Additive neurorestorative effects of exercise and docosahexaenoic acid intake in a mouse model of Parkinson’s disease

There is a need to develop interventions to slow or reverse the degeneration of dopamine neurons in Parkinson’s disease after diagnosis.Given that preclinical and clinical studies suggest benefits of dietary n-3 polyunsaturated fatty acids,such as docosahexaenoic acid,and exercise in Parkinson’s disease,we investigated whether both could synergistically interact to induce recovery of the dopaminergic pathway.First,mice received a unilateral stereotactic injection of 6-hydroxydopamine into the striatum to establish an animal model of nigrostriatal denervation.Four weeks after lesion,animals were fed a docosahexaenoic acid-enriched or a control diet for the next 8 weeks.During this period,the animals had access to a running wheel,which they could use or not.Docosahexaenoic acid treatment,voluntary exercise,or the combination of both had no effect on(i)distance traveled in the open field test,(ii)the percentage of contraversive rotations in the apomorphine-induction test or(iii)the number of tyrosine-hydroxylase-positive cells in the substantia nigra pars compacta.However,the docosahexaenoic acid diet increased the number of tyrosine-hydroxylase-positive terminals and induced a rise in dopamine concentrations in the lesioned striatum.Compared to docosahexaenoic acid treatment or exercise alone,the combination of docosahexaenoic acid and exercise(i)improved forelimb balance in the stepping test,(ii)decreased the striatal DOPAC/dopamine ratio and(iii)led to increased dopamine transporter levels in the lesioned striatum.The present results suggest that the combination of exercise and docosahexaenoic acid may act synergistically in the striatum of mice with a unilateral lesion of the dopaminergic system and provide support for clinical trials combining nutrition and physical exercise in the treatment of Parkinson’s disease.

AB047.Quebec database of clinical data and biological material for research on uveal melanoma

Background:Sharing biological material and clinical data from patients with uveal melanoma.Methods:Uveal melanoma is the most common intraocular malignancy in the adult population.Because uveal melanoma is primarily a sporadic cancer and familial cases are rare,it is difficult to prevent or detect it.Despite effective treatment of ocular tumors,more than 50%of patients develop incurable liver metastases mainly in the 5-10 years following the detection of the primary tumor.This cancer is relatively rare and the obtained biopsies are very small.About 20 samples are taken each year in Quebec.This provincial infrastructure is made of biological material from donors with uveal melanoma and a large clinical database.Collected tumor biopsies are used for culturing cell lines and the creation of a DNA/RNA library used for genomic and genetic studies.Results:This infrastructure plays an important role in the achievement of various research programs for a better understanding of genetic and environmental factors involved in the development of melanoma and the spread of metastasis.It allows collaboration with other researchers at a provincial,national and international level in order to make progress in basic and clinical research on uveal melanoma.Conclusions:The biological material and clinical data of this infrastructure are available upon request to VHRN members whose research project was approved by the ethics committee of the institution.

AB030. Characterization of the interactions between hepatic stellate cells and tumor cells during uveal melanoma metastatic progression

