Long-term in vitro monitoring of AAV-transduction efficiencies in real-time with Hoechst 33342.

Adeno-associated viral transduction allows the introduction of nucleic fragments into cells and is widely used to modulate gene expressions in vitro and in vivo. It enables the study of genetic functions and disease mechanisms and, more recently, serves as a tool for gene repair. To achieve optimal transduction performance for a given cell type, selecting an appropriate serotype and the number of virus particles per cell, also known as the multiplicity of infection, is critical. Fluorescent proteins are one of the common reporter genes to visualize successfully transduced cells and assess transduction efficiencies. Traditional methods of measuring fluorescence-positive cells are endpoint analysis by flow cytometry or manual counting with a fluorescence microscope. However, the flow cytometry analysis does not allow further measurement in a test run, and manual counting by microscopy is time-consuming. Here, we present a method that repeatedly evaluates transduction efficiencies by adding the DNA-stain Hoechst 33342 during the transduction process combined with a microscope or live-cell imager and microplate image analysis software. The method achieves fast, high-throughput, reproducible, and real-time post-transduction analysis and allows for optimizing transduction parameters and screening for a proper approach.

A simple dissection method for the isolation of mouse trabecular meshwork cells.

PURPOSE: The outflow pathway, especially trabecular meshwork (TM), plays an essential role in glaucoma, and the availability of TM cells is crucial for in vitro research. So far, the isolation of TM cells from mice has been anything but manageable due to the small size of the eye. Direct isolation using a stereomicroscope and forceps requires a high grade of dexterity. Indirect isolation is based on the phagocytic properties of TM cells and involves injecting magnetic microspheres into the anterior chamber of live mice followed by isolation. Therefore, a simpler, less expensive, and nonexperimental strategy for isolating mouse TM cells would be desirable. METHODS: After enucleation, the eyes were cut in half anterior-to-posteriorly. The lens and posterior segment were removed. Iris and the attached ciliary body were gently pulled backward and disconnected from the remaining tissue to expose the TM. By incising through the cornea anteriorly and posteriorly of the TM, the cornea/TM stripe could be isolated. The cornea/TM stripe was cultured with the pigmented side down in a 6-well. The outgrowing pigmented cells were analyzed by immunocytochemistry and mRNA expression for previously described TM cell markers. The phagocytic properties of the cells were additionally confirmed using fluorescent microspheres. RESULTS: Pigmented phagocytic cells were the first to grow out of the cornea/TM strips after approximately 4-7 days. Cells were positive for Collagen IV, Fibronectin1, Vimentin, and Actin alpha 2 and could phagocytize fluorescent microbeads. Cross-linked actin networks were visible after 9 days of exposure to TGFB2 (transforming growth factor-beta 2). Additionally, treatment with 500 nM Dexamethasone for one week increased myocilin expression, as previously reported for TM cells. In addition, we proved that this method can also be used in albino mice, which lack pigmentation of the trabecular meshwork. CONCLUSIONS: The isolated cells show phagocytic properties and specific expression of markers reported in TM cells. Therefore, our dissection-based method is inexpensive and reproducible for isolating TM cells in mice.

Breaking a Dogma: High-Throughput Live-Cell Imaging in Real-Time with Hoechst 33342.

Automated high-throughput live cell imaging (LCI) enables investigation of substance effects on cells in vitro. Usually, cell number is analyzed by phase-contrast imaging, which is reliable only for a few cell types. Therefore, an accurate cell counting method, such as staining the nuclei with Hoechst 33342 before LCI, will be desirable. However, since the mid-1980s, the dogma exists that Hoechst can only be used for endpoint analyses because of its cytotoxic properties and the potentially phototoxic effects of the excitation light. Since microscopic camera sensitivity has significantly improved, this study investigates whether this dogma is still justified. Therefore, exposure parameters are optimized using a 4× objective, and the minimum required Hoechst concentration is evaluated, allowing LCI at 30-min intervals over 5 days. Remarkably, a Hoechst concentration of only 57 × 10-9 m significantly inhibits proliferation and thus impairs cell viability. However, Hoechst concentrations between 7 × 10-9  and 28 × 10-9 m can be determined, which are neither cytotoxic nor impacting cell viability, proliferation, or signaling pathways. The method can be adapted to regular inverted fluorescence microscopes and allows, for example, to determine the cytotoxicity of a substance or the transduction efficiency, with the advantage that the analysis can be repeated at any desired time point.

