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PloS One 2024Many newborn screening programs worldwide have introduced screening for diseases using DNA extracted from dried blood spots (DBS). In Germany, DNA-based assays are...
BACKGROUND
Many newborn screening programs worldwide have introduced screening for diseases using DNA extracted from dried blood spots (DBS). In Germany, DNA-based assays are currently used to screen for severe combined immunodeficiency (SCID), spinal muscular atrophy (SMA), and sickle cell disease (SCD).
METHODS
This study analysed the impact of pre-analytic DNA carry-over in sample preparation on the outcome of DNA-based newborn screening for SCID and SMA and compared the efficacy of rapid extraction versus automated protocols. Additionally, the distribution of T cell receptor excision circles (TREC) on DBS cards, commonly used for routine newborn screening, was determined.
RESULTS
Contaminations from the punching procedure were detected in the SCID and SMA assays in all experimental setups tested. However, a careful evaluation of a cut-off allowed for a clear separation of true positive polymerase chain reaction (PCR) amplifications. Our rapid in-house extraction protocol produced similar amounts compared to automated commercial systems. Therefore, it can be used for reliable DNA-based screening. Additionally, the amount of extracted DNA significantly differs depending on the location of punching within a DBS.
CONCLUSIONS
Newborn screening for SMA and SCID can be performed reliably. It is crucial to ensure that affected newborns are not overlooked. Therefore a carefully consideration of potential contaminating factors and the definition of appropriate cut-offs to minimise the risk of false results are of special concern. It is also important to note that the location of punching plays a pivotal role, and therefore an exact quantification of TREC numbers per μl may not be reliable and should therefore be avoided.
Topics: Humans; Neonatal Screening; Infant, Newborn; Muscular Atrophy, Spinal; Severe Combined Immunodeficiency; DNA; Dried Blood Spot Testing; High-Throughput Screening Assays; Polymerase Chain Reaction
PubMed: 38941330
DOI: 10.1371/journal.pone.0306329 -
Annals of Intensive Care Jun 2024A notable increase in severe cases of COVID-19, with significant hospitalizations due to the emergence and spread of JN.1 was observed worldwide in late 2023 and early...
Clinical phenotypes and outcomes associated with SARS-CoV-2 Omicron sublineage JN.1 in critically ill COVID-19 patients: a prospective, multicenter cohort study in France, November 2022 to January 2024.
BACKGROUND
A notable increase in severe cases of COVID-19, with significant hospitalizations due to the emergence and spread of JN.1 was observed worldwide in late 2023 and early 2024. However, no clinical data are available regarding critically-ill JN.1 COVID-19 infected patients.
METHODS
The current study is a substudy of the SEVARVIR prospective multicenter observational cohort study. Patients admitted to any of the 40 participating ICUs between November 17, 2022, and January 22, 2024, were eligible for inclusion in the SEVARVIR cohort study (NCT05162508) if they met the following inclusion criteria: age ≥ 18 years, SARS-CoV-2 infection confirmed by a positive reverse transcriptase-polymerase chain reaction (RT-PCR) in nasopharyngeal swab samples, ICU admission for acute respiratory failure. The primary clinical endpoint of the study was day-28 mortality. Evaluation of the association between day-28 mortality and sublineage group was conducted by performing an exploratory multivariable logistic regression model, after systematically adjusting for predefined prognostic factors previously shown to be important confounders (i.e. obesity, immunosuppression, age and SOFA score) computing odds ratios (OR) along with their corresponding 95% confidence intervals (95% CI).
RESULTS
During the study period (November 2022-January 2024) 56 JN.1- and 126 XBB-infected patients were prospectively enrolled in 40 French intensive care units. JN.1-infected patients were more likely to be obese (35.7% vs 20.8%; p = 0.033) and less frequently immunosuppressed than others (20.4% vs 41.4%; p = 0.010). JN.1-infected patients required invasive mechanical ventilation support in 29.1%, 87.5% of them received dexamethasone, 14.5% tocilizumab and none received monoclonal antibodies. Only one JN-1 infected patient (1.8%) required extracorporeal membrane oxygenation support during ICU stay (vs 0/126 in the XBB group; p = 0.30). Day-28 mortality of JN.1-infected patients was 14.6%, not significantly different from that of XBB-infected patients (22.0%; p = 0.28). In univariable logistic regression analysis and in multivariable analysis adjusting for confounders defined a priori, we found no statistically significant association between JN.1 infection and day-28 mortality (adjusted OR 1.06 95% CI (0.17;1.42); p = 0.19). There was no significant between group difference regarding duration of stay in the ICU (6.0 [3.5;11.0] vs 7.0 [4.0;14.0] days; p = 0.21).
