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Neuromedin U (NMU) is an evolutionary conserved neuropeptide that has been implicated in multiple processes, such as circadian regulation, energy homeostasis, reward processing and stress coping. Although central expression of NMU has been addressed previously, the lack of specific and sensitive tools has prevented a comprehensive characterization of NMU-expressing neurons in the brain. We have generated a knock-in mouse model constitutively expressing Cre recombinase under theNmupromoter. We have validated the model using a multi-level approach based on quantitative reverse-transcription polymerase chain reactions,in situhybridization, a reporter mouse line and an adenoviral vector driving Cre-dependent expression of a fluorescent protein. Using the Nmu-Cre mouse, we performed a complete mapping of NMU expression in adult mouse brain, unveiling a potential midline NMU modulatory circuit with the ventromedial hypothalamic nucleus (VMH) as a key node. Moreover, immunohistochemical analysis suggested that NMU neurons in the VMH mainly constitute a unique population of hypothalamic cells. Taken together, our results suggest that Cre expression in the Nmu-Cre mouse model largely reflects NMU expression in the adult mouse brain, without altering endogenous NMU expression. Thus, the Nmu-Cre mouse model is a powerful and sensitive tool to explore the role of NMU neurons in mice.

Photoporation is an up-and-coming technology for the gentle and efficient transfection of cells. Inherent to the application of photoporation is the optimization of several process parameters, such as laser fluence and sensitizing particle concentration, which is typically done one factor at a time (OFAT). However, this approach is tedious and runs the risk of missing a global optimum. Therefore, in this study, we explored whether response surface methodology (RSM) would allow for more efficient optimization of the photoporation procedure. As a case study, FITC-dextran molecules of 500 kDa were delivered to RAW264.7 mouse macrophage-like cells, making use of polydopamine nanoparticles (PDNPs) as photoporation sensitizers. Parameters that were varied to obtain an optimal delivery yield were PDNP size, PDNP concentration and laser fluence. Two established RSM designs were compared: the central composite design and the Box-Behnken design. Model fitting was followed by statistical assessment, validation, and response surface analysis. Both designs successfully identified a delivery yield optimum five- to eight-fold more efficiently than when using OFAT methodology while revealing a strong dependence on PDNP size within the design space. In conclusion, RSM proves to be a valuable approach to efficiently optimize photoporation conditions for a particular cell type.

Monocyte-derived macrophages (Mφs) are crucial regulators during muscularis inflammation. However, it is unclear which micro-environmental factors are responsible for monocyte recruitment and anti-inflammatory Mφ differentiation in this paradigm. Here, we investigate Mφ heterogeneity at different stages of muscularis inflammation and determine how environmental cues can attract and activate tissue-protective Mφs. Results showed that muscularis inflammation induced marked alterations in mononuclear phagocyte populations associated with a rapid infiltration of Ly6c+ monocytes that locally acquired unique transcriptional states. Trajectory inference analysis revealed two main pro-resolving Mφ subpopulations during the resolution of muscularis inflammation, i.e. Cd206+ MhcIIhi and Timp2+ MhcIIlo Mφs. Interestingly, we found that damage to the micro-environment upon muscularis inflammation resulted in EGC activation, which in turn stimulated monocyte infiltration and the consequent differentiation in anti-inflammatory CD206+ Mφs via CCL2 and CSF1, respectively. In addition, CSF1-CSF1R signaling was shown to be essential for the differentiation of monocytes into CD206+ Mφs and EGC proliferation during muscularis inflammation. Our study provides a comprehensive insight into pro-resolving Mφ differentiation and their regulators during muscularis inflammation. We deepened our understanding in the interaction between EGCs and Mφs, thereby highlighting pro-resolving Mφ differentiation as a potential novel therapeutic strategy for the treatment of intestinal inflammation. ispartof: MUCOSAL IMMUNOLOGY vol:15 issue:6 pages:1296-1308 ispartof: location:United States status: published

Approximately 20% of sleeping sickness patients exhibit respiratory complications, however, with a largely unknown role of the parasite. Here we show that tsetse fly-transmitted Trypanosoma brucei parasites rapidly and permanently colonize the lungs and occupy the extravascular spaces surrounding the blood vessels of the alveoli and bronchi. They are present as nests of multiplying parasites exhibiting close interactions with collagen and active secretion of extracellular vesicles. The local immune response shows a substantial increase of monocytes, macrophages, dendritic cells and gamma delta and activated alpha beta T cells and a later influx of neutrophils. Interestingly, parasite presence results in a significant reduction of B cells, eosinophils and natural killer cells. T. brucei infected mice show no infection-associated pulmonary dysfunction, mirroring the limited pulmonary clinical complications during sleeping sickness. However, the substantial reduction of the various immune cells may render individuals more susceptible to opportunistic infections, as evident by a co-infection experiment with respiratory syncytial virus. Collectively, these observations provide insights into a largely overlooked target organ, and may trigger new diagnostic and supportive therapeutic approaches for sleeping sickness. A number of human African trypanosomiasis, or sleeping sickness, patients suffer from respiratory symptoms commonly attributed to cardiac insufficiency. Here, the authors characterise the role of pulmonary Trypanosoma brucei in respiratory infection.

Objective: sensorineural hearing loss (SNHL) and bilateral vestibulopathy (BV) have been associated with cognitive decline and incident dementia. Our aim was to investigate the combined effect of profound SNHL and BV on spatial cognition and hippocampal neurogenesis in adult mice. Methods: Single oral intake of allylnitrile produces otovestibular failure in less than a week. Behavioral assessment included recording of spontaneous activity, motor activity, spatial cognition, etc. Evaluation of hippocampal neurogenesis was performed 8 weeks after treatment by quantification of neural precursor cells and proliferating cells in the dentate gyrus by staining with doublecortin (Dcx) and Ki67, respectively. Results: Profound SNHL and BV were confirmed in the allylnitrile-treated mice respectively by means of auditory brainstem response (ABR) and acoustic startle response, and several vestibular tests. Spatial cognitive deficits, i.e. higher latency to target, were observed with the Barnes maze. In the right hemisphere, no statistically significant difference was observed between groups. In the left hemisphere, the difference in mean cell densities of Dcx positive cells was statistically significant when compared to the control group, whereas the difference in mean cell density of Ki67 positive cells did not differ significantly. Conclusion: Spatial cognitive deficits and decreased immunoreactivity to DCX in the left hippocampus were observed 8 weeks after adult mice acquired profound SNHL and BV.