MiR-302d inhibits TGFB-induced EMT and promotes MET in primary human RPE cells.

PURPOSE: Transforming growth factor-beta (TGFB)-mediated epithelial-mesenchymal transition (EMT) plays a crucial role in the pathogenesis of retinal fibrosis, which is one of the leading causes of impaired vision. Current approaches to treating retinal fibrosis focus, among other things, on inhibiting the TGFB signaling pathway. Transient expression of microRNAs (miRNAs) is one way to inhibit the TGFB pathway post-transcriptionally. Our previous study identified the miRNA miR-302d as a regulator of multiple TGFB-related genes in ARPE-19 cells. To further explore its effect on primary cells, the effect of miR-302d on TGFB-induced EMT in primary human retinal pigment epithelium (hRPE) was investigated in vitro. METHODS: hRPE cells were extracted from patients receiving enucleation. Transfection of hRPE cells with miR-302d was performed before or after TGFB1 stimulation. Live-cell imaging, immunocytochemistry staining, Western blot, and ELISA assays were utilized to identify the alterations of cellular morphology and EMT-related factors expressions in hRPE cells. RESULTS: hRPE cells underwent EMT by TGFB1 exposure. The transfection of miR-302d inhibited the transition with decreased production of mesenchymal markers and increased epithelial factors. Meanwhile, the phosphorylation of SMAD2 activated by TGFB1 was suppressed. Moreover, miR-302d expression promoted TGFB1-induced fibroblast-like hRPE cells to revert towards an epithelial stage. As confirmed by ELISA, miR-302d reduced TGFB receptor 2 (TGFBR2) and vascular endothelial growth factor A (VEGFA) levels 48 hours after transfection. CONCLUSIONS: The protective effect of miR-302d might be a promising approach for ameliorating retinal fibrosis and neovascularization. MiR-302d suppresses TGFB-induced EMT in hRPE cells via downregulation of TGFBR2, even reversing the process. Furthermore, miR-302d reduces the constitutive secretion of VEGFA from hRPE cells.

Alpha1-antitrypsin counteracts heme-induced endothelial cell inflammatory activation, autophagy dysfunction and death.

Free heme toxicity in the vascular endothelium is critical for the pathogenesis of hemolytic disorders including sickle cell disease. In the current study, it is demonstrated that human alpha1-antitrypsin (A1AT), a serine protease inhibitor with high binding-affinity for heme, rescues endothelial cell (EC) injury caused by free heme. A1AT provided endothelial protection against free heme toxicity via a pathway that differs from human serum albumin and hemopexin, two prototypical heme-binding proteins. A1AT inhibited heme-mediated pro-inflammatory activation and death of ECs, but did not affect the increase in intracellular heme levels and up-regulation of the heme-inducible enzyme heme oxygenase-1. Moreover, A1AT reduced heme-mediated generation of mitochondrial reactive oxygen species. Extracellular free heme led to an increased up-take of A1AT by ECs, which was detected in lysosomes and was found to reduce heme-dependent alkalization of these organelles. Finally, A1AT was able to restore heme-dependent dysfunctional autophagy in ECs. Taken together, our findings show that A1AT rescues ECs from free heme-mediated pro-inflammatory activation, cell death and dysfunctional autophagy. Hence, A1AT therapy may be useful in the treatment of hemolytic disorders such as sickle cell disease.

Inferior vena cava-syndrome.

Inferior vena cava syndrome (IVCS) is caused by agenesis, compression, invasion, or thrombosis of the IVC, or may be associated with Budd-Chiari syndrome. Its incidence and prevalence are unknown. Benign IVCS is separated from malignant IVCS. Both cover a wide clinical spectrum reaching from asymptomatic to highly symptomatic cases correlated to the underlying cause, the acuity, the extent of the venous obstruction, and the recruitment and development of venous collateral circuits. Imaging is necessary to determine the underlying cause of IVCS and to guide clinical decisions. Interventional therapy has changed the therapeutic approach in symptomatic patients. This article provides an overview over IVCS and focuses on interventional therapeutic methods and results.