CONCLUSIONS
Critically-ill patients with Omicron JN.1 infection showed a different clinical phenotype than patients infected with the earlier XBB sublineage, including more frequent obesity and less immunosuppression. Compared with XBB, JN.1 infection was not associated with higher day-28 mortality.
PubMed: 38940865
DOI: 10.1186/s13613-024-01319-w -
Alternative Therapies in Health and... Jun 2024Osteoporosis (OP) is a chronic skeletal disorder characterized by low bone mass and microarchitectural deterioration of bone tissue, resulting in increased bone...
Study on the Mechanism of Xianling Gubao Capsule Regulating Runt-Related Transcription Factor 2 (RUNX2) and Promoting Osteoblast Differentiation by N6-Methyladenosine (m6A) Methyltransferase-Like 3 (METTL3).
BACKGROUND
Osteoporosis (OP) is a chronic skeletal disorder characterized by low bone mass and microarchitectural deterioration of bone tissue, resulting in increased bone fragility and a higher risk of fractures. It is a significant public health concern, particularly among postmenopausal women and older adults. The imbalance between bone formation and resorption is the fundamental cause of OP. Current clinical drugs for OP have limited efficacy and can cause side effects. Therefore, there is a need to explore alternative treatments and investigate their mechanisms to improve OP management. The Xianling Gubao capsule, a traditional Chinese medicine, is commonly used to treat OP by tonifying the kidney. However, the specific mechanism of action of the Xianling Gubao capsule in improving OP remains unclear, necessitating further research in this area.
METHODS
The N6-methyladenosine (m6A) content was evaluated by dot blot and m6A ribonucleic acid (RNA) methylation assay kit. The contents of methyltransferase-like 3 (METTL3), runt-related transcription factor 2 (RUNX2), alkaline phosphatase (ALP), and bone gamma-carboxyglutamate protein (BGLAP) were appraised by quantitative Reverse Transcription polymerase chain reaction (qRT-PCR) and western blot. The bilateral ovariectomy (OVX) method was used to establish an animal model of OP. OP bone marrow mesenchymal stem cells (OP-BMSCs) were extracted from mice in the OVX group by the whole bone marrow method. METTL3 overexpression and control vectors were transfected to OP-BMSCs using X-tremeGENE HP DNA Transfection Reagent. The ALP activity in OP-BMSCs was assessed by ALP staining. The calcium nodules in OP-BMSCs were detected by Alizarin Red S (ARS) assay. The Xianling Gubao capsule solution was employed to gavage mice, and the drug-containing serum was used to treat OP-BMSCs. Dot blot allows for the assessment of relative levels of m6A modification. The m6A RNA methylation assay kit is a specialized kit designed to quantitatively measure m6A levels in RNA samples. qRT-PCR allows for the measurement of mRNA levels of target genes. Western blot is used to detect and quantify specific proteins in a sample, and provides information about protein expression levels. OVX mimics the hormonal changes occurring in postmenopausal women and leads to bone loss and osteoporotic conditions in animals. This model allows for the investigation of the effects of the Xianling Gubao capsule on OP in a controlled experimental setting.
RESULTS
The m6A modification and METTL3, RUNX2, ALP, and BGLAP levels were reduced in bone samples of patients with OP and OVX mice compared with the corresponding control groups. Upregulated METTL3 enhanced the osteogenic ability of OP-BMSCs. METTL3 overexpression obviously increased m6A modification and METTL3, RUNX2, ALP, and BGLAP levels in OP-BMSCs. Xianling Gubao capsule treatment could weaken the impact of OP in mice by regulating the m6A modification and METTL3, RUNX2, ALP, and BGLAP levels. Serum containing Xianling Gubao capsule could enhance the osteogenic capability of OP-BMSCs and boost METTL3, RUNX2, ALP, and BGLAP levels. Treatment with the Xianling Gubao capsule shows promising effects in attenuating the impact of OP. The capsule is found to regulate m6A modification and increase the levels of METTL3, RUNX2, ALP, and BGLAP in OP-BMSCs. This indicates that the Xianling Gubao capsule may rescue the diminished osteogenic capability of OP-BMSCs by modulating METTL3. These findings suggest that the Xianling Gubao capsule has the potential to be an effective drug for the treatment of OP.
