Spatial transcriptomics reveals gene interactions and signaling pathway dynamics in rat embryos with anorectal malformation.

Anorectal malformation (ARM) is a prevalent early pregnancy digestive tract anomaly. The intricate anatomy of the embryonic cloaca region makes it challenging for traditional high-throughput sequencing methods to capture location-specific information. Spatial transcriptomics was used to sequence libraries of frozen sections from embryonic rats at gestational days (GD) 14 to 16, covering both normal and ARM cases. Bioinformatics analyses and predictions were performed using methods such as WGCNA, GSEA, and PROGENy. Immunofluorescence staining was used to verify gene expression levels. Gene expression data was obtained with anatomical annotations of clusters, focusing on the cloaca region's location-specific traits. WGCNA revealed gene modules linked to normal and ARM cloacal anatomy development, with cooperation between modules on GD14 and GD15. Differential gene expression profiles and functional enrichment were presented. Notably, protein levels of Pcsk9, Hmgb2, and Sod1 were found to be downregulated in the GD15 ARM hindgut. The PROGENy algorithm predicted the activity and interplay of common signaling pathways in embryonic sections, highlighting their synergistic and complementary effects. A competing endogenous RNA (ceRNA) regulatory network was constructed from whole transcriptome data. Spatial transcriptomics provided location-specific cloaca region gene expression. Diverse bioinformatics analyses deepened our understanding of ARM's molecular interactions, guiding future research and providing insights into gene regulation in ARM development.

Exploring the origin of a unique mutant allele in twin-tail goldfish using CRISPR/Cas9 mutants.

Artificial selection has been widely applied to genetically fix rare phenotypic features in ornamental domesticated animals. For many of these animals, the mutated loci and alleles underlying rare phenotypes are known. However, few studies have explored whether these rare genetic mutations might have been fixed due to competition among related mutated alleles or if the fixation occurred due to contingent stochastic events. Here, we performed genetic crossing with twin-tail ornamental goldfish and CRISPR/Cas9-mutated goldfish to investigate why only a single mutated allele-chdS with a E127X stop codon (also called chdAE127X)-gives rise to the twin-tail phenotype in the modern domesticated goldfish population. Two closely related chdS mutants were generated with CRISPR/Cas9 and compared with the E127X allele in F2 and F3 generations. Both of the CRISPR/Cas9-generated alleles were equivalent to the E127X allele in terms of penetrance/expressivity of the twin-tail phenotype and viability of carriers. These findings indicate that multiple truncating mutations could have produced viable twin-tail goldfish. Therefore, the absence of polymorphic alleles for the twin-tail phenotype in modern goldfish likely stems from stochastic elimination or a lack of competing alleles in the common ancestor. Our study is the first experimental comparison of a singular domestication-derived allele with CRISPR/Cas9-generated alleles to understand how genetic fixation of a unique genotype and phenotype may have occurred. Thus, our work may provide a conceptual framework for future investigations of rare evolutionary events in domesticated animals.

A prospective and consecutive study assessing short-term clinical and radiographic outcomes of Chinese domestically manufactured 3D printing trabecular titanium acetabular cup for primary total hip arthroplasty: evaluation of 236 cases.

Purpose: We prospectively evaluate the short-term clinical and radiographic outcomes of the only Chinese domestically produced trabecular titanium acetabular cup(3D ACT™ cup) in primary total hip arthroplasty (THA), aiming to provide evidence-based support for its clinical application. Methods: A total of 236 patients, who underwent primary THA using 3D ACT™ cup in the Department of Joint Surgery at our hospital between January 2017 and June 2019, were included in this study. General patient data, imaging information, functional scores, and complications were collected to evaluate the early clinical efficacy. Results: All patients were followed up for 33-52 months, with an average of (42.2 ± 9.2) months. At the last follow-up, the preoperative HHS score increased significantly from 43.7 ± 6.8 to 85.6 ± 9.3 points (P < 0.01). Similarly, the preoperative WOMAC scores showed significant improvement from 59.2 ± 5.8 to 13.1 ± 3.5 points (P < 0.01). 92.3% of the patients expressed satisfaction or high satisfaction with the clinical outcome. Furthermore, 87.7% of the acetabular cups were positioned within the Lewinnek safe zone, achieving successful reconstruction of the acetabular rotation center. The cup survival rate at the last follow-up was 100%. Conclusions: The utilization of the only Chinese domestically manufactured 3D printing trabecular titanium acetabular cup in primary THA demonstrated favorable short-term clinical and radiographic outcomes. The acetabular cup exhibits excellent initial stability, high survival rate, and favorable osseointegration, leading to a significant enhancement in pain relief and functional improvement. In the future, larger sample sizes and multicenter prospective randomized controlled trials will be required to validate the long-term safety and effectiveness of this 3D ACT™ cup.

