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European journal of pharmaceutical sciences : official journal of the European Federation for Pharmaceutical Sciences, 129, 181--189

Multi- and unilamellar liposomal encapsulation of ciprofloxacin as ways to modify its phototoxicity and photodegradation.

Zgadzaj, A, Giebułtowicz, J, Gubernator, J, Podbielska, M, Sommer, S, Zaremba-Czogalla, M, Nałęcz-Jawecki, G

Liposomes are vesicular preparations that improve bioavailability of many pharmaceuticals, used even in ocular therapy. In addition, it is well documented that vesicular carriers could affect the photodegradation of molecules encapsulated inside, which is especially important for drugs that may exhibit phototoxicity when they are applied topically on sensitive light-exposed tissues. In this study, we investigated the effect of ciprofloxacin encapsulation into liposomes on its photodegradation, phototoxicity and photogenotoxicity in vitro at the concentration ranges applied in ophthalmology. We tested two variants of liposomes: large unilamellar vesicles (LUV) and multilamellar vesicles (MLV) in comparison to antibiotic solutions without phospholipids (CPX). On the basis of our research, the kinetics of ciprofloxacin photolysis was the fastest in formulations with vesicles with low drug-to-lipid ratio. Depending on vesicles type (drug-to-lipid ratio, MLV or LUV) and time of irradiation different degradants were produced. We proposed structures of the novel ciprofloxacin photolysis products characteristic for vesicles. We did not notice any photoprotective effect of application of ciprofloxacin encapsulation into liposomes, but it significantly affected the photodegradation product profile of the drug and the Photo-Irritation-Factor of the vesicular preparations. In the MTT and micronucleus assays impact of encapsulation was not as clearly visible.

Digital object identifier (DOI): 10.1016/j.ejps.2019.01.006

The journal of pathology. Clinical research, 5, 63--78

Combined epithelial marker analysis of tumour budding in stage II colorectal cancer.

Slik, Khadija, Blom, Sami, Turkki, Riku, Välimäki, Katja, Kurki, Samu, Mustonen, Harri, Haglund, Caj, Carpén, Olli, Kallioniemi, Olli, Korkeila, Eija, Sundström, Jari, Pellinen, Teijo

Tumour budding predicts survival of stage II colorectal cancer (CRC) and has been suggested to be associated with epithelial-to-mesenchymal transition (EMT). However, the underlying molecular changes of tumour budding remain poorly understood. Here, we performed multiplex immunohistochemistry (mIHC) to phenotypically profile tumours using known EMT-associated markers: E-cadherin (adherence junctions), integrin β4 (ITGB4; basement membrane), ZO-1 (tight junctions), and pan-cytokeratin. A subpopulation of patients showed high ITGB4 expression in tumour buds, and this coincided with a switch of ITGB4 localisation from the basal membrane of intact epithelium to the cytoplasm of budding cells. Digital image analysis demonstrated that tumour budding with high ITGB4 expression in tissue microarray (TMA) cores correlated with tumour budding assessed from haematoxylin and eosin (H&E) whole sections and independently predicted poor disease-specific survival in two independent stage II CRC cohorts (hazard ratio [HR] = 4.50 (95% confidence interval [CI] = 1.50-13.5), n = 232; HR = 3.52 (95% CI = 1.30-9.53), n = 72). Furthermore, digitally obtained ITGB4-high bud count in random TMA cores was better associated with survival outcome than visual tumour bud count in corresponding H&E-stained samples. In summary, the mIHC-based phenotypic profiling of human tumour tissue shows strong potential for the molecular characterisation of tumour biology and for the discovery of novel prognostic biomarkers.

Digital object identifier (DOI): 10.1002/cjp2.119

Cells, 8

Oxidative Stress Induces Telomere Dysfunction and Senescence by Replication Fork Arrest.