Study question Can diet normalization or caloric restriction (CR) for two weeks be used as a preconception care intervention in obese Swiss mice to restore oocyte development and quality Summary answer Diet normalization or CR as short-term preconception care interventions in obese mice only partially restored oocyte quality but did improve overall developmental competence. What is known already Maternal metabolic disorders like obesity and metabolic syndrome may result in decreased oocyte and embryo quality, and thus reproductive failure. Overweight and obese patients are advised to lose weight before conception to increase the chance of a healthy pregnancy. However, as human studies show no univocal guidelines, more fundamental research might provide additional answers. In order to avoid interference with increased maternal age, the question remains if oocyte quality can be restored after only a short preconception care intervention (PCCI). Study design, size, duration Outbred mice were fed a control (CTRL) or high-fat/high-sugar (HF) diet for seven weeks. Afterwards, HF-mice were put on different PCCIs for two weeks, resulting in four treatment groups: control diet (9w; CTRL_CTRL), HF diet (9w; HF_HF), switch from HF (7w) to an ad libitum control diet for 2w (HF_CTRL) or to a 30% CR diet for 2w (HF_CR). Oocyte developmental competence (n = 357) and quality (12-16 oocytes /treatment, scored blinded) were determined, using 6-8 mice/treatment. Participants/materials, setting, methods Body weight changes were recorded. In vivo matured oocytes were collected after superovulation and analysed for quality or in vitro fertilized and cultured. Oocyte quality was determined by staining for lipid content (Bodipy) and mitochondrial inner membrane potential and active mitochondria localization (JC-1). Oocyte developmental competence [cleavage (24h p.i.) and blastocyst rates (5 days p.i.)] was scored. Categorical and numerical data were analysed using binary logistic regression and ANOVA, respectively and corrected for multiple testing. Main results and the role of chance Compared to the CTRL group, HF diet increased body weight after 7 weeks by 24.19% (P 0.1) were not different. HF_CR, but not HF_CTRL, oocytes showed higher cleavage rates (68.48%, P Limitations, reasons for caution Although using a mouse model has several advantages, translating these results to the human setting is a limitation of this study. However, to improve this translatability, an outbred mouse model was used. Additional data will be collected to gain more information regarding the best preconception care intervention advice. Wider implications of the findings This research aims to provide fundamental insights in order to be able to formulate clear preconception guidelines to obese women planning for pregnancy. In addition, we aim to find the shortest possible intervention period to improve fertility. Trial registration number Not applicable

ObjectiveMonocyte-derived macrophages (Mφs) are crucial regulators during muscularis inflammation. However, it is unclear which microenvironmental factors are responsible for monocyte recruitment and neurotrophic Mφ differentiation in this paradigm. Here, we investigate Mφ heterogeneity at different stages of muscularis inflammation and determine how environmental cues can attract and activate tissue protective Mφs.DesignSingle cell RNA sequencing was performed on immune cells from the muscularis of wild-type and CCR2-/- mice at different timepoints after muscularis inflammation. CX3CR1GFP/+ and CX3CR1CreERT2 R26YFP mice were analyzed by flow cytometry and immunofluorescence. The transcriptome of enteric glial cells (EGCs) was investigated using PLPCreERT2 Rpl22HA mice. In addition, we assessed the effect of supernatant from neurosphere-derived EGCs on monocyte differentiation based on the expression of pro- and anti-inflammatory factors.ResultsMuscularis inflammation induced marked alterations in mononuclear phagocyte populations associated with a rapid infiltration of Ly6c+ monocytes that locally acquired unique transcriptional states. Trajectory inference analysis revealed two main pro-resolving Mφ subpopulations during the resolution of muscularis inflammation, i.e. Cd206+ MhcIIhi and Timp2+ MhcIIlo Mφs, which were both derived from CCR2+ monocytes. Interestingly, we found that EGCs were able to sense damage to the muscularis to stimulate monocyte recruitment and differentiation towards pro-resolving Mφs via CCL2 and CSF1, respectively.ConclusionOur study provides a comprehensive insight into pro-resolving Mφ differentiation and their regulators during muscularis inflammation. We deepened our understanding in the interaction between EGCs and Mφs, thereby highlighting pro-resolving Mφ differentiation as a potential novel therapeutic strategy for the treatment of intestinal inflammation.

Although plant organ shapes are defined by spatio-temporal variations of directional tissue expansion, this is a little characterized aspect of organ growth regulation. Although it is well known that the plant hormone gibberellin increases the leaf length/with ratio, its effects on cell expansion in the growing leaf are largely unknown. To understand how variations in rate and anisotropy of growth establish the typical monocotelydonous leaf shape, we studied the leaf growth zone of maize (Zea mays) with a kinematic analysis of cell expansion in the three directions of growth: proximo-distal, medio-lateral, and dorso-ventral. To determine the effect of gibberellin, we compared a gibberellin-deficientdwarf3mutant and the overproducing UBI:GA20OX-1 line with their wild types. We found that, as expected, longitudinal growth was dominant throughout the growth zone. The highest degree of anisotropy occurred in the division zone, where relative growth rates in width and thickness were almost zero. Growth anisotropy was smaller in the elongation zone, due to higher lateral and dorso-ventral growth rates. Growth in all directions stopped at the same position. Gibberellin increased the size of the growth zone and the degree of growth anisotropy by stimulating longitudinal growth rates. Inversely, the duration of expansion was negatively affected, so that mature cell length was unaffected, while width and height of cells were reduced. Our study provides a detailed insight in the dynamics of growth anisotropy in the maize leaf and demonstrates that gibberellin specifically stimulates longitudinal growth rates throughout the growth zone.

Vascular damage has been reported to contribute to atresia formation in several diseases including biliary atresia. This study focused on the extrahepatic biliary plexus in experimental biliary atresia. Newborn BALB/cAnNCrl-pups were infected with rhesus rotavirus within 24 hr after birth to induce experimental biliary atresia. The extrahepatic biliary plexus was examined by confocal microscopy on whole-mount preparations, scored by three independent researchers, and further evaluated at the subcellular level with transmission electron microscopy. Imaging results revealed a progressive destruction of the extrahepatic biliary vascular plexus in the course of experimental biliary atresia induced by rotavirus infection. Endothelial cell damage was already visible as cell swelling and necrosis in the first days after infection and a damaged microcirculation that rapidly deteriorated with progression of obliterative cholangiopathy, was observed in the infected mice as early as 72 hr after birth. In experimental biliary atresia, the destruction of the extrahepatic biliary vascular plexus starts already in the first days postinfection and clearly precedes the morphological symptoms of atresia. The deterioration of the vascular bed architecture continues with disease progression. Therefore, we conclude that the (ultra)structural changes in the extrahepatic biliary microvasculature occurring before the visible onset of atresia has a predictive diagnostic value and this impairment in blood supply to the extrahepatic bile duct may be an important contributing factor to the pathogenesis of acquired biliary atresia. Anat Rec, 2018. (c) 2018 Wiley Periodicals, Inc. Anat Rec, 302:818-824, 2019. (c) 2018 Wiley Periodicals, Inc.