Rack1-mediated ferroptosis affects hindgut development in rats with anorectal malformations: Spatial transcriptome insights.

Anorectal malformation (ARM), a common congenital anomaly of the digestive tract, is a result of insufficient elongation of the urorectal septum. The cytoplasmic protein Receptor of Activated C-Kinase 1 (Rack1) is involved in embryonic neural development; however, its role in embryonic digestive tract development and ARM formation is unexplored. Our study explored the hindgut development and cell death mechanisms in ARM-affected rats using spatial transcriptome analysis. We induced ARM in rats by administering ethylenethiourea via gavage on gestational day (GD) 10. On GDs 14-16, embryos from both normal and ARM groups underwent spatial transcriptome sequencing, which identified key genes and signalling pathways. Rack1 exhibited significant interactions among differentially expressed genes on GDs 15 and 16. Reduced Rack1 expression in the ARM-affected hindgut, verified by Rack1 silencing in intestinal epithelial cells, led to increased P38 phosphorylation and activation of the MAPK signalling pathway. The suppression of this pathway downregulated Nqo1 and Gpx4 expression, resulting in elevated intracellular levels of ferrous ions, reactive oxygen species (ROS) and lipid peroxides. Downregulation of Gpx4 expression in the ARM hindgut, coupled with Rack1 co-localisation and consistent mitochondrial morphology, indicated ferroptosis. In summary, Rack1, acting as a hub gene, modulates ferrous ions, lipid peroxides, and ROS via the P38-MAPK/Nqo1/Gpx4 axis. This modulation induces ferroptosis in intestinal epithelial cells, potentially influencing hindgut development during ARM onset.

Epidemiology of spinal cord injury and spinal cord injury-induced urinary tract stones in Taiwan: A 2005-2015 population-based cohort study.

CONTEXT: Patients with spinal cord injury (SCI) can develop urinary tract stones (UTSs) up to years after the injury, which is especially common in the first few months. However, relevant epidemiological studies and up-to-date epidemiological data for SCI in Taiwan are lacking. PURPOSE: To estimate SCI and SCI-induced UTS incidence and trauma severity, neurological deficits, and injury site in patients with SCI-induced UTSs in Taiwan. DESIGN: Retrospective cohort study.Patient sample: Taiwan National Health Insurance Research Database (NHIRD) data and death data from the Department of Health and Welfare Data Science Center (HWDC) collected over 2005-2015 from 13,977 patients with SCI aged >18 years. OUTCOME MEASURES: Cumulative incidence (CI), incidence density (ID), relative ratios (RRs), odds ratios (ORs), and hazard ratios (HRs) were measured. METHODS: By using Cox regression, we assessed UTS risk in patients with SCI. RESULTS: Although standardized SCI incidence demonstrated a decreasing trend annually, the average annual incidence remained at 60.4 per million. Most (65.7%) of the included patients were men. SCI incidence was 1.98 times higher in men than in women. The most common injury site was the cervical spine (63.8%); the incidence at this site was 2.83 times higher in men than in women. Most (76.1%) of the patients had traumatic SCI (TSCI), and the standardized incidence of TSCI and non-TSCI was 45.9 and 14.4 per million, respectively. 46.1% of the patients had severe SCI (RISS ≥ 16). Over the 11-year follow-up period, UTSs occurred in 10.4% of the patients, with a standardized incidence of 2.39 per 100 person-years, and UTS risk was 1.56 times higher in men than in women. Age of 45-65 years, SCIs at multiple sites, and neurological deficits (e.g. paraplegia) were noted to be UTS risk factors. Finally, UTS onset mainly occurred in the first year after SCI. CONCLUSION: The risk of UTS among patients with SCI is influenced by age, sex, injury site, and paraplegia but not by paralysis resulting from other neurological deficits. Even though SCI incidence is declining annually, severe SCI remains a significant issue. Therefore, continuing to reduce SCI incidence and strengthening urinary tract management in patients with SCI are essential for reducing UTS occurrence and their impact on health.