Coluzzi, Elisa, Leone, Stefano, Sgura, Antonella

Oxidative DNA damage, particularly 8-oxoguanine, represents the most frequent DNA damage in human cells, especially at the telomeric level. The presence of oxidative lesions in the DNA can hinder the replication fork and is able to activate the DNA damage response. In this study, we wanted to understand the mechanisms by which oxidative damage causes telomere dysfunction and senescence in human primary fibroblasts. After acute oxidative stress at telomeres, our data demonstrated a reduction in TRF1 and TRF2, which are involved in proper telomere replication and T-loop formation, respectively. Furthermore, we observed a higher level of γH2AX with respect to 53BP1 at telomeres, suggesting a telomeric replication fork stall rather than double-strand breaks. To confirm this finding, we studied the replication of telomeres by Chromosome Orientation-FISH (CO-FISH). The data obtained show an increase in unreplicated telomeres after hydrogen peroxide treatment, corroborating the idea that the presence of 8-oxoG can induce replication fork arrest at telomeres. Lastly, we analyzed the H3K9me3 histone mark after oxidative stress at telomeres, and our results showed an increase of this marker, most likely inducing the heterochromatinization of telomeres. These results suggest that 8-oxoG is fundamental in oxidative stress-induced telomeric damage, principally causing replication fork arrest.

Digital object identifier (DOI): 10.3390/cells8010019

Journal of cell science, 132

Synthetic lethality of cytolytic HSV-1 in cancer cells with ATRX and PML deficiency.

Han, Mingqi, Napier, Christine E, Frölich, Sonja, Teber, Erdahl, Wong, Ted, Noble, Jane R, Choi, Eugene H Y, Everett, Roger D, Cesare, Anthony J, Reddel, Roger R

Cancers that utilize the alternative lengthening of telomeres (ALT) mechanism for telomere maintenance are often difficult to treat and have a poor prognosis. They are also commonly deficient for expression of ATRX protein, a repressor of ALT activity, and a component of promyelocytic leukemia nuclear bodies (PML NBs) that are required for intrinsic immunity to various viruses. Here, we asked whether ATRX deficiency creates a vulnerability in ALT cancer cells that could be exploited for therapeutic purposes. We showed in a range of cell types that a mutant herpes simplex virus type 1 (HSV-1) lacking ICP0, a protein that degrades PML NB components including ATRX, was ten- to one thousand-fold more effective in infecting ATRX-deficient cells than wild-type ATRX-expressing cells. Infection of co-cultured primary and ATRX-deficient cancer cells revealed that mutant HSV-1 selectively killed ATRX-deficient cells. Sensitivity to mutant HSV-1 infection also correlated inversely with PML protein levels, and we showed that ATRX upregulates PML expression at both the transcriptional and post-transcriptional levels. These data provide a basis for predicting, based on ATRX or PML levels, which tumors will respond to a selective oncolytic herpesvirus.

Digital object identifier (DOI): 10.1242/jcs.222349

Annals of laboratory medicine, 39, 91--95

Dose Estimation Curves Following In Vitro X-ray Irradiation Using Blood From Four Healthy Korean Individuals.

Jang, Mi Ae, Han, Eun Ae, Lee, Jin Kyung, Cho, Kwang Hwan, Shin, Hee Bong, Lee, You Kyoung

Cytogenetic dosimetry is useful for evaluating the absorbed dose of ionizing radiation based on analysis of radiation-induced chromosomal aberrations. We created two types of dose-response calibration curves for dicentric chromosomes (DC) and translocations (TR) induced by X-ray irradiation, using an electron linear accelerator, which is the most frequently used medical device in radiotherapy. We irradiated samples from four healthy Korean individuals and compared the resultant curves between individuals. Aberration yields were studied in a total of 31,800 and 31,725 metaphases for DC and TR, respectively, obtained from 11 X-ray irradiation dose-points (0, 0.05, 0.1, 0.25, 0.5, 0.75, 1, 2, 3, 4, and 5 Gy). The dose-response relationship followed a linear-quadratic equation, Y=C+αD+βD², with the coefficients C=0.0011 for DC and 0.0015 for TR, α=0.0119 for DC and 0.0048 for TR, and β=0.0617 for DC and 0.0237 for TR. Correlation coefficients between irradiation doses and chromosomal aberrations were 0.971 for DC and 0.6 for TR, indicating a very strong and a moderate correlation, respectively. This is the first study implementing cytogenetic dosimetry following exposure to ionizing X-radiation.

Digital object identifier (DOI): 10.3343/alm.2019.39.1.91

Chemosphere, 215, 703--709

Nanomaterials induce DNA-protein crosslink and DNA oxidation: A mechanistic study with RTG-2 fish cell line and Comet assay modifications.