Superior vena cava syndrome.

The superior vena cava syndrome (SVCS) is caused by compression, invasion, and/or thrombosis of the superior vena cava and/or the brachiocephalic veins. Benign SVCS is separated from malignant SVCS. SVCS comprises a broad clinical spectrum reaching from asymptomatic cases to rare life-threatening emergencies with upper airway obstruction and increased intracranial pressure. Symptoms are correlated to the acuity and extent of the venous obstruction and inversely correlated to the development of the venous collateral circuits. Imaging is necessary to determine the exact underlying cause and to guide further interventions. Interventional therapy has widely changed the therapeutic approach in symptomatic patients. This article provides an overview over this complex syndrome and focuses on interventional therapeutic methods and results.

The microRNAs miR-302d and miR-93 inhibit TGFB-mediated EMT and VEGFA secretion from ARPE-19 cells.

The transforming growth factor-beta (TGFB) plays an essential role in the pathogenesis of some ophthalmologic diseases, including neovascular age-related macular degeneration (nAMD) and proliferative vitreoretinopathy (PVR). TGFB activates the transcription factors SMAD2 and SMAD3 via the TGFB receptor, which together activate several genes, including VEGFA. TGFB treated ARPE-19 cells show an increased proliferation rate and undergo epithelial to mesenchymal transition (EMT). Since microRNAs (miRNAs) are capable of inhibiting the translation of multiple genes, we screened for miRNAs that regulate the TGFB signalling pathways at multiple levels. In this study, we focused on two miRNAs, miR-302d and miR-93, which inhibit TGFB signalling pathway and therefore TGFB-induced EMT transition as well as VEGFA secretion from ARPE-19 cells. Furthermore, we could show that both miRNAs can retransform TGFB-stimulated mesenchymal ARPE-19 cells towards the morphological epithelial-like state. Taken together, transient overexpression of these miRNAs in RPE cells might be a promising approach for further translational strategies.

Human and murine macrophages exhibit differential metabolic responses to lipopolysaccharide - A divergent role for glycolysis.

Macrophages adopt different phenotypes in response to microenvironmental changes, which can be principally classified into inflammatory and anti-inflammatory states. Inflammatory activation of macrophages has been linked with metabolic reprogramming from oxidative phosphorylation to aerobic glycolysis. In contrast to mouse macrophages, little information is available on the link between metabolism and inflammation in human macrophages. In the current report it is demonstrated that lipopolysaccharide (LPS)-activated human peripheral blood monocyte-derived macrophages (hMDMs) fail to undergo metabolic reprogramming towards glycolysis, but rely on oxidative phosphorylation for the generation of ATP. By contrast, activation by LPS led to an increased extracellular acidification rate (glycolysis) and decreased oxygen consumption rate (oxidative phosphorylation) in mouse bone marrow-derived macrophages (mBMDMs). Mitochondrial bioenergetics after LPS stimulation in human macrophages was unchanged, but was markedly impaired in mouse macrophages. Furthermore, treatment with 2-deoxyglucose, an inhibitor of glycolysis, led to cell death in mouse, but not in human macrophages. Finally, glycolysis appeared to be critical for LPS-mediated induction of the anti-inflammatory cytokine interleukin-10 in both human and mouse macrophages. In summary, these findings indicate that LPS-induced immunometabolism in human macrophages is different to that observed in mouse macrophages.

Ethyl 2-((4-Chlorophenyl)amino)thiazole-4-carboxylate and Derivatives Are Potent Inducers of Oct3/4.

The octamer-binding transcription factor 4 (Oct3/4) is a master gene in the transcriptional regulatory network of pluripotent cells. Repression of Oct3/4 in embryonic stem cells (ESCs) is associated with cell differentiation and loss of pluripotency, whereas forced overexpression in cooperation with other transcriptional factors, such as Nanog, Sox2, and Lin28, can reprogram somatic cells back into pluripotent cells, termed induced pluripotent stem cells (iPSCs). However, random integration and potential tumorigenic transformation caused by viral transduction limit the clinical application of iPSCs. By performing a cell-based high throughput screening (HTS) campaign, we identified several potential small molecules as inducers of Oct3/4 expression. Here we report a lead structure ethyl 2-((4-chlorophenyl)amino)-thiazole-4-carboxylate, termed O4I2, showing high activity in enforcing Oct3/4 expression. On the basis of chemical expansion, we further identified derivatives having increased activities toward Oct3/4 induction. Thus, O4I2 and its derivatives should provide a new class of small molecules suitable for iPSC generation.

