Factors Associated With Perception of Stigma Among Parents of Children With Cleft Lip and Palate: Cross-Sectional Study.

BACKGROUND: Parents of children with cleft lip with or without cleft palate (CL/P) often face stigmatization, which has a significant impact on their quality of life and mental health. However, to date, there is a lack of comprehensive, multicenter empirical research on parents of children with CL/P in China, particularly those with large-scale samples. OBJECTIVE: This study aimed to identify major factors that contribute to the perception of stigma experienced by parents of children with CL/P. METHODS: A cross-sectional survey was conducted. A total of 104 parents of children diagnosed with CL/P in 2 hospitals were selected by convenience sampling. Demographics and disease information, the Chinese Perception of Stigma Questionnaire, the Center for Epidemiological Studies Depression Scale, and the Social Anxiety Scale were used in this study. Descriptive statistics, t tests, and one-way ANOVA were used to compare the differences between participants' demographic information and perception of stigma. Multivariable linear regression was performed to assess associations between demographic factors, social anxiety, depression, and perception of stigma. RESULTS: The mean scores for the dimensions of perception of stigma, depression, and social anxiety were 22.97 (SD 9.21), 38.34 (SD 8.25), and 22.86 (SD 6.69), respectively. Depression and social anxiety were positively associated with discrimination, while surgery status was a negatively associated variable. Parents with a college education or higher had significantly lower levels of perceived stigma compared to parents with a junior high school education (all P values <.05). These 4 factors explained 40.4% of the total model variance (F8=9.726; P<.001; R2=0.450; adjusted R2=0.404). CONCLUSIONS: Our findings highlight a concerning trend of diminished quality of life among parents of children with CL/P. Factors such as parents' education level, surgery status, depression, and social anxiety are shown to influence the level of stigma experienced. Implementing comprehensive nursing care and providing presurgical support are effective strategies for alleviating parents' social anxiety, reducing perceived stigma, and preventing depression.

Compact Cje3Cas9 for Efficient In Vivo Genome Editing and Adenine Base Editing.

Many therapeutic applications of CRISPR-Cas9 gene editing rely on delivery using the highly versatile adeno-associated virus (AAV) vector. The smallest type II Cas9 ortholog-Cje1Cas9, derived from Campylobacter jejuni with <1,000 amino acids-is particularly attractive for AAV delivery. However, the complex protospacer adjacent motif (PAM) of Cje1Cas9 (N3VRYAC) greatly restricts the density of recognition sequences in human genome. In this study, we identify two compact CjeCas9 orthologs designated as Cje2Cas9 and Cje3Cas9, whose PAM-interacting residues are different from those of the well-known Cje1Cas9. They can induce efficient genome editing in human cells, and their simpler trinucleotide PAM (N4CYA) requirements expand the scope of targeting. Moreover, Cje3Cas9 efficiently disrupts the Tyr gene in mice after being micro-injected into zygotes with the corresponding sgRNA. It also successfully disrupts the Pcsk9 gene in 8-week-old mouse liver after delivery with an sgRNA using an all-in-one AAV delivery vehicle. The gene-edited mice showed lower cholesterol level than wild-type mice. Notably, the 8e-nCje3-ABE and an sgRNA targeting Pcsk9 were successfully packaged into a single AAV vector for genome editing in adult mouse liver, with editing efficiency up to 12%. Thus, simple PAMs and a compact size enable Cje2/3Cas9 to expand the target scope of CRISPR-Cas9 toolsets, exhibiting considerable potential for therapeutic applications.

PHF13 epigenetically activates TGFß driven epithelial to mesenchymal transition.

Epigenetic alteration is a pivotal factor in tumor metastasis. PHD finger protein 13 (PHF13) is a recently identified epigenetic reader of H3K4me2/3 that functions as a transcriptional co-regulator. In this study, we demonstrate that PHF13 is required for pancreatic-cancer-cell growth and metastasis. Integrative analysis of transcriptome and epigenetic profiles provide further mechanistic insights into the epigenetic regulation of genes associated with cell metastasis during the epithelial-to-mesenchymal transition (EMT) induced by transforming growth factor ß (TGFß). Our data suggest PHF13 depletion impairs activation of TGFß stimulated genes and correlates with a loss of active epigenetic marks (H3K4me3 and H3K27ac) at these genomic regions. These observations argue for a dependency of TGFß target activation on PHF13. Furthermore, PHF13-dependent chromatin regions are enriched in broad H3K4me3 domains and super-enhancers, which control genes critical to cancer-cell migration and invasion, such as SNAI1 and SOX9. Overall, our data indicate a functional and mechanistic correlation between PHF13 and EMT.

