Profiles of Circulating Exosomal Mirna in Patients with SAPHO Patients by High-Throughput Sequencing.

BACKGROUND: Synovitis, acne, pustulosis, hyperostosis, and osteitis (SAPHO) syndrome is a rare disease that is characterized by autoinflammatory lesions on both bones and skin. The diverse manifestations and limited understanding of its etiology have hindered the diagnosis and treatment of this condition. SAPHO syndrome is also classified as a primary inflammatory osteitis. The onset of osteoarticular involvement in this disease is typically gradual, and the identification of associated biomarkers may be crucial for accurate diagnosis, effective treatment, and a better understanding of its underlying mechanisms. METHODS: We enrolled a total of 6 SAPHO patients and 3 healthy volunteers for this study. The miRNA expression profile in circulating exosomes was analyzed using next-generation sequencing. A total of 45 miRNAs were found to be differentially expressed in SAPHO patients. Linear discriminant analysis effect size analysis and Wilcoxon rank-sum test were employed to identify biomarkers based on these differentially expressed miRNAs. Among them, we selected 4 miRNAs as biomarkers for SAPHO syndrome, resulting in an area under the receiver operating characteristic curve of 1. RESULTS: The differentially expressed miRNAs indicated enrichment in immune system and endocrine system-related KEGG pathways, as well as infectious diseases and cancers. Furthermore, the most significantly enriched molecular functions in GO analysis were protein binding and catalytic activity. CONCLUSION: The exosomal miRNA profile in SAPHO syndrome exhibited significant changes, suggesting its potential as a candidate biomarker for diagnostic assistance, although further investigation is warranted to elucidate their role in the pathology.

Comparison of transcriptome-wide N6-methyladenosine profiles from healthy trio families reveals regulator-mediated methylation alterations.

The N6-methyladenosine (m6A) modification is a highly conserved RNA modification found in eukaryotic messenger RNAs (mRNAs). It plays a vital role in regulating various biological processes. Dysregulation of m6A modifications has been linked to a range of complex genetic diseases in humans. However, there has been a lack of comprehensive characterization and comparison of m6A modifications at the transcriptome-wide level within families. To address this gap, we profiled transcriptome-wide m6A methylation in 18 individuals across 6 Yoruba trio families. The m6A methylomes of these 18 individuals revealed that m6A modifications in children showed greater similarity to each other than to their parents. This suggests that m6A modifications are influenced by multiple factors rather than solely determined by genetic factors. Additionally, we found that mRNAs exhibiting m6A modifications specific to children were enriched in cell cycle control processes, while those with m6A modifications specific to parents were associated with chromatin modifications. Furthermore, our analysis on the interactions between differentially expressed m6A-related regulatory genes and age-related genes suggested that age might be one of the factors influencing m6A modifications. In summary, our study provided a valuable dataset that highlighted the differences and functional diversity of m6A modifications within and between trio families.

Association of PTGER4 and PRKAA1 genetic polymorphisms with gastric cancer.

BACKGROUND: Gastric cancer (GC) is one of the most common malignancies, affected by several genetic loci in the clinical phenotype. This study aimed to determine the association between PTGER4 and PRKAA1 gene polymorphisms and the risk of GC. METHODS: A total of 509 GC patients and 507 age and sex-matched healthy controls were recruited to explore the association between PTGER4 and PRKAA1 genetic polymorphisms and GC susceptibility. Logistic regression analysis was used to study the correlation between these SNPs and GC, with odd ratio (OR) and 95% confidence interval (CI) as indicators. Multifactor dimensionality reduction was utilized to analyze the genetic relationships among SNPs. was conducted to predict gene expression, the impact of SNPs on gene expression, and the signaling pathways involved in PTGER4 and PRKAA1. RESULTS: Overall, rs10036575 in PTGER4 (OR = 0.82, p = 0.029), rs10074991 (OR = 0.82, p = 0.024) and rs13361707 (OR = 0.82, p = 0.030) in PRKAA1 were associated with susceptibility to GC. Stratification analysis revealed that the effects of these SNPs in PTGER4 and PRKAA1 on GC susceptibility were dependent on smoking and were associated with a reduced risk of adenocarcinoma (p < 0.05). Bioinformatics analysis showed an association between SNPs and corresponding gene expression (p < 0.05), and PRKAA1 may affect GC by mediating RhoA. CONCLUSION: This study suggests that PTGER4 and PRKAA1 SNPs might affect the susceptibility of GC, providing a new biological perspective for GC risk assessment, pathogenesis exploration, and personalized treatment.

