Piscine reovirus (PRV) is the causative agent of heart and skeletal muscle inflammation (HSMI), which is detrimental to Atlantic Salmon (AS) aquaculture, but so far has not been cultivatable, which impedes studying the disease and developing a vaccine. Homogenates of head kidney and red blood cells (RBC) from AS in which PRV-1 had been detected were applied to fish cell lines. The cell lines were from embryos, and from brain, blood, fin, gill, gonads, gut, heart, kidney, liver, skin, and spleen, and had the shapes of endothelial, epithelial, fibroblast, and macrophage cells. Most cell lines were derived from the Neopterygii subclass of fish, but one was from subclass Chondrostei. Cultures were examined by phase contrast microscopy for appearance, and by quantitative polymerase chain reaction (qPCR) for PRV-1 RNA amplification and for the capacity to transfer any changes to new cultures. No changes in appearance and Ct values were observed consistently or transferable to new cultures. Therefore, 31 cell lines examined were unable to support PRV-1 amplification and are described as belonging to the non-supportive PRV-1 invitrome. However, these investigations and cell lines can contribute to understanding PRV-1 cellular and host tropism, and the interactions between virus-infected and bystander cells. The purpose of this work was to screen fish cell lines for their ability to support the life cycle of, and amplify, piscine reovirus (PRV), in the hope that at least one would be found and then could be used to study heart and skeletal muscle inflammation (HSMI). PRV is the primary causative agent of HSMI in farmed Atlantic salmon (AS) ,2,3. To Screening cell lines for their ability to produce a new virus is challenging in part because of the surprisingly large number of animal cell lines. The online data base, Cellosaurus, has over 100,000 cell lines, which includes over 500 from fish . To makeTo date, the ability to support PRV replication has been investigated in cell lines from AS , rainbow trout (RTG-2), Chinook salmon (CHSE-214), common carp (CCB), fathead minnow  and orange spotted grouper (GF-1) ,9,10,11.Another way that the fish invitrome can be subdivided is on the basis of the properties of the cell lines after they have been established (heading II in Vulnerability to a particular virus can be expected to vary between the cell lines of a cell line collection (invitrome) . The lifCell lines producing infectious virions belong to the supportive invitrome for a particular virus and can be described as being either lytically or persistently permissive . LyticalThe partially supportive invitrome is made up of cell lines that become infected but fail to produce infectious virions and has two branches . One braFinally, some cell lines show no evidence of viral replication or gene expression and belong to the non-supportive viral invitrome . These cTo date, attempts to cultivate PRV from fish homogenates have been equivocal. The first tries used ASK, CHSE-214, FHM, EPC and GF-1 with homogenates of heart and kidney from Norwegian farmed AS that had been diagnosed with HSMI ,10,11. STherefore, the search for a cell culture system to support the PRV-1 life cycle has been expanded. Homogenates of primarily head kidney and RBC from PRV-1-infected AS were applied to cell lines from 14 fish species and 11 organs, as well as from embryos. Additionally, a cell line was derived from a PRV-1 infected Atlantic salmon. PRV-1 amplification was evaluated by monitoring cultures for increasing viral RNA quantity (Ct) and/or changes in cellular appearance over time. From these, the 31 cell lines were concluded to be non-supportive to PRV-1. With a few of these, the potential value of non-supportive cell lines for studying infected cell/bystander cell interactions in HSMI was demonstrated by measuring, through Western blotting, the induction of myxovirus-resistant protein (Mx), a classic antiviral effector , by RBC This section provides an overview of the screening of 30 cell lines for capacity to support PRV-1 amplification in vitro. The detailed screening results will be described in individual subsections below. Three isolates of PRV-1  were screened for capacity to replicate and amplify in 30 fish cell lines  HK homogenate on 11 cells lines using the standard supernatant transfer sub-cultivation protocol recommended by the OIE Manual of Diagnostic Tests for Aquatic Animals for isolation of disease causing viruses, such as viral haemorrhagic septicaemia virus, infectious haematopoietic necrosis virus, and infectious salmon anaemia virus. Additionally, a long-term screening experiment was done with the ASimf20 cell line exposed to Chilean (PRV-1-unknown subtype) HK homogenate. This is a single cell culture passage isolation assay, but instead of terminating early, the exposed cells were kept alive with weekly fresh medium changes for up to 3 months. Replicate cultures were collected at 2 h , 1 month, and 3 months for analysis by RT-qPCR. Full description of results for the initial screening experiment is described in Subsequently, screening of 26 cell lines for capacity to support Canadian (PRV-1a) and Norwegian (PRV-1b) replication and amplification was performed but more comprehensively, using up to five virus isolation assays. This increased screening rigor is due to the lack of PRV-1 amplification from the initial screening of cell lines with the Chilean (PRV-1-unknown subtype) HK homogenate briefly mentioned above. The virus isolation assays  were: (1) single cell culture passage, (2) sub-cultivation of exposed cells with fresh cells, (3) sub-cultivation of only exposed cells, (4) cell lysate transfer, and (5) supernatant transfer. At least one virus isolation method was examined in each cell line, and for some cell lines, all five virus isolation methods were attempted. The homogenate source for Canadian PRV-1a and Norwegian PRV-1b experiments was RBCs from infected fish. Full description of results for screening Canadian (PRV-1a) and Norwegian (PRV-1b) amplification is described in In addition to monitoring for PRV-1 replication by RT-qPCR in the above screening experiments, the potential appearance of CPE in the inoculated cell lines was also monitored and will be described in A total of 11 cell lines from five fish species were tested for capacity to support PRV-1 amplification upon exposure to Chilean (PRV-1 unknown subtype) HK homogenate. In these experiments, the supernatant transfer virus isolation assay was used . Specifically, for these cell lines, supernatant was transferred (passaged) to new cell cultures in a serial manner at day 7, 14, 21, and 28, for a total of four passages and seven days between each passage. Cell samples were collected for RT-qPCR analysis at the same time points. For RTL-W1, PRV-1 was not detected at any time point, as the cycle threshold was not crossed . For RTHTo determine whether PRV-1 replicated and amplified in 26 cell lines, the Ct value of samples collected at the final time point was compared against the Ct value of samples collected at the initial time point. In virus isolation assay with only one passage, the initial time point is during an early day (day 2) post-inoculation, and the final time point is day 14 post-inoculation. In virus isolation assays with multiple passages, the initial time point is at the end of the first infection passage and the final time point at the end of the second passage . In mostCell lines screened with either Chilean (PRV-1 unknown subtype), Canadian (PRV-1a), or Norwegian (PRV-1b) in screening experiments described above were regularly monitored for appearance of CPE. As viewed by phase contrast microscopy, the appearance of cell cultures with control and PRV-1 homogenates occasionally changed, but any change occurred erratically, with no consistent association with viral homogenates. In cultures of the monocyte/macrophage cell line, RTS11, some cells are also in suspension and others are loosely adherent on the flask surface. With homogenates, floating RTS11 cells formed clumps A. All thThree co-administration experiments of cell lines were performed to determine if the properties of these other viruses can enhance PRV-1 replication, since cell lines exposed to individual PRV-1 isolates did not promote PRV-1 amplification in the previous screening experiments. First, cell lines were co-administered with two different isolates of PRV-1, the Norwegian (PRV-1b) and Canadian (PRV-1a) isolates, with the aim of allowing for possible reassortment of PRV-1 genomes in infected cells, since the PRV-1 genome is fragmented and consists of 10 segments. Second, cell lines were co-administered with PRV-1 and CSV, with the aim of again allowing for possible reassortment, since CSV is also a reovirus, and to determine if the syncytia inducing capacity of CSV can aid PRV-1 dissemination in vitro. Third, cell lines were co-administered with PRV-1 and IPNV to determine if the lytic capacity of IPNV can promote PRV-1 dissemination in cultures. Co-administration of cell lines with two PRV-1 isolates was done on seven cell lines and monitored at two time points, day 2 and day 14, over one passage . The Ct Co-administration of cell lines with either PRV-1 and CSV or PRV-1 and IPNV was done on eight cell lines over two passages. In the first passage, samples were collected between 13 to 18 days post-inoculation, and in the second passage, samples were collected between 12 to 14 days post-passaging, which is between 26 to 32 cumulative days post-initial inoculation in passage one. In all cases, the Ct value was either higher or not detected in samples collected in the second passage . This reThe interaction between PRV-1 homogenates and cell lines has been described using only two observational indicators so far, the changes in Ct values and CPE; therefore, the potential induction of an antiviral response in cell lines by PRV-1 homogenates was used as a third indicator of interaction. Three salmonid cell lines  were examined. In this case, only salmonid cell lines were chosen because the anti-Mx antibody was developed from rainbow trout and has known cross-reactivity with Atlantic salmon Mx but unknown cross-reactivity with other fish species. The cell lines were ASHe and BAASf from Atlantic salmon and RTHDF from rainbow trout. In all three cell lines, both the Norwegian (PRV-1b) and Canadian (PRV-1a) RBC homogenates induced Mx protein expression at levels that were higher than control RBC homogenates from uninfected fish . FurtherThus far, exposure of previously established cell lines to three PRV-1 isolates resulted in no dramatic amplification of PRV-1, particularly over multiple passages and regardless of the virus isolation assay used. Since PRV-1 cannot be amplified when exposed to the currently available cell lines examined in this work, attempts were made to develop new cell lines from fish already infected with PRV-1, with hopes of successfully obtaining new cell lines already or persistently infected with PRV-1. To accomplish this task, two in vivo infection challenges were performed to produce PRV-1-infected Atlantic salmon. The first infection challenge was done with Canadian PRV-1a at PBS-DFO in Nanaimo, B.C., and the second was done with Chilean PRV-1 (unknown subtype) at a level 3 facility that