CircJag1 promotes apoptosis of ethylene thiourea-exposed anorectal malformations through sponging miR-137-3p by regulating Sox9 and suppressing Wnt/ß-catenin pathway during the hindgut development of rat embryos.

Anorectal malformations (ARMs) are common birth defects involving congenital structural anomalies of the gastrointestinal tract. As an important component of non-coding RNAs, circular RNAs (circRNAs) widely participate in the digestive system development; however, the specific molecular mechanism of their involvement in ARM occurrence remains obscure. Herein, we generated rat models of ARMs induced by ethylene thiourea. A novel circRNA (circJag1) was screened and identified by RNA-Seq, which is remarkably upregulated in hindgut tissues of ARM rat embryos. In vivo experiments, colocation analysis via fluorescence in situ hybridization, and immunofluorescence further demonstrated that the disordered circJag1/miR-137-3p/Sox9 expression caused a spatiotemporal imbalance in the urorectal septum (URS) of ARMs. In vitro, functional assays confirmed that circJag1 upregulation resulted in the degradation of nuclear ß-catenin, C-myc, and Cyclin D1 in rat intestinal epithelial cells, as well as the promotion of apoptosis and suppression of cell proliferation. Mechanistically, dual-luciferase reporter assay and RNA immunoprecipitation assay indicated that circJag1 acted as a miR-137-3p sponge, thereby inhibiting its repressive effect on its target Sox9. Further experiments showed that a loss of Sox9 abolished the circJag1-mediated increase in apoptosis. In conclusion, aberrantly high circJag1 expression promotes epithelial apoptosis by suppressing the canonical Wnt/ß-catenin pathway via the miR-137-3p/Sox9 axis, which leads to fusion failure of the URS and cloacal membrane, and eventually contributed to ARMs. Our achievements might boost the comprehension of ARM pathogenesis and could provide a novel candidate target for the development of therapies for ARMs to complement surgical treatment.

miR-141-3p affects ß-catenin signaling and apoptosis by targeting Ubtd2 in rats with anorectal malformations.

Anorectal malformations (ARMs) are the most common gastrointestinal malformations. miR-141-3p was obtained from whole-transcriptome sequencing, and Ub domain-containing protein 2 (Ubtd2) was predicted as the target gene. An ARM rat model was induced using ethylenethiourea. Fluorescence in situ hybridization and immunofluorescence were used to detect the spatiotemporal expression of miR-141-3p and Ubtd2, respectively. A dual-luciferase reporter assay confirmed their targeting relationship, and cell proliferation and apoptosis were investigated after transfection in the intestinal epithelium (IEC-6). Additionally, western blotting and co-immunoprecipitation were used to examine the protein levels and the endogenous binding relationship. miR-141-3p was downregulated in the ARM group, whereas Ubtd2 increased and colocalized with TUNEL-positive cells. After miR-141-3p inhibition, protein expression of USP5 and ß-catenin was affected via Ubtd2, and USP5 could bind to both Ubtd2 and ß-catenin. Flow cytometry analysis and caspase 3/7 staining demonstrated that downregulated miR-141-3p promoted cell apoptosis through Ubtd2. In summary, targeting Ubtd2 decreased in miR-141-3p and promoted apoptosis of intestinal epithelium and regulated ß-catenin expression. This may cause aberrant apoptosis during hindgut development and mediate the imbalance of ß-catenin signaling in the cloaca, further affecting the occurrence of ARMs.

