They offer a low throughput but high content in vitro experimental platform. Using mouse models as surrogates for three common human solid tumours, we describe a standardised workflow for systematic comparison of tumour slice cultivation methods and a tissue microarray-based method to archive them. Cultivated slices were compared to their in vivo source tissue using immunohistochemical and transcriptional biomarkers, particularly of cellular stress. Mechanical slicing induced minimal stress. Cultivation of tumour slices required organotypic support materials and atmospheric oxygen for maintenance of integrity and was associated with significant temporal and loco-regional changes in protein expression, for example HIF-1α. We recommend adherence to the robust workflow described, with recognition of temporal-spatial changes in protein expression before interrogation of tumour slices by pharmacological or other means.Precision-cut slices of  However, highly reductionist models, especially established cell lines growing in two dimensions on plastic6in vitro has opened up new opportunities for the study of heterogeneous tissue and tumour architecturein vivo is only partially reconstructed in vitro by laboratory manipulation of established cell lines or pluripotent stem cells and components of the stroma are often absent.Cancers have been appropriately described as pathological organs13in vitro representation of tumour architecture, heterogeneity and the complexity of a tumour in situ is provided by precision-cut slices . Such 200–300 micron precision-cut slices capture both the three-dimensional architecture and full heterogeneity of the tumour of interest, in addition to potentially retaining important native cellular elements such as immune, vascular and mesenchymal cells9101112in vivo tumour. Although the study has drug target validation and drug discovery in view (www.predect.eu), it is relevant to tumour biology in general, where it is increasingly clear that biochemical circuitry is influenced by tumour architecture and the microenvironment. Towards the goal of characterising precision-cut slices, we first asked what were the biochemical consequences, measured by both selected transcript profiling and immunohistochemistry, of the transport after surgical removal of the in situ tumour, the subsequent manipulations necessary to provide precision-cut slices and then of the incubation of the slices themselves under various conditions in vitro with time. Such fundamental questions need to be answered before a slice can be interrogated as an appropriate surrogate for an in situ tumour; the potential trauma of removing, transporting and then slicing the tumour in addition to its subsequent incubation in vitro may “rewire” the biochemical circuitry of both the tumour and stromal cells. A major concern was the possible imposition of tissue stress responses on the biochemistry of slices and we asked, if so, whether this stress could be ameliorated.A potentially more complete We here address the technical issues regarding preparation and maintenance of precision-cut slices, using readily available tumour tissue from mouse models of human breast, prostate and lung cancers derived from both cell lines (CDX) and patient-derived human tumours (PDX) as xenografts, as well as a genetically engineered mouse model (GEMM) of non-small cell lung cancer. These models were necessary surrogates for intensive technical studies that could not be readily performed with fresh human tumours, given their limited availability, intra-tumoural heterogeneity and reproducibility. We describe a workflow for slice preparation, cultivation and analysis that is amenable to bespoke optimisation. We propose this as a prerequisite baseline prior to planning any model-pertinent functional studies, such as drug perturbation. Additionally, to permit continuing interrogation of protein expression in slices by immunohistochemistry, we describe a novel method for preparing tumour slices as tissue microarrays (TMAs), permitting systematic archiving of large numbers of slice samples.in vivo models were cultivated for 48–96 h, either immersed in media (floating), or on a Millicell® filter support at an air-liquid interface (filter). Tumour slices were cultivated in either low oxygen conditions  or in atmospheric oxygen   . Qualitarphology . In contrphology , arrows.rphology . This warphology . The neerphology . Analogorphology . Finallyrphology .+) breast tumours showed that the importance of using a filter support for primary tissue viability was variable. Slices from one tumour required a filter support for retention of ER+ cells. However, ER+ cells were retained in slices from a second tumour cultivated both with and without filter supports  and pathologies , is best maintained when slices are cultivated on organotypic filter supports with atmospheric oxygen. Preliminary experiments with two primary human ER positive  supporting good inter-experimental reproducibility.The reproducibility of ER expression in tissue slices of four ER positive breast cancer MCF-7 CDX tumours cultivated on organotypic filter inserts with atmospheric oxygen is illustrated in To complement the histological analysis of slices and to have a wider view of the impact of slice cultivation on tissue viability, we analysed changes in expression of genes thought to be biomarkers related to cellular stress in tumour slices using Fluidigm microfluidic dynamic qPCR arrays (48.48 and 96.96 chip formats), in combination with 134 TaqMan® assays. The choice of transcripts was either based on a known role of the respective proteins in cellular stress pathways (<15% of the transcripts) or according to the following criteria (>85%): (i) they are typical targets of stress-activated transcription factors (such as p53 or NFkB), (ii) they are differentially regulated upon inhibitors/siRNA treatment specifically blocking certain stress pathways (such as the p38/JNK pathway) or (iii) the promoter regions of their genes have been described to contain stress response elements (such as endoplasmic reticulum (er) stress response elements). A full list of transcripts and their functional classification can be found in in vivo and day 0 samples except for MCF-7  and Euclidian distance scores . Importaor MCF-7 . This wa samples  and by t samples . The folAlthough changes in biomarkers were generally consistent between the different models, we identified changes specific to individual models that would require consideration when using slices for functional studies. For example, in our hands, MCF-7 CDX tissue slices cultivated floating without filter supports were observed to undergo a substantial loss of tissue architecture and cell viability . As a coClassification of stress-related transcripts according to their involvement in cellular processes  revealedA tissue slice is a complex structure of finite depth and in order to evaluate loco-regional changes across the slice, formalin-fixed tumour slices were embedded in paraffin wax and either sectioned horizontally  or vertiin vivo and this variability was captured in the slices where, on cultivation under optimal conditions, hypoxia increased significantly (+ 55.2  μm (n = 7) estimated by using this software. MCF-7 slices were also cut at thicknesses between 175 μm and 250 μm and the expression of HIF1α assessed   and γH2Aficantly . The invficantly . After 4assessed . The expAs an assessment of mouse host infiltrate, IHC was performed with an antibody specific to mouse macrophages (F4/80 positive cells). This revealed that host stroma was retained in MCF-7 tumour slices, with macrophages detectable largely at the oxygenated region of the slice close to the air interface after 48 h . The disThe loco-regional changes in biomarkers that developed as slices from human xenograft tumours (CDX and PDX) and a GEMM were incubated with a filter support in atmospheric oxygen, were also observed when the work-flow was applied to samples of human patient tumours . In tissHaving established that filter-supported cultivation was optimal, although associated with loco-regional changes in biomarker expression, we next investigated whether alternative support materials and incubation methodology influenced the loco-regional changes. Changes in the type of the slice support material and methods of incubation indeed influenced the slice architecture and stress biomarker profile. Two types of Alvetex adherent filter supports and a tissue slice incubation unit that permits intermittent immersion of the slice into the media, were tested . IntermiTo permit further interrogation of tumour tissue slices by IHC and other FFPE methods, we tailored a method to punch tissue slice FFPE blocks and produce a tissue microarray (TMA). Using standard TMA punching to generate a tissue slice TMA from horizontally embedded tumour slices was challenging, as only small amounts of tissue material were present at the sectioning face, as shown in ex vivo slices obtained from one unique human tumour. Across four independent laboratories, we have developed a unified workflow using surrogate tumours from GEMM and xenograft models, a workflow which can also be applied to primary human tumours and may be adapted to other cancer pathologies. The consistent availability of this surrogate source of accessible and homogenous tumour material allowed us to systematically compare the in vivo tumour with tissue slices immediately after preparation and upon cultivation under various conditions. The use of such models to prepare slices is anyway pertinent to fulfilling the needs of investigators when multiple, repeated studies of tumour biochemistry or pharmacology are to be performed or when intensive efforts in industry are required, such as for target validation. We established a biomarker platform to determine how preparation and/or cultivation might influence the activity of stress related pathways in tumour slices. In our hands, a single palpable murine tumour (minimum of 400mm3) was able to provide around 20 slices, permitting multiple investigations ex vivo of a single complex in vivo tumour, with the potential to reduce the use of animals in some studies. Importantly, both vertically and horizontally embedded slices were interrogated by microscopy and immunohistochemistry, permitting an analysis of the status of the interior of the slice and not only of its surface, as reported in other studies.Tissue slice culture has the potential to permit the interrogation of complex, heterogeneous material from either human tumours or appropriate models whilst the tumour microenvironment is maintained. There are diverse publications suggesting that tumour tissue slice culture can be utilised productively using different cultivation protocols  and <0.1% (severe hypoxia)The kinetics of the onset of hypoxia in incubated slices was variable amongst the models and pathologies studied here, with slices from the MCF-7 CDX showing expression of HIF1α after only 24 h. The diffusion of oxygen and its utilisation will be controlled by several variables in a slice lacking a functional vasculature. Perhaps surprisingly, slice thickness was not a major determinant . Other pin vitro models. Although advances have been made in generating cell lines from human cancers, for example from drug-resistant lung cancers21in vitro studies, and avoids the use of disaggregation systems. Tissue slice culture can be a valuable addition to the suite of ex vivo or in vitro assays that are used currently to study tumour biology, pharmacology and for the validation of tumour biomarkers on primary human tissue.Our standardised workflow