bluuebunny/crossref_metadata_2025_split huggingface dataset:
Rescue of common and rare exon 2 skipping variants of the GAA gene using modified U1 snRNA
Authors: Peruzzo Paolo , Bergamin Natascha , Bon Martina , Cappelli Sara , Longo Alessandra , Goina Elisa , Stuani Cristiana , Buratti Emanuele , Dardis Andrea 
Published: February 2025

Abstract

Background
Pompe disease (PD) is an autosomal recessive lysosomal storage disorder caused by the deficient activity of acid alpha glucosidase (GAA) enzyme due to mutations in the GAA gene. As a result, undigested glycogen accumulates within lysosomes causing their dysfunction. From a clinical point of view, the disease can be classified in infantile-onset (IO) and late-onset (LO) forms. The common GAA c.-32-13T>G variant, found in 40–70% of LO-PD alleles, is a leaky splicing mutation interfering with the correct GAA exon 2 recognition by the spliceosome leading to the production of non-functional GAA transcripts. In this study, we used modified, GAA-tailored U1 snRNAs to correct the aberrant splicing determined by the c.-32-13T>G and other GAA exon 2-skipping mutations.

Methods
A set of constructs expressing 5 different engineered U1 snRNAs was generated. A functional splicing assay using a GAA hybrid minigene carrying different variants known to affect GAA exon 2 splicing was used to test the effect of engineered U1 snRNAs on exon 2 inclusion. The effect on endogenously expressed GAA transcript and GAA enzymatic activity was assessed by transfecting patient-derived fibroblasts bearing the common c.-32-13T>G with the best performing modified U1 snRNA.

Results
Modified U1-3, U1+1 and U1+6 snRNAs were all able to increase, in a dose-dependent manner, the inclusion of exon 2 within the transcript derived from the GAA minigene harbouring the c.-32-13T>G variant. The U1+1 was the most effective one (2,5 fold increase). Moreover, U1+1 snRNA partially rescued the correct splicing of GAA minigenes harbouring mutations that affect the 3’ss (c.-32-3C>G, c.-32-2A>G) and the 5’ss (c.546G>A, c.546G>C, c.546G>T). Notably, the treatment of patient-derived fibroblasts carrying the c.-32-13T>G mutation with the U1+1 snRNA increased the amount of normal GAA mRNA by 1,8 fold and the GAA enzymatic activity by 70%.

Conclusions
we provide the proof-of-concept for the use of modified GAA-tailored U1 snRNAs, designed to potentiate the recognition of the GAA exon 2 5’ss, as therapeutic tools to correct the aberrant transcripts carrying variants that affect exon 2 splicing, including the common c.-32-13T>G variant.
TAB2 deficiency induces dilated cardiomyopathy by promoting mitochondrial calcium overload in human iPSC-derived cardiomyocytes
Authors: Sun Wenrui , Zhang Jianchao , Li Shuang , Fu Wanrong , Liu Yangyang , Liu Mengduan , Dong Jianzeng , Zhao Xiaoyan , Li Xiaowei 
Published: February 2025

Abstract

Background
TGF-β-activated kinase 1 binding protein 2 (TAB2) is an intermediary protein that links Tumor necrosis factor receptor 1 (TNFR1) and other receptor signals to the TGF-β-activated kinase 1 (TAK1) signaling complex. TAB2 frameshift mutations have been linked to dilated cardiomyopathy (DCM), while the exact mechanism needs further investigation.

Methods
In this study, we generated a TAB2 compound heterozygous knockout cell line in induced pluripotent stem cells (iPSCs) derived from a healthy individual using CRISPR/Cas9 technology. IPSCs are not species-dependent, are readily accessible, and raise fewer ethical concerns.

Results
TAB2 disruption had no impact on the cardiac differentiation of iPSCs and led to confirmed TAB2 deficiency in human iPSC-derived cardiomyocytes (hiPSC-CMs). TAB2-deficient hiPSC-CMs were found to develop phenotypic features of DCM, such as distorted sarcomeric ultrastructure, decreased contractility and energy production, and mitochondrial damage at day 30 post differentiation. Paradoxically, TAB2 knockout cell lines showed abnormal calcium handling after 40 days, later than reduced contractility, suggesting that the main cause of impaired contractility was abnormal energy production due to mitochondrial damage. As early as day 25, TAB2 knockout cardiomyocytes showed significant mitochondrial calcium overload, which can lead to mitochondrial damage. Furthermore, TAB2 knockout activated receptor-interacting protein kinase 1 (RIPK1), leading to an increase in mitochondrial calcium uniporter (MCU) expression, thereby augmenting the uptake of mitochondrial calcium ions. Finally, the application of the RIPK1 inhibitor Nec-1s prevents the progression of these phenotypes.