RBFOX2 recognizes N6-methyladenosine to suppress transcription and block myeloid leukaemia differentiation.

N6-methyladenosine (m6A) methylation can be deposited on chromatin-associated RNAs (caRNAs) by the RNA methyltransferase complex (MTC) to regulate chromatin state and transcription. However, the mechanism by which MTC is recruited to distinct genomic loci remains elusive. Here we identify RBFOX2, a well-studied RNA-binding protein, as a chromatin factor that preferentially recognizes m6A on caRNAs. RBFOX2 can recruit RBM15, an MTC component, to facilitate methylation of promoter-associated RNAs. RBM15 also physically interacts with YTHDC1 and recruits polycomb repressive complex 2 (PRC2) to the RBFOX2-bound loci for chromatin silencing and transcription suppression. Furthermore, we found that this RBFOX2/m6A/RBM15/YTHDC1/PRC2 axis plays a critical role in myeloid leukaemia. Downregulation of RBFOX2 notably inhibits survival/proliferation of acute myeloid leukaemia cells and promotes their myeloid differentiation. RBFOX2 is also required for self-renewal of leukaemia stem/initiation cells and acute myeloid leukaemia maintenance. Our study presents a pathway of m6A MTC recruitment and m6A deposition on caRNAs, resulting in locus-selective chromatin regulation, which has potential therapeutic implications in leukaemia.

An in-library ligation strategy and its application in CRISPR/Cas9 screening of high-order gRNA combinations.

Simultaneous targeting multiple genes is a big advantage of CRISPR (clustered regularly interspaced short palindromic repeats) genome editing but challenging to achieve in CRISPR screening. The crosstalk among genes or gene products is a common and fundamental mechanism to ensure cellular stability and functional diversity. However, the screening approach to map high-order gene combinations to the interesting phenotype is still lacking. Here, we developed a universal in-library ligation strategy and applied it to generate multiplexed CRISPR library, which could perturb four pre-designed targets in a cell. We conducted in vivo CRISPR screening for potential guide RNA (gRNA) combinations inducing anti-tumor immune responses. Simultaneously disturbing a combination of three checkpoints in CD8+ T cells was demonstrated to be more effective than disturbing Pdcd1 only for T cell activation in the tumor environment. This study developed a novel in-library ligation strategy to facilitate the multiplexed CRISPR screening, which could extend our ability to explore the combinatorial outcomes from coordinated gene behaviors.

Salubrinal Protects Against Cisplatin-Induced Cochlear Hair Cell Endoplasmic Reticulum Stress by Regulating Eukaryotic Translation Initiation Factor 2α Signalling.