for tumour tissue slice preparation and culture, using filter support in atmospheric oxygen, enables implementation of a robust and routine platform. Importantly, when establishing tissue slice cultures it is essential to generate a baseline biomarker profile of the model of interest, analysing vertical sections of the slice, so that multiple samples from the same tissue of origin or from different pathologies can be compared. From this baseline it should be possible to develop the platform further, integrating developments in microfluidics and perfusion systems in order to optimise oxygen supply and further reduce cultivation-induced cell stress, thus permitting longer-term culture. The platform permits future evaluation of the fidelity of signalling circuitry and the role of the native stroma in a heterogeneous cancer by immunohistochemical methods, using the archived TMAs. This offers a very powerful technique to investigate tumour biology and pharmacology in the context of a tissue, something that is missing from alternative All animal facilities and handling protocols were approved by the local committees on ethics of animal experiments, as required in each country, and adhered to the European Convention for Protection of Vertebrate Animals used for Experimental Purposes (Directive 2010/63/EU). Experiments carried out with mice at AstraZeneca were compliant with the UK Animals (Scientific Procedures) Act, which is consistent with EU Directive 2010/63/EU and had undergone internal ethical review. Experiments with mice performed at Oncotest were scrutinised by the Committee on the Ethics of Animal Experiments of the regional council . In Helsinki, studies with mice followed guidelines from the Finnish National Board of Animal Experimentation, and were approved by the Experimental Animal Committee of the University of Helsinki and the State Provincial Office of Southern Finland (Licence number ESAVI-2010-04855/Ym-23). In Rotterdam, the protocols for the use of mice were approved by the Animal Experiments Committee, DECConsult, an independent committee responsible for accreditation of all animal experimentation under the Dutch Experiments on Animals Act and all protocols were reviewed so to adhere to the European Convention for Protection of Vertebrate Animals used for Experimental Purposes.6 MCF-7 cells (American Type Culture Collection) per 0.1 ml in 50:50 basal media and Matrigel (BD Biosciences) into the left flank of male SCID mice (SCID/CB17). Animals were implanted with 0.5 mg/21 day 17β oestradiol pellets (Innovative Research of America) one day prior to cell implant.The CDX MCF-7 was derived by subcutaneous injection of 5 × 106 NCI-H1437 cells (American Type Culture Collection) into the flank of 4–6 weeks old NMRI nu/nu mice.The CDX NCI-H1437 was derived by subcutaneous injection of 5 × 10PDX from lung cancer (LXFA 1647) was derived from a metastasis in the adrenal gland of a 53 year old woman. 4–6 week old female NMRI nu/nu mice under isoflurane anaesthesia receive tumour implants subcutaneously in the flank.PDX of prostate cancer (PC295) was established as described previouslyLSL-KrasG12D/+; Lkb1fl/fl  GEMM was used and maintained on a mixed genetic background. LSL-KrasG12D/+Lkb1fl/fl7 pfu/mouse; Gene Transfer Vector Core) in 60 μL volume, as previously describedThe conditional 3. Animals were euthanised by cervical dislocation and tumours were excised. A small sample of the tumour was either snap frozen or fixed in 10% neutral buffered formalin immediately after resection. Tumours were placed into ice-cold Oncostore Tumour storage solution (OncoScience AG) or ice cold Leibovitz L15 medium (Lonza) prior to tissue slicing. NSCLC GEMMs were euthanised by cervical dislocation when they showed laboured breathing. Lungs en bloc were harvested for tissue slicing or fixation with formalin.Xenografted tumours were harvested when the volume was between 0.4–1 cmBreast tumour samples were obtained with fully informed consent from Wythenshawe hospital and transferred to AstraZeneca in ice-cold Oncostore tumour storage solution (OncoScience AG) on the day of surgical excision. The use of these samples was approved by AstraZeneca and The Manchester Cancer Research Centre Biobank (http://www.mcrc.manchester.ac.uk/biobank/), which operates under HTA licence number 30004 and NRES ethics number 07/H1003/161+5 in accordance with the approved guidelines. Fresh sterile tumour tissue of NSCLC patients was obtained as surgical waste from Klinik Schillerhöhe, Stuttgart immediately after surgical resection and maintained in organ transportation medium  until use. The investigation was approved by the local ethics committee (159/2011BO2) and informed consent was obtained from the patients.Tumours were mounted onto the magnetic specimen holder of a Leica VT1200S vibrating blade microtome using cyanoacrylate adhesive. Tumours of small volume were first embedded in low temperature gelling agarose (Thermo) prior to mounting on the specimen holder. Tumour tissue slices were prepared at a thickness of 200 μm (mouse lung), 250 μm (lung and breast) and 300 μm (prostate) using the precision-cut vibrating-blade microtome (Leica VT1200S). Slices of <175 μm were found to be too fragile to manipulate. Slices were visually inspected whilst being cut to ensure the tissue was not compressed or torn, resulting in inconsistent slice thickness. It is of note that 3/21 primary human breast tumours received were unable to be sliced; the dense consistency of these tumours prevented the blade from cutting the tumour. Lung PDX and CDX slices were cultivated in RPMI 1640, GEMM tumour slices were cultivated in F12 (Gibco), MCF-7 CDX slices were cultivated in DMEM (Sigma). Medium was supplemented with glutamine , penicillin , streptomycin  and 10% foetal bovine serum . Prostate PDX slices were cultivated in DMEM/F12 medium  supplemented with 2% FBS (Gibco), 1% insulin-transferrin-selenium (Gibco BRL), 0.01% bovine serum albumin (Boehringer Mannheim), 10 ng/ml epidermal growth factor (Sigma-Aldrich), penicillin/streptomycin antibiotics  plus the following additions: 100 ng/ml fibronectin (Harbor Bio-Products), 20 μg/ml fetuine, 50 ng/ml cholera toxin, 0.1 mM phosphoethanolamine, 0.6 ng/ml triiodothyronine, 500 ng/ml hydrocortisone and 0.1 nM synthetic androgen R1881 2 in a humidified atmosphere under low oxygen (3–5% oxygen) or atmospheric oxygen (21% oxygen) conditions. Tissue slices were maintained submerged in medium (floating), on Millicell Cell Culture inserts , on Alvetex Strata membranes , on Alvetex Polaris membranes  or loaded onto titanium grids and rotated using a tissue slice incubation unit29Cultivation was performed at 37°C and 5% COTissue slices were collected as FFPE blocks from all tumour samples and archived centrally at The Institute for Molecular Medicine Finland (FIMM) as tissue microarrays (TMA). The TMA archive is used for comparison of protein expression profiles across the various PREDECT consortium platforms by centralised IHC. TMAs were constructed by punching two 0.6 mm cores per sample from 30 vertically embedded tissue slices using a semi-automatic punching device (MiniCore). After arraying, we sectioned the TMA in 3.5 μm sections on Superfrost objective glasses (Kindler O Gmbh) using Microm 355S microtome (Thermo Scientific). Sections were dried and stored at +4°C. TMA sections were scanned at 0.22 micron resolution using Pannoramic P250 Flash whole slide scanner (3DHistech) equipped with 20× objective.For examination of histopathology, paraffin sections (3–4 μm) were stained with hematoxylin and eosin. IHC staining was carried out by standard protocols. Briefly, sections were de-waxed in xylene and rehydrated in graded ethanol, heat mediated antigen retrieval of tissue sections was carried out before being allowed to cool. Specific antigen retrieval details for each tissue type are contained in IHC scoring was carried out manually or using computer supported IHC quantification. Computer aided quantification was performed in whole tissue sections using the image analysis software Tissue Studio from Definiens or Aperio image analysis software . These programs allow quantification of the number of positively stained cells and the level of relative staining intensity in user defined regions of interest (ROI). Within these ROIs, algorithms were used that detect nuclei, membranous and cytoplasmic staining. Images were manually checked to ensure exclusion of mistakenly identified objects. Mean percentages of positively stained tumour cells from computer aided and manual IHC quantification were calculated across tumour types from at least three independent experiments. Statistical analysis was carried out using the student’s t test in GraphPad Prism (GraphPad Software). Spatial analysis of IHC images was carried out using bespoke in-house image processing algorithm for spatial feature detection in Matlab 13b (MathWorks). Slice images were compartmentalised arbitrarily into 50 layers, with each layer having an average thickness of 7.1 μm ± a standard deviation of 1.1 μm. ER H score was generated from ER staining quantified by image analysis software; it allows generation of a single figure that incorporates both the frequency of positively stained cells and the intensity of the staining. H scores were generated using the following formula: (1*per cent positive cells with intensity score 1) + (2*percent positive cells with intensity score 2) + (3*per cent positive cells with intensity score 3).in vivo tumours, 0 h, and cultivated slices were manually drawn after pathologist’s review. A graphics-editing program  was used to draw the masks (i.e. ROI and tumour necrosis) on top of a downscaled (0.88 μm/pixel) versions of the original samples. During the process of drawing the masks, the full resolution whole-slide images were available in parallel to better discriminate the tissue morphology. The final tumour viability in the selected ROI was defined as a ratio of viable tumour tissue area in respect to the whole ROI area. The Matlab scientific computing environment was used to calculate the tumour viability measurements by importing the manually drawn image masks.Necrotic regions in Total RNA was extracted from snap-frozen tissue slices. Tissue lysates were prepared using lysing matrix D with the FastPrep sample preparation system . Subsequently, RNA extraction was carried out using the RNeasy Mini kit (Qiagen) and on-column DNase (Qiagen) digestion was performed to eliminate genomic DNA contamination.cDNA was generated using MMLV reverse transcriptase (Promega). Expression analysis was performed on the BioMark HD System (Fluidigm) according to manufacturer’s instructions. TaqMan assays were purchased from Applied Biosystems .limma package in R statistics environmentGene expression qPCR data was normalised to a housekeeping gene and converted to log2 values. Differentially expressed genes were found using in vivo situation. Before calculating Euclidean distances and doing Principal Component Analysis (PCA)removeBatchEffect from limma R package was used to remove the effect of the tumour number. PCA was done using prcomp function in base R. Genes containing any missing values were removed before calculating principal components. First two components  were shown on the scatterplot.Euclidean distance (square root of the sum of square differences) was used to measure the difference of the slice models from the How to cite this article: Davies, E. J. et al. Capturing complex tumour biology in vitro: histological and molecular characterisation of precision cut slices. Sci. Rep. 5, 17187; doi: 10.1038/srep17187 (2015).