Background:Uveal melanoma(UM)is the most common primary eye tumor in adults,and the most frequent site of malignant transformation of the melanocytes after the skin.It spreads to the liver in half of the cases,and the death rate following the report of metastasis is 92%at 2 years.Hepatic tropism of UM cannot simply be explained by the blood circulatory system organization,and illustrates the“seed and soil”hypothesis that describes an interaction between tumor cells(seed)and a specific microenvironment(soil).We decided to focus our study on the synergic interaction between UM cells and hepatic stellate cells(HSC),whose role has been previously described in the metastatic progression of colon and pancreatic cancers.Furthermore,HSC have been found surrounding UM liver metastasis,and the UM secretome contains activating cytokines of hepatic stromal cells.Our hypothesis is that HSC provide a specific microenvironment in the liver enhancing the growth of UM cells and increasing their therapeutic resistance.Using an in vitro 3D model and an original xenograft mouse model,we aim to decipher the mechanisms of UM metastatic progression,in order to elaborate new therapeutic strategies.Methods:First,using an agar coating,spheroids were generated with UM cells and were allowed to grow for 72 h.These tumor spheroids were then embedded in Matrigel and the HSC conditioned medium was used to evaluate the impact of the HSC secretome on UM invasion.Next,an original in vivo xenograft mouse model was generated,in which metastatic UM cells were injected alone or with human HSC in the spleen of immunodeficient mice.This model allows us to evaluate in 3-6 weeks the metastatic potential of each cell population,and thus to determine the cooperation between HSC and UM cells in the liver.Results:The HSC conditioned medium increased the invasion of UM spheroids compared to non-conditioned medium in our in vitro model.In addition,UM cells inoculated in the mouse spleen alone or with human HSC were able to metastasize to the liver,and the host HSC were also recruited by UM metastases.Conclusions:Our preliminary results strongly suggest that the secretome of HSC provides a permissive microenvironment for UM metastatic progression.We now have to confirm these results by characterizing the secretome of HSC,in order to identify cytokines or growth factors that increase the invasion of the liver by UM.Our models can be used to test the efficacy of new therapeutic strategies targeting the UM microenvironment.

Considerations for the clinical use of stem cells in genitourinary regenerative medicine

The genitourinary tract can be affected by several pathologies which require repair or replacement to recover biological functions.Current therapeutic strategies are challenged by a growing shortage of adequate tissues.Therefore,new options must be considered for the treatment of patients,with the use of stem cells(SCs)being attractive.Two different strategies can be derived from stem cell use:Cell therapy and tissue therapy,mainly through tissue engineering.The recent advances using these approaches are described in this review,with a focus on stromal/mesenchymal cells found in adipose tissue.Indeed,the accessibility,high yield at harvest as well as anti-fibrotic,immunomodulatory and proangiogenic properties make adipose-derived stromal/SCs promising alternatives to the therapies currently offered to patients.Finally,an innovative technique allowing tissue reconstruction without exogenous material,the self-assembly approach,will be presented.Despite advances,more studies are needed to translate such approaches from the bench to clinics in urology.For the 21st century,cell and tissue therapies based on SCs are certainly the future of genitourinary regenerative medicine.

AB027.Varying pattern of proteases secretion in Fuchs corneal endothelial dystrophy

Background:The goal of this project was to analyze the relationship between cell morphology and proteases/proteases inhibitors(PIs)secretion profile in fuchs endothelial corneal dystrophy(FECD)corneal endothelial cells(CECs).Methods:Cell morphology was determined using a circularity index(4π×area/perimeter2)for each CECs population extracted from surgical FECD specimens(N=2)and healthy Eye bank corneas(N=3).CECs were cultured 28 days post-confluency.Supernatant was collected and analysed using Proteome Profiler Array detecting 35 proteases and 32 PIs(R&D Systems).Proteome signal was analyzed using Image Studio Lite and correlated with the population’s circularity index.Results:Calculation of circularity index reported different morphologies among FECD populations(0.59±0.18 and 0.64±0.17)and healthy populations(0.44±0.18,0.66±0.13 and 0.71±0.11).Proteome arrays revealed the presence of 10 proteases(ADAMTS1,Cathepsin A,B,D,and X/Z/P,DPPIV/CD26,MMP-2,3 and 12,uPA/Urokinase)and 10 PIs(Protease Nexin II,Cystatin B and C,EMMPRIN/CD147,Latexin,Lipocalin-1,Serpin E1,TFPI,TFPI-2,TIMP-1,2 and 4).Healthy and FECD specimens showed similar variation patterns according to morphology for secretion of ADAMTS1,MMP-3 and 12.However,opposing patterns between healthy and FECD populations were observed for Cathepsin B and D.Moreover,some proteins did not show variation according to phenotype in healthy CECs,but did in FECD CECs:Cathepsin A,Cystatin C,TFPI-2 and total TIMPs.For the other proteins,secretion did not vary according to morphology or no specific pattern was distinguishable.Conclusions:To conclude,our results suggest that cell phenotype is linked to the secretion of certain proteases/PIs in both groups.However,there seems to be differences in secretion of particular proteases and PIs between FECD and healthy specimens as morphology did not have a similar influence.These differences might initiate an imbalance between proteases and PIs explaining the irregular thickening of the Descemet membrane seen in FECD.