previously belonged to Elanco Canada in Victoria, P.E.I. The results of these two attempts are described in detail below. Since the end goal is to obtain permanent cell lines, PRV-1 screening was not performed on the initial individual primary culture explants, as this would interfere with cell growth and reduce the potential and probability for these primary cultures to develop into permanent cell lines downstream. However, PRV-1 was confirmed systemically in the fish used to develop primary cultures.For the experimental infection trial with PRV-1a at PBS-DFO in Nanaimo, B.C., infected fish produced high levels of PRV-1a . Week 2 For the experimental infection trial with Chilean PRV-1 (unknown subtype) at Elanco Canada’s previously owned level 3 facility, Atlantic salmon were successfully infected with PRV-1 but the amount of virus in fish was low at both week 2 and week 3 post-infection . PrimaryCell line cultures from 14 fish species and multiple organs were exposed in different ways and conditions to RBC and kidney homogenates from AS with PRV-1 and examined for up to 3 months for changes in appearance and PRV-1 Ct values. These were largely unchanged, indicating that the cell lines did not support PRV-1 amplification. In the context of None of the cultures exposed to PRV-1 homogenates acquired the defining properties of lytically permissive cell cultures: viral replication, significant culture deterioration, and capacity to transfer or passage to new cultures the same properties. On a few occasions, substantial monolayer deterioration was seen, with one example occurring with sturgeon brain SB3, but this was not consistently found and cannot be attributed to PRV-1 because over the time of monolayer deterioration, Ct values were unchanged. One possibility is that the SB3 cells were responding to another virus. However, this is unlikely to be a known salmon virus, such as IPNV, IHNV, VHSV, or CSV, because the same homogenates elicited no CPE in CHSE-214, EPC, and RTG-2, which are cell lines known to detect them . Also, aThe occasional vacuolization of cultures after exposure to PRV-1 homogenates hints at the possibility that some cell lines might be partially permissive to PRV-1, with the vacuoles being the visible indicator (CPE) of infection. Previously, after exposure to PRV or HSMI homogenates, cultures of ASK, GF-1, EPC, and FHM were reported to become vacuolated, but in all these cases vacuolization disappeared with passaging ,10,11. AWhether some cell lines could be persistently infected is interesting to consider. Recently, AS were described with a persistent, low-activity but productive PRV infection, and PRV RNA persisted in erythroid progenitor cells and macrophages, especially in the kidney but also in the spleen . In the With the other possibilities being unlikely, the cell lines of this study are concluded to be in the non-supportive PRV invitrome. In the future, several research paths can be taken to determine whether this invulnerability is a fundamental property of the cells or caused by experimental inadequacies. For example, the cell lines might be screened with PRV purified from RBCs to make sure that something in the homogenates has not interfered with infection, and also to be able to test at a high multiplicity of infection, which might allow cellular antiviral mechanisms to be overwhelmed . AdditioPRV appears to have a rather narrow cellular tropism, as judged by the fact that cell lines from multiple tissues and organs were found to be non-supportive and by the histological examination of others on PRV-infected fish that showed restricted localization of PRV RNA and polypeptides. RTS11 from the spleen and ASHe from the heart represented macrophage and endothelial cells respectively, whereas the other cell lines provided homogeneous cell populations of either fibroblast or epithelial cells from a wide range of anatomical sites, including brain, gut, heart, liver, and skin. Presumably, the cell lines lack specific receptors or internalization capabilities for PRV-1 entry and/or intracellular components for PRV-1 gene expression. In PRV-infected fish, susceptible cell types appear to be erythroid progenitors and possibly cardiomyocytes and hepatocytes ,44. InasMallotus villosus, Atlantic horse mackerel Trachurus trachurus, Atlantic herring Clupea harenugs and great silver smelt Argenita silus [(Acipenser fulvescens), with the rest from the subclass Neopterygii, including one from the infraclass, Holostei, longnose gar (Lepisosteus osseus). The salmonid invitrome was the most comprehensively investigated family, with four species: AS , rainbow trout (Oncorhynchus mykiss), Chinook salmon (O. tshawytscha), and Arctic char . For life histories, five species were anadromous: AS, Rainbow trout, Chinook salmon, Arctic char, and the threespine stickleback (Gasterosteus aculeatus). One was catadromous, the American eel (Anguilla rostrata). Six species were from freshwater: Lake sturgeon, Longnose gar, Fathead minnow , Walleye (Sander vitreus), Yellow perch (Perca flavescens) and Zebrafish (Danio rerio). The two marine species were Pacific herring  and haddock Melanogrammus aeglefinus). Geographically, five species were from the North American Great Lakes basin: Fathead minnow, Lake sturgeon, Longnose gar, Walleye, and Yellow perch. Three species  were from the Northeast Pacific and three  from the North Atlantic. Finally, the natural geographical locations of the Arctic char and zebrafish are, respectively, the Arctic ocean and South Asian freshwater.From the perspective of possible hosts, the results suggest that surveying cell lines from more fish species seems unlikely to uncover PRV-1 supportive cell lines. No supportive cell lines were found in invitromes from 14 species of ray-finned fish (Class Actinoptergyii), representing a wide range of taxonomies, life histories, and geographies. The range is wider than the range of species in which PRV has been detected. To date, PRV has been found in salmonids and in 4 marine species, capelin ta silus ,55. For One potential use of non-supportive cell lines is as surrogates for bystander cells in order to study in vitro how bystanders of different anatomical and tissue origins interact with and respond to virus-infected cells. In PRV-infected fish, the RBCs and their progenitors are susceptible ,44,56 anIn conclusion, no fish cell lines from a range of organs and species were found that supported the amplification of PRV-1 from Atlantic salmon homogenates, as evidenced by no consistent change in the appearance and no consistent decrease in Ct values of cell cultures. These cell lines are referred to as the non-supportive PRV-1 invitrome and should be useful for studying the basis of the cellular and host tropism of PRV-1, and for understanding bystander interactions with infected cells. Future searches for supportive lines should focus on the erythroblast lineage.The cell lines are provided in PRV-1 inoculums were prepared from either head kidney tissues or whole blood. Head kidney tissues were aseptically harvested from farmed Atlantic salmon in Chile that exhibited clinical signs and mortality consistent with HSMI, based on histopathology and qPCR analyses, after transfer into a freshwater facility. The kidneys were homogenized using a Dounce homogenizer and sonicated for one minute in intervals of 10 s at 30% intensity. Homogenized samples were centrifuged for 10 min and the pellets were suspended in Minimum Essential Medium (MEM) media with the same volume as the removed supernatant.g for 5 min at 4 °C. Plasma was removed and cell pellet suspended in 10×  L15 supplemented with 50 μg/mL gentamicin (GS). For example, if original blood volume was 1 mL then added 10 mL of L15 to the pellet. Suspended pellet was sonicated with a Branson Sonifier  on ice for 1 min and 20 s in 10 s bursts with 30 s rests and then spun at 2000× g for 5 min at 4 °C to pellet cellular debris. The clarified supernatant was kept for use as inoculum. Whole blood inoculum was also prepared from a few Atlantic salmon that had no history of exposure to PRV-1 or HSMI homogenates and had negative Ct values for PRV-1. These are referred to as control homogenates.Whole blood was sourced from Atlantic salmon experimentally infected with PRV-1. Blood was collected from the caudle vein of infected fish via 22-gauge needles and syringes, and immediately transferred to heparinized vacutainer tubes. The blood was stored frozen as either whole blood or blood in RNAlater. RNAlater was not removed from the mixture because it could contain materials released from lysed blood cells after thawing. Blood was homogenized by diluting the blood/RNAlater mixture 1 to 10 in L15 containing antibiotics and homogenized with a dounce homogenizer. The tubes were spun at 2000× Two experimental infection trials were performed to infect Atlantic salmon with PRV-1a and PRV-1 (unknown genotype) and to develop cell cultures from the infected fish. The trials were at Fisheries and Oceans Canada, Pacific Biological Station  and a level 3 R&D facility in Victoria, PE  that previously belonged to Elanco Canada. The experimental protocols varied between the two trials and are described in detail below. The PRV-1 RNA level  for the infected fish is listed in 2, washed once, then transferred into T25 flasks, with each flask containing 1 mL of growth medium and approximately 12 pieces of minced tissue. For the week three infected fish, primary culture was initiated using 500 μg/mL of either collagenase type II or IV digestion. Tissues were minced into sizes ranging from 1 to 3 mm2. Heart tissues were transferred into micro-centrifuge tubes containing 1 mL of collagenase type II. The same was done for spleen and head kidney tissues, except with collagenase type IV. Tissues were incubated overnight at 4 °C to allow for digestion. The next day, tubes were centrifuged at 1000× g for 5 min to pellet cells and tissues. Tissues were then suspended in 1 mL of growth medium and added to T25 flasks. Flasks were incubated at 15 °C. For the week four infected fish, primary cultures were initiated using trypsin digestion. Tissues were minced into sizes ranging from 1 to 3 mm2 and incubated in trypsin overnight at 4 °C. After digestion, the transfer of tissues into T25 flasks was done as described for the collagenase treated tissues.In trial #1, Atlantic salmon (approximately 50 g weight) held in 10–12 °C brackish water (10 ppm) were anesthetized with MS-222 and injected with 100 μL of blood PRV-1a inoculum as described previously [2 and added to T12.5 flasks containing 0.5 mL of growth medium. All flasks were incubated at 20 °C. The next day, 4 mL of growth medium was added to each flask and the cultures were allowed to develop.In trial #2, Atlantic salmon (approximately 147 g weight) were infected by intraperitoneal injection of 100 μL of Chilean head kidney tissue homogenate containing PRV-1 (unknown subtype). Fish were held in a 400 L circular tank, with UV filtered saltwater, in a flow-through system, with a salinity range 29.9–34.5 ‰, and at a temperature range of 10.0–12.4 °C for 21 days post-exposure. At two- and three-weeks post-infection, ten fish were euthanized with an overdose of MS-222, and head kidney and spleen tissues were collected for establishment of