Objective: Cisplatin is a broad-spectrum anti-tumour drug commonly used in clinical practice. However, its ototoxicity greatly limits its clinical application, and no effective method is available to prevent this effect. Endoplasmic reticulum stress (ERS) is reportedly involved in cisplatin ototoxicity, but the exact mechanism remains unclear. Therefore, this study aimed to investigate the role of eukaryotic translation initiation factor 2α (eIF2α) signalling and its dephosphorylation inhibitor salubrinal in cisplatin ototoxicity. Methods: We evaluated whether salubrinal could protect against cisplatin-induced damage in House Ear Institute-Organ of Corti 1 (HEI-OC1) cells and mouse cochlear explants. By knocking down eIF2α, we elucidated the vital role of eIF2α in cisplatin-induced damage in HEI-OC1 cells. Whole-mount immunofluorescent staining and confocal microscopy of mouse cochlear explants and HEI-OC1 cells were performed to analyse cisplatin-induced damage in cochlear hair cells and the auditory cell line. Results: Data suggested salubrinal attenuated cisplatin-induced hair cell injury by inhibiting apoptosis. In addition, salubrinal significantly reduced ERS levels in hair cells via eIF2α signalling, while eIF2α knockdown inhibited the protective effect of salubrinal. Significance: Salubrinal and eIF2α signalling play a role in protecting against cisplatin-induced ototoxicity, and pharmacological inhibition of eIF2α-mediated ERS is a potential treatment for cisplatin-induced damage in the cochlea and HEI-OC1 cells.

Intestinal Flora: A Potential Mechanism by Which Yinlai Decoction Treats Lipopolysaccharide-Induced Pneumonia.

Background: We intended to explore the mechanism of Yinlai decoction in the treatment of lipopolysaccharide (LPS)-induced pneumonia from the perspective of intestinal flora. Methods: Thirty Sprague-Dawley rats were randomly assigned to the blank control group (N), the pneumonia group (P), and the Yinlai decoction group (PT). The rat pneumonia model was established using LPS inhalation (0.5 mg/mL, 5 mL, 30 min/day, 3 days). Yinlai decoction was administered intragastrically (2 mL/100 g, 3 days). Lung tissue pathology, organ indexes, serum inflammatory factors, tumor necrosis factor-alpha (TNF-α), and intestinal flora changes were measured. Results: Lung tissue inflammation was prevented by Yinlai decoction. IL-6 levels showed a higher tendency to be higher, and IL-12 and TNF-α were significantly higher in the PT group than in the P group. The structure of the intestinal flora in the P differed from that in the N. The relative abundance of 10 out of 12 microflora was significantly higher in the P group than in the N and PT groups. In the PT group, the structure and the distribution of microbial groups were like those of the N group. Conclusions: Yinlai decoction inhibited LPS-induced lung and systemic inflammation in rats and may help the intestinal flora restore equilibrium by inhibiting the colonization of pathogenic bacteria and adjusting the ratio between probiotics and pathogenic bacteria. Intestinal flora may serve as a mediator of Yinlai decoction's effect on LPS-induced pneumonia.

New Chromatin Run-On Reaction Enables Global Mapping of Active RNA Polymerase Locations in an Enrichment-free Manner.

The development of a simple and cost-effective method to map the distribution of RNA polymerase II (RNPII) genome-wide at a high resolution is highly beneficial to study cellular transcriptional activity. Here we report a mutation-based and enrichment-free global chromatin run-on sequencing (mGRO-seq) technique to locate active RNPII sites genome-wide at near-base resolution. An adenosine triphosphate (ATP) analog named N6-allyladenosine triphosphate (a6ATP) was designed and could be incorporated into nascent RNAs at RNPII-located positions during a chromatin run-on reaction. By treatment of the run-on RNAs with a mild iodination reaction and subjection of the products to reverse transcription into complementary DNA (cDNA), base mismatch occurs at the original a6A incorporation sites, thus making the RNPII locations detected in the high-throughput cDNA sequencing. The mGRO-seq yields both the map of RNPII sites and the chromatin RNA abundance and holds great promise for the study of single-cell transcriptional activity.

Single-cell transcriptome analysis uncovers the molecular and cellular characteristics of thin endometrium.