The analysis of cerebrospinal fluid (CSF) is usually done under steady-state conditions, when proteins  reach diffusion equilibrium between blood and CSF. However, little data has been published on CSF analysis under non-steady-state conditions after therapeutic apheresis. By reducing serum proteins , while leaving CSF unchanged, therapeutic apheresis might cause spuriously altered intrathecal immunoglobulin fractions.Based on the incidental finding of plasma exchange-induced increased intrathecal immunoglobulin fractions in a cohort of 12 unsystematically selected patients with various neurological disorders, we retrospectively investigated CSF results that had been raised during routine diagnostic work-up from 41 consecutive neurological patients  treated with plasmapheresis or immunoadsorption in a tertiary care university hospital in whom lumbar puncture (LP) was performed after a varying number of treatments of therapeutic apheresis.Only when LP was performed 1 day after therapeutic apheresis, spurious quantitative intrathecal immunoglobulin (Ig) synthesis of at least one subclass  was found in 68.4 % of the patients, irrespective of the number of treatments, in all age groups and independent of other previous immunotherapies . This phenomenon occurred only transiently and was almost always accompanied by an elevation of the IgG index. In one patient, an elevated IgG index was noticed even 2 days after plasmapheresis. Neither quantitative Ig synthesis, nor elevated IgG index was observed when the LP was performed three or more days after therapeutic apheresis.Spurious quantitative intrathecal Ig synthesis and increased IgG index are common findings shortly after plasmapheresis or immunoadsorption due to altered serum immunoglobulin levels. Knowledge of this phenomenon is needed for clinicians to prevent false interpretations leading to unnecessary diagnostic and therapeutic procedures. Misdiagnoses can be avoided by considering the characteristic CSF constellation including absence of oligoclonal bands and the close temporal relation to therapeutic apheresis. IgG). In some inflammatory neurological disorders of infectious or autoimmune origin , the disturbance of immunoglobulin (Ig) and albumin quotients leads to an increase of the quantitatively calculated intrathecal Ig fractions indicative of an intrathecal antibody production by local B-cells. In this regard, intrathecal Ig synthesis strongly suggests a neuroinflammatory process  × 100 [%] established by Reiber et al. [In some patients LP was performed within, in others after a complete apheresis cycle consisting of up to six treatments. The methodology of CSF and serum analysis was equivalent in both centers and according to the German Society for Cerebrospinal Fluid Diagnostics and Clinical Neurochemistry. The following routine laboratory CSF data were collected from medical records: time interval between the last treatment of PE or IADS and LP; total protein concentration; albumin concentration in CSF and serum; albumin quotient; immunoglobulin  concentrations in CSF and in serum; immunoglobulin  quotients, as well as a quantitative analysis of intrathecal immunoglobulin  fractions  according to the formula Igr et al. ; IgG indr et al. ; and finU test was used for statistical analyses comparing CSF results before and after therapeutic apheresis within the Freiburg study group. A p-value of < 0.05 was regarded as statistically significant.Because of the unsystematic patient selection of the Berlin study cohort we did not pool data with the Freiburg study group. The two-tailed Mann–Whitney Of the 12 patients treated between 2013 and 2014, ten were females, and two were males. The average age was 46 years (range 22–77 years). Eight patients were diagnosed with autoimmune encephalitis, two with neuromyelitis optica, one with multiple sclerosis, and one with myelitis. In these 12 patients, between five and ten treatments of PE were conducted. Six of the 12 patients had an LP 1 day after PE that showed increased quantitative intrathecal immunoglobulin fractions , supporting the hypothesis of spurious intrathecal Ig synthesis. By contrast, the remaining six patients who had an LP more than 1 day after PE with a median time interval to CSF analysis of 18.0 days (range 3–26 days) did not demonstrate increased intrathecal immunoglobulin fractions. Of the six patients with spurious intrathecal Ig synthesis, one patient with autoimmune encephalitis and IgG as well as IgA synthesis had persistence of an increased IgA fraction in the further course, whereas the other five  had a CSF analysis before PE without increased intrathecal immunoglobulin fractions. In these six patients LP showing increased intrathecal Ig fractions was performed after a median number of three apheresis treatments (range 1–6 treatments).At University Medical Center Freiburg, 346 neurological patients who were treated with either PE or IADS between 2005 and 2014 were identified. The vast majority (234 cases) had to be excluded since no CSF analysis had been performed within two months after therapeutic apheresis; an additional 50 had incomplete CSF data . In addition, eight had neurological disorders with intrinsic intrathecal Ig synthesis  and had no other CSF analysis showing the absence of quantitative intrathecal Ig synthesis. In 13 patients, data on therapeutic apheresis were not available. Finally, 41 patients fulfilled all study criteria.The mean age at the time of therapeutic apheresis was 58 years (range 24–80 years); gender distribution was balanced , and in a variety of neurological diagnoses (Table data not shown). According to quantitative IF analysis (data not shown), none of the 22 patients who underwent LP more than 1 day after therapeutic apheresis showed elevated intrathecal Ig fractions. In one patient, an elevated IgG index was noticed 2 days after PE, but in none of the patients with a CSF analysis three or more days after therapeutic apheresis (data not shown). Strikingly, positive or predominant OCBs in CSF were detected in none of the 19 cases with elevated intrathecal Ig fractions or an elevated IgG index up to 2 days after therapeutic apheresis.Elevated intrathecal immunoglobulin fractions were seen after one or more treatments, in patients from all age groups, independent of other previous immunotherapies es Table . Elevatees Table . Five ofBerlin study cohort), as well as in 13 of 19 patients (68.4 %) from a well-defined, independent, confirmatory cohort of patients with various neurological disorders (the Freiburg study group). Additionally, 18 of these 19 patients (94.7 %) had spuriously elevated IgG indexes. While being well-acknowledged among CSF experts, to our knowledge this is the first systematic study on these spurious phenomena.In this study, we describe spurious intrathecal immunoglobulin (Ig) synthesis of all three subclasses  occurring 1 day after therapeutic apheresis in 6 of 12 selected patients  with respect to molecular size [Freiburg study group mainly consisted of patients with neurological diagnoses typically not associated with intrinsic intrathecal Ig synthesis or elevated IgG index  [. (3) Elevated intrathecal Ig fractions and elevated IgG index occurred only transiently, i.e., no more than 1 day after PE or IADS for intrathecal Ig fractions and 2 days for IgG index, respectively, and disappeared in the further course, indicating a “steady-state equilibrium” of CSF and serum proteins over time (data not shown) [The assumption that this phenomenon is spurious is supported by several facts: 1) The yndrome) . In case The yndrt shown) . As statTo ensure correct interpretation, CSF and serum samples should be analyzed under steady-state conditions allowing proteins  to reach a diffusion equilibrium, since the Reibergrams commonly used for CSF analysis were developed under steady-state serum/CSF conditions , 2. HencFreiburg study group. Additionally, we included both patient groups receiving plasmapheresis or immunoadsorption, which are different therapeutic measures [A limitation of this study is its retrospective character and the small sample size of only 41 systematically selected patients in the measures . BrieflyTransiently elevated intrathecal Ig fractions and increased IgG index , both due to dropped serum Ig levels, are frequent phenomena occurring in the first 2 days following therapeutic apheresis. To avoid unnecessary diagnostic and therapeutic procedures, clinicians should be aware of this spurious phenomenon. Recognition of “spurious intrathecal synthesis” is possible when taking into account the absolute concentrations of CSF and serum Ig. In addition, OCBs should be considered to distinguish between “real” and “spurious” Ig synthesis, as they remain negative in the CSF despite spuriously elevated intrathecal Ig fractions and/or IgG indexes. If CSF analysis of intrathecal Ig synthesis is indicated after therapeutic apheresis, we recommend a minimum time interval of 3 days between apheresis and LP.