AB022.Membrane binding properties of the C-terminal segment of retinol dehydrogenase 8

Background:Retinol dehydrogenase 8(RDH8)is a 312-amino acid(aa)protein involved in the visual cycle.Bound to the outer segment disk membranes of photoreceptors,it reduces all-trans-retinal to all-trans-retinol1 as one of the rate-limiting steps of the visual cycle2.RDH8 is a member of the short-chain dehydrogenase/reductase family.Its C-terminal segment allows its membrane-anchoring through the postulated presence of an amphipathicα-helix and of 1 to 3 acyl groups at positions 299,302 and 3043.The secondary structure and membrane binding characteristics of RDH8 and its C-terminal segment have not yet been described.Methods:To evaluate the membrane binding of RDH8,the full-length protein(aa 1-312),a truncated form(aa 1-296),its C-terminal segment(aa 281-312 and 297-312)as well as different additional variants of this segment were used.The truncated protein binds membranes less efficiently than the full-length form.Thus,the C-terminal segment of RDH8 is essential for the binding and has thus been further examined.The intrinsic fluorescence of tryptophan residues at positions 289 and 310 of the wild-type C-terminal segment of RDH8 and the mutants W289F,W310F and W310R have thus been used to determine their extent of binding to lipid vesicles and to monitor their local environment.Unilamellar lipid vesicles composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine(POPC)or a mixture of POPC and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine(POPS)were used to mimic the phospholipid content of the outer segment disk membranes of photoreceptors.Results:An increase in fluorescence intensity and in fluorescence lifetime is observed upon increasing the concentration of lipid vesicles.These data allowed calculating values of partition coefficient of the C-terminal segment of RDH8 varying between Kp=1.1 E6 to 1.7 E6.It is noteworthy that the observation of a more intense shift to lower wavelengths upon membrane binding of the mutant W310R and W310F indicates a deeper incorporation of the remaining tryptophan residue at position 289 into the lipid bilayer.The secondary structure of the C-terminal segment of RDH8 observed by circular dichroism and infrared spectroscopy shows a superposition ofα-helical,β-turn and unordered structures.Conclusions:The peptides derived from the C-terminal segment of RDH8 show a strong binding to lipid vesicles.These strength of binding is independent of the type of lipid and the presence of a mutation.

AB021.Discovery of a new role for the WNK1 kinase in corneal wound healing

Background:Damage to the corneal epithelium triggers important changes in the composition of the extracellular matrix(ECM)to which the basal human corneal epithelial cells(hCECs)attach.These changes are perceived by integrins,a family of trans-membrane receptors that activate different intracellular signalling pathways,ultimately leading to re-epithelialization of the injured epithelium.Our goal is to study the impact of the pharmacological inhibition/activation of specific signal transduction mediators on corneal wound healing using both monolayers of hCECs and tissue-engineered human corneas(hTECs)as in vitro models.Methods:hTECs were produced by the self-assembly approach and wounded with a 8-mm biopsy punch.Total RNA and proteins were isolated from the wounded and unwounded hTECs to conduct gene profiling analyses and protein kinase arrays.The wounded tissues were then incubated either with the lysine deficient protein kinase 1(WNK1)inhibitor WNK463,the WNK1 indirect agonist AM1241,or with the vehicle alone(DMSO;negative control)and wound healing was monitored for 6 days.The impact of WNK1 inhibition/activation on hCECs monolayers was determined using scratch wound assays.Results:Gene profiling analyses and protein kinases arrays revealed that expression and activity of several mediators from the integrin-dependent signalling pathways were altered in response to the ECM changes taking place during corneal wound healing.Phosphorylation of the WNK1 kinase turned out to be the most striking activation event occurring during wound healing.Since the pharmacological inhibition of WNK1 by WNK463 significantly reduced the rate of corneal wound closure in our hTECs and hCECs monolayers compared to their respective negative controls,we believe that the pharmacological activation of WNK1 could turn out to be an interesting avenue to accelerate corneal wound closure.Conclusions:These results will contribute to a better understanding of the cellular and molecular mechanisms involved in corneal wound healing.Furthermore,they identified a new function for the WNK1 kinase in corneal wound healing and might lead to the identification of a new therapeutic target in the field of corneal wounds.