Infertility is a social and medical problem around the world and the incidence continues to rise. Thin endometrium (TE) is a great challenge of infertility treatment, even by in vitro fertilization and embryo transfer. It is widely believed that TE impairs endometrium receptivity. However, only a few studies have explained the molecular mechanism. Herein, in order to reveal the possible mechanism, we sampled endometrium from a TE patient and a control volunteer and got a transcriptomic atlas of 18 775 individual cells which was constructed using single-cell RNA sequencing, and seven cell types have been identified. The cells were acquired during proliferative and secretory phases, respectively. The proportion of epithelial cells and stromal cells showed a significant difference between the TE group and the control group. In addition, differential expressed genes (DEGs) in diverse cell types were revealed, the enriched pathways of DEGs were found closely related to the protein synthesis in TE of both proliferative and secretory phases. Some DEGs can influence cell-type ratio and impaired endometrial receptivity in TE. Furthermore, divergent expression of estrogen receptors 1 and progesterone receptors in stromal and epithelial cells were compared in the TE sample from the control. The cellular and molecular heterogeneity found in this study provided valuable information for disclosing the mechanisms of impaired receptivity in TE.

The circular RNA hsa_circ_000780 as a potential molecular diagnostic target for gastric cancer.

BACKGROUND: The present study aimed to identify a specific circular RNA (circRNA) for early diagnosis of gastric cancer (GC). METHODS: Totally 82 patients with GC, 30 with chronic nonatrophic gastritis and 30 with chronic atrophic gastritis were included in this study. Four of the 82 GC patients were selected for screening. Total RNA from malignant and adjacent tissue samples was extracted, and circRNAs in four patients were screened. According to the screening results, the eight most upregulated and downregulated circRNAs with a statistically significant association with GC were identified by real-time fluorescent quantitative polymerase chain reaction (PCR). Then, the most regulated circRNA was selected for further sensitivity and specificity assessments. CircRNA expression was examined by quantitative reverse transcriptase PCR in 78 GC (21 and 57 early and advanced GC, respectively) and adjacent tissue samples, as well as in gastric fluid samples from 30 patients with chronic nonatrophic gastritis, 30 with chronic atrophic gastritis, and 78 GC. RESULTS: A total of 445 circRNAs, including 69 upregulated and 376 downregulated circRNAs, showed significantly altered expression in GC tissue samples. Hsa_circ_000780 was significantly downregulated in 80.77% of GC tissue samples, with levels in GC tissue samples correlating with tumor size, tumor stage, T stage, venous invasion, carcinoembryonic antigen amounts, and carbohydrate antigen 19-9 levels. Strikingly, this circRNA was found in the gastric fluid of patients with early and advanced GC. CONCLUSIONS: The present study uncovered a new circRNA expression profile in human GC, with hsa_circ_000780 significantly downregulated in GC tissue and gastric fluid specimens. These findings indicate that hsa_circ_000780 should be considered a novel biomarker for early GC screening.

Berberine-Based Heterogeneous Linear Supramolecules Neutralized the Acute Nephrotoxicity of Aristolochic Acid by the Self-Assembly Strategy.

Aristolochic acid (AA) has been reported to cause a series of health problems, including aristolochic acid nephropathy and liver cancer. However, AA-containing herbs are highly safe in combination with berberine (Ber)-containing herbs in traditional medicine, suggesting the possible neutralizing effect of Ber on the toxicity of AA. In the present study, in vivo systematic toxicological experiments performed in zebrafish and mice showed that the supramolecule self-assembly formed by Ber and AA significantly reduced the toxicity of AA and attenuated AA-induced acute kidney injury. Ber and AA can self-assemble into linear heterogenous supramolecules (A-B) via electrostatic attraction and π-π stacking, with the hydrophobic groups outside and the hydrophilic groups inside during the drug combination practice. This self-assembly strategy may block the toxic site of AA and hinder its metabolism. Meanwhile, A-B linear supramolecules did not disrupt the homeostasis of gut microflora as AA did. RNA-sequence analysis, immunostaining, and western blot of the mice kidney also showed that A-B supramolecules almost abolished the acute nephrotoxicity of AA in the activation of the immune system and tumorigenesis-related pathways.

Tumor Fibroblast-Derived FGF2 Regulates Expression of SPRY1 in Esophageal Tumor-Infiltrating T Cells and Plays a Role in T-cell Exhaustion.