CONCLUSION
Taken together, the m6A modification and contents of osteogenic-related factors were reduced in OP. Upregulated METTL3 improved the osteogenic ability, m6A modification, and osteogenic-related factor abundances in OP-BMSCs. Xianling Gubao capsule rescued the diminished osteogenic capability of OP-BMSCs by modulating METTL3 and might serve as an effective drug for OP. The Xianling Gubao capsule, as a traditional Chinese medicine, could potentially complement existing therapeutic approaches for OP. By targeting the m6A modification pathway and promoting osteogenic differentiation, the capsule may help to expedite bone formation and repair, which are critical for managing OP and reducing the risk of fractures.
PubMed: 38940781
DOI: No ID Found -
Cancer Biology & Medicine Jun 2024Radiotherapy has achieved remarkable effects in treating non-small cell lung cancer (NSCLC). However, radioresistance remains the major obstacle to achieving good...
OBJECTIVE
Radiotherapy has achieved remarkable effects in treating non-small cell lung cancer (NSCLC). However, radioresistance remains the major obstacle to achieving good outcomes. This study aims at identifying potential targets for radiosensitizing NSCLC and elucidating the underlying mechanisms.
METHODS
Lentivirus-based infection and CRISPR/Cas9 technology were used to modulate the expression of microRNA-384 (miR-384). Cell clonogenic formation assays and a xenograft tumor model were used to analyze radiosensitivity in NSCLC cells. Fluorescence-activated cell sorting was used to assess the cell cycle and cell death. Immunofluorescence staining, Comet assays, and homologous recombination or non-homologous end-joining I-SceI/GFP reporter assays were used to study DNA damage and repair. Western blotting and quantitative real-time polymerase chain reaction were used to identify the targets of miR-384. Chromatin immunoprecipitation and polymerase chain reaction were performed to evaluate upstream regulators of miR-384.
RESULTS
MiR-384 was downregulated in NSCLC. Overexpression of miR-384 increased the radiosensitivity of NSCLC cells and , whereas knockout of miR-384 led to radioresistance. Upregulation of miR-384 radiosensitized NSCLC cells by decreasing G2/M cell cycle arrest, inhibiting DNA damage repair, and consequently increasing cell death; miR-384 depletion had the opposite effects. Further investigation revealed that ATM, Ku70, and Ku80 were direct targets of miR-384. Moreover, miR-384 was repressed by NF-κB.
CONCLUSIONS
MiR-384 is an ionizing radiation-responsive gene repressed by NF-κB. MiR-384 enhances the radiosensitivity of NSCLC cells targeting ATM, Ku80, and Ku70, which impairs DNA damage repair. Therefore, miR-384 may serve as a novel radiosensitizer for NSCLC.
PubMed: 38940672
DOI: 10.20892/j.issn.2095-3941.2024.0146 -
Current Eye Research Jun 2024Posterior capsule opacification (PCO) is the major complication of visual impairment after cataract surgery. Circular RNAs (circRNAs) are involved in the development of...
PURPOSE
Posterior capsule opacification (PCO) is the major complication of visual impairment after cataract surgery. Circular RNAs (circRNAs) are involved in the development of many diseases. The purpose of this study was to explore the role and molecular mechanism of circ_0000099 in PCO.
METHODS
SRA01/04 cells were treated with TGF-β2 to establish a PCO cell model. The expression of circ_0000099, miR-223-3p, and connective tissue growth factor (CTGF) mRNA was determined by real-time quantitative polymerase chain reaction (qRT-PCR). Western blot assay was used to analyze the protein expression. Cell proliferation, migration, and invasion were analyzed by (4-5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), 5-ethynyl-2 '-Deoxyuridine (EdU), transwell, and wound healing tests. The circ_0000099/miR-223-3p/CTGF relationship was verified by dual luciferase reporter gene and RNA binding protein immunoprecipitation (RIP) assays.