Conclusions
In summary, TAB2 abatement cardiomyocytes mimic dilated cardiomyopathy in vitro. This finding emphasizes the importance of using a human model to study the underlying mechanisms of this specific disease. More importantly, the discovery of a unique pathogenic pathway introduces a new notion for the future management of dilated cardiomyopathy.
Collagen turnover biomarkers to predict outcome of patients with biliary cancer
Authors: Kaps Leonard , Genc Muhammed A. , Moehler Markus , Grabbe Stephan , Schattenberg Jörn M. , Schuppan Detlef , Pedersen Rasmus Sund , Karsdal Morten A. , Mildenberger Philipp , Maderer Annett , Willumsen Nicholas 
Published: February 2025

Abstract

Background
The collagen-rich tumor stroma plays a crucial role in biliary tract cancer (BTC). Collagen biomarkers of type I collagen (reC1M), type III collagen (PRO-C3), type IV collagen (C4G), type VIII collagen (PRO-C8), type XI collagen (PRO-C11), type XVII collagen (PRO-C17) and type VIII collagen (TUM) may be used as potential non-invasive biomarkers.

Methods
We measured the seven biomarkers of collagen turnover in sera of 72 patients with BTC at baseline and after first and second chemotherapy cycle (CTX). Markers were also assessed in sera of 50 healthy controls and compared to levels of patients at baseline. The diagnostic and prognostic value of the markers was evaluated for overall survival (OS) and progression-free survival (PFS).

Results
Patients had a median age of 65 years (IQR 57–70), while healthy controls were younger, with a median age of 46 years (IQR 38–54). The majority of patients (62%) were diagnosed with intrahepatic bile duct adenocarcinoma. Except C4G, all collagen turnover markers were significantly (p < 0.001) increased in serum from patients with BTC compared to healthy controls. PRO-C3 was the best marker to discriminate between patients with BTC and controls, reaching an area under a receiver operating characteristic (AUROC) of 0.98 (95% CI 0.95; 0.99) with a sensitivity (92%) and specificity (94%) balanced cutoff of 77.3 ng/ml. Patients with high levels (cohort separated by median split) of PRO-C8 (HR 2.85, 95% CI 1.42; 5.73) followed by C3M (HR 2.33, 95% CI 1.2; 4.5), PRO-C3 (HR 3.09, 95% CI 1.5; 6.36) and CA 19–9 (HR 2.52, 95% CI 1.37; 4.64) as reference biomarker had a shorter OS. Notably, only the novel marker PRO-C8 was also predictive of PFS (HR 3.26, 95% CI 1.53; 6.95). Associations with survival outcomes remained significant after adjusting for relevant risk factors (CA 19–9 and CEA at baseline, age, presence of metastases, weight, height and gender).

Conclusion
The collagen turnover markers PRO-C8, C3M, PRO-C3 and the established biomarker CA 19–9 were prognostic for OS in patients with BTC while only PRO-C8 was also predictive for PFS. PRO-C3 showed the best diagnostic performance to discriminate between patients with BTC and controls.

Trial registration

Trial registration number and date of registration

NCT00661830 (NCT number)
15 April 2008

Trial registry

The complete registry can found under:

https://clinicaltrials.gov/study/NCT00661830?tab=table#administrative-information (last accessed 01/2025)

Principal investigator and study sponsor

Markus Moehler, MD
Johannes Gutenberg University Mainz
Assessment of in vitro skin permeation and accumulation of phenolic acids from honey and honey-based pharmaceutical formulations
Authors: Nowak Anna , Muzykiewicz-Szymańska Anna , Perużyńska Magdalena , Kucharska Edyta , Kucharski Łukasz , Jakubczyk Karolina , Niedźwiedzka-Rystwej Paulina , Stefanowicz-Hajduk Justyna , Droździk Marek , Majtan Juraj 
Published: February 2025

Abstract

Background
Honey has been successfully used in wound care and cosmetics because of its effective biological properties, including antibacterial, antioxidant, and anti-inflammatory activities. Polyphenols, particularly phenolic acids, are key honey components responsible for these beneficial effects. In recent years, there has been a growing demand for natural, ecologically friendly, and biodegradable products in the modern cosmetics and wound care market. This study aimed to identify and quantify phenolic acids in four Polish honey samples of different botanical origins (heather, buckwheat, linden and rapeseed) and to assess for the first time the permeation of the identified phenolic acids through the skin and their accumulation after the application of pure honey samples, as well as honey-based hydrogel and emulsion formulations.