Analysis of Differential Diagnosis of Benign and Malignant Partially Cystic Thyroid Nodules Based on Ultrasound Characterization With a TIRADS Grade-4a or Higher Nodules.

Partially cystic thyroid nodules (PCTNs) are a kind of thyroid nodule with both solid and cystic components, and are usually misdiagnosed as benign nodules. The objective of this study was to determine the ultrasound (US) characterizations with a TIRADS Grade-4a or higher partially cystic thyroid nodules (PCTNs) which are associated with being malignant or benign. In this study, 133 PCTNs with a TIRADS Grade-4a or higher were enrolled; 83 were malignant and 50 were benign. TI-RADS classification can detect malignant PCTNs, and its sensitivity, specificity, positive predictive value, negative predictive value, and accuracy are 39.8%, 96.0%, 94.3%, 49.0%, and 60.9%, respectively. Univariate analyses revealed that nodule shape, margin, and structure were related to PCTNs' being benign and malignant, among which nodules taller-than-wide, with an irregular shape, non-smooth margin, eccentric sharp angle, or edge sharp angle were significantly associated with malignancy while ovoid to round nodules, smooth margins, multiple separation, and eccentric obtuse angle structures were significantly associated with a benign nature. For the solid part of PCTNs, its free margin, echo, and calcification are related to benign and malignant PCTNs. Among them, the free margin of the solid part is non-smooth, hypoechoic, and microcalcification, which are related to malignant PCTNs, while the free margin of the solid part is smooth, isoechoic, macrocalcification, non-calcification and are related to benign PCTNs. Calcification of solid part and free margin are important factors for predicting malignant PCTNs. In addition, nodules' composition, blood flow signal, and other factors had nothing to do with PCTNs' being benign or malignant. In the multivariate Logistic regression analysis, solid part calcification (OR: 17.28; 95%CI: 5.14~58.08) and free margin (OR: 3.18; 95%CI: 1.01~10.00) were revealed to be the strongest independent predictors for malignancy (P<0.05). Our study indicated that understanding the ultrasound characteristics of malignant PCTNs, to avoid misdiagnosed PCTNs patients, is important to make a precise diagnosis and prognosis of PCTNs.

Probiotic in the prevention of ventilator-associated pneumonia in critically ill patients: evidence from meta-analysis and trial sequential analysis of randomized clinical trials.

BACKGROUND: Probiotic might have a role in the prevention of ventilator-associated pneumonia (VAP) among mechanically ventilated patients, but the efficacy and safety remained inconsistent. The aim of this systematic review and meta-analysis was to evaluate the efficacy and safety of probiotic (prebiotic, synbiotic) versus placebo in preventing VAP in critically ill patients undergoing mechanical ventilation. METHODS: PubMed, Embase and the Cochrane library databases were searched to 10 October 2021 without language restriction for randomized or semi-randomized controlled trials evaluating probiotic (prebiotic, synbiotic) vs. placebo in prevention of VAP in critically ill mechanically ventilated patients. The pooled relative risk (RR) along with 95% confidence intervals (CI) were combined using a random-effects model. Furthermore, the trial sequential analysis (TSA) and subgroup analyses were performed. Statistical significance was regarded as P < 0.05. RESULTS: Twenty-three trials involving 5543 patients were eligible for this meta-analysis. The combined RR of decreasing the risk of VAP by probiotic was 0.67 (0.56, 0.81) for all eligible studies, 0.69 (n = 5136; 95% CI = 0.57 to 0.84; P < 0.01) for adults studies and 0.55 (n = 407; 95%CI = 0.31 to 0.99; P = 0.046) for neonates/children studies. Additionally, the above-mentioned positive finding in 20 adults studies was verified by the results of TSA, subgroup analyses and cumulative meta-analysis. Ample evidences demonstrated a 31% decrease in RR of incidence of VAP was noted when prophylactic probiotic therapy was administrated among adult patients. Finally, there were no effects on the ICU/hospital/28-/90-day mortality, bacteremia, CRBSI, diarrhea, ICU-acquired infections, infectious complications, pneumonia, UTI and wound infection between two groups (P > 0.05 for all). CONCLUSIONS: Based on the results of our study, the current evidences suggested that prophylactic administration of probiotic might be utilized as a preventive method for VAP in neonates/children and adults patients who required mechanical ventilation. However, further large, high-quality RCTs are warranted to assess the efficacy and safety of probiotic treatment in critically ill patients, especially for the neonates/children studies and the long-term consequences of this therapy.

