diagnosis of 15 consanguineous Pakistani families led to the identification of a total of 5 novel variants contributing to genetic heterogeneity of LCA-associated genes and helped to provide genetic counseling to the affected families. Full article

To report the spectrum of Leber congenital amaurosis (LCA) associated genes in a large German cohort and to delineate their associated phenotype. Local databases were screened for patients with a clinical diagnosis of LCA and for patients with disease-causing variants in known LCA-associated genes independent of their clinical diagnosis. Patients with a mere clinical diagnosis were invited for genetic testing. Genomic DNA was either analyzed in a diagnostic-genetic or research setup using various capture panels for syndromic and non-syndromic IRD (inherited retinal dystrophy) genes. Clinical data was obtained mainly retrospectively. Patients with genetic and phenotypic information were eventually included. Descriptive statistical data analysis was performed. A total of 105 patients (53 female, 52 male, age 3–76 years at the time of data collection) with disease-causing variants in 16 LCA-associated genes were included. The genetic spectrum displayed variants in the following genes: CEP290 (21%), CRB1 (21%), RPE65 (14%), RDH12 (13%), AIPL1 (6%), TULP1 (6%), and IQCB1 (5%), and few cases harbored pathogenic variants in LRAT, CABP4, NMNAT1, RPGRIP1, SPATA7, CRX, IFT140, LCA5, and RD3 (altogether accounting for 14%). The most common clinical diagnosis was LCA (53%, 56/105) followed by retinitis pigmentosa (RP, 40%, 42/105), but also other IRDs were seen (cone-rod dystrophy, 5%; congenital stationary night blindness, 2%). Among LCA patients, 50% were caused by variants in CEP290 (29%) and RPE65 (21%), whereas variants in other genes were much less frequent (CRB1 11%, AIPL1 11%, IQCB1 9%, and RDH12 7%, and sporadically LRAT, NMNAT1, CRX, RD3, and RPGRIP1). In general, the patients showed a severe phenotype hallmarked by severely reduced visual acuity, concentric narrowing of the visual field, and extinguished electroretinograms. However, there were also exceptional cases with best corrected visual acuity as high as 0.8 (Snellen), well-preserved visual fields, and preserved photoreceptors in spectral domain optical coherence tomography. Phenotypic variability was seen between and within genetic subgroups. The study we are presenting pertains to a considerable LCA group, furnishing valuable comprehension of the genetic and phenotypic spectrum. This knowledge holds significance for impending gene therapeutic trials. In this German cohort, CEP290 and CRB1 are the most frequently mutated genes. However, LCA is genetically highly heterogeneous and exhibits clinical variability, showing overlap with other IRDs. For any therapeutic gene intervention, the disease-causing genotype is the primary criterion for treatment access, but the clinical diagnosis, state of the retina, number of to be treated target cells, and the time point of treatment will be crucial. Full article

Biallelic pathogenic variants in TULP1 are mostly associated with severe rod-driven inherited retinal degeneration. In this study, we analyzed clinical heterogeneity in 17 patients and characterized the underlying biallelic variants in TULP1. All patients underwent thorough ophthalmological examinations. Minigene assays and structural analyses were performed to assess the consequences of splice variants and missense variants. Three patients were diagnosed with Leber congenital amaurosis, nine with early onset retinitis pigmentosa, two with retinitis pigmentosa with an onset in adulthood, one with cone dystrophy, and two with cone-rod dystrophy. Seventeen different alleles were identified, namely eight missense variants, six nonsense variants, one in-frame deletion variant, and two splice site variants. For the latter two, minigene assays revealed aberrant transcripts containing frameshifts and premature termination codons. Structural analysis and molecular modeling suggested different degrees of structural destabilization for the missense variants. In conclusion, we report the largest cohort of patients with TULP1-associated IRD published to date. Most of the patients exhibited rod-driven disease, yet a fraction of the patients exhibited cone-driven disease. Our data support the hypothesis that TULP1 variants do not fold properly and thus trigger unfolded protein response, resulting in photoreceptor death. Full article