Carnitine palmitoyltransferase (CPT) deficiencies are a common autosomal recessive disorder resulting in a defect in mitochondrial fatty acid oxidation. The CPT system is made up of two separated proteins located in the outer CPT1 and inner CPT2 mitochondrial membranes. CPT1 catalyses the formation of acylcarnitine from carnitine and long chain fatty acyl-CoA. Acylcarnitine then crosses the inner mitochondrial membrane where it combines with CoA to form acyl-CoA, a process catalyzed by CPT2. Acyl-CoA is then available to undergo beta-oxidation. Deficiencies of both CPT1 and 2 have been described and leave patients unable to derive energy from fatty acid oxidation. Once immediate glucose and glycogen stores are exhausted, hypoglycemia may occur. This process conducts to rhabdomyolysis with muscle pains, hyperkalemia, metabolic acidosis and myoglobinuria. In severe severe cases acute renal failure, cardiac arrest and death ensure.A 35 years old woman was referred for a consultation to our malignant hyperthermia center before anesthesia for in vitro fertilization treatment (IVF). She never had an anesthesia. Her parents had 2 other healthy children. All family's members had had general anesthesia without problems. At 18 year old, after massive rhabdomyolysis during an interactive viral infection, the patient had a DNA analysis that revealed 1237Arg and Ser113Leu, mutations in CPT 2gene. The patient's was diagnosed as CPT2 deficiency. From these date she over went 4 crises of massive rhabdomyolisis, during which the CPK levels exceed 90.000 IU/l. The patient is in good physical status, renal functions are normal, liver enzymes are mildly elevated.1. Is there indication for muscle biopsy and in vitro contracture test (IVCT)? What is the risk and what is the benefit from IVCT?pregnancy?2. Does this patient need a special genetic counseling before 3. IVF needs a great number of sedations - what is the sedation plan?4. The pregnancy may aggravates her general status and the delivery may be a challenge for patient as well as for us - anesthesia plans.


Induction chemotherapy is a common therapeutic option for patients with locoregionally-advanced head and neck cancer (HNC), but it remains unclear which patients will benefit. In this study, we searched for biomarkers predicting the response of patients with locoregionally-advanced HNC to induction chemotherapy by evaluating the expression pattern of DNA repair proteins.Expression of a panel of DNA-repair proteins in formalin-fixed paraffin embedded specimens from a cohort of 37 HNC patients undergoing platinum-based induction chemotherapy prior to definitive chemoradiation were analyzed using quantitative immunohistochemistry.We found that XPF (an ERCC1 binding partner) and phospho-MAPKAP Kinase 2 (pMK2) are novel biomarkers for HNSCC patients undergoing platinum-based induction chemotherapy. Low XPF expression in HNSCC patients is associated with better response to induction chemoradiotherapy, while high XPF expression correlates with a worse response (p = 0.02). Furthermore, low pMK2 expression was found to correlate significantly with overall survival after induction plus chemoradiation therapy (p = 0.01), suggesting that pMK2 may relate to chemoradiation therapy.We identified XPF and pMK2 as novel DNA-repair biomarkers for locoregionally-advanced HNC patients undergoing platinum-based induction chemotherapy prior to definitive chemoradiation. Our study provides insights for the use of DNA repair biomarkers in personalized diagnostics strategies. Further validation in a larger cohort is indicated. Head and neck squamous cell carcinoma (HNSCC) is the 6th most common malignant neoplasm worldwide and accounts for 45.000 new cases in the US every year A meta-analysis also confirmed a small survival advantage with induction chemotherapy despite heterogeneity of the included therapies DNA repair proteins play an essential role in maintaining genome stability and have been implicated in tumorigenesis. Patients with chromosomal instability syndromes such as Fanconi anemia (FA), ataxia telangiectasia (AT), Bloom's syndrome or Werner syndrome show defects in DNA repair and an associated increased risk and poor prognosis for cancer including head and neck cancers Therefore, it is crucial to identify DNA repair biomarkers that can predict which patients benefit from induction chemotherapy in locoregionally-advanced head and neck cancer.in vivo and is responsible for the 5′ incision during nucleotide excision repair Previous reports suggest that ERCC1 is a potential biomarker for platinum-based therapy In this study, we investigated a panel of DNA repair proteins in five major DNA repair pathways using immunohistochemistry (IHC) and a digital pathology platform to evaluate whether the expression pattern of DNA repair proteins at the biopsy stage can predict tumor response in patients with locoregionally-advanced HNSCC undergoing induction chemotherapy prior to definitive chemoradiation. Our study shows that XPF is highly variable among head and neck cancers with a wide dynamic range: Low levels of expression of XPF correlate with better response to induction chemoradiotherapy, while high levels of XPF expression are associated with a worse response. Furthermore, pMK2, a kinase that has been reported to be critical for post-transcriptional regulation of gene expression as part of DNA damage response Biopsy specimens  from 37 patients with stage IV locoregionally-advanced HNSCC treated at the University of Chicago were evaluated from whole sections. The HNSCC patient biopsies had been obtained from a primary excision or biopsy prior to therapy. Written informed consent was obtained from all donors or the next of kin for the use of these samples in research approved under University of Chicago IRB protocol 8980 and 15410A. All patients had been treated with induction chemotherapy consisting of two cycles of paclitaxel and carboplatin for a total of eight weeks. We subsequently performed an interim assessment, followed by paclitaxel, 5-fluorouracil, hydroxyurea and radiotherapy-based regimens (FHX) based chemoradiotherapy and finally we evaluated for response Response evaluation was performed in the interval between induction chemotherapy and consecutive chemoradiotherapy by CT scan and/or clinical examination by an ENT specialist and best response was assessed. Response criteria were defined as complete response (CR) The simian virus 40-transformed fibroblasts GM08437  cells and HeLa cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal calf serum (FCS) in a humidified 5% CO2 incubator at 37°C.The whole sections of the samples were stained by IHC using antibodies against XPF (SPM228)/ERCC1 (8F1) (AbCam), FANCD2 (Santa Cruz), PAR, γH2AX (Millipore), MLH1 (AbSerotec) and phospho-MAPKAP Kinase2 (pMK2) . Tissue sections were deparaffinized/rehydrated using standard techniques. Heat-induced epitope retrieval was performed and the tissues were stained with antibodies overnight at 4°C. Primary antibodies were omitted for negative controls. Hematoxylin was used as nuclear counterstain. Two-fold antibody dilution ranges were established, and antigen retrieval conditions were set such that antibody was in excess and discriminated between control cancer tissues and between low and high expression levels. Renaissance TSATM  Biotin System (Perkin Elmer) was used for detection of XPF and FANCD2. Super Sensitive TM IHC Detection System (BioGenex) was used for detection of PAR, PARP1, MLH1, pMK2, γH2AX and ERCC1.The IHC stained tissues on the slides were scanned into a digital pathology platform (Aperio). Quality of staining pattern was pathology reviewed. Intensity of nuclear staining, and/or localization of the marker into both nuclear and cytoplasmic compartments was determined. Three tumor regions of interest in a whole section were selected by pathologists in order to minimize the effects of IHC staining variation. Scanned slides were then evaluated by pathologists and machine-based digital image analysis (Aperio). The percentage (0–100%) of tumor cells with positive staining Quantity (Q) and intensity (I) for each marker were independently scored by two trained pathologists , who were blinded from clinical history. A nuclear score was reported for XPF, ERCC1, FANCD2, MLH1, PARP1, PAR and γH2AX. The nuclear and cytoplasmic compartments were scored separately for pMK2. Staining quantity (Q) was scored 0 to 4: no nuclear staining  = 0; 1–9% of cells with nuclear stain  = 1; 10–39%  = 2; 40–69% = 3; and 70–100% = 4. Staining intensity (I) was classified from 0 to 3, with 0 = negative, 1+ = weak, 2+ = intermediate, 3+ = strong. Final scores were obtained by multiplying the quantity and staining intensity scores (IxQ) Immunoblotting for XPF, ERCC1, and β-Actin was done using standard methodology as previously described Biomarker scoring was correlated with clinical data to assess for correlation with outcome. A set of optimal threshold marker values was determined by univariate analysis for each marker that yielded the highest discrimination to separate Complete response (CR), Partial Response (PR), Stable Disease (SD) groups for induction chemotherapy and overall survival. Multivariate analysis was not feasible due to the small sample number. Univariate Cox proportional hazards models were constructed for each of the markers (single marker models) to examine their potential predictive powers. Discriminant and partition analysis was also conducted to maximally separate the dataset samples into groups.Statistical outputs for p-value , Apparent Error Rate (AER), Receiver Operator Characteristics (ROC) and Area Under Curve (AUC). ROC is a graphical plot of the sensitivity vs. (1-specificity) for a binary classifier system as its discrimination of true positives, in this case, it is 1-specificity  versus sensitivity . AUC is a measure of how well two classes of data separate under a testing scheme. Sensitivity, Specificity, Positive Predictive Power, Negative Predictive Power, Relative Risk (RR) and Odds Ratio were computed in the alternative models.To assess the association of the biomarker scores to overall patient survival, thresholds for each biomarker were determined, which separated patients into two groups. These thresholds were selected by choosing the biomarker value that generated the minimum survival curve p-value when patients with scores above the threshold were compared to patients below the threshold. Thresholds that created a minimum group size of less than 10% of all samples were not considered reliable and excluded from analysis.Survival curves for the low- and high-risk groups were compared using Kaplan-Meier models and the p-value reported. Additionally, the AER, AUC, ROC curve, sensitivity, specificity, positive predictive value, negative predictive value, and relative risk are reported.DNA repair pathways are important for the cellular response to chemotherapy and radiation. Eight selected DNA repair proteins in five major DNA repair pathways were evaluated by IHC in a cohort of 37 patients; an IHC staining example for each