primary cultures. The growth media used to initiate primary culture was L15 medium supplemented with 1% PS, 1% Amp. B, 0.1% GS, and 20% FBS. The wash solution used was DPBS supplemented with 1% PS, 1% Amp. B, and 0.1% GS. On the day of primary culture initiation, head kidneys and spleens were minced into sizes ranging from 1 to 3 mmPRV-1 replication in cell lines was evaluated and monitored using five different virus isolation assays. Detailed methods of the assays are provided in their independent subsections below. One assay involved only a single cell culture passage (single passage isolation). The other four virus isolation assays involved at least two cell culture passages. They are termed: sub-cultivation of exposed cells with fresh cells, sub-cultivation of only exposed cells, cell lysate transfer, and supernatant transfer.In single cell culture passage isolation assays, cell lines were exposed to PRV-1 inoculums, then PRV-1 replication was monitored early at day 2 post-inoculation and late at day 14 post-inoculation by RT-qPCR. Replicate inoculations were done where some of the replicates were collected at day 2, and then the remaining replicates were collected at day 14 for RT-qPCR analysis. Detailed experimental methods are described below. Confluent cell cultures in T25 flasks were exposed to 4 mL of Canadian PRV-1a RBC homogenate (1:500 in 2% FBS/L15) or Norwegian PRV-1b RBC homogenate (1:500 in 2% FBS/L15) continuously for up to 2 weeks. The cultures were incubated at 14 °C. Samples were collected for RNA extraction at 2 d, and then at 14 d post-exposure.In sub-cultivation of exposed cells with fresh cells assays, cell lines were exposed to PRV-1 inoculums for 14 d (passage 1), then at day 14, 20% of cells were collected as viable cells and added to a new culture vessel containing a monolayer of fresh cells (passage 2). The remaining 80% of passage 1 cells were collected for RT-qPCR analysis. Fourteen days after the beginning of passage 2, all cells in passage 2 were collected for RT-qPCR analysis. Detailed experimental methods are described below. Confluent cell cultures in T25 flasks were exposed to 4 mL of Canadian PRV-1a RBC homogenate (1:100 in 2% FBS/L15) continuously for up to 2 weeks (passage 1). The cultures were incubated at 14 °C. At 14 d post-exposure, cells were collected using ~0.125% trypsin, and 20% of collected cells (diluted in 4 mL of 2% FBS/L15) were added to a new flask containing a monolayer of fresh cells (passage 2). The remaining 80% of passage 1 cells were collected for RT-qPCR analysis. At 14 days into passage 2, all cells were collected for RT-qPCR analysis.In sub-cultivation of only exposed cells assays, cell lines were exposed to PRV-1 inoculums for 14–22 d (passage 1) depending on the cell line, then at that day, half of the cells were collected as viable cells and added to a new culture vessel (passage 2). The remaining half of passage 1 were collected for RT-qPCR analysis. From 21 to 30 d (depending on the cell line) after the beginning of passage 2, all cells in passage 2 were collected for RT-qPCR analysis. Detailed experimental methods are described below. Confluent cell cultures in T25 flasks were exposed to 4 mL of Canadian PRV-1a RBC homogenate (1:100 to 1:160 dilution in 15% FBS/L15) or Norwegian PRV-1b RBC homogenate (1:80 to 1:100 dilution in 15% FBS/L15) continuously for up to 22 d (passage 1). High FBS concentration was used because the cells were being propagated. After 14–22 days (depending on the cell line), old medium was then removed, and the cells were washed once with 2 mL DPBS. One mL of ~0.125% trypsin was added to detach cells. After cellular detachment, 1 mL of 15% FBS/L15 was added to inactivate trypsin and mix cells. Half the cells (1 mL) was collected for RNA extraction and the second half was mixed with 3 mL of 15% FBS/L15 and added to a new T25 flask to propagate in passage 2. For this part of the experiment, homogenate availability was limited at the time, so this resulted in variable dilutions of PRV-1 homogenate used throughout this period.In cell lysate transfer assays, cell lines were exposed to PRV-1 inoculums for 14 days (passage 1), then, at day 14, cells were lysed using water and freeze-thaw lysis. A portion of the lysate was added to a new culture vessel containing a monolayer of fresh cells (passage 2). The remaining cell lysates of passage 1 were collected for RT-qPCR analysis. Fourteen days after the beginning of passage 2, all cells in passage 2 were collected for RT-qPCR analysis. Detailed experimental methods are described below. Cell cultures in T25 flasks were exposed to Canadian PRV-1a or Norwegian PRV-1b RBC homogenate (1:100 dilutions in 4 mL of 2% FBS/L15) continuously for 14 d (passage 1). Subsequently, the supernatant was removed, and 2 mL of cell culture water was added to each flask. The flasks were incubated with water for at least 5 min before being placed at −80 °C for at least 5 min (until the liquid was frozen). The flasks were then left to thaw at room temperature and mixed by pipetting, and cellular lysis was confirmed under an inverted phase contrast microscope. After freeze-thaw lysis, 500 μL of lysate mixture was added to a new confluent flask of fresh cells containing 4.5 mL of 2% FBS/L15 (passage 2). The remaining 1.5 mL of lysate mixture was frozen at −80 °C and 200 μL of that was later used for RNA extraction.The supernatant transfer assays were done with two types of PRV-1 homogenates from different tissues. One was the Canadian PRV-1a and Norwegian PRV-1b RBC homogenate, and the other was the Chilean PRV-1 (unknown subtype) head kidney homogenate. For the RBC homogenates, cell lines were exposed to PRV-1 inoculums for 14 to 26 d depending on the cell line (passage 1), then, at that day, a portion of the supernatant was collected and was added to a new culture vessel containing a monolayer of fresh cells (passage 2). All cells of passage 1 were collected for RT-qPCR analysis. Fourteen days after the beginning of passage 2, all cells in passage 2 were collected for RT-qPCR analysis. For the head kidney homogenate experiments, the protocol was similar, except that up to four passages were done for each cell line and the time between each passage (supernatant transfer) was 7 d. Detailed experimental methods are described below.Cells were seeded into T25 flasks and allowed to become confluent or near confluent. The cultures were exposed to either 2 mL Canadian PRV-1a RBC homogenate (1:20 in 2% FBS/L15), 2 mL Norwegian PRV-1b RBC homogenate (1:20 in 2% FBS/L15), or 2 mL Chilean PRV-1 (unknown subtype) head kidney homogenate (1:10 in 2% FBS/L15). Cultures were incubated overnight before the inoculums were removed and cell monolayer washed once with 2 mL DPBS. Four mL of fresh 2% FBS/L15 was then added to each flasks and flasks were incubated at 14 °C for 14 to 26 d (for RBC homogenates) and 7 d (for head kidney homogenates) before supernatant was collected to inoculated fresh cells in passage 2 and cells were collected for RT-qPCR analysis. After 14 d (for RBC homogenates) and 7 d (for head kidney homogenates) of incubation in passage 2, cells were collected for RT-qPCR analysis. At this point, the experiments with RBC homogenates ended, but the experiments with head kidney homogenates continued for two additional passages up to passage four.For the possibility that cytopathic effect (CPE) would develop, primary and cell line cultures were observed by phase-contrast microscopy shortly after the addition of PRV-1 homogenates in all infection experiments described and for the duration of those experiments. The longest observation period was for 3 months.For the co-administration of cell cultures with mixed PRV-1 isolates, mixed PRV-1 inoculum was prepared by mixing one part of Canadian PRV-1a RBC homogenate (1:500 in 2% FBS/L15) with one part of Norwegian PRV-1b RBC homogenate (1:500 in 2% FBS/L15). Confluent cell cultures in T25 flasks were exposed to 4 mL of mixed PRV-1 inoculums continuously for up 14 d. The cultures were incubated at 14 °C. Samples were collected for RT-qPCR analysis at 2 d and 14 d post-exposure. 4.3 TCID50/mL) or 1:10 IPNV (108.1 TCID50/mL). Confluent cell cultures in T25 flasks were exposed to 4 mL of the virus mixture continuously for 13 to 18 d (depending on the cell line) at 14 °C (passage 1). Afterwards, the supernatant of the exposed culture was used to inoculate fresh cells in new flasks (passage 2), and cell/supernatant mixture collected for RT-qPCR analysis. Supernatant transfer assays were used in this case because CSV and IPNV was able to develop cytopathic effects in the cultures and lyse infected cells. At 12 to 14 d (depending on the cell line) after passaging, cell/supernatant mixtures were collected for RT-qPCR analysis.For the co-administration of cell cultures with either PRV-1 and CSV or PRV-1 and IPNV, 10 mL of PRV-1 RBC homogenate (1:10 dilution in 2% FBS/L15) were mixed with 2 mL of either stock CSV or IPNV and 8 mL of 2% FBS/L15. This created a final mixture that contained 1:20 PRV-1 RBC homogenate with either 1:10 CSV  according to the manufacturer’s protocol. Following extraction, a one-step quantitative reverse transcription PCR (RT-qPCR) was used to amplify target sequences of the L1 gene in PRV ,60. The Data for PRV-1 samples in tables is shown as either a single biological replicate  or an average of two or three biological replicates. For average Ct calculations of biological replicates, if at least one of the replicates had no Ct values or Ct values greater than 35.0, then that replicate was not included in the calculation and indicated as such in the results. Samples with no Ct value or Ct values greater than 35 are labeled as “negative”. All direct Ct comparisons between cell lines and time points were conducted on the same plate (96/384 wells) to eliminate the between plate variability. Also, a standard curve was run on all plates using a serial dilution of gBlocks Gene Fragments .https://www.bio-rad.com/en-ca/product/image-lab-software?ID=KRE6P5E8Z). Three cell lines, ASHe, BAASf, and RTHDF, confluent in 6-well plates were exposed to either control, Canadian (PRV-1a), or Norwegian (PRV-1b) RBC homogenate at 1:100 dilution in 2 mL of 15%FBS/L15 per well in triplicate wells. Additionally, BAASf and RTHDF were also exposed to heat-inactivated Canadian PRV-1a RBC homogenate (1:100 dilution in 2 mL of 15%FBS/L15). Heat inactivation was done by boiling the homogenate in a water bath for 15 min. At days 1, 3, and 5 post-inoculation, protein samples were extracted using radioimmunoprecipitation assay buffer (RIPA buffer) and stored at −20 °C until used. Detailed protein extraction, protein quantification, SDS-PAGE, and Western blot procedures were done as previously described . The priAll work with animals was performed in strict accordance with the recommendations in the Canadian Council on Animal Care (CCAC) Guide to the Care and Use of Experimental Animals and use of live animals in experiments conducted at the PBS-DFO, were approved by the Pacific Region Animal Care Committee under protocol #16-013. The study NAH-16-025 at Elanco Canada’s R&D facility was conducted under animal use approval EIAC-0504, granted by the Elanco Animal Health Institutional Animal Care and Use Committee on 02Feb2017.