N⁶-methyladenosine (m⁶A) is the most abundant modification in linear RNA molecules. Over the last few years, interestingly, many circRNA molecules are also found to have extensive m⁶A modification sites with temporal and spatial specific expression patterns. To date, however, little information is available concerning the expression profiling and functional regulatory characteristics of m⁶A modified circRNAs (m⁶A-circRNAs) in secondary hair follicles (SHFs) of cashmere goats. In this study, a total of fifteen m⁶A-circRNAs were identified and characterized in the skin tissue of cashmere goats. Of these, six m⁶A-circRNAs were revealed to have significantly higher expression in skin at anagen compared with those at telogen. The constructed ceRNA network indicated a complicated regulatory relationship of the six anagen up-regulated m⁶A-circRNAs through miRNA mediated pathways. Several signaling pathways implicated in the physiological processes of hair follicles were enriched based on the potential regulatory genes of the six anagen up-regulated m⁶A-circRNAs, such as TGF-beta, axon guidance, ribosome, and stem cell pluripotency regulatory pathways, suggesting the analyzed m⁶A-circRNAs might be essentially involved in SHF development and cashmere growth in cashmere goats. Further, we showed that four m⁶A-circRNAs had highly similar expression trends to their host genes in SHFs of cashmere goats including m⁶A-circRNA-ZNF638, -TULP4, -DNAJB6, and -CAT. However, the expression patterns of two m⁶A-circRNAs (m⁶A-circRNA-STAM2 and -CAAP1) were inconsistent with the linear RNAs from their host genes in the SHFs of cashmere goats. These results provide novel information for eluci-dating the biological function and regulatory characteristics of the m⁶A-circRNAs in SHF development and cashmere growth in goats. Full article

Glioblastoma is an aggressive cancer of the nervous system that accounts for the majority of brain cancer-related deaths. Through cross-species transcriptome studies, we found that Engrailed 1 (EN1) is highly expressed in serum-free cultured glioma cells as well as glioma tissues, and increased expression level predicts a worse prognosis. EN1 controls glioma cell proliferation, colony formation, migration, and tumorigenic capacity in vivo. It also influences sensitivity of glioma cells to γ-ray irradiation by regulating intracellular ROS levels. Mechanistically, EN1 influences Hedgehog signaling by regulating the level of Gli1 as well as primary cilia length and the primary cilia transport-related protein TULP3. In conclusion, we demonstrate that EN1 acts as an oncogenic regulator that contributes to glioblastoma pathogenesis and could serve as a diagnostic/prognostic marker and therapeutic target for glioblastoma. Full article

Photoreceptors are highly compartmentalized cells with large amounts of proteins synthesized in the inner segment (IS) and transported to the outer segment (OS) and synaptic terminal. Tulp1 is a photoreceptor-specific protein localized to the IS and synapse. In the absence of Tulp1, several OS-specific proteins are mislocalized and synaptic vesicle recycling is impaired. To better understand the involvement of Tulp1 in protein trafficking, our approach in the current study was to physically isolate Tulp1-containing photoreceptor compartments by serial tangential sectioning of retinas and to identify compartment-specific Tulp1 binding partners by immunoprecipitation followed by liquid chromatography tandem mass spectrometry. Our results indicate that Tulp1 has two distinct interactomes. We report the identification of: (1) an IS-specific interaction between Tulp1 and the motor protein Kinesin family member 3a (Kif3a), (2) a synaptic-specific interaction between Tulp1 and the scaffold protein Ribeye, and (3) an interaction between Tulp1 and the cytoskeletal protein microtubule-associated protein 1B (MAP1B) in both compartments. Immunolocalization studies in the wild-type retina indicate that Tulp1 and its binding partners co-localize to their respective compartments. Our observations are compatible with Tulp1 functioning in protein trafficking in multiple photoreceptor compartments, likely as an adapter molecule linking vesicles to molecular motors and the cytoskeletal scaffold. Full article

Photoreceptor disc component (PRCD) is a small protein which is exclusively localized to photoreceptor outer segments, and is involved in the formation of photoreceptor outer segment discs. Mutations in PRCD are associated with retinal degeneration in humans, mice, and dogs. The purpose of this work was to identify PRCD-binding proteins in the retina. PRCD protein-protein interactions were identified when implementing the Ras recruitment system (RRS), a cytoplasmic-based yeast two-hybrid system, on a bovine retina cDNA library. An interaction between PRCD and tubby-like protein 1 (TULP1) was identified. Co-immunoprecipitation in transfected mammalian cells confirmed that PRCD interacts with TULP1, as well as with its homolog, TUB. These interactions were mediated by TULP1 and TUB highly conserved C-terminal tubby domain. PRCD localization was altered in the retinas of TULP1- and TUB-deficient mice. These results show that TULP1 and TUB, which are involved in the vesicular trafficking of several photoreceptor proteins from the inner segment to the outer segment, are also required for PRCD exclusive localization to photoreceptor outer segment discs. Full article

