IGF2BP1 overexpression causes fetal-like hemoglobin expression patterns in cultured human adult erythroblasts.

Here we investigated in primary human erythroid tissues a downstream element of the heterochronic let-7 miRNA pathway, the insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1), for its potential to affect the hemoglobin profiles in human erythroblasts. Comparison of adult bone marrow to fetal liver lysates demonstrated developmental silencing in IGF2BP1. Erythroid-specific overexpression of IGF2BP1 caused a nearly complete and pancellular reversal of the adult pattern of hemoglobin expression toward a more fetal-like phenotype. The reprogramming of hemoglobin expression was achieved at the transcriptional level by increased gamma-globin combined with decreased beta-globin transcripts resulting in gamma-globin rising to 90% of total beta-like mRNA. Delta-globin mRNA was reduced to barely detectable levels. Alpha-globin levels were not significantly changed. Fetal hemoglobin achieved levels of 68.6 ± 3.9% in the IGF2BP1 overexpression samples compared with 5.0 ± 1.8% in donor matched transduction controls. In part, these changes were mediated by reduced protein expression of the transcription factor BCL11A. mRNA stability and polysome studies suggest IGF2BP1 mediates posttranscriptional loss of BCL11A. These results suggest a mechanism for chronoregulation of fetal and adult hemoglobin expression in humans.

Yin Yang 1-mediated epigenetic silencing of tumour-suppressive microRNAs activates nuclear factor-κB in hepatocellular carcinoma.

Enhancer of zeste homolog 2 (EZH2) catalyses histone H3 lysine 27 trimethylation (H3K27me3) to silence tumour-suppressor genes in hepatocellular carcinoma (HCC) but the process of locus-specific recruitment remains elusive. Here we investigated the transcription factors involved and the molecular consequences in HCC development. The genome-wide distribution of H3K27me3 was determined by chromatin immunoprecipitation coupled with high-throughput sequencing or promoter array analyses in HCC cells from hepatitis B virus (HBV) X protein transgenic mouse and human cell models. Transcription factor binding site analysis was performed to identify EZH2-interacting transcription factors followed by functional characterization. Our cross-species integrative analysis revealed a crucial link between Yin Yang 1 (YY1) and EZH2-mediated H3K27me3 in HCC. Gene expression analysis of human HBV-associated HCC specimens demonstrated concordant overexpression of YY1 and EZH2, which correlated with poor survival of patients in advanced stages. The YY1 binding motif was significantly enriched in both in vivo and in vitro H3K27me3-occupied genes, including genes for 15 tumour-suppressive microRNAs. Knockdown of YY1 reduced not only global H3K27me3 levels, but also EZH2 and H3K27me3 promoter occupancy and DNA methylation, leading to the transcriptional up-regulation of microRNA-9 isoforms in HCC cells. Concurrent EZH2 knockdown and 5-aza-2'-deoxycytidine treatment synergistically increased the levels of microRNA-9, which reduced the expression and transcriptional activity of nuclear factor-κB (NF-κB). Functionally, YY1 promoted HCC tumourigenicity and inhibited apoptosis of HCC cells, at least partially through NF-κB activation. In conclusion, YY1 overexpression contributes to EZH2 recruitment for H3K27me3-mediated silencing of tumour-suppressive microRNAs, thereby activating NF-κB signalling in hepatocarcinogenesis.

A CCRK-EZH2 epigenetic circuitry drives hepatocarcinogenesis and associates with tumor recurrence and poor survival of patients.