Methods
The honey samples’ antioxidant activity and total phenolic content were determined using the DPPH and ABTS assays and the Folin–Ciocalteu method, respectively. Phenolic acids and volatile compounds were identified and quantified in honey samples using the HPLC-UV and GC-MS method, respectively. The biocompatibility of the honey samples was evaluated using a murine fibroblast cell line (L929). A Franz-type vertical diffusion cell with porcine skin was used to assess phenolic acid’s permeation and skin accumulation from different honey-based pharmaceutical formulations. The biodegradability of the prepared formulations was also characterised.

Results
Gallic acid, 3,4-dihydroxybenzoic acid, 2,5-dihydroxybenzoic acid, coumaric acid, and 3-hydroxybenzoic acid were identified and quantified in the honey samples. Heather honey exhibited significantly higher antioxidant activity and total polyphenol content than the other honey samples. Heather, linden and buckwheat honey samples significantly decreased cell viability at concentrations of 5% and 2.5%, while rapeseed honey sample markedly reduced fibroblast viability only at 5%. Among the tested formulations - pure honey, hydrogel, and emulsion - higher skin permeation and accumulation rates of phenolic acids were observed with the prepared honey-based hydrogels than with the pure honeys and emulsions. Additionally, the prepared formulations were classified as partially biodegradable.

Conclusions
The obtained results confirmed the effectiveness of two pharmaceutical formulations in the form of a hydrogel or emulsion containing honey after applied topically. The inclusion of honey in the vehicle, in particular hydrogel increased the penetration of phenolic acids through the skin.
Immune microenvironment spatial landscapes of tertiary lymphoid structures in gastric cancer
Authors: Xie Yi , Peng Haoxin , Hu Yajie , Jia Keren , Yuan Jiajia , Liu Dan , Li Yanyan , Feng Xujiao , Li Jian , Zhang Xiaotian , Sun Yu , Shen Lin , Chen Yang 
Published: February 2025

Abstract

Background
Tertiary lymphoid structures (TLS) correlate with tumour prognosis and immunotherapy responses in gastric cancer (GC) studies. However, understanding the complex and diverse immune microenvironment within TLS requires comprehensive analysis.

Methods
We examined the prognostic impact of TLS within the tumour core (TC) of 59 GC patients undergoing immunotherapy. Multispectral fluorescence imaging was employed to evaluate variations in immune cell infiltration across different TLS sites among 110 GC patients, by quantifying immune cell density and spatial characteristics. We also generated a single-cell transcriptomic atlas of TLS-positive (n = 4) and TLS-negative (n = 8) microenvironments and performed spatial transcriptomics (ST) analysis on two samples.

Results
TLS presence in the TC significantly correlated with improved immune-related overall survival (P = 0.049). CD8+LAG-3−PD-1+TIM-3−, CD4+PD-L1+, and CD4+FoxP3− T cell densities were significantly higher in the TLS within TC compared to tumour and stromal regions. Immune cells within TLS exhibited closer intercellular proximity than those outside TLS. Five key density and spatial characteristics of immune cells within TLS in the TC were selected to develop the Density and Spatial Score risk model. Single-cell RNA sequencing revealed strong intercellular interactions in the presence of TLS within the microenvironment. However, TLS-absent environment facilitated tumour cell interactions with immune cells through MIF- and galectin-dependent pathways, recruiting immunosuppressive cells. ST analysis confirmed that T and B cells co-localise within TLS, enhancing immune response activation compared to cancer nests and exerting a strong anti-tumour effect.