Mesenchymal Stromal Cells (MSC) are multipotent cells characterized by self-renewal, multilineage differentiation, and immunomodulatory properties. To obtain a gene regulatory profile of human MSCs, we generated a compendium of more than two hundred cell samples with genome-wide expression data, including a homogeneous set of 93 samples of five related primary cell types: bone marrow mesenchymal stem cells (BM-MSC), hematopoietic stem cells (HSC), lymphocytes (LYM), fibroblasts (FIB), and osteoblasts (OSTB). All these samples were integrated to generate a regulatory gene network using the algorithm ARACNe (Algorithm for the Reconstruction of Accurate Cellular Networks; based on mutual information), that finds regulons (groups of target genes regulated by transcription factors) and regulators (i.e., transcription factors, TFs). Furtherly, the algorithm VIPER (Algorithm for Virtual Inference of Protein-activity by Enriched Regulon analysis) was used to inference protein activity and to identify the most significant TF regulators, which control the expression profile of the studied cells. Applying these algorithms, a footprint of candidate master regulators of BM-MSCs was defined, including the genes EPAS1, NFE2L1, SNAI2, STAB2, TEAD1, and TULP3, that presented consistent upregulation and hypomethylation in BM-MSCs. These TFs regulate the activation of the genes in the bone marrow MSC lineage and are involved in development, morphogenesis, cell differentiation, regulation of cell adhesion, and cell structure. Full article

Variants in more than 271 different genes have been linked to hereditary retinal diseases, making comprehensive genomic approaches mandatory for accurate diagnosis. We explored the genetic landscape of retinal disorders in consanguineous families from North-Western Pakistan, harboring a population of approximately 35 million inhabitants that remains relatively isolated and highly inbred (~50% consanguinity). We leveraged on the high degree of consanguinity by applying genome-wide high-density single-nucleotide polymorphism (SNP) genotyping followed by targeted Sanger sequencing of candidate gene(s) lying inside autozygous intervals. In addition, we performed whole-exome sequencing (WES) on at least one proband per family. We identified 7 known and 4 novel variants in a total of 10 genes (ABCA4, BBS2, CNGA1, CNGA3, CNGB3, MKKS, NMNAT1, PDE6B, RPE65, and TULP1) previously known to cause inherited retinal diseases. In spite of all families being consanguineous, compound heterozygosity was detected in one family. All homozygous pathogenic variants resided in autozygous intervals ≥2.0 Mb in size. Putative founder variants were observed in the ABCA4 (NM_000350.2:c.214G>A; p.Gly72Arg; ten families) and NMNAT1 genes (NM_022787.3:c.25G>A; p.Val9Met; two families). We conclude that geographic isolation and sociocultural tradition of intrafamilial mating in North-Western Pakistan favor both the clinical manifestation of rare “generic” variants and the prevalence of founder mutations. Full article

The aim of this article is to propose the 2-tuple picture fuzzy linguistic aggregation operators and a decision-making model to deal with uncertainties in the form of 2-tuple picture fuzzy linguistic sets; 2-tuple picture fuzzy linguistic operators have more flexibility than general fuzzy set. We proposed a number of aggregation operators, namely, 2-TPFLWA, 2-TPFLOWA, 2-TPFLHA, 2-TPFLWG, 2-TPFLOWG, and 2-TPFLHG operators. The distinguished feature of the developed operators are studied. At that point, we used these operators to design a model to deal with multiple attribute decision-making issues under the 2-tuple picture fuzzy linguistic information. Then, a practical application of robot selection by manufacturing unit is given to prove the introduced technique and to show its practicability and effectiveness. Besides this, a systematic comparison analysis with other existent approaches is conducted to reveal the advantage of our developed method. Results indicate that the proposed method is suitable and effective for decision-making problems. Full article

Moraea pallida Bak. (yellow tulp) poisoning is the most important cardiac glycoside-induced intoxication in ruminants in South Africa. The toxic principle, 1α, 2α-epoxyscillirosidine, is a bufadienolide. To replace the use of sentient animals in toxicity testing, the aim of this study was to evaluate the cytotoxic effects of epoxyscillirosidine on rat embryonic cardiomyocytes (H9c2 cell line). This in vitro cell model can then be used in future toxin neutralization or toxico-therapy studies. Cell viability, evaluated with the methyl blue thiazol tetrazolium (MTT) assay, indicated a hormetic dose/concentration response, characterized by a biphasic low dose stimulation and high dose inhibition. Increased cell membrane permeability and leakage, as expected with necrotic cells, were demonstrated with the lactate dehydrogenase (LDH) assay. The LC50 was 382.68, 132.28 and 289.23 μM for 24, 48, and 72 h respectively. Numerous cytoplasmic vacuoles, karyolysis and damage to the cell membrane, indicative of necrosis, were observed at higher doses. Ultra-structural changes suggested that the cause of H9c2 cell death, subsequent to epoxyscillirosidine exposure, is necrosis, which is consistent with myocardial necrosis observed at necropsy. Based on the toxicity observed, and supported by ultra-structural findings, the H9c2 cell line could be a suitable in vitro model to evaluate epoxyscillirosidine neutralization or other therapeutic interventions in the future. Full article