BACKGROUND & AIMS: Aberrant chromatin modification is a key feature of hepatocellular carcinoma (HCC), which is characterized by strong sexual dimorphism. Both enhancer of zeste homolog 2 (EZH2) and cell cycle-related kinase (CCRK) contribute to hepatocarcinogenesis, yet whether the two oncogenic factors have functional crosstalk is unknown. METHODS: Cellular proliferation and tumorigenicity upon transgenic expression and RNA interference were determined by colony formation and soft agar assays, xenograft, orthotopic and diethylnitrosamine-induced HCC models. Gene regulation was assessed by chromatin immunoprecipitation, site-directed mutagenesis, luciferase reporter, co-immunoprecipitation and expression analyses. Protein levels in clinical specimens were correlated with clinicopathological parameters and patient survival rates. RESULTS: Ectopic CCRK expression in immortalized human liver cells increased EZH2 and histone H3 lysine 27 trimethylation (H3K27me3) to stimulate proliferation and tumor formation. Conversely, knockdown of CCRK reduced EZH2/H3K27me3 levels and decreased HCC cell growth, which could be rescued by EZH2 over-expression. Mechanistically, GSK-3ß phosphorylation by CCRK activated a ß-catenin/TCF/E2F1/EZH2 transcriptional feedback loop to epigenetically enhance androgen receptor (AR) signaling. Simultaneously, the phosphorylation of AKT/EZH2 by CCRK facilitated the co-occupancy of CCRK promoter by EZH2-AR and its subsequent transcriptional activation, thus forming a self-reinforcing circuitry. Lentiviral-mediated knockdown of CCRK, which abrogated the phosphorylation-transcriptional network, prevented diethylnitrosamine-induced tumorigenicity. More importantly, the hyperactivation of the CCRK-EZH2 circuitry in human HCCs correlated with tumor recurrence and poor survival. CONCLUSIONS: These findings uncover an epigenetic vicious cycle in hepatocarcinogenesis that operates through reciprocal regulation of CCRK and EZH2, providing novel therapeutic strategy for HCC.

Cell cycle-related kinase mediates viral-host signalling to promote hepatitis B virus-associated hepatocarcinogenesis.

BACKGROUND: Androgen receptor (AR) signalling contributes to male predominance in hepatocellular carcinoma (HCC), which is more pronounced in HBV-endemic areas. Cell cycle-related kinase (CCRK) is essential for AR-induced hepatocarcinogenesis but its molecular function in HBV-associated HCC remains obscure. OBJECTIVE: To determine the molecular function of CCRK in HBV-associated HCC. DESIGN: Transcriptional regulation was assessed by chromatin immunoprecipitation, promoter mutation and luciferase reporter assays. Hepatocellular proliferation and tumourigenesis were examined by colony formation, soft agar assays and using HBV X protein (HBx) transgenic mice with low-dose exposure to diethylnitrosamine. Protein expressions were examined in clinical samples and correlated with patient survival by log-rank Mantel-Cox test. RESULTS: Overexpression of CCRK, but not its kinase-defective mutant, activated ß-catenin/T cell factor signalling through phosphorylation of glycogen synthase kinase-3ß (GSK-3ß) at Ser9, led to upregulation of AR transcriptional activity and, subsequently, expression of HBx. The viral transactivator in turn induced CCRK expression through enhanced AR signalling, thus forming a positive regulatory loop. RNA interference silencing of CCRK, which suppressed the CCRK/GSK-3ß/ß-catenin/AR regulatory loop, significantly suppressed HBx-induced hepatocellular proliferation (p=0.001) and transformation (p<0.001) and remarkably reduced >80% diethylnitrosamine-mediated hepatocarcinogenesis in HBx transgenic mice. Finally, patients with HBV-associated HCC with concordant overexpression of CCRK, GSK-3ß phosphorylation at Ser9, active dephosphorylated ß-catenin and AR phosphorylation at Ser81 had poorer overall (HR=31.26, p<0.0001) and disease-free (HR=3.60, p<0.01) survival rates. CONCLUSIONS: Our findings highlight the critical role of CCRK in a self-reinforcing circuitry that regulates HBV-associated hepatocarcinogenesis. Further characterisation of this intricate viral-host signalling may provide new prognostic biomarkers and therapeutic targets for HCC treatment.

Helicobacter pylori causes epigenetic dysregulation of FOXD3 to promote gastric carcinogenesis.

BACKGROUND & AIMS: Deregulation of forkhead box (Fox) proteins, an evolutionarily conserved family of transcriptional regulators, leads to tumorigenesis. Little is known about their regulation or functions in the pathogenesis of gastric cancer. Promoter hypermethylation occurs during Helicobacter pylori-induced gastritis. We investigated whether the deregulated genes contribute to gastric tumorigenesis. METHODS: We used integrative genome-wide scans to identify concomitant hypermethylated genes in mice infected with H pylori and human gastric cancer samples. We also analyzed epigenetic gene silencing in gastric tissues from patients with H pylori infection and gastritis, intestinal metaplasia, gastric tumors, or without disease (controls). Target genes were identified by chromatin immunoprecipitation microarrays and expression and luciferase reporter analyses. RESULTS: Methylation profile analyses identified the promoter of FOXD3 as the only genomic region with increased methylation in mice and humans during progression of H pylori-associated gastric tumors. FOXD3 methylation also correlated with shorter survival times of patients with gastric cancer. Genome demethylation reactivated FOXD3 expression in gastric cancer cell lines. Transgenic overexpression of FOXD3 significantly inhibited gastric cancer cell proliferation and invasion, and reduced growth of xenograft tumors in mice, at least partially, by promoting tumor cell apoptosis. FOXD3 bound directly to the promoters of, and activated transcription of, genes encoding the cell death regulators CYFIP2 and RARB. Levels of FOXD3, CYFIP2, and RARB messenger RNAs were reduced in human gastric tumor samples, compared with control tissues. CONCLUSIONS: FOXD3-mediated transcriptional control of tumor suppressors is deregulated by H pylori infection-induced hypermethylation; this could perturb the balance between cell death and survival. These findings identify a pathway by which epigenetic changes affect gastric tumor suppression.

