Role of FMRP in AKT/mTOR pathway-mediated hippocampal autophagy in fragile X syndrome.

Fragile X syndrome (FXS) is caused by epigenetic silencing of the Fmr1 gene, leading to the deletion of the coding protein FMRP. FXS induces abnormal hippocampal autophagy and mTOR overactivation. However, it remains unclear whether FMRP regulates hippocampal autophagy through the AKT/mTOR pathway, which influences the neural behavior of FXS. Our study revealed that FMRP deficiency increased the protein levels of p-ULK-1 and p62 and decreased LC3II/LC3I(LC3-II/I) level in Fmr1 knockout (KO) mice. The mouse hippocampal neuronal cell line HT22 with knockdown of Fmr1 by lentivirus showed that the protein levels of p-ULK-1 and p62 were increased, whereas LC3II/LC3I was unchanged. Further observations revealed that FMRP deficiency obstructed autophagic flow in HT22 cells. Therefore, FMRP deficiency inhibited autophagy in the mouse hippocampus and HT22 cells. Moreover, FMRP deficiency increased reactive oxygen species (ROS) level, decreased the co-localization between the mitochondrial outer membrane proteins TOM20 and LC3 in HT22 cells, and caused a decrease in the mitochondrial autophagy protein PINK1 in HT22 cells and Fmr1 KO mice, indicating that FMRP deficiency caused mitochondrial autophagy disorder in HT22 cells and Fmr1 KO mice. To explore the mechanism by which FMRP deficiency inhibits autophagy, we examined the AKT/mTOR signaling pathway in the hippocampus of Fmr1 KO mice, found that FMRP deficiency caused overactivation of the AKT/mTOR pathway. Rapamycin-mediated mTOR inhibition activated and enhanced mitochondrial autophagy. Finally, we examined whether rapamycin affected the neurobehavior of Fmr1 KO mice. The Fmr1 KO mice exhibited stereotypical behavior, impaired social ability, and learning and memory impairment, while rapamycin treatment improved behavioral disorders in Fmr1 KO mice. Thus, our study revealed the molecular mechanism by which FMRP regulates autophagy function, clarifying the role of hippocampal neuron mitochondrial autophagy in the pathogenesis of FXS, and providing novel insights into potential therapeutic targets of FXS.

The alteration of the structure and macroscopic mechanical response of porcine patellar tendon by elastase digestion.

Background: The treatment of patellar tendon injury has always been an unsolved problem, and mechanical characterization is very important for its repair and reconstruction. Elastin is a contributor to mechanics, but it is not clear how it affects the elasticity, viscoelastic properties, and structure of patellar tendon. Methods: The patellar tendons from six fresh adult experimental pigs were used in this study and they were made into 77 samples. The patellar tendon was specifically degraded by elastase, and the regional mechanical response and structural changes were investigated by: (1) Based on the previous study of elastase treatment conditions, the biochemical quantification of collagen, glycosaminoglycan and total protein was carried out; (2) The patellar tendon was divided into the proximal, central, and distal regions, and then the axial tensile test and stress relaxation test were performed before and after phosphate-buffered saline (PBS) or elastase treatment; (3) The dynamic constitutive model was established by the obtained mechanical data; (4) The structural relationship between elastin and collagen fibers was analyzed by two-photon microscopy and histology. Results: There was no statistical difference in mechanics between patellar tendon regions. Compared with those before elastase treatment, the low tensile modulus decreased by 75%-80%, the high tensile modulus decreased by 38%-47%, and the transition strain was prolonged after treatment. For viscoelastic behavior, the stress relaxation increased, the initial slope increased by 55%, the saturation slope increased by 44%, and the transition time increased by 25% after enzyme treatment. Elastin degradation made the collagen fibers of patellar tendon become disordered and looser, and the fiber wavelength increased significantly. Conclusion: The results of this study show that elastin plays an important role in the mechanical properties and fiber structure stability of patellar tendon, which supplements the structure-function relationship information of patellar tendon. The established constitutive model is of great significance to the prediction, repair and replacement of patellar tendon injury. In addition, human patellar tendon has a higher elastin content, so the results of this study can provide supporting information on the natural properties of tendon elastin degradation and guide the development of artificial patellar tendon biomaterials.

