Comprehensive genetic analysis of facioscapulohumeral muscular dystrophy by Nanopore long-read whole-genome sequencing.

BACKGROUND: Facioscapulohumeral muscular dystrophy (FSHD) is a high-prevalence autosomal dominant neuromuscular disease characterized by significant clinical and genetic heterogeneity. Genetic diagnosis of FSHD remains a challenge because it cannot be detected by standard sequencing methods and requires a complex diagnosis workflow. METHODS: We developed a comprehensive genetic FSHD detection method based on Oxford Nanopore Technologies (ONT) whole-genome sequencing. Using a case-control design, we applied this procedure to 29 samples and compared the results with those from optical genome mapping (OGM), bisulfite sequencing (BSS), and whole-exome sequencing (WES). RESULTS: Using our ONT-based method, we identified 59 haplotypes (35 4qA and 24 4qB) among the 29 samples (including a mosaic sample), as well as the number of D4Z4 repeat units (RUs). The pathogenetic D4Z4 RU contraction identified by our ONT-based method showed 100% concordance with OGM results. The methylation levels of the most distal D4Z4 RU and the double homeobox 4 gene (DUX4) detected by ONT sequencing are highly consistent with the BSS results and showed excellent diagnostic efficiency. Additionally, our ONT-based method provided an independent methylation profile analysis of two permissive 4qA alleles, reflecting a more accurate scenario than traditional BSS. The ONT-based method detected 17 variations in three FSHD2-related genes from nine samples, showing 100% concordance with WES. CONCLUSIONS: Our ONT-based FSHD detection method is a comprehensive method for identifying pathogenetic D4Z4 RU contractions, methylation level alterations, allele-specific methylation of two 4qA haplotypes, and variations in FSHD2-related genes, which will all greatly improve genetic testing for FSHD.

METTL14 is decreased and regulates m6 A modification of α-synuclein in Parkinson's disease.

N6-methyladenosine (m6A), an emerging modification of messenger RNA, has been implicated in many biological processes. However, its role in Parkinson's disease (PD) remains largely unknown. Here, we investigated the role of m6A modification and its underlying mechanism in PD. First, 86 individuals with PD and 86 healthy controls were recruited from a pilot multicenter cohort. Levels of m6A and its modulators in peripheral blood mononuclear cells of patients with PD and controls were measured using an m6A RNA methylation quantification kit and quantitative real-time PCR. The underlying mechanism of m6A modification in PD was investigated in vitro through RNA immunoprecipitation assay, RNA stability assay, gene silencing or overexpression, western blot, and confocal immunoassay. The results show that mRNA levels of m6A, METTL3, METTL14, and YTHDF2 in patients with PD were significantly lower than in healthy controls, and METTL14 was the main factor involved in abnormal m6A modification. Area under the curve (AUC) analysis suggests METTL14 may provide excellent diagnostic capability for PD, especially when combined with plasma α-synuclein (α-syn). Spearman correlation analysis identified that METTL14 was moderately negatively correlated with plasma α-syn and the motor function of PD. Mechanistic experiments demonstrated that Mettl14 targets and regulates the expression of the α-syn gene using its methylation function. Overexpression of Mettl14 dramatically increased m6 A modification of α-syn mRNA and weakened its stability. Further results suggest that α-syn mRNA was modified by Mettl14 binding of an m6 A motif in the coding region of α-syn mRNA, while the reading protein Ythdf2 was involved in recognizing m6 A-modified α-syn mRNA. Taken together, our results reveal the potential of METTL14 as a novel diagnostic biomarker for PD and identify modification of pathogenic α-syn protein by METTL14 via an m6 A-YTHDF2-dependent mechanism.

Whole genome sequencing vs chromosomal microarray analysis in prenatal diagnosis.