A material-sparing simplified buoyancy method for determining the true density of solids.

True density is an important physical property of powdered materials, especially in the context of powder compaction. Currently available methods for true density determination either require a significant amount of materials or are laborious. Hence, a material-sparing and efficient method for true density determination is of value. In this work, we detail a simplified buoyancy-based method capable of fast determination of true density with accuracy comparable to helium pycnometry. This miniaturized method only uses a few milligrams of a powder with data collection process expedited by centrifugation in a laboratory centrifuge. This method can be easily adapted in a laboratory since determination of true density only requires a balance, a micropipette, a laboratory centrifuge, and standard stock liquids of low and high densities. Hence, it is a useful addition to the materials characterization tool kit critical for pharmaceutical formulation development.

Pyoderma Gangrenosum: An Updated Literature Review on Established and Emerging Pharmacological Treatments.

Pyoderma gangrenosum is a rare inflammatory skin disease classified within the group of neutrophilic dermatoses and clinically characterized by painful, rapidly evolving cutaneous ulcers with undermined, irregular, erythematous-violaceous edges. Pyoderma gangrenosum pathogenesis is complex and involves a profound dysregulation of components of both innate and adaptive immunity in genetically predisposed individuals, with the follicular unit increasingly recognized as the putative initial target. T helper 17/T helper 1-skewed inflammation and exaggerated inflammasome activation lead to a dysregulated neutrophil-dominant milieu with high levels of tumor necrosis factor-α, interleukin (IL)-1ß, IL-1α, IL-8, IL-12, IL-15, IL-17, IL-23, and IL-36. Low-evidence studies and a lack of validated diagnostic and response criteria have hindered the discovery and validation of new effective treatments for pyoderma gangrenosum. We review established and emerging treatments for pyoderma gangrenosum. A therapeutic algorithm based on available evidence is also provided. For emerging treatments, we review target molecules and their role in the pathogenesis of pyoderma gangrenosum.

Development and Industrial-Scale Fabrication of Next-Generation Low-Energy Membranes for Desalination.

Spiral-wound modules have been the most common configuration of packing flat-sheet membranes since the early development of polyamide (PA) membranes for water treatment applications. Conventional spiral-wound modules (SWMs) for desalination applications typically consist of several leaf sets, with each leaf set comprising feed spacers, membranes, and a permeate carrier (PC) wrapped around a permeate-collecting tube. The membrane area that can be packed into a given module diameter is limited by the overall leaf set thickness, restricting module productivity for a given membrane permeability. We describe here a novel industrial-scale method for successfully coating the polysulfone (PSf) ultrafiltration (UF) support layer directly onto a permeate carrier, instead of conventional non-woven fabric, as a precursor to the polyamide TFC coating, resulting in twofold benefits: (a) drastically simplifying the membrane fabrication process by eliminating the use of non-woven fabric and (b) increasing the throughput of each membrane module by facilitating the packing of a larger membrane area in a standard module housing. By combining the permeate carrier and membrane into a single sheet, the need for the non-woven support layer was eliminated, leading to a significantly reduced leaf set thickness, enabling a much larger membrane area to be packed in a given volume, leading to lower energy consumption per cubic meter of produced water. Molecular-weight cutoff (MWCO) values in the range of 36-96 kDa were found to be dependent on PC thickness and material. Nevertheless, the reinforced membranes were successfully fabricated with a ~9% reduction in membrane leaf thickness compared to a conventional membrane. Preliminary trials of coating a thin-film composite PA layer resulted in defect-free reverse osmosis (RO) membranes with a salt rejection of 94% and a flux of 40 L m-2 h-1 when tested against a 2000 mg/L NaCl feed solution at an operating pressure of 15 bar. Results from the testing of the 1812 and 2514 elements validated the novel concept and paved the way for further improvements towards full-scale RO membranes with the potential to be the next low-energy workhorse of the water industry.