RESULTS
TGF-β2 treatment promoted SRA01/04 cell proliferation invasion, migration, and EMT. Circ_0000099 expression was increased in POC patients and TGF-β2-treated SRA01/04 cells.Knockdown of circ_0000099 suppressed TGF-β2-induced proliferation, invasion, migration, and EMT in SRA01/04 cells. miR-223-3p was identified as the target of circ_0000099, and miR-223-3p inhibitor might partly abolish the repression of circ_0000099 silencing on TGF-β2-triggered SRA01/04 cell disorders. MiR-223-3p directly targeted CTGF. Knockdown of CTGF suppressed TGF-β2-induced SRA01/04 cell injury. Circ_0000099 can regulate CTGF expression by targeting miR-223-3p.
CONCLUSIONS
Circ_0000099 silencing might relieve TGF-2-induced SRA01/04 cell injury by the miR-223-3p/CTGF axis, providing new avenues for the prevention and treatment of PCO.
PubMed: 38940233
DOI: 10.1080/02713683.2024.2357600 -
Annals of Agricultural and... Jun 2024Intestinal parasitoses are important causes of morbidity and mortality, especially in immunocompromised individuals. In patients with chronic renal insufficiency (CRI),...
INTRODUCTION AND OBJECTIVE
Intestinal parasitoses are important causes of morbidity and mortality, especially in immunocompromised individuals. In patients with chronic renal insufficiency (CRI), the accumulation of non-excreted metabolites leads to uraemia, which induces a state of immunodeficiency, increasing the incidence of infections. The aim of the study was molecular screening for enteric protozoa in patients with chronic renal insufficiency.
MATERIAL AND METHODS
A total of 53 samples were collected in January 2023 from patients undergoing dialysis at Logman Ltd. Nephrodialysis Centre in Košice, Slovakia. Samples were examined by polymerase chain reaction (PCR) for the presence of / , , Microsporidia spp., and sp.
RESULTS
From the 53 samples, the only pathogen identified by PCR was Blastocystis sp., in 13 patients (24.5 %). Sequence analyses confirmed that the most prevalent subtype (ST) among patients was ST 3 (n=9, 69.2%), followed by ST 1 (n=3, 23.1%) and ST 2 (n=1, 7.7%).
CONCLUSIONS
Molecular methods for the detection of microscopic enteric parasites are not used as a first-line diagnostic method in Slovakia. In immunocompromised patients, diarrhoea can be caused not only by a chronic disease or therapy but can also be a result of an ongoing underdiagnosed infection. Early diagnosis leads to targeted therapy and subsequent partial improvement of the quality of life. This study also shows the first insights into sp. subtype distribution in humans in Slovakia.
Topics: Humans; Slovakia; Blastocystis; Male; Female; Middle Aged; Blastocystis Infections; Renal Dialysis; Aged; Polymerase Chain Reaction; Adult; Renal Insufficiency, Chronic; Feces; Intestinal Diseases, Parasitic; Aged, 80 and over
PubMed: 38940102
DOI: 10.26444/aaem/185634 -
Frontiers in Bioscience (Landmark... Jun 2024Persistent hyperuricemia can lead to the generation and deposition of monosodium urate (MSU) crystals. This can trigger gouty arthritis (GA), which in turn induces...
BACKGROUND
Persistent hyperuricemia can lead to the generation and deposition of monosodium urate (MSU) crystals. This can trigger gouty arthritis (GA), which in turn induces inflammation. Activation of the Nod-like receptor pyrin domain containing 3 (NLRP3) inflammasome plays a critical role in the onset and progression of GA. Autophagy may have a dual effect on GA with regard to the NLRP3 inflammasome. Therefore, the present study aimed to gain a deeper comprehension of the interaction between autophagy and NLRP3 inflammasome activation is imperative for developing more efficacious treatments for GA.