BACKGROUND: Emerging studies suggest that whole genome sequencing provides additional diagnostic yield of genomic variants when compared with chromosomal microarray analysis in the etiologic diagnosis of infants and children with suspected genetic diseases. However, the application and evaluation of whole genome sequencing in prenatal diagnosis remain limited. OBJECTIVE: This study aimed to evaluate the accuracy, efficacy, and incremental yield of whole genome sequencing in comparison with chromosomal microarray analysis for routine prenatal diagnosis. STUDY DESIGN: In this prospective study, a total of 185 unselected singleton fetuses with ultrasound-detected structural anomalies were enrolled. In parallel, each sample was subjected to whole genome sequencing and chromosomal microarray analysis. Aneuploidies and copy number variations were detected and analyzed in a blinded fashion. Single nucleotide variations and insertions and deletions were confirmed by Sanger sequencing, and trinucleotide repeats expansion variants were verified using polymerase chain reaction plus fragment-length analysis. RESULTS: Overall, genetic diagnoses using whole genome sequencing were obtained for 28 (15.1%) cases. Whole genome sequencing not only detected all these aneuploidies and copy number variations in the 20 (10.8%) diagnosed cases identified by chromosomal microarray analysis, but also detected 1 case with an exonic deletion of COL4A2 and 7 (3.8%) cases with single nucleotide variations or insertions and deletions. In addition, 3 incidental findings were detected including an expansion of the trinucleotide repeat in ATXN3, a splice-sites variant in ATRX, and an ANXA11 missense mutation in a case of trisomy 21. CONCLUSION: Compared with chromosomal microarray analysis, whole genome sequencing increased the additional detection rate by 5.9% (11/185). Using whole genome sequencing, we detected not only aneuploidies and copy number variations, but also single nucleotide variations and insertions and deletions, trinucleotide repeat expansions, and exonic copy number variations with high accuracy in an acceptable turnaround time (3-4 weeks). Our results suggest that whole genome sequencing has the potential to be a new promising prenatal diagnostic test for fetal structural anomalies.

Optical genome mapping for detection of chromosomal aberrations in prenatal diagnosis.

INTRODUCTION: Chromosomal aberrations are the most important etiological factors for birth defects. Optical genome mapping is a novel cytogenetic tool for detecting a broad range of chromosomal aberrations in a single assay, but relevant clinical feasibility studies of optical genome mapping in prenatal diagnosis are limited. MATERIAL AND METHODS: We retrospectively performed optical genome mapping analysis of amniotic fluid samples from 34 fetuses with various clinical indications and chromosomal aberrations detected through standard-of-care technologies, including karyotyping, fluorescence in situ hybridization, and/or chromosomal microarray analysis. RESULTS: In total, we analyzed 46 chromosomal aberrations from 34 amniotic fluid samples, including 5 aneuploidies, 10 large copy number variations, 27 microdeletions/microduplications, 2 translocations, 1 isochromosome, and 1 region of homozygosity. Overall, 45 chromosomal aberrations could be confirmed by our customized analysis strategy. Optical genome mapping reached 97.8% concordant clinical diagnosis with standard-of-care methods for all chromosomal aberrations in a blinded fashion. Compared with the widely used chromosomal microarray analysis, optical genome mapping additionally determined the relative orientation and position of repetitive segments for seven cases with duplications or triplications. The additional information provided by optical genome mapping will be conducive to characterizing complex chromosomal rearrangements and allowing us to propose mechanisms to explain rearrangements and predict the genetic recurrence risk. CONCLUSIONS: Our study highlights that optical genome mapping can provide comprehensive and accurate information on chromosomal aberrations in a single test, suggesting that optical genome mapping has the potential to become a promising cytogenetic tool for prenatal diagnosis.

Comparative Biomechanical Analysis of Anterior Process Locking Plate Alone versus Combined with Percutaneous Cannulated Screw Fixation for Sanders Type II Calcaneal Fractures: A Finite Element Study.

BACKGROUND Calcaneal fractures are the most common tarsal bone fractures, and account for 75% of intra-articular fractures. The purpose of this study was to compare the biomechanical stability of the anterior process locking plate combined with the percutaneous cannulated screw fixation (screw group) versus the anterior process locking plate fixation alone (plate group) for the treatment of Sanders type II calcaneal fractures using finite element analysis to provide a theoretical basis for clinical work. MATERIAL AND METHODS We established a 3D model of Sanders type II calcaneal fracture; assigned material properties to the internal fixation systems; applied loads; set up analysis criteria; analyzed the displacement of the fracture, relative displacement, stress state of bone tissue, and internal fixation; and compared mechanical stability. RESULTS For Sanders type II A, II B, and II C calcaneal fractures, the degree of displacement and relative displacement of the fracture in the screw group was less than that of the plate group. For all subtypes of Sanders type II calcaneal fractures, the screw group had better mechanical stability than the plate group. CONCLUSIONS Both fixation methods (screw and plate group) were within a reasonable range for restoring the levelling effect of the joint surface and maintaining the strength of fixation, and both had good mechanical stability. Finite element analysis is a relatively reliable method, and biomechanics and clinical studies must further verify the experimental results.