This study aimed to investigate the influence of physical activity in innate immunity to conduce to an effective antitumoral immune response analyzing the phenotype and activation status of infiltrating cells. We analysed the intracellular cytokines and the transcription factors of tumor infiltrating lymphocytes (TILS) and spleen leukocytes. The Nos2 gene expression was evaluated in spleen cells and futhermore the ROS production was measured and spleen cells; another cell evaluated was dendritic cells (TIDCs), their cytokines expression and membrane molecules; finally to understood the results obtained, we analysed the dendritic cells obtained from bone marrow. Were used female Balb/c mice divided into 4 groups: two controls without tumor, sedentary (GI) and trained (GII) and two groups with tumor, sedentary (GIII) or trained (GIV). The physical activity (PA) was realized acoording swimming protocol. Tumor was induced by injection of 4T1 cells. All experiments were performed in biological triplicate. After the experimental period, the tumor was removed and the cells were identified by flow cytometry with labeling to CD4, CD8, CD11c, CD11b, CD80, CD86 and Ia, and intracelular staining IL-10, IL-12, TNF-alpha, IFN-gamma, IL-17, Tbet, GATA3, ROR gamma t and FoxP3. The bone marrow of the animals was obtained to analyse the derivated DCs by flow cytometry and culture cells to obtain the supernatant to measure the cytokines. Our results demonstrated that the PA inhibit the tumoral growth although not to change the number of TILS, but reduced expression of GATA-3, ROR-gamma T, related with poor prognosis, and TNF-a intracellular; however occur one significantly reduction in TIDCS, but these cells expressed more co-stimulatory and presentation molecules. Furthermore, we observed that the induced PA stimulated the gene expression of Tbet and the production of inflammatory cytokines suggesting an increase of Th1 systemic response. The results evaluating the systemic influence in DCs showed that the PA improve significantly the number of those cells in bone marrow as well the number of co-stimulatory molecules. Therefore, we could conclude that PA influence the innate immunity by interfering to promote in process of maturation of DCs both in tumor and systemically, that by its turn promote a modification in acquired immune cells, representing by T helper to induce an important alteration transcription factors that are responsible to maintain a suppressive microenviroment, and thereby, allowing the latter cells can thus activate antitumor immune response. The PA was able improve the Th1 systemic response by enhance to Tbet gene expression, promote a slightly increased of Th1-type cytokines and decrease Gata3 and Foxp3 gene expression in which can inhibit the Th1 immune response.

The genus Lactobacillus is known to be extremely diverse and consists of different phylogenetic groups that show a diversity that is roughly equal to the expected diversity of a typical bacterial genus. One of the most prominent phylogenetic groups within this genus is the Lactobacillus plantarum group, which contains the understudied Lactobacillus mudanjiangensis species. Before this study, only one L. mudanjiangensis strain, DSM 28402T, had been described, but without whole-genome analysis. In this study, three strains classified as L. mudanjiangensis were isolated from three different carrot juice fermentations and their whole-genome sequence was determined, together with the genome sequence of the type strain. The genomes of all four strains were compared with publicly available L. plantarum group genome sequences. This analysis showed that L. mudanjiangensis harboured the second largest genome size and gene count of the whole L. plantarum group. In addition, all members of this species showed the presence of a gene coding for a cellulose-degrading enzyme. Finally, three of the four L. mudanjiangensis strains studied showed the presence of pili on scanning electron microscopy (SEM) images, which were linked to conjugative gene regions, coded on a plasmid in at least two of the strains studied.

Although an increasing number of beneficial microbiome members are characterized for the human gut and vagina, beneficial microbes are underexplored for the human upper respiratory tract (URT). In this study, we demonstrate that taxa from the beneficial Lactobacillus genus complex are more prevalent in the healthy URT than in patients with chronic rhinosinusitis (CRS). Several URT-specific isolates are cultured, characterized, and further explored for their genetic and functional properties related to adaptation to the URT. Catalase genes are found in the identified lactobacilli, which is a unique feature within this mostly facultative anaerobic genus. Moreover, one of our isolated strains, Lactobacillus casei AMBR2, contains fimbriae that enable strong adherence to URT epithelium, inhibit the growth and virulence of several URT pathogens, and successfully colonize nasal epithelium of healthy volunteers. This study thus demonstrates that specific lactobacilli are adapted to the URT and could have a beneficial keystone function in this habitat. De Boeck et al. show that lactobacilli are decreased in patients with chronic rhinosinusitis and enriched in healthy controls. They isolate one strain adapted to the nasopharyngeal mucosa that shows beneficial probiotic effects and safety in the form of a nasal spray in healthy volunteers.

The genusLactobacillusis known to be extremely diverse and consists of different phylogenetic groups that show a diversity roughly equal to the expected diversity of a typical bacterial genus. One of the most prominent phylogenetic groups within this genus is theLactobacillus plantarumgroup which contains the understudiedLactobacillus mudanjiangensisspecies. Before this study, only oneL. mudanjiangensisstrain, DSM 28402T, was described but without whole-genome analysis. In this study, three strains classified asL. mudanjiangensis, were isolated from three different carrot juice fermentations and their whole-genome sequence was determined, together with the genome sequence of the type strain. The genomes of all four strains were compared with publicly availableL. plantarumgroup genome sequences. This analysis showed thatL. mudanjiangensisharbored the second largest genome size and gene count of the wholeL. plantarumgroup. In addition, all members of this species showed the presence of a gene coding for a putative cellulose-degrading enzyme. Finally, three of the fourL. mudanjiangensisstrains studied showed the presence of pili on scanning electron microscopy (SEM) images, which were linked to conjugative gene regions, coded on plasmids in at least two of the strains studied.Author summaryLactobacillus mudanjiangensisis an understudied species within theLactobacillus plantarumgroup. Since its first description, no other studies have reported its isolation. Here, we present the first four genome sequences of this species, which include the genome sequence of the type strain and three newL. mudanjiangensisstrains isolated from fermented carrot juice. The genomes of all four strains were compared with publicly availableL. plantarumgroup genome sequences. We found that this species harbored the second largest genome size and gene count of the wholeL. plantarumgroup. Furthermore, we present the first scanning electron microscopy (SEM) images ofL. mudanjiangensis, which showed the formation of pili in three strains that we linked to genes related to conjugation. Finally, we found the presence of a unique putative cellulose-degrading enzyme, opening the door for different industrial applications of theseLactobacillusstrains.

Maternal metabolic stress conditions are of growing importance in both human and dairy cattle settings as they can have significant repercussions on fertility. Upregulated lipolysis is a common trait associated with metabolic disorders and results in systemically elevated concentrations of non-esterified fatty acids (NEFAs). The effects of high NEFA concentrations on the follicular environment, oocyte and embryo development is well documented. However, knowledge on the effects of NEFAs within the oviduct, representing the initial embryonic growth environment, is currently lacking. Therefore, the experiments outlined here were designed to obtain fundamental insights into both the direct and indirect interactions between NEFAs, bovine oviductal cells and developing zygotes. Hence, zygotes were co-cultured with NEFA-pre-exposed bovine oviductal cells or subjected to simultaneous NEFA exposure during the co-culture period. The outcome parameters assessed were embryo development with cleavage (48h post insemination (pi)), morula (120–126h pi) and blastocyst (192h pi) rates, as well as morula intracellular lipid content and blastocyst quality using Bodipy and differential staining respectively. Our data suggest a direct embryotoxicity of NEFAs as well as impaired embryo development through a reduced oviductal ability to support and protect early embryo development.