Versatile and efficient in vivo genome editing with compact Streptococcus pasteurianus Cas9.

Compact CRISPR-Cas9 systems that can be packaged into an adeno-associated virus (AAV) show promise for gene therapy. However, the requirement of protospacer adjacent motifs (PAMs) restricts the target scope. To expand this repertoire, we revisited and optimized a small Cas9 ortholog derived from Streptococcus pasteurianus (SpaCas9) for efficient genome editing in vivo. We found that SpaCas9 enables potent targeting of 5'-NNGYRA-3' PAMs, which are distinct from those recognized by currently used small Cas9s; the Spa-cytosine base editor (CBE) and Spa-adenine base editor (ABE) systems efficiently generated robust C-to-T and A-to-G conversions both in vitro and in vivo. In addition, by exploiting natural variation in the PAM-interacting domain, we engineered three SpaCas9 variants to further expand the targeting scope of compact Cas9 systems. Moreover, mutant mice with efficient disruption of the Tyr gene were successfully generated by microinjection of SpaCas9 mRNA and the corresponding single guide RNA (sgRNA) into zygotes. Notably, all-in-one AAV delivery of SpaCas9 targeting the Pcsk9 gene in adult mouse liver produced efficient genome-editing events and reduced its serum cholesterol. Thus, with distinct PAMs and a small size, SpaCas9 will broaden the CRISPR-Cas9 toolsets for efficient gene modifications and therapeutic applications.

Immune Cell Infiltration Landscape of Ovarian Cancer to Identify Prognosis and Immunotherapy-Related Genes to Aid Immunotherapy.

Ovarian cancer (OC) is the second leading cause of death in gynecological cancer. Multiple study have shown that the efficacy of tumor immunotherapy is related to tumor immune cell infiltration (ICI). However, so far, the Immune infiltration landscape of tumor microenvironment (TME) in OC has not been elucidated. In this study, We organized the transcriptome data of OC in the Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases, evaluated the patient's TME information, and constructed the ICI scores to predict the clinical benefits of patients undergoing immunotherapy. Immune-related genes were further used to construct the prognostic model. After clustering analysis of ICI genes, we found that patients in ICI gene cluster C had the best prognosis, and their tumor microenvironment had the highest proportion of macrophage M1 and T cell follicular helper cells. This result was consistent with that of multivariate cox (multi-cox) analysis. The prognostic model constructed by immune-related genes had good predictive performance. By estimating Tumor mutation burden (TMB), we also found that there were multiple genes with statistically different mutation frequencies in the high and low ICI score groups. The model based on the ICI score may help to screen out patients who would benefit from immunotherapy. The immune-related genes screened may be used as biomarkers and therapeutic targets.

Establishing a novel lens opacities classification system based on ultrasound biomicroscopy (UBM) for pediatric cataracts: reliability and availability.

BACKGROUND: This study sought to develop and validate a lens opacities classification system based on ultrasound biomicroscopy (UBM) imaging to grade pediatric cataracts. METHODS: The study was conducted at Guangzhou Children's Hospital, Guangzhou Women and Children's Medical Center. UBM images of patients at the hospital from September 2013 to November 2014 were used in this study. We summarized the characteristics of lenticular opacification in each of the following 4 zones: the anterior capsule (A); the cortex (C); the nucleus (N); and the posterior capsule (P). The UBM data and intraoperative videos were compared, and sensitivity, specificity, accuracy, and positive and negative predictive values were determined for our Lens Opacities Classification System based on UBM for Pediatric Cataracts (LOCS-UP) detection. Two physicians classified pediatric cataracts (anterior capsule, cortex, and posterior capsule) by extracting 146 images from the UBM database. Patients' data were recorded to calculate the kappa coefficients. The LOCS-UP was developed. RESULTS: Under this standard, all types of pediatric cataracts can be classified and acquired a code by the LOCS-UP. The LOCS-UP had the highest sensitivity (100%) and specificity (98.96%) in naming the anterior capsule and the lowest sensitivity (50%) and specificity (89.59%) in naming the posterior capsule. Its consistency at naming the anterior capsule was satisfactory (Kappa coefficient: 0.70), and it was also able to name the nucleus, cortex, and posterior capsule (0.56, 0.58, and 0.48, respectively). CONCLUSIONS: LOCS-UP could name pediatric cataracts by providing an unique digital encoding, which could reflect characteristics exactly for different local lens anomalies to all kinds of pediatric cataract patients. This method provides detailed and accurate information about Patients' lenses.

