Core planar cell polarity genes VANGL1 and VANGL2 in predisposition to congenital vertebral malformations.

Congenital scoliosis (CS), affecting approximately 0.5 to 1 in 1,000 live births, is commonly caused by congenital vertebral malformations (CVMs) arising from aberrant somitogenesis or somite differentiation. While Wnt/ß-catenin signaling has been implicated in somite development, the function of Wnt/planar cell polarity (Wnt/PCP) signaling in this process remains unclear. Here, we investigated the role of Vangl1 and Vangl2 in vertebral development and found that their deletion causes vertebral anomalies resembling human CVMs. Analysis of exome sequencing data from multiethnic CS patients revealed a number of rare and deleterious variants in VANGL1 and VANGL2, many of which exhibited loss-of-function and dominant-negative effects. Zebrafish models confirmed the pathogenicity of these variants. Furthermore, we found that Vangl1 knock-in (p.R258H) mice exhibited vertebral malformations in a Vangl gene dose- and environment-dependent manner. Our findings highlight critical roles for PCP signaling in vertebral development and predisposition to CVMs in CS patients, providing insights into the molecular mechanisms underlying this disorder.

Molecular oxidation-reduction junctions for artificial photosynthetic overall reaction.

Constructing redox semiconductor heterojunction photocatalysts is the most effective and important means to complete the artificial photosynthetic overall reaction (i.e., coupling CO2 photoreduction and water photo-oxidation reactions). However, multiphase hybridization essence and inhomogeneous junction distribution in these catalysts extremely limit the diverse design and regulation of the modes of photogenerated charge separation and transfer pathways, which are crucial factors to improve photocatalytic performance. Here, we develop molecular oxidation-reduction (OR) junctions assembled with oxidative cluster (PMo12, for water oxidation) and reductive cluster (Ni5, for CO2 reduction) in a direct (d-OR), alternant (a-OR), or symmetric (s-OR) manner, respectively, for artificial photosynthesis. Significantly, the transfer direction and path of photogenerated charges between traditional junctions are obviously reformed and enriched in these well-defined crystalline catalysts with monophase periodic distribution and thus improve the separation efficiency of the electrons and holes. In particular, the charge migration in s-OR shows a periodically and continuously opposite mode. It can inhibit the photogenerated charge recombination more effectively and enhance the photocatalytic performance largely when compared with the traditional heterojunction models. Structural analysis and density functional theory calculations disclose that, through adjusting the spatial arrangement of oxidation and reduction clusters, the energy level and population of the orbitals of these OR junctions can be regulated synchronously to further optimize photocatalytic performance. The establishment of molecular OR junctions is a pioneering important discovery for extremely improving the utilization efficiency of photogenerated charges in the artificial photosynthesis overall reaction.

Homochiral Nanopropeller via Chiral Active Surface Growth.

Under the control of chiral ligand glutathione and in the presence of hexadecyltrimethylammonium bromide, Au deposition on Au seeds is known to give chiral nanostructures. We have previously shown that the protruding chiral patterns, as opposed to flat facets, are likely caused by active surface growth, where nonuniform ligand coverage could be responsible for the focused growth at a few active sites. By pushing the limit of such a growth mode, here, we use decahedral seeds to prepare homochiral nanopropellers with intricate patterns of deep valleys and protruding ridges. Control experiments show that the focused growth depends on the rates of Au deposition by changing either the seed concentration or the reductant concentration, consistent with the proposed mechanism. The dynamic growth competition between the ligand-deficient active sites and the ligand-rich surfaces gradually focuses the growth onto a few active sites, causing the expansion of grooves, squeezing of steep ridges, and a surprising 36° rotation of the pentagonal outline. The imbalanced deposition on the prochiral slopes is responsible for the tilted grooves, the twisted walls, and thus the well-separated and distorted blades, which become the origin of the chiroptical responses.

Perturbations of genes essential for Müllerian duct and Wölffian duct development in Mayer-Rokitansky-Küster-Hauser syndrome.