T-cell exhaustion was initially identified in chronic infection in mice and was subsequently described in humans with cancer. Although the distinct signature of exhausted T (TEX) cells in cancer has been well investigated, the molecular mechanism of T-cell exhaustion in cancer is not fully understood. Using single-cell RNA sequencing, we report here that TEX cells in esophageal cancer are more heterogeneous than previously clarified. Sprouty RTK signaling antagonist 1 (SPRY1) was notably enriched in two subsets of exhausted CD8+ T cells. When overexpressed, SPRY1 impaired T-cell activation by interacting with CBL, a negative regulator of ZAP-70 tyrosine phosphorylation. Data from the Tumor Immune Estimation Resource revealed a strong correlation between FGF2 and SPRY1 expression in esophageal cancer. High expression of FGF2 was evident in fibroblasts from esophageal cancer tissue and correlated with poor overall survival. In vitro administration of FGF2 significantly upregulated expression of SPRY1 in CD8+ T cells and attenuated T-cell receptor-triggered CD8+ T-cell activation. A mouse tumor model confirmed that overexpression of FGF2 in fibroblasts significantly upregulated SPRY1 expression in TEX cells, impaired T-cell cytotoxic activity, and promoted tumor growth. Thus, these findings identify FGF2 as an important regulator of SPRY1 expression involved in establishing the dysfunctional state of CD8+ T cells in esophageal cancer. SIGNIFICANCE: These findings reveal FGF2 as an important regulator of SPRY1 expression involved in establishing the dysfunctional state of CD8+ T cells and suggest that inhibition of FGF2 has potential clinical value in ESCC. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/24/5583/F1.large.jpg.

Cold-Inducible RNA-Binding Protein Prevents an Excessive Heart Rate Response to Stress by Targeting Phosphodiesterase.

RATIONALE: The stress response of heart rate, which is determined by the plasticity of the sinoatrial node (SAN), is essential for cardiac function and survival in mammals. As an RNA-binding protein, CIRP (cold-inducible RNA-binding protein) can act as a stress regulator. Previously, we have documented that CIRP regulates cardiac electrophysiology at posttranscriptional level, suggesting its role in SAN plasticity, especially upon stress conditions. OBJECTIVE: Our aim was to clarify the role of CIRP in SAN plasticity and heart rate regulation under stress conditions. METHODS AND RESULTS: Telemetric ECG monitoring demonstrated an excessive acceleration of heart rate under isoprenaline stimulation in conscious CIRP-KO (knockout) rats. Patch-clamp analysis and confocal microscopic Ca2+ imaging of isolated SAN cells demonstrated that isoprenaline stimulation induced a faster spontaneous firing rate in CIRP-KO SAN cells than that in WT (wild type) SAN cells. A higher concentration of cAMP-the key mediator of pacemaker activity-was detected in CIRP-KO SAN tissues than in WT SAN tissues. RNA sequencing and quantitative real-time polymerase chain reaction analyses of single cells revealed that the 4B and 4D subtypes of PDE (phosphodiesterase), which controls cAMP degradation, were significantly decreased in CIRP-KO SAN cells. A PDE4 inhibitor (rolipram) abolished the difference in beating rate resulting from CIRP deficiency. The mechanistic study showed that CIRP stabilized the mRNA of Pde4b and Pde4d by direct mRNA binding, thereby regulating the protein expression of PDE4B and PDE4D at posttranscriptional level. CONCLUSIONS: CIRP acts as an mRNA stabilizer of specific PDEs to control the cAMP concentration in SAN, maintaining the appropriate heart rate stress response.

In Vivo Observation of Lens Regeneration in Rat Using Ultra-Long Scan Depth Optical Coherence Tomography.