METHODS
Peripheral blood monocytes (PBMCs) were first isolated from GA patients and healthy controls and underwent bulk RNA sequencing analysis. Overexpression and knockdown of dual specificity phosphatase 1 (DUSP1) was performed in THP-1 monocytes to investigate its role in the immune response and mitochondrial damage. The luciferase assay and Western blot analysis were used to study the interaction between autophagy and NLRP3 inflammasome activation.
RESULTS
Bulk RNA sequencing analysis showed significant upregulation of DUSP1 expression in PBMCs from GA patients compared to healthy controls. This result was subsequently verified by reverse transcription quantitative polymerase chain reaction (RT-qPCR). DUSP1 expression in human THP-1 monocytes was also shown to increase after MSU treatment. Downregulation of DUSP1 expression increased the secretion of inflammatory cytokines after MSU treatment, whereas the overexpression of DUSP1 decreased the secretion levels. Lipopolysaccharides (LPS) combined with adenosine-triphosphate (ATP) led to mitochondrial damage, which was rescued by overexpressing DUSP1. DUSP1 overexpression further increased the level of autophagy following MSU treatment, whereas downregulation of DUSP1 decreased autophagy. Treatment with the autophagy inhibitor 3-Methyladenine (3-MA) restored inflammatory cytokine secretion levels in the DUSP1 overexpression group. MSU caused pronounced pathological ankle swelling . However, DUSP1 overexpression significantly mitigated this phenotype, accompanied by significant downregulation of inflammatory cytokine secretion levels in the joint tissues.
CONCLUSIONS
This study revealed a novel function and mechanism for DUSP1 in promoting autophagy to mitigate the MSU-induced immune response in GA. This finding suggests potential diagnostic biomarkers and anti-inflammatory targets for more effective GA therapy.
Topics: Humans; Autophagy; Dual Specificity Phosphatase 1; Arthritis, Gouty; Uric Acid; NLR Family, Pyrin Domain-Containing 3 Protein; Inflammasomes; THP-1 Cells; Male; Monocytes; Case-Control Studies; Female; Leukocytes, Mononuclear; Middle Aged
PubMed: 38940057
DOI: 10.31083/j.fbl2906222 -
Frontiers in Bioscience (Landmark... Jun 2024Under fasting conditions, the pathway converting gluconeogenesis precursors into muscle glycogen becomes crucial due to reduced glycogen reserves. However, there is...
BACKGROUND
Under fasting conditions, the pathway converting gluconeogenesis precursors into muscle glycogen becomes crucial due to reduced glycogen reserves. However, there is limited research on skeletal muscle gluconeogenesis and the impact of fasting on gluconeogenic gene expression.
METHODS
Sheep fetal skeletal muscle cells cultured were used to study the effects of varying lactic acid concentrations (0 to 30 mM) and 2.5 mM glucose on the expression of gluconeogenesis-related genes after 6 h of fasting. The effects on mRNA and protein expression of key genes involved in skeletal muscle gluconeogenesis were measured by quantitative real time polymerase chain reaction (qRT-PCR), immunofluorescence, and western blotting at 48 h.
RESULTS
Fasting increased the expression of key gluconeogenic genes, fructose-1,6-bisphosphatase 2 (), glucose-6-phosphatase 3 (), pyruvate kinase M (), monocarboxylate transporter1 (), glucose transporter type 4 (), pyruvate carboxylase (), and lactate dehydrogenase A (). The mRNA levels of , , and significantly decreased with glucose addition. Additionally, 10 mM lactic acid significantly promoted the expression of , , , , , and while inhibiting phosphoenolpyruvate carboxykinase () expression. At the protein level, 10 mM lactic acid significantly increased FBP2 and PKM protein expression.
CONCLUSIONS
This study shows that fasting regulates key gluconeogenic gene expression in sheep skeletal muscle cells and highlights the role of lactic acid in inducing these gene expressions.
Topics: Animals; Gluconeogenesis; Sheep; Muscle, Skeletal; Gene Expression Regulation; Glucose; Cells, Cultured; Lactic Acid; Fructose-Bisphosphatase
PubMed: 38940053
DOI: 10.31083/j.fbl2906237 -
Frontiers in Bioscience (Landmark... Jun 2024Hormone receptors exert their function through binding with their ligands, which results in cellular signaling activation mediated by genomic or non-genomic mechanisms....