Upregulation of PPPDE1 contributes to anorectal malformations via the mitochondrial apoptosis pathway during hindgut development in rats.

Congenital anorectal malformations (ARMs) are among the most prominent deformities of the gastrointestinal tract; however, their precise aetiology remains obscure. Immunohistochemistry demonstrated that, in the ARM group, the PPPDE1-positive cells were widely distributed in the hindgut epithelial tissue from GD13 to GD16. Immunofluorescence revealed that most TUNEL-, Bax-, and Cytochrome C (Cyt C)-positive cells overlapped with PPPDE1-positive cells in the urorectal septum (URS). Western blotting and quantitative real-time RT-PCR revealed that PPPDE1 levels were significantly higher in the ARM group from GD13 to GD14 (p < 0.05). IEC-6 cells were transfected with PPPDE1 overexpression plasmid/NC (negative control) or si-PPPDE1/si-NC. Flow cytometry analysis and CCK-8 assay (used to detect apoptosis and proliferation, respectively), as well as western blotting, showed that the levels of PPPDE1 were positively correlated with the pro-apoptotic molecules Bax and Cyt C. Accordingly, aberrantly high expression of PPPDE1 caused a spatiotemporal imbalance in foetal rats with ARMs during hindgut development. Therefore, the upregulation of PPPDE1 may promote epithelial apoptosis and reduce proliferation in the hindgut via the mitochondrial apoptotic pathway. This could affect the fusion of the URS and cloacal membrane, ultimately inhibiting the hindgut development and resulting in ARMs.

RNA-Seq Profiling of Circular RNAs During Development of Hindgut in Rat Embryos With Ethylenethiourea-Induced Anorectal Malformations.

Anorectal malformations (ARMs) are among the most common congenital terminal digestive tract malformations. Circular RNAs (circRNAs), a novel type of endogenous non-coding RNAs, play roles in the development of the digestive system; however, their contributions to the pathogenesis of ARMs are not well-established. In this study, we explored the mechanism underlying ethylenethiourea (ETU)-induced ARMs by profiling circRNA expression via RNA-seq and constructing a regulatory circRNA-miRNA-mRNA network. Nine pregnant rats were gavage-fed a single dose of 125 mg/kg 1% ETU (ARM group) on gestational day 10 (GD10), and another 9 pregnant rats received a similar dose of saline (normal group) as a control. Embryos were obtained by cesarean section on the key time-points of anorectal development (GD14, GD15, and GD16). Hindgut samples isolated from the fetuses were evaluated by high-throughput sequencing and differentially expressed circRNAs were validated by reverse transcription-quantitative polymerase chain reaction, agarose gel electrophoresis, and Sanger cloning and sequencing. A total of 18295 circRNAs were identified in the normal and ARM groups. Based on the 425 differentially expressed circRNAs (|Fc| > 2, p < 0.05), circRNA-miRNA and miRNA-mRNA pairs were predicted using miREAP, miRanda, and TargetScan. A total of 55 circRNAs (14 up- and 41 downregulated in the ARM group compared to the normal group) were predicted to bind to 195 miRNAs and 947 mRNAs. Competing endogenous RNA networks and a Kyoto Encyclopedia of Genes and Genomes analysis revealed that novel_circ_001042 had the greatest connectivity and was closely related to ARM-associated signaling pathways, such as the Wingless Type MMTV integration site family, mitogen-activated protein kinase, and transforming growth factor-ß pathways. These results provide original insight into the roles of circRNAs in ARMs and provide a valuable resource for further analyses of molecular mechanisms and signaling networks.