Identification of 2-[4-[(4-Methoxyphenyl)methoxy]-phenyl]acetonitrile and Derivatives as Potent Oct3/4 Inducers.

Reprogramming somatic cells into induced-pluripotent cells (iPSCs) provides new access to all somatic cell types for clinical application without any ethical controversy arising from the use of embryonic stem cells (ESCs). Established protocols for iPSCs generation based on viral transduction with defined factors are limited by low efficiency and the risk of genetic abnormality. Several small molecules have been reported as replacements for defined transcriptional factors, but a chemical able to replace Oct3/4 allowing the generation of human iPSCs is still unavailable. Using a cell-based High Throughput Screening (HTS) campaign, we identified that 2-[4-[(4-methoxyphenyl)methoxy]phenyl]acetonitrile (1), termed O4I1, enhanced Oct3/4 expression. Structural verification and modification by chemical synthesis showed that O4I1 and its derivatives not only promoted expression and stabilization of Oct3/4 but also enhanced its transcriptional activity in diverse human somatic cells, implying the possible benefit from using this class of compounds in regenerative medicine.

miR-128 regulates neuronal migration, outgrowth and intrinsic excitability via the intellectual disability gene Phf6.

miR-128, a brain-enriched microRNA, has been implicated in the control of neurogenesis and synaptogenesis but its potential roles in intervening processes have not been addressed. We show that post-transcriptional mechanisms restrict miR-128 accumulation to post-mitotic neurons during mouse corticogenesis and in adult stem cell niches. Whereas premature miR-128 expression in progenitors for upper layer neurons leads to impaired neuronal migration and inappropriate branching, sponge-mediated inhibition results in overmigration. Within the upper layers, premature miR-128 expression reduces the complexity of dendritic arborization, associated with altered electrophysiological properties. We show that Phf6, a gene mutated in the cognitive disorder Börjeson-Forssman-Lehmann syndrome, is an important regulatory target for miR-128. Restoring PHF6 expression counteracts the deleterious effect of miR-128 on neuronal migration, outgrowth and intrinsic physiological properties. Our results place miR-128 upstream of PHF6 in a pathway vital for cortical lamination as well as for the development of neuronal morphology and intrinsic excitability.

miR-27 negatively regulates pluripotency-associated genes in human embryonal carcinoma cells.

Human embryonic stem cells and human embryonal carcinoma cells have been studied extensively with respect to the transcription factors (OCT4, SOX2 and NANOG), epigenetic modulators and associated signalling pathways that either promote self-renewal or induce differentiation in these cells. The ACTIVIN/NODAL axis (SMAD2/3) of the TGFß signalling pathway coupled with FGF signalling maintains self-renewal in these cells, whilst the BMP (SMAD1,5,8) axis promotes differentiation. Here we show that miR-27, a somatic-enriched miRNA, is activated upon RNAi-mediated suppression of OCT4 function in human embryonic stem cells. We further demonstrate that miR-27 negatively regulates the expression of the pluripotency-associated ACTIVIN/NODAL axis (SMAD2/3) of the TGFß signalling pathway by targeting ACVR2A, TGFßR1 and SMAD2. Additionally, we have identified a number of pluripotency-associated genes such as NANOG, LIN28, POLR3G and NR5A2 as novel miR-27 targets. Transcriptome analysis revealed that miR-27 over-expression in human embryonal carcinoma cells leads indeed to a significant up-regulation of genes involved in developmental pathways such as TGFß- and WNT-signalling.

Comparative molecular portraits of human unfertilized oocytes and primordial germ cells at 10 weeks of gestation.