Oxytocin receptor induces mammary tumorigenesis through prolactin/p-STAT5 pathway.

Oxytocin receptor (OXTR) is involved in social behaviors, thermoregulation, and milk ejection, yet little is known about its role in breast cancer. To investigate the role of OXTR in mammary gland development and tumorigenesis, a transgenic mouse model of OXTR overexpression (++Oxtr) was used. Overexpression of OXTR-induced progressive mammary hyperplasia, unexpected milk production, and tumorigenesis in females. OXTR-induced mammary tumors showed ERBB2 upregulation and mixed histological subtypes with predomination of papillary and medullary carcinomas. OXTR overexpression led to an activation of prolactin (PRL)/p-STAT5 pathway and created a microenvironment that promotes mammary-specific tumorigenesis. PRL inhibitor bromocriptine (Br) could mitigate OXTR-driven mammary tumor growth. The study demonstrates Oxtr is an oncogene and a potential drug target for HER2-type breast cancer.

Ultrasound biomicroscopy for the assessment of postoperative complications after congenital cataract surgery.

BACKGROUND: The aim of the present study was to investigate the role of ultrasound biomicroscopy (UBM) in the evaluation of postoperative complications in children with congenital cataracts. METHODS: A retrospective study was conducted between September 2012 and December 2016 at Guangzhou Women and Children's Medical Center. Red reflex test and high-resolution bag/balloon UBM were performed to evaluate postoperative congenital cataracts. The red reflex test results were recorded, and UBM imaging results were recorded and analyzed. Different postoperative complications were classified based on the UBM imaging features, and a second procedure was performed accordingly. The UBM images were compared with the images captured from the intraoperative videos. RESULTS: In total, we looked at 120 eyes in 96 patients (65 males and 31 females) in the present study. The age of the cohort was 3-76 months. A total of 51 eyes with poor red reflex were included. There were complications in 46 eyes after congenital cataract surgery, as detected by UBM, including posterior capsular opacification (n=29 eyes), pupil block (n=8 eyes), synechia (n=5 eyes), hyphema (n=1 eye), and abnormal intraocular lens (IOL) placement (n=3 eyes). UBM images showed specific features of postoperative complications. CONCLUSIONS: UBM is a valuable tool for the early evaluation of postoperative complications of congenital cataracts, especially for those with media opacities or when pupil dilation is not possible.

Application effect of initiation of enteral nutrition at different time periods after surgery in neonates with complex congenital heart disease: A retrospective analysis.

ABSTRACT: Early enteral nutrition (EN) promotes the recovery of critically ill patients, but the initiation time for EN in neonates after cardiac surgery remains unclear.This study aimed to investigate the effect of initiation time of EN after cardiac surgery in neonates with complex congenital heart disease (CHD).Neonates with complex CHD admitted to the CICU from January 2015 to December 2017 were retrospectively analyzed. Patients were divided into the 24-hour Group (initiated at 24 hours after surgery in 2015) (n = 32) and 6-hour Group (initiated at 6 hours after surgery in 2016 and 2017) (n = 66). Data on the postoperative feeding intolerance, nutrition-related laboratory tests (albumin, prealbumin, retinol binding protein), and clinical outcomes (including duration of mechanical ventilation, CICU stay, and postoperative hospital stay) were collected.The incidence of feeding intolerance was 56.3% in 24-hour Group and 39.4%, respectively (P = .116). As compared to 24-hour Group, prealbumin and retinol binding protein levels were higher (160.7 ±â€Š64.3 vs 135.2 ±â€Š28.9 mg/L, P = .043 for prealbumin; 30.7 ±â€Š17.7 vs 23.0 ±â€Š14.1 g/L P = .054 for retinol-binding protein). The duration of CICU stay (9.4 ±â€Š4.5 vs 13.3 ±â€Š10.4 day, P = .049) and hospital stay (11.6 ±â€Š3.0 vs 15.8 ±â€Š10.3 day, P = .028) were shorter in 6-hour Group.Early EN improves nutritional status and clinical outcomes in neonates with complex CHD undergoing cardiac surgery, without significant feeding intolerance.