Mayer-Rokitansky-Küster-Hauser syndrome (MRKHS) is associated with congenital absence of the uterus, cervix, and the upper part of the vagina; it is a sex-limited trait. Disrupted development of the Müllerian ducts (MD)/Wölffian ducts (WD) through multifactorial mechanisms has been proposed to underlie MRKHS. In this study, exome sequencing (ES) was performed on a Chinese discovery cohort (442 affected subjects and 941 female control subjects) and a replication MRKHS cohort (150 affected subjects of mixed ethnicity from North America, South America, and Europe). Phenotypic follow-up of the female reproductive system was performed on an additional cohort of PAX8-associated congenital hypothyroidism (CH) (n = 5, Chinese). By analyzing 19 candidate genes essential for MD/WD development, we identified 12 likely gene-disrupting (LGD) variants in 7 genes: PAX8 (n = 4), BMP4 (n = 2), BMP7 (n = 2), TBX6 (n = 1), HOXA10 (n = 1), EMX2 (n = 1), and WNT9B (n = 1), while LGD variants in these genes were not detected in control samples (p = 1.27E-06). Interestingly, a sex-limited penetrance with paternal inheritance was observed in multiple families. One additional PAX8 LGD variant from the replication cohort and two missense variants from both cohorts were revealed to cause loss-of-function of the protein. From the PAX8-associated CH cohort, we identified one individual presenting a syndromic condition characterized by CH and MRKHS (CH-MRKHS). Our study demonstrates the comprehensive utilization of knowledge from developmental biology toward elucidating genetic perturbations, i.e., rare pathogenic alleles involving the same loci, contributing to human birth defects.

Phase 1 study of safety and preliminary efficacy of intranasal transplantation of human neural stem cells (ANGE-S003) in Parkinson's disease.

BACKGROUND: Intranasal transplantation of ANGE-S003 human neural stem cells showed therapeutic effects and were safe in preclinical models of Parkinson's disease (PD). We investigated the safety and tolerability of this treatment in patients with PD and whether these effects would be apparent in a clinical trial. METHODS: This was a 12-month, single-centre, open-label, dose-escalation phase 1 study of 18 patients with advanced PD assigned to four-time intranasal transplantation of 1 of 3 doses: 1.5 million, 5 million or 15 million of ANGE-S003 human neural stem cells to evaluate their safety and efficacy. RESULTS: 7 patients experienced a total of 14 adverse events in the 12 months of follow-up after treatment. There were no serious adverse events related to ANGE-S003. Safety testing disclosed no safety concerns. Brain MRI revealed no mass formation. In 16 patients who had 12-month Movement Disorder Society-Unified Parkinson's Disease Rating Scale (MDS-UPDRS) data, significant improvement of MDS-UPDRS total score was observed at all time points (p<0.001), starting with month 3 and sustained till month 12. The most substantial improvement was seen at month 6 with a mean reduction of 19.9 points (95% CI, 9.6 to 30.3; p<0.001). There was no association between improvement in clinical outcome measures and cell dose levels. CONCLUSIONS: Treatment with ANGE-S003 is feasible, generally safe and well tolerated, associated with functional improvement in clinical outcomes with peak efficacy achieved at month 6. Intranasal transplantation of neural stem cells represents a new avenue for the treatment of PD, and a larger, longer-term, randomised, controlled phase 2 trial is warranted for further investigation.

Conditional loss of Ube3d in the retinal pigment epithelium accelerates age-associated alterations in the retina of mice.

Several studies have suggested a correlation between the ubiquitin-proteasome system (UPS) and age-related macular degeneration (AMD), with its phenotypic severity ranging from mild visual impairment to blindness, but the mechanism for UPS dysfunction contributing to disease progression is unclear. In this study, we investigated the role of ubiquitin protein ligase E3D (UBE3D) in aging and degeneration in mouse retina. Conditional knockout of Ube3d in the retinal pigment epithelium (RPE) of mice led to progressive and irregular fundus lesions, attenuation of the retinal vascular system, and age-associated deterioration of rod and cone responses. Simultaneously, RPE-specific Ube3d knockout mice also presented morphological changes similar to the histopathological characteristics of human AMD, in which a defective UPS led to RPE abnormalities such as phagocytosis or degradation of metabolites, the interaction with photoreceptor outer segment, and the transport of nutrients or waste products with choroidal capillaries via Bruch's membrane. Moreover, conditional loss of Ube3d resulted in aberrant molecular characterizations associated with the autophagy-lysosomal pathway, oxidative stress damage, and cell-cycle regulation, which are implicated in AMD pathology. Thus, our findings strengthen and expand the impact of UPS dysfunction on retinal pathophysiology during aging, indicating that genetic Ube3d deficiency in the RPE could lead to the abnormal formation of pigment deposits and secondary fundus alterations. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Association between long-term exposure to air pollution and diabetic retinopathy: Evidence from the Fujian Eye Study.