Cholangiocarcinoma (CCA) is a fatal cancer with high resistance to anticancer drugs. The development of new drugs or compounds to be used alone or in combination with currently available chemotherapeutic agents to improve the treatment of CCA is needed. Compound A (CpdA), which is a small plant-derived glucocorticoid receptor modulator, strongly inhibited the growth and survival of several cancers. However, the effect of CpdA on cholangiocarcinoma has not been elucidated. The aim of this study was to investigate the effect of CpdA on CCA.  Cytotoxicity of CpdA was tested in primary cells including peripheral blood mononuclear cells (PBMCs), fibroblasts, and human umbilical vein endothelial cells (HUVECs), as well as on CCA cell lines  was examined. Cell cycle distribution and IL-6 expression was assessed by flow cytometry and real-time polymerase chain reaction, respectively. The effect of combination CpdA and cisplatin was evaluated by cell viability assay. CpdA significantly inhibited cell cycle at G1 phase in CCA cell lines, and reduced IL-6 mRNA expression. However, combination CpdA and cisplatin did not enhance the inhibitory effect. TGFβR-II expression was increased in CCA cells after the combination treatment. These results indicate the potential of CpdA for CCA treatment. However, combination treatment with CpdA and cisplatin increased CCA cell survival. The molecular mechanism is likely attributable to promotes cell survival via the TGFβR-II signaling pathway. The combination of CpdA with other anticancer drugs for CCA treatment should be further examined. Cholangiocarcinoma (CCA) is an aggressive and lethal cancer that originates from biliary epithelia within the biliary tract . This cancer is rare worldwide, but its incidence is increasing and it is the most common liver cancer in Northeastern Thailand . Infection with the liver fluke Opisthorchis viverrini is the most common risk factor for CCA in Thailand . CCA is normally difficult to diagnose until the disease becomes advanced. Surgery is the only curative treatment for patients with CCA. However, CCA is well known to recur after surgery, and the mortality rate is high . The studies that investigated the use of chemotherapy to treat CCA were small, the results they reported were conflicting, and the patient outcomes were poor . The aggressive and unresponsive nature of this disease highlights the need to urgently identify new drugs or compounds to be used alone or in combination with currently available chemotherapeutic agents to improve the treatment of CCA.The carcinogenesis of CCA is a multistep cellular process . CCA cells express altered molecular mechanisms that enhance cell proliferation, decrease apoptosis, and increase the capacity of tissue invasion. Inflammatory cytokines, such as IL-6, play an important role in the pathogenesis and growth of CCA by activating several pathways involved in survival signaling, and they also contribute to chemoresistance . Glucocorticoids (GC) are potent anti-inflammatory agents that are used in chronic inflammatory diseases and in many cancers. Dexamethasone, which is a synthetic GC, was used to treat CCA in a phase II clinical trial, but various metabolic side effects were observed . A small plant-derived glucocorticoid receptor (GR) modulator, 2-(4-acetoxyphenyl)-2-chloro-N-methylethylammonium-chloride, also called compound A (CpdA), was recently shown to prevent GR dimerization, preserve glucocorticoid anti-inflammatory activity, and have fewer side effects compared with glucocorticoids. CpdA is a plant-derived phenyl aziridine precursor that is extracted from Salsola tuberculatiformis Botschantzev, a Namibian shrub . CpdA also demonstrates anti-inflammatory activity , and it strongly inhibits the growth and survival of prostate cancer and multiple myeloma cells by reducing the release of inflammatory cytokines via inhibition of NF-kB and AP-1 transcriptional factors . The fact that CpdA strongly interacts with GR, and that it instigates inhibition of cancer cell progression suggests that CpdA may have inhibitory effects on CCA.In this study, we set forth to evaluate the anticancer effect of CpdA on CCA cell lines. The cytotoxicity of CpdA on primary cells, including peripheral blood mononuclear cells (PBMCs), fibroblasts, and human umbilical vein endothelial cells (HUVECs), as well as on CCA cell lines  was examined. The effect of CpdA at sublethal dose on cell cycle distribution and IL-6 expression was determined by real-time polymerase chain reaction (PCR) and flow cytometry, respectively. The anticancer activities of CpdA on cell survival were investigated using CpdA alone and in combination cisplatin, which is a conventional anticancer agent. The possible mechanism of CpdA-induced cisplatin-resistance was also studied.CCA cell lines and skin fibroblast cells 2. Cell lines were subcultured twice a week using a standard trypsinization protocol. Skin fibroblast cells (SF-A4) were a gift from Associate Professor Dr. Chanitra Thuwajit, Department of Immunology, Faculty of Medicine Siriraj Hospital, Mahidol University. Human CCA cell lines KKU-100 (JCRB1568), KKU-055 (JCRB1551), and KKU-213 (JCRB1557) were obtained from the Japanese Collection of Research Bioresources Cell Bank (JCRB), National Institute of Biomedical Innovation, Osaka Japan. These cell lines were opisthorchiasis-associated CCA that were established and characterized at the Liver Fluke and Cholangiocarcinoma Research Center, Khon Kaen University, Khon Kaen, Thailand. These cell lines were deposited at the JCRB for long-term storage and for availability as research bioresources. Cell lines were maintained in Dulbecco’s Modified Eagle Medium (DMEM)  containing 10% fetal bovine serum , 100 units/ml penicillin, and 100 μg/ml streptomycin at 37°C with 5% COPreparation of peripheral blood mononuclear cells and human umbilical vein endothelial cellsThe protocols for the collection of peripheral blood mononuclear cells (PBMCs) and human umbilical vein endothelial cells (HUVECs) were approved by the Siriraj Institutional Review Board (SIRB) (COA No. Si405/2013). PBMCs from healthy donors were isolated by Ficoll-Hypaque Density Gradient Media . PBMCs were subsequently resuspended in Roswell Park Memorial Institute (RPMI) 1640 Medium . HUVECs were isolated from healthy term pregnancies. HUVECs were isolated from a 6-inch umbilical cord segment via trypsin digestion. HUVECs were cultured using M199 . Cytotoxicity analysis4 cells/well, while PBMCs and HUVECs were seeded at 1x105 cells/well of a 96-well plate for 24 h at 37°C and 5%CO2. The cells were exposed to the compound A (CpdA) [2-(4-acetoxyphenyl)-2-chloro-N-methylethylammonium-chloride], which was a gift from Dr. Irina Budunova, Department of Dermatology, Northwestern University, Chicago, Illinois, USA, with concentrations ranging between 1.56 µM and 250 µM. The microtitre plate was incubated for further 24 and 48 h and PrestoBlue® Cell Viability Reagent was added. The plates were incubated for further 2 h where after the absorbance of the colour complex was read at 570 nm with a reference wavelength set at 600 nm for PrestoBlue® Cell Viability Reagent, using a BIO-TEK multiwell plate reader.Cytotoxicity was measured using PrestoBlue® Cell Viability Reagent . CCA cell lines and skin fibroblast were seeded in a 96-well microtitre plate at a concentration of 1x10Cell cycle distribution analysis5 cells/well in 2 ml of culture medium and incubated for 24 h. Cells were then treated with CpdA at indicated concentrations for 24 h. After CpdA treatment, cell pellets were collected, washed with cold phosphate-buffered saline (PBS), and fixed with ice-cold 70% ethanol. After the fixative was decanted, the cell pellets were resuspended in staining solution containing DNase-free RNase and PI. Cell cycle was determined using FACSortTM Flow Cytometer , and the data was analyzed using CellQuestTM software .Cell cycle distributions were analyzed by measuring DNA contents that were stained with propidium iodide (PI) . CCA cell lines were seeded in 6-well plates at a density of 1×10Real-time reverse transcription-polymerase chain reaction (RT-PCR)® 480 SYBR Green I Master . The reactions were recorded and analyzed using a LightCycler® 480 Instrument equipped with a 96-well thermal cycler . Relative mRNA expression was normalized against GAPDH mRNA level (forward primer sequence: 5’-CGACCACTTTGTCAAGCTCA-3’ and reverse primer sequence: 5’-AGGGGTCTACAT GGCAACTG-3’) using a comparative Ct method (delta delta Ct).To examine IL-6 transcription, total RNA was prepared from CCA cells untreated or treated with CpdA using Trizol™ Reagent , following the manufacturer’s recommended procedure. IL-6 mRNA was quantified by real-time RT-PCR technique using specific primers (forward primer sequence: 5’-GTACATCCTCGACG GCATC-3’ and reverse primer sequence: 5’-AGCCACTGGTTCTGTGCCT-3’). To examine TGFβR-II transcription, total RNA was prepared from CCA cells untreated or treated with CpdA and cisplatin as indicated by using Trizol™ Reagent , following the manufacturer’s recommended procedure. TGFβR-II mRNA was quantified by real-time RT-PCR technique using specific primers (forward primer sequence: 5’-ACGTTCAG AAGTCGGATGTGG-3’ and reverse primer sequence: 5’-TGCACTTTGGAGAAGCA GCA-3’). For both genes, cDNA synthesis was performed using Superscript®III Reverse Transcriptase , following the manufacturer’s recommended guidelines. Amplification of cDNA by real-time PCR was performed in a LightCyclerCell proliferation assay® Cell Viability Reagent . CCA cells were seeded at 1×104 cells/well in 96-well microtiter plates. After 24-h incubation, the cells were treated with indicated concentrations of CpdA and cisplatin  for 24 h at 37oC in 5% CO2. Cell culture media was not refreshed during this period. At the end of incubation, PrestoBlueTM Cell Viability Reagent was added, after which the cells were incubated at 37°C until the reagent color