Conclusions
TLS presence facilitates frequent cell-to-cell communication, forming an active immune microenvironment, highlighting the prognostic value of TLS.
Cervical pedicle screw fixation with the Tianji orthopedic surgical robot
Authors: Shen Hao , Zhou Jinlong , Yu Lipeng 
Published: February 2025

Abstract

Objective
To compare the accuracy and safety of implanting cervical pedicle screws (CPS) between orthopedic surgical robot-assisted technique and traditional fluoroscopy-assisted free-hand technique.

Methods
Retrospective analysis of 95 patients treated with posterior cervical spinal surgery using either Tianji orthopedic surgical robot-assisted or traditional fluoroscopy-assisted free-hand pedicle screw implantation technology from March 2021 to March 2024, including 44 cases in the orthopedic surgical robot group and 51 cases in the traditional fluoroscopy group.

Results
Compared with the traditional fluoroscopy group, the orthopedic surgical robot group had better accuracy in screw implantation that is, a higher acceptable rate of screws (p = 0.0083). In addition, compared with the traditional fluoroscopy group, postoperative hospital stay was shorter in the orthopedic surgical robot group (p = 0.0447), but operation duration was longer (p = 0.0038). There was no significant difference in intraoperative blood loss between groups (p = 0.0872). There were 2 cases of cerebrospinal fluid leakage and 1 case of decreased left handgrip strength in the traditional fluoroscopy group, while only 1 case of cerebrospinal fluid leakage occurred in the orthopedic surgical robot group.

Conclusions
In this retrospective study, the accuracy of spine surgery with CPS implantation assisted by orthopedic surgical robot is often superior to that of spine surgery using traditional fluoroscopy-guided CPS implantation technique, while maintaining comparable safety.
Metabolites and lipid species mediate the associations of adiposity in childhood and early adulthood with mammographic breast density in premenopausal women
Authors: Getz Kayla R. , Jeon Myung Sik , Liu Lili , Liu Lei , Zhang Haixiang , Luo Chongliang , Luo Jingqin , Toriola Adetunji T. 
Published: February 2025

Abstract

Background
Mammographic breast density (MBD), a strong predictor of breast cancer, is highly influenced by body mass index (BMI) in childhood and early adulthood, but the mechanisms underlying these associations are not fully understood. Our goal is to identify biomarkers that mediate the associations of BMI at ages 10 and 18 with MBD in premenopausal women.

Methods
This study consists of 705 premenopausal women who had their screening mammogram at Washington University in St. Louis, MO, and provided a fasting blood sample. Our comprehensive metabolomic and lipidomic profiling yielded complete data for 828 metabolites and 857 lipid species after imputation. We used Volpara to determine volumetric measures of MBD. We performed high dimensional mediation analysis using the HIMA R package, adjusted for confounders, to determine whether lipid species and metabolites mediate the associations of BMI at 10 and 18 with MBD. We applied a false discovery rate (FDR) p-value < 0.1.

Results
Four metabolites (glutamate, β-cryptoxanthin, cortolone glucuronide (1), phytanate) significantly mediated the association of BMI at 10 with volumetric percent density (VPD), and two (glutamate, β-cryptoxanthin) mediated the association of BMI at 18 with VPD. Glutamate was the strongest mediator across time points. Glutamate mediated 6.7% (FDR p-value = 0.06) and 9.3% (FDR p-value = 0.008) of the association between BMI at age 10 and 18, respectively. Four lipid species (CER(18:0), LCER(14:0), LPC(18:1), PC(18:1/18:1)), mediated the association of BMI at 10 with VPD, while five lipid species (CER(18:0), LCER(14:0), PC(18:1/18:1), TAG56:5-FA22:5, TAG52:2-FA16:0) mediated the association of BMI at 18 with VPD. The strongest mediator was PC(18:1/18:1), which mediated 9.7%, (FDR-p = 0.009) and 7.7%, (FDR-p = 0.04) of the association of BMI at age 10 and 18 with VPD, respectively.

Conclusions
Metabolites in amino acid, lipid, cofactor/vitamin, and xenobiotic super-pathways as well as lipid species across the phospholipid, neutral complex lipid and sphingolipid super-pathways mediated the associations of BMI in early-life and MBD in premenopausal women. This study offers insight into the biological mechanisms underlying the link between early-life adiposity and MBD, which can support future research into breast cancer prevention.
Merck Open Global Health Library in vitro screening against Schistosoma mansoni identified two new substances with antischistosomal activities for further development
Authors: Ueberall Monique Evelyn , Berchthold Martina , Häberli Cécile , Lindemann Sven , Spangenberg Thomas , Keiser Jennifer , Grevelding Christoph G. 
Published: February 2025

Abstract

Background
Schistosomiasis, which is caused by the parasite Schistosoma mansoni as well as other species of the trematode genus Schistosoma, leads to chronic inflammation and finally to liver fibrosis. If untreated, the disease can cause life-threatening complications. The current treatment of schistosomiasis relies on a single drug, praziquantel (PZQ). However, there is increasing concern about emerging resistance to PZQ due to its frequent use.