Coexisting White and Dark Without Pressure Abnormalities Surrounding Congenital Hypertrophy of the Retinal Pigment Epithelium.

The authors report a case of coexisting white and dark without pressure abnormalities surrounding a small congenital hypertrophy of the retinal pigment epithelium and showing corresponding hyperreflectivity and hyporeflectivity of the ellipsoid layer on optical coherence tomography. [J Pediatr Ophthalmol Strabismus. 2019;56:e5-e7.].

Psychological and Genetic Predictors of Pain Tolerance.

Previous studies have shown associations between genetic polymorphisms and pain tolerance, but psychological evaluations are seldom measured. The objective of this study was to determine the independent effects of demographic, psychological, and genetic predictors of cold noxious pain tolerance. Healthy subjects (n = 89) completed the Pain Catastrophizing Scale (PCS) and Fear of Pain Questionnaire (FPQ-III), underwent genotyping for candidate single nucleotide polymorphisms (SNPs), and completed a cold-pressor test in a 1-2°C water bath for a maximum of 3 minutes. The primary outcome measure was pain tolerance, defined as the maximum duration of time subjects left their nondominant hand in the cold-water bath. Cox proportional hazards regression indicated that female sex, Asian race, and increasing PCS and FPQ-III scores were associated with lower pain tolerance. No candidate SNP was significantly associated with pain tolerance. Future genetic studies should include demographic and psychological variables as confounders in experimental pain models.

Enoxaparin dosing errors in the emergency department.

BACKGROUND: The study aimed to determine the frequency of enoxaparin dosing errors for patients who had a measured emergency department (ED) weight compared to those who did not have a measured ED weight, and to determine if demographic variables (e.g., weight, height, age, English-speaking, race) impact the likelihood of receiving an inappropriate dose. METHODS: This is a retrospective, electronic chart review of patients who received a dose of enoxaparin in the ED between January 1, 2008 and July 1, 2013. We identified all patients >18 years who received a dose of enoxaparin while in the ED, were admitted, and had at least one inpatient weight within the first four days of hospitalization. Patients were excluded if they received enoxaparin for prophylaxis or a dose of more than 1.25 mg/kg. RESULTS: A total of 1,944 patients were included. Patients were more likely to experience an error if they did not have a measured ED weight. Over-doses of >10 mg were more likely to occur in patients without a measured ED weight. Patients with no documented ED weight or with a staff-estimated ED weight were more likely to experience a dosing error than those with a patient-stated weight. Patients were more likely to experience an error if their first inpatient weight was more than 96 kg, they were more than 175-cm tall, or were English speaking. CONCLUSION: Dosing errors are more likely to occur when patients are not weighed in the ED. Modifications to current workflows to incorporate weighing those patients who receive weight-dosed medications may be warranted.

Applying the health belief model to analyze intention to participate in preventive pulmonary tuberculosis chest X-ray examinations among indigenous nursing students.

The purpose of this study was to investigate knowledge, perceptions of tuberculosis (TB) and intent to receive chest X-ray screening among indigenous (Taiwanese of ethnic Malayo-Polynesian descent) nursing students in Taiwan. A convenience sample was chosen from an technical institute in northern Taiwan that maintained admission quotas and provided scholarships for indigenous students. This school had over 150 classes and around 7,000 students, about 20% -25% of whom were ethnically indigenous. A power analysis based on a pilot study showed that a large effect size required a minimum sample of 485. To attain this number, 50 classes from years one through five were randomly selected based on an expected sampling of 20 students per classroom. In all, 1,000 questionnaires were distributed and 865 returned, for a response rate of 86.5%. A cross-sectional study design was used, and this study was run between the months of February and June 2003. Survey responses were self-reported in two questionnaires that gathered information on respondent knowledge and perceptions regarding TB and intent to take chest X-ray tests. Study results showed a moderate level of general knowledge about TB, misunderstandings regarding transmission vectors, and low perceptions regarding susceptibility. The knowledge score was associated with perceived benefits and barriers to preventing TB. Different places of residence influenced perceived barriers to preventing and treating TB. Age was negatively associated with the perceived benefits of receiving a chest X-ray exam for TB. Indigenous nursing students with higher perceptions of susceptibility and severity and lower perception of barriers on preventing TB had a higher level of intent to take the X-ray exam. Nursing schools must address the subject of TB in-depth and design age-specific curricula that incorporate multifaceted strategies for different students. Additionally, health educators should design health education programs targeted to dispel misconceptions and improve both knowledge and levels of awareness of tuberculosis among the indigenous population in order to enhance their willingness and ability to detect TB.