changed. Metabolically active cells were stained. Absorbance was read at excitation of 570 nm and emission of 600 nm as a reference wavelength for normalization, and the percentage of cell survival was calculated. Data are reported as the percentage of cell survival compared to the percentage in CCA cells without treatment (set at 100%). The effect of CpdA and cisplatin on CCA cell proliferation was determined using PrestoBlueStatistical analysisAll statistical analyses were performed using GraphPad Prism 6 Software . Mean and standard deviation (SD) from three independent experiments was calculated. The difference in experiment results was analyzed by one-way analysis of variance (ANOVA), followed by Tukey’s pos hoc test. A p-value less than 0.05 was considered statistically significant.Cytoxicity of compound A on primary cells To examine the cytotoxicity of compound A (CpdA) on primary normal cells, three types of primary cells, including skin fibroblast cells (SF-A4), peripheral blood mononuclear cells (PBMCs), and human umbilical vein endothelial cells (HUVECs), were tested. The effect of CpdA on cell survival was determined to observe the 50% cytotoxic concentration (CC50) of CpdA. After initial tests to determine appropriate concentrations, SF-A4 cells, PBMCs, and HUVECs were treated with CpdA at concentrations of 0-250 µM, 0-100 µM, and 0-25 µM, respectively, for 24 h and 48 h. The cell viability of each well was determined using PrestoBlueTM Cell Viability Reagent. The average cell survival obtained from triplicate determinations at each concentration was plotted as a dose-response curve. The CC50 of CpdA was defined as the lowest concentration that reduced cell growth by 50% in treated culture compared to untreated culture. At 24 h after treatment, the CC50 of CpdA for SF-A4, PBMCs, and HUVECs was 15.44±1.5, 49.86±3.33, and 3.22±0.61 µM, respectively. At 48 h after treatment, the CC50 of CpdA for SF-A4, PBMCs, and HUVECs was 13.23±3.38, 49.82±2.16, and 3.20±0.71 µM, respectively .Cytotoxicity of compound A on CCA cell linesThe cytotoxicity of CpdA on CCA cells was investigated in three CCA cell lines. KKU-100, KKU-055, and KKU-213 were treated with CpdA at concentration 0-50 µM for 24 h and 48 hr. PrestoBlueTM Cell Viability Reagent was then added, and the plate was incubated for an additional 2 h. The absorbance of the color complex was read at 570 nm with the reference wavelength set at 600 nm using a multiwell plate reader. As shown in Effect of compound A on cell cycle distribution in CCA cell linesThe effect of CpdA on cell cycle distribution in CCA cell lines was investigated using propidium iodide (PI) staining. KKU-100 was treated with CpdA at 0.62, 1.25, and 2.5 µM, and KKU-055 and KKU213 were treated with CpdA at 0.32, 0.62, and 1.25 µM. After 24 h of incubation, treated cells were fixed and stained with PI. The G1, S, and G2-M phases of cell cycle were analyzed by flow cytometry. After CpdA treatment, the numbers of cells in G1 phase of KKU-100, KKU-055, and KKU213 cells were all significantly increased . These rEffect of compound A on IL-6 expression in CCA cell linesTo examine the effect of CpdA on IL-6 expression in CCA cell lines, sublethal doses of CpdA were used to treat the cells for 24 h. KKU-100 was treated with CpdA at 0.62, 1.25, and 2.5 µM, and KKU-055 and KKU213 were treated with CpdA at 0.32, 0.62, and 1.25 µM. After 24 h of incubation, IL-6 expression was determined by real-time PCR. As shown in Effect of combination compound A and cisplatin treatment on CCA cell survivalIL-6 plays an important role in the pathogenesis and growth of CCA via the activation of several pathways involved in cell survival signaling. In previous experiments, it was observed that CpdA could reduce IL-6 expression and effectuate cell cycle arrest during G1 phase, and cisplatin is used for treatment of CCA. We were, therefore, inspired to investigate the combined effect of CpdA and cisplatin on CCA cell lines. Cell survival was determined using PrestoBlueTM Cell Viability Reagent. CpdA and cisplatin were combined and tested in the KKU-100, KKU-055, and KKU-213 CCA cell lines. CpdA at concentrations of 0.62, 1.25, and 2.5 µM for KKU-100, and at concentrations of 0.32, 0.62, and 1.25 µM for KKU-055 and KKU-213 were used in combination with cisplatin at 1, 10, and 100 µM. Combination CpdA and cisplatin treatments showed higher numbers of KKU-100 cell survival than the survival rates of cells treated with cisplatin alone . The comEffect of compound A and cisplatin on TGFβReceptor-II expressionIt was previously reported that glucocorticoid (GC) could upregulate TGFβ receptor II (TGFβR-II) expression, which induced cancer cell resistance to ciplatin . Since CpdA has the same effect as GC, TGFβR-II expression was examined in the present study. Ten µM of cisplatin was used alone or in combination with CpdA at the indicated concentrations. TGFβR-II expression in treated CCA cells was determined by real-time PCR. As shown in CCA is a lethal human cancer that evolves from a multistep carcinogenesis that involves chronic inflammation and alterations in cellular processes . Proinflammatory cytokines, such as IL-6, play important roles in these altered cellular processes, in the onset of inflammation, and in the growth of CCA via activation of several survival signaling pathways . Dexamethasone, which is a synthetic glucocorticoid (GC), is a potent anti-inflammatory drug that is used for treatment of many chronic inflammatory diseases and cancers. GCs exhibit therapeutic effects by interacting with glucocorticoid receptor (GR) and cellular transcription factors   that decrease proinflammatory, antiapoptotic, and survival gene expression, and these effects ultimately lead to tumor cell death . Dexamethasone was used to treat CCA, but several adverse metabolic side effects were observed . The side effects of GCs were found to be mediated by interaction of GR homodimer with responsive elements in promoters and enhancers of the genes implicated in metabolic controls . In contrast to GCs, compound A (CpdA) is a small plant-derived GR modulator that harbors anti-inflammatory activities with fewer side effects . CpdA strongly inhibits the growth and survival of prostate cancer and multiple myeloma cells by reducing proinflammatory cytokines . In the present study, the inhibitory effects of CpdA on CCA cell proliferation and IL-6 production were investigated. The combined effect of CpdA and the anticancer drug cisplatin was also evaluated. Initially, the cytotoxicity of CpdA was tested on primary normal cells, including skin fibroblasts (SF-A4), PBMCs, and HUVECs, and on the KKU-100, KKU-055, and KKU-213 CCA cell lines  and 2 toThe effect of CpdA on cell proliferation was then investigated. The numbers of cells at the G1 phase of the cell cycle were significantly increased in CCA cell lines at 24 h after CpdA treatment . These rCpdA has also been reported to inhibit cytokine expression and secretion in many inflammatory diseases . Several studies demonstrated that CpdA acts as anti-inflammatory agent by reducing the production of proinflammatory cytokines, such as IL-4, IL-5, and IL-13 . Regarding the molecular mechanism, CpdA binds to GR and induces its nuclear translocation . Subsequently, it suppresses the expression of proinflammatory cytokine genes encoding IL-6 and matrix metalloproteinase-1 . Moreover, the anti-inflammatory effect of CpdA was reported to play a role in reducing DNA-binding activity and interferences with the transactivation potential of NF-λB . A 2013 study reported that CpdA reduced TNFα-stimulated IκBα degradation and NF-kB p65 nuclear translocation in a heat shock factor 1-dependent manner in A549 lung epithelial cells. This effect diminished the expression of NF-kB-driven genes, such as IL-6 and IL-8 . IL-6 is one of the cytokines that increases and promotes CCA cell proliferation in CCA patients . The effect of CpdA on IL-6 expression in the KKU-100, KKU-055, and KKU-213 CCA cell lines was examined and it was significantly decreased in CpdA-treated CCA cells , which sThe anticancer drug resistance reported in CCA patients result in a need for increased doses of the drugs used. We hypothesized that CpdA combined with cisplatin might be a more effective treatment for CCA. The results of our experiments revealed no increased anticancer benefit from combination CpdA and cisplatin in CCA. Importantly and in contrast, the combination of CpdA and cisplatin had the effect of increasing CCA cell survival . This fiThe results of this study demonstrated that a sublethal dose of CpdA inhibited CCA cell cycle at G1 phase, and reduced IL-6 expression in CCA cell lines. However, instead of producing an increased anticancer effect, combination treatment with CpdA and cisplatin actually increased CCA cell survival. The molecular mechanism of the resistance of CCA cells to the combined treatment is likely attributable to increased TGFβR-II expression that promotes cell survival via the TGFβR-II signaling pathway. Combination CpdA and cisplatin is, therefore, not recommended for the treatment of CCA. The combination of CpdA with other anticancer drugs for CCA treatment should be further investigated.


Nilaparvata lugens  resulted in reproductive sterility due to egg retention  and octopamine beta 2 (Octβ2) receptor in D. melanogaster, have also been linked to reproductive physiology in both insects  using X-tremeGENE® HP DNA Transfection Reagent . The cells were incubated for 72 h and used for the functional cell assay.”“A HEK293/CNG cell line that stably expresses a modified cyclic nucleotide-gated channel (CNG)  were raised in Dulbecco's Modified Eagle Medium Nutrient Mixture F12-Ham (DMEM/F-12)  supplemented with 10% heat-inactivated fetal bovine serum, 1% penicillin and streptomycin, and 100 μg/mL G418. The cells were incubated at 37°C in 5% COThe authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated.