Klingelfus, T, Disner, G R, Voigt, C L, Alle, L F, Cestari, M M, Leme, D M

Genotoxic effects of nanomaterials (NMs) have been controversially reported in literature, and the mode of action (MoA) via DNA oxidation is cited as the main damage caused by them. Evidence of nano-silver as a crosslinker has been previously reported by the present research team in an in vivo fish genotoxicity study. Thus, aiming to confirm the evidence about NMs as crosslinker agent, the present investigation elucidated the genotoxic potential of NMs and their genotoxic MoA through in vitro assay with RTG-2 cells line (rainbow trout gonadal) by exposure to nano-silver (PVP-coated) and nano-titanium. The types and levels of DNA damage were assessed by the Comet assay (standard alkaline, hOGG1-modified alkaline, and two crosslink-modified alkaline versions). It was demonstrated that the use of the standard alkaline Comet assay alone may inaccurately predict the genotoxicity of NMs since oxidative and crosslink DNA damages were also verified in RTG-2 cells when assessed by the modified versions of the alkaline protocol. More importantly, it was confirmed that both nano-silver and nano-titanium acted as DNA-protein crosslinkers through the Comet assay version with proteinase K. As both nano-silver and nano-titanium present a great risk to aquatic life, these findings reinforce the need of genotoxicity testing strategies that encompass the assessment of different types of DNA damage, in order to ensure an accurate prediction of the genotoxic potential of NMs.

Digital object identifier (DOI): 10.1016/j.chemosphere.2018.10.118

Biochemical and biophysical research communications, 511, 658--664

Distinctive Krebs cycle remodeling in iPSC-derived neural and mesenchymal stem cells.

Benlamara, Sarah, Aubry, Laetitia, Fabregue, Julien, Bénit, Paule, Rustin, Pierre, Rak, Malgorzata

Mitochondria play a vital role in proliferation and differentiation and their remodeling in the course of differentiation is related to the variable energy and metabolic needs of the cell. In this work, we show a distinctive mitochondrial remodeling in human induced pluripotent stem cells differentiated into neural or mesenchymal progenitors. While leading to upregulation of the citrate synthase-α-ketoglutarate dehydrogenase segment of the Krebs cycle and increased respiratory chain activities and respiration in the mesenchymal stem cells, the remodeling in the neural stem cells resulted in downregulation of α-ketoglutarate dehydrogenase, upregulation of isocitrate dehydrogenase 2 and the accumulation of α-ketoglutarate. The distinct, lineage-specific changes indicate an involvement of these Krebs cycle enzymes in cell differentiation.

Digital object identifier (DOI): 10.1016/j.bbrc.2019.02.033

Microbiological research, 221, 28--35

Muscodor brasiliensis sp. nov. produces volatile organic compounds with activity against Penicillium digitatum.

Pena, Lorena C, Jungklaus, Gustavo H, Savi, Daiani C, Ferreira-Maba, Lisandra, Servienski, André, Maia, Beatriz H L N S, Annies, Vinicius, Galli-Terasawa, Lygia V, Glienke, Chirlei, Kava, Vanessa

Endophytic fungi belonging to Muscodor genus are considered as promising alternatives to be used in biological control due to the production of volatile organic compounds (VOCs). The strains LGMF1255 and LGMF1256 were isolated from the medicinal plant Schinus terebinthifolius and, by morphological data and phylogenetic analysis, identified as belonging to Muscodor genus. Phylogenetic analysis suggests that strain LGMF1256 is a new species, which is herein introduced as Muscodor brasiliensis sp. nov. The analysis of VOCs production revealed that compounds phenylethyl alcohol, α-curcumene, and E (β) farnesene until now has been reported only from M. brasiliensis, data that supports the classification of strain LGMF1256 as a new species. M. brasiliensis completely inhibited the phytopathogen P. digitatum in vitro. We also evaluated the ability of VOCs from LGMF1256 to inhibit the development of green mold symptoms by inoculation of P. digitatum in detached oranges. M. brasiliensis reduced the severity of diseases in 77%, and showed potential to be used for fruits storage and transportation to prevent the green mold symptoms development, eventually reducing the use of fungicides.

Digital object identifier (DOI): 10.1016/j.micres.2019.01.002

BMC plant biology, 19, 183

Development and characterisation of interspecific hybrid lines with genome-wide introgressions from Triticum timopheevii in a hexaploid wheat background.