While an increasing number of beneficial microbiome members is characterized for the human gut and vagina, beneficial microbes are underexplored for the human upper respiratory tract (URT). In this study, we demonstrate that taxa from the beneficial Lactobacillus genus might have a role as keystone species in the healthy URT. Several URT-specific Lactobacillus isolates were cultured, characterized, and further explored for their genetic and functional properties related to adaptation to the URT. Catalase genes were found in the identified Lactobacillus taxa, which is a unique feature within this mostly facultative anaerobic genus. Moreover, one of our isolated strains, Lactobacillus casei AMBR2, contained unique fimbriae enabling strong adherence to URT epithelium, inhibited the growth and virulence of several URT pathogens, and successfully colonized nasal epithelium of healthy volunteers. This study thus demonstrates that specific Lactobacillus taxa are adapted to the URT and could have a beneficial keystone function in this habitat.

The preservation of the viability of microorganisms in probiotic formulations is the most important parameter ensuring the adequate concentration of live microorganisms at the time of administration. The formulation and processing techniques used to produce these probiotic formulations can influence the preservation of the microbial viability. However, it is also required that the bacteria maintain their key probiotic capacities during processing, formulation and shelf life. In this study, we investigated the impact of spray-drying on different cell wall properties of the model probiotic strain Lactobacillus rhamnosus GG, including its adherence to intestinal epithelial cells. The dltD gene knock-out mutant, L. rhamnosus GG CMPG5540, displaying modified cell wall lipoteichoic acids, showed significantly increased colony-forming units after spray-drying and subsequent storage under standard conditions compared to wild-type L. rhamnosus GG. In contrast, disruption of the biosynthesis of exopolysaccharides or pili expression did not impact survival. However, spray-drying did significantly affect the adherence capacity of L. rhamnosus GG. Scanning electron microscopy confirmed that the pili, key surface factors for adherence to intestinal cells and mucus, were sheared off during the spray-drying process. These data thus highlight that both the functionality and viability of probiotics should be assessed during the spray-drying process and subsequent storage.

Persistent high-risk human papillomavirus (HPV) infection is strongly associated with development of high-grade cervical intraepithelial neoplasia or cancer (CIN3+). In single type infections, serial type-specific viral-load measurements predict the natural history of the infection. In infections with multiple HPV-types, the individual type-specific viral-load profile could distinguish progressing HPV-infections from regressing infections. A case-cohort natural history study was established using samples from untreated women with multiple HPV-infections who developed CIN3+ (n = 57) or cleared infections (n = 88). Enriched cell pellet from liquid based cytology samples were subjected to a clinically validated real-time qPCR-assay (18 HPV-types). Using serial type-specific viral-load measurements (≥3) we calculated HPV-specific slopes and coefficient of determination (R(2) ) by linear regression. For each woman slopes and R(2) were used to calculate which HPV-induced processes were ongoing (progression, regression, serial transient, transient). In transient infections with multiple HPV-types, each single HPV-type generated similar increasing (0.27copies/cell/day) and decreasing (-0.27copies/cell/day) viral-load slopes. In CIN3+, at least one of the HPV-types had a clonal progressive course (R(2) ≥ 0.85; 0.0025copies/cell/day). In selected CIN3+ cases (n = 6), immunostaining detecting type-specific HPV 16, 31, 33, 58 and 67 RNA showed an even staining in clonal populations (CIN3+), whereas in transient virion-producing infections the RNA-staining was less in the basal layer compared to the upper layer where cells were ready to desquamate and release newly-formed virions. RNA-hybridization patterns matched the calculated ongoing processes measured by R(2) and slope in serial type-specific viral-load measurements preceding the biopsy. In women with multiple HPV-types, serial type-specific viral-load measurements predict the natural history of the different HPV-types and elucidates HPV-genotype attribution.

Background The intestinal wall has a complex topographical architecture. The multi-layered network of the enteric nervous system and its intercellular interactions are difficult to map using traditional section-based or whole-mount histology. With the advent of optical clearing techniques, it has become feasible to visualize intact tissue and organs in 3D. However, as yet, a gap still needs to be filled in that no in-depth analysis has been performed yet on the potential of different clearing techniques for the small intestine. Aim The goal of this study was to identify an optimal clearing protocol for in toto imaging of mouse intestinal tissue. Methods Five aqueous-based clearing protocols (SeeDB2, CUBIC, ScaleS, Ce3D, and UbasM) and four organic reagent-based clearing protocols (3DISCO, iDISCO+, uDISCO, and Visikol® ) were assessed in segments of small intestine from CX3CR1GFP/GFP and wild-type mice. Following clearing, optical transparency, tissue morphology, green fluorescent protein (GFP) fluorescence retention, and compatibility with (immuno-)labeling were analyzed. Key results All organic reagent-based clearing protocols-except for Visikol-rendered tissue highly transparent but led to substantial tissue shrinkage and deformation. Of the aqueous-based protocols, only Ce3D yielded full-thickness tissue transparency. In addition, Ce3D displayed excellent GFP retention and preservation of tissue morphology. Conclusions Ce3D emerged as a most efficient protocol for enabling rapid full-thickness 3D mapping of the mouse intestinal wall.

The aim of the study was to identify specific human papillomavirus (HPV) type responsible for malignancy in penile tissue samples using laser micro-dissection and TaqMan quantitative real-time PCR (qPCR). The study was based on two pre-malignant and seven malignant penile tissue samples and laser micro-dissection was performed on all. Genotyping was performed on whole tissue sections and laser micro-dissection samples using qPCR. Two whole tissue section samples were HPV negative while seven were HPV positive. In four samples that were single HPV infections with whole tissue section PCR, identical HPV types were confirmed with laser micro-dissection PCR. Clearly confirming that the single HPV type detected is responsible for malignancy. In two samples that had multiple HPV infections with whole tissue section PCR, only one HPV type with the highest viral load was detected with laser micro-dissection PCR, suggesting that the HPV type with the highest viral load is most likely the cause of that particular lesion. HPV 11 and/or HPV 16 were the only types detected with laser micro-dissection PCR in these cases, compared to multiple HPV types (HPV 11, HPV 16, HPV 18, HPV 31, HPV 33, HPV 35, and HPV 39) initially detected with whole tissue section PCR. HPV 11 was associated with verrucous lesions while HPV 16 was associated with squamous cell carcinoma and PIN 3 lesions. This study confirms that laser micro-dissection and qPCR are essential tools in identifying the HPV types responsible for malignancy in penile lesions, particularly in samples with multiple infections. J. Med. Virol. 87:1761–1768, 2015. © 2015 Wiley Periodicals, Inc.