Bladder carcinoma (BC) incidence and mortality rates are increasing worldwide. The development of novel therapeutic strategies is required to improve clinical management of this cancer. Aberrant protein expression may lead to cancer initiation and progression. Therefore, the identification of these potential protein targets and limiting their expression levels would provide alternative treatment options. In this study, we utilized a liquid-chromatography tandem mass spectrometry-based global proteomics approach to identify differentially expressed proteins in bladder cancer cell lines. A total of 3913 proteins were identified in this study, of which 479 proteins were overexpressed and 141 proteins were downregulated in 4 out of 6 BC cell lines when compared with normal human urothelial cell line (TERT-NHUC). We evaluated the role of UDP-N-acetylhexosamine pyrophosphorylase (UAP1) in bladder cancer pathogenesis. The silencing of UAP1 led to reduction in proliferation, invasion, colony formation and migration capability of bladder cancer cell lines. Thus, our study reveals UAP1 as a promising therapeutic target for bladder cancer. Bladder carcinoma (BC) is the most common malignancy of the urinary tract. Majority of the patients are diagnosed as non-muscle invasive NMIBC; 60%) with a recurrence rate of 50%–70% and about 20% of patients are diagnosed as muscle invasive bladder cancer (MIBC) . The ris0% with aSeveral FDA approved immune checkpoint inhibitors, such as atezoliumab, nivoluma, pembrolizmab, durlumab and avelumab, were used to treat bladder cancer, with response rates ranging from 15% to 25% ,8. MoreoProteomics is a high-throughput technology that can be used to identify differentially expressed proteins that potentially play an important role in pathogenesis. A rapidly evolving technology platform is known to have the potential to identify novel proteins involved in key biological processes in the cell that may serve as potential drug targets. Therefore, an unbiased investigation of the proteomic alterations in BC using high resolution mass spectrometry-based approach will aid in identifying alternative therapeutic targets.In this study, we investigated six BC cell lines , which were previously characterized as luminal, basal and non-type subtype . PreviouTM keratinocyte growth medium containing supplements . All the cell lines were maintained at 37 °C in a humidified 5% CO2 incubator.BC cell lines SW780, RT112, VMCUB1, T24, J82 and UMUC3 and normal human urothelial cell line TERT-NHUC (JTERT) were received from Prof. Jean Paul Thiery . The non-type , luminal  and basal (VMCUB-1) BC cell lines were cultured using DMEM medium supplemented with 10% Fetal Bovine Serum (FBS) and 1% Penicillin/Streptomycin. TERT-NHUC was grown in KGM goldg for 20 min. Protein estimation of the clarified lysate was done using bicinchoninic acid assay (BCA)  according to manufacturer’s instructions. Equal amount of protein from all the cell lines were reduced, using 10 mM Dithiotheritol (DTT) for 30 min at 60 °C, followed by alkylation using 20 mM Iodoacetamide (IAA) in dark for 10 min at room temperature. Reduced and alkylated protein lysate was subjected to acetone precipitation, using 7 volumes of pre-chilled acetone to remove SDS from the solution. Protein digestion was performed using sequencing grade trypsin  at 1:20 enzyme:substrateratio at 37 °C for 12–14 h. The tryptic peptides were vacuum dried and stored until further use.Cells were lysed in lysis buffer (50 mM triethyl ammonium bicarbonate (TEABC) pH 8.0, 2% SDS, 1 mM sodium orthovanadate, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM sodium fluoride), sonicated and centrifuged at 16,000× TMT labeling was done according to the manufacturer’s instructions, with minor modifications. Briefly, the TMT labels were reconstituted in 41µL of anhydrous acetonitrile (ACN) and trypsin digested peptide samples were reconstituted in 100 µL of 50 mM TEABC (pH 8.0). Tandem mass tag (TMT) labels 128N, 128C, 129C, 130N, 130C and 131 were used for labeling bladder cancer cell line samples, and TMT label 126 was used for TERT-NHUC control cell line. TMT labels were mixed with respective samples and the reaction was incubated for 1 h at room temperature. After incubation, the reaction was quenched with 8 µL of 5% hydroxylamine. The labeled peptides were lyophilized and subjected to basic pH reverse phase chromatography (bRPLC). Lyophilized samples were reconstituted in bRPLC solvent A  and were separated on XBridge BEH C18 Column , using solvent B . The column was equilibrated at 5% Solvent A from 0-to-5 min; the solvent B percentage was gradually increased from 5% to 55% in the following 60 min and then increased from 55–90% for the following 10 min, and then maintained at 90% Solvent B for 10 min before being decreased to 5% for 2 min on an Agilent 1100 Liquid Chromatography (LC) system, with a flow rate of 1 mL/min. A total of 96 fractions were collected over a period of 60 min gradient and later concatenated into 12 fractions. Fractionated samples were lyophilized before LC-MS/MS analysis.LC-MS/MS analysis was done on an Orbitrap Fusion mass spectrometer , interfaced with Easy-nLC1000 nanoflow LC system . The peptides were reconstituted in 0.1% formic acid and loaded onto a trap column . Peptides were resolved on an analytical column ), at a flow rate of 300 nL/min, using a linear gradient of 7–35% solvent B (0.1% formic acid in 100% ACN) over 100 min. The total run time including sample loading and column reconditioning was 120 min. Data-dependent acquisition with full scans in 350–1700 m/z range was carried out using an Orbitrap mass analyzer at a mass resolution of 120,000 at 400 m/z. Most intense precursor ions from a survey scan were selected for MS/MS fragmentation using higher energy collision dissociation (HCD) fragmentation, with 35% normalized collision energy and detected at a mass resolution of 30,000 at 400 m/z. AGC target value was set to 50,000, with maximum ion injection time of 150 ms. For MS3 analysis, synchronous precursor selection was enabled and 10 precursor ions were selected for fragmentation with 55% HCD collision energy.The mass spectrometry data were searched for using MASCOT (version 2.2.0) and SEQUEST search algorithms against Human RefSeq database (version 89), using Proteome Discoverer . The search parameters for both algorithms included carbamidomethylation of cysteine residues (57.02 Da), TMT modification at peptide N-terminus and lysine side chain as a fixed modification, oxidation of methionine (15.99 Da) as dynamic modification. MS/MS spectra were searched with a precursor mass tolerance of 10 ppm and fragment mass tolerance of 0.1 Da. Trypsin was specified as the protease, and a maximum of two missed cleavages were allowed. The data was searched against target decoy database and the false discovery rate was set to 1% at the peptide level. The TMT ratio for each peptide-spectrum match was calculated by the quantitation node.https://string-db.org; version: 11.0; University of Zurich, Zurich, Switzerland) [Interaction network was analyzed using the STRING functional protein association network (zerland) . The inpCell lines were cultured up to 70% confluency and cells were harvested using RIPA lysis buffer  and sonicated to extract proteins. Western blot analysis was performed as previously described, using 30 µg protein lysates . NitroceON-TARGETplusSMARTpool control siRNA and UAP1siRNA  were procured from Dharmacon , and BC cell lines  were transfected with controlandUAP1siRNA using RNAiMAX reagent , according to the manufacturer’s instructions. Cells were subjected to invasion assays and colony formation assays 48 h post-transfection, unless otherwise stated.Cell proliferation assays were carried out as described previously . Cells fColony formation assays were carried out as described previously, with minor modifications . UMUC3, The wound healing assays were performed as described previously . Briefly4 cells per 500 μL of media on the Matrigel-coated PET membrane in the upper compartment. The lower compartment was filled with complete growth media and the plates were maintained at 37 °C for 48 h. At the end of the incubation time, the upper surface of the membrane was wiped with a cotton-tip applicator, to remove non migratory cells. Cells that migrated to the lower side of membrane were fixed and stained using 4% methylene blue. The membranes were cut out from the transwell and mounted on glass slide using Dibutylphthalate Polystyrene Xylene (DPX) and covered with a microscope cover slip. The number of cells that penetrated was counted for six randomly selected viewing fields at 10x magnification.Invasion assays were performed in a transwell system (BD Biosciences), with Matrigel-coated filters and cellular invasion was evaluated after 48 h, as described previously . BrieflyStatistical analyses were carried out using GraphPad Prism version 6 . For cell culture-based assays  non-parametric test (Mann–Whitney U test) was used to assess statistical significance. For proteomics data, a statistical analysis was done using one-way ANOVA for individual proteins.We studied the protein expression of BC cell lines  and normal human urothelial carcinoma cell line (TERT-NHUC) using tandem mass tag (TMT)-based labeling technology, coupled with high-resolution mass spectrometry to identify differentially expressed proteins. The experimental workflow used for differential proteomic expression analysis in this study is depicted in We compared the protein expression pattern of the non-type/basal and luminal subtypes of bladder carcinoma cell lines. Unsupervised clustering based on both rows and columns was conducted with one minus Kendall’s correlation with average linkage and we observed that the luminal cell lines showed a distinct expression pattern A. We comWe identified 122 proteins to be overexpressed and 34 proteins to be downregulated across all the 6 BC cell lines, compared to the TERT-NHUC (control) cell line A. 479 prWe sought to identify the proteins that show higher expression in the basal and non-type subtype of bladder carcinoma, which might be attributed to the aggressive phenotype in these cells. We identified 39 proteins which were overexpressed in non-type and basal subtype . We seleWestern blotting analysis confirmed the overexpression of UAP1 in most of the BC cell lines compared with the TERT-NHUC cell line A. We selAfter demonstrating the role of UAP1 in cellular proliferation, we continued to study the role of UAP1 in the colony forming ability of the BC cells. UAP1 siRNA transfected cells and control siRNA transfected cells were seeded onto 6 well plates at 1000 cells per well density and allowed to grow for 7 days. The colonies formed were fixed, stained, counted under microscope and photographed for representation. Silencing of UAP1 led to reduction in the number of colonies formed and reduced the size of colonies formed in UAP1 knockdown non-type BC cells A,B. Our We further examined whether UAP1 has a role in BC invasiveness. Invasion assays were performed after silencing the endogenous expression of UAP1 using siRNA. BC cells transfected with UAP1 siRNA or control siRNA were transferred to the matrigel-coated PET membrane in the upper compartment and the plates were incubated at 37 °C for 48 h. We fixed and stained the migrated cells and counted the cells under a microscope. siRNA mediated silencing of UAP1 showed reduction in invasive capability of all the non-type BC cell lines C,D. Our Since the silencing of UAP1 reduces both the cell proliferation and colony forming ability of non-type subtype BC cells, we decided to explore whether UAP1 has any role in tumor cell motility. We performed scratch wound assays using non-type subtype BC cells with or without UAP1 silencing. Cells were grown till full confluence in 