Identification of seven variants in the col4a1 gene that alter RNA splicing by minigene assay.

Type IV collagen is an integral component of basement membranes. Mutations in COL4A1, one of the key genes encoding Type IV collagen, can result in a variety of diseases. It is clear that a significant proportion of mutations that affect splicing can cause disease directly or contribute to the susceptibility or severity of disease. Here, we analyzed exonic mutations and intronic mutations described in the COL4A1 gene using bioinformatics programs and identified candidate mutations that may alter the normal splicing pattern through a minigene system. We identified seven variants that induce splicing alterations by disrupting normal splice sites, creating new ones, or altering splice regulatory elements. These mutations are predicted to impact protein function. Our results help in the correct molecular characterization of variants in COL4A1 and may help develop more personalized treatment options.

Androgen levels in autism spectrum disorders: a systematic review and meta-analysis.

Background: Accumulating evidence suggests that the autism spectrum disorder (ASD) population exhibits altered hormone levels, including androgens. However, studies on the regulation of androgens, such as testosterone and dehydroepiandrosterone (DHEA), in relation to sex differences in individuals with ASD are limited and inconsistent. We conducted the systematic review with meta-analysis to quantitatively summarise the blood, urine, or saliva androgen data between individuals with ASD and controls. Methods: A systematic search was conducted for eligible studies published before 16 January 2023 in six international and two Chinese databases. We computed summary statistics with a random-effects model. Publication bias was assessed using funnel plots and heterogeneity using I2 statistics. Subgroup analysis was performed by age, sex, sample source, and measurement method to explain the heterogeneity. Results: 17 case-control studies (individuals with ASD, 825; controls, 669) were assessed. Androgen levels were significantly higher in individuals with ASD than that in controls (SMD: 0.27, 95% CI: 0.06-0.48, P=0.01). Subgroup analysis showed significantly elevated levels of urinary total testosterone, urinary DHEA, and free testosterone in individuals with ASD. DHEA level was also significantly elevated in males with ASD. Conclusion: Androgen levels, especially free testosterone, may be elevated in individuals with ASD and DHEA levels may be specifically elevated in males.

Coordination-Driven Self-Assembly of Metal Ion-Antisense Oligonucleotide Nanohybrids for Chronic Bacterial Infection Therapy.

Bacterial infection poses a significant challenge to wound healing and skin regeneration, leading to substantial economic burdens on patients and society. Therefore, it is crucial to promptly explore and develop effective methodologies for bacterial infections. Herein, we propose a novel approach for synthesizing nanostructures based on antisense oligonucleotides (ASOs) through the coordination-driven self-assembly of Zn2+ with ASO molecules. This approach aims to provide effective synergistic therapy for chronic wound infections caused by Staphylococcus aureus (S. aureus). The resulting hybrid nanoparticles successfully preserve the structural integrity and biological functionalities of ASOs, demonstrating excellent ASO encapsulation efficiency and bioaccessibility. In vitro antibacterial experiments reveal that Zn-ASO NPs exhibit antimicrobial properties against Escherichia coli, Staphylococcus aureus, and Bacillus subtilis. This antibacterial ability is attributed to the high concentration of metal zinc ions and the generation of high levels of reactive oxygen species. Additionally, the ftsZ-ASO effectively inhibits the expression of the ftsZ gene, further enhancing the antimicrobial effect. In vivo antibacterial assays demonstrate that the Zn-ASO NPs promote optimal skin wound healing and exhibit favorable biocompatibility against S. aureus infections, resulting in a residual infected area of less than 8%. This combined antibacterial strategy, which integrates antisense gene therapy and metal-coordination-directed self-assembly, not only achieves synergistic and augmented antibacterial outcomes but also expands the horizons of ASO coordination chemistry. Moreover, it addresses the gap in the antimicrobial application of metal-coordination ASO self-assembly, thereby advancing the field of ASO-based therapeutic approaches.