BACKGROUND: Diabetic retinopathy (DR), one of the most common microvascular complications of diabetes mellitus (DM), is a major contributor of vision impairment and blindness worldwide. Studies have shown that air pollution exposure is adversely associated with DM. However, evidence is scarce regarding how air pollution exposure affects DR. This study aimed to investigate the association between ambient air pollution exposure and DR risk. METHODS: The study population was based on the Fujian Eye Study (FJES), an ophthalmologic, epidemiologic survey investigating the eye health condition of residents in Fujian Province from 2018 to 2019. Daily average concentrations of ambient air pollutants (PM2.5, PM10, SO2, NO2, and O3) were acquired from a high-resolution air quality dataset in China from 2013 to 2018. We used a logistic regression model to examine the associations between DR risk and long-term air pollution at various exposure windows. RESULTS: A total of 2405 out of the 8211 participants were diagnosed with diabetes, among whom 183 had DR. Ambient air pollution, especially particulate matter (i.e., PM2.5 and PM10) and NO2 were positively associated with DR prevalence among all the study subjects. Ambient SO2 and O3 concentrations were not associated with DR prevalence. PM2.5 and NO2 seemed to be borderline significantly associated with increased prevalence of DR in subjects with DM, especially under the model adjusted for sex, age, BMI, SBP, and DBP. CONCLUSIONS: These findings showed that long-term exposure to ambient particulate matter and NO2 was associated with a high DR risk in Fujian province, where ambient air pollution is relatively low.

A Covalent Organic Nanoribbon: Preparation, Single Crystal Structure with Chinese Luban Lock Configuration, and Photocatalytic Behavior.

The multiple mortise-and-tenon joint parts are the core factors to provide the structural stability and diversity of Chinese Luban locks, however, constructing such structures is very challenging. Herein, single crystals of covalent organic nanoribbon (named CityU-27) are prepared through the assembly of hexahydroxytriphenylene (HHTP), 4,4'-Vinylenedipyridine (BYE), and phenylboronic acid (BA) together via dative boron←nitrogen (B←N) bonds. The single crystal X-ray diffraction analysis indicates that CityU-27 has covalent organic nanoribbon, where each nanoribbon forms multiple and tight π-π interactions with four neighboring others to generate a Luban lock-like configuration. CityU-27 has been demonstrated as an efficient photocatalyst in a one-pot tandem reaction of hydrogen evolution reaction (HER) and semi-hydrogenation reaction of alkynes in series to produce olefins without any additional photosensitizers and co-catalysts (metal-free).

Dissection of mendelian predisposition and complex genetic architecture of craniovertebral junction malformation.

The craniovertebral junction (CVJ) is an anatomically complex region of the axial skeleton that provides protection of the brainstem and the upper cervical spinal cord. Structural malformation of the CVJ gives rise to life-threatening neurological deficits, such as quadriplegia and dyspnea. Unfortunately, genetic studies on human subjects with CVJ malformation are limited and the pathogenesis remains largely elusive. In this study, we recruited 93 individuals with CVJ malformation and performed exome sequencing. Manual interpretation of the data identified three pathogenic variants in genes associated with Mendelian diseases, including CSNK2A1, MSX2, and DDX3X. In addition, the contribution of copy number variations (CNVs) to CVJ malformation was investigated and three pathogenic CNVs were identified in three affected individuals. To further dissect the complex mutational architecture of CVJ malformation, we performed a gene-based rare variant association analysis utilizing 4371 in-house exomes as control. Rare variants in LGI4 (carrier rate = 3.26%, p = 3.3 × 10-5) and BEST1 (carrier rate = 5.43%, p = 5.77 × 10-6) were identified to be associated with CVJ malformation. Furthermore, gene set analyses revealed that extracellular matrix- and RHO GTPase-associated biological pathways were found to be involved in the etiology of CVJ malformation. Overall, we comprehensively dissected the genetic underpinnings of CVJ malformation and identified several novel disease-associated genes and biological pathways.