RNF40 exerts stage-dependent functions in differentiating osteoblasts and is essential for bone cell crosstalk.

The role of histone ubiquitination in directing cell lineage specification is only poorly understood. Our previous work indicated a role of the histone 2B ubiquitin ligase RNF40 in controlling osteoblast differentiation in vitro. Here, we demonstrate that RNF40 has a stage-dependent function in controlling osteoblast differentiation in vivo. RNF40 expression is essential for early stages of lineage specification, but is dispensable in mature osteoblasts. Paradoxically, while osteoblast-specific RNF40 deletion led to impaired bone formation, it also resulted in increased bone mass due to impaired bone cell crosstalk. Loss of RNF40 resulted in decreased osteoclast number and function through modulation of RANKL expression in OBs. Mechanistically, we demonstrate that Tnfsf11 (encoding RANKL) is an important target gene of H2B monoubiquitination. These data reveal an important role of RNF40-mediated H2B monoubiquitination in bone formation and remodeling and provide a basis for exploring this pathway for the treatment of conditions such as osteoporosis or cancer-associated osteolysis.

LncRNA ZEB2-AS1 contributes to the tumorigenesis of gastric cancer via activating the Wnt/ß-catenin pathway.

Studies have shown that long noncoding RNA Zinc finger E-box-binding homeobox 2 antisense RNA 1 (ZEB2-AS1) is involved in the progression of lung cancer, bladder cancer, and hepatocellular carcinoma. However, its role in the pathogenesis of gastric cancer remains unknown. The Wnt/ß-catenin pathway contributes to the development of gastric cancer. ZEB2-AS1 expression was firstly detected in the gastric carcinoma tissue samples as well as in gastric cancer cells. Knockdown of ZEB2-AS1 was performed by ZEB2-AS1-shRNA, and the viability, migration, invasion, and apoptosis of gastric cancer cells were determined by CCK-8, scratch assay, transwell, and flow cytometry, respectively. Furthermore, levels of Ki-67, PCNA, VEGF, MMP9, epithelial-mesenchymal transition (EMT) markers (E-cadherin, Vimentin and ZEB2), cleaved caspase 3/8/9 and PARP, active ß-catenin, c-Myc, cyclinD1, and AXIN2 were assayed by Western blot or real-time PCR. Additionally, the role and mechanism of ZEB2-AS1 were confirmed in a xenograft nude mouse model. We found ZEB2-AS1 expression was increased in gastric carcinoma samples, and it was correlated with tumor progression. Also, its expression was elevated in gastric cancer cells. Knockdown of ZEB2-AS1 reduced the proliferation, migration, invasion, and EMT, but increased the apoptosis of gastric carcinoma cells. Furthermore, ZEB2-AS1 downregulation remarkably suppressed the expression of Ki-67, PCNA, VEGF and MMP9, and the activation of Wnt/ß-catenin signaling, whereas elevated the levels of cleaved caspase 3/8/9 and PARP in gastric cancer cells. And ZEB2 overexpression reversed the effects of ZEB2-AS1 downregulation on the proliferation, EMT and inactivation of Wnt/ß-catenin signaling. Additionally, ZEB2-AS1 knockdown inhibited tumor growth, Ki-67 staining, and the expression of VEGF, MMP9, active ß-catenin, c-Myc, cyclinD1, and AXIN2 in mice. In conclusion, ZEB2-AS1 promotes the tumorigenesis of gastric carcinoma that is related to the upregulation of ZEB2 and the activation of the Wnt/ß-catenin pathway.

CHD1 regulates cell fate determination by activation of differentiation-induced genes.