In Africa, data is limited on quantitation of human papillomavirus (HPV) types in women with multiple infections. This study applied a real time PCR (qPCR) assay for detection, genotyping and quantitation of multiple HPV infections in 90 tissue blocks of South African women with cervical squamous cell carcinoma. One sample with multiple HPV types was subjected to laser micro-dissection and qPCR. Four samples were negative for β-globin and these were excluded from the analysis. The HPV DNA positivity rate was 93.0% (80/86). All 80 positives showed the presence of HR HPV types; HPV 68 was the only type negative in all the samples. Overall, HPV 16 was positive in most of the samples (88.8%), followed by HPV 56 (28.7%), HPV 18 (20.0%) and HPV 39 (18.7%). More than half of the samples (65.0%) had multiple infections. HPV 16 was present in majority of single (85.7%) and multiple infections (90.4%). HPV 16 showed higher viral loads in 70.3% of the HPV 16 co-infected samples. In one multiple infected sample laser micro-dissection and qPCR identified HPV 18 with higher viral load as the most likely cause of the invasive lesion. There is large number of multiple HPV infections in South African women with cervical squamous cell carcinoma. HPV 16 is the most frequently detected type and often presents with higher viral load, suggesting it could be responsible for pathogenesis of the lesions in the majority of cases.

Background The pulmonary neuroepithelial body (NEB) microenvironment (ME) consists of innervated cell clusters that occur sparsely distributed in the airway epithelium, an organization that has so far hampered reliable selective gene expression analysis. Although the NEB ME has been suggested to be important for airway epithelial repair after ablation, little is known about their potential stem cell characteristics in healthy postnatal lungs. Here we report on a large-scale selective gene expression analysis of the NEB ME. Methods A GAD67-GFP mouse model was used that harbors GFP-fluorescent NEBs, allowing quick selection and pooling by laser microdissection (LMD) without further treatment. A panel of stem cell-related PCR arrays was used to selectively compare mRNA expression in the NEB ME to control airway epithelium (CAE). For genes that showed a higher expression in the NEB ME, a ranking was made based on the relative expression level. Single qPCR and immunohistochemistry were used to validate and quantify the PCR array data. Results Careful optimization of all protocols appeared to be essential to finally obtain high-quality RNA from pooled LMD samples of NEB ME. About 30% of the more than 600 analyzed genes showed an at least two-fold higher expression compared to CAE. The gene that showed the highest relative expression in the NEB ME, Delta-like ligand 3 (Dll3), was investigated in more detail. Selective Dll3 gene expression in the NEB ME could be quantified via single qPCR experiments, and Dll3 protein expression could be localized specifically to NEB cell surface membranes. Conclusions This study emphasized the importance of good protocols and RNA quality controls because of the, often neglected, fast RNA degradation in postnatal lung samples. It was shown that sufficient amounts of high-quality RNA for reliable complex gene expression analysis can be obtained from pooled LMD-collected NEB ME samples of postnatal lungs. Dll3 expression, which has also been reported to be important in high-grade pulmonary tumor-initiating cells, was used as a proof-of-concept to confirm that the described methodology represents a promising tool for further unraveling the molecular basis of NEB ME physiology in general, and its postnatal stem cell capacities in particular. Electronic supplementary material The online version of this article (doi:10.1186/s12931-017-0571-4) contains supplementary material, which is available to authorized users.

The emergence of antibiotic-resistant Helicobacter pylori strains impacts the efficacy of eradication therapy and promotes the development of alternative treatment strategies. Apocynin inhibits neutrophil NADPH oxidase and hence may decrease reactive oxygen species-mediated tissue damage in H. pylori-infected stomach tissue. Apocynin was tested in vitro for its cytotoxic and direct antibacterial effects. The therapeutic efficacy of orally administered apocynin (100 mg/kg/day through drinking water or 200 mg/kg/day through combined administration of drinking water and slow-release formulation) was assessed at 9 weeks after infection in the Mongolian gerbil model. Bacterial burdens were quantified by viable plate count and quantitative PCR. Histopathological evaluation of antrum and pylorus provided insight into mucosal inflammation and injury. Apocynin showed no cytotoxic or direct antibacterial effects in vitro or in vivo. Nine weeks of apocynin treatment at 200 mg/kg/day reduced active H. pylori gastritis as neutrophil infiltration in the mucous neck region and pit abscess formation decreased significantly. In our gerbil model, prolonged high-dose apocynin treatment significantly improved H. pylori-induced pit abscess formation without indications of drug toxicity and thus further investigation of the dosage regimen and formulation and the long-term impact on neoplastic development should be carried out.

We hypothesized that elevated non-esterified fatty acids (NEFA) modify in vitro bovine oviduct epithelial cell (BOEC) metabolism and barrier function. Hereto, BOECs were studied in a polarized system with 24-h treatments at Day 9: (1) control (0 µM NEFA + 0% EtOH), (2) solvent control (0 µM NEFA + 0.45% EtOH), (3) basal NEFA (720 µM NEFA + 0.45% EtOH in the basal compartment) and (4) apical NEFA (720 µM NEFA + 0.45% EtOH in the apical compartment). FITC-albumin was used for monolayer permeability assessment and related to transepithelial electric resistance (TER). Fatty acid (FA), glucose, lactate and pyruvate concentrations were measured in spent medium. Intracellular lipid droplets (LD) and FA uptake were studied using Bodipy 493/503 and immunolabelling of FA transporters (FAT/CD36, FABP3 and CAV1). BOEC-mRNA was retrieved for qRT-PCR. Results revealed that apical NEFA reduced relative TER increase (46.85%) during treatment and increased FITC–albumin flux (27.59%) compared to other treatments. In basal NEFA, FAs were transferred to the apical compartment as free FAs: mostly palmitic and oleic acid increased respectively 56.0 and 33.5% of initial FA concentrations. Apical NEFA allowed no FA transfer, but induced LD accumulation and upregulated FA transporter expression (↑CD36, ↑FABP3 and ↑CAV1). Gene expression in apical NEFA indicated increased anti-apoptotic (↑BCL2) and anti-oxidative (↑SOD1) capacity, upregulated lipid metabolism (↑CPT1, ↑ACSL1 and ↓ACACA) and FA uptake (↑CAV1). All treatments had similar carbohydrate metabolism and oviduct function-specific gene expression (OVGP1, ESR1 and FOXJ1). Overall, elevated NEFAs affected BOEC metabolism and barrier function differently depending on NEFA exposure side. Data substantiate the concept of the oviduct as a gatekeeper that may actively alter early embryonic developmental conditions.