The impact of artificial intelligence on retinal disease management: Vision Academy retinal expert consensus.

PURPOSE OF REVIEW: The aim of this review is to define the "state-of-the-art" in artificial intelligence (AI)-enabled devices that support the management of retinal conditions and to provide Vision Academy recommendations on the topic. RECENT FINDINGS: Most of the AI models described in the literature have not been approved for disease management purposes by regulatory authorities. These new technologies are promising as they may be able to provide personalized treatments as well as a personalized risk score for various retinal diseases. However, several issues still need to be addressed, such as the lack of a common regulatory pathway and a lack of clarity regarding the applicability of AI-enabled medical devices in different populations. SUMMARY: It is likely that current clinical practice will need to change following the application of AI-enabled medical devices. These devices are likely to have an impact on the management of retinal disease. However, a consensus needs to be reached to ensure they are safe and effective for the overall population.

Current status and practical considerations of artificial intelligence use in screening and diagnosing retinal diseases: Vision Academy retinal expert consensus.

PURPOSE OF REVIEW: The application of artificial intelligence (AI) technologies in screening and diagnosing retinal diseases may play an important role in telemedicine and has potential to shape modern healthcare ecosystems, including within ophthalmology. RECENT FINDINGS: In this article, we examine the latest publications relevant to AI in retinal disease and discuss the currently available algorithms. We summarize four key requirements underlining the successful application of AI algorithms in real-world practice: processing massive data; practicability of an AI model in ophthalmology; policy compliance and the regulatory environment; and balancing profit and cost when developing and maintaining AI models. SUMMARY: The Vision Academy recognizes the advantages and disadvantages of AI-based technologies and gives insightful recommendations for future directions.

Recent advances in targeted delivery of non-coding RNA-based therapeutics for atherosclerosis.

Cardiovascular disease (CVD) has overtaken infectious illnesses as the leading cause of mortality and disability worldwide. The pathology that underpins CVD is atherosclerosis, characterized by chronic inflammation caused by the accumulation of plaques in the arteries. As our knowledge about the microenvironment of blood vessel walls deepens, there is an opportunity to fine-tune treatments to target the mechanisms driving atherosclerosis more directly. The application of non-coding RNAs (ncRNAs) as biomarkers or intervention targets is increasing. Although these ncRNAs play an important role in driving atherosclerosis and vascular dysfunction, the cellular and extracellular environments pose a challenge for targeted transmission and therapeutic regulation of ncRNAs. Specificity, delivery, and tolerance have hampered the clinical translation of ncRNA-based therapeutics. Nanomedicine is an emerging field that uses nanotechnology for targeted drug delivery and advanced imaging. Recently, nanoscale carriers have shown promising results and have introduced new possibilities for nucleic acid targeted drug delivery, particularly for atherosclerosis. In this review, we discuss the latest developments in nanoparticles to aid ncRNA-based drug development, particularly miRNA, and we analyze the current challenges in ncRNA targeted delivery. In particular, we highlight the emergence of various kinds of nanotherapeutic approaches based on ncRNAs, which can improve treatment options for atherosclerosis.

Construction of Prognostic Nomogram in Patients with N3-Stage Nasopharyngeal Carcinoma.

