Critical design parameters to develop biomimetic organ-on-a-chip models for the evaluation of the safety and efficacy of nanoparticles.

INTRODUCTION: Organ-on-a-chip (OOC) models are based on microfluidics and can recapitulate the healthy and diseased microstructure of organs1 and tissues and the dynamic microenvironment inside the human body. However, the use of OOC models to evaluate the safety and efficacy of nanoparticles (NPs) is still in the early stages. AREAS COVERED: The different design parameters of the microfluidic chip and the mechanical forces generated by fluid flow play a pivotal role in simulating the human environment. This review discusses the role of different key parameters on the performance of OOC models. These include the flow pattern, flow rate, shear stress (magnitude, rate, and distribution), viscosity of the media, and the microchannel dimensions and shape. We also discuss how the shear stress and other mechanical forces affect the transport of NPs across biological barriers, cell uptake, and their biocompatibility. EXPERT OPINION: We describe several good practices and design parameters to consider for future OOC research. We submit that following these recommendations will help realize the full potential of the OOC models in the preclinical evaluation of novel therapies, including NPs.

Placental Nanoparticle Uptake-On-a-Chip: The Impact of Trophoblast Syncytialization and Shear Stress.

The placenta is a dynamic and complex organ that plays an essential role in the health and development of the fetus. Placental disorders can affect the health of both the mother and the fetus. There is currently an unmet clinical need to develop nanoparticle-based therapies to target and treat placental disorders. However, little is known about the interaction of nanoparticles (NPs) with the human placenta under biomimetic conditions. Specifically, the impact of shear stress exerted on the trophoblasts (placental epithelial cells) by the maternal blood flow, the gradual fusion of the trophoblasts along the gestation period (syncytialization), and the impact of microvilli formation on the cell uptake of NPs is not known. To this end, we designed dynamic placenta-on-a-chip models using BeWo cells to recapitulate the micro-physiological environment, and we induced different degrees of syncytialization via chemical induction with forskolin. We characterized the degree of syncytialization quantitatively by measuring beta human chorionic gonadotropin (ß-hCG) secretion, as well as qualitatively by immunostaining the tight junction protein, ZO-1, and counter nuclear staining. We also characterized microvilli formation under static and dynamic conditions via F-actin staining. We used these models to measure the cell uptake of chondroitin sulfate a binding protein (CSA) conjugated and control liposomes using confocal microscopy, followed by image analysis. Interestingly, exposure of the cells to a dynamic flow of media intrinsically induced syncytialization and microvilli formation compared to static controls. Under dynamic conditions, BeWo cells produced more ß-hCG in conditions that increased the cell exposure time to forskolin (p < 0.005). Our cell uptake results clearly show a combined effect of the exerted shear stress and forskolin treatment on the cell uptake of liposomes as uptake increased in forskolin exposed conditions (p < 0.05). Overall, the difference in the extent of cell uptake of liposomes among the different conditions clearly displays a need for the development of dynamic models of the placenta that consider the changes in the placental cell phenotype along the gestation period, including syncytialization, microvilli formation, and the expression of different transport and uptake receptors. Knowledge generated from this work will inform future research aiming at developing drug delivery systems targeting the placenta.

Development and validation of a novel evaporation setup-assisted TLC method with fluorescence detection for determination of flibanserin in pharmaceutical and biological samples.

A specific and sensitive thin layer chromatographic method coupled with fluorescence detection for determination of flibanserin (FLN) that treats woman hypoactive sexual desire disorder was developed. The proposed method depends on the enhancement of FLN native fluorescence intensity via the exposure of the developed TLC plate to concentrated hydrochloric acid vapors. Herein, an evaporation setup needed for HCl vapors exposure step was designed for the first time to ensure a uniform distribution of the vapors throughout the developed bands on the plate. Chloroform: methanol (9.5: 0.5, v/v) was the optimum mobile phase that gave a compact band (Rf= 0.44 ± 0.02) using TLC aluminium plates precoated with silica gel G 60F254 as a stationary phase. After exposure of the developed TLC plate to HCl vapors, the FLN bands emission intensities were measured after excitation at 275 nm. Conferring ICH guidelines, the linearity range was 20.0 - 1500.0 ng/band with a good linear relationship (r= 0.9998). Detection and quantitation limits were 5.12 and 15.50 ng/band, respectively. Also, the method was validated for accuracy, precision, robustness, specificity and selectivity. Statistical analysis verified the suitability of the proposed method for estimation of FLN in tablets and in human plasma with acceptable recoveries (98.07-101.45%).