Persistent high-risk human papillomavirus (HPV) infection is strongly associated with development of high-grade cervical intraepithelial neoplasia or cancer (CIN3+). In single type infections, serial type-specific viral-load measurements predict the natural history of the infection. In infections with multiple HPV-types, the individual type-specific viral-load profile could distinguish progressing HPV-infections from regressing infections. A case-cohort natural history study was established using samples from untreated women with multiple HPV-infections who developed CIN3+ (n = 57) or cleared infections (n = 88). Enriched cell pellet from liquid based cytology samples were subjected to a clinically validated real-time qPCR-assay (18 HPV-types). Using serial type-specific viral-load measurements (≥3) we calculated HPV-specific slopes and coefficient of determination (R(2) ) by linear regression. For each woman slopes and R(2) were used to calculate which HPV-induced processes were ongoing (progression, regression, serial transient, transient). In transient infections with multiple HPV-types, each single HPV-type generated similar increasing (0.27copies/cell/day) and decreasing (-0.27copies/cell/day) viral-load slopes. In CIN3+, at least one of the HPV-types had a clonal progressive course (R(2) ≥ 0.85; 0.0025copies/cell/day). In selected CIN3+ cases (n = 6), immunostaining detecting type-specific HPV 16, 31, 33, 58 and 67 RNA showed an even staining in clonal populations (CIN3+), whereas in transient virion-producing infections the RNA-staining was less in the basal layer compared to the upper layer where cells were ready to desquamate and release newly-formed virions. RNA-hybridization patterns matched the calculated ongoing processes measured by R(2) and slope in serial type-specific viral-load measurements preceding the biopsy. In women with multiple HPV-types, serial type-specific viral-load measurements predict the natural history of the different HPV-types and elucidates HPV-genotype attribution.

Contains fulltext : 80661.pdf (Publisher’s version ) (Closed access) Age-related hearing impairment (ARHI), or presbycusis, is the most prevalent sensory impairment in the elderly. ARHI is a complex disease caused by an interaction between environmental and genetic factors. Here we describe the results of the first whole genome association study for ARHI. The study was performed using 846 cases and 846 controls selected from 3434 individuals collected by eight centers in six European countries. DNA pools for cases and controls were allelotyped on the Affymetrix 500K GeneChip for each center separately. The 252 top-ranked single nucleotide polymorphisms (SNPs) identified in a non-Finnish European sample group (1332 samples) and the 177 top-ranked SNPs from a Finnish sample group (360 samples) were confirmed using individual genotyping. Subsequently, the 23 most interesting SNPs were individually genotyped in an independent European replication group (138 samples). This resulted in the identification of a highly significant and replicated SNP located in GRM7, the gene encoding metabotropic glutamate receptor type 7. Also in the Finnish sample group, two GRM7 SNPs were significant, albeit in a different region of the gene. As the Finnish are genetically distinct from the rest of the European population, this may be due to allelic heterogeneity. We performed histochemical studies in human and mouse and showed that mGluR7 is expressed in hair cells and in spiral ganglion cells of the inner ear. Together these data indicate that common alleles of GRM7 contribute to an individual's risk of developing ARHI, possibly through a mechanism of altered susceptibility to glutamate excitotoxicity.

The aim of the study was to identify specific human papillomavirus (HPV) type responsible for malignancy in penile tissue samples using laser micro-dissection and TaqMan quantitative real-time PCR (qPCR). The study was based on two pre-malignant and seven malignant penile tissue samples and laser micro-dissection was performed on all. Genotyping was performed on whole tissue sections and laser micro-dissection samples using qPCR. Two whole tissue section samples were HPV negative while seven were HPV positive. In four samples that were single HPV infections with whole tissue section PCR, identical HPV types were confirmed with laser micro-dissection PCR. Clearly confirming that the single HPV type detected is responsible for malignancy. In two samples that had multiple HPV infections with whole tissue section PCR, only one HPV type with the highest viral load was detected with laser micro-dissection PCR, suggesting that the HPV type with the highest viral load is most likely the cause of that particular lesion. HPV 11 and/or HPV 16 were the only types detected with laser micro-dissection PCR in these cases, compared to multiple HPV types (HPV 11, HPV 16, HPV 18, HPV 31, HPV 33, HPV 35, and HPV 39) initially detected with whole tissue section PCR. HPV 11 was associated with verrucous lesions while HPV 16 was associated with squamous cell carcinoma and PIN 3 lesions. This study confirms that laser micro-dissection and qPCR are essential tools in identifying the HPV types responsible for malignancy in penile lesions, particularly in samples with multiple infections.

Elevated non-esterified fatty acids (NEFAs) have been recognized as an important link between lipolytic metabolic conditions and impaired fertility in high-yielding dairy cows. However, NEFA effects on the oviductal micro-environment currently remain unknown. We hypothesize that elevated NEFAs may contribute to the complex pathology of subfertility by exerting a negative effect on bovine oviductal epithelial cell (BOEC) physiology. Therefore, the objectives of this study were to elucidate direct NEFA effects on BOEC physiology in three different in vitro cell culture systems. Bovine oviductal epithelial cells (four replicates) were mechanically isolated, pooled, and cultured as conventional monolayers, as explants, and in a polarized cell culture system with Dulbecco's modified Eagle's medium/F12based culture medium. Bovine oviductal epithelial cells were exposed to an NEFA mixture of oleic, stearic, and palmitic acids for 24 hours at both physiological and pathologic concentrations. A control (0 μM NEFA) and a solvent control (0 μM NEFA + 0.45% ethanol) group were implemented. Bovine oviductal epithelial cells physiology was assessed by means of cell number and viability, a sperm binding assay, transepithelial electric resistance (TER), and a wound-healing assay. Bovine oviductal epithelial cell morphology was assessed by scanning electron microscopy on cell polarity, presence of microvilli and cilia, and monolayer integrity. Bovine oviductal epithelial cell number was negatively affected by increasing NEFAs, however, cell viability was not. Sperm binding affinity significantly decreased with increasing NEFAs and tended (P = 0.051) to be more affected by the direction of NEFA exposure in the polarized cell culture system. The absolute TER increase after NEFA exposure in the control (110 ± 11 Ω.cm2) was significantly higher than that in all the other treatments and was also different depending on the exposure side. Bidirectional exposed monolayers were even associated with a significant TER reduction (−15 ± 10 Ω.cm2; P < 0.05). Cell proliferation capacity showed a decreased cell migration with increasing NEFA concentrations but was irrespective of the exposure side. Bovine oviductal epithelial cell morphology was not affected. In conclusion, in an in vitro setting, NEFAs exert a negative effect on BOEC physiology but not morphology. Ultimately, these physiological alterations in its microenvironment may result in suboptimal development of the pre-implantation embryo and a reduced reproductive outcome in dairy cattle.

The mouse mutant Ozzy, originating from an ENU-mutagenesis programme, displays a head bobbing phenotype. We report here that Ozzy mice show a clear deficit in vestibulo-ocular reflex (VOR). Micro-CT scanning of the inner ears showed narrowing and truncations of at least one of the semicircular canals and loss of the ampullae. Frequency-specific auditory-evoked brainstem response (ABR) tests revealed a slight threshold increase in the middle frequency range compared to wild-type littermates. Linkage analysis localised the gene in a 5.5-cM region on chromosome 2. Subsequently, a 499 T--A missense mutation was identified in Jag1, leading to a substitution of an evolutionary conserved tryptophane (W167R). Mutations in the human homologue of Jag1 cause Alagille syndrome (AGS), an autosomal dominant disorder associated with liver, heart, eye and skeletal abnormalities, accompanied by a characteristic facies. In human patients, it occasionally affects other organ systems like the kidney or the inner ear. Liver disease is the main diagnostic factor for AGS. Ozzy mice showed significantly less intrahepatic bile ducts than wild-type littermates. Thirty-seven percent of Ozzy mice showed heart defects. No eye or vertebral abnormalities could be detected. In conclusion, Ozzy mice show two of the major and one minor characteristic of AGS.