INTRODUCTION: The aim of the study was to retrospectively identify the metastatic influence factors and predict the prognosis and develop an individualized prognostic prediction model for patients with N3-stage nasopharyngeal carcinoma (NPC). METHODS: The study collected 446 NPC patients with N3 stage from the Surveillance, Epidemiology, and End Results database between 2010 and 2015. The patients were classified into subgroups based on the histological types and metastatic status. Multivariable logistic, Cox regression, and Kaplan-Meier method with the log-rank test were performed. The nomogram model was created using the prognostic factors identified from Cox regression analysis. The predictive accuracy was determined based on the concordance index (c-index) and calibration curves. RESULTS: The 5-year overall survival (OS) of the NPC patients with N3 stage was 43.9%, and the prognosis of patients without any distant metastases was largely longer than that with metastases. No difference was observed between different pathological types in the entire cohort. However, patients with non-keratinized squamous cell carcinoma had a better OS than that of the patients with keratinized squamous cell carcinoma in a nonmetastatic subgroup. Using the Cox regression analysis results, the nomogram successfully classified these patients into low- and high-risk subgroups and presented the survival difference. The c-index of the nomogram for predicting the prognosis was satisfactory. CONCLUSION: This study identified metastatic risk factors and developed a convenient clinical tool for the prognosis of NPC patients. This tool can be used for individualized risk classification and decision-making regarding treatment of NPC patients with N3 stage.

Efficacy and Safety of Polycaprolactone in Treating Nasolabial Folds: A Prospective, Multicenter, and Randomized Controlled Trial.

Nasolabial folds (NLFs) are the most pronounced sign of facial aging. This study explored the efficacy and safety of polycaprolactone gel in treating Chinese patients with moderate-to-severe NLFs. Patients with moderate-to-severe NLF who wished to be treated by dermal fillers were recruited from three centers between July 2017 and September 2019. The randomizing ratio was 1:1 in the polycaprolactone group (polycaprolactone injection) or control group (sodium hyaluronate gel injection). The primary endpoint was the effectiveness rate of Wrinkle Severity Rating Score (WSRS) scores at 12 months after injection. The full-analysis set (FAS) and safety sets had 80 patients in the polycaprolactone group and control group, respectively. In the FAS, the effectiveness rate at 12 months in the polycaprolactone group was 88.8% compared with 23.8% in controls (P < 0.001). The improvement in WSRS sustained during 12 months in the polycaprolactone group, while gradually vanished in the control group since 3 months after surgery. The global aesthetic improvement scale (GAIS) by investigator assessments was improved, much improved, or very much improved in all patients during follow-up, while the proportion of patients with a "no change" assessment gradually increased during follow-up after 6 months in the control group. The rates of injection-related adverse event (AE) and serve injection-related AE were 8.8 versus 11.3% and 0 versus 1.3% in the polycaprolactone group and control groups, respectively. Polycaprolactone gel injection is effective and safe to treat moderate-to-severe NLFs in Chinese patients.

CBL/CAP Is Essential for Mitochondria Respiration Complex I Assembly and Bioenergetics Efficiency in Muscle Cells.

CBL is rapidly phosphorylated upon insulin receptor activation. Mice whole body CBL depletion improved insulin sensitivity and glucose clearance; however, the precise mechanisms remain unknown. We depleted either CBL or its associated protein SORBS1/CAP independently in myocytes and assessed mitochondrial function and metabolism compared to control cells. CBL- and CAP-depleted cells showed increased mitochondrial mass with greater proton leak. Mitochondrial respiratory complex I activity and assembly into respirasomes were reduced. Proteome profiling revealed alterations in proteins involved in glycolysis and fatty acid degradation. Our findings demonstrate CBL/CAP pathway couples insulin signaling to efficient mitochondrial respiratory function and metabolism in muscle.

Synthesis and Antineoplastic Activity of a Dimer, Spiroindolinone Pyrrolidinecarboxamide.