Polarized M2 macrophages induced by glycosylated nano-hydroxyapatites activate bone regeneration in periodontitis therapy.

AIM: To investigate the association between periodontal macrophage polarization states and the alveolar bone levels, and to assess whether glycosylated nano-hydroxyapatites (GHANPs) could improve bone regeneration in periodontitis by inducing macrophage M2 polarization. MATERIALS AND METHODS: The change of macrophage polarization state in inflammatory periodontal tissues (with bone loss) was examined using clinical gingival samples. The relationship between macrophage phenotype and bone level in periodontal bone loss and repair was evaluated using a mouse periodontitis model. The effect of GHANPs on macrophage polarization was assessed by the in vitro model of lipopolysaccharide (LPS)-stimulated inflammation. The polarization-related markers were detected by immunofluorescence staining, real-time polymerase chain reaction and enzyme-linked immunosorbent assay analysis. The therapeutic effect of GHANPs on alveolar bone loss was explored in experimental periodontitis by histological staining and micro-CT analysis. RESULTS: A lower macrophage M2/M1 ratio was observed in periodontitis-affected human gingival tissues. The results of animal experiments demonstrated a positive correlation between a lower Arg-1/iNOS ratio and accelerated alveolar bone loss; also, the proportion of Arg-1-positive macrophages increased during bone repair and regeneration. The administration of GHANPs partially restored M2 macrophage polarization after LPS stimulation. GHANPs increased alveolar bone repair and regeneration in experimental periodontitis induced by ligation, potentially related to their macrophage M2 transition regulation. CONCLUSIONS: The findings of this study indicate that the induction of macrophage M2 polarization can be considered a viable approach for enhancing inflammatory bone repair. Additionally, GHANPs show potential in the clinical treatment of periodontitis.

Facile preparation of hollow covalent organic frameworks as superior and universal matrix clean-up micro-structures for high throughout determination of food hazards.

The complicated food matrix seriously limits the one-time test for the potential food hazards in non-targeted analysis. Accordingly, developing advanced sample pretreatment strategy to reduce matrix effects is of great significance. Herein, newly-integrated hollow-structured covalent organic frameworks (HCOFs) with large internal adsorption capacity and target-matched pore size were synthesized via etching the core-shell structured COFs. The as-prepared HCOFs could be directly applied for matrix clean-up of vegetable samples, while further modification of polydopamine (PDA) network facilitated application for animal samples. Both HCOFs and HCOFs@PDA with the comparable sizes to the matrix interference gave excellent adsorption performance to targets, achieving satisfied recoveries (70%-120%) toward 90 pesticides and 44 veterinary drugs in one-test, respectively. This work showed the great potential of the facile-integrated HCOFs with high stability and customized size to remove interference matrix and offered a universal strategy to achieve simultaneous screening of hazards with considerable quantity in high-throughput non-targeted analysis.

Targeting starvation therapy for diabetic bacterial infections with endogenous enzyme-triggered hyaluronan-modified nanozymes in the infection microenvironment.

The high-glycemic microenvironment of diabetic wounds promotes bacterial proliferation, leading to persistent infections and delayed wound healing. This poses a significant threat to human health, necessitating the development of new nanodrug visualization platforms. In this study, we designed and synthesized cascade nano-systems modified with targeted peptide and hyaluronic acid for diabetic infection therapy. The nano-systems were able to target the site of infection using LL-37, and in the microenvironment of wound infection, the hyaluronic acid shell of the nano-systems was degraded by endogenous hyaluronidase. This precise degradation released a cascade of nano-enzymes on the surface of the bacteria, effectively destroying their cytoskeleton. Additionally, the metals in the nano-enzymes provided a photo-thermal effect, accelerating wound healing. The cascade nano-visualization platform demonstrated excellent bactericidal efficacy in both in vitro antimicrobial assays and in vivo diabetic infection models. In conclusion, this nano-system employs multiple approaches including targeting, enzyme-catalyzed therapy, photothermal therapy, and chemodynamic therapy to kill bacteria and promote healing. The Ag@Pt-Au-LYZ/HA-LL-37 formulation shows great potential for the treatment of diabetic wounds.