biomarker is shown in In our study, we determined specificity of the XPF (SPM228) and ERCC1 (8F1) antibodies by IHC using formalin fixed, paraffin-embedded blocks of HeLa (positive control) and XPF deficient cell pellets. Other XPF and ERCC1 antibodies were evaluated (data not shown/proprietary). SPM228 was chosen due to high degree of specificity, and 8F1 chosen as it is the most widely used ERCC1 antibody. Our result showed that specific nuclear staining by a monoclonal antibody against XPF (SPM228) was detected in HeLa cells but not in XPF deficient cells, in contrast, nuclear staining by the ERCC1 8F1 antibody was found in both HeLa and XPF deficient cells, indicating that this SPM228 antibody is XPF specific and suitable for detection of XPF by IHC, and ERCC1 8F1 recognizes additional non-specific nuclear proteins and is unable to specifically detect ERCC1 in specimens .We evaluated XPF expression in both, p16(+) and p16(−) samples and did not detect a significant difference .We then measured the level of XPF expression in lysates of nine HNSCC cell lines by immunoblot. Two bands of XPF at 110 kD and 75 kD were found consistently, with the 75 kD band recognizing full length XPF and the other band representing a cleavage product of XPF (XPF breakdown) . We alsoEight DNA repair biomarkers stained on 37 patient specimens by IHC were analyzed for their ability to predict response to induction chemotherapy. Of the 37 HNC patients treated with induction chemotherapy in the study, complete response (CR) We then evaluated association of the DNA repair biomarkers to overall survival for this cohort of patients. pMK2 did not correlate with response to induction chemotherapy , but it Chemotherapy induces DNA-damage in tumor cells. Therefore the ability to repair such damage using specific DNA repair pathways is likely predictive of drug sensitivity/resistance, and treatment outcome. Thus, diagnostic DNA repair biomarkers hold potential to significantly change diagnostic strategies and affect therapeutic decision-making and treatment planning for patients with head and neck cancer. In our study, we evaluated eight DNA repair biomarkers in five different DNA repair pathways by immunohistochemistry in locoregionally-advanced head and neck cancer. Significant variations in multiple DNA repair pathways were observed in HNSCC tumors suggesting that clinical decisions may be influenced by a DNA repair biomarker profile , 2. AmonWhile patient response to induction chemotherapy is a potential predictor of good overall outcome as reported by several groups Given the heterogeneity of head and neck cancer, and the intricately connected network of six major DNA repair pathways, it is unreasonable to anticipate that meaningful diagnostic testing can rely on a single, specific marker. As our study suggests, markers for induction and chemoradiation are likely different. Furthermore, compensation of DNA repair in the absence of one repair pathway by another pathway suggests the possibility that multiple markers may be necessary to optimally assess responsiveness. Such a DNA repair response signature will have to be evaluated by our group, using a larger cohort and may allow improved assessment of HNC heterogeneity and complexities of DNA repair networks.In conclusion, our study provides an established method to measure DNA repair biomarkers and other biomarkers using quantitative immunohistochemistry to identify and evaluate functional changes to DNA repair and damage signaling pathways as a valuable tool for personalized diagnostics. Our results indicate usefulness of XPF as a biomarker to predict which patients benefit from which treatments with induction chemotherapy. Specifically XPF proved superior to ERCC1 (8F1) testing. XPF may also have value to predict overall treatment success, which potentially relates to its role for prediction of induction response


Faithful transcription of DNA is constantly threatened by different endogenous and environmental genotoxic effects. Transcription coupled repair (TCR) has been described to stop transcription and quickly remove DNA lesions from the transcribed strand of active genes, permitting rapid resumption of blocked transcription. This repair mechanism has been well characterized in the past using individual target genes. Moreover, numerous efforts investigated the fate of blocked RNA polymerase II (Pol II) during DNA repair mechanisms and suggested that stopped Pol II complexes can either backtrack, be removed and degraded or bypass the lesions to allow TCR. We investigated the effect of a non-lethal dose of UVB on global DNA-bound Pol II distribution in human cells. We found that the used UVB dose did not induce Pol II degradation however surprisingly at about 93% of the promoters of all expressed genes Pol II occupancy was seriously reduced 2–4 hours following UVB irradiation. The presence of Pol II at these cleared promoters was restored 5–6 hours after irradiation, indicating that the negative regulation is very dynamic. We also identified a small set of genes , where the UVB-induced Pol II clearing did not operate. Interestingly, at promoters, where Pol II promoter clearance occurs, TFIIH, but not TBP, follows the behavior of Pol II, suggesting that at these genes upon UVB treatment TFIIH is sequestered for DNA repair by the TCR machinery. In agreement, in cells where the TCR factor, the Cockayne Syndrome B protein, was depleted UVB did not induce Pol II and TFIIH clearance at promoters. Thus, our study reveals a UVB induced negative regulatory mechanism that targets Pol II transcription initiation on the large majority of transcribed gene promoters, and a small subset of genes, where Pol II escapes this negative regulation. Our genome is continuously exposed to genotoxic attacks that generate aberrant DNA structures. These can block the transcribing DNA-dependent RNA polymerase II (Pol II) enzyme and can lead to deleterious cellular processes. Cells have developed several mechanisms to stop Pol II, repair the roadblocks and to restore normal polymerase traffic. Numerous efforts investigated the fate of blocked Pol II during DNA repair mechanisms and suggested that stopped Pol II complexes can either backtrack, be removed or bypass the lesions to allow repair. We carried out a genome-wide analysis of Pol II behavior upon a DNA damaging stress, UVB, which is relevant from the public health standpoint. Thus, we could follow UVB-induced Pol II behavior changes on every human gene over time. We uncovered a novel UV induced negative regulatory mechanism, which inhibits the recruitment of Pol II to the promoters of about 93% of all transcribed genes, and a small subset of gene  that escapes this negative regulation, probably because their gene products are required during/after UVB irradiation. Thus, we uncover how a cell induces a global negative regulation at the level of transcription initiation in response to a genotoxic stress. Analyses of short transcribed RNA molecules showed that these arrested polymerases are predominantly in a transcriptionally engaged state end of the annotated genes (EAGs) representing Pol IIs that have finished transcribing the pre-mRNA and are slowly transcribing and approaching the termination site often 4–6 kb away from the 3′end of the gene Proper cell homeostasis and function requires expression of the DNA encoded information. Maintenance of genome integrity and accurate replication is crucial for correctly regulated gene expression. Transcription of thousands of coding and non-coding RNAs by the RNA polymerase II (Pol II) is a regulated multistep process that can be divided into five stages: pre-initiation, initiation, promoter clearance, elongation and termination. Based on numerous genome-wide studies analyzing Pol II transcription in several metazoan organisms using chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) it is now clear that on different regions of an expressed gene, distinct types of Pol II occupancy signals can be detected. The “canonical” Pol II occupancy ChIP-seq profile on an average expressed gene displays Pol II molecules engaged in the major phases of transcription Damage or alterations of the DNA structure can threaten the progression of transcription. Indeed, Pol II driven transcription has been reported to be disturbed by “roadblocks” on the DNA template, which arises from both environmental and endogenous sources, such as special DNA sequences, non-canonical DNA structures, topological constrains and DNA lesions UV light is one of the most genotoxic environmental sources of transcription-blocking DNA damages. Different wavelengths of the UV light can generate a wide range of lesions in the DNA template. Based on its wavelength, UV light can be divided into UVA (315–400 nm), UVB (280–315 nm) and UVC (<280 nm). UVC has higher energy level than UVA and UVB due to its shorter wavelength; however, UVC radiation is not relevant from a public health standpoint as it is absorbed by the ozone layer. UV-related lesions in living organisms are induced mostly by UVA and UVB.The ionizing energy of UVB and UVC radiation is directly absorbed by the DNA and can cause cyclobutane pyrimidine dimers (CPDs), pyrimidine 6-4 pyrimidone photoproducts (6-4PPs), which are the most persistent and predominant lesions In vitro the half-life of an arrested Pol II at a CPD can be ∼20 hours, and it covers 10 nt upstream and 25 nt downstream of the CPD It seems that cells have evolved several solutions to deal with the persistently stopped Pol II elongation complexes: i) Pol II can bypass the lesions, but this process is slow and extremely inefficient Our knowledge about Pol II transcription inhibition after UV irradiation is based on studies carried out on a few model genes and in experiments often using lethal UVC doses. Moreover, many studies have investigated the fate of Pol II elongation complexes during TCR, but very little information has been obtained on how TCR influences the whole Pol II transcription cycle during TCR. Therefore, the mechanism by which Pol II transcription is affected genome-wide upon sublethal doses of UV irradiation is not yet well understood. To investigate the fate of Pol II during TCR processes, we have investigated Pol II occupancy at a genome wide level following UVB treatment over time in human MCF7 cells. Our results show that on about 93% of the promoters of expressed genes Pol II occupancy is seriously reduced 2–4 hours following UVB irradiation, and that the presence of Pol II is restored to “normal”, or even sometimes higher, levels 5–6 hours after irradiation. We also identified a smaller set of genes, where the presence of Pol II at the promoter regions does not decrease, but rather increases at the promoters and also throughout the entire transcription units of these genes after UVB irradiation. Thus, our study reveals a global negative regulatory mechanism that targets RNA polymerase II transcription initiation on the large majority of transcribed genes following sublethal UV irradiation and a small subset of key regulatory genes, where Pol II escapes