The mutation or function loss of tumour suppressor p53 plays an important role in abnormal cell proliferation and cancer generation. Murine Double Minute 2 (MDM2) is one of the key negative regulators of p53. p53 reactivation by inhibiting MDM2-p53 interaction represents a promising therapeutic option in cancer treatment. Here, to develop more effective MDM2 inhibitors with lower off-target toxicities, we synthesized a dimer, spiroindolinone pyrrolidinecarboxamide XR-4, with potent MDM2-p53 inhibition activity. Western blotting and qRT-PCR were performed to detect the impact of XR-4 on MDM2 and p53 protein levels and p53 downstream target gene levels in different cancers. Cancer cell proliferation inhibition and clonogenic activity were also investigated via the CCK8 assay and colony formation assay. A subcutaneous 22Rv1-derived xenografts mice model was used to investigate the in vivo anti-tumour activity of XR-4. The results reveal that XR-4 can induce wild-type p53 accumulation in cancer cells, upregulate the levels of the p53 target genes p21 and PUMA levels, and then inhibit cancer cell proliferation and induce cell apoptosis. XR-4 can also act as a homo-PROTAC that induces MDM2 protein degradation. Meanwhile, the in vivo study results show that XR-4 possesses potent antitumour efficacy and a favourable safety property. In summary, XR-4 is an interesting spiroindolinone pyrrolidinecarboxamide-derivative dimer with effective p53 activation activity and a cancer inhibition ability.

Chinese Guideline on the Management of Polypoidal Choroidal Vasculopathy (2022).

Background In mainland China, patients with neovascular age-related macular degeneration (nAMD) have approximately an 40% prevalence of polypoidal choroidal vasculopathy (PCV). This disease leads to recurrent retinal pigment epithelium detachment (PED), extensive subretinal or vitreous hemorrhages, and severe vision loss. China has introduced various treatment modalities in the past years and gained comprehensive experience in treating PCV.Methods A total of 14 retinal specialists nationwide with expertise in PCV were empaneled to prioritize six questions and address their corresponding outcomes, regarding opinions on inactive PCV, choices of anti-vascular endothelial growth factor (anti-VEGF) monotherapy, photodynamic therapy (PDT) monotherapy or combined therapy, patients with persistent subretinal fluid (SRF) or intraretinal fluid (IRF) after loading dose anti-VEGF, and patients with massive subretinal hemorrhage. An evidence synthesis team conducted systematic reviews, which informed the recommendations that address these questions. This guideline used the GRADE (Grading of Recommendations, Assessment, Development, and Evaluation) approach to assess the certainty of evidence and grade the strengths of recommendations. Results The panel proposed the following six conditional recommendations regarding treatment choices. (1) For patients with inactive PCV, we suggest observation over treatment. (2) For treatment-na?ve PCV patients, we suggest either anti-VEGF monotherapy or combined anti-VEGF and PDT rather than PDT monotherapy. (3) For patients with PCV who plan to initiate combined anti-VEGF and PDT treatment, we suggest later/rescue PDT over initiate PDT. (4) For PCV patients who plan to initiate anti-VEGF monotherapy, we suggest the treat and extend (T&E) regimen rather than the pro re nata (PRN) regimen following three monthly loading doses. (5) For patients with persistent SRF or IRF on optical coherence tomography (OCT) after three monthly anti-VEGF treatments, we suggest proceeding with anti-VEGF treatment rather than observation. (6) For PCV patients with massive subretinal hemorrhage (equal to or more than four optic disc areas) involving the central macula, we suggest surgery (vitrectomy in combination with tissue-plasminogen activator (tPA) intraocular injection and gas tamponade) rather than anti-VEGF monotherapy. Conclusions Six evidence-based recommendations support optimal care for PCV patients' management.

Cornuside inhibits glucose-induced proliferation and inflammatory response of mesangial cells.

Cornuside is a secoiridoid glucoside compound extracted from the fruits of Cornus officinalis. Cornuside has immunomodulatory and anti-inflammatory properties; however, its potential therapeutic effects on diabetic nephropathy (DN) have not been completely explored. In this study, we established an in vitro model of DN through treating mesangial cells (MMCs) with glucose. MMCs were then treated with different concentrations of cornuside (0, 5, 10, and 30 µM). Cell viability was determined using cell counting kit-8 and 5-ethynyl-2'-deoxyuridine assays. Levels of proinflammatory cytokines, including interleukin (IL)-6, tumor necrosis factor-α, and IL-1ß were examined using enzyme-linked immunosorbent assay. Reverse transcription quantitative real-time polymerase chain reaction and Western blotting were performed to detect the expression of AKT and nuclear factor-kappa B (NF-κB)-associated genes. We found that cornuside treatment significantly reduced glucose-induced increase in MMC viability and expression of pro-inflammatory cytokines. Moreover, cornuside inhibited glucose-induced phosphorylation of AKT and NF-κB inhibitor alpha, decreased the expression of proliferating cell nuclear antigen and cyclin D1, and increased the expression of p21. Our study indicates that the anti-inflammatory properties of cornuside in DN are due to AKT and NF-κB inactivation in MMCs.

miR-564: A potential regulator of vascular smooth muscle cells and therapeutic target for aortic dissection.