Devi, Urmila, Grewal, Surbhi, Yang, Cai-Yun, Hubbart-Edwards, Stella, Scholefield, Duncan, Ashling, Stephen, Burridge, Amanda, King, Ian P, King, Julie

Triticum timopheevii (2n = 4x = 28; A A GG), is an important source for new genetic variation for wheat improvement with genes for potential disease resistance and salt tolerance. By generating a range of interspecific hybrid lines, T. timopheevii can contribute to wheat's narrow gene-pool and be practically utilised in wheat breeding programmes. Previous studies that have generated such introgression lines between wheat and its wild relatives have been unable to use high-throughput methods to detect the presence of wild relative segments in such lines. A whole genome introgression approach, exploiting homoeologous recombination in the absence of the Ph1 locus, has resulted in the transfer of different chromosome segments from both the A and G genomes of T. timopheevii into wheat. These introgressions have been detected and characterised using single nucleotide polymorphism (SNP) markers present on a high-throughput Axiom® Genotyping Array. The analysis of these interspecific hybrid lines has resulted in the detection of 276 putative unique introgressions from T. timopheevii, thereby allowing the generation of a genetic map of T. timopheevii containing 1582 SNP markers, spread across 14 linkage groups representing each of the seven chromosomes of the A and G genomes of T. timopheevii. The genotyping of the hybrid lines was validated through fluorescence in situ hybridisation (FISH). Comparative analysis of the genetic map of T. timopheevii and the physical map of the hexaploid wheat genome showed that synteny between the two species is highly conserved at the macro-level and confirmed the presence of inter- and intra-genomic translocations within the A and G genomes of T. timopheevii that have been previously only detected through cytological techniques. In this work, we report a set of SNP markers present on a high-throughput genotyping array, able to detect the presence of T. timopheevii in a hexaploid wheat background making it a potentially valuable tool for marker assisted selection (MAS) in wheat pre-breeding programs. These valuable resources of high-density molecular markers and wheat-T. timopheevii hybrid lines will greatly enhance the work being undertaken for wheat improvement through wild relative introgressions.

Digital object identifier (DOI): 10.1186/s12870-019-1785-z

Cell death \& disease, 10, 186

Type 3 inositol 1,4,5-trisphosphate receptor has antiapoptotic and proliferative role in cancer cells.

Rezuchova, Ingeborg, Hudecova, Sona, Soltysova, Andrea, Matuskova, Miroslava, Durinikova, Erika, Chovancova, Barbora, Zuzcak, Michal, Cihova, Marina, Burikova, Monika, Penesova, Adela, Lencesova, Lubomira, Breza, Jan, Krizanova, Olga

Although the involvement of type 1 (IP R1) and type 2 (IP R2) inositol 1,4,5-trisphosphate receptors in apoptosis induction has been well documented in different cancer cells and tissues, the function of type 3 IP R (IP R3) is still elusive. Therefore, in this work we focused on the role of IP R3 in tumor cells in vitro and in vivo. We determined increased expression of this receptor in clear cell renal cell carcinoma compared to matched unaffected part of the kidney from the same patient. Thus, we hypothesized about different functions of IP R3 compared to IP R1 and IP R2 in tumor cells. Silencing of IP R1 prevented apoptosis induction in colorectal cancer DLD1 cells, ovarian cancer A2780 cells, and clear cell renal cell carcinoma RCC4 cells, compared to apoptosis in cells treated with scrambled siRNA. As expected, silencing of IP R3 and subsequent apoptosis induction resulted in increased levels of apoptosis in all these cells. Further, we prepared a DLD1/IP R3_del cell line using CRISPR/Cas9 gene editing method. These cells were injected into nude mice and tumor's volume was compared with tumors induced by DLD1 cells. Lower volume of tumors originated from DLD1/IP R3_del cells was observed after 12 days, compared to wild type DLD1 cells. Also, the migration of these cells was lesser compared to wild type DLD1 cells. Apoptosis under hypoxic conditions was more pronounced in DLD1/IP R3_del cells than in DLD1 cells. These results clearly show that IP R3 has proliferative and anti-apoptotic effect in tumor cells, on contrary to the pro-apoptotic effect of IP R1.

Digital object identifier (DOI): 10.1038/s41419-019-1433-4

Journal of applied genetics, 60, 63--70

Structural and copy number chromosome abnormalities in canine cutaneous mast cell tumours.