6 well plates and wounds were made in uniform size. After incubation for 8 h, control BC cells showed increased migration compared to UAP1 silenced BC cells. We took the images of wounds at 0 h and 8 h and calculated the distance covered by cells using Image J software I A,B. Our BC management is challenging due to its presentation, histological subtypes and high recurrence rates. Surgical intervention and systemic chemotherapy are the preferred treatment options by clinicians, due to lack of bladder cancer specific targeted therapies . The deeCancer cell lines provide abundant information about cancer cell biology and alterations in protein repertoire in disease pathogenesis. In this study, we identified 122 proteins that were overexpressed across all the BC cell lines. These proteins might play an important role in cancer pathogenesis. The protein-protein interaction network revealed interaction between MCM proteins which formed the hub proteins. These proteins include a panel of minichromosome maintenance complex component (MCM) proteins; namely MCM2, MCM3, MCM4, MCM5, MCM6 and MCM7 ,27. MCM Apart from MCM family proteins, we also identified thymidine kinase (TK1), aurora kinase B (AURKB), cell division cycle protein 20 (CDC20), DNA topoisomerase 2-α (TOP2A), exportin-5 (XPO5) and several other proteins significantly overexpressed in our study ,35,36,37Our proteomics data identified 39 proteins which were specifically overexpressed more than 2-fold in basal and non-type subtype cell lines compared to the luminal subtype (table A). Among the list, only few molecules have been previously studied in bladder cancer tumorigenesis. Annexin-6 was overexpressed in pT1 grade3 in urothelial carcinoma . ExpressAberrant glycosylation has been gaining importance as one of the hallmarks of cancer. Alteration in metabolism and dysregulation in cellular energetics leads to cancer progression . A tightIn our data, we identified that UDP-N-acetylhexosamine pyrophosphorylase (UAP1) was overexpressed more than 2-fold in all the basal and non-type subtypes of bladder cancer cell lines. Although the lack of biological replicates for proteomic analysis of each cell line is a limitation, UAP1 was chosen for further characterization, as it was overexpressed in four out of six cell lines analyzed. Functional assays to evaluate role of UAP1 in regulating proliferation, cell migration and colony formation were carried out in biological replicates across three different cell lines. UAP1 is the final enzyme in the hexosamine biosynthetic pathway (HBP), producing UDP-N-Acetylglucosamine (UDP-GlcNAc), which is a substrate for protein glycosylation. Abnormal protein glycosylation was previously shown to be associated with poor prognosis in bladder cancer patients . A previIn conclusion, comprehensive quantitative proteomic analysis of urinary BC cell lines identified the overexpression of UAP1 for the first time. Knockdown of UAP1 reduced BC cell proliferation, invasion, colony formation and migration ability of these cell lines. Our data suggests that UAP1 could be a promising therapeutic target for aggressive urinary bladder cancer.


Spacer design in spiral-wound membranes (SWMs) significantly affects the axial pressure drop in the flow channel but also the deposit layer removal. However, the effects of the spacer design and feed flow distribution in the module on the filtration performance have not yet been investigated during the highly fouling-susceptible fractionation of proteins from skim milk by SWMs. Therefore, a parallel spacer with no turbulence promotion and a less homogeneous feed flow distribution in the SWM was compared to a diamond spacer with regard to its impact on deposit formation and filtration performance. The experiments were conducted in a flat sheet test cell and in SWMs. The parallel spacer induced a more homogeneous deposit layer formation. However, no difference in filtration performance could be observed in the experiments with the test cell. Even though deposit layer formation dominates the microfiltration, its amount and spatial distribution could not be directly linked to the filtration performance. Furthermore, both spacers were assessed in SWM. Despite the higher crossflow velocity applicable in the more open channels of the parallel spacer, the performance of the parallel spacer was inferior to the diamond spacer. This was independent of the viscosity of the feed. Due to the high curvature of the membrane sheets close to the permeate collection tube, the cross-section of the flow channels in the SWM equipped with the parallel spacer was reduced. This resulted in a distinctly lower deposit layer control and performance, which could not be compensated by the resulting higher crossflow velocity far from the permeate collection tube. Deposit layer formation is one of the major obstacles in the application of membrane technology, since filtration performance is significantly affected by the fouling layer on the membrane surface. Thus, a number of works have investigated the causes and mechanisms of deposition ,2,3,4. ISince spacers define the flow channel in SWM and the local flow regime in close vicinity of the spacer strands, their effect on the homogeneity of the feed flow distribution and the resulting filtration performance should be investigated. It is known that the spacer causes an axial pressure drop, which increases disproportionally with the crossflow velocity . The preThis is especially relevant for the fractionation of skim milk by microfiltration (MF). In this process, the skim milk feed needs to be concentrated to obtain the optimum fractionation performance with regard to the feed composition . This seApart from the influence of the spacer design on the pressure drop, the spacer geometry also affects the flow pattern in the SWM. It was shown that the spacer geometry can cause eddies, i.e., an inhomogeneous wall shear stress distribution in spacer-filled channels . Thus, dSince the deposit layer acts as a secondary filtration layer on the membrane, the amount of deposited protein and thus, the geometry of the spacer should not only affect the flux but also protein permeation. The amount of deposited protein is referred to as the quantity of protein, which is bond to the membrane surface and can be detected by a method described previously .Our goal was to assess the deposit formation and filtration performance of a standard diamond spacer and a parallel spacer during skim milk MF. On the one hand, the influence of the form drag  on the pThe experiments were conducted on a filtration test cell and a pilot-scale filtration plant for SWMs. The 0.1-µm polyvinylidene fluoride (PVDF) membrane  was either used as a flat sheet or manufactured to a SWM using the same membrane material and the same spacer design in both cases.Details on the test cell filtration rig, the geometry of the filtration unit, and the experimental design are described elsewhere . In brieL) of the plant was set to 1.0 bar m−1 and the ΔpTM was adjusted. The axial pressure drop resulted in a mean crossflow velocity of 0.21 and 0.14 m s−1 (0.26 and 0.19 m s−1 considering the reduction of the flow channel by the presence of the spacer) for the diamond and the parallel spacer, respectively. The mean crossflow velocity v was calculated according to Equation (1) with the feed volume flow Prior to filtration, a new flat sheet membrane was soaked in deionized water for 24 h at 4 °C. The membrane was inserted into the test cell with the selective layer pointing downwards and then conditioned . A volume of 2.5 L of UF milk concentrate (CF 3) was heated to 25 °C and sheared for 30 min to restore the viscosity directly after the concentration. Afterwards, the concentrate was cooled to the filtration temperature of 10 °C. In order to flush the filtration plant, 0.5 L of concentrate were drained to remove the mixed phase before retentate and permeate were recirculated. The axial pressure drop , whereas the composition of all other solutes was equal to milk at CF 1. The composition of the major protein fractions can be found in Pasteurized skim milk  was obtained from a local dairy . For experiments on the pilot plant, skim milk was used and if necessary concentrated in a first processing step by MF as described above. Due to the low active membrane area, filtration experiments on the test cell were conducted with skim milk concentrated to CF 3 by UF  prior to the experiment. This milk UF-concentrate contained a three-fold amount of proteins (approximately 120 g Lr and in the permeate cp according to Equation (3):The permeation of whey proteins was assessed by determination of its main component β-lg. Permeation P was calculated by the concentration of β-lg in the retentate ctot and the volume of the spacer Vspacer [In this study, the influence of the spacer design on the spatial distribution of the deposit layer, the crossflow velocity distribution in the SWM, and the filtration performance at varying CF values was investigated. Two spacer geometries were tested: a parallel and a diamond-shaped spacer, both with a height of 46 mil (milli-inch) or 1.17 mm. The parallel spacer a was mad Vspacer :(4)ε=VtoIt has to be noted that the backbone divided the feed channel in two sections. Excluding the backbone of the spacer, the void volume fraction was 0.93.The diamond spacer was manufactured by Intermas  b. It is 2) was completely dissolved in 5 mL of a guanidine buffer  described by Dumpler et al. [The quantification of proteinaceous deposit layers was done by RP-HPLC. To do so, the deposit layer on part of the membrane . The calibration of the staining method can be found in Hartinger et al. .A spatially resolved analysis of the deposit layer formation was performed. Using ImageJ, the gray values were correlated to a color spectrum. The spectrum referred to the correlation function depicting the deposit layer´s spatial distribution. The lookup table (LUT) can be found elsewhere . In a fuSince false color and topographic images are hard to compare in terms of deposit layer formation, the gray value and thus the amount of deposit layer were evaluated parallel and orthogonally to the spacer strands. A pattern of parallel lines describes the interspace between the spacer strands .The gray values were transformed into the amount of deposited protein by a correlation function. As a statement of place, the relative length (Rl) and relative width (Rw) were used. A relative length or relative width of 1 refers to the distance between the centers of two spacer strands or ribs in the direction parallel and perpendicular to the spacer strand or ribs, respectively. Note that the absolute lengths of the relative length units differ between the spacer designs.Data was plotted using OriginPro 2017G . The program also fitted the data with an exponential function using a Levenberg Marquardt as an iteration algorithm or via b-spline. Error bars show the standard deviations of two individual experiments. The plots of the graphical analysis are a single determination.Deposit layer formation is known to reduce the filtration performance of membranes tremendously. Therefore, we investigated the influence of the spacer geometry on the spatial distribution of protein deposition on the membrane surface during skim milk filtration. The deposit layer mainly consisted of casein compare  as alrea−1, beyond which no further differentiation of the amount of protein is possible. The cumulative distribution can be found in the −2. These areas are randomly distributed along the membrane-spacer contact zone. This allows two conclusions: Firstly, no full contact between the membrane and spacer exists. Thus, parts of the membrane in the contact zone close to the spacer contribute to the filtration. Due to the low crossflow velocities in the contact zone, flux and protein permeation are