Purpose: To evaluate morphologic changes of lens regeneration in rats in vivo after extracapsular lens extraction (ECLE) by ultra-long scan depth optical coherence tomography (UL-OCT). Methods: A total of 42 Sprague-Dawley rats were used in this study. We performed ECLE on the right eyes of animals in the surgery group (n = 34). Biomicroscopy and UL-OCT scans were carried out for the surgery group immediately (within 1 hour postoperatively) and at days 1 and 3, weeks 1 and 2, and months 1, 2, and 3 postoperatively. After in vivo examination, three animals of the surgery group were euthanized at each time point for histology study, while the other 10 animals were examined continuously at those time points. The regenerated lens was evaluated in OCT images at 2 and 3 months postoperatively. The control group consisted of eight untreated rats that had OCT examination at the age of 5 months. Results: Lens regeneration could be observed from 2 weeks postoperatively. Regeneration was mainly at the peripheral capsular bag in the first month and central region thereafter. The average thickness of regenerated lenses was 2222 ± 309 and 2324 ± 352 µm at 2 and 3 months, respectively. Regeneration was faster in the first 2 months and slowed down thereafter. Although anterior capsule opening and posterior capsule adhesion and wrinkling existed, the regenerated lens still could form a relatively regular shape, however, the size was much smaller than that of the normal lenses from rats with the same age. Conclusions: Ultra-long OCT provides longitudinal data of the process of lens regeneration on a single individual rat in vivo, which may allow one to follow and compare the lens regenerative process under different interventions or therapy after ECLE in rats.

Epithelium-on corneal collagen crosslinking for management of advanced keratoconus.

PURPOSE: To report the 1-year visual and keratometric results of epithelium-on corneal collagen crosslinking (CXL) for advanced keratoconus (median maximum keratometry [K] ≥58.0 diopters [D]). SETTING: School of Ophthalmology and Optometry and Eye Hospital of Wenzhou Medical College, Wenzhou, China. DESIGN: Prospective case series. METHODS: Patients with bilateral progressive keratoconus had tetracaine-enhanced epithelium-on CXL. The worse eye had CXL, and the fellow eye was not treated. Results were reported 1, 3, 6, and 12 months postoperatively. The outcomes were compared with those in the fellow untreated eyes. RESULTS: Twenty-one eyes of 21 patients with a median age of 20.4 years (interquartile range [IQR], 9.5 years) were treated. A significant improvement in postoperative uncorrected distance visual acuity was observed at 12 months (P = .002). Postoperative corrected distance visual acuity improved at 6 months and 12 months (P ≤ .009) compared with baseline values. The maximum K decreased by 1.63 D from the median preoperative maximum K of 62.7 D (IQR, 12.9 D) at 12 months (P < .001). The reduction in maximum K was higher after CXL than in untreated eyes at the end of 12 months (P = .001). Correlation analysis between the preoperative maximum K values and the change over 6 to 12 months between different studies showed a significant correlation (r = -0.764, P < .001; Spearman correlation). CONCLUSIONS: Epithelium-on CXL was an effective treatment for patients with advanced keratoconus. A higher preoperative maximum K value correlated with greater corneal flattening after CXL. FINANCIAL DISCLOSURE: None of the authors has a financial or proprietary interest in any material or method mentioned.

Atomic structure of the apoptosome: mechanism of cytochrome c- and dATP-mediated activation of Apaf-1.

The apoptotic protease-activating factor 1 (Apaf-1) controls the onset of many known forms of intrinsic apoptosis in mammals. Apaf-1 exists in normal cells as an autoinhibited monomer. Upon binding to cytochrome c and dATP, Apaf-1 oligomerizes into a heptameric complex known as the apoptosome, which recruits and activates cell-killing caspases. Here we present an atomic structure of an intact mammalian apoptosome at 3.8 Å resolution, determined by single-particle, cryo-electron microscopy (cryo-EM). Structural analysis, together with structure-guided biochemical characterization, uncovered how cytochrome c releases the autoinhibition of Apaf-1 through specific interactions with the WD40 repeats. Structural comparison with autoinhibited Apaf-1 revealed how dATP binding triggers a set of conformational changes that results in the formation of the apoptosome. Together, these results constitute the molecular mechanism of cytochrome c- and dATP-mediated activation of Apaf-1.