Annals of Eye Science, 2018 7 © Annals of Eye Science. All rights reserved. Ann Eye Sci 2018;3:AB007 aes.amegroups.com

Background:The goal of this study was to engineer an epithelialized and endothelialized pigmented choroidal substitute using the self-assembly approach of tissue engineering.Methods:Cells from human choroids were isolated and cultured.Culture purity was assessed using immunostaining(CD31,HMB45,vimentin,keratins 8/18).To engineer the choroid,fibroblasts were cultured in the presence of serum and ascorbic acid to promote extracellular matrix(ECM)assembly.Endothelial cells,melanocytes or RPE cells were separately seeded on the stromal substitutes.Choroidal substitutes were further characterized by histology,mass spectrometry,immunostaining,and compared to native human choroids.Results:The technique used to isolate choroidal cells yielded pure cultures of fibroblasts,melanocytes and vascular endothelial cells.The stromal substitutes engineered using the self-assembly approach were composed of collagen(types I,VI,XII and XIV),proteoglycans(decorin,lumican)and other ECM proteins.Protein expression was confirmed using immunostaining.Endothelial cells spontaneously assembled into capillary-like structures and vascular networks when cocultured with fibroblast-containing ECM sheets.Conclusions:This study shows that the self-assembly approach of tissue engineering can be used to reconstruct a choroid using native cells.This model represents a unique tool to better understand the crosstalk between the different choroidal cell types and cell-ECM interactions.

AB093.Ocular tissues bank for vision health research

Background:This infrastructure delivers biological material necessary for several research projects to Vision Health Research Network investigators(VHRN).Methods:Héma-Québec is the organism in charge obtaining consent and retrieving donor eyes for patient treatment or for research.In Quebec City,donor eyes are sent to the eye bank of the“Centre Universitaire d’Ophtalmologie”(CUO)of Saint-Sacrement hospital.Technicians at the eye bank evaluate the quality of the tissues.Those unfit for graft are transferred to the infrastructure where the coordinator encodes samples prior to their distribution.Results:Between 2013 and 2017,27 fundamental investigators,clinical investigators and collaborators supported by 60 students,trainees and laboratory assistants used this infrastructure to move forward their projects.Since 2013,results from those projects generated 21 scientific publications and 232 presentations.The infrastructure helped VHRN investigators obtain near 4 million dollars in grants from many organisms(CIHR,NSERC,Foundations,etc.).These grants allowed recruitment and formation of highly qualified personnel.Last year(April 2016 to March 2017),189 corneas and 23 eyes transited through the infrastructure.Conclusions:This infrastructure is available for all investigators that are members of the VHRN.Many original projects have been elaborated thanks to the human ocular tissues provided by this infrastructure.These projects will advance our knowledge in vision health.A better understanding of eye functions will lead to new treatments for eye diseases.

Ultrastretchable and wearable conductive multifilament enabled by buckled polypyrrole structure in parallel

Stretchable conductive fibers have attracted much attention due to their potential use in wearable electronics.However,the ultrahigh strain insensitive conductivity is hindered by mechanical mismatch in Young’s modulus and failure of stretchable structures under large deformation.This challenge is addressed with a conductive and elastic multifilament made of the polyurethane monofilaments that are surface-coated with buckled polypyrrole(PPy)of which flexibility is improved by sodium sulfosalicylate.Such parallel conductive monofilaments with PPy buckling on surface reduce the influence of cracks in the conductive coating on the overall conductivity,displaying an ultra-high strain insensitive behavior(quality factor Q=10.9).Remarkably,various complex forms of wearable electronic textiles made by this conductive multifilament maintain the strain-insensitive behavior of the original multifilament,even upon the large deformation of human joint.This multifilament with wrinkled PPy has attractive advantages in the application of super-stretched wearable electronic devices.