Systematic analysis of migration factors by MigExpress identifies essential cell migration control genes in non-small cell lung cancer.

Cell migration is an essential process in health and in disease, including cancer metastasis. A comprehensive inventory of migration factors is nonetheless lacking-in part due to the difficulty in assessing migration using high-throughput technologies. Hence, there are currently very few screens that systematically reveal factors controlling cell migration. Here, we introduce MigExpress as a platform for the 'identification of Migration control genes by differential Expression'. MigExpress exploits the combination of in-depth molecular profiling and the robust quantitative analysis of migration capacity in a broad panel of samples and identifies migration-associated genes by their differential expression in slow- versus fast-migrating cells. We applied MigExpress to investigate non-small cell lung cancer (NSCLC), which is the most frequent cause of cancer mortality mainly due to metastasis. In 54 NSCLC cell lines, we comprehensively determined mRNA and protein expression. Correlating the transcriptome and proteome profiles with the quantified migration properties led to the discovery and validation of FLNC, DSE, CPA4, TUBB6, and BICC1 as migration control factors in NSCLC cells, which were also negatively correlated with patient survival. Notably, FLNC was the least expressed filamin in NSCLC, but the only one controlling cell migration and correlating with patient survival and metastatic disease stage. In our study, we present MigExpress as a new method for the systematic analysis of migration factors and provide a comprehensive resource of transcriptomic and proteomic data of NSCLC cell lines related to cell migration.

Electrospun vancomycin-loaded nanofibers for management of methicillin-resistant Staphylococcus aureus-induced skin infections.

Skin damage exposes the underlying layers to bacterial invasion, leading to skin and soft tissue infections. Several pathogens have developed resistance against conventional topical antimicrobial treatments and rendered them less effective. Recently, several nanomedical strategies have emerged as a potential approach to improve therapeutic outcomes of treating bacterial skin infections. In the current study, nanofibers were utilized for topical delivery of the antimicrobial drug vancomycin and evaluated as a promising tool for treatment of topical skin infections. Vancomycin-loaded nanofibers were prepared via electrospinning technique, and vancomycin-loaded nanofibers of the optimal composition exhibited nanosized uniform smooth fibers (ca. 200 nm diameter), high drug entrapment efficiency and sustained drug release patterns over 48 h. In vitro cytotoxicity assays, using several cell lines, revealed the biocompatibility of the drug-loaded nanofibers. In vitro antibacterial studies showed sustained antibacterial activity of the vancomycin-loaded nanofibers against methicillin-resistant Staphylococcus aureus (MRSA), in comparison to the free drug. The nanofibers were then tested in animal model of superficial MRSA skin infection and demonstrated a superior antibacterial efficiency, as compared to animals treated with the free vancomycin solution. Hence, nanofibers might provide an efficient nanodevice to overcome MRSA-induced skin infections and a promising topical delivery vehicle for antimicrobial drugs.

A pan-cancer analysis reveals nonstop extension mutations causing SMAD4 tumour suppressor degradation.

Nonstop or stop-loss mutations convert a stop into a sense codon, resulting in translation into the 3' untranslated region as a nonstop extension mutation to the next in-frame stop codon or as a readthrough mutation into the poly-A tail. Nonstop mutations have been characterized in hereditary diseases, but not in cancer genetics. In a pan-cancer analysis, we curated and analysed 3,412 nonstop mutations from 62 tumour entities, generating a comprehensive database at http://NonStopDB.dkfz.de. Six different nonstop extension mutations affected the tumour suppressor SMAD4, extending its carboxy terminus by 40 amino acids. These caused rapid degradation of the SMAD4 mutants via the ubiquitin-proteasome system. A hydrophobic degron signal sequence of ten amino acids within the carboxy-terminal extension was required to induce complete loss of the SMAD4 protein. Thus, we discovered that nonstop mutations can be functionally important in cancer and characterize their loss-of-function impact on the tumour suppressor SMAD4.

The oligodendrocyte lineage transcription factor 2 (OLIG2) is epigenetically regulated in acute myeloid leukemia.