A Novel Splice Site Mutation in the FBN2 Gene in a Chinese Family with Congenital Contractural Arachnodactyly.

Congenital contractural arachnodactyly (CCA) is a rare connective tissue disorder characterized by arachnodactyly, multiple joint contractures, progressive kyphoscoliosis, pectus deformity and abnormal crumpled ears. FBN2 is the only gene currently known to be associated with CCA. In this study, we report on a prenatal case presented with skeletal, cardiac and spinal malformations. And his father had elongated limbs, contractures of the proximal interphalangeal joints, high myopia and scoliosis. We conducted whole exome sequencing (WES) on the fetus-parental trio and a heterozygous variant (hg19 chr5:127,673,685, c.3598 + 4A > G, NM_001999.4) in intron 27 of the FBN2 gene was successfully identified, inherited from the father. Reverse transcriptase-polymerase chain reaction (RT-PCR) was performed to evaluate the potential splicing effect of this variant, which confirmed that the variant caused a deletion of exon 27 (126 bp) by disrupting the splice-donor site and destroyed the 17th calcium-binding epidermal growth factor-like (cbEGF) domain. Our research not only finds the etiology of the disease in affected individuals and expands the mutation spectrum of FBN2 gene, but also provides genetic counseling and fertility guidance for this family.

miR-29a-3p Regulates Autophagy by Targeting Akt3-Mediated mTOR in SiO2-Induced Lung Fibrosis.

Silicosis is a refractory pneumoconiosis of unknown etiology that is characterized by diffuse lung fibrosis, and microRNA (miRNA) dysregulation is connected to silicosis. Emerging evidence suggests that miRNAs modulate pulmonary fibrosis through autophagy; however, its underlying molecular mechanism remains unclear. In agreement with miRNA microarray analysis, the qRT-PCR results showed that miR-29a-3p was significantly decreased in the pulmonary fibrosis model both in vitro and in vivo. Increased autophagosome was observed via transmission electron microscopy in lung epithelial cell models and lung tissue of silicosis mice. The expression of autophagy-related proteins LC3α/ß and Beclin1 were upregulated. The results from using 3-methyladenine, an autophagy inhibitor, or rapamycin, an autophagy inducer, together with TGF-ß1, indicated that autophagy attenuates fibrosis by protecting lung epithelial cells. In TGF-ß1-treated TC-1 cells, transfection with miR-29a-3p mimics activated protective autophagy and reduced alpha-smooth muscle actin and collagen I expression. miRNA TargetScan predicted, and dual-luciferase reporter experiments identified Akt3 as a direct target of miR-29a-3p. Furthermore, Akt3 expression was significantly elevated in the silicosis mouse model and TGF-ß1-treated TC-1 cells. The mammalian target of rapamycin (mTOR) is a central regulator of the autophagy process. Silencing Akt3 inhibited the transduction of the mTOR signaling pathway and activated autophagy in TGF-ß1-treated TC-1 cells. These results show that miR-29a-3p overexpression can partially reverse the fibrotic effects by activating autophagy of the pulmonary epithelial cells regulated by the Akt3/mTOR pathway. Therefore, targeting miR-29a-3p may provide a new therapeutic strategy for silica-induced pulmonary fibrosis.

Axenfeld-Rieger syndrome-associated mutants of the transcription factor FOXC1 abnormally regulate NKX2-5 in model zebrafish embryos.