BACKGROUND
Hormone receptors exert their function through binding with their ligands, which results in cellular signaling activation mediated by genomic or non-genomic mechanisms. The intrinsic molecular communication of tick and its host comprises an endocrine regulation involving hormones. In the present study, we performed a molecular and analysis of a Membrane Associated Progesterone Receptor in (RmMAPRC).
METHODS
The RmMAPRC protein sequence was analyzed with bioinformatics tools, and its structure was characterized by three-dimensional (3D) modeling and molecular docking. A semi-quantitative reverse transcription and polymerase chain reaction (sqRT-PCR) assessed the gene presence and relative expression in tick organs and embryonic cells.
RESULTS
relative expression in salivary glands, ovaries, and embryonic cells showed overexpression of 3%, 13%, and 24%, respectively. Bioinformatic analysis revealed that RmMAPRC corresponded to a Progesterone Receptor Membrane Component 1 (RmPGRMC1) of ~23.7 kDa, with an N-terminal transmembrane domain and a C-terminal Cytochrome b5-like heme/steroid binding domain. The docking results suggest that RmPGRMC1 could bind to progesterone (P4), some progestins, and P4 antagonists. The phylogenetic reconstruction showed that spp. MAPRC receptors were clustered in a clade that includes , , and (RmMAPRC), and mammals and helminths MAPRC receptors clustered in two separated clades away from ticks.
CONCLUSIONS
The presence of RmPGRMC1 highlights the importance of transregulation as a conserved adaptive mechanism that has succeeded for arthropod parasites, making it a target for tick control.
Topics: Animals; Rhipicephalus; Receptors, Progesterone; Progesterone; Cattle; Molecular Docking Simulation; Host-Parasite Interactions; Female; Amino Acid Sequence; Protein Binding; Phylogeny
PubMed: 38940045
DOI: 10.31083/j.fbl2906238 -
Frontiers in Bioscience (Landmark... Jun 2024L-Theanine, a nonproteinogenic amino acid derived from green tea, is being recognized as an anti-cancer candidate. However, it's roles in the development of cancer...
BACKGROUND
L-Theanine, a nonproteinogenic amino acid derived from green tea, is being recognized as an anti-cancer candidate. However, it's roles in the development of cancer chemoresistance is still unknown and the molecular mechanism is urgently to be explored.
METHODS
The effects of L-Theanine on lung cancer chemoresistance were validated by Cell Counting Kit-8 (CCK-8) assay, transwell assay, and tumor spheroid formation assay; the expression of proteins was detected by using polymerase chain reaction (PCR) and western blotting. RNA-sequencing (RNA-seq) and bioinformatics analysis were used to identify differentially expressed genes induced by L-Theanine. knockdown and overexpression were constructed by using a lentivirus-mediated transfection system.
RESULTS
L-Theanine improved the chemoresistance to -diamminedichloroplatinum (DDP) and inhibited stemness of DDP-resistant lung cancer cells but not non-resistant lung cancer cells. The results from RNA-seq analysis showed that STAT3/NOTCH1 pathway was a potential dominant signaling involved in L-Theanine improving the chemoresistance in DDP-resistant lung cancer. Mechanistically, L-Theanine impeded migration and stemness activation of DDP-resistant lung cancer cells via regulating the expression of STAT3/NOTCH1/BMAL1 signaling-induced stemness markers as well as inhibiting the expression levels of drug resistance-related genes. In addition, a combination of L-Theanine and Stat3 blockade synergistically improved the chemoresistance in DDP-resistant lung cancer.
CONCLUSION
L-Theanine improves the chemoresistance by regulating STAT3/NOTCH1/BMAL1 signaling, reducing stemness, and inhibiting the migration of DDP-resistant lung cancer cells. The finding might provide some evidence for therapeutic options in overcoming the chemoresistance in cancers, including lung cancer.
Topics: Humans; Glutamates; Drug Resistance, Neoplasm; Lung Neoplasms; Cisplatin; STAT3 Transcription Factor; Signal Transduction; Receptor, Notch1; Cell Line, Tumor; ARNTL Transcription Factors; Antineoplastic Agents; Gene Expression Regulation, Neoplastic; A549 Cells; Cell Movement
PubMed: 38940036
DOI: 10.31083/j.fbl2906226