Methods
To identify potential alternative drugs for repurposing, the Open Global Health Library (OGHL) was screened in vitro, using two different screening workflows at two institutions, against adult S. mansoni couples and newly transformed schistosomula. This was followed by confirmation of the effects of the lead structures against adult worms.

Results
In vitro screening at one of the institutions identified two fast-acting substances affecting worm physiology (OGHL00022, OGHL00121). The effects of the two lead structures were investigated in more detail by confocal laser scanning microscopy and 5-ethynyl 2´-deoxyuridine (EdU) assays to assess morphological effects and stem cell effects. Both substances showed negative effects on stem cell proliferation in S. mansoni but no further morphological changes. The EC50values of both compounds were determined, with values for compound OGHL00022 of 5.955 µM for pairing stability, 10.88 µM for attachment, and 18.77 µM for motility, while the values for compound OGHL00121 were 7.088 µM for pairing stability, 8.065 µM for attachment, and 6.297 µM for motility 24 h after treatment. Furthermore, S. mansoni couples were treated in vitro with these two lead structures simultaneously to check for additive effects, which were found with respect to reduced motility. The second in vitro screening, primarily against newly transformed schistosomula and secondarily against adult worms, identified four lead structures in total (OGHL00006, OGHL00022, OGHL00169, OGHL00217). In addition, one of the tested analogues of the hits OGHL00006, OGHL00169, and OGHL00217 showed effects on both stages.

Conclusions
In two independent in vitro screening approaches against two stages of S. mansoni one common interesting structure with rapid effects was identified, OGHL00022, which provides opportunities for further development.

Graphical Abstract
Sex differences in the neuroinflammatory signaling pathway: effect of miRNAs on fatty acid synthesis in microglia
Authors: Zheng Haolin , Mizokami Akiko , Romera-Giner Sergio , Llera-Oyola Jaime , Yamawaki Yosuke , Sano Tomomi , Jimi Eijiro , García-García Francisco , Kanematsu Takashi 
Published: February 2025

Abstract

Background
Significant sex differences exist in the prevalence and incidence of Alzheimer’s disease (AD). Notably, testosterone has been reported to regulate cognitive functions in the brain, with low serum testosterone levels correlating with increased AD risk. However, the specific mechanisms underlying this relationship remain unclear. Recent studies have demonstrated that microglia, the primary innate immune cells in the brain, play a crucial role in AD development. Therefore, this study aimed to explore sex differences in microglial function, specifically focusing on the role of testosterone in miRNA-mediated regulation of microglial gene expression.

Methods
Microglia were isolated from pooled hippocampal tissue of five 8-month-old male and female mice. Total RNA was extracted and subjected to miRNA microarray analysis. The mouse microglial cell line MG6 was used for in vitro experiments. Following testosterone treatment, miRNA, gene, and protein expression levels were investigated. An inflammatory response was induced using lipopolysaccharide (LPS) stimulation, and subsequent p65 phosphorylation was assessed.

Results
Sex-dependent differences were observed in miRNA-mediated biological processes, with males exhibiting greater changes. Male-enriched miRNAs were associated with fatty acid synthesis and metabolism pathways. In MG6 cells, testosterone treatment upregulated the expression of several miRNAs enriched in male microglia, particularly those targeting genes related to fatty acid synthesis. Additionally, testosterone significantly reduced the gene expression of fatty acid synthase (FASN). This testosterone-induced inhibition of FASN expression attenuated NF-κB/p65 phosphorylation. Consequently, the suppression of FASN expression led to reduced expression and secretion of tumor necrosis factor-alpha following LPS stimulation in MG6 cells.

Conclusions
These findings suggest that testosterone modulates inflammation in male microglia by regulating fatty acid synthesis, potentially contributing to the observed sex differences in AD pathogenesis.