the negative regulation.2 of UVB irradiation on the DNA damage response proficient MCF7 human breast cancer cell line, containing wild type p53 , along the GBs of the genes (from TSS +100 bp to EAG) and downstream from EAG (from EAG to EAG +4 kb) regions of the 4500 expressed genes from the control data set and the six UVB irradiated samples . In addiFrom the heat maps it is visible that the generated clusters represent genes with distinct, unique Pol II transcription responses following UVB treatment, as we can observe well-defined differences in the changes of Pol II distribution at the different regions of the annotated genes. We found that the 4500 expressed genes can be sorted into two main groups, hereafter called A and B. Group A contains about 93% of the examined genes  and showInterestingly, in many sub-clusters Pol II occupancy increased at 1 hour and then again at 5 and 6 hours following UVB treatment at distinct regions of the transcription units . CompareIn contrast to group A, the relatively small set of genes in group B  and genes, out of which many regulate DNA damage response, signal transduction by p53 and apoptosis, where Pol II presence is increased in the TSS, GB and slightly at EAG+4000 regions . Note however that most of the NER factors are abundant in the nucleus DNA ligase IV (ATP-dependent), cyclin D2, p21 , GADD45A and GADD45B, and TP53AIP1, SESN2, FAS, BBC3, which are regulators of repair, cell growth or survival pathways further validating the biological significance of the present Pol II occupancy study.In the above detailed global analysis of Pol II behavior on expressed genes upon UVB treatment we have observed that many genes in group B, belonging to the GO categories ‘response to UV’ and ‘DNA damage response’, have increased Pol II occupancy throughout their whole transcription unit. Thus, we analyzed Pol II distribution at annotated repair and UV-responsive genes existing in the KEGG database To evaluate whether the above observed massive Pol II promoter clearance at 2–4 hour time points in Group A  is due tAdditionally we tested the level of the phosphorylation of the CTD of Rpb1 using antibodies that recognize different and specifically phosphorylated forms of the CTD heptapeptide repeats . In thisTo test whether the degradation of additional PIC subunits would be responsible for the important Pol II clearance from the promoters upon UVB irradiation, we tested the global protein levels of TBP, TFIIB and the kinase subunit of TFIIH, CDK7, which is known to phosphorylate the CTD of Pol II 2 UVB irradiation. Pol II signals were quantified on the promoters of two randomly selected genes from group A (rplp1 and ubc) and B (p21 and wdr24). Negative/mock control ChIP was carried out with Sepharose G beads alone (NoAb), and for an additional control, oligonucleotides were designed to target an intergenic region, where no Pol II binding is expected . To this end we used anti-Pol II ChIP coupled qPCR detection on non-treated samples and on samples incubated for 3 h and 6 h after 55 J/mexpected .p21) or increased (wdr24) Pol II enrichment at the promoters at 3 hours after UVB treatment compared to the control. In the case of wdr24 gene Pol II enrichment increases up to 6 hours post UVB treatment. These ChIP-qPCR validations are in good agreement with our ChIP-seq, bioinformatics and IF results.The ChIP-qPCR experiment confirmed the different Pol II behavior patterns at the promoters of the selected genes. At the promoters of genes from group A we observed a decreased Pol II occupancy in the sample that was harvested 3 hours following UVB irradiation when compared to the control . As expeNext, to better understand the mechanisms that may regulate the opposite Pol II behavior on gene promoters belonging to either group A or B, we investigated whether certain PIC subunit enrichments would also be affected upon UVB irradiation. To this end we carried out ChIP-qPCR detection using antibodies against subunits of two GTFs, the TATA-box binding protein (TBP) a subunit of TFIID, and p62, a subunit of the TFIIH . SurprisCSB is known to trigger the recruitment of NER factors, including TFIIH, to UV-induced DNA lesions to carry out the repair process see . Thus, tIn the past decades a lot of efforts have been devoted to understand how Pol II transcription is recovered after genotoxic stress, such as UV irradiation. However, our knowledge about Pol II transcription inhibition during TCR and the subsequent recovery of RNA synthesis is based only on a handful of model genes. Note that most of the previous studies that investigated the link between repair and transcription used much higher, often lethal, UVC doses 2 dose of UVB irradiation used had a wide effect on the genome of MCF7 cells, as it induced CPD lesions genome-wide, which persisted up to, or even longer than 24 hours  Pol II assembled in a PIC and pausing at or close to the TSSs runs in the gene body, ii) new PIC formation does not occur, or iii) existing PICs containing paused Pol II are actively removed from TSSs. Nevertheless, our observations strongly suggest the existence of a rapid negative regulatory mechanism, which seems to act on almost all expressed gene promoters during TCR to either remove existing PICs, containing paused Pol IIs, and/or prevent Pol II to enter in initiation, or re-initiation, complexes at promoters, when transcription-blocking lesions may still be present on the ORFs. The fact that promoter-proximal pausing of Pol II at group A genes 2–4 h after UVB irradiation decreases everywhere , indepenIn agreement, an active global transcriptional repression process, rather than a physical blocking of transcription, has already been suggested It is crucial for the cell that DNA lesions are repaired in the different transcription units with equal efficiencies, in order to avoid that short genes with low damage probability are repaired and transcribed quicker than long genes with a high likelihood of carrying many UV-induced CPD lesions. We do not know whether this negative “preventive” transcription regulatory mechanism can operate at several levels separately (i.e. remove existing PICs containing paused Pol II and/or block Pol II incorporation in new PICs) or whether it would inhibit both mechanisms by blocking the function of the same factor for example. Interestingly, it has been observed that the loss of de novo RNA synthesis occurs only at the UV-irradiated area of the nucleus The transcription factor TFIIH was shown to play an essential role in both transcription initiation and DNA repair In line with the sequestration model by the TC-NER machinery, earlier work showed that TFIIH engagement in transcription initiation takes only a few seconds, whereas when it engages in NER it takes minutes for every NER event, which may explain why the pool of TFIIH is quickly depleted and shifted towards repair The detected synchronized Pol II reappearance on the TSSs of most of the transcribed genes 5–6 hours after UVB irradiation suggests that transcription-coupled repair is largely completed by this time in MCF7 cells; TFIIH complexes are released from the repair sites and again available for new PIC formation on the transcribed genes. Moreover, our results investigating the presence of TBP, a DNA-binding component of TFIID, showed that the presence of TBP at the TSSs of genes is not changing dramatically over time after UVB irradiation. This observation suggests that on the transcribed genes during UVB irradiation, and following the potential TFIIH sequestration by the repair machinery, a minimal TFIID/TBP-containing re-initiation complex may stay bookmarking the TSSs where transcription has to be resumed once TCR is completed.While the Pol II clearance and recovery seems to be synchronized at the promoters of group A genes, we found very diverse patterns of Pol II behavior on the GB and on the EAG+4000 regions of the expressed genes during recovery after UVB irradiation . Based oAnother common characteristic exists on the GBs of group A transcription units, namely that in most of the cases there is an increase of Pol II occupancy at 1 hour after irradiation on the GBs of these genes, which show Pol II clearance from their promoters  and 3. TMoreover, Pol II changes on the GBs and EAG+4000 regions did not always parallel the homogenous decrease in Pol II binding at the promoters of group A genes following UVB irradiation. This suggests that the UVB-induced global negative regulatory mechanisms affecting Pol II transcription initiation, elongation and termination may be distinct and are not always interconnected.We also detected a set of genes, which show no decreased Pol II presence at their promoter regions, but rather a general increase of Pol II density throughout their three analyzed gene regions after UVB irradiation . Genes bp21/cdkn1a model gene that p53 uses unorthodox mechanisms to activate p21 when Pol II CTD phosphorylating kinases are inhibited, the recruitment of specific elongation factors are defective and mRNA synthesis from certain individual genes is inhibited It is known that p53 can regulate a subset of genes under compromised physiological conditions, when for example DNA is damaged. It is has been suggested using the Studies investigating the effect of genotoxic stresses on transcription reported two possible pathways by which Pol II protein levels may be affected: either the hyperphosphorylation of the CTD or the degradation of the largest subunit of Pol II Surprisingly, none of the antibodies raised against different phosphorylated forms of the CTD hepta-peptide repeat  revealed increased levels of phosphorylation of the Rpb1 CTD upon UVB stress that would explain the shift from the Pol IIA to the Pol IIO form . These rTaken together all our observations it seems that the massive Pol II clearance on group A genes upon UVB irradiation is not due to Pol II degradation, but due to the incapability of the Pol II complex to initiate transcription. It seems that during TCR the general transcription factor TFIIH is sequestered away from the TSSs by the repair machinery and this in turn would either dismantle genome-wide existing PICs and/or block the formation of new PICs on the majority of the transcribed genes. In contrast, on a small subset of key regulatory genes this negative mechanism does not work either because transcription initiation on these genes is not dependent on the repair-competent form of TFIIH or these genes are repaired much faster than the inhibited genes. Further experiments, will be needed to distinguish amongst all these different exciting possibilities. In conclusion, our study for the first time gives a genome-wide view about the mechanistic details of UVB induced Pol II transcription changes during TCR.The MCF7 human cell line  was grown in Dulbecco's Modified Eagle Medium  supplemented with 10% foetal calf serum (FCS). The medium contained insulin (0.6 µg/ml) and gentamicin (40 µg/ml). For assessing global and ongoing transcription cells were incubated with 1 mM 5-fluorouridine (Sigma-Aldrich) for 20 minutes. Incorporation of the modified nucleotide was monitored by indirect immunofluorescence using an anti 5-FU antibody from Sigma-Aldrich.2 UVB dose at 312 nm wavelength (Vilber T-15M lamp) or with with 55, 100 and 200 Joules/m2 , GAPD and non-targeting control  were transfected to MCF7 cells using Dharmafect Transfection Reagent 1  according to the manufacturers protocol. Cells were harvested for western blot analysis or ChIP 72 hours after the transfection.MCF7 cells were plate into a 6 well plate (300 cells/well) one day prior to UV treatment. In the following days, the survived cells will form colonies. 