DNA methylation differences between normal tissue and cancerous tissue resulting in differential expression of genes are a hallmark of acute myeloid leukemia (AML) and can provide malignant cells with a growth advantage via silencing of specific genes, for example, transcription factors. Oligodendrocyte lineage transcription factor 2 (OLIG2) was reported to be differentially methylated and associated with prognosis in AML and, as reported for acute lymphoblastic leukemia and malignant glioma, may play a role in malignant transformation. We report that DNA methylation of OLIG2 is associated with decreased expression of mRNA in AML cell lines and patients. Moreover, in cell lines, decreased mRNA expression also translated into decreased OLIG2 protein expression. Treatment of non-expressing cell lines PL-21 and U-937 with the demethylating agent decitabine resulted in robust re-expression of OLIG2 on mRNA and protein levels. Furthermore, stable overexpression of OLIG2 in non-expressing cell lines Kasumi-1 and U-937, using a lentiviral vector system, led to moderate growth inhibition after 4 days and resulted in signs of differentiation in U-937 cells. Interestingly, although CD34 + cells from healthy donors and 10 of 12 AML patients exhibited no protein expression, OLIG2 was expressed in two patients, both bearing the translocation t(15;17), corresponding to OLIG2 expression in NB-4 cells, also harboring t(15;17). In conclusion, we provide first evidence that OLIG2 is epigenetically regulated via DNA methylation and expressed in a subset of AML patients. OLIG2 may exert antiproliferative activity in leukemia cell lines, and its potential leukemia-suppressing role in AML warrants further investigation.

Evaluating the impact of genetic and epigenetic aberrations on survival and response in acute myeloid leukemia patients receiving epigenetic therapy.

Treatment with hypomethylating agents such as decitabine, which results in overall response rates of up to 50%, has become standard of care in older patients with acute myeloid leukemia (AML) who are not candidates for intensive chemotherapy. However, there still exists a lack of prognostic and predictive molecular biomarkers that enable selection of patients who are likely to benefit from epigenetic therapy. Here, we investigated distinct genetic (FLT3-ITD, NPM1, DNMT3A) and epigenetic (estrogen receptor alpha (ERα), C/EBPα, and OLIG2) aberrations in 87 AML patients from the recently published phase II decitabine trial (AML00331) to identify potential biomarkers for patients receiving hypomethylating therapy. While FLT3-ITD and NPM1 mutational status were not associated with survival or response to therapy, patients harboring DNMT3A R882 mutations showed a non-significant association towards shorter overall survival (hazard ratio (HR) 2.15, 95% confidence interval (CI) 0.91-5.12, p = 0.08). Promoter DNA methylation analyses using pyrosequencing also revealed a non-significant association towards shorter overall survival of patients with higher levels of methylation of ERα (HR 1.50, CI 0.97-2.32, p = 0.07) and OLIG2 CpG4 (HR 1.52, CI 0.96-2.41, p = 0.08), while DNA methylation of C/EBPα showed no association with outcome. Importantly, in multivariate analyses adjusted for clinical baseline parameters, the impact of ERα and OLIG2 CpG4 methylation was conserved (HR 1.76, CI 1.01-3.06, p = 0.05 and HR 1.67, CI 0.91-3.08, p = 0.10, respectively). In contrast, none of the investigated genetic and epigenetic markers was associated with response to treatment. Additional to the previously reported adverse prognostic clinical parameters such as patients' age, reduced performance status, and elevated lactate dehydrogenase levels, DNMT3A R882 mutation status, as well as ERα and OLIG2 CpG4 DNA methylation status, may prove to be molecular markers in older AML patients prior to hypomethylating therapy.

Tracing the development of acute myeloid leukemia in CBL syndrome.

We describe the development of acute myeloid leukemia (AML) in an adult with CBL syndrome caused by a heterozygous de novo germline mutation in CBL codon D390. In the AML bone marrow, the mutated CBL allele was homozygous after copy number-neutral loss-of-heterozygosity and amplified through a chromosomal gain; moreover, an inv(16)(p13q22) and, as assessed by whole-exome sequencing, 12 gene mutations (eg, in CAND1, NID2, PTPRT, DOCK6) were additionally acquired. During complete remission of the AML, in the presence of normal blood counts, the hematopoiesis stably maintained the homozygous CBL mutation, which is reminiscent of the situation in children with CBL syndrome and transient juvenile myelomonocytic leukemia. No additional mutations were identified by whole-exome sequencing in granulocytes during complete remission. The study highlights the development of AML in an adult with CBL syndrome and, more generally, in genetically aberrant but clinically inconspicuous hematopoiesis.