[The role of submerged group ecological media box in repairing black and odorous water body]. / 潜水式生态介质箱对黑臭水体的修复效果.

As a conventional technology, the ecological floating bed has been widely used to repair waste water body. However, it can only repair the surface oxygen-enriched water, and has limited ability to repair the lower anoxic water. To meet the needs of the restoration of black and odorous water body, we designed a submersible ecological media box (submerged group) and compared it with traditional ecological floating bed (floating bed group). Water quality of black and odorous water before and after the restoration was examined, with the growth status of aquatic plants and the accumulation ability of N and P being investigated. The results showed that with the prolongation of repairing time, the removal rate of each pollutant increased gradually in both treatments. The removal ability of the submerged group for TN, NH4+-N, TP was better than that of the floating bed group, but its ability to remove CODMn was slightly inferior than that of the floating bed group. Plants (Vallisneria natans) in the submerged group grew better than that in the floating bed group (Acorus cala-mus), with similar patterns of the absorption and accumulation capacity and removal rate of TN and TP. In addition, the plasma membrane permeability and malondialdehyde content of V. natans were lower than that of A. calamus and the chlorophyll content of A. calamus was higher than that of V. natans, indicating that V. natans is more suitable for planting in black and odorous water bodies. Thus, the submersible ecological media box is a new in-situ integrated remediation device, which is more suitable to repair the black and odorous water.

Low level of Cyclin-D1 correlates with worse prognosis of clear cell renal cell carcinoma patients.

Cyclin-D1 (CCND1) belongs to the highly conserved cyclin family whose members are characterized by abundant expression during the cell cycle. As an oncogene, high level of CCND1 was observed and related to poor prognosis and tumor recurrence in many cancers. In this study, we focused on the role of CCND1 in the clinical outcome of clear cell renal cell carcinoma (ccRCC). Gene Expression Omnibus database, The Cancer Genome Atlas database, and immunohistochemical staining were used. The mRNA and protein levels of CCND1 were significantly enhanced in ccRCC tumor tissues. However, the low level of CCND1, but not high level of CCND1, was related to poor prognosis and tumor recurrence in ccRCC. Further analysis showed that CCND1 mRNA level decreased with increasing ccRCC tumor grades and the rate of recurrence in ccRCC patients. In a nomogram model, the CCND1 mRNA level was shown to help predict ccRCC patient recurrence. CCND1 is a strong determinant for prediction of recurrence. The patients with high CCND1 level appear to have a more favorable prognosis together with more frequent low-grade tumors and low rate of recurrence. This is the first study to investigate the prognostic roles of CCND1 in ccRCC and discovered that CCND1 had an unconventional positive impact on the clinical outcome of ccRCC patients.

Overexpression of KIF2A is Suppressed by miR-206 and Associated with Poor Prognosis in Ovarian Cancer.

BACKGROUND/AIMS: This study aimed to investigate the expression and prognostic value of kinesin family member 2A (KIF2A) and the suppression effects of microRNA-206 (miR-206) on KIF2A in ovarian cancer. METHODS: Ovarian cancer tissues from patients and ovarian cancer cell lines (A2780 and SKOV3) were used in this study. miR-206 mimics and control were transiently transfected into cells. RT-qPCR was performed to detect KIF2A mRNA and miR-206 expression levels, Western blot was performed to detect KIF2A protein levels, Dual-Luciferase Reporter Assay was used to examine the inhibition effects of miR-206 on KIF2A mRNA, immunohistochemical staining was used to examine the expression of KIF2A in tissue sections. CCK-8, transwell and Annexin-V-FITC/Propidium Iodide staining with flow cytometry were used to detect the cell proliferation, migration/invasion, and apoptosis respectively. RESULTS: Our study explored the expression profiles of KIF2A and miR-206 in the patients with ovarian cancer. We found that overexpression of KIF2A was associated with a poor prognosis in ovarian cancer. We also found that KIF2A mRNA contains two target sites for miR-206 binding and confirmed that miR-206 directly suppresses KIF2A; inhibits ovarian cancer cell proliferation, migration, and invasion; and induces apoptosis. CONCLUSION: The results suggest KIF2A could serve a valuable prognostic indicator in ovarian cancer and provide a rationale for treatment of ovarian cancer by targeting KIF2A via miR-206.