Using data from‘visible’populations to estimate the size and importance of‘hidden’populations in an epidemic:A modelling technique

We used reported behavioural data from cisgender men who have sex with men and transgender women(MSM/TGW)in Bangalore,mainly collected from‘hot-spot’locations that attract MSM/TGW,to illustrate a technique to deal with potential issues with the representativeness of this sample.A deterministic dynamic model of HIV transmission was developed,incorporating three subgroups of MSM/TGW,grouped according to their reported predominant sexual role(insertive,receptive or versatile).Using mathematical modelling and data triangulation for‘balancing’numbers of partners and role preferences,we compared three different approaches to determine if our technique could be useful for inferring characteristics of a more‘hidden’insertive MSM subpopulation,and explored their potential importance for the HIV epidemic.Projections for 2009 across all three approaches suggest that HIV prevalence among insertive MSM was likely to be less than half that recorded in the surveys(4.5e6.5%versus 13.1%),but that the relative size of this subgroup was over four times larger(61e69%of all MSM/TGW versus 15%).We infer that the insertive MSM accounted for 10e20%of all prevalent HIV infections among urban males aged 15e49.Mathematical modelling can be used with data on‘visible’MSM/TGW to provide insights into the characteristics of‘hidden’MSM.A greater understanding of the sexual behaviour of all MSM/TGW is important for effective HIV programming.More broadly,a hidden subgroup with a lower infectious disease prevalence than more visible subgroups,has the potential to contain more infections,if the hidden subgroup is considerably larger in size.

Focus-tunable microscope for imaging small neuronal processes in freely moving animals

Miniature single-photon microscopes have been widely used to image neuronal assemblies in the brain of freely moving animals over the last decade. However,these systems have important limitations for imaging in-depth fine neuronal structures. We present a subcellular imaging single-photon device that uses an electrically tunable liquid crystal lens to enable a motion-free depth scan in the search of such structures. Our miniaturized microscope is compact (10 mm×17 mm×12 mm) and lightweight (≈1.4 g),with a fast acquisition rate (30–50 frames per second),high magnification (8.7×),and high resolution (1.4μm) that allow imaging of calcium activity of fine neuronal processes in deep brain regions during a wide range of behavioral tasks of freely moving mice.

Association between genome-wide epigenetic and genetic alterations in breast cancer tissue and response to HER2-targeted therapies in HER2- positive breast cancer patients: new findings and a systematic review

Recent evidence suggests that genetic and epigenetic mechanisms might be associated with acquired resistance to cancer therapies.The aim of this study was to assess the association of genome-wide genetic and epigenetic alterations with the response to anti-HER2 agents in HER2-positive breast cancer patients.PubMed was screened for articles published until March 2021 on observational studies investigating the association of genome-wide genetic and epigenetic alterations,measured in breast cancer tissues or blood,with the response to targeted treatment in HER2-positive breast cancer patients.Sixteen studies were included in the review along with ours,in which we compared the genome-wide DNA methylation pattern in breast tumor tissues of patients who acquired resistance to treatment (case group, n = 6) to that of patients who did not develop resistance (control group, n =6). Among genes identified as differentially methylated between the breast cancer tissue of cases and controls, oneof them, PRKACA, was also reported as differentially expressed in two studies included in the review. Althoughincluded studies were heterogeneous in terms of methodology and study population, our review suggests thatgenes of the PI3K pathway may play an important role in developing resistance to anti-HER2 agents in breastcancer patients. Genome-wide genetic and epigenetic alterations measured in breast cancer tissue or blood mightbe promising markers of resistance to anti-HER2 agents in HER2-positive breast cancer patients. Further studiesare needed to confirm these data.