Epigenetic priming of AML blasts for all-trans retinoic acid-induced differentiation by the HDAC class-I selective inhibitor entinostat.

All-trans retinoic acid (ATRA) has only limited single agent activity in AML without the PML-RARα fusion (non-M3 AML). In search of a sensitizing strategy to overcome this relative ATRA resistance, we investigated the potency of the HDAC class-I selective inhibitor entinostat in AML cell lines Kasumi-1 and HL-60 and primary AML blasts. Entinostat alone induced robust differentiation of both cell lines, which was enhanced by the combination with ATRA. This "priming" effect on ATRA-induced differentiation was at least equivalent to that achieved with the DNA hypomethylating agent decitabine, and could overall be recapitulated in primary AML blasts treated ex vivo. Moreover, entinostat treatment established the activating chromatin marks acH3, acH3K9, acH4 and H3K4me3 at the promoter of the RARß2 gene, an essential mediator of retinoic acid (RA) signaling in different solid tumor models. Similarly, RARß2 promoter hypermethylation (which in primary blasts from 90 AML/MDS patients was surprisingly infrequent) could be partially reversed by decitabine in the two cell lines. Re-induction of the epigenetically silenced RARß2 gene was achieved only when entinostat or decitabine were given prior to ATRA treatment. Thus in this model, reactivation of RARß2 was not necessarily required for the differentiation effect, and pharmacological RARß2 promoter demethylation may be a bystander phenomenon rather than an essential prerequisite for the cellular effects of decitabine when combined with ATRA. In conclusion, as a "priming" agent for non-M3 AML blasts to the differentiation-inducing effects of ATRA, entinostat is at least as active as decitabine, and both act in part independently from RARß2. Further investigation of this treatment combination in non-M3 AML patients is therefore warranted, independently of RARß2 gene silencing by DNA methylation.

Public health significance of dermatophytes in Ismailia and Port Said Provinces, Egypt.

BACKGROUND: [corrected] Dermatophytes are common in both developed and developing countries, the species involved and the resulting clinical entities vary geographically. AIM OF STUDY: To determine public health importance of dermatophytes with special regard to the distribution of the zoophilic species among the examined human cases. METHODS: Patient specimens (dermatophytosis-like lesions) were mycologically examined using direct microscopic and cultural examinations. RESULTS: The overall dermatophyte infection rates among the examined patients (260) were 81.5% and 61.9% by direct microscopic and cultural examinations, respectively. A total of 161 dermatophyte isolates were obtained from culturally positive human specimens. The most common isolated species were Trichophyton violaceum (60, 37.3%) and Microsporum canis (46, 28.6%), followed by Trichophyton rubrum (20, 12.4%), Trichophyton tonsurans (16, 9.9%) and Trichophyton mentagrophytes (11, 6.8%). The less frequently isolated species were Trichophyton verrucosum (3, 1.9%), Epidermophyton floccosum (3, 1.9%), Microsporum gypseum (1, 0.6%) and Microsporum audouinii (1, 0.6%). The current study further revealed that out of 161 culturally positive patients, 61 (37.9%) had contact with pet animals, 6 (3.7%) had contact with farm animals, 7 (4.3%) had a history of rodent presence in or around their home and 87 (54%) had no contact with animals. CONCLUSIONS AND RECOMMENDATIONS: Although the anthropophilic species dominated the aetiology of human dermatophytosis in present study, the zoophilic species represented nearly one third of the totally obtained isolates, with the most important organisms being M. canis, T.mentagrophytes var. mentagrophytes and T. verrucosum. These findings indicate the necessity of human beings taking appropriate control measures when they are in contact with animals.

MeDIP coupled with a promoter tiling array as a platform to investigate global DNA methylation patterns in AML cells.