Conformational switch of polyglutamine-expanded huntingtin into benign aggregates leads to neuroprotective effect.

The abundant accumulation of inclusion bodies containing polyglutamine-expanded mutant huntingtin (mHTT) aggregates is considered as the key pathological event in Huntington's disease (HD). Here, we demonstrate that FKBP12, an isomerase that exhibits reduced expression in HD, decreases the amyloidogenicity of mHTT, interrupts its oligomerization process, and structurally promotes the formation of amorphous deposits. By combining fluorescence-activated cell sorting with multiple biophysical techniques, we confirm that FKBP12 reduces the amyloid property of these ultrastructural-distinct mHTT aggregates within cells. Moreover, the neuroprotective effect of FKBP12 is demonstrated in both cellular and nematode models. Finally, we show that FKBP12 also inhibit the fibrillization process of other disease-related and aggregation-prone peptides. Our results suggest a novel function of FKBP12 in ameliorating the proteotoxicity in mHTT, which may shed light on unraveling the roles of FKBP12 in different neurodegenerative diseases and developing possible therapeutic strategies.

Evaluation of the efficacy of excimer laser ablation of cross-linked porcine cornea.

BACKGROUND: Combination of riboflavin/UVA cross-linking (CXL) and excimer laser ablation is a promising therapy for treating corneal ectasia. The cornea is strengthened by cross-linking, while the irregular astigmatism is reduced by laser ablation. This study aims to compare the efficacy of excimer laser ablation on porcine corneas with and without cross-linking. METHODS AND FINDINGS: The porcine cornea was de-epithelialized and treated with 0.1% riboflavin solution for 30 minutes. A half of the cornea was exposed to UVA-radiation for another 30 minutes while the controlled half of the cornea was protected from the UVA using a metal shield. Photo therapeutic keratectomy (PTK) was then performed on the central cornea. Corneal thickness of 5 paired locations on the horizontal line, ± 0.5, ± 1.0, ± 1.5, ± 2.0, and ± 2.5 mm from the central spot, were measured using optical coherence tomography prior to and after PTK. The ablation depth was then determined by the corneal thickness. There was a 9% difference (P<0.001) in the overall ablation depth between the CXL-half corneas (158 ± 22 µm) and the control-half corneas (174 ± 26 µm). The ablation depths of all 5 correspondent locations on the CXL-half were significantly smaller (P<0.001). CONCLUSION: The efficacy of the laser ablation seems to be lower in cross-linked cornea. Current ablation algorithms may need to be modified for cross-linked corneas.

[Effect of small interfering RNA targeting p63 on the proliferation and invasiveness of human cholangiocarcinoma cells in vitro].

OBJECTIVE: To construct a small interfering RNA (siRNA) vector targeting p63 and observe its effect on the proliferation and invasiveness of human cholangiocarcinoma cells in vitro. METHODS: Real-time PCR was used to examine the expression of p63 in human cholangiocarcinoma QBC939 cells. The recombinant lentivirus shRNA-p63 vector was constructed and transfected into QBC939 cells via Lipofectamine 2000 to establish a cholangiocarcinoma cell line with stable expression of siRNA-p63. The interfering efficiency of the siRNA targeting p63 was assessed using Western blotting. MTT and soft agar colony formation assays were used to evaluate the changes in the cell proliferation, and Boyden test was employed to observe the cell invasiveness after the transfection. RESULTS: QBC939 cells showed a high expression of p63. The recombinant lentivirus shRNA-p63 vector was successfully constructed as verified by sequencing. Transfection with the vector significantly suppressed the proliferation and invasiveness of QBC939 cells. CONCLUSION: Down-regulation of p63 can inhibit the proliferation and invasiveness of human cholangiocarcinoma QBC939 cells in vitro.