Vozdova, Miluse, Kubickova, Svatava, Cernohorska, Halina, Fröhlich, Jan, Fictum, Petr, Rubes, Jiri

Mast cell tumours (MCTs) are the most common skin tumours in dogs. Their clinical behaviour is variable and their aetiology remains largely unknown. We performed a metaphase fluorescence in situ hybridisation (FISH) with whole chromosome painting probes, and interphase FISH with BAC probes for 14 cancer-related genes to reveal clonal structural chromosome rearrangements and copy number variants (CNVs) in canine cutaneous MCTs. The metaphase FISH performed in three MCTs revealed several clonal monosomies and trisomies and two different chromosome rearrangements. No centric fusions were detected. The interphase FISH showed a variety of low frequency CNVs for the individual cancer-related genes. The heterogeneous character of the detected abnormalities indicates increased chromosome instability in canine MCTs. The clonal gain of chromosome 11 was detected in 81% (13/16) of the MCTs. Further research is needed to evaluate the significance of this abnormality as prognostic factor for the survival time or recurrence risk assessments in canine cutaneous MCTs.

Digital object identifier (DOI): 10.1007/s13353-018-0471-4

The journal of obstetrics and gynaecology research

Utility and performance of bacterial artificial chromosomes-on-beads assays in chromosome analysis of clinical prenatal samples, products of conception and blood samples.

Rose, Rajiv, Venkatesh, Aishwarya, Pietilä, Sanna, Jabeen, Gazala, Jagadeesh, Sujatha M, Seshadri, Suresh

Chromosome analysis of prenatal samples and products of conception (POC) has conventionally been done by karyotyping (KT). Shortcomings of KT like high turnaround time and culture failure led to technology innovations, such as the bacterial artificial chromosomes (BAC)s-on-Beads (BoBs)-based tests, Prenatal BoBs (prenatal samples) and KaryoLite BoBs (POC samples). In the present study, we validated and evaluated the utility of each test on prenatal, POC and blood samples. Study A (n = 305; 259 prenatal + 46 blood/POC) and Study B (n = 176; 146 POC/chorionic vill + 30 blood/amniotic fluid) samples were analyzed using Prenatal and KaryoLite BoBs kits, respectively. KT, array-based Comparative Genomic Hybridization (arrayCGH) and fluorescence in situ hybridization (FISH) were used for comparison of results. Ability of KaryoLite BoBs to identify ring chromosomes was tested. Prenatal BoBs had zero test failure rate and results of all samples were concordant with KT results. Totally four microdeletions were identified by Prenatal BoBs but not by KT. In Study B, all but two POC samples (one triploid and one tetraploid) were concordant with KT and arrayCGH. Partial chromosomal imbalance detection rate was ~64% and KaryoLite BoBs indicated the presence of a ring chromosome in all four cases. The failure rate of KaryoLite BoBs was 3%. We conclude that Prenatal BoBs (common aneuploidies and nine microdeletions) together with KT constitutes more comprehensive prenatal testing compared to FISH and KT. KaryoLite BoBs for aneuploidies of all chromosomes is highly successful in POC analysis and the ability to indicate presence of ring chromosomes improves its clinical sensitivity. Both tests are robust and could also be used for different specimens.

Digital object identifier (DOI): 10.1111/jog.13920

Journal of cellular biochemistry, 120, 4804--4812

Optimization of prostate cancer cell detection using multiplex tyramide signal amplification.

Roy, Sounak, Axelrod, Haley D, Valkenburg, Kenneth C, Amend, Sarah, Pienta, Kenneth J

Approximately 29 000 men die of prostate cancer (PCa) each year in the United States, and 90% to 100% of them are due to incurable bone metastasis. It is difficult to determine (1) when PCa disseminates in the natural history of the disease; (2) where cancer cell disseminates before becoming overt metastatic lesions; and (3) which tumors are aggressive and which are indolent. Tumor tissue and liquid (blood and bone marrow) biopsies provide important information to answer these questions, but significant limitations exist for immunostaining strategies that assess protein expression in these tissues. Classic immunohistochemistry (IHC) assays can typically assess expression of one or two proteins per tissue section. We have developed a novel immunofluorescence staining protocol to detect a panel of seven proteins on PCa tissue from primary tumor biopsies and metastatic lesion autopsy tissue, as well as cancer cells from liquid biopsies. We used a tyramide-based system to amplify the true signal and optimized the protocol to reduce background signal, thereby boosting the signal-to-noise ratio. Any protein-specific antibody in this protocol can be exchanged for a different validated antibody. This protocol therefore, represents a highly informative and flexible assay that can be used to provide important information about cancer tissue for the purpose of improving detection, diagnosis, and treatment.

Digital object identifier (DOI): 10.1002/jcb.28016

Molecular cytogenetics, 11, 4

Is cancer progression caused by gradual or simultaneous acquisitions of new chromosomes?