Pyrrole-containing hybrids as potential anticancer agents: An insight into current developments and structure-activity relationships.

Cancer poses a significant threat to human health. Therefore, it is urgent to develop potent anti-cancer drugs with excellent inhibitory activity and no toxic side effects. Pyrrole and its derivatives are privileged heterocyclic compounds with significant diverse pharmacological effects. These compounds can target various aspects of cancer cells and have been applied in clinical settings or are undergoing clinical trials. As a result, pyrrole has emerged as a promising drug scaffold and has been further probed to get novel entities for the treatment of cancer. This article reviews recent research progress on anti-cancer drugs containing pyrrole. It focuses on the mechanism of action, biological activity, and structure-activity relationships of pyrrole derivatives, aiming to assist in designing and synthesizing innovative pyrrole-based anti-cancer compounds.

H6N2 reassortant avian influenza virus isolate in wild birds in Jiangxi Province, China.

H6 avian influenza virus is widely prevalent in wild birds and poultry and has caused human infection in 2013 in Taiwan, China. During our active influenza surveillance program in wild waterfowl at Poyang Lake, Jiangxi Province, an H6N2 AIV was isolated and named A/bean goose/JiangXi/452-4/2013(H6N2). The isolate was characterized as a typical low pathogenic avian influenza virus (LPAIV) due to the presence of the amino acid sequence PQIETR↓GLFGAI at the cleavage site of the hemagglutinin (HA) protein. The genetic evolution analysis revealed that the NA gene of the isolate originated from North America and exhibited the highest nucleotide identity (99.29%) with a virus recovered from wild bird samples in North America, specifically A/bufflehead/California/4935/2012(H11N2). Additionally, while the HA and PB1 genes belonged to the Eurasian lineage, they displayed frequent genetic interactions with the North American lineage. The remaining genes showed close genetic relationships with Eurasian viruses. The H6N2 isolate possessed a complex genome, indicating it is a multi-gene recombinant virus with genetic material from both Eurasian and North American lineages.

A novel heterozygous variant of the SALL1 gene with atypical Townes-Brocks syndrome phenotypes in Chinese family.

Townes-Brocks syndrome (TBS) is an autosomal dominant disorder characterised by the triad of anorectal, thumb, and ear malformations. It may also be accompanied by defects in kidney, heart, eyes, hearing, and feet. TBS has been demonstrated to result from heterozygous variants in the SALL1 gene, which encodes zinc finger protein believed to function as a transcriptional repressor. The clinical characteristics of an atypical TBS phenotype patient from a Chinese family are described, with predominant manifestations including external ear dysplasia, unilateral renal hypoplasia with mild renal dysfunction, and hearing impairment. A novel heterozygous variant c.3060T>A (p.Tyr1020*) in exon 2 of the SALL1 gene was identified in this proband. Pyrosequencing of the complementary DNA of the proband revealed that the variant transcript accounted for 48% of the total transcripts in peripheral leukocytes, indicating that this variant transcript has not undergone nonsense-mediated mRNA decay. This variant c.3060T > A is located at the terminal end of exon 2, proximal to the 3' end of the SALL1 gene, and exerts a relatively minor impact on protein function. We suggest that the atypical TBS phenotype observed in the proband may be attributed to the truncated protein retaining partial SALL1 function.

A nursing report on a corneal contact lens wearer receiving keratoplasty due to corneal ulcer and perforation caused by Pythium insidiosum infection: A case report.