Background: Three mutations in the DFNA5 gene have been described in three families with autosomal dominant non-syndromic hearing impairment. Although these mutations are different at the genomic DNA level, they all lead to skipping of exon 8 at the mRNA level. We hypothesise that hearing impairment associated with DFNA5 is caused by a highly unusual mechanism, in which skipping of one specific exon leads to disease that is not caused by other mutations in this gene. We hypothesise that this represents a very specific “gain of function” mutation, with the truncated protein exerting a deleterious new function. Methods: We performed transfection experiments in mammalian cell lines (HEK293T and COS-1) with green fluorescent protein (GFP) tagged wildtype and mutant DFNA5 and analysed cell death with flow cytometry and fluorescence microscopy. Results: Post-transfection death of HEK293T cells approximately doubled when cells were transfected with mutant DFNA5 –GFP compared with wildtype DFNA5–GFP. Cell death was attributed to necrotic events and not to apoptotic events. Conclusion: The transfection experiments in mammalian cell lines support our hypothesis that the hearing impairment associated with DFNA5 is caused by a “gain of function” mutation and that mutant DFNA5 has a deleterious new function.

A mutation in DFNA5 leads to a type of hearing loss that closely resembles the frequently observed age-related hearing impairment (ARHI). The hearing loss is sensorineural, progressive and starts at the high frequencies. As DFNA5 was considered an excellent candidate ARHI susceptibility gene, we performed linkage analysis to a quantitive measure of high frequency hearing loss. However, no significant linkage between ARHI and microsatellite markers from the DFNA5 region could be detected. Subsequently, the DFNA5 coding region was analysed for single nucleotide polymorphisms (SNPs). Two SNPs leading to amino-acid substitutions (P142H and V207M) were selected for further analysis. Using these SNPs, an association study based on a collection of random individuals, and a case-control association study were performed. No significant differences in genotypes between good hearing and hearing impaired individuals could be detected in either study design. We conclude that there exists no strong association between DFNA5 and ARHI.

In Africa, data is limited on quantitation of human papillomavirus (HPV) types in women with multiple infections. This study applied a real time PCR (qPCR) assay for detection, genotyping and quantitation of multiple HPV infections in 90 tissue blocks of South African women with cervical squamous cell carcinoma. One sample with multiple HPV types was subjected to laser micro-dissection and qPCR. Four samples were negative for β-globin and these were excluded from the analysis. The HPV DNA positivity rate was 93.0% (80/86). All 80 positives showed the presence of HR HPV types; HPV 68 was the only type negative in all the samples. Overall, HPV 16 was positive in most of the samples (88.8%), followed by HPV 56 (28.7%), HPV 18 (20.0%) and HPV 39 (18.7%). More than half of the samples (65.0%) had multiple infections. HPV 16 was present in majority of single (85.7%) and multiple infections (90.4%). HPV 16 showed higher viral loads in 70.3% of the HPV 16 co-infected samples. In one multiple infected sample laser micro-dissection and qPCR identified HPV 18 with higher viral load as the most likely cause of the invasive lesion. There is large number of multiple HPV infections in South African women with cervical squamous cell carcinoma. HPV 16 is the most frequently detected type and often presents with higher viral load, suggesting it could be responsible for pathogenesis of the lesions in the majority of cases.

Faunal evidence from the Fayum Neolithic is often cited in the framework of early stock keeping in Egypt. However, the data suffer from a number of problems. In the present paper, large faunal datasets from new excavations at Kom K and Kom W (4850-4250 BC) are presented. They clearly show that, despite the presence of domesticates, fish predominate in the animal bone assemblages. In this sense, there is continuity with the earlier Holocene occupation from the Fayum, starting ca. 7350 BC. Domesticated plants and animals appear first from approximately 5400 BC. The earliest possible evidence for domesticates in Egypt are the very controversial domesticated cattle from the 9th/8th millennium BC in the Nabta Playa-Bir Kiseiba area. The earliest domesticates found elsewhere in Egypt date to the 6th millennium BC. The numbers of bones are generally extremely low at this point in time and only caprines are present. From the 5th millennium BC, the numbers of sites with domesticates dramatically increase, more species are also involved and they are usually represented by significant quantities of bones. The data from the Fayum reflect this two phase development, with very limited evidence for domesticates in the 6th millennium BC and more abundant and clearer indications in the 5th millennium BC. Any modelling of early food production in Egypt suffers from poor amounts of data, bias due to differential preservation and visibility of sites and archaeological remains, and a lack of direct dates for domesticates. In general, however, the evidence for early stock keeping and accompanying archaeological features shows large regional variation and seems to be mainly dependent on local environmental conditions. The large numbers of fish at Kom K and Kom W reflect the proximity of Lake Qarun. ispartof: PLoS One vol:9 issue:10 ispartof: location:United States status: published

The role of vegetation and soil factors on the biodiversity of two soil dwelling, saprophagous, fly families (Sphaeroceridae and Lonchopteridae) in a heathland ecosystem were investigated. The fly community is primarily affected by soil humidity and the amount of organic matter while the vegetation structure and species composition only indirectly influence the fly communities. There was no correlation between plant species richness and the fly diversity indices. Based on our results and literature data, we hypothesise that the direct effects of the vegetation is more evident for herbivorous insects than for species that do not feed on plants. The investigated families show a clear response to microhabitat differences in the soil factors, which makes them promising indicators for soil health and as tool for monitoring environmental changes.

Background and Methods: In Paget’s disease of the breast, the epidermis of the nipple is infiltrated by large neoplastic cells of glandular origin. It has been hypothesized that the spread of Paget cells through the nipple epidermis is induced by a motility factor that acts via the HER2/NEU receptor. To test this hypothesis, we characterized and purified a motility factor released by keratinocytes and identified its target receptors in specimens from patients with Paget’s disease and in SK-BR-3 breast adenocarcinoma cells, which overexpress HER2/NEU. Results: We isolated the motility factor from keratinocyte-conditioned medium and sequenced tryptic peptides. These sequences were used to identify the motility factor as heregulin-a, which is released by skin keratinocytes. Heregulin-a induces spreading, motility, and chemotaxis of SK-BR-3 cells, as does motility factor. Motility factor activities of heregulin-a are inhibited by monoclonal antibody AB2, directed against the extracellular domain of HER2/NEU, which blocks the binding of heregulin-a .W e used in situ hybridization to show that normal epidermal cells produce heregulin-a messenger RNA and that heregulin receptors, HER3 and/or HER4, as well as their coreceptor HER2/NEU, are expressed by Paget cells. Conclusions: Heregulin-a is a motility factor that is produced and released by normal epidermal keratinocytes and thus plays a key role in the pathogenesis of Paget’s disease. Paget cells express heregulin receptors HER2/NEU, as well as HER3 and/or HER4, both of which function as a co-receptor of HER2/NEU. Binding of heregulin-a to the receptor complex on Paget cells results in the chemotaxis of these breast cancer cells, which eventually migrate into the overlying nipple epidermis. [J Natl Cancer Inst 2000;92:622‐8]