Bloomfield, Mathew, Duesberg, Peter

Foulds defined, "Tumor progression (as a) permanent, irreversible qualitative change in one or more of its characters" (Cancer Res. 1954). Accordingly progressions, such as metastases and acquired drug-resistance, were since found to be subspecies of cancers with conserved and numerous new chromosomes. Here we ask whether cancers acquire numerous new chromosomes gradually or simultaneously in progressions. The currently prevailing theory of Nowell (Science, 1976) holds that unexplained "genetic instability" generates "variant sublines (with) changes in chromosome number" and that "clonal" progressions arise by "stepwise selection of more aggressive sublines". The literature, however, contains many examples of "immediate" selections of progressions with numerous new chromosomes - notably experimentally initiated fusions between cancers and heterologous cells. Furthermore, the stepwise progression theory predicts intermediate sublines of cancers with multiple non-clonal additions of new chromosomes. However, the literature does not describe such intermediates. In view of these inconsistencies with stepwise progression we test here a saltational theory, in which the inherent variability of cancer-specific aneuploidy generates "immediate" progressions with individual clonal karyotypes, transcriptomes and phenotypes in single steps. Using cell fusion as an established controllable model of "immediate" progression, we generated seven immortal murine hybridomas by fusing immortal murine myeloma cells and normal antibody-producing B-cells with polyethylene glycol within a few minutes. These immortal hybridomas contained individual sets of 71 to 105 clonal chromosomes, compared to the 52 chromosomes of the parental myeloma. Thus the myeloma had gained 19 to 53 new clonal chromosomes in seven individual hybridomas in a single step. Furthermore, no stable intermediates were found, as would be predicted by a saltational process. We conclude that random fusions between myelomas and normal B-cells generate clonal hybridomas with multiple, individual chromosomes in single steps. Similar single-step mechanisms may also generate the "late" clonal progressions of cancers with gains of numerous new chromosomes and thus explain the absence of intermediates. Latency would reflect the low probability of rare stochastic progressions. In conclusion, the karyotypic clonality of hybridomas and spontaneous progressions suggests karyotypic alterations as proximate causes of neoplastic progressions. Since cancer-specific aneuploidy catalyzes karyotypic variation, the degree of aneuploidy predicts the clinical risk of neoplastic progression onfirming classical predictions based on DNA content

Digital object identifier (DOI): 10.1186/s13039-017-0350-4

Cancer letters, 412, 99--107

Quantified postsurgical small cell size CTCs and EpCAM, [email protected], circulating tumor stem cells with cytogenetic abnormalities in hepatocellular carcinoma patients determine cancer relapse.

Wang, Liang, Li, Yilin, Xu, Jing, Zhang, Aiqun, Wang, Xuedong, Tang, Rui, Zhang, Xinjing, Yin, Hongfang, Liu, Manting, Wang, Daisy Dandan, Lin, Peter Ping, Shen, Lin, Dong, Jiahong

Detection of hepatocellular carcinoma circulating tumor cells performed with conventional strategies, is significantly limited due to inherently heterogeneous and dynamic expression of EpCAM, as well as degradation of cytokeratins during epithelial-to-mesenchymal transition, which inevitably lead to non-negligible false negative detection of such "uncapturable and invisible" CTCs. A novel SE-iFISH strategy, improved for detection of HCC CTCs in this study, was applied to comprehensively detect, in situ phenotypically and karyotypically characterize hepatocellular and cholangiocarcinoma CTCs (CD45 /CD31 ) in patients subjected to surgical resection. Clinical significance of diverse subtypes of CTC was systematically investigated. Existence of small cell size CTCs (≤5 μm of WBCs) with cytogenetic abnormality of aneuploid chromosome 8, which constituted majority of the detected CTCs in HCC patients, was demonstrated for the first time. The stemness marker EpCAM aneuploid circulating tumor stem cells (CTSCs), and EpCAM small CTCs with trisomy 8, promote tumor growth. Postsurgical quantity of small triploid CTCs (≥5 cells/6 ml blood), multiploid (≥pentasomy 8) CTSCs or CTM (either one ≥ 1) significantly correlated to HCC patients' poor prognosis, indicating that detection of those specific subtypes of CTCs and CTSCs in post-operative patients help predict neoplasm recurrence.

Digital object identifier (DOI): 10.1016/j.canlet.2017.10.004

PloS one, 13, e0190970

Doxorubicin-provoked increase of mitotic activity and concomitant drain of G0-pool in therapy-resistant BE(2)-C neuroblastoma.