BACKGROUND: To report the nursing experience of a case of corneal contact lens wearer receiving the 2nd keratoplasty due to corneal ulcer and perforation caused by Pythium insidiosum infection. METHODS: A 30-year-old female patient had blurred vision after deep anterior lamellar keratoplasty for a right corneal ulcer. At the 5th week, the right eye appeared the symptoms, such as redness and pain. The anterior segment photography was performed on the eye, and the result showed that the epithelium was missing in the right eye lesion area, and a large number of longitudinal and transversal streaks were visible from the epithelium to the stroma, with fungus filaments to be discharged. Upon macro-genome sequencing of the corneal secretion, a P. insidiosum infection was observed. Then, the patient underwent the keratoplasty, and 3 weeks later, the corneal implant showed a tendency to dissolve, the sutures were partially loosened, and the eye was almost blind. Subsequently, the patient was admitted to our hospital and subject to the 2nd penetrating keratoplasty of the right eye (allograft). After surgery, linezolid and azithromycin injections were given through intravenous drip and local drip of the eye for anti-inflammation, and tacrolimus eye drops for antirejection. RESULTS: Postoperatively, the patient showed signs of recovery with slight corneal edema and visible pupil, leading to discharge with improved vision. The corneal implant was normal 1 week after surgery and the vision of the right eye was hand move/before eye at the 6th month of follow-up. Continuous care and removal of sutures 3 months post-surgery contributed to a successful outcome, with the patient achieving hand motion vision 6 months after the procedure. CONCLUSION: Corneal ulcer caused by P. insidiosum infection not only needs timely and effective keratoplasty intervention, but also requires perfect nursing measures.

Euchromatin histone-lysine N-methyltransferase 2 regulates the expression of potassium-sodium-activated channel subfamily T member 1 in primary sensory neurons and contributes to remifentanil-induced pain sensitivity.

Intraoperative remifentanil administration has been linked to increased postoperative pain sensitivity. Recent studies have identified the involvement of euchromatic histone-lysine N-methyltransferase 2 (Ehmt2/G9a) in neuropathic pain associated with the transcriptional silencing of many potassium ion channel genes. This study investigates whether G9a regulates the potassium sodium-activated channel subfamily T member 1 (Slo2.2) in remifentanil-induced post-incisional hyperalgesia (RIH) in rodents. We performed remifentanil infusion (1 µg·kg-1·min-1 for 60 min) followed by plantar incision to induce RIH in rodents. Our results showed that RIH was accompanied by increased G9a and H3K9me2 production and decreased Slo2.2 expression 48 h postoperatively. Deletion of G9a rescued Slo2.2 expression in DRG and reduced RIH intensity. Slo2.2 overexpression also reversed this hyperalgesia phenotype. G9a overexpression decreased Slo2.2-mediated leak current and increased excitability in the small-diameter DRG neurons and laminal II small-diameter neurons in the spinal dorsal horn, which was implicated in peripheral and central sensitization. These results suggest that G9a contributes to the development of RIH by epigenetically silencing Slo2.2 in DRG neurons, leading to decreased central sensitization in the spinal cord. The findings may have implications for the development of novel therapeutic targets for the treatment of postoperative pain.

Identification of marker genes associated with N6-methyladenosine and autophagy in ulcerative colitis.

BACKGROUND: Both N6-methyladenosine (m6A) methylation and autophagy are considered relevant to the pathogenesis of ulcerative colitis (UC). However, a systematic exploration of the role of the com-bination of m6A methylation and autophagy in UC remains to be performed. AIM: To elucidate the autophagy-related genes of m6A with a diagnostic value for UC. METHODS: The correlation between m6A-related genes and autophagy-related genes (ARGs) was analyzed. Finally, gene set enrichment analysis (GSEA) was performed on the characteristic genes. Additionally, the expression levels of four characteristic genes were verified in dextran sulfate sodium (DSS)-induced colitis in mice. RESULTS: GSEA indicated that BAG3, P4HB and TP53INP2 were involved in the inflammatory response and TNF-α signalling via nuclear factor kappa-B. Furthermore, polymerase chain reaction results showed significantly higher mRNA levels of BAG3 and P4HB and lower mRNA levels of FMR1 and TP53INP2 in the DSS group compared to the control group. CONCLUSION: This study identified four m6A-ARGs that predict the occurrence of UC, thus providing a scientific reference for further studies on the pathogenesis of UC.