The coordinated temporal and spatial activation of gene expression is essential for proper stem cell differentiation. The Chromodomain Helicase DNA-binding protein 1 (CHD1) is a chromatin remodeler closely associated with transcription and nucleosome turnover downstream of the transcriptional start site (TSS). In this study, we show that CHD1 is required for the induction of osteoblast-specific gene expression, extracellular-matrix mineralization and ectopic bone formation in vivo. Genome-wide occupancy analyses revealed increased CHD1 occupancy around the TSS of differentiation-activated genes. Furthermore, we observed that CHD1-dependent genes are mainly induced during osteoblast differentiation and are characterized by higher levels of CHD1 occupancy around the TSS. Interestingly, CHD1 depletion resulted in increased pausing of RNA Polymerase II (RNAPII) and decreased H2A.Z occupancy close to the TSS, but not at enhancer regions. These findings reveal a novel role for CHD1 during osteoblast differentiation and provide further insights into the intricacies of epigenetic regulatory mechanisms controlling cell fate determination.

BRD4 promotes p63 and GRHL3 expression downstream of FOXO in mammary epithelial cells.

Bromodomain-containing protein 4 (BRD4) is a member of the bromo- and extraterminal (BET) domain-containing family of epigenetic readers which is under intensive investigation as a target for anti-tumor therapy. BRD4 plays a central role in promoting the expression of select subsets of genes including many driven by oncogenic transcription factors and signaling pathways. However, the role of BRD4 and the effects of BET inhibitors in non-transformed cells remain mostly unclear. We demonstrate that BRD4 is required for the maintenance of a basal epithelial phenotype by regulating the expression of epithelial-specific genes including TP63 and Grainy Head-like transcription factor-3 (GRHL3) in non-transformed basal-like mammary epithelial cells. Moreover, BRD4 occupancy correlates with enhancer activity and enhancer RNA (eRNA) transcription. Motif analyses of cell context-specific BRD4-enriched regions predicted the involvement of FOXO transcription factors. Consistently, activation of FOXO1 function via inhibition of EGFR-AKT signaling promoted the expression of TP63 and GRHL3. Moreover, activation of Src kinase signaling and FOXO1 inhibition decreased the expression of FOXO/BRD4 target genes. Together, our findings support a function for BRD4 in promoting basal mammary cell epithelial differentiation, at least in part, by regulating FOXO factor function on enhancers to activate TP63 and GRHL3 expression.

Clinical Characteristics and Surgical Procedures for Children with Congenital Membranous Cataract.

OBJECTIVE: In a group case series, the clinical characteristics of congenital membranous cataract in children were studied to establish a system of classification and determine the surgical method suited for each type. METHODS: Children (18 eyes) with congenital membranous cataract were examined by slit lamp, ultrasound biomicroscopy, and operating microscopy to classify cataracts. The clinical characteristics of congenital membranous cataract and its feature related to the surgical method were analyzed. RESULTS: Five major types of congenital membranous cataracts were classified. All of the surgeries were successful. Anterior and posterior capsulorhexis was performed using Klöti RF capsulotomy tips. The capsular flap was removed, and anterior vitrectomy was performed using a vitrectomy cutter. Postoperative complications included posterior capsule opacification in 16.7% of the patients. CONCLUSION: Ultrasound biomicroscopy was used successfully to classify congenital membranous cataracts prior to surgery. Anterior and posterior capsulorhexis was performed using Klöti RF capsulotomy tips, and capsulectomy was performed using a vitrectomy cutter. These were effective techniques and should be considered for congenital membranous cataract removal surgery. This trial is registered with registration number chiCTR-OOC-17010913.

Image features of lens opacity in pediatric cataracts using ultrasound biomicroscopy.