7 days after UV irradiation, the cells were washed with PBS then stained with Crystal violet solution . The staining was removed from the cells with 1% sodium dodecyl sulphate (SDS) containing MQ water. The crystal violet amount in both UV treated and in control samples were measured with spectrophotometer at 595 nm. Results were calculated from biological triplicatesCells were plated in 6 cm plates and UV-treated at 80–90% confluence. After incubation for 1, 2, 4, 6, 8, 16, 24 hours DNA was extracted from samples as well as from non-treated control with SIGMA GenElute Mammalian Genomic DNA Miniprep Kit. DNA content was quantified with Nanodrop. 250 ng of DNA was diluted in 2× SSC  buffer from each sample and was transferred to Amersham Hybond-N+ membrane with Slot-blot vacuum chamber. The membrane was treated with Denaturizing buffer  then Neutralizing buffer . The membrane was then treated according to Western blot protocol: blocked with PBS +2% milk, and then incubated with mouse IgG monoclonal anti-CPD (TDM2)  primary antibody overnight in 4°C.2 UVB. The irradiated and control cells 1, 2, 3, 4, 5, 6, hours later after incubation were washed twice with ice-cold PBS containing complete protease inhibitor cocktail (1×), phenylmethylsulfonyl fluoride (PMFS)(0.5 mM), β-glycerophosphate (10 mM), sodium orthovanadate (1 mM), sodium fluoride (20 mM) inhibitors. Cells were scraped in PBS containing protease inhibitor cocktail and frozen-thawed three times. Samples were sonicated for 10 cycles  (Bioruptor) then boiled for 5 min in loading solution. Protein samples were separated by a 6–10% SDS-PAGE, transferred and western blot assays were carried out by using standard methods. The following antibodies were used: RNA polymerase II N-terminal H-224× (Santa Cruz), Pol II Ser2, AB: Covance, (MMS-129R), Pol II Ser5 AB: Abcam, (ab5131) Pol II Ser 7 AB: MCF7 cells were plated in 6 cm plates prior treatment. At 80–90% confluence, cells were treated with 55 J/mUbc/NM_021009, Rplp1/NM_001003, p21/cdkn1a/NM_000389 and WDR24/NM_032259 genes. Intergenic region was selected as a negative control region. The ChIP experiments were repeated at least twice, and all the qPCR reactions were done in triplicates.MCF7 cells were plated in 15 cm dishes. At 80–90% confluence cells were UVB treated as described above. After 1, 2, 3, 4, 5, or 6 hours of incubation cells were quickly washed with PBS, and cross-linked with 1% formaldehyde for 20 minutes at room temperature. ChIP experiments were carried out as described earlier http://www.ncbi.nlm.nih.gov/gds) database. The 32 base pair tags generated from Hi-seq 2000 were mapped to the human genome (UCSC hg19) using the eland program allowing two mismatches. Only sequences that mapped uniquely to the genome with maximum of two mismatches were used for further analysis. To eliminate non-specific binding sites, we used our control ChIP-seq dataset, which was generated using an antibody that does not recognize any human proteins (GSE34001).The sample preparation for ChIP-seq was the same as described https://genome.ucsc.edu/), human genome Build 37 (hg19 assembly). Gene definitions were given by the refseq genes track Genome annotations were downloaded from the UCSC Genome Browser (http://www.bioconductor.org/packages/2.9/bioc/vignettes/DESeq/inst/doc/DESeq.pdf). We carried out analyses on all genes found in the refseq database and on 4500 expressed genes, based on the published RNA-seq dataset for MCF-7 cell line Intergenic regions (around 8000 regions) were selected that are far away from genes about 20 kb and tag numbers were counted on 2 kb interval in the middle of them for all the 7 samples. These values were used as an input for DESeq Bioconductor package, which normalizes the samples based on their median values.  was used for GO analyses and gene ID conversion. Further GO enrichment analyses were also performed in Manteia using its statistical module (http://manteia.igbmc.fr/). During the analyses only GO categories with lower than 0.01 p-values  were considered as positive results.The Database for Annotation, Visualization and Integrated Discovery (D.A.V.I.D.)  together with the survival of non-treated/control cells. (B) Slot Blot assay was carried out with a CPD specific antibody to test the genotoxicity of 55 J/m2 of UVB irradiation. The appearance and the persistence of the CPDs were tested at the indicated time points up to 24 hours after UVB treatment. (C) Western blot assays were carried out to test whether 55 J/m2 UVB induces detectable DNA-repair response in MCF7 cells. We tested the induction of two well-characterized regulators of the UV-response at the indicated time points: phospho-Chk1 and phospho-p53, respectively. Tubulin-α was used as loading control.Effects of sublethal dose of UVB on MCF7 cells. Experiments were carried out to find and characterize the sublethal dose of UVB irradiation for our study. (A) Crystal violet assay was carried out to compare the effect of different doses of UVB on MCF7 cells. The results are represented as a bar chart. The % of surviving cells are shown corresponding to each dose of UVB irradiation (55 J/m(PDF)Click here for additional data file.Figure S2Global Pol II tag density changes dynamically in the gene body regions of all transcribed genes following UVB irradiation. Mean Pol II read values (Y axis) were calculated for each ChIP-seq sample (as indicated on the x axis) from the GB region of the 4500 expressed genes. Each P value was calculated applying pairwise T-tests between control sample and each of the UVB treated samples (as indicated).(PDF)Click here for additional data file.Figure S3(First and second part): Different Pol II behavior patterns in time after UVB irradiation on selected genes from each subgroup of (PDF)Click here for additional data file.Figure S4CSB depletion using siRNA transfection. Scrambled siRNA (lane 1) and ERCC6 (CSB) siRNA (lane 2) transfection in MCF7 cells was carried out as described in (PDF)Click here for additional data file.Table S1Results of gene ontology analyses done with the Manteia software to identify the potential differential gene-function categories for the detected gene groups with distinct Pol II behavior patterns from (PDF)Click here for additional data file.Table S2List of genes represented in (XLSX)Click here for additional data file.


Mycobacterium ulcerans, the causal agent of Buruli ulcer, an emerging human disease. A survey was carried out over two years in Cameroon. Morphological analyses were done in two steps. A first step consisted in separating the specimens based on broadly shared characters into morphotypes. The specimens were then separated into two independent batches containing each the same representation of each morphotype. One batch (309 specimens) was used by taxonomy experts on aquatic bugs for species level identification and/or to reconcile nymph with their corresponding adult species. The second batch (188 specimens) was used to define species based on the COI DNA sequences (standard sequence used for “DNA barcoding”) and using the Automatic Barcode Gap Discovery (ABGD) method. The first morphological analysis step separated the specimens into 63 different morphotypes (49 adults and 14 nymphs), which were then found to belong to 54 morphological species in the infra-orders Gerromorpha and Nepomorpha based on the species-level morphological identification, and 41–45 putative molecular species according to the gap value retained in the ABGD. Integrating morphology and “DNA barcoding” reconciled all the specimens into 62 aquatic bug species in Cameroon. Generally, we obtained a good congruence between species a priori identified based on morphology from adult morphotypes and molecular putative species. Moreover, molecular identification has allowed the association of 86% of nymphs with adults. This work illustrates the importance of integrative taxonomy.Aquatic and semi-aquatic bugs (Heteroptera) represent a remarkable diversity and a resurging interest has been given to documenting at the species level these insects inhabiting Cameroon in Central Africa due to their potential implication in the transmission of the bacterium  Mycobacterium ulcerans, the causal agent of an emerging human disease named Buruli ulcer [Aquatic and sub-aquatic true bugs (Heteroptera), comprised in the Leptopodomorpha, the Gerromorpha and the Nepomorpha infra-orders, represent a remarkable species diversity of the aquatic biota with 4,656 species from 326 genera and 20 families found worldwide except in Antarctica . Severalli ulcer .et al. [The current taxonomy of bugs is mostly based on morphological characters of adults, which are more or less reliable because of their intraspecific variability. Moreover, immature forms are difficult to identify based only on morphology because of the lack of discriminating characters . Therefoet al.  have shoet al. –16. Seveet al. –19, but et al. .Initial work , 21 repoWith the growing implementation of DNA barcoding, it is now possible to not only assign a sample to a pre-existing classification but also to identify unknown species and to decide whether species should be separated or merged using various delimitation methods. Two main classes of methods exist: distance-based methods which consist in clustering sequences in Molecular Operational Units (MOTUs), e.g. the Automatic Barcoding Gap Discovery method (ABGD)  and phylMycobacterium ulcerans.Here, we integrate both methods based on morphology and molecular data. The ABGD method with COI sequences was applied for the molecular species delimitation. This study aims to contribute to better understand the Cameroon’s biodiversity of aquatic and sub-aquatic bugs putatively involved in the transmission to humans of the environmentally-persistent bacteria 2 and at different depth levels (down to a depth of 1 m), and indirectly by using light trapping to capture winged imagoes. Aquatic sampling, which included a large variety of streams, rivers, swamps and flooded areas, was performed in triplicate at each survey session, on three consecutive mornings. Light traps were installed twice at each survey session in each site from 6:30PM to 11:00PM. Two survey periods were realized in 2011 (March to June) and 2012–2013 (September 2012 to February 2013) in the endemic (4 times and 6 times respectively at each survey period) and non-endemic zones (2 times at each survey period). After collection, adults and nymphs (L5) were selected, counted, and preserved in 70% ethanol, which was