expected to be rather low compared to the rest of the membrane. Additionally, excessive mixing between the branch currents streaming in the gap between the spacer ribs is not to be expected. Secondly, a removal of the spacer from the membrane prior to staining does not detach parts of the deposit layer. Otherwise, sharp edges would be expected. Hence, the deposition patterns shown in In the areas in which the spacer is in direct contact with the membrane, the deposit layer is predominantly low. As no protein was detected to be remaining on the spacer after removal from the membrane, this indicates that no filtration took place below the spacer ribs. Otherwise, protein deposition would be expected to be intense, since particle-removing forces are low due to a lack of crossflow below the spacer ribs. Nevertheless, certain areas in the membrane-spacer contact zone are fouled with up to 10 g m−2 of protein are deposited, since a boundary layer forms. The crossflow velocity is reduced due to frictional energy losses caused by the spacer. Thus, the wall shear stress decreases toward the spacer, causing a more intense particle deposition.The effect of the spacer can also be observed close to the contact zone between spacer rib and membrane. More than 10 g m−2. Due to the lack of baffles, the flow is undisturbed. Wall shear stress values are expected to be constant along the flow lines parallel to the ribs, causing a homogeneous deposition. This can be concluded from Except for the areas close to the spacer, the deposit layer is homogeneously distributed all over the membrane with about 2 to 4 g mContrary to the observations along the relative length axis, inhomogeneities in the deposit layer formation perpendicular to the feed flow can be observed b. Close It was found that the deposit layer pattern of the parallel spacer is homogeneous along the flow path except for areas close to the spacer contact zone. This is due to the lack of flow restrictions and the lack of eddies. In turn, the crossflow velocity and wall shear stress can be assumed to be practically constant in the areas of low fouling. Furthermore, we assume that friction causes lower crossflow velocities close to the spacer. This increases the deposition of proteins since wall shear stress values are lower compared to the areas of undisturbed flow. Therefore, we can confirm that the local flow regime directly affects deposition.Comparable to the approach for the parallel spacer, we analyzed the deposit layer distribution caused by the diamond spacer in the test cell. The results on the parallel spacer show the influence of the flow regime on the deposition of proteins. This can also be expected from the diamond spacer as it causes eddies in the feed flow on a small scale. In this study, we investigated a non-woven spacer with two layers of spacer strands orthogonal to one another. Only one layer of spacer strands is in contact with one membrane surface and spatial crossflow velocities and wall shear stresses differ strongly in the spacer net. The protein deposition also changes along the flow path, which can be seen in Since the wall shear stress is also low in front of the spacer strand with membrane contact, much protein is deposited in this area as well. In the membrane–spacer contact zone, much lower amounts of proteins are deposited, as already shown with the parallel spacer, due to the lack of filtration.−2 determined by HPLC). Due to a narrowing of the flow channel, the crossflow velocity  increases. In turn, the wall shear stress is increased, reducing deposit layer formation. Koutsou et al. [Apart from those areas adjacent to or covered by the spacers, the deposit layer distribution on the membrane in the area between the spacer strands is also inhomogeneous. In the areas close to the spacer without membrane contact, deposit layer formation was found to be lower than average . Thus, the amount of deposited protein decreases to a lowest value of 2 g m−2 and the width of the area with a deposition above 10 g m−2 decreases. Apart from that, the deposit layer decreases with distance from the spacer strands with membrane contact  of 0.5 a, the am contact b. As repThe results show that the diamond-shaped spacer causes a characteristic deposit layer pattern on the membrane surface directly reflecting the spacer configuration. Thereby, areas with high crossflow velocity and wall shear stress, respectively, are less susceptible to fouling, but at the same time, areas of very poor deposit control exist.The cumulative fouling distribution of the diamond can be found in the As reported above, the spacer design significantly affects the deposit layer pattern. As the deposit layer is known to dominate the filtration during skim milk fractionation, we investigated the influence of the spacer design on the filtration performance. For this purpose, we analyzed flux and protein permeation of the parallel and diamond-shaped spacer during skim milk fractionation to obtain a casein and a whey protein fraction in a flat channel test cell. −1 (mean crossflow velocity of 0.5 m s−1), the pressure drop was 1.45 and 0.65 bar m−1 for the diamond and the parallel spacer, respectively. This can also be attributed to frictional losses. As already described by Da Costa et al. [As expected, the pressure drop increases with increasing crossflow velocity due to enhanced friction. Apart from that, the parallel spacer caused a significantly lower pressure drop. At 80 L ha et al.  and Kavia et al. , the feeIt was shown by Samuelsson et al.  that higThe expectation was that the more homogeneous and lower deposition resulting from the parallel spacer leads to a better filtration performance in terms of flux and protein permeation compared to the diamond spacer. TM, the flux increased until the limiting flux was reached at about 5 L m−2 h−1. Interestingly, and against expectation, the flux did not differ between the parallel and the diamond spacer when operated at the same pressure drop of 1.0 bar m−1 even though the feed flow differed distinctly between the two spacer designs. In contrast to the pressure drop in water, the diamond spacer allowed a higher crossflow velocity at CF 3 compared to the parallel spacer .With increasing ΔpSimilar to the flux, protein permeation in the test cell was also not dependent on the spacer design .TM was 32% and 30% for the diamond and the parallel spacer, respectively, and decreased to 8% and 8% at 2.0 bar. Again, the spacer design had no decisive influence on the filtration performance when they were operated at the same pressure drop in the flat sheet test cell. Even though the deposit layer formation was distinctly different between the two spacer geometries, no significant effect on the filtration performance was observed in our study. Presumably, the effect of the spacer geometry on the axial pressure drop counterbalances the differences in the mass flow coefficient of both spacer geometries. Since no effect of the spacer design was observed within the test cell, another set of experiments was conducted within SWMs with their curved feed flow channels and more complex architecture.As expected, the protein permeation decreased with increasing transmembrane pressure due to enhanced deposit layer compression . The perThe observations on the deposit layer pattern and amount did not go along with the obtained results from the test cell. Apart from that, the curvature of the SWM could not be depicted by the test cell. Therefore, the test cell experiments were also conducted in SWMs at concentration factors of 1 to 3. For the application of spacers, frictional losses in the membrane module have to be traded off against the positive effect of turbulence promotion on the mass transfer. In this regard, a change in viscosity is expected to shift the geometrical optimum of the spacer geometry from a more turbulence-generating shape  to a less turbulence-generating shape .The axial pressure drop at varying CF values is shown in The expectation was that the lower amount of flow constrictions would result in a significantly lower pressure drop with the parallel spacer compared to the diamond spacer. This can be observed for low concentration factors. However, the difference between both spacer geometries becomes lower with increasing feed concentration. This means the main positive aspect of the parallel spacer having a lower axial pressure drop and allowing higher crossflow velocities vanishes at high CF values. The difference in behavior between the test cell and the SWM with regard to the axial pressure drop is probably due to the differences in the feed inlet (compare ).In contrast to the results of the test cell, the diamond spacer showed a higher flux in the SWM, not only at low CF but also at higher CF b. This iTherefore, the geometry of the flow channels in the SWM and radial distribution of the crossflow velocity have to affect the filtration performance. In the outer part of the module, the membranes and the spacer form a flow path for the feed. In the inner part of the module, however, the spacer is pressed into the membranes. Due to indentation of the spacer into the membrane, the active membrane area decreases. This has already been described by Karabelas et al. . AdditioTM at CF 1–3.Due to the poor deposit layer control in the inner channels of the SWM, the flux is reduced. The higher crossflow velocity in the outer parts of the module cannot compensate for that and the overall performance of the module is lower compared to the module with the diamond spacer. This is affirmed by the results on the protein permeation. It is shown in TM of 0.5 bar), respectively. At CF 3, however, the permeation of the parallel spacer was distinctly lower with 39% compared to 48% for the diamond spacer. Although the mean crossflow velocity at CF 3 was similar in both SWMs (compare The discrepancy in protein permeation between the parallel and the diamond spacer becomes more intense with increasing CF, i.e., higher feed viscosity. At CF 1, the permeation of β-lg was almost similar with both spacers with 56% and 60% for the diamond and the parallel spacer (at a Δp compare a, the crThe results of this study showed that staining by CBB is an effective method to characterize the deposit layer with regard to its spatial distribution. Although the sensitivity of the staining method is limited at high amounts of deposited material, the staining method can be used to visualize the effect of the spacer design on the deposition and thus can be an indicator of the wall shear stress distribution. This can be used for the verification of CFD results. However, the pattern on the membrane and the amount of deposited protein did not correlate with the filtration performance of the SWM during milk protein fractionation. This could be attributed to the observation that the parallel spacer does not keep the inner flow channels of the SWM open. Thus, the deposit layer control in the inner parts of the SWM is poor. Additionally, the active membrane surface is reduced and compensates the possible advantages of the more homogeneous deposit layer formation of the parallel spacer in comparison to the diamond spacer.Furthermore, our results confirmed that the curvature of the feed channel and its influence on the radial distribution of the crossflow velocity have to be considered in the assessment of spacers. Although both spacers had an equal filtration performance in the flat channel test cell, the performance of the diamond spacer was significantly better in the SWM. Apart from the turbulence promotion, the more homogeneous feed flow distribution in the radial direction of the SWM was positive to the performance during skim milk protein fractionation. In this regard, investing effort to induce a homogeneous feed flow distribution in SWMs seems to be beneficial to the module performance.