PURPOSE: To study lens opacity in pediatric cataract images captured using ultrasound biomicroscopy (UBM). METHODS: The medical records of patients operated on from September 2012 to October 2013 were reviewed retrospectively. Prior to surgery, patients were placed in the supine position under sedation with oral chloral hydrate for UBM imaging. Lens morphology was evaluated by UBM examination with a 50 MHz probe that was equipped with a water bag instead of the standard plastic shell. UBM images were compared to images captured from intraoperative videos. RESULTS: UBM examination was performed in 50 patients (including 10 infants) aged 2 months to 6 years. The UBM ecographic images showed features specific to pediatric cataract lenses. These features were used to define 2 types of anterior capsule of the lens, 4 types of cortex and nucleus of the lens, 3 types of posterior capsule of the lens, and membranous cataracts. CONCLUSIONS: Capsule morphology and the cortex density of pediatric cataracts could be evaluated before surgery using UBM imaging. Adoption of this technique could provide useful preoperative information to surgeons.

Histone Chaperone SSRP1 is Essential for Wnt Signaling Pathway Activity During Osteoblast Differentiation.

Cellular differentiation is accompanied by dramatic changes in chromatin structure which direct the activation of lineage-specific transcriptional programs. Structure-specific recognition protein-1 (SSRP1) is a histone chaperone which is important for chromatin-associated processes such as transcription, DNA replication and repair. Since the function of SSRP1 during cell differentiation remains unclear, we investigated its potential role in controlling lineage determination. Depletion of SSRP1 in human mesenchymal stem cells elicited lineage-specific effects by increasing expression of adipocyte-specific genes and decreasing the expression of osteoblast-specific genes. Consistent with a role in controlling lineage specification, transcriptome-wide RNA-sequencing following SSRP1 depletion and the induction of osteoblast differentiation revealed a specific decrease in the expression of genes involved in biological processes related to osteoblast differentiation. Importantly, we observed a specific downregulation of target genes of the canonical Wnt signaling pathway, which was accompanied by decreased nuclear localization of active ß-catenin. Together our data uncover a previously unknown role for SSRP1 in promoting the activation of the Wnt signaling pathway activity during cellular differentiation. Stem Cells 2016;34:1369-1376.

Histone deacetylase inhibitor significantly improved the cloning efficiency of porcine somatic cell nuclear transfer embryos.

Valproic acid (VPA), a histone deacetylase inbibitor, has been shown to generate inducible pluripotent stem (iPS) cells from mouse and human fibroblasts with a significant higher efficiency. Because successful cloning by somatic cell nuclear transfer (SCNT) undergoes a full reprogramming process in which the epigenetic state of a differentiated donor nuclear is converted into an embryonic totipotent state, we speculated that VPA would be useful in promoting cloning efficiency. Therefore, in the present study, we examined whether VPA can promote the developmental competence of SCNT embryos by improving the reprogramming state of donor nucleus. Here we report that 1 mM VPA for 14 to 16 h following activation significantly increased the rate of blastocyst formation of porcine SCNT embryos constructed from Landrace fetal fibroblast cells compared to the control (31.8 vs. 11.4%). However, we found that the acetylation level of Histone H3 lysine 14 and Histone H4 lysine 5 and expression level of Oct4, Sox2, and Klf4 was not significantly changed between VPA-treated and -untreated groups at the blastocyst stage. The SCNT embryos were transferred to 38 surrogates, and the cloning efficiency in the treated group was significantly improved compared with the control group. Taken together, we have demonstrated that VPA can improve both in vitro and in vivo development competence of porcine SCNT embryos.

Vitamin C enhances in vitro and in vivo development of porcine somatic cell nuclear transfer embryos.

The reprogramming of differentiated cells into a totipotent embryonic state through somatic cell nuclear transfer (SCNT) is still an inefficient process. Previous studies revealed that the generation of induced pluripotent stem (iPS) cells from mouse and human fibroblasts could be significantly enhanced with vitamin C treatment. Here, we investigated the effects of vitamin C, to our knowledge for the first time, on the in vitro and in vivo development of porcine SCNT embryos. The rate of blastocyst development in SCNT embryos treated with 50µg/mL vitamin C 15h after activation (36.0%) was significantly higher than that of untreated SCNT embryos (11.5%). The enhanced in vitro development rate of vitamin C-treated embryos was associated with an increased acetylation level of histone H4 lysine 5 and higher Oct4, Sox2 and Klf4 expression levels in blastocysts, as determined by real-time PCR. In addition, treatment with vitamin C resulted in an increased pregnancy rate in pigs. These findings suggest that treatment with vitamin C is beneficial for enhancement of the in vitro and in vivo development of porcine SCNT embryos.