International audience; DFNA5 was first identified as a gene causing autosomal dominant hearing loss. Different mutations have been found, all exerting a highly specific gain of function effect, in which skipping of exon 8 causes the hearing loss. Later reports revealed the involvement of the gene in different types of cancer. Epigenetic silencing of DFNA5 in a large percentage of gastric, colorectal and breast tumors and p53 dependent transcriptional activity have been reported, concluding that DFNA5 acts as a tumor suppressor gene in different frequent types of cancer. Despite these data, the molecular function of DFNA5 has not been investigated properly. Previous transfection studies with mutant DFNA5 in yeast and in mammalian cells showed a toxic effect of the mutant protein which was not seen after transfection of the wild type protein. Here, we demonstrate that DFNA5 is composed of two domains, separated by a hinge region. The first region induces apoptosis when transfected in HEK293T cells, the second region masks and probably regulates this apoptosis inducing capability. Moreover, the involvement of DFNA5 in apoptosis related pathways in a physiological setting was demonstrated through gene expression microarray analysis using Dfna5 knockout mice. In view of its important role in carcinogenesis, this finding is expected to lead to new insights on the role of apoptosis in many types of cancer. In addition, it provides a new line of evidence supporting an important role for apoptosis in monogenic and complex forms of hearing loss.

A complex mutation in DFNA5, resulting in exon 8 skipping, causes autosomal dominant hearing impairment, which starts in the high frequencies between 5 and 15 years of age and progressively affects all frequencies. To study its function in vivo, Dfna5 knockout mice were generated through the deletion of exon 8, simultaneously mimicking the human mutation. To test the hearing impairment, frequency-specific Auditory Brainstem Response (ABR) measurements were performed at different ages in two genetic backgrounds (C57Bl/6J and CBA/Ca), but no differences between Dfna5-/- and Dfna5+/+ mice could be demonstrated. Morphological studies demonstrated significant differences in the number of fourth row outer hair cells between Dfna5-/- mice and their wild-type littermates. These results were obtained in both genetic backgrounds, albeit with opposite effects. In contrast to the results obtained in Dfna5-/- zebrafish, we did not observe different UDP-glucose dehydrogenase and hyaluronic acid levels in Dfna5-/- mice when compared to Dfna5+/+ mice.

The urokinase plasminogen activator (uPA) system is a proteolytic cascade involved in tumor invasion and metastasis. uPA and its inhibitor PAI-1 are described as biomarkers for breast cancer with the highest level of evidence. For the screening of uPA in breast tumors with commercially available ELISA kits that determine the total uPA content (active and inactive), fresh tumor tissue is required. Molecular imaging with activity-based uPA probes might overcome this limitation. Therefore we have developed some highly selective, non-peptidic and specific uPA activity-based probes based on the irreversible uPA inhibitor UAMC-00150. In this study we describe the evaluation of a fluorescent Cy5 labeled and a 18F labeled PET probe in the high uPA expressing MDA-MB-231 breast cancer mouse model to make the step towards clinical translation of this imaging biomarker. Orthotopic MDA-MB-231 tumors were established in nude mice and subjected to uPA imaging at a tumor volume of 150mm3. For the fluorescent imaging, three test groups were considered (n=5): mice from the first and the second experimental group received a single dose of equimolar amounts (0.23 μMol/kg) Cy5-uPA-probe or its inactivated hydrolyzed variant, respectively. The third group received an injection with unlabeled uPA inhibitor, 30 min before administration of Cy5-uPA probe, in a concentration that was 70-fold higher than the probe concentration. Mice were imaged at different time points for 48 h post injection (p.i.) and were subsequently sacrificed for ex vivo imaging and histology. PET imaging (n=15) was performed after i.v. injection of the radiotracer with a maximum of 0,2ml and/or 0,5mCi per injection, resulting in a range of 0,15-0,5mCi/injection. Animals were scanned at 15, 45, 90, 240 and 360 minutes p.i. At each time point, 3 mice were sacrificed to generate ex vivo biodistribution data. The Cy5-uPA-probe demonstrated good tumor-targeting properties in the high uPA-expressing breast tumor model, with fluorescent intensities reaching a maximum at 24 h post-probe administration in mice treated with Cy5-uPA-probe alone. The groups treated with the inactivated probe or the unlabeled inhibitor showed a significant decrease in the fluorescent tumor signal (p>0,016), which was also confirmed on histology. PET imaging with the 18F-uPA-probe showed a peak tumor uptake of 2.76 ± 0.37 %ID/g at 4 h p.i. Further in vitro PPB and ex vivo HPLC data revealed a high affinity of the tracer for blood proteins and a high metabolisation rate potentially explaining the rather moderate to low tumor uptake of the current PET uPa biomarker. In conclusion, fluorescent imaging data clearly indicate that the Cy5-uPA-probe enables non-invasive NIR-imaging of uPA expression in tumors in vivo. The first PET experiments indicate translational capabilities, for which we are now improving the pharmacokinetics of this and future uPa radiotracers. Citation Format: Johan Ides, Christel Vangestel, Jonas Messagie, Dieter Verzele, David Thomae, Sofie Thys, An Wouters, John-Paul Bogers, Sigrid Stroobants, Jurgen Joossens, Pieter Van der Veken, Marc Peeters, Filip Lardon, Steven Staelens, Koen Augustyns. Targeting urokinase plasminogen activator: evaluation of activity-based imaging probes in an orthotopic breast cancer model. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3910. doi:10.1158/1538-7445.AM2013-3910

Heterozygous mutations in the WFS1 gene are responsible for autosomal dominant low frequency hearing loss at the DFNA6/14 locus, while homozygous or compound heterozygous mutations underlie Wolfram syndrome. In this study we examine expression of wolframin, the WFS1-gene product, in mouse inner ear at different developmental stages using immunohistochemistry and in situ hybridization. Both techniques showed compatible results and indicated a clear expression in different cell types of the inner ear. Although there were observable developmental differences, no differences in staining pattern or gradients of expression were observed between the basal and apical parts of the cochlea. Double immunostaining with an endoplasmic reticulum marker confirmed that wolframin localizes to this organelle. A remarkable similarity was observed between cells expressing wolframin and the presence of canalicular reticulum, a specialized form of endoplasmic reticulum. The canalicular reticulum is believed to be involved in the transcellular movements of ions, an important process in the physiology of the inner ear. Although there is nothing currently known about the function of wolframin, our results suggest that it may play a role in inner ear ion homeostasis as maintained by the canalicular reticulum.