Down-regulated miR-148b increases resistance to CHOP in diffuse large B-cell lymphoma cells by rescuing Ezrin.

BACKGROUND: Aberrant microRNA (miRNAs) have recently been proposed as important regulators in acquiring resistance to cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) in diffuse large B-cell lymphoma (DLBCL). The purpose of this study was to establish the role of miR-148b in the development of CHOP resistance in DLBCL. METHODS: The expression patterns of miR-148b, HDAC6, and Ezrin were detected in CHOP-resistant clinical specimens and a DLBCL cell line. miR-148b, HDAC6, and Ezrin in DLBCL cells were manipulated by cell transfection to explore the functional correlation between them. Cell viability was determined using a CCK-8 assay. RESULTS: We found that miR-148b levels were markedly reduced and that the protein expressions of HDAC6 and Ezrin were increased in DLBCL CHOP-resistant clinical specimens and the cell line CRL2631/CHOP. Indeed, HDAC6 decreased the acetylation of histones H3 and H4 in the miR-148b promoter to inhibit miR-148b expression in DLBCL. Moreover, down-regulated miR-148b encouraged CHOP resistance in CRL2631 cells and miR-148b sensitized CRL2631 cells. We further revealed that Ezrin was negatively regulated by miR-148b and that the knockdown of Ezrin significantly attenuated CHOP resistance in CRL2631 cells induced by miR-148b silencing. MiR-148b also sensitized CRL2631/CHOP cell xenografts to CHOP in mice. CONCLUSION: Our data indicated that the high level of HDAC6 inhibited miR-148b via maintaining the low acetylation of histones H3 and H4 in the miR-148b promoter, thus rescuing Ezrin expression and promoting CHOP resistance in DLBCL.

Increased histone deacetylase 6 expression serves as a favorable prognostic factor for diffuse large B-cell lymphoma.

OBJECTIVE: This study aims to investigate ectopic expression of histone deacetylase 6 (HDAC6) in diffuse large B-cell lymphoma (DLBCL). METHODS: This study analyzed patients with DLBCL (n=132) and reactive lymph node hyperplasia (n=32) diagnosed in our hospital from December 2007 to May 2016. Correlation between HDAC6 expression and clinical pathologic features was analyzed by χ2 test. The significant differences between the 5-year overall survival (OS) or progression-free survival (PFS) and high HDAC6 expression as well as DLBCL clinic-pathological features including age, International Prognostic Index (IPI) score, Eastern Cooperative Oncology Group score, lactate dehydrogenase (LDH), and germinal center B-cell-like were assessed by univariate and multivariate analyses. RESULTS: HDAC6 high-expression percentage in DLBCL was significantly higher than that in the control group. The proportion of IPI score of 0-2, 5-year OS, and PFS in the high-expression group, which had lower percentage of patients with increased LDH and ß2-microglobulin, were significantly higher than those in the low-expression group. Moreover, HDAC6 mRNA expression in HDAC6 protein low expression was markedly lower than that in protein high expression. The multivariate analysis demonstrated that HDAC6 high expression was an independent prognostic factor for patients with DLBCL. CONCLUSION: HDAC6 high expression might be a prognostic factor for DLBCL.

ß-defensin 1 expression in HCV infected liver/liver cancer: an important role in protecting HCV progression and liver cancer development.