FOXC1 is a member of the forkhead family of transcription factors, and whose function is poorly understood. A variety of FOXC1 mutants have been identified in patients diagnosed with the autosomal dominant disease Axenfeld-Rieger syndrome, which is mainly characterized by abnormal development of the eyes, particularly those who also have accompanying congenital heart defects (CHD). However, the role of FOXC1 in CHD, and how these mutations might impact FOXC1 function, remains elusive. Our previous work provided one clue to possible function, demonstrating that zebrafish foxc1a, an orthologue of human FOXC1 essential for heart development, directly regulates the expression of nkx2.5, encoding a transcriptional regulator of cardiac progenitor cells. Abnormal expression of Nkx2-5 leads to CHD in mice and is also associated with CHD patients. Whether this link extends to the human system, however, requires investigation. In this study, we demonstrate that FOXC1 does regulate human NKX2-5 expression in a dose-dependent manner via direct binding to its proximal promoter. A comparison of FOXC1 mutant function in the rat cardiac cell line H9c2 and zebrafish embryos suggested that the zebrafish embryos might serve as a more representative model system than the H9c2 cells. Finally, we noted that three of the Axenfeld-Rieger syndrome FOXC1 mutations tested increased, whereas a fourth repressed the expression of NKX2-5 These results imply that mutant FOXC1s might play etiological roles in CHD by abnormally regulating NKX2-5 in the patients. And zebrafish embryos can serve as a useful in vivo platform for rapidly evaluating disease-causing roles of mutated genes.

The dorsal and the ventral side of hypoglossal motor nucleus showed different response to chronic intermittent hypoxia in rats.

PURPOSE: To study neurochemical reactions to chronic intermittent hypoxia (CIH) in the hypoglossal nucleus (HN) of rats. METHODS: Adult male Sprague-Dawley rats (n = 12) were randomly divided into two groups (the CIH and the control group). The CIH rats were housed in a hypoxic chamber with the fraction of oxygen volume alternating between 21% and 5% by providing air for 60 s and then providing nitrogen for 60 s from 8:30 am to 16:30 pm each day for 35 days. The control group was housed in a cabin with normal oxygen levels. We studied the expression of c-fos protein, 5-hydroxytryptamine (5-HT) positive terminals, and its 2A receptors in hypoglossal nuclei by immunohistochemistry. RESULTS: The expression of c-fos, 5-HT positive terminals, and accordingly 5-HT 2A receptors in the CIH group were significantly higher than that in the controls (p < 0.05). The ventral side of the HN showed a clearly higher expression of 5-HT and its 2A receptors than the dorsal side (p < 0.05). CONCLUSION: There were 2 responses of the HN to CIH. First, CIH induced a higher expression of 5-HT positive terminals and its 2A receptors, and second, this reaction was much more evident in ventral side than in the dorsal side. We postulate that these responses may serve to be a protective and compensatory mechanism for CIH.

The transcription factor Foxc1a in zebrafish directly regulates expression of nkx2.5, encoding a transcriptional regulator of cardiac progenitor cells.

Cardiogenesis is a tightly controlled biological process required for formation of a functional heart. The transcription factor Foxc1 not only plays a crucial role in outflow tract development in mice, but is also involved in cardiac structure formation and normal function in humans. However, the molecular mechanisms by which Foxc1 controls cardiac development remain poorly understood. Previously, we reported that zebrafish embryos deficient in foxc1a, an ortholog of mammalian Foxc1, display pericardial edemas and die 9-10 days postfertilization. To further investigate Foxc1a's role in zebrafish cardiogenesis and identify its downstream target genes during early heart development, we comprehensively analyzed the cardiovascular phenotype of foxc1a-null zebrafish embryos. Our results confirmed that foxc1a-null mutants exhibit disrupted cardiac morphology, structure, and function. Performing transcriptome analysis on the foxc1a mutants, we found that the expression of the cardiac progenitor marker gene nkx2.5 was significantly decreased, but the expression of germ layer-patterning genes was unaffected. Dual-fluorescence in situ hybridization assays revealed that foxc1a and nkx2.5 are co-expressed in the anterior lateral plate mesoderm at the somite stage. Chromatin immunoprecipitation and promoter truncation assays disclosed that Foxc1a regulates nkx2.5 expression via direct binding to two noncanonical binding sites in the proximal nkx2.5 promoter. Moreover, functional rescue experiments revealed that developmental stage-specific nkx2.5 overexpression partially rescues the cardiac defects of the foxc1a-null embryos. Taken together, our results indicate that during zebrafish cardiogenesis, Foxc1a is active directly upstream of nkx2.5.

ß-Asarone Regulates ER Stress and Autophagy Via Inhibition of the PERK/CHOP/Bcl-2/Beclin-1 Pathway in 6-OHDA-Induced Parkinsonian Rats.