changed weekly, for morphological identification and molecular analyses. The 22,375 specimens collected were first classified by family using the Heteroptera classification given by Schuh and Slater [According to The catch insects’ authorization has been issued by the Director of Wildlife and Protected Areas of the Ministry of Forests and Wildlife which is responsible for the field studies nationwide Decision N° 0859/PCBS/MINFOF/SG/DFAP/SDVEF/SC, insects were collected in 10 locations in Cameroon including two existing endemic zones for Buruli ulcer (Akonolinga and Bankim) and eight non-endemic zones with the same ecological characteristics that the two endemic ones  . A map  was maded Slater  and thenThe 309 specimens were identified down to the species or genus using the pioneer taxonomic work of Poisson on Afrotropical aquatic Heteroptera –5,28–33,5’-GGTCAACAAATCATAAAGATATTGG-3’ and HCO 5’-TAAACTTCAGGGTGACCAAA AAATCA-3’[A total of 188 specimens were analyzed among which 45 nymphs and 143 adults. About four individuals per morphotype were sampled for DNA sequence analysis. Total DNA was extracted from legs or full body for small insects, using the NucleoSpin Tissue XS  accordinAAATCA-3’.PCR always started with a denaturation step of 94°C for five min, followed by 25 cycles comprising denaturation at 94°C, for one min, annealing at 50°C for 1.5 min and elongation at 72°C for one min, and ended with a ten min final elongation at 72°C. PCR products were cleaned by Exosap IT , a singlThe cleaned products were then sequenced to 690 bp nucleotides. Multiple alignments were made using Clustal W according to the default settings: full multiple alignments with bootstrap number equal to 1000. Finally, we obtained a data set of 171 COI homologous sequences of 669 bp. We corroborated the (99%) homology of our sequences with COI sequences obtained from BLAST-GenBank and added to our data set some Genbank reference sequences of COI  = 1, Nb bins = 20) and with K2P distances. The data can be partitioned into finer and finer partitions until there is no further partitioning. Tree different partitions were used in this study: p1 (p = 0.0359); p2 (p = 0.0046); p3 (p = 0.0028).Molecular putative species limits were explored with the Automatic Barcode Gap Discovery (ABGD) . This meMacrocoris flavicollis flavicollis and Macrocoris laticollis laticollis, Mor16 separated into Laccotrephes dilatatus and Laccotrephes fabricii, Mor17 separated into Laccotrephes calcaratus and Laccotrephes armatus, Mor18 separated into Laccotrephes latimanus and Laccotrephes sp., Mor21 separated into Anisops (Micranisops) apicalis and Anisops (Micranisops) parvulus, Mor25 separated into Anisops (s. str.) sardeus and Anisops sp., Mor31 separated into Limnogonus (s. str.) cereiventris and Limnogonus (Limnogoïdes) poissoni, Mor33 separated into Neogerris severini and Gerris swakopensis, Mor38 separated into Hydrometra sp.1 and Hydrometra huntchinsoni and Mor39 separated into Hydrometra albolineata and Hydrometra sp.2 , these morphotypes were dispatched in 54 species belonging to 11 different families Tables  and 3. Itra sp.2 . And in brus sp. . NoticeaNeonychia congoensis congoensis Hungerford, 1946. For all remaining nymphal morphotypes it was impossible to attribute a specific name based on the morphology.In addition, MorL14 initially designated as a nymphal morphotype was identified as an adult of the species Using the ABGD method, we explored the limits between the different species using the COI sequence from 188 specimens including 49 adult and 14 nymphal morphotypes. The COI amplification failed for some adult morphotypes , certainly due to DNA degradation. The molecular data set obtained for the 45 adult morphotypes were partitioned into 41 or 45 putative molecular species according to the gap value retained corresponding to distance values of 0.0359 and 0.0028, respectively , S1 and Figs. Tha posteriori species names associated to the corresponding adult determined based on morphology. According to the gap-value used, 11 (p = 0.0359) to 17 (p = 0.0028) molecular species could be recognized for the nymph morphotypes , Notonectidae (Mor26-27) and Hebridae (Mor30-49). By contrast, two species were recognized by the two methods for a single a priori morphological species in Gerridae , Hydrometridae (Mor38). However, some discrepancies are noticed for Gerridae (Mor31-32) and Mesoveliidae (Mor40-41-42).For the first set of samples , 54 species were determined and for the second set (based on molecular criteria), 52–62 species were putatively delimited using COI molecular marker. Nine species were lacking from the molecular sampling and if we consider the partition p3, 8 putative molecular species have no morphological species associated with them  yield a total of 62 aquatic bugs species in Cameroon.The species biodiversity of aquatic bugs varies among identified families. The most diversified families are Notonectidae (13 species), Belostomatidae (11 species), Gerridae (10 species) and Nepidae (8 species). Other families with intermediate diversity are: Micronectidae (2 species), Mesoveliidae (3 species), Hydrometridae and Veliidae (4 species for each family) and Naucoridae (5 species). Corixidae and Hebridae are the least diversified families with only one identified species each .This study complements the work realized by Poisson –5 on thea priori in the field by non-experts relying on basic identification keys and the morphological analysis carried out by taxonomists from the museum specialized in aquatic bugs. This difference in expertise explains why different morphological species could not be distinguished in the field as different morphotypes and vice versa. In bugs, identification at the species level is difficult in some families, such as Mesoveliidae and Veliidae, and usually requires the presence of males. Some species have different morphological characteristics in males and females for example at A. sardeus and E. glauca males and females were initially classified into different morphotypes  used for the molecular analysis compared to the number of specimens (309) used for the morphological analyses. In other words, the molecular samples might not have been as representative a sample as desired. An explanation for sharing the same molecular putative species between different morphological species is that using a single gene could miss instances of very recent speciation events caused by selection at a few number of loci because a drastic change in morphology could be due to a few genes among which COI is not included. In other words, the “neutral” marker COI might not carry any record of species divergence in that case. Another explanation could be the fact that COI is a mitochondrial gene; it could have introgressed into another species after some hybridization events between the two species. A last hypothesis is that the species based on molecular marker are real but several morphotypes are present within one species.et al. [et al. [It is better to complement this type of study with phylogenetic analyses to determine relationships between species or group of species, but due to the limited sampling size of our study and the use of a single mitochondrial gene that is not intended for building phylogeny, we performed only the separation of the molecular species within the families of aquatic bugs identified. Previous works based on many loci described the phylogenetic relationships of the aquatic bugs more accurately. Hebsgaard et al.  used 16S [et al.  used 37 This study improves our knowledge on the diversity and distribution of aquatic bugs in Cameroon and confirms that COI can reliably be used to identify species in most families of aquatic bugs described here apart from the few exceptions observed. In the near future, molecular identification could also help to routinely identify aquatic bug species of importance in the transmission of the bacillus causing Buruli ulcer in human in the tropical region.This pioneering study will be extended to other Afrotropical region to better document the biodiversity of aquatic bugs in this part of the world.S1 FigEach color represents one ABGD putative specie delimited with associated nymph corresponding to code situated in front of vertical line respectively families’ initial name followed by putative specie number and ML followed by putative nymph specie number, before vertical line Mor = adult morphotype code following by morphotype number and individual number, ML = nymph morphotype code following by morphotype number and individual number.(TIFF)Click here for additional data file.S2 FigEach color represents one ABGD putative specie delimited with associated nymph corresponding to code situated in front of vertical line respectively families’ initial name followed by putative specie number and ML followed by putative nymph specie number, before vertical line Mor = adult morphotype code following by morphotype number and individual number, ML = nymph morphotype code following by morphotype number and individual number.(TIFF)Click here for additional data file.S1 FileSequence name corresponds to families initial name followed by individual code number.(FAS)Click here for additional data file.


Although cardiac magnetic resonance (CMR) has good diagnostic performance in myocarditis, an endomyocardial biopsy (EMB) is still required when treatment against the underlying etiology is considered. Many patients however have a good prognosis following myocarditis and will therefore not benefit from an EMB. The ability of CMR to predict outcomes in these patients may therefore guide selective EMB.All patients admitted with an acute coronary syndrome (ACS)-like presentation of myocarditis, with confirmed diagnosis at baseline according to standard CMR criteria and follow-up examination available, were retrospectively identified in our database. CMR images were analyzed to determine left ventricular (LV) dimensions, LV mass and total amount of late gadolinium enhancement. LGE was quantified using signal intensity >5 SD above normal myocardium and was expressed as percentage of LV mass. Improvement of LV function (LVF) was defined as in increase in ejection fraction (EF) of ≥5%.Fifteen consecutive patients were included in the present study. Follow-up CMR was performed at a mean of 282 ± 187 days after admission. At baseline, mean EF was 51 ± 7 % and was impaired in 11 patients (47 ± 4 %) . EF improved in 8 of these 11 patients (73%). Total indexed myocardial mass (TMM) and the extent of LGE at baseline did not differ between patients that improved and those that did not . At follow-up, the extent of LGE decreased in patients with and patients without LV improvement . However, TMM decreased in patients that showed EF improvement, and not in patients without improvement .The majority of patients with an ACS-like presentation of myocarditis have a good prognosis. The improvement of LVF was accompanied by a decrease in myocardial mass. This decrease in myocardial mass likely reflects the resolution of acute inflammatory changes in acute myocarditis.None.