ß-defensin family plays a role in host defense against viral infection, however its role in HCV infection is still unknown. In this study, we demonstrated that ß-defensin 1 was significantly reduced in HCV-infected liver specimens. Treatment with interferon and ribavirin upregulated ß-defensin-1, but not other ß-defensin tested, with the extent and duration of upregulation associated with treatment response. We investigated ß-defensin family expression in liver cancer in publicly available datasets and found that among all the ß-defensins tested, only ß-defensin 1 was significantly downregulated, suggesting ß-defensin 1 plays a crucial role in liver cancer development. Further analysis identified E-cadherin as the top positive correlated gene, while hepatocyte growth factor-regulated tyrosine kinase substrate as the top negative correlated gene. Expression of two proteoglycans were also positively correlated with that of ß-defensin 1. We have also identified small molecules as potential therapeutic agents to reverse ß-defensin 1-associated gene signature. Furthermore, the downregulation of ß-defensin 1 and E-cadherin, and upregulation of hepatocyte growth factor-regulated tyrosine kinase substrate, were further confirmed in liver cancer and adjacent normal tissue collected from in-house Chinese liver cancer patients. Together, our results suggest ß-defensin 1 plays an important role in protecting HCV progression and liver cancer development.

Multivalent Histone and DNA Engagement by a PHD/BRD/PWWP Triple Reader Cassette Recruits ZMYND8 to K14ac-Rich Chromatin.

Elucidation of interactions involving DNA and histone post-translational-modifications (PTMs) is essential for providing insights into complex biological functions. Reader assemblies connected by flexible linkages facilitate avidity and increase affinity; however, little is known about the contribution to the recognition process of multiple PTMs because of rigidity in the absence of conformational flexibility. Here, we resolve the crystal structure of the triple reader module (PHD-BRD-PWWP) of ZMYND8, which forms a stable unit capable of simultaneously recognizing multiple histone PTMs while presenting a charged platform for association with DNA. Single domain disruptions destroy the functional network of interactions initiated by ZMYND8, impairing recruitment to sites of DNA damage. Our data establish a proof of principle that rigidity can be compensated by concomitant DNA and histone PTM interactions, maintaining multivalent engagement of transient chromatin states. Thus, our findings demonstrate an important role for rigid multivalent reader modules in nucleosome binding and chromatin function.

Promiscuous targeting of bromodomains by bromosporine identifies BET proteins as master regulators of primary transcription response in leukemia.

Bromodomains (BRDs) have emerged as compelling targets for cancer therapy. The development of selective and potent BET (bromo and extra-terminal) inhibitors and their significant activity in diverse tumor models have rapidly translated into clinical studies and have motivated drug development efforts targeting non-BET BRDs. However, the complex multidomain/subunit architecture of BRD protein complexes complicates predictions of the consequences of their pharmacological targeting. To address this issue, we developed a promiscuous BRD inhibitor [bromosporine (BSP)] that broadly targets BRDs (including BETs) with nanomolar affinity, creating a tool for the identification of cellular processes and diseases where BRDs have a regulatory function. As a proof of principle, we studied the effects of BSP on leukemic cell lines known to be sensitive to BET inhibition and found, as expected, strong antiproliferative activity. Comparison of the modulation of transcriptional profiles by BSP after a short exposure to the inhibitor resulted in a BET inhibitor signature but no significant additional changes in transcription that could account for inhibition of other BRDs. Thus, nonselective targeting of BRDs identified BETs, but not other BRDs, as master regulators of context-dependent primary transcription response.

Beating the odds: BETs in disease.

Bromodomains (BRDs) are evolutionarily conserved protein interaction modules that specifically recognise acetyl-lysine on histones and other proteins, facilitating roles in regulating gene transcription. BRD-containing proteins bound to chromatin loci such as enhancers are often deregulated in disease leading to aberrant expression of proinflammatory cytokines and growth-promoting genes. Recent developments targeting the bromo and extraterminal (BET) subset of BRD proteins demonstrated remarkable efficacy in murine models providing a compelling rationale for drug development and translation to the clinic. Here we summarise recent advances in our understanding of the roles of BETs in regulating gene transcription in normal and diseased tissue as well as the current status of their clinical translation.