ß-Asarone (1,2,4-trimethoxy-5-[(Z)-prop-1-enyl]benzene) is an essential component of Acorus tatarinowii Schott volatile oil. Previous research has observed that ß-asarone effectively attenuated symptoms in parkinsonian rats and improved their performance, but the mechanism of this effect remains unclear. Other research has shown that endoplasmic reticulum (ER) stress plays an important role in the pathogenesis of Parkinson's disease (PD). The protein kinase RNA-like endoplasmic reticulum kinase (PERK) was observed in the nigrostriatal dopaminergic neurons of patients with PD. However, our group observed that ER stress and autophagy occurred in 6-hydroxy dopamine (6-OHDA)-induced parkinsonian rats, and ER stress might induce autophagy. We assume that the protective role of ß-asarone in parkinsonian rats is mediated via the ER stress-autophagy pathway. To support this hypothesis, we investigated the expressions of glucose regulated protein 78 (GRP78), PERK phosphorylation (p-PERK), C/EBP homologous binding protein (CHOP), Bcl-2 and Beclin-1 in 6-OHDA-induced parkinsonian rats after ß-asarone treatment. The results showed that the ß-asarone group and PERK inhibitor group had lower levels of GRP78, p-PERK, CHOP and Beclin-1 while having higher levels of Bcl-2. We deduced that ß-asarone might regulate the ER stress-autophagy via inhibition of the PERK/CHOP/Bcl-2/Beclin-1 pathway in 6-OHDA-induced parkinsonian rats.

Zebrafish Znfl1 proteins control the expression of hoxb1b gene in the posterior neuroectoderm by acting upstream of pou5f3 and sall4 genes.

Transcription factors play crucial roles in patterning posterior neuroectoderm. Previously, zinc finger transcription factor znfl1 was reported to be expressed in the posterior neuroectoderm of zebrafish embryos. However, its roles remain unknown. Here, we report that there are 13 copies of znfl1 in the zebrafish genome, and all the paralogues share highly identical protein sequences and cDNA sequences. When znfl1s are knocked down using a morpholino to inhibit their translation or dCas9-Eve to inhibit their transcription, the zebrafish gastrula displays reduced expression of hoxb1b, the marker gene for the posterior neuroectoderm. Further analyses reveal that diminishing znfl1s produces the decreased expressions of pou5f3, whereas overexpression of pou5f3 effectively rescues the reduced expression of hoxb1b in the posterior neuroectoderm. Additionally, knocking down znfl1s causes the reduced expression of sall4, a direct regulator of pou5f3, in the posterior neuroectoderm, and overexpression of sall4 rescues the expression of pou5f3 in the knockdown embryos. In contrast, knocking down either pou5f3 or sall4 does not affect the expressions of znfl1s Taken together, our results demonstrate that zebrafish znfl1s control the expression of hoxb1b in the posterior neuroectoderm by acting upstream of pou5f3 and sall4.

ß-asarone inhibited cell growth and promoted autophagy via P53/Bcl-2/Bclin-1 and P53/AMPK/mTOR pathways in Human Glioma U251 cells.

Glioma is the most common type of primary brain tumor and has an undesirable prognosis. Autophagy plays an important role in cancer therapy, but it is effect is still not definite. P53 is an important tumor suppressor gene and protein that is closely to autophagy. Our aim was to study the effect of ß-asarone on inhibiting cell proliferation in human glioma U251 cells and to detect the effect of the inhibition on autophagy through the P53 signal pathway. For cell growth, the cells were divided into four groups: the model, ß-asarone, temozolomide (TMZ), and co-administration groups. For cell autoghapy and the P53 pathway, the cells were divided into six groups: the model, ß-asarone, 3MA, Rapa, Pifithrin-µ, and NSC groups. The counting Kit-8 assay and flow cytometry (FCM) were then used to measure the cell proliferation and cycle. Electron microscopy was used to observe autophagosome formation. Cell immunohistochemistry/-immunofluorescence, FCM and Western blot (WB) were used to examine the expression of Beclin-1 and P53. The levels of P53 and GAPDH mRNA were detected by RT-PCR. Using WB, we determined autophagy-related proteins Beclin-1, LC3-II/I, and P62 and those of the P53 pathway-related proteins P53, Bcl-2, mTOR, P-mTOR, AMPK, P-AMPK, and GAPDH. We got the results that ß-asarone changed the cellular morphology, inhibited cell proliferation, and enhanced the expression of P53, LC3-II/I, Beclin-1, AMPK, and pAMPK while inhibiting the expression of P62, Bcl-2, mTOR, and pmTOR. All the data suggested that ß-asarone could reduce the cell proliferation and promote autophagy possible via the P53 pathway in U251 cells.