EEG rhythm separation and time-frequency analysis of fast multivariate empirical mode decomposition for motor imagery BCI.

Motor imagery electroencephalogram (EEG) is widely employed in brain-computer interface (BCI) systems. As a time-frequency analysis method for nonlinear and non-stationary signals, multivariate empirical mode decomposition (MEMD) and its noise-assisted version (NA-MEMD) has been widely used in the preprocessing step of BCI systems for separating EEG rhythms corresponding to specific brain activities. However, when applied to multichannel EEG signals, MEMD or NA-MEMD often demonstrate low robustness to noise and high computational complexity. To address these issues, we have explored the advantages of our recently proposed fast multivariate empirical mode decomposition (FMEMD) and its noise-assisted version (NA-FMEMD) for analyzing motor imagery data. We emphasize that FMEMD enables a more accurate estimation of EEG frequency information and exhibits a more noise-robust decomposition performance with improved computational efficiency. Comparative analysis with MEMD on simulation data and real-world EEG validates the above assertions. The joint average frequency measure is employed to automatically select intrinsic mode functions that correspond to specific frequency bands. Thus, FMEMD-based classification architecture is proposed. Using FMEMD as a preprocessing algorithm instead of MEMD can improve the classification accuracy by 2.3% on the BCI Competition IV dataset. On the Physiobank Motor/Mental Imagery dataset and BCI Competition IV Dataset 2a, FMEMD-based architecture also attained a comparable performance to complex algorithms. The results indicate that FMEMD proficiently extracts feature information from small benchmark datasets while mitigating dimensionality constraints resulting from computational complexity. Hence, FMEMD or NA-FMEMD can be a powerful time-frequency preprocessing method for BCI.

Calcium signal regulated carbohydrate metabolism in wheat seedlings under salinity stress.

This study aimed to explore the mechanism by which calcium (Ca) signal regulated carbohydrate metabolism and exogenous Ca alleviated salinity toxicity. Wheat seedlings were treated with sodium chloride (NaCl, 150 mM) alone or combined with 500 µM calcium chloride (CaCl2), lanthanum chloride (LaCl3) and/or ethylene glycol tetraacetic acid (EGTA) to primarily analyse carbohydrate starch and sucrose metabolism, as well as Ca signaling components. Treatment with NaCl, EGTA, or LaCl3 alone retarded wheat-seedling growth and decreased starch content accompanied by weakened ribulose-1,5-bisphosphate carboxylation/oxygenase (Rubisco) and Rubisco activase activities, as well as enhanced glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, alpha-amylase, and beta-amylase activities. However, it increased the sucrose level, up-regulated the sucrose phosphate synthase (SPS) and sucrose synthase (SuSy) activities and TaSPS and TaSuSy expression together, but down-regulated the acid invertase (SA-Inv) and alkaline/neutral invertase (A/N-Inv) activities and TaSA-Inv and TaA/N-Inv expression. Except for unchanged A/N-Inv activities and TaA/N-Inv expression, adding CaCl2 effectively blocked the sodium salt-induced changes of these parameters, which was partially eliminated by EGTA or LaCl3 presence. Furthermore, NaCl treatment also significantly inhibited Ca-dependent protein kinases and Ca2+-ATPase activities and their gene expression in wheat leaves, which was effectively relieved by adding CaCl2. Taken together, CaCl2 application effectively alleviated the sodium salt-induced retardation of wheat-seedling growth by enhancing starch anabolism and sucrose catabolism, and intracellular Ca signal regulated the enzyme activities and gene expression of starch and sucrose metabolism in the leaves of sodium salt-stressed wheat seedlings.