Hultman, Isabell, Haeggblom, Linnea, Rognmo, Ingvild, Jansson Edqvist, Josefin, Blomberg, Evelina, Ali, Rouknuddin, Phillips, Lottie, Sandstedt, Bengt, Kogner, Per, Shirazi Fard, Shahrzad, Ährlund-Richter, Lars

In this study chemotherapy response in neuroblastoma (NB) was assessed for the first time in a transplantation model comprising non-malignant human embryonic microenvironment of pluripotent stem cell teratoma (PSCT) derived from diploid bona fide hESC. Two NB cell lines with known high-risk phenotypes; the multi-resistant BE(2)-C and the drug sensitive IMR-32, were transplanted to the PSCT model and the tumour growth was exposed to single or repeated treatments with doxorubicin, and thereafter evaluated for cell death, apoptosis, and proliferation. Dose dependent cytotoxic effects were observed, this way corroborating the experimental platform for this type of analysis. Notably, analysis of doxorubicin-resilient BE(2)-C growth in the PSCT model revealed an unexpected 1,5-fold increase in Ki67-index (p<0.05), indicating that non-cycling (G0) cells entered the cell cycle following the doxorubicin exposure. Support for this notion was obtained also in vitro. A pharmacologically relevant dose (1μM) resulted in a marked accumulation of Ki67 positive BE(2)-C cells (p<0.0001), as well as a >3-fold increase in active cell cycle (i.e. cells positive staining for PH3 together with incorporation of EdU) (p<0.01). Considering the clinical challenge for treating high-risk NB, the discovery of a therapy-provoked growth-stimulating effect in the multi-resistant and p53-mutated BE(2)-C cell line, but not in the drug-sensitive p53wt IMR-32 cell line, warrants further studies concerning generality and clinical significance of this new observation.

Digital object identifier (DOI): 10.1371/journal.pone.0190970

Investigative ophthalmology \& visual science, 59, 561--571

A Novel C-Terminal Mutation in Gsdma3 (C+/H-) Leads to Alopecia and Corneal Inflammatory Response in Mice.

Swirski, Sebastian, Röger, Carsten, Pienkowska-Schelling, Aldona, Ihlenburg, Cynthia, Fischer, Gösta, May, Oliver, Vorm, Mariann, Owczarek-Lipska, Marta, Neidhardt, John

Mutations in the gene encoding Gasdermin A3 (Gsdma3) have been described to cause severe skin phenotypes, including loss of sebaceous glands and alopecia, in mice. We discovered a novel C-terminal mutation in Gsdma3 in a new mouse line and characterized a less frequently reported corneal phenotype, likely caused by degeneration of Meibomian glands of the inner eyelid. We used histologic methods to evaluate the effects of the C+/H- mutation on sebaceous gland and skin morphology as well as Meibomian glands of the inner eyelid and corneal tissue. Chromosomal aberrations were excluded by karyogram analyses. The mutation was identified by Sanger sequencing of candidate genes. Analyses of skin samples from affected mice confirmed the frequently reported phenotypes associated with mutations in Gsdma3: Degeneration of sebaceous glands and complete loss of pelage. Immunologic staining of corneal samples suggested an inflammatory response with signs of neovascularization in half of the affected older mice. While the corneal phenotype was observed at irregular time points, mainly after 6 months, its appearance coincided with a degeneration of Meibomian glands in the eyelids of affected animals. The mutation described herein is associated with inflammation and neovascularization of corneal tissue. Simultaneous degeneration of Meibomian glands in affected animals suggested a change in tear-film composition as the underlying cause for the corneal phenotype. Our data further support that different pathogenic mechanisms underlie some of the reported mutations in Gsdma3.

Digital object identifier (DOI): 10.1167/iovs.17-22658

Journal of phycology

Quantitative comparison of taxa and taxon concepts in the diatom genus Fragilariopsis: a case study on using slide scanning, multi-expert image annotation and image analysis in taxonomy.