BACKGROUND: Aortic dissection (AD) is a lethal cardiac disorder and one of the most concerning cardiovascular diseases (CVDs). Increasing evidence indicates that human aortic vascular smooth muscle cells (VSMCs) play a crucial role in the pathogenesis of AD, especially related to phenotypic transformation. And notablely, the development of AD is also accompanied by inflammation. METHODS: By using quantitative real-time PCR and fluorescence in situ hybridization (FISH), we detected the expression levels of miR-564 in vitro and in vivo. The effects of miR-564 proliferation and migration were investigated in VSMCs. The downstream targets of miR-564 were found by bioinformatics analyse, and verified in the regulation on VSMCs. An AD murine model was constructed and clinical evaluation was performed to explore the critical roles of miR-564 in vivo. At the same time, the level of inflammation was detected using quantitative real-time PCR and immunofluorescence. RESULTS: Overexpression of miR-564 inhibited cell proliferation and migration, as well as phenotype switch, with or without platelet-derived growth factor BB (PDGF-BB) treatment, whereas downregulation of miR-564 led to opposite results. Mechanistically, miR-564 directly interacted with the target genes proto-oncogene (SKI) and neurogranin (NRGN) to regulate the biological functions of VSMCs. In particular, animal experiments demonstrated that miR-564 can alleviate the progression of AD mainly through mediating phenotypic swithing and inflammation which was consistent with clinical evaluation. CONCLUSIONS: Our study identified miR-564 as a significant molecule that attenuates AD progression by inhibiting inflammation and VSMCs proliferation, migration and phenotypic transformation, suggesting that it may be a potential therapeutic target for AD.

Functional characteristics of a broad spectrum of TBX6 variants in Mayer-Rokitansky-Küster-Hauser syndrome.

PURPOSE: Mayer-Rokitansky-Küster-Hauser syndrome (MRKHS) is characterized by congenital absence of the uterus, cervix, and the upper part of the vagina in females. Whole-gene deletion and loss-of-function variants in TBX6 have been identified in association with MRKHS. We aimed to expand the spectrum of TBX6 variants in MRKHS and explore the biological effect of the variant alleles. METHODS: Rare variants in TBX6 were called from a combined multiethnic cohort of 622 probands with MRKHS who underwent exome sequencing or genome sequencing. Multiple in vitro functional experiments were performed, including messenger RNA analysis, western blotting, transcriptional activity assay, and immunofluorescence staining. RESULTS: We identified 16 rare variants in TBX6 from the combined cohort, including 1 protein-truncating variant reported in our previous study and 15 variants with unknown effects. By comparing the prevalence of TBX6 variants in the Chinese MRKHS cohort vs 1038 female controls, we observed a significant mutational burden of TBX6 in affected individuals (P = .0004, odds ratio = 5.25), suggesting a causal role of TBX6 variants in MRKHS. Of the 15 variants with uncertain effects, 7 were shown to induce a loss-of-function effect through various mechanisms. The c.423G>A (p.Leu141=) and c.839+5G>A variants impaired the normal splicing of TBX6 messenger RNA, c.422T>C (p.Leu141Pro) and c.745G>A (p.Val249Met) led to decreased protein expression, c.10C>T (p.Pro4Ser) and c.400G>A (p.Glu134Lys) resulted in perturbed transcriptional activity, and c.356G>A (p.Arg119His) caused protein mislocalization. We observed incomplete penetrance and variable expressivity in families carrying deleterious variants, which indicates a more complex genetic mechanism than classical Mendelian inheritance. CONCLUSION: Our study expands the mutational spectrum of TBX6 in MRKHS and delineates the molecular pathogenesis of TBX6 variants, supporting the association between deleterious variants in TBX6 and MRKHS.