Zebrafish foxc1a plays a crucial role in early somitogenesis by restricting the expression of aldh1a2 directly.

Foxc1a is a member of the forkhead transcription factors. It plays an essential role in zebrafish somitogenesis. However, little is known about the molecular mechanisms underlying its controlling somitogenesis. To uncover how foxc1a regulates zebrafish somitogenesis, we generated foxc1a knock-out zebrafish using TALEN (transcription activator-like effector nuclease) technology. The foxc1a null embryos exhibited defective somites at early development. Analyses on the expressions of the key genes that control processes of somitogenesis revealed that foxc1a controlled early somitogenesis by regulating the expression of myod1. In the somites of foxc1a knock-out embryos, expressions of fgf8a and deltaC were abolished, whereas the expression of aldh1a2 (responsible for providing retinoic acid signaling) was significantly increased. Once the increased retinoic acid level in the foxc1a null embryos was reduced by knocking down aldh1a2, the reduced expression of myod1 was partially rescued by resuming expressions of fgf8a and deltaC in the somites of the mutant embryos. Moreover, a chromatin immunoprecipitation assay on zebrafish embryos revealed that Foxc1a bound aldh1a2 promoter directly. On the other hand, neither knocking down fgf8a nor inhibiting Notch signaling affected the expression of aldh1a2, although knocking down fgf8a reduced expression of deltaC in the somites of zebrafish embryos at early somitogenesis and vice versa. Taken together, our results demonstrate that foxc1a plays an essential role in early somitogenesis by controlling Fgf and Notch signaling through restricting the expression of aldh1a2 in paraxial mesoderm directly.

ß-Asarone Inhibits IRE1/XBP1 Endoplasmic Reticulum Stress Pathway in 6-OHDA-Induced Parkinsonian Rats.

Parkinson's disease (PD) is a neurodegenerative disease, with genetics and environment contributing to the disease onset. The limited pathological cognize of the disease restrained the approaches to improve the clinical treatment. Recently, studies showed that endoplasmic reticulum (ER) stress played an important role in the pathogenesis of PD. There was a neuroprotective effect partly mediated by modulating ER stress. ß-Asarone is the essential constituent of Acorus tatarinowii Schott volatile oil. Our team observed that ß-asarone could improve the behavior of parkinsonian rats; increase the HVA, Dopacl, and 5-HIAA levels; and reduce α-synuclein levels. Here we assumed that the protective role of ß-asarone on parkinsonian rats was mediated via ER stress pathway. To prove the hypothesis we investigated the mRNA levels of glucose regulated protein 78 (GRP78) and C/EBP homologous binding protein (CHOP) in 6-hydroxy dopamine (6-OHDA) induced parkinsonian rats after ß-asarone treatment. Furthermore, the inositol-requiring enzyme 1/X-Box Binding Protein 1 (IRE1/XBP1) ER stress pathway was also studied. The results showed that ß-asarone inhibited the mRNA levels of GRP78 and CHOP, accompanied with the delined expressions of phosphorylated IER1 (p-IRE1) and XBP1. We deduced that ß-asarone might have a protective effect on the 6-OHDA induced parkinsonian rats via IRE1/XBP1 Pathway. Collectively, all data indicated that ß-asarone might be a potential candidate of medicine for clinical therapy of PD.

ß-asarone inhibits autophagy by activating the PI3K/Akt/mTOR pathway in a rat model of depression in Parkinson's disease.