Beszteri, Bánk, Allen, Claire, Almandoz, Gastón O, Armand, Leanne, Barcena, María Ángeles, Cantzler, Hannelore, Crosta, Xavier, Esper, Oliver, Jordan, Richard W, Kauer, Gerhard, Klaas, Christine, Kloster, Michael, Leventer, Amy, Pike, Jennifer, Rigual Hernández, Andrés S

Semi-automated methods for microscopic image acquisition, image analysis and taxonomic identification have repeatedly received attention in diatom analysis. Less well studied is the question whether and how such methods might prove useful for clarifying the delimitation of species that are difficult to separate for human taxonomists. To try to answer this question, three very similar Fragilariopsis species endemic to the Southern Ocean were targeted in this study: F. obliquecostata, F. ritscheri, and F. sublinearis. A set of 501 extended focus depth specimen images were obtained using a standardized, semi-automated microscopic procedure. Twelve diatomists independently identified these specimen images in order to reconcile taxonomic opinions and agree upon a taxonomic gold standard. Using image analyses, we then extracted morphometric features representing taxonomic characters of the target taxa. The discriminating ability of individual morphometric features was tested visually and statistically, and multivariate classification experiments were performed to test the agreement of the quantitatively-defined taxa assignments with expert consensus opinion. Beyond an updated differential diagnosis of the studied taxa, our study also shows that automated imaging and image analysis procedures for diatoms are coming close to reaching a broad applicability for routine use. This article is protected by copyright. All rights reserved.

Digital object identifier (DOI): 10.1111/jpy.12767

Radiology, 288, 529--535

Abdominopelvic 1.5-T and 3.0-T MR Imaging in Healthy Volunteers: Relationship to Formation of DNA Double-Strand Breaks.

Suntharalingam, Saravanabavaan, Mladenov, Emil, Sarabhai, Theresia, Wetter, Axel, Kraff, Oliver, Quick, Harald H, Forsting, Michael, Iliakis, Georg, Nassenstein, Kai

Purpose To investigate the relationship between abdominopelvic magnetic resonance (MR) imaging and formation of DNA double-strand breaks (DSBs) in peripheral blood lymphocytes among a cohort of healthy volunteers. Materials and Methods Blood samples were obtained from 40 healthy volunteers (23 women and 17 men; mean age, 27.2 years [range, 21-37 years]) directly before and 5 and 30 minutes after abdominopelvic MR imaging performed at 1.5 T (n = 20) or 3.0 T (n = 20). The number of DNA DSBs in isolated blood lymphocytes was quantified after indirect immunofluorescent staining of a generally accepted DSB marker, ?-H2AX, by means of high-throughput automated microscopy. As a positive control of DSB induction, blood lymphocytes from six volunteers were irradiated in vitro with x-rays at a dose of 1 Gy (70-90 keV). Statistical analysis was performed by using a Friedman test. Results No significant alteration in the frequency of DNA DSB induction was observed after MR imaging (before imaging: 0.22 foci per cell, interquartile range [IQR] = 0.54 foci per cell; 5 minutes after MR imaging: 0.08 foci per cell, IQR = 0.39 foci per cell; 30 minutes after MR imaging: 0.09 foci per cell, IQR = 0.63 foci per cell; P = .057). In vitro radiation of lymphocytes with 1 Gy led to a significant increase in DSBs (0.22 vs 3.43 foci per cell; P = .0312). The frequency of DSBs did not differ between imaging at 1.5 T and at 3.0 T (5 minutes after MR imaging: 0.23 vs 0.06 foci per cell, respectively [P = .57]; 30 minutes after MR imaging: 0.12 vs 0.08 foci per cell [P = .76]). Conclusion Abdominopelvic MR imaging performed at 1.5 T or 3.0 T does not affect the formation of DNA DSBs in peripheral blood lymphocytes.

Digital object identifier (DOI): 10.1148/radiol.2018172453

Journal of Alzheimer's disease : JAD, 61, 899--905

A Novel Antibody Targeting Tau Phosphorylated at Serine 235 Detects Neurofibrillary Tangles.

Brici, David, Götz, Jürgen, Nisbet, Rebecca M

Alzheimer's disease is characterized by two main pathological hallmarks in the human brain: the extracellular deposition of amyloid-β as plaques and the intracellular accumulation of the hyperphosphorylated protein tau as neurofibrillary tangles (NFTs). Phosphorylated tau (p-tau) specific-antibodies and silver staining have been used to reveal three morphological stages of NFT formation: pre-NFTs, intraneuronal NFTs (iNFTs), and extraneuronal NFTs (eNFTs). Here we characterize a novel monoclonal antibody, RN235, which is specific for tau phosphorylated at serine 235, and detects iNFTs and eNFTs in brain tissue, suggesting that phosphorylation at this site is indicative of late stage changes in tau.

Digital object identifier (DOI): 10.3233/JAD-170610