Primordial germ cells (PGCs) are precursors of gametes and share several features in common with pluripotent stem cells, such as alkaline phosphatase activity and the expression of pluripotency-associated genes such as OCT4 and NANOG. PGCs are able to differentiate into oocytes and spermatogonia and establish totipotency after fertilization. However, our knowledge of human germ cell development is still fragmentary. In this study, we have carried out genome-wide comparisons of the transcriptomes and molecular portraits of human male PGCs (mPGCs), female PGCs (fPGCs) and unfertilized oocytes. We detected 9210 genes showing elevated expression in fPGCs, 9184 in mPGCs and 9207 in oocytes, with 6342 of these expressed in common. As well as known germ cell-related genes such as BLIMP1/PRDM1, PIWIL2, VASA/DDX4, DAZL, STELLA/DPPA3 and LIN28, we also identified 465 novel non-annotated genes with orthologs in the mouse. A plethora of olfactory receptor-encoding genes were detected in all samples, which would suggest their involvement not only in sperm chemotaxis, but also in the development of female germ cells and oocytes. We anticipate that our data might increase our meagre knowledge of the genes and associated signaling pathways operative during germ cell development. This in turn might aid in the development of strategies enabling better differentiation and molecular characterisation of germ cells derived from either embryonic or induced pluripotent stem cells. Ultimately, this would have a profound relevance for reproductive as well as regenerative medicine.

The let-7 target gene mouse lin-41 is a stem cell specific E3 ubiquitin ligase for the miRNA pathway protein Ago2.

The let-7 miRNA and its target gene Lin-28 interact in a regulatory circuit controlling pluripotency. We investigated an additional let-7 target, mLin41 (mouse homologue of lin-41), as a potential contributor to this circuit. We demonstrate the presence of mLin41 protein in several stem cell niches, including the embryonic ectoderm, epidermis and male germ line. mLin41 colocalized to cytoplasmic foci with P-body markers and the miRNA pathway proteins Ago2, Mov10 and Tnrc6b. In co-precipitation assays, mLin41 interacted with Dicer and the Argonaute proteins Ago1, Ago2 and Ago4. Moreover, we show that mLin41 acts as an E3 ubiquitin ligase in an auto-ubiquitylation assay and that mLin41 mediates ubiquitylation of Ago2 in vitro and in vivo. Overexpression and depletion of mLin41 led to inverse changes in the level of Ago2 protein, implicating mLin41 in the regulation of Ago2 turnover. mLin41 interfered with silencing of target mRNAs for let-7 and miR-124, at least in part by antagonizing Ago2. Furthermore, mLin41 cooperated with the pluripotency factor Lin-28 in suppressing let-7 activity, revealing a dual control mechanism regulating let-7 in stem cells.

Incidence of symptomatic vertebral fractures in patients after percutaneous vertebroplasty.

The aim of this study was to evaluate the incidence of secondary symptomatic vertebral compression fractures (VCFs) in patients previously treated by percutaneous vertebroplasty (VTP). Three hundred sixteen patients with 486 treated VCFs were included in the study according to the inclusion criteria. Patients were kept in regular follow-up using a standardized questionairre before, 1 day, 7 days, 6 months, and 1 year after, and, further on, on a yearly basis after VTP. The incidence of secondary symptomatic VCF was calculated, and anatomical distribution with respect to previous fractures characterized. Mean follow-up was 8 months (6-56 months) after VTP. Fifty-two of 316 (16.4 %) patients (45 female, 7 male) returned for treatment of 69 secondary VCFs adjacent to (35/69; 51%) or distant from (34/69; 49%) previously treated levels. Adjacent secondary VCF occurred significantly more often compared to distant secondary VCF. Of the total 69 secondary VCFs, 35 of 69 occurred below and 27 of 69 above pretreated VCFs. Of the 65 sandwich levels generated, in 7 of 65 (11%) secondary VCFs were observed. Secondary VCF below pretreated VCF occurred significantly earlier in time compared to VCF above and compared to sandwich body fractures. No major complication occurred during initial or follow-up intervention. We conclude that secondary VCFs do occur in individuals after VTP but the rate found in our study remains below the level expected from epidemiologic studies. Adjacent fractures occur more often and follow the cluster distribution of VCF as expected from the natural history of the underlying osteoporosis. No increased rate of secondary VCF after VTP was observed in this retrospective analysis. In accordance with the pertinent literature, short-term and also midterm clinical results are encouraging and provide further support for the usefulness and the low complication rate of this procedure as an adjunct to the spectrum of pain management in patients with severe midline back pain due to osteoporotic spine fractures.