Buying a Wealthy Dream: Determinants of Rural Residents' Lottery Purchase Behavior in China.

The expansion of illicit lottery activities has caused significant harm to both agricultural production and the livelihood of rural residents. An analysis of the factors that influence rural residents' participation in underground lotteries can provide crucial insight for regulating the lottery industry's development. This study examines the present state of rural residents' participation in underground lotteries, investigates the factors that impact their participation using the Double-Hurdle model, and further employs the ISM model to evaluate the correlations and hierarchical structure among the factors, using field survey data collected from 603 rural residents in S Province, China. The findings reveal that 53.07% of the respondents have participated in underground lotteries. Risk preference, information acquisition, social networks, age, education, family burden, percentage of agricultural labor, agricultural business scale, and household income significantly affect whether residents purchase lotteries (WPL). All factors, except the percentage of agricultural labor and agricultural business scale, also significantly impact underground lottery spending (LS). Among the significant influencing factors, rural residents' risk preference is the direct surface factor, whereas agricultural business scale, household income, information acquisition, and social networks are the middle indirect factors, and residents' age, education, family burden, and percentage of agricultural labor are the deep-rooted factors. It is recommended to regulate the lottery industry's development can be achieved by enhancing entertainment and cultural activities, expanding economic opportunities, enhancing rural education, increasing law awareness, and improving the public welfare lottery business model. The conclusions offer a valuable reference point for the standardized development of the lottery industry and the promotion of social stability in rural areas.

Regulation and resilience: Panarchy analysis in forest socio-ecosystem of Northeast National Forest Region, China.

This paper examines the socio-ecological resilience within China's Northeast National Forest Region (NNFR), focusing on the implications of climate change for forest management and carbon sequestration. It offers a critical assessment of the Natural Forest Protection Program (NFPP) and the associated logging ban policy, recognizing their pivotal contributions to forest conservation but also identifying the shortcomings of a one-size-fits-all approach. Integrating panarchy theory, the study proposes sustainable management practices that align ecological dynamics with societal needs, emphasizing nature-based solutions. The overarching aim is to bolster the long-term resilience and enhance the carbon sequestration potential of the NNFR's forests. It aims to inform global environmental strategy with lessons from the NNFR, advocating for integrated approaches that ensure both ecological sustainability and community prosperity. This approach seeks to provide a comprehensive and effective strategy for addressing environmental challenges, ensuring both ecological integrity and community well-being.

Three exonic variants in the COL4A5 gene alter RNA splicing in a minigene assay.

BACKGROUND: X-linked Alport syndrome (XLAS) is an inherited renal disease caused by rare variants of COL4A5 on chromosome Xq22. Many studies have indicated that single nucleotide variants (SNVs) in exons can disrupt normal splicing process of the pre-mRNA by altering various splicing regulatory signals. The male patients with XLAS have a strong genotype-phenotype correlation. Confirming the effect of variants on splicing can help to predict kidney prognosis. This study aimed to investigate whether single nucleotide substitutions, located within three bases at the 5' end of the exons or internal position of the exons in COL4A5 gene, cause aberrant splicing process. METHODS: We analyzed 401 SNVs previously presumed missense and nonsense variants in COL4A5 gene by bioinformatics programs and identified candidate variants that may affect the splicing of pre-mRNA via minigene assays. RESULTS: Our study indicated three of eight candidate variants induced complete or partial exon skipping. Variants c.2678G>C and c.2918G>A probably disturb classic splice sites leading to corresponding exon skipping. Variant c.3700C>T may disrupt splicing enhancer motifs accompanying with generation of splicing silencer sequences resulting in the skipping of exon 41. CONCLUSION: Our study revealed that two missense variants positioned the first nucleotides of the 5' end of COL4A5 exons and one internal exonic nonsense variant caused aberrant splicing. Importantly, this study emphasized the necessity of assessing the effects of SNVs at the mRNA level.