Epigenetic silencing of miR-490-3p reactivates the chromatin remodeler SMARCD1 to promote Helicobacter pylori-induced gastric carcinogenesis.

Chromatin remodeling has emerged as a hallmark of gastric cancer, but the regulation of chromatin regulators other than genetic change is unknown. Helicobacter pylori causes epigenetic dysregulation to promote gastric carcinogenesis, but the roles and functions of microRNAs (miRNA) in this multistage cascade are not fully explored. In this study, miRNA expression in preneoplastic and neoplastic lesions in murine stomachs induced by H. pylori and N-methyl-N-nitrosourea (MNU) was profiled by miRNA expression array. miR-490-3p exhibited progressive downregulation in gastritis, intestinal metaplasia, and adenocarcinoma during H. pylori and MNU-induced gastric carcinogenesis. Significant downregulation of miR-490-3p was confirmed in human gastric cancer tissues in which its regulatory region was found to be hypermethylated. miR-490-3p exerted growth- and metastasis-suppressive effects on gastric cancer cells through directly targeting SMARCD1, a SWItch/Sucrose NonFermentable (SWI/SNF) chromatin remodeling complex subunit. Knockdown of SMARCD1 significantly attenuated the protumorigenic effects of miR-490-3p inhibitor, whereas enforced expression of SMARCD1 promoted in vitro and in vivo oncogenic phenotypes of gastric cancer cells. SMARCD1 was markedly upregulated in gastric cancer in which its high expression was associated with shortened patients' survival independent of TNM staging. In conclusion, hypermethylation-mediated silencing of miR-490-3p reactivates SMARCD1 to confer malignant phenotypes, mechanistically linking H. pylori, chromatin remodeling, and gastric carcinogenesis.

Evidence of octacalcium phosphate and Type-B carbonated apatites deposited on the surface of explanted acrylic hydrogel intraocular lens.

Fourier-transform infrared (FTIR) microspectroscopy combining with attenuated total reflection (ATR) microsampling technique and micro-Raman spectrophotometer were used to detect the deposited materials on the surface of acrylic hydrogel intraocular lens (IOL) with or without ocular implantation. Surface morphology and the interface of this IOL were further examined by a confocal laser scanning microscope. The brand-new IOL exhibited a very smooth, transparent and featureless surface, but the explanted IOL had an irregular cerebriform-like opaque appearance. Both FTIR/ATR and Raman microspectroscopic analyses showed the deposits on the surface of acrylic hydrogel IOL after ocular implantation to consist of octacalcium phosphate (OCP) and Type B carbonated apatites, leading to the opalescence of acrylic hydrogel IOL. Both vibrational microspectroscopic examinations also confirmed the mineralization still in progress on the surface of acrylic hydrogel IOL after ocular implantation for 2 years.

Synthesis and characterization of two-photon chromophores based on a tetrasubstituted tetraethynylethylene scaffold.

A new series of model dye molecules composed of three multibranched analogues based on the tetrasubstituted tetraethynylethylene structural motif have been synthesized and experimentally shown to possess strong and widely dispersed two-photon absorption (2PA) in the near-IR region. It was found that the spectral position of the major 2PA band could be tuned by the electronic nature of the selected substitution units. The studied model fluorophores also exhibited fairly low photodegradation of their fluorescence intensity even under prolonged UV-light irradiation, which is beneficial for the development of fluorescence probes that are needed for long-term light exposure. Furthermore, representative chromophores were selected to demonstrate the power-control properties within the femtosecond and nanosecond time domains.

Degenerate two-photon absorption and effective optical-power-limiting properties of multipolar chromophores derived from 2,3,8-trisubstituted indenoquinoxaline.

Two analogous multipolar chromophores (1 and 2) that contained 2,3,8-trisubstituted indenoquinoxaline moieties have been synthesized and characterized for their two-photon absorption properties, both in the femtosecond and nanosecond time regimes. We demonstrated that their multi-branched framework structures, which incorporated appropriately functionalized indenoquinoxaline units, afforded large molecular nonlinear absorptivities within the studied spectroscopic range. Effective optical-power-limiting and stabilization behaviors in the nanosecond regime of dye molecule (2) were also investigated and the results indicated that such a structural motif could be a useful approach to the molecular design of highly active two-photon systems for quick-response and related broadband optical-suppressing applications, in particular for confronting laser pulses of a long duration.