Detection of cement leakage after vertebroplasty with a non-flat-panel angio unit compared to multidetector computed tomography--an ex vivo study.

The purpose of this study was to investigate the detection of cement leakages after vertebroplasty using angiographic computed tomography (ACT) in a non-flat-panel angio unit compared to multidetector computed tomography (MDCT). Vertebroplasty was performed in 19 of 33 cadaver vertebrae (23 thoracic and 10 lumbar segments). In the angio suite, ACT (190 degrees ; 1.5 degrees per image) was performed to obtain volumetric data. Another volumetric data set of the specimen was obtained by MDCT using a standard algorithm. Nine multiplanar reconstructions in standardized axial, coronal, and sagittal planes of every vertebra were generated from both data sets. Images were evaluated on the basis of a nominal scale with 18 criteria, comprising osseous properties (e.g., integrity of the end plate) and cement distribution (e.g., presence of intraspinal cement). MDCT images were regarded as gold standard and analyzed by two readers in a consensus mode. Rotational acquisitions were analyzed by six blinded readers. Results were correlated with the gold standard using Cohen's j-coefficient analysis. Furthermore, interobserver variability was calculated. Correlation with the gold standard ranged from no correlation (osseous margins of the neuroforamen, j = 0.008) to intermediate (trace of vertebroplasty canula; j = 0.615) for criteria referring to osseous morphology. However, there was an excellent correlation for those criteria referring to cement distribution, with kappa values ranging from 0.948 (paravertebral cement distribution) to 0.972 (intraspinal cement distribution). With a minimum of j = 0.768 ("good correlation") and a maximum of j = 0.91 ("excellent"), interobserver variability was low. In conclusion, ACT in an angio suite without a flat-panel detector depicts a cement leakage after vertebroplasty as well as MDCT. However, the method does not provide sufficient depiction of osseous morphology.

A feedback loop comprising lin-28 and let-7 controls pre-let-7 maturation during neural stem-cell commitment.

miRNA populations, including mammalian homologues of lin-4 (mir-125) and let-7, undergo a marked transition during stem-cell differentiation. Originally identified on the basis of their mutational phenotypes in stem-cell maturation, mir-125 and let-7 are strongly induced during neural differentiation of embryonic stem (ES) cells and embryocarcinoma (EC) cells. We report that embryonic neural stem (NS) cells express let-7 and mir-125, and investigate post-transcriptional mechanisms contributing to the induction of let-7. We demonstrate that the pluripotency factor Lin-28 binds the pre-let-7 RNA and inhibits processing by the Dicer ribonuclease in ES and EC cells. In NS cells, Lin-28 is downregulated by mir-125 and let-7, allowing processing of pre-let-7 to proceed. Suppression of let-7 or mir-125 activity in NS cells led to upregulation of Lin-28 and loss of pre-let-7 processing activity, suggesting that let-7, mir-125 and lin-28 participate in an autoregulatory circuit that controls miRNA processing during NS-cell commitment.

Interventional management of renal bleeding after partial nephrectomy.

OBJECTIVE: Partial nephrectomy (PN) has emerged as a serious alternative to nephrectomy in oncologic therapy of renal tumours. While complications are rare in general, renal hemorrhage may occur und necessitate angiographic embolization. In this retrospective study, we evaluate the clinical, imaging and procedural findings of seven interventions in five patients with renal hemorrhage after PN. In four out of five patients (80%) the bleeding could be treated successfully by embolotherapy. CONCLUSION: Angiographic embolization in patients with renal hemorrhage after PN is feasible and has a high success rate. The procedure might facilitate avoidance of nephrectomy.

Complications in percutaneous vertebroplasty associated with puncture or cement leakage.

Due to the minimally invasive character and excellent clinical outcome of percutaneous vertebroplasty (PVP), the procedure is being performed in greatly increasing numbers. While PVP has a low complication rate in general, severe complications can occur. We focus on the imaging appearance of complications of PVP associated with puncture or cement leakage--from harmless to life-threatening.