OBJECTIVE: It is unclear whether ß-asarone has a good antidepressant effect and what is the main mechanism in Depression in Parkinson's disease (DPD) model rats. METHODS: In this study, DPD model rats were screened from 6-OHDA induced rats by sucrose preference test (SPT) and forced swimming test (FST). DPD model rats were divided into eight groups: model group, pramipexole group, ß-asarone low-dose group (ß-asarone 7.5 group), ß-asarone medium-dose group (ß-asarone 15 group), ß-asarone high-dose group (ß-asarone 30 group), 3-MA group, rapamycin group, and PI3K inhibitor group. 28 days after the end of treatment, open field test (OFT), SPT and FST were conducted in rats. The level of α-synuclein (α-syn) in the striatum was determined by enzyme-linked immunosorbent assay (ELISA). The expression of Beclin-1, p62 in the striatum was determined by western blot. The expression of PI3K, p-PI3K, Akt, p-Akt, mTOR, p-mTOR, Beclin-1, and p62 in the hippocampus was determined by western blot. The spine density of neurons in the hippocampus was detected by golgi staining. RESULTS: The results showed that 4-week oral administration of ß-asarone improve the motor and depressive symptoms of DPD model rats, and decrease the content of α-syn in the striatum. ß-asarone inhibited the expression of autophagy in the striatum of DPD model rats. Furthermore, ß-asarone decreased the levels of Beclin-1 protein, increased the expression of p62, p-PI3K, p-AKT, and p-mTOR, and improved the density of neuron dendritic spine in the hippocampus. CONCLUSIONS: We concluded that ß-asarone might improve the behavior of DPD model rats by activating the PI3K/Akt/mTOR pathway, inhibiting autophagy and protecting neuron.

Effects of intermittent overload doses of oral vitamin D3 on serum 25(OH)D concentrations and the incidence rates of fractures, falls, and mortality in elderly individuals: A systematic review and meta-analysis.

Vitamin D is commonly used to prevent and treat osteoporosis, with studies indicating its potential to reduce fractures, falls, and mortality. However, meta-analyses present inconsistent findings regarding its efficacy, particularly reflecting significant variability in data and outcomes related to various dosing regimens. In this meta-analysis, we assessed the impact of high-dose intermittent oral administration of vitamin D3 on serum 25(OH)D levels, fractures, falls, and mortality among elderly individuals. We included 14 randomized controlled trials (RCTs) and employed Review Manager 5.4 for statistical analysis. Our findings indicate that intermittent monthly administration of vitamin D3 (over 800 IU per day) significantly raised serum 25(OH)D levels at all timepoints after six months, maintaining levels above 75 nmol/L throughout the year. This regimen showed no increase in all-cause mortality, with a risk ratio (95% CI) of 0.95 (0.87-1.04). Likewise, it did not significantly reduce the risks of falls and fractures, with risk ratios of 1.02 (0.98-1.05) and 0.95 (0.87-1.04) respectively. Although one-year intermittent administration significantly increased the concentration of 25(OH)D in serum, further research is needed to determine if this method would increase the incidence of falls. Therefore, it is not recommended at this stage due to the lack of demonstrated safety in additional relevant RCTs. This study had been registered on PROSPERO (CRD42022363229).

The potential protective role of Parkinson's disease against hypothyroidism: co-localisation and bidirectional Mendelian randomization study.

Background: The association between hypothyroidism and Parkinson's disease (PD) has sparked intense debate in the medical community due to conflicting study results. A better understanding of this association is crucial because of its potential implications for both pathogenesis and treatment strategies. Methods: To elucidate this complex relationship, we used Bayesian co-localisation (COLOC) and bidirectional Mendelian randomization (MR) analysis. COLOC was first used to determine whether hypothyroidism and PD share a common genetic basis. Subsequently, genetic variants served as instrumental variables in a bidirectional MR to explore causal interactions between these conditions. Results: COLOC analysis revealed no shared genetic variants between hypothyroidism and PD, with a posteriori probability of hypothesis 4 (PPH4) = 0.025. Furthermore, MR analysis indicated that hypothyroidism does not have a substantial causal effect on PD (OR = 0.990, 95% CI = 0.925, 1.060, p = 0.774). Conversely, PD appears to have a negative causal effect on hypothyroidism (OR = 0.776, 95% CI = 0.649, 0.928, p = 0.005). Conclusion: Our findings suggest the absence of shared genetic variants between hypothyroidism and PD. Interestingly, PD may inversely influence the risk of developing hypothyroidism, a finding that may inform future research and clinical approaches.