Hypermethylation of CpGs in promoter regions and subsequent changes in gene expression are common features in acute myeloid leukemia (AML). Genome-wide studies of the methylome are not only useful to understand changes in DNA methylation and gene regulation but also to identify potential targets for antileukemic treatment. Here we performed methylated DNA immunoprecipitation (MeDIP) in the AML cell line HL-60 and donor-derived CD34+ cells, followed by hybridization on a human promoter tiling array. The comparative analysis of HL-60 versus CD34+ cells revealed differentially methylated promoter regions including genes that are frequently methylated in AML, such as p15/INK4B, OLIG2, RARß2 and estrogen receptor. Microarray data was validated by quantitative pyrosequencing. We corroborate previous reports that MeDIP, in our study combined with a promoter tiling array (MeDIP-Chip), is a robust method to identify genes that are differentially methylated in AML cells in a genome-wide manner, and is thus useful to identify new epigenetic targets for therapeutic or prognostic research.

HDAC6 as a target for antileukemic drugs in acute myeloid leukemia.

Inhibition of histone deacetylases (HDACs) by drugs such as vorinostat or depsipeptide has become treatment strategy under study in acute myeloid leukemia. Most preclinically and clinically investigated HDACi target classes I, II and IV, but only few are selective in inhibiting specific HDACs. Here we analyzed the in vitro antileukemic activity of three novel hydroxamate derivatives, the pan-HDAC-inhibitors ST13, ST34 and the known HDAC6 inhibitor ST80, treating leukemia cell lines HL60, Kasumi-1, NB-4, THP-1, K562, U937, Jurkat as well as primary AML blasts. In cell lines all three compounds exerted a strong growth-inhibitory effect at low micromolar concentrations. ST13 increased acetylation of H3, H4 and α-tubulin, while ST34 preferentially acetylated histones H3 and H4. Interestingly, ST80 preferentially induced α-tubulin acetylation at low micromolar doses, confirming a selective inhibition of HDAC6 by ST80 in leukemic cells. These observations were also confirmed in primary AML blasts cultured ex vivo. Growth-inhibition by ST80 was independent of pre-treatment HDAC6 protein expression and in contrast to ST13 and ST34, ST80 did not result in induction of p21/WAF. Immunofluorescence imaging confirmed that ST80 treatment both increased the abundance and resulted in unilateral local accumulation of acetylated α-tubulin. In conclusion, the three novel HDACi show potent antileukemic activity in myeloid cell lines and primary AML blasts at low micromolar concentrations. Preferential acetylation of α-tubulin implies that ST80 might exert its antileukemic effect not through histone reacetylation but rather through inhibition of HDAC6.

Reversal of p15/INK4b hypermethylation in AML1/ETO-positive and -negative myeloid leukemia cell lines.

In vitro and in vivo, myeloid leukemic and preleukemic cells exhibit variable sensitivity to the antiproliferative and proapoptotic effects induced already at low concentrations of DNA methyltransferase (DNMT) inhibitors. The molecular mechanisms underlying this variable sensitivity of leukemic blasts to azanucleosides such as 5-azacytidine and 5-aza-2'-deoxycytidine (DAC) may involve modifier effects of specific fusion proteins such as AML1/ETO. The cyclin-dependent kinase inhibitor p15/INK4b is one potential target of DNA demethylating activity in AML and MDS where it is frequently silenced by hypermethylation. To study sensitivity to DAC in myeloid leukemia cells, we chose the myeloid cell lines Kasumi-1 (expressing AML1/ETO), KG-1 and KG-1a (both AML1/ETO-negative) all of which a highly methylated p15/INK4b gene. Treatment with DAC resulted in dose-dependent regional demethylation of p15/INK4b in Kasumi-1 and KG-1, but only to a modest degree in KG-1a cells. Demethylation was associated with induction of p15/INK4b protein expression. Growth-inhibitory and proapoptotic activity of DAC was significantly higher in Kasumi-1 than in KG-1a cells, and sensitization of cells to a cooperating effect of All-trans retinoic acid and of the histone deacetylase (HDAC) inhibitor Trichostatin A was observed. DAC-induced growth inhibition and apoptosis were enhanced when AML1/ETO was conditionally expressed in AML1/ETO-negative U-937 cells. In conclusion, hypomethylation and reactivation of p15/INK4b in myeloid cell lines are among the molecular events associated with DAC-induced growth arrest and apoptosis. Further studies of AML1/ETO as a modifier of the epigenotype and sensitivity of myeloid